{ "40659489": { "pmid": "40659489", "title": "A lyophilized open-source RT-LAMP assay for molecular diagnostics in resource-limited settings.", "abstract": "A critical bottleneck for equitable access to population-scale molecular diagnostics is the limited availability of rapid, inexpensive point-of-care tests, especially in low- and middle-income countries. Here, we developed an open-source reverse transcription loop-mediated isothermal amplification (RT-LAMP) molecular assay for pathogen detection. It is based on nonproprietary enzymes, namely, HIV-1 reverse transcriptase, Bst LF DNA polymerase, and UDG BMTU thermolabile uracil-DNA glycosylase. Formulated as liquid or lyophilized reaction mixtures, these reagents enable sensitive colorimetric detection of respiratory samples without the need for prior nucleic acid isolation. We evaluated our lyophilized RT-LAMP assay on clinical samples with suspected COVID-19 infection, demonstrating high sensitivity and 100% specificity compared with the gold-standard RT-qPCR. Reaction performance was unaffected by prolonged storage of lyophilized reagents at ambient or elevated temperatures. As a proof of concept, we evaluated the robustness and ease of use of lyophilized RT-LAMP reaction mixes through independent laboratory testing of COVID-19 samples in Ghana. Overall, our open-source RT-LAMP assay provides a flexible and scalable point-of-care test that can be adapted for rapid detection of various pathogens in resource-limited settings.", "journal": "Life science alliance", "year": "2025", "doi": "10.26508/lsa.202403167", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Matl M", "dirs": [ "A", "C" ] }, "42226661": { "pmid": "42226661", "title": "Towards deployable CRISPR-based nucleic acid detection.", "abstract": "Deployable diagnostics are necessary for the control and treatment of infectious diseases, with significant unmet needs revealed during the COVID-19 pandemic. Nucleic acid diagnostics remain among the most sensitive and specific forms of detection, yet their reliance on laboratory equipment and trained personnel limits their deployment in resource limited settings. CRISPR-based diagnostics are uniquely positioned to enable rapid, affordable, and highly accurate nucleic acid testing at both the point-of-care and the point-of-need. In this review, we discuss advances toward deployable CRISPR-based diagnostics. We begin by examining innovations in sample processing methods, emphasizing strategies that reduce equipment requirements and enhance compatibility across diverse sample types and pathogens. We then explore developments in one-pot isothermal and amplification-free approaches, comparing the benefits and tradeoffs associated with each, as well as multiplexing strategies for simultaneous detection of multiple pathogens. Finally, we consider additional factors that impact assay deployability, including reagent lyophilization to minimize cold chain dependence and readout technologies that enable detection in resource-limited settings. We conclude by outlining remaining challenges and opportunities for future progress.", "journal": "Progress in biomedical engineering (Bristol, England)", "year": "2026", "doi": "10.1088/2516-1091/ae6e0b", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't", "Research Support, N.I.H., Extramural" ], "first_author": "Guo A", "dirs": [ "A" ] }, "40985812": { "pmid": "40985812", "title": "An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of Haemophilus influenzae.", "abstract": "Timely and accurate diagnosis is essential for the effective treatment of Haemophilus influenzae (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. The aim of this study was to establish a one-pot detection device for HI DNA detection using CRISPR/Cas13a that would be applicable for POCT. We established a two-step polymerase chain reaction (PCR)-CRISPR assay, a two-step recombinase-aided amplification (RAA)-CRISPR assay, and a one-pot RAA-CRISPR assay. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct an extraction-free one-pot detection device, named EFORCA (extraction-free one-pot RAA-CRISPR/Cas13a assay). Validation was performed on 90 simulated samples and 77 clinical samples. The two-step PCR/RAA-CRISPR assay for HI DNA detection was established and optimized, with a detection limit of 1 copy/μL and 100% specificity. The sensitivity and specificity of the two-step PCR-CRISPR assay for the 90 simulated clinical samples were 96.7% and 95%, respectively, and for the 77 clinical samples, 97.5% and 100%, respectively. The one-pot RAA-CRISPR assay and the EFORCA detected 50 and 25 CFU/mL HI in bacterial suspensions within 30 min, respectively. The valuation results for 77 clinical samples demonstrated that the sensitivity of both methods was 95%, which was greater than the sensitivity of quantitative real-time PCR (qPCR) (92.5%) with the same specificity of 100%. We developed an extraction-free one-pot RAA-CRISPR/Cas13a assay for HI detection, which effectively performs a POCT test and is a valuable tool for the early detection and monitoring of HI infection.IMPORTANCETimely and accurate diagnosis is essential for the effective treatment of Haemophilus influenzae (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. In this study, sensitive two-step polymerase chain reaction (PCR)-CRISPR and recombinase-aided amplification (RAA)-CRISPR assays were developed, followed by the establishment of the one-pot RAA-CRISPR assay through integration of RAA, CRISPR, and rapid lysis for HI detection. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct the extraction-free one-pot RAA-CRISPR/Cas13a assay (EFORCA). With its high sensitivity, specificity, rapid turnaround time, and operational simplicity, this assay shows great potential as a practical diagnostic tool for HI infection in small laboratory settings.", "journal": "Journal of clinical microbiology", "year": "2025", "doi": "10.1128/jcm.00535-25", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Cao Y", "dirs": [ "A" ] }, "41087332": { "pmid": "41087332", "title": "A fully automated rotary microfluidic platform for high-throughput multiplex detection of respiratory tract pathogens.", "abstract": "Rapid, high-throughput, timely, multiplex diagnosis of respiratory-tract infections still relies on laboratory infrastructure, sequential assays, and trained personnel, thereby delaying targeted therapy and outbreak containment. In this study, a Fully Automated rotary microfluidic platform (FA-RMP) for high-throughput multiplex respiratory tract pathogens detection was presented. FA-RMP enables a true \"sample-in, result-out\" workflow through the integration of swab lysis, reagent partitioning, lyophilized reverse transcription loop-mediated isothermal amplification (RT-LAMP), and moving-probe fluorescence read-out, all encapsulated with a disposable microfluidic cartridge and paired with a 9 kg, four-channel benchtop reader. The FA-RMP enables parallel processing of 16 independent reactions within 30 min, supporting simultaneous detection of up to 4 distinct clinical samples. Analytical validation using serially diluted Mycoplasma pneumoniae (MP) DNA established a limit of detection (LoD) of 50 copies µL-1 and a log-linear correlation between threshold time and template load (R2 = 0.9528). Testing with eight non-target respiratory pathogens yielded no amplification, confirming high analytical specificity. FA-RMP successfully detected the clinical samples with influenza A, influenza B, and MP, further demonstrating its robust multiplex detection capability. By integrating automated sample preparation, multiplex isothermal amplification and quantitative detection into a portable, high-throughput system, the platform delivers laboratory-grade performance at the point of care, serving as a scalable tool for routine respiratory pathogens screening and rapid epidemic response.", "journal": "Microsystems & nanoengineering", "year": "2025", "doi": "10.1038/s41378-025-01044-9", "ptypes": [ "Journal Article" ], "first_author": "Zhang D", "dirs": [ "A", "C" ] }, "40956287": { "pmid": "40956287", "title": "Enhanced One-Pot Dual-CRISPR-Based Assay Lyophilized on a 3D-Printed Disc for Field-Deployable Multiplex Bacteria Detection.", "abstract": "One-pot CRISPR-based detection combining recombinase polymerase amplification (RPA) enables rapid and accurate nucleic acid testing but faces challenges in performance, multiplexing, and field-ready lyophilization. Here, an enhanced one-pot, helicase-assisted RPA (hRPA)-combined, dual-CRISPR/uAsCas12a (EOD-CRISPR) assay is described which can be lyophilized on a 3D-printed microfluidic disc to achieve field-deployable multiplex bacteria detection. In EOD-CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D-printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD-CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point-of-need testing, EOD-CRISPR-lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e., Bacillus cereus, Salmonella enterica, Staphylococcus aureus, and Escherichia coli O157:H7), the onsite detection platform is validated and the sensitivity (80%-88.9%) and specificity (92.3%-100%) are comparable to those of standard PCR methods. Therefore, the field-deployable multiplex EOD-CRISPR assays holds great potentials for onsite bacteria detection and beyond.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2025", "doi": "10.1002/advs.202509355", "ptypes": [ "Journal Article" ], "first_author": "Shen Y", "dirs": [ "A", "C" ] }, "40779684": { "pmid": "40779684", "title": "Programmable 3DP microfluidic bio-reaction system: automated LAMP-on-a-chip.", "abstract": "Point-of-care (PoC) devices have revolutionized healthcare by enabling remote diagnostics and therapeutics, with microfluidic systems playing a pivotal role in their advancement. This study focuses on the detailed engineering and characterization of three-dimensional hydrophobic valves to form novel programmable bio-reaction reservoirs. Using 3D-printed soft lithography, we meticulously investigated the effects of channel dimensions and surface properties on the burst pressures of these reservoirs, which ranged from 6.4 to 44.8 mbar. The bio-reaction reservoirs were demonstrated in both series and parallel configurations, offering versatile platforms for the miniaturization and automation of biological processes. Our findings highlight the capability of these reservoirs to program flows in a variety of fluid samples, including water, blood and serum. Additionally, a portable pressure pump was developed to leverage the functionality of these hydrophobic valves, enabling precise control of fluid dynamics in PoC applications. The study culminated in the design of a microfluidic chip integrating two consecutive reservoirs for the PoC execution of loop-mediated isothermal amplification (LAMP) for detection of the Mpox virus. Primers were lyophilized within the bio-reservoirs, and the system successfully enabled visible colorimetric detection via the LAMP assay.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d5lc00003c", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Birtek MT", "dirs": [ "A", "C" ] }, "41979265": { "pmid": "41979265", "title": "Cell-Free Protein Synthesis-Based Biosensing Platforms for Clinical Diagnostics.", "abstract": "Cell-free protein synthesis (CFPS) is an in vitro platform that enables rapid protein production using cell extracts, energy sources, and genetic templates. Owing to its fast response, elimination of cell culture, open reaction environment, lyophilization compatibility, and high programmability, CFPS has emerged as a versatile engine for diagnostic sensing. Recent advances have integrated CFPS with modular genetic circuits, CRISPR-based detection, isothermal amplification, and portable formats such as paper-based devices and microfluidic chips, enabling sensitive and specific detection of viral nucleic acids, pathogen antigens, and small-molecule targets. These platforms further support multiplexed and point-of-care testing, substantially reducing assay time, cost, and infrastructure requirements. Despite remaining challenges in biosensor design for novel targets, analytical sensitivity in complex samples, batch-to-batch reproducibility, and clinical translation, continued engineering optimization is rapidly improving CFPS performance and robustness. This review summarizes the fundamental principles of CFPS, its major technological platforms, recent progress in diagnostic applications, and key challenges and opportunities for future development.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c04869", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sun X", "dirs": [ "A", "C" ] }, "40705463": { "pmid": "40705463", "title": "Lyophilised Reverse Transcriptase and Polymerase for Point-of-Care Diagnostics of SARS-CoV-2.", "abstract": "Early diagnosis of pathogens is key to reducing their spreading and to preventing severe health risks. The COVID-19 pandemic showed us the need for rapid point-of-care tests. Here, we describe the preparation of an amplification master mix for point-of-care diagnostics. Therefore, two off-patent amplification enzymes were designed, expressed and purified. The preparation of the key components enables independence from delivery issues, manufacturer portfolio and product information's. For long-term storage and cold-free transport, our fabricated amplification mix was lyophilised. Finally, we applied our lyophilised master mix on an integrated point-of-care diagnostic system and could detect 10 copies/μL COVID-19 RNA. The combination of stable, cold-free reagents with the mobile and low-cost device will allow molecular diagnostics of pathogens in a field or home setting.", "journal": "Microbial biotechnology", "year": "2025", "doi": "10.1111/1751-7915.70200", "ptypes": [ "Journal Article" ], "first_author": "Mutter N", "dirs": [ "A" ] }, "40076433": { "pmid": "40076433", "title": "Optimization and Benchmarking of RT-LAMP-CRISPR-Cas12a for the Detection of SARS-CoV-2 in Saliva.", "abstract": "Resource-limited settings and supply chain difficulties faced throughout the COVID-19 pandemic prompted the development of rapid and alternative methods of detecting SARS-CoV-2. These methods include reverse-transcription loop-mediated isothermal amplification (RT-LAMP), reverse-transcription recombinase polymerase amplification (RT-RPA), and CRISPR-Cas12a fluorescence detection. We describe RT-LAMP, RT-RPA, and CRISPR-Cas12a assays for the detection of the N and E-gene amplicons of SARS-CoV-2 and the optimization of various assay components, including incubation temperatures, Cas12a enzymes, reporter molecules, and the use of a lyophilized RT-LAMP master mix. We also describe the testing of a one-tube RT-LAMP-CRISPR-Cas12a assay. The one-tube assay showed promise in reducing hands-on time and improving time-to-result. We found no improvements in assay sensitivity with RT-RPA, but did achieve detection at a lower copy number with the lyophilized RT-LAMP master mix compared to liquid reagent (50 vs. 100 copies at 20 min). When used to detect the presence of SARS-CoV-2 RNA in clinical saliva samples from 75 infected patients, the discriminatory ability of the optimized RT-LAMP-CRISPR Cas12a assay was found to be comparable with RT-qPCR, with a minor reduction in sensitivity.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms26051806", "ptypes": [ "Journal Article" ], "first_author": "Lynch CRH", "dirs": [ "A", "C" ] }, "42153664": { "pmid": "42153664", "title": "Rapid Cas13a-based penA genotyping for cefixime susceptibility in Neisseria gonorrhoeae.", "abstract": "Antimicrobial resistance in Neisseria gonorrhoeae is an urgent public health threat. Resistance-guided therapy can assure appropriate treatment and reintroduce alternative therapeutic options by identifying genetic predictors of resistance. Mosaicism at codons 375-377 of the penA gene is associated with cefixime resistance. Rapid, field-deployable assays for predicting cefixime susceptibility are lacking. We used a machine-learning algorithm to develop a CRISPR Cas13a-based assay to detect the absence of mosaicism at codons 375-377 of the penA gene, combined with isothermal amplification in a single reaction. We integrated the assay onto a portable fluorescence-based platform. We evaluated performance using cultured isolates and compared results with PCR genotyping and phenotypic antimicrobial susceptibility testing. We also assessed the feasibility of reagent lyophilization of an existing porA N. gonorrhoeae detection assay, which can support cold-chain-independent deployment. Among 40 N. gonorrhoeae isolates, the Cas13a penA assay demonstrated 100% concordance with PCR genotyping and 92.5% (95% confidence interval 79.6%-98.4%) concordance with phenotypic cefixime susceptibility. Median time to detection of the penA mosaic assay was 12 min (interquartile range [IQR] 5 min). The lyophilized porA N. gonorrhoeae detection system detected all 12 isolates with a median time to detection of 45 min (IQR 40-45) compared to 45 min (IQR 35-50) for the positive aqueous control, although peak fluorescence was higher for the aqueous control (P < 0.01). The Cas13a assay was rapid and demonstrated strong correlation with genotypic and phenotypic cefixime susceptibility in N. gonorrhoeae, while a lyophilized assay retained functionality. Antimicrobial resistance in Neisseria gonorrhoeae is an urgent threat to public health. Recent reports have highlighted the continued rise in ceftriaxone resistance, our last-line empiric treatment option. Molecular assays that detect the genetic determinants of resistance can improve antibiotic stewardship and increase the therapeutic options for cases of gonorrhea. As a result, those tests can reduce the pressure toward the emergence of ceftriaxone resistance. However, most molecular assays cannot be deployed in low-resource settings due to a lack of infrastructure. We report on the development of a point-of-care assay for predicting resistance to an oral first-line treatment option, cefixime. The assay provided results in under 30 min and demonstrated strong correlation with phenotypic and genotypic resistance. Furthermore, we report on the proof-of-concept freeze-drying of assay reagents, which could permit cold-chain-independent use. Collectively, our study provides the groundwork for developing and deploying such molecular resistance assays in low-resource settings.", "journal": "mSphere", "year": "2026", "doi": "10.1128/msphere.00182-26", "ptypes": [ "Journal Article" ], "first_author": "Nguyen THY", "dirs": [ "A" ] }, "41494257": { "pmid": "41494257", "title": "Novel nucleic acid detection based on the guide-independent nuclease activity of TbAgo.", "abstract": "In addition to the well-characterized programmable nucleic acid cleavage activity, prokaryotic Argonaute proteins (pAgos) possess a guide-independent nuclease activity capable of degrading nucleic acids into small fragments in the absence of exogenous guide strands, a function commonly referred to as \"chopping\", which remains poorly understood. Here, we report the robust chopping activity of Thermus brockianus Argonaute (TbAgo) and leverage this activity to develop chop-NAD (chopping-based Nucleic Acid Detection), a minimal guide-free platform for sequence-specific nucleic acid sensing. When integrated with loop-mediated isothermal amplification (LAMP), chop-NAD enables sensitive, specific, and multiplexed detection of Bordetella pertussis, Bordetella parapertussis, and Mycoplasma pneumoniae in a single tube, with detection limits of 2.38 genome equivalents (gEq)/μL, 2.04 gEq/μL, and 59.7 copies/μL, respectively. Furthermore, a paraffin-encapsulated, lyophilized one-pot format facilitates contamination-resistant, sample-to-answer testing, showing 100 % concordance with clinical PCR results. This one-pot chop-NAD platform offers reduced reagent costs, simplified assay adaptation, improved biosafety, and a streamlined workflow, collectively establishing it as a versatile and scalable diagnostic platform with broad potential for point-of-care testing.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.118370", "ptypes": [ "Journal Article" ], "first_author": "Fang M", "dirs": [ "A", "C" ] }, "41745936": { "pmid": "41745936", "title": "Advances in CRISPR-Cas12a/13a-Based Nucleic Acid Detection for Porcine Viral Diseases: A Comprehensive Review.", "abstract": "The global swine industry suffers persistent economic losses and health challenges due to major viral pathogens such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine circovirus (PCV). Traditional diagnostic methods, including virus isolation, serology, and quantitative PCR (qPCR), are limited by time, equipment requirements, and field applicability. Recent advances in CRISPR-based diagnostics, particularly those leveraging the collateral cleavage activity of Cas12a and Cas13a, have enabled rapid, sensitive, and field-deployable nucleic acid detection. This review outlines the principles of CRISPR-Cas12a/13a systems, their integration with isothermal amplification techniques, and their application in detecting major swine viruses. Cas12a-based platforms (e.g., DETECTR) and Cas13a-based systems (e.g., SHERLOCK) achieve detection limits as low as single-copy/μL within 25-60 min at 37 °C, offering high specificity and compatibility with visual readouts. Applications include ASFV, PRRSV, CSFV, PCV, foot-and-mouth disease virus (FMDV), porcine rotavirus (PoRV), and porcine parvovirus 7 (PPV7). Despite significant advances, challenges remain, notably the reliance on nucleic acid extraction and the need for fully integrated \"sample-in, result-out\" systems. Ongoing innovations in extraction-free methods, lyophilized reagents, and multiplex detection will strengthen the role of CRISPR diagnostics in swine disease surveillance and control. From an application standpoint, the technology offers a low-capital, field-adaptable alternative to qPCR, with its value proposition rooted in early outbreak containment and loss prevention. Its adoption pathway is expected to vary across production systems-serving as a sentinel tool in intensive settings, a leapfrogging solution in rapidly intensifying regions, and through shared-service models in resource-limited contexts. However, translation to routine use still requires overcoming standardization hurdles, regulatory validation, and workflow integration.", "journal": "Veterinary sciences", "year": "2026", "doi": "10.3390/vetsci13020141", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhang X", "dirs": [ "A" ] }, "40078652": { "pmid": "40078652", "title": "Human Umbilical Cord Mesenchymal Stem Cells-Derived Exosomes Attenuates Experimental Periodontitis in Mice Partly by Delivering miRNAs.", "abstract": "Periodontitis is the most common non-communicable disease in humans. The main challenge in the treatment of periodontitis is to effectively control periodontal inflammation and promote tissue repair. Human umbilical cord mesenchymal stem cells-derived exosomes (hucMSCs-exo) have been reported to modulate inflammatory responses and promote tissue repairment mainly through miRNAs in several diseases. However, the effect of hucMSCs-exo on periodontitis remains unknown. In this study, we hypothesized that hucMSCs-exo could inhibit bone destruction in periodontitis mice. In this study, we constructed and characterized the exo@H drug delivery platform. Lipopolysaccharide was used to construct an inflammatory microenvironment in vitro to detect MC3T3-E1 cells proliferation and bone regeneration capacity. Ligation induced to construct an experimental periodontitis mouse model. The distance of the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) was measured for bone resorption evaluation. Hematoxylin-eosin (H&E) staining and Tartrate resistant acid phosphatase (TRAP) staining were used to observe periodontal tissue changes. MicroRNA (miRNA) sequencing was used to detect differential genes and for bioinformatics analysis. Real-time quantitative polymerase chain reaction (qRT-PCR). WB assay and dual luciferase assay were used to further validate the screened differentially expressed miRNAs and the targeted binding relationship with the corresponding target genes. We found that lyophilized hucMSCs-exo promoted the proliferation and osteogenic differentiation of MC3T3-E1 cells, and showed more significant proliferative and osteogenic differentiation abilities in combination with the hydrogel (P < 0.05). Using periodontitis mice, bone resorption evaluation revealed a significant reduction in alveolar bone resorption in the exo@H group compared to the hydrogel group (P < 0.01), and exo@H was able to reduce the inflammatory response of periodontal tissues and the number of osteoclasts on the surface of the alveolar bone compared to the hydrogel group. Moreover, 59 miRNAs were upregulated, such as let-7f-5p and miR-203-3p, which positively targeted IL-13 and Nit2, respectively. These results suggest that exo@H provides protection against periodontitis partly by delivering miRNAs to periodontal tissue. Our results confirm the feasibility of the exo@H delivery platform we constructed and the effectiveness of its use for periodontitis treatment, and this study provides a promising approach for the treatment of periodontitis via miRNA.", "journal": "International journal of nanomedicine", "year": "2025", "doi": "10.2147/IJN.S502192", "ptypes": [ "Journal Article" ], "first_author": "Li K", "dirs": [ "A" ] }, "41581987": { "pmid": "41581987", "title": "High-throughput screening of biomolecules using cell-free systems.", "abstract": "High-throughput screening (HTS) has revolutionized the identification and evaluation of biomolecules by enabling the parallel testing of large libraries of compounds, nucleic acids, and proteins against biological targets. Traditionally conducted in live cells, HTS faces limitations such as cellular toxicity, metabolic interference, and regulatory constraints. Cell-free systems (CFS), which operate in vitro using reconstituted transcription-translation machinery, have emerged as powerful alternatives. These systems circumvent the constraints of cellular physiology, allowing for rapid and tunable expression of biomolecules directly from DNA or RNA templates. This chapter explores the principles, platforms, and applications of CFS-based HTS, highlighting its transformative impact on synthetic biology, drug discovery, diagnostics, and protein engineering. Several cell-free systems are detailed, including those derived from E. coli, wheat germ, rabbit reticulocytes, and the defined PURE system. The integration of CFS with high-throughput platforms such as microplates, droplet microfluidics, and paper-based devices enables cost-effective, scalable, and multiplexed assays. Analytical readouts, including fluorescence, luminescence, mass spectrometry, and digital PCR, provide real-time, sensitive detection of biochemical outputs. Furthermore, automation and machine learning are increasingly incorporated through robotic liquid handling and data-driven DBTL cycles, accelerating discovery and design processes. Despite challenges such as high reagent costs and limited post-translational modifications, innovations such as lyophilized CFS kits, artificial cells, and AI-integrated closed-loop platforms are expanding the frontiers of HTS. Altogether, CFS-based HTS offers a flexible, rapid, and accessible approach for next-generation biomolecular screening and therapeutic development.", "journal": "Progress in molecular biology and translational science", "year": "2026", "doi": "10.1016/bs.pmbts.2025.11.001", "ptypes": [ "Journal Article", "Review" ], "first_author": "Singh B", "dirs": [ "A" ] }, "39725227": { "pmid": "39725227", "title": "Trehalose and mannitol based lyoprotection of Taq DNA polymerase for cold-chain-free long-term storage.", "abstract": "Polymerase chain reactions (PCR) are most reliable and precise means for nucleic acid analysis of biological samples. A cold-chain system with temperature at around -20 °C is generally necessary for storage and transportation of PCR-related reagents. In order to facilitate ambient temperature storage and transportation, this study prepared Taq DNA polymerase and 5 × HS-Taq Mix (as low as 0.5 U/sample) into stable solid formulations using an optimized freeze-drying process and lyoprotectant formulations comprising trehalose dihydrate (3.3∼5%, w/v) and mannitol (10%, w/v). The lyocakes were characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In the optimized freeze-drying process, trehalose dihydrate mainly formed an amorphous structure and acted as both cryoprotectant and lyoprotectant, while mannitol crystallized to serve as a bulking agent. The enzyme activities of Taq and 5 × HS-Taq Mix samples were measured via real-time quantitative PCR (qPCR). Long-term storage stability test demonstrated that freeze-dried samples with optimized formulations showed no remarkable reduction in amplification efficiencies for target sequence compared to freshly prepared corresponding samples after being stored at 37 °C and 55% relative humidity (RH) for 0, 1, 4, 8 and 12 weeks.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2024.12.022", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Jin J", "dirs": [ "A" ] }, "39788671": { "pmid": "39788671", "title": "Development of a portable multi-step microfluidic device for point-of-care nucleic acid diagnostics.", "abstract": "The COVID-19 pandemic has significantly affected global health, economies, and societies, and highlighted the urgent need for rapid, sensitive, affordable, and portable diagnostic devices for respiratory diseases, especially in areas with limited resources. In recent years, there has been rapid development in integrated equipments using microfluidic chips and biochemical detection technologies. However, these devices are expensive and complex to operate, showing limited feasibility for in point of care tests (PoCTs). This study aims to develop a cost-effective, portable, and practical microfluidic nucleic acid PoCT device for rapid virus diagnosis. Here, we developed a device based on freeze-dried reverse transcription loop-mediated isothermal amplification (RT-LAMP) reagents for rapid nucleic acid diagnostics. Homebrew RT-LAMP reagents were optimized to eliminate non-specific amplification. The multi-step device combines nucleic acid extraction, RT-LAMP and fluorescence detection in an integrated microfluidic chip, enabling sample-in, result-out diagnosis. This device showed satisfactory sensitivity in detecting SARS-CoV-2 and Trichomonas vaginalis RNA samples, with a limit of detection (LOD) of 400 copies/μL and 80 copies/μL respectively in 45 min. The LOD of cultured Trichomonas vaginalis samples were 0.32 cells/μL in 50 min. Additionally, the freeze-dried homebrew RT-LAMP can be stored at 4 °C for up to 30 days while still maintaining high sensitivity and detection capabilities. The cost of diagnosis was reduced to as low as 0.45 $ per reaction. Overall, by integrating freeze-dried homebrew RT-LAMP and microfluidic chip, the device achieves ready-to-use, laboratory-free, quick, resource-independent and cost-effective nucleic acid detection, and provides a feasible alternative to complex equipments. The device shows potentials for point-of-care testing of SARS-CoV-2 and other respiratory diseases in remote or resource-limited areas with proper implementation.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2024.343518", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Shi L", "dirs": [ "A", "C" ] }, "41005481": { "pmid": "41005481", "title": "A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases.", "abstract": "The importance of rapid and convenient pathogen detection has been emphasized by the alarming threat of the Coronavirus Disease 2019 (COVID-19) pandemic since 2019. Point-of-care testing (POCT) provides rapid diagnostic results directly at the sampling site. However, isothermal amplification-based POCT faces technical challenges including primer design complexity and false-positive rates. To address these limitations, we developed the Thermostatic Step Amplification (TSA)/Clustered regularly interspaced short palindromic repeats (CRISPR) One-Pot System (TCOPS). This sensitive, rapid, and efficient platform specifically detects Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Mpox virus (MPXV) and Rabíes virus (RV) through integrated amplification and CRISPR-based detection. Our integrated TCOPS overcomes the technical challenges through single-tube reactions combining thermostatic amplification and CRISPR detection, reducing contamination while maintaining high accuracy for field applications. TCOPS enables single-tube CRISPR detection of high-risk viruses, with 10 copies/μL sensitivity shown using cloned DNA template for RV. In evaluations against Quantitative Polymerase Chain Reaction (qPCR) using 50 clinical samples, TCOPS incorporating freeze-dried reagents and a newly developed miniature fluorescence system (Q max) demonstrated >90 % sensitivity and 100 % specificity. Combined with the portable Q max device and its lyophilized reagent kit, TCOPS enables simple, rapid detection of multiple zoonotic viruses (SARS-CoV-2, MPXV, and RV) at the point of care. This integrated system achieves high sensitivity and specificity while establishing a practical, field-deployable prototype for next-generation POCT applications in resource-limited settings.", "journal": "Journal of microbiological methods", "year": "2025", "doi": "10.1016/j.mimet.2025.107277", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Gong Z", "dirs": [ "A", "C" ] }, "39714558": { "pmid": "39714558", "title": "Isolation and Characterization of Probiotic Bacteria from Traditional Foods.", "abstract": "A probiotic is a live bacterium that, when given in sufficient proportions, helps to improve the host's gut health. Kimchi and pearl millet, two traditional foods, were used to isolate probiotic bacterial strains. This study's primary goals were to isolate, identify and analyse the microorganisms for potential probiotic traits, tolerance to gastrointestinal environments, and antimicrobial activity, and produce probiotic capsule. The present investigation resulted with identification of two probiotic strains (KAC1 and PAC1) from conventional foods, such as kimchi and pearl millet porridge. The isolated probiotics were identified as Enterobacteriaceae family by 16S rRNA sequencing and are deposited in GenBank (NCBI), accession numbers OQ629827 (KAC1) and OQ629828 (PAC1), respectively. These strains exhibited the characteristics of possible probiotic traits, such as the ability to tolerate simulated gastric juice, inhibits the growth of pathogenic bacteria, auto-aggregation, co-aggregation, and hydrophobicity. Furthermore, spectroscopic analysis divulges some critical findings which corroborate the results obtained. Finally, capsules containing freeze-dried  probiotics was successfully produced.", "journal": "Applied biochemistry and biotechnology", "year": "2025", "doi": "10.1007/s12010-024-05125-9", "ptypes": [ "Journal Article" ], "first_author": "Davoodbasha M", "dirs": [ "A" ] }, "41885508": { "pmid": "41885508", "title": "Lyophilised colourimetric LAMP for visual readout with dual colour indicators.", "abstract": "Loop-mediated isothermal amplification (LAMP) is increasingly recognised as a practical alternative to PCR for pathogen detection, offering rapid turnaround time, a constant operating temperature, and compatibility with a wide range of detection methods. Colourimetric LAMP has gained popularity due to its potential for instrument-free readout, making it suitable for molecular diagnostics in low-resource settings. Despite these advantages, its adoption at the point-of-care remains limited as it has been widely used in liquid format and therefore restricted to the availability of cold-chain storage and trained personnel. This work introduces the development and optimisation of ready-to-use lyophilised colourimetric LAMP (lyo-cLAMP) that does not require cold-chain, additional reagents, or manual intervention beyond the addition of extracted nucleic acids from a sample, which indicates the presence of a target of interest by colour change after amplification. A variety of pH and metal indicators were screened and combined to evaluate their synergy, identifying four combinations with high discrimination between positive and negative amplification. The performance of lyo-cLAMP was assessed with synthetic SARS-CoV-2 RNA, comparing it to the liquid format and achieving the same limit-of-detection. Lastly, the translation of lyo-cLAMP to diagnostic applications was demonstrated by screening positive SARS-CoV-2 residual clinical samples, achieving high accuracy. The developed lyo-cLAMP is compatible with any LAMP assay, allowing for rapid adaptation to new targets, which is particularly valuable in outbreak scenarios.", "journal": "The Analyst", "year": "2026", "doi": "10.1039/d5an01238d", "ptypes": [ "Journal Article" ], "first_author": "Malpartida-Cardenas K", "dirs": [ "A" ] }, "40381422": { "pmid": "40381422", "title": "Lyophilized nasal swabs for COVID-19 detection by ATR-FTIR spectroscopy: Machine learning-based approach.", "abstract": "The COVID-19 pandemic continues to pose challenges for global health. The disease burden and diagnostic pressure has forced scientists to explore alternate diagnostic tools beyond the standard PCR testing. One such promising tool is the use of spectroscopy-based diagnostics. The objective of this study is to assess the potential of ATR-FTIR spectroscopy, applied to lyophilized nasal swab samples to discriminate between healthy and infected COVID-19 patients. Equal number (55 each) of positive and negative freeze-dried nasal swab samples were analyzed. After pre-processing, average mean spectra (600-4000 cm-1) showed significant variations between healthy and infected sample types. Clear spectral variations were recorded at 17 locations, of which, 13 peaks were observed in COVID-19 spectra while 4 peaks were observed in negative sample spectra. Statistical discrimination was done using principal component analysis (PCA), linear discriminant analysis (LDA) and support vector machine (SVM). The first two principal components (PCs) showed a combined variance of 76 %. Classification accuracy of 100 % were observed in the LDA graph using Quadratic kernel. Similarly, SVM model with both internal validation and external validation confirmed the robustness with a 100 % classification accuracy. These results show that lyophilized nasal swab samples are the ideal sample choice for FTIR-based analysis of COVID-19. This sample preparation method coupled with spectroscopy can serve as a robust and accessible diagnostic tool for post-covid testing.", "journal": "Biophysical chemistry", "year": "2025", "doi": "10.1016/j.bpc.2025.107459", "ptypes": [ "Journal Article" ], "first_author": "Shahid Z", "dirs": [ "A" ] }, "41037144": { "pmid": "41037144", "title": "Shedding light on deception: LAMP as a tool for detecting adulterants in herbal medicine.", "abstract": "The integrity of herbal products is frequently undermined by both intentional and unintentional adulteration, leading to substantial health risks and economic losses. Loop-mediated isothermal amplification (LAMP), a DNA-based molecular technique, has emerged as a formidable solution due to its simplicity, specificity, sensitivity, and ability to operate under isothermal conditions. The review critically evaluates the application of LAMP in authenticating herbal materials, showcasing its enhanced efficiency and user-friendliness compared to conventional techniques. The LAMP technique employs four to six primers that target six to eight distinct regions of the target DNA, ensuring unparalleled specificity. The amplification at a constant temperature negates the need for thermal cyclers, thus rendering it highly suitable for point-of-care applications and field-based authentication. The article presents case studies that illustrate LAMP's efficacy in detecting adulteration across traditional medicines, dietary supplements, and crude drug materials. Visualization methods in LAMP, such as turbidity, colorimetry, and fluorescence, greatly enhance its accessibility and ease of use, making it well-suited for both laboratory and field applications. Although there are limitations, such as primer design complexity and contamination risks, recent innovations, including the use of lyophilized reagents, multiplexing capabilities, and integration with mobile detection platforms, are significantly advancing the practicality of LAMP assays. This review underscores the potential of LAMP in both regulatory and commercial contexts, promoting the authenticity, safety, and quality of herbal products, thereby making a vital contribution to consumer protection and the sustainability of the herbal medicine trade.", "journal": "Protoplasma", "year": "2026", "doi": "10.1007/s00709-025-02117-2", "ptypes": [ "Journal Article", "Review" ], "first_author": "Anju PB", "dirs": [ "A", "C" ] }, "40455278": { "pmid": "40455278", "title": "Impact of Ilex paraguariensis extract on osteoblastic phenotype expression.", "abstract": "The use of dietary supplements and the functionalization of biomaterials with natural compounds have been explored to enhance bone regeneration. While Ilex paraguariensis St. Hil (yerba mate) has shown benefits in various tissues, its effects on bone tissue remain underexplored. This study evaluated the impact of Ilox® Micro extract on in vitro osteoblastic differentiation. To this end, osteoblasts from the UMR-106 cell line were exposed to 0 (control), 10, 30, and 60 µg/mL of lyophilized Ilox® Micro extract. The antioxidant capacity and total phenolic content were determined using the colorimetric Folin-Ciocalteu method. Cell viability was assessed using the Live/Dead kit (3 days) and the resazurin reduction method (3 and 5 days). The expression of Runx-2, Sp7, Col1a1, Alpl, Ibsp and Bglap genes was quantified by real-time PCR (5 days), and in situ alkaline phosphatase (ALP) activity and mineralized matrix were evaluated using Fast Red (5 days) and Alizarin Red (7 days) staining, respectively. Quantitative data were analyzed using ANOVA (α = 5%). Live/Dead staining demonstrated that adhered and spread cells were predominantly viable, with cell density increasing proportionally to the Ilox® Micro extract concentration. The resazurin reduction assay indicated higher values at 30 and 60 µg/mL. While Sp7, Col1a1, Alpl, and Ibsp showed no significant differences, Runx2 and Bglap were upregulated at 60 µg/mL. Additionally, 30 µg/mL exhibited greater ALP staining, and a trend toward increased mineralization was observed at 10 and 30 µg/mL. Based on the obtained results, it can be concluded that Ilox® Micro extract can positively modulate some key phenomena associated with the acquisition of the osteoblastic phenotype in vitro.", "journal": "Molecular biology reports", "year": "2025", "doi": "10.1007/s11033-025-10647-4", "ptypes": [ "Journal Article" ], "first_author": "Martorano AS", "dirs": [ "A" ] }, "41669836": { "pmid": "41669836", "title": "Fast and Simplified CRISPR-Cas13d Assay for Sensitive Detection of Porcine Deltacoronavirus.", "abstract": "Rapid and portable diagnostic technologies are essential for controlling infectious diseases. Here, we describe RAPID (Rapid Automated Portable Integrated Detection), a single-step, extraction-free CRISPR-Cas13d-based assay for sensitive and specific detection of porcine deltacoronavirus (PDCoV) RNA. RAPID integrates isothermal recombinase polymerase amplification with EsCas13d-mediated collateral cleavage in a one-pot reaction, enabling sample-to-answer detection within 30 min. A brief room-temperature lysis step allows direct RNA release from unextracted samples, simplifying sample preparation and reducing equipment requirements. Lyophilized reagents enhance stability during refrigerated storage (≤4 °C) and facilitate simplified transportation using conventional cooling measures, thereby reducing reliance on strict cold-chain logistics. The assay operates optimally at 37 °C and remains functional under ambient (∼25 °C) conditions with reduced sensitivity, permitting instrument-free operation when temperature control is unavailable. Detection is achieved via in-tube fluorescence or lateral-flow readouts. Clinical validation using porcine samples showed complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. RAPID provides a practical point-of-care diagnostic platform for on-farm surveillance and deployment in resource-limited settings.", "journal": "ACS synthetic biology", "year": "2026", "doi": "10.1021/acssynbio.5c00909", "ptypes": [ "Journal Article" ], "first_author": "Dai YM", "dirs": [ "A" ] }, "41284608": { "pmid": "41284608", "title": "One-Pot Photocontrolled CRISPR-Cas12b Coupled with Loop-Mediated Isothermal Amplification Assay for Point-of-Care Test of Mycoplasma pneumonia.", "abstract": "Mycoplasma pneumoniae (MP) is a highly prevalent respiratory pathogen, making the development of point-of-care testing (POCT) methods for its detection essential. The integration of loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b systems demonstrates remarkable specificity and offers promising potential for MP POCT application. However, the current one-pot LAMP/CRISPR-Cas12b system (HOLMESv2) faces the challenge of low sensitivity due to the premature cleavage of the template by CRISPR, which limits its practical utility. To address this, this study introduces a photocontrolled HOLMESv2 (pHOLMESv2) assay using gRNA with an NPOM-modified spacer region. This modification prevents full base pairing between the gRNA and MP DNA, thereby keeping the CRISPR-Cas12b system in an inactive condition during the LAMP reaction and avoiding unintended cleavage of the DNA template. After completion of the LAMP reaction, light irradiation eliminates the NPOM group from the gRNA, restoring its activity to cleave the LAMP products, resulting in a fluorescence signal. The pHOLMESv2 assay successfully addresses the issue of premature DNA template cleavage, improving the limit of detection (LoD) by 133-fold (7.5 copies/μL). Additionally, this method enables direct detection of samples treated with nucleic acid release agents, eliminating the need for complex extraction, and features lyophilized reagents for enhanced stability, storage, and transport. The efficacy of pHOLMESv2 was assessed with 160 clinical MP samples, achieving a sensitivity of 99.0% and a specificity of 100.0%. The pHOLMESv2 assay, when combined with the developed smartphone-based amplification reader, provides a highly sensitive, specific, portable, and cost-effective MP detection, indicating its potential for significant diagnostic use.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.5c01679", "ptypes": [ "Journal Article" ], "first_author": "Zhu W", "dirs": [ "A", "C" ] }, "42036713": { "pmid": "42036713", "title": "Lyophilized mesenchymal stromal cell-derived extracellular vesicles for the oral treatment of inflammatory bowel disease: a novel cell-free therapeutic strategy.", "abstract": "Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn's disease (CD), is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. Current standard treatments, including aminosalicylates, corticosteroids, immunomodulators, and biologic therapies, can induce and maintain remission; however, a considerable proportion of patients show limited responsiveness or experience adverse effects. This highlights the urgent need for safer and more effective therapeutic alternatives. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) represent a promising cell-free therapeutic modality owing to their immunomodulatory and reparative capabilities. These vesicles replicate many of the therapeutic benefits of mesenchymal stromal cells (MSCs) while avoiding the risks associated with conventional cell-based treatments. Nevertheless, clinical translation of MSC-EVs for IBD management remains constrained by issues such as limited storage stability and invasive delivery routes. Extracellular vesicles (EVs) were isolated from the culture supernatant of human umbilical cord-derived MSCs via tangential flow filtration (TFF) followed by ultracentrifugation, and were characterized using transmission electron microscopy, western blotting, and nanoflow cytometry. To enhance their stability, MSC-EVs were lyophilized with trehalose and mannitol serving as cryoprotectants. The lyophilized powder was subsequently encapsulated in enteric-coated capsules for oral administration. The therapeutic efficacy of this formulation was evaluated in a dextran sulfate sodium (DSS)-induced rat colitis model. Disease activity was recorded daily, and post-experimental analyses included histopathology, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and serum biochemistry to evaluate hepatorenal function. A robust lyophilization protocol was successfully established to enhance MSC-EV stability, along with an innovative oral delivery system for IBD therapy. In DSS-induced colitis rats, lyophilized MSC-EVs markedly alleviated disease severity, as indicated by a reduced disease activity index (DAI), preserved colon length, and improved histological architecture. MSC-EV administration restored intestinal barrier integrity, modulated cytokine expression profiles, mitigated systemic inflammation, and improved hepatic and renal function. This study demonstrated the therapeutic efficacy of a lyophilized, orally administered MSC-EV formulation in experimental colitis. This approach not only enhances vesicle stability and simplifies storage but also provides a non-invasive, patient-friendly route of administration. These findings underscore the strong translational potential of lyophilized MSC-EVs as a practical and effective cell-free therapeutic strategy for IBD.", "journal": "Stem cell research & therapy", "year": "2026", "doi": "10.1186/s13287-026-05033-1", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Yang S", "dirs": [ "A" ] }, "40825624": { "pmid": "40825624", "title": "An integrated biosensor and mobile workstation for on-site species and sex identification from bloodstains.", "abstract": "Bloodstains are the most common biological specimens at a crime scene. Rapid and sensitive species and sex identification from bloodstains can expedite crime investigation. Nevertheless, time-consuming protocols and specialized equipment are often required in forensic practice. Taking advantage of speediness and convenience, loop-mediated isothermal amplification (LAMP) has been used for bloodstain analysis. However, only a single characteristic gene can be targeted per reaction, leading to specimen consumption and limited throughput. Herein, a portable biosensor is first developed for on-site simultaneous species and sex identification from bloodstains without DNA purification. By combining multiplex LAMP and a triplex lateral flow device (LFD), minute amounts of specimen can be identified without any sophisticated apparatus. Using gold magnetic nanoparticle as nanoprobe for amplicon hybridization, the results can be interpreted with naked eye. Meanwhile, the relative intensity of tinctorial signal can be quantitated using a smartphone to eliminate subjective bias. Besides, carryover contamination from uncapping is thoroughly prevented by the uracil-DNA glycosylase-based anti-aerosol strategy while holding constant reaction temperature. Based on the simplicity of the whole \"sample to result\" workflow, a mobile workstation containing lyophilized LAMP reaction mixtures and a compact heater is customized for on-site detection, rendering this biosensor competent in various scenarios. As a demonstration, species and sex can be identified within 1 h by targeting cytochrome b gene and Y-chromosomal amelogenin gene. Even bloodstains stored for over 6 years can be accurately identified, indicating the significant potential of this biosensor for forensic nucleic acid analysis.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344398", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhou H", "dirs": [ "A", "C" ] }, "40333119": { "pmid": "40333119", "title": "Promising Role of Fruitless Wolfberry Bud Tea in Combating Nakaseomyces glabratus Resistance.", "abstract": "The rising antifungal resistance in Nakaseomyces glabratus, especially to azole drugs like fluconazole, itraconazole, and voriconazole, presents a significant clinical challenge. Plant-derived compounds with synergistic antifungal effects offer a promising solution. Fruitless wolfberry bud tea, rich in flavonoids from a Lycium barbarum L. hybrid, shows potential but is underexplored in antifungal therapies. This study assessed FWE's antifungal efficacy alone and with azoles against resistant N. glabratus isolates, exploring mechanisms like efflux pump inhibition and gene expression changes. A total of 52 clinical isolates were tested. Fruitless wolfberry bud tea was methanol-extracted (FWE) and lyophilized. Antifungal susceptibility was evaluated using broth microdilution, and synergistic effects were analyzed with checkerboard assays. Growth inhibition, rhodamine 6G efflux, and qRT-PCR for resistance-related genes were conducted. FWE demonstrated inhibitory activity with MICs ranging from 16 to 32 μg/mL. When combined with ITR or VRC, synergistic or additive effects were observed, reducing MICs by 2-8-fold. FWE + VRC exhibited synergy (FICI ≤ 0.5) in 50% of isolates, while FWE + ITR showed synergy in 37.5%. Efflux pump activity, measured by rhodamine 6G, significantly decreased in combination groups (11.4-14.6%) compared to monotherapy (17.3-17.5%). qRT-PCR indicated downregulation of CgCDR1, CgERG11, and CgPDR1 in FWE-treated Cg 1 isolate, with greater suppression in combination groups. FWE might boost the bacteriostatic impact of azole antifungal drugs by blocking efflux pumps and altering the expression of resistance genes. This study identifies FWE as a potent adjuvant to overcome cross-resistance, supporting its inclusion in antifungal strategies. Further research to identify bioactive compounds in FWE and in vivo validation is necessary for clinical application.", "journal": "Pathogens (Basel, Switzerland)", "year": "2025", "doi": "10.3390/pathogens14040351", "ptypes": [ "Journal Article" ], "first_author": "Zhang L", "dirs": [ "A" ] }, "42027461": { "pmid": "42027461", "title": "Integrating isothermal amplification with magnetic flocculation and DNA-guided PfAgo for SARS-CoV-2 detection.", "abstract": "The recurrent threat of respiratory infectious disease outbreaks creates an urgent need for diagnostic tests that are rapid, portable, and user-friendly, applicable across diverse settings-from central laboratories and hospitals to field deployments, resource-limited regions, and home use. In response, we developed three novel nucleic acid detection platforms-RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo-that integrate reverse transcription-recombinase-aided amplification (RT-RAA) with distinct instrument-free readout mechanisms. These systems utilize custom-designed primers and guide DNA (gDNA) targeting the SARS-CoV-2 N and ORF genes. Performance evaluation demonstrated high analytical sensitivity, with detection limits of 102 copies/μL for RT-RAA-MPs and 5 copies/μL for both RT-RAA-F-PfAgo and RT-RAA-L-PfAgo. All assays exhibited excellent specificity, showing no cross-reactivity with nine common respiratory pathogens. Clinical validation using 23 patient samples revealed perfect agreement with RT-qPCR (100% concordance), supported by a Kappa value of 1.00, indicating outstanding diagnostic consistency and statistical robustness. With lyophilized reagent compatibility and minimal equipment requirements, these platforms are readily adaptable for a wide range of applications, including clinical diagnosis at all hospital levels, point-of-care screening, border control inspection, and environmental monitoring. This work establishes a versatile, reliable, and deployable toolkit for effective outbreak response and decentralized molecular diagnostics.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1792924", "ptypes": [ "Journal Article" ], "first_author": "Zhou X", "dirs": [ "A" ] }, "42007994": { "pmid": "42007994", "title": "Programmable CRISPR-Cas diagnostic platforms for rapid detection of uropathogens and antimicrobial resistance.", "abstract": "Urinary tract infections (UTIs) represent a global burden, impacting more than 150 million individuals annually, and are a major contributor to the antimicrobial resistance (AMR) crisis because of the rampant empirical use of antibiotics. Although microbial culture remains the gold standard of diagnosis, its long turnaround time (48–72 h) limits timely, actionable decision-making for effective antimicrobial stewardship. This review is a critical analysis of the development of CRISPR-Cas biosensing as a better analytical platform to detect uropathogens in a rapid and point-of-care (POC) format. The diagnostic mechanisms of Class II Cas effectors (Cas12, Cas13, and Cas14) are discussed in detail, including programmable collateral trans-cleavage activity that allows programmable attomolar sensitivity and single-nucleotide specificity in the identification of specific resistance markers. New engineering advancements that have brought these assays from the bench to the bedside are heavily emphasized, particularly the integration of isothermal pre-amplification techniques (LAMP/RPA) into streamlined one-pot systems that lower the risk of contamination. Additionally, the review assesses the advancement of instrument-free formats, e.g., lyophilized reagents, paper-based microfluidics, and smartphone-based colorimetric readouts that would be suitable in resource-constrained environments. Lastly, the manuscript discusses important analytical issues, including sample preparation in complicated urine samples and multiplexing. It concludes that next-generation CRISPR diagnostics, when satisfying the requirements of the World Health Organization in its category of REASSURED, can fill the gap between molecular accuracy and decentralized patient care, and this will provide a powerful answer to the global AMR threat.", "journal": "Archives of microbiology", "year": "2026", "doi": "10.1007/s00203-026-04908-4", "ptypes": [ "Journal Article", "Review" ], "first_author": "Tiwari P", "dirs": [ "A", "C" ] }, "40864204": { "pmid": "40864204", "title": "Designing the homogeneous competitive bioluminescence-based assay for tick-borne encephalitis virus (TBEV) point-of-care detection.", "abstract": "Tick-borne encephalitis virus (TBEV), a highly pathogenic infectious agent that causes serious damage to the nervous system is mainly transmitted by Ixodidae ticks. The laboratory methods (immunoassay and the PCR-based one) are successfully used to detect the virus in tick samples thereby avoiding unwarranted immunoprophylaxis. However, there is a need to determine the tick infection outside the laboratory conditions. In this study, we have developed a one-stage (of mix-and-read type) method for detecting virus in biological samples based on split NanoLuc complementation assay. Artificial NanoLuc luciferase split fragments NLuc(N-residue), 17.6 kDa, and NLuc(C-residue), 11 a.a., were genetically fused with the protein prED3 (fragment of the TBEV capsid protein E) or mouse anti-TBEV single-chain antibody 14D5a in all possible variants. The corresponding hybrid proteins were synthesized in E. coli recombinant cells, purified and studied. Assembling of the luciferase fragments into a bioluminescent complex proceeded due to antigen-antibody affinity interaction. The most efficient luciferase complementation was observed for the pair 14D5a-NLucCter + prED3-NLucNter: the integral bioluminescence of the complex was 2.4% of that of the intact luciferase. Using this complex, a single-phase competitive enzyme immunoassay of TBEV-associated targets was developed. A large number of native ticks were analyzed and a statistically significant difference was shown between \"healthy\" and virus-carrying ticks. Lyophilized all-in-one reagents, reconstituted upon addition of the sample matrices, were developed and tested in model assay. The results offer a basis for the development of a point-of-need portable device for rapid tick detection.", "journal": "Analytical and bioanalytical chemistry", "year": "2025", "doi": "10.1007/s00216-025-06090-w", "ptypes": [ "Journal Article" ], "first_author": "Kudryavtsev AN", "dirs": [ "A" ] }, "42486811": { "pmid": "42486811", "title": "Lyophilized LAMP Assay Combined with Lateral Flow Biosensor for Cold-Chain-Free Detection of Mycobacterium tuberculosis.", "abstract": "To decentralize tuberculosis surveillance in resource-limited and tropical regions, a rapid, robust, and cold-chain-independent near-point-of-care biosensor is urgently required to overcome the instability of conventional liquid-formulation diagnostics. Herein, we developed a ready-to-use lyophilized reaction mix Loop-Mediated Isothermal Amplification (LAMP) matrix integrated with a Lateral Flow Biosensor (LFB) for the instrument-free visual detection of Mycobacterium tuberculosis (M. tuberculosis). The freeze-dried reagents, stabilized with an optimized trehalose-BSA matrix, were hermetically packaged and systematically evaluated for long-term thermal stability across four regulated profiles (-20 °C, 4 °C, 25 °C, and 37 °C) over a 12-month period. Diagnostic viability was further validated in a double-blinded study using 191 clinical sputum specimens against gold-standard culture and line probe assays. The stabilized platform demonstrated an analytical sensitivity down to 102 CFU/mL and maintained full functional activity for up to 12 months at -20 °C and 4 weeks at 4 °C, with robust short-term resilience under tropical stress (25 °C) for up to 1 week. However, the formulation was unable to withstand prolonged exposure to harsh environmental conditions at 37 °C, resulting in a progressive decline in amplification performance following phase-transition instability. Crucially, clinical validation yielded an outstanding diagnostic sensitivity of 98.92% and a positive predictive value (PPV) of 100.00%. These findings validate the lyophilized LAMP-LFB platform as a highly reliable, sustainable, and distributable molecular diagnostic tool capable of overcoming the cold-chain bottleneck in field-based tuberculosis detection. Although the developed platform shows strong potential for near-point-of-care deployment, further optimization of the lyophilization formulation and process will be necessary to enable prolonged storage and transportation under ambient-temperature conditions.", "journal": "ACS infectious diseases", "year": "2026", "doi": "10.1021/acsinfecdis.6c00520", "ptypes": [ "Journal Article" ], "first_author": "Ckumdee J", "dirs": [ "A", "C", "D" ] }, "42413880": { "pmid": "42413880", "title": "Nucleic acid amplification tests for Yersinia pestis detection: A comprehensive technical and field-oriented review.", "abstract": "Plague, caused by Yersinia pestis, remains a highly fatal zoonosis with endemic foci on three continents. Rapid and accurate diagnosis is essential for patient survival and outbreak containment. Nucleic acid amplification tests (NAATs) have evolved from conventional PCR to real-time PCR, isothermal methods (LAMP, RPA), droplet digital PCR, and CRISPR-based platforms. This 5000-word review provides an in-depth technical comparison of these technologies, their analytical performance (limit of detection [LoD], sensitivity, specificity), clinical validation in human specimens, environmental and vector applications, and point-of-care (POC) readiness. We critically analyze sample preparation challenges, genetic target selection, and multiplexing strategies. Four comprehensive tables summarize target genes, real-time PCR assays, isothermal methods, and point-of-care platforms. Future directions include AI-integrated devices, wearable sensors, and lyophilized CRISPR tests. This review serves as a definitive guide for clinical microbiologists, field epidemiologists, and diagnostic developers.", "journal": "Journal of microbiological methods", "year": "2026", "doi": "10.1016/j.mimet.2026.107618", "ptypes": [ "Journal Article", "Review" ], "first_author": "Khosrojerdi M", "dirs": [ "A", "C" ] }, "40355941": { "pmid": "40355941", "title": "External quality assessment for molecular detection of sand fly-borne phleboviruses circulating in the Mediterranean Basin.", "abstract": "Sand fly-borne phleboviruses (SbPV) are globally distributed and pose potential public health risks. Despite increased detection in recent decades, detailed knowledge of their ecology, characteristics and clinical relevance remains limited. Many cases of SbPV infection likely go unreported or misdiagnosed due to limited awareness and the lack of standardized screening. The External Quality Assessment (EQA) reported herein was organized within the framework of the European Union CLIMOS (EU Climate Monitoring and Decision Support Framework for Sand Fly-borne Diseases Detection and Mitigation) project. The aim of this EQA was to standardize the detection of phleboviruses in order to provide comparable data to feed mathematical models for the surveillance of the impact of climate changes and environmental parameters on the kinetics and diversity of sand fly species and on sand fly-borne microorganisms. Nine laboratories from seven countries participated in the EQA. Each laboratory was provided with eight vials, each containing an anonymous sample; two vials of lyophilized primers and probes to be used for the detection of Toscana virus (TOSV) and several Sandfly fever Sicilian virus (SFSV) species with a reverse-transcriptase PCR (RT-PCR) assay; and one vial of lyophilized primers for the detection of generic phleboviruses with a RT-PCR assay along with the standard operating procedure. The laboratories were instructed to submit their results together with details on the techniques employed. All nine laboratories successfully detected the two TOSV- and the one SFSV-positive samples. Only one laboratory, using a generic phlebovirus assay, detected all of the targeted phleboviruses. All participating laboratories successfully identified the two TOSV and one SFSV using the proposed RT-qPCR assays, albeit with some variations in cycle threshold values across laboratories. The detection rate of SbPV was lower with the generic Phlebovirus assay than with the specific real-time RT-qPCR assays. This EQA aimed to assess the SbPV detection capabilities of molecular tools and strengthen their use, thereby supporting the involvement of laboratories in virus discovery and surveillance beyond their core expertise.", "journal": "Parasites & vectors", "year": "2025", "doi": "10.1186/s13071-025-06785-0", "ptypes": [ "Journal Article" ], "first_author": "Ayhan N", "dirs": [ "A" ] }, "40136193": { "pmid": "40136193", "title": "Salmonella spp. detection in fresh and freeze-dried frog meat: a comparison of methods.", "abstract": "This study examined the presence of Salmonella spp. in fresh frog meat and freeze-dried seasoned frog meat stored for up to 90 days at room temperature. The samples were analyzed in triplicate using the conventional official method, by polymerase chain reaction (PCR) and mass spectrometry technique (MALDI -TOF) - for a comparison of the results obtained by the three tested methods. Mass spectrometry analyses confirmed the presence of Salmonella spp. in 100% of the fresh frog meat samples. In the analysis by the conventional method, the percentages of detection were 67% and 100% in samples B2 and B3, whereas the PCR method indicated 100% and 67% presence of the bacteria in the respective samples. In the analyses of freeze-dried none of the three methods detected the presence of Salmonella spp. The different analysis methods tested in this study were efficient in determining the presence or absence of Salmonella spp. in the samples, and the comparison between methods revealed a high percentage of compatibility of results. Adoption of efficient detection methods, such as PCR and MALDI-TOF, is essential, not only for public health, but also for compliance with Brazilian regulations, ensuring that food products are free from Salmonella spp. contamination and safe for consumption.", "journal": "Anais da Academia Brasileira de Ciencias", "year": "2025", "doi": "10.1590/0001-3765202520230552", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "Mello SCRP", "dirs": [ "A" ] }, "42660094": { "pmid": "42660094", "title": "Multiplex crRNAs powered CRISPRCas13a detection for rifampicin-resistant mycobacterium tuberculosis.", "abstract": "Rapid and accessible detection of Mycobacterium tuberculosis (MTB) and rifampicin resistance is essential for timely treatment and transmission control. However, widely used drug-resistance testing methods, including qPCR and gene sequencing, are time-consuming and require specialized instrumentation, limiting their utility for rapid testing in primary-care and resource-limited settings. Therefore, rapid, sensitive and readily deployable assays are urgently needed for the simultaneous diagnosis of tuberculosis (TB) and screening for drug resistance. A multiplex crRNA system was integrated with isothermal amplification, lyophilized reagents and a lateral flow readout to enable detection of 4 common rpoB mutation sites in a single reaction. Analytical sensitivity and specificity were evaluated using mutant plasmids, rifampicin-resistant clinical isolates, clinically relevant bacterial pathogens and non-tuberculous mycobacteria (NTM). Clinical performance was assessed using sputum-derived nucleic acids, with qPCR and Sanger sequencing as reference methods. This detection assay can accurately identify nucleic acids from 10 clinical rifampicin-resistant isolates carrying four types of target mutations. For nucleic acids extracted from 21 clinical sputum samples, the assay achieved a sensitivity and specificity of 100%, with detection results fully consistent with those of qPCR and gene sequencing. The limit of detection (LOD) of the assay for all mutant plasmids was 10 copies/μL. In addition, this study realized highly sensitive and specific detection of MTB, with a LOD as low as 1 copy/μL, and successfully identified nucleic acids from 42 clinical TB samples. No cross-reactivity was observed between this assay and 5 pathogenic bacteria with clinical symptoms overlapping with MTB, as well as 8 NTM strains. Amplification and visual lateral flow readout were completed within 1 h. This multiplex CRISPR-Cas13a assay enables rapid, sensitive and visually interpretable detection of MTB and common rifampicin resistance-associated mutations with limited instrumentation, offers a novel detection method for rifampicin-resistant tuberculosis (RR-TB) and exhibits potential in improving TB diagnosis.", "journal": "Journal of infection and public health", "year": "2026", "doi": "10.1016/j.jiph.2026.103348", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Huang Z", "dirs": [ "A", "C" ] }, "39304108": { "pmid": "39304108", "title": "A stable thymidine kinase 1 tetramer for improved quality control of serum level quantification.", "abstract": "DNA synthesis is a critical process for cell growth and division. In cancer patients, an enzyme called thymidine kinase 1 (TK1) is often elevated in the blood, making it a valuable biomarker for cancer diagnosis and treatment. However, previous studies have shown that recombinant TK1 can exist in unstable mixtures of tetramers and dimers, leading to inconsistent results and potentially affecting accuracy. To address this issue, we hypothesized that incorporating tetrameric coiled-coil peptides could enhance TK1 self-assembly into stable tetramers without requiring additional adenosine triphosphate. In this study, we successfully expressed a recombinant TK1 tetramer protein in the Escherichia coli system. We optimized the induction conditions, significantly increasing protein expression levels, functionality, and solubility. Size exclusion chromatography confirmed the formation of a tetrameric structure in the expressed TK1 protein, with a molecular weight of 127.2 KDa, consistent with our expectations. We also found that the TK1 tetramer exhibited higher affinity with anti-TK1 IgY than wild-type TK1, as shown by enzyme-linked immunosorbent assay experiments. Moreover, the TK1 tetramer demonstrated good stability against heating, freeze-thawing and lyophilization with almost no immunoactivity lost. These findings suggest that recombinant TK1 tetramers have the potential to serve as calibrators in diagnostic assay kits, becoming promising candidates for quality control of clinical laboratory and in vitro diagnostic reagents.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2025", "doi": "10.1016/j.cca.2024.119967", "ptypes": [ "Journal Article" ], "first_author": "Feng X", "dirs": [ "A" ] }, "40469727": { "pmid": "40469727", "title": "Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system.", "abstract": "Largemouth bass virus (LMBV), the causative agent of largemouth bass ulcerative syndrome, poses a significant economic threat to the aquaculture industry. Rapid, simple, and reliable detection methods are essential for the timely identification of LMBV infections, enabling effective prevention and control measures. In this study, a detection platform utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system was developed for LMBV. CRISPR RNA (crRNA) and recombinase polymerase amplification (RPA) primers were designed to target the highly conserved region of the major capsid protein (MCP) gene. Additionally, a one-pot method and lyophilization strategy were optimized for field applications. The RPA-CRISPR/Cas12a system achieved a sensitivity of 50 copies/reaction within 40 minutes, without requiring specialized equipment, and exhibits high specificity for LMBV. Validation with 42 clinical samples of suspected LMBV infection demonstrated 100% concordance among the RPA-CRISPR/Cas12a method, quantitative PCR and lateral flow strip assay. The one-pot method and lyophilization strategy demonstrated consistent detection results with two-step RPA-CRISPR methods in clinical sample testing, offering more convenient and stable application characteristics for on-site detection. This study establishes an efficient process for detecting LMBV nucleic acids in fish clinical samples, culminating in a CRISPR-based fluorescent readout, offering significant advantages for viral diagnosis and monitoring.", "journal": "Frontiers in microbiology", "year": "2025", "doi": "10.3389/fmicb.2025.1599006", "ptypes": [ "Journal Article" ], "first_author": "Li N", "dirs": [ "A" ] }, "42461754": { "pmid": "42461754", "title": "Paper-Based Chip Platform: PCR-SERS-LFA Integration for On-Site Coastal Harmful Microorganism Detection.", "abstract": "Harmful algal blooms (HABs) and pathogenic microorganisms threaten coastal ecosystems and public health, requiring rapid field-deployable detection. Herein, we present an integrated paper-based platform combining polymerase chain reaction (PCR), surface-enhanced Raman scattering (SERS), and lateral flow assay (LFA) for on-site nucleic acid detection. The platform integrates a miniature thermal cycler, lyophilized paper-based reaction chambers, and a capillary-driven detection pen for contamination-free transfer and dual-mode readout. The platform achieved limits of detection of 101-102 copies μL-1 by SERS and 103-104 copies μL-1 by visualization for Enterococcus spp., Escherichia coli, Alexandrium tamarense, and Noctiluca scintillans, with complete amplification and detection within 30 min. Field validation at a coastal bathing beach showed agreement with laboratory-based quantitative PCR (qPCR), with only one discordant result out of 36 environmental samples, demonstrating comparable performance to the current gold-standard method while offering portability and rapid on-site response. Furthermore, the platform identified Ulva prolifera (a key driver of green tide) micropropagules as small as 600 μm and enabled large-scale mapping of overwintering zones across the Yellow-Bohai Sea. This platform supports rapid environmental monitoring and public health surveillance.", "journal": "Environmental science & technology", "year": "2026", "doi": "10.1021/acs.est.6c06094", "ptypes": [ "Journal Article" ], "first_author": "Wang Q", "dirs": [ "A" ] }, "42508912": { "pmid": "42508912", "title": "A smartphone-integrated isothermal amplification-based point-of-care testing platform for ultrasensitive detection of severe fever with thrombocytopenia syndrome virus.", "abstract": "Severe fever with thrombocytopenia syndrome (SFTS), caused by the severe fever with thrombocytopenia syndrome virus (SFTSV), poses a serious public health threat due to its high fatality rate, and delayed diagnosis is strongly associated with disease progression and increased mortality. Conventional laboratory diagnosis still relies largely on quantitative real-time PCR (qPCR), which requires specialized instruments, trained personnel, and centralized testing facilities. These challenges underscore the urgent need for rapid, sensitive, and portable diagnostic methods for SFTSV detection in clinical and on-site settings. Here, we developed an integrated isothermal amplification-based point-of-care testing (IAPOCT) platform that incorporates a recombinase polymerase amplification (RPA)-based sensing and interpretation strategy with a smartphone-interfaced portable device. This platform demonstrated low-copy analytical detection capability, achieving a limit of detection (LOD) as low as 10 copies/μL. The core amplification and fluorescence readout steps can be completed within 20 min, while the complete serum testing workflow from thermal lysis to result interpretation requires approximately 25 min. Preliminary evaluation in a cohort of 24 clinical serum specimens showed complete qualitative agreement with qPCR, correctly identifying 20 positive and 4 negative samples. Endpoint fluorescence signals were analyzed through the smartphone-based interface, enabling portable and objective qualitative interpretation of clinical samples. The lyophilized reagent format was incorporated to facilitate handling and integration with the portable testing workflow for routine on-site molecular testing in resource-limited settings. This work provides a practical platform for rapid SFTSV detection with portable fluorescence readout, with potential applications in clinical screening and on-site epidemiological surveillance. By integrating RPA-based amplification, lyophilized reagents, smartphone-assisted interpretation, and a portable device, the platform offers a compact analytical strategy for point-of-care molecular testing of infectious diseases.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345815", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Li W", "dirs": [ "A" ] }, "40665979": { "pmid": "40665979", "title": "Research on the construction of corneal endothelium transplantation with acellular amniotic membrane as a scaffold.", "abstract": "This study aimed to develop a human acellular amniotic membrane (HAAM) scaffold suitable for corneal endothelial transplantation. The HAAM was engineered using sequential chemical treatments and physical agitation to remove cellular components while preserving the extracellular matrix structure. The study sought to evaluate the biocompatibility and functional properties of the HAAM when seeded with immortalized human corneal endothelial cells (HCECs), with the ultimate goal of providing a potential therapeutic option for corneal endothelial dysfunction. The HAAM was fabricated through a series of chemical treatments involving trypsin/EDTA, Triton X-100, sodium deoxycholate, and peracetic acid/ethanol, combined with physical agitation. Following lyophilization, the HAAM was sterilized and coated with fibronectin and chondroitin sulfate (FNC) to enhance cell adhesion. HCECs were then seeded onto the HAAM scaffold. Biocompatibility was assessed by evaluating cell adhesion using microscopy, cell viability using CCK-8 and EdU assays, and cell proliferation. Functional validation included immunofluorescence detection of tight junction proteins (ZO-1), transcriptome sequencing (RNA-seq), and quantitative PCR (qPCR) to analyze the expression of genes regulating barrier function, ion transport, and extracellular matrix synthesis. Additionally, the expression of key genes critical for endothelial function was assessed to validate the functionality of the HAAM-based corneal endothelial transplantation membrane. The HAAM was successfully prepared, maintaining an intact collagen fiber structure. HCECs adhered closely to the HAAM scaffold, forming a continuous monolayer. The HAAM promoted cell viability and proliferation, as evidenced by positive expression of tight junction proteins and upregulation of key functional genes. Transcriptome analysis identified genes involved in proliferation and matrix synthesis, further supporting the biocompatibility and functional properties of the HAAM. The HAAM scaffold demonstrated excellent transparency, mechanical properties, and biocompatibility, making it suitable for the attachment and proliferation of HCECs. The effective maintenance of key functional gene expression levels suggests that the HAAM functionally mimics the characteristics of the natural corneal endothelial layer. These findings provide experimental evidence for the potential clinical application of the HAAM in corneal endothelial transplantation, offering a promising therapeutic option for patients with corneal endothelial dysfunction. Further studies are warranted to explore the long-term efficacy and safety of the HAAM in preclinical and clinical settings.", "journal": "Frontiers in medicine", "year": "2025", "doi": "10.3389/fmed.2025.1592123", "ptypes": [ "Journal Article" ], "first_author": "Chen YN", "dirs": [ "A" ] }, "41797144": { "pmid": "41797144", "title": "Hemoglobin-based high-throughput assessment of lyoprotectant efficacy.", "abstract": "Biologics, including vaccines, therapeutic proteins, blood products, and cell therapies, are susceptible to degradation during drying and therefore require the incorporation of protective additives (lyoprotectants) into the processing formulation to maintain their desired biological activities after reconstitution. However, assays to quantitatively assess lyoprotectant efficacy remain limited. A hemoglobin oxidation assay was developed into a high-throughput platform for screening the protective properties of compounds during freeze-drying. Hemoglobin, a redox-sensitive protein, oxidizes upon drying and rehydration, providing a robust reporter of desiccation-induced protein denaturation. Concentration-dependent protection by saccharides and polymers was quantified as the concentration required to inhibit 50% of hemoglobin oxidation (IC50) after drying and rehydration. When comparing carbohydrates, raffinose exhibited the strongest protection on a molar basis (IC50 = 2.5 ± 0.5 mM), but when expressed as mass-based concentration (% w/v), the IC50 values showed little to no difference across carbohydrates (IC50 = 0.12 ± 0.02 % w/v). To complement experimental results, all-atom molecular dynamics simulations of hemoglobin were employed in the presence of glucose, trehalose, and raffinose under hydrated and frozen conditions. Simulations demonstrated that larger carbohydrates form more hydrogen bonds with hemoglobin and accumulate more extensively on its surface, displacing hydration water and providing a protective \"coat\" during freezing. Together, these results implicate water replacement and surface coverage as principal mechanisms by which sugars stabilize biologics during drying, likely operating coordinately with vitrification. The integrated experimental-computational framework establishes a quantitative, mechanistically informed approach for evaluating, advancing discovery, and characterizing lyoprotectants, and for designing optimal stabilizers for freeze-dried red blood cells.", "journal": "Cryobiology", "year": "2026", "doi": "10.1016/j.cryobiol.2026.105600", "ptypes": [ "Journal Article" ], "first_author": "Elder CA", "dirs": [ "A" ] }, "41048035": { "pmid": "41048035", "title": "Thermodynamic Delivery of Trehalose into Extracellular Vesicles for Enhanced Lyophilization.", "abstract": "Extracellular vesicles (EVs) hold great potential as therapeutic agents and drug carriers. Conventionally, EVs are cryopreserved at ultra-low temperatures, with substantial cryoinjury associated with the freeze/thaw cycle. Lyophilization has emerged as a promising alternative approach; however, suboptimal outcomes remain owing to the challenge of lyoprotectant delivery into EVs. Here, the atypical transport property of the EV membrane is unraveled, and lyoprotective trehalose is delivered into EVs by combining substantial hypotonicity (below intravesicular colloid osmolality) and mild heat shock (42 °C). Consequently, the lyophilization of trehalose-laden EVs is notably enhanced, with less EV loss, more RNA and protein retention, and superior therapeutic efficacy. Moreover, the EVs can be co-reconstituted and co-lyophilized with hyaluronic and methylcellulose (HAMC) hydrogel carriers, yielding excellent preservation outcomes and controlled EV release. Lyophilized EVs or HAMC-EVs maintain Treg cell regulation in vitro and experimental autoimmune encephalomyelitis disease treatment in vivo. Overall, not only are EVs and EV-hydrogel constructs efficiently lyophilized for widespread application, but a thermodynamic approach is also developed to safely and uniformly deliver various hydrophilic molecules into EVs.", "journal": "Advanced healthcare materials", "year": "2026", "doi": "10.1002/adhm.202500522", "ptypes": [ "Journal Article" ], "first_author": "Wang T", "dirs": [ "A" ] }, "40582521": { "pmid": "40582521", "title": "Development of a stable inhalable dry powder formulation with osteoglycin fragment for alveolar epithelial repair.", "abstract": "Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation and progressive lung function decline, with a critical need for innovative treatments that can repair damaged lung tissue. The active fragment of osteoglycin (OGN) has previously shown significant regenerative potential in activating growth of alveolar epithelial cells, making it a promising candidate for therapeutic development. In this study, we developed an inhalable dry powder formulation of the OGN fragment using spray drying, incorporating inulin or mannitol as excipients and leucine as a dispersion enhancer. The formulations were assessed for their ability to support lung organoid formation and differentiation in vitro. After spray drying with inulin or mannitol, the OGN fragment maintained its ability to induce organoid formation, but only the formulation containing inulin supported the differentiation towards alveolar-type organoids (surfactant protein C positive). After 28 days of storage at high temperatures (60 °C) inulin was found to provide superior stability compared to mannitol. The dry powder formulation with OGN fragment and inulin demonstrated favorable aerodynamic properties, meeting the requirements for deep lung deposition when dispersed via a dry powder inhaler. Additionally, we showed that adding 4 % leucine or inulin sweeper particles significantly improved the dispersion behavior of the formulation and reduced dry powder inhaler retention. This study highlights the potential of an inhalable OGN fragment formulation as a novel therapeutic approach for lung tissue repair in COPD. The findings support further development and clinical evaluation of this formulation, particularly with inulin as the preferred stabilizing excipient.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125913", "ptypes": [ "Journal Article" ], "first_author": "van der Koog L", "dirs": [ "A" ] }, "42182009": { "pmid": "42182009", "title": "Mechanisms of viability loss in dehydrated Metarhizium anisopliae blastospores.", "abstract": "The commercial use of Metarhizium anisopliae blastospores is significantly limited by their poor storage stability, mainly due to viability loss over time. Understanding the mechanisms underlying this decline is therefore essential to improve blastospore-based products. In this study, we investigated factors associated with viability loss in blastospores dried to two water activity levels, aw = 0.1 (BS01) and aw = 0.3 (BS03), representing more severely and moderately desiccated states, respectively. Samples were stored at 28 °C for 90 days and analyzed every 30 days using viability assays, flow cytometry, protein oxidation analysis, GC-MS, HPLC, and Raman spectroscopy. Viability declined progressively by approximately three orders of magnitude during storage, with BS01 showing a markedly faster reduction than BS03 during the first 30 days. Early storage stages were characterized by high oxidative stress, including elevated reactive oxygen species, lipid peroxidation, and impaired membrane integrity, all of which declined over time as metabolic activity decreased. In contrast, protein carbonylation increased continuously, reaching 11.02 ± 2.23 nmol/mg protein after 90 days. Metabolic analysis of BS01 revealed disrupted cellular homeostasis, including trehalose depletion and glucose accumulation, suggesting impaired metabolic capacity. Raman spectroscopy confirmed progressive molecular alterations in proteins, lipids, and polysaccharides. Together, these findings indicate that cumulative oxidative damage, particularly irreversible protein carbonylation, combined with progressive metabolic dysfunction, is a central driver of blastospore viability loss during storage.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1764301", "ptypes": [ "Journal Article" ], "first_author": "Iwanicki NSA", "dirs": [ "A" ] }, "40576576": { "pmid": "40576576", "title": "Harnessing Microneedles for Delivery and Preservation of Natural Killer Cell-Derived Extracellular Vesicles.", "abstract": "Natural killer cell-derived extracellular vesicles (NK-EVs) have demonstrated anti-inflammatory properties similar to those of their parent cells. EVs have been commonly delivered via intravenous (IV) administration, which can be invasive and is not ideal for chronic treatment. Another limitation of nanotherapy is its storage requirements, as EVs are commonly stored at -80 °C to preserve EV cargo and stability. In order to address these limitations, we explored dissolvable microneedles (MNs) as a promising alternative method for the administration of EVs. MNs have been used to deliver drugs, vaccines, and biomolecules, offering a convenient, noninvasive route of administration while preserving the therapeutic efficacy of EVs for extended periods, even at room temperature. Thus, we hypothesize that MN has the potential to sustain NK-EV stability and successfully deliver NK-EVs with minimal invasion. To test our hypothesis, we first developed an optimal MN formulation composed of hyaluronic acid and trehalose, both protein-protective materials that are biocompatible and biodegradable. After preparing MNs, we evaluated their stiffness, EV release profile, and ability to puncture pig skin. Additionally, the long-term storage stability of the EVs in MNs in inflammatory models in vitro and in vivo was evaluated. The MN successfully maintained EV efficacy even after storage after six months at room temperature, reducing the pro-inflammatory cytokine IL-6 by about 70% in inflamed human fibroblast cells relative to nontreated groups. Furthermore, EV-loaded MN treatment reduced both pro-inflammatory cytokines (IL-6 and TNFα) and psoriasis markers (Ki67 and IL-17) expression in a psoriasis model of chronic inflammation by about 40% compared to nontreated groups. Herein, our MN demonstrates the potential for easy-to-administer NK-EV therapies with long-term storage capabilities that preserve the NK-EV's anti-inflammatory properties.", "journal": "ACS biomaterials science & engineering", "year": "2025", "doi": "10.1021/acsbiomaterials.5c00760", "ptypes": [ "Journal Article" ], "first_author": "Lim SA", "dirs": [ "A" ] }, "42511171": { "pmid": "42511171", "title": "Development and Evaluation of Candied Pumpkin Produced with Jerusalem Artichoke Syrup.", "abstract": "This study developed candied pumpkin using Jerusalem artichoke syrup as an alternative to sucrose and evaluated its physicochemical properties, microstructure, sensory quality, and storage stability. Five formulations with Jerusalem artichoke syrup-to-pumpkin ratios of 1.25:1.00-1.30:1.00 were compared with a sucrose-based control. Syrup-based samples contained more protein (0.96-0.99 g/100 g) and fewer carbohydrates (21.25-23.35 g/100 g) than the control (0.40 and 40.40 g/100 g, respectively). Their caloric values ranged from 90.37 to 99.34 kcal/100 g, compared with 163.20 kcal/100 g in the control. Initial hardness was lower in syrup-based samples (30-40 kPa) than in the control (45 kPa), indicating a less rigid texture. Microstructural analysis showed preserved fibrous organization and a more uniform matrix, particularly in Sample 3 (1.27:1.00). This formulation also showed favorable sensory characteristics and higher contents of vitamin C, pantothenic acid, niacin, phosphorus, magnesium, sodium, and iron. During storage, moisture content decreased and water activity increased gradually but remained below 0.60. Microbial counts remained within permissible limits for 9 months, with no detectable coliforms or pathogenic microorganisms. Therefore, Jerusalem artichoke syrup may be considered a promising sucrose substitute for producing candied pumpkin with improved nutritional value and acceptable storage stability.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15142424", "ptypes": [ "Journal Article" ], "first_author": "Smolnikova F", "dirs": [ "A" ] }, "41421043": { "pmid": "41421043", "title": "Temperature-scanning infrared spectroscopic analysis of sugar glasses using principal component analysis.", "abstract": "Sugars, such as sucrose and trehalose, can protect biological materials during drying. Protective properties of sugars have been attributed to their ability to form hydrogen bonds and facilitate glass formation during dehydration. The aim of this study was to identify intermolecular interactions in amorphous sugar matrices that are involved in glass formation and determine thermophysical properties. Therefore, we studied sugar glasses composed of sucrose, trehalose, and sucrose/albumin mixtures using temperature-scanning infrared spectroscopy (FTIR) empowered by principal component analysis (PCA). Spectral pre-processing procedures included selection of spectral regions, baseline correction, and vector-normalization. PCA revealed that the glass transition coincides with notable changes in the high wavenumber region of the spectra, i.e., in the OH-stretching region. This indicates that particularly hydrogen bonding interactions change during the glass transition. The shape of the broad OH-stretching band depicts strong and weak hydrogen bonding interactions, at respectively low and high νOH wavenumber positions. The glass transition is associated with a change in the thermal expansion coefficient of hydrogen bonding interactions, i.e., the length of intermolecular hydrogen bonds, which increases during the glass transition. Besides deriving the glass transition temperature, Tg, PCA on temperature-dependent spectra revealed differences in hydrogen-bonding networks amongst sucrose and trehalose glasses. Also, protein addition alters the hydrogen-bonding network of a sugar glass, which coincides with an increase in Tg and molecular packing. Taken together, FTIR combined with PCA is a promising tool to study molecular physical characteristics of sugar glasses used for dry preservation of biomolecules and cells.", "journal": "Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy", "year": "2026", "doi": "10.1016/j.saa.2025.127346", "ptypes": [ "Journal Article" ], "first_author": "Liu D", "dirs": [ "A" ] }, "41019173": { "pmid": "41019173", "title": "Protective Effects Proanthocyanidin Nanoliposome Freeze-Dried Powder on Oxidative Injury in a p31-43 Induced Celiac Disease Cell Model.", "abstract": "Proanthocyanidins play a crucial role in celiac disease (CD) through their antioxidant and anti-inflammatory properties, effectively regulating oxidative stress and inflammatory responses. However, the poor stability and low bioavailability of proanthocyanidins significantly limit their therapeutic applications. The aim of this study was to investigate the effects of lyoprotectants on proanthocyanidin nanoliposome freeze-dried powder (PCNL-FD), characterize its structural properties, evaluate its in vitro antioxidant capacity and explore its protective effects in a celiac disease cell model. The addition of 4% trehalose as a lyoprotectant effectively maintained the encapsulation efficiency and redispersion stability of PCNL-FD. FTIR and TEM analyses confirmed the successful incorporation of proanthocyanidins into the phospholipid bilayer. In vitro antioxidant assays showed that PCNL-FD exhibited significantly higher DPPH, ABTS, and hydroxyl radical scavenging activities compared to free proanthocyanidins. In the p31-43 peptide-induced celiac disease Caco-2 cell model, PCNL-FD significantly reduced oxidative stress by decreasing ROS and MDA levels while increasing GSH levels and antioxidant enzyme activities (SOD and CAT). The formulation showed anti-inflammatory effects by inhibiting pro-inflammatory cytokines (TNF-α, IL-6) and promoting anti-inflammatory cytokines (IL-4, IL-10). In addition, PCNL-FD activated the Keap1/Nrf2 signaling pathway and upregulated the expression of NQO-1 and HO-1. This nanoliposomal delivery system effectively overcame the bioavailability and stability limitations of free proanthocyanidins, providing new insights into the management of oxidative stress in inflammatory diseases such as celiac disease.", "journal": "Food science & nutrition", "year": "2025", "doi": "10.1002/fsn3.71000", "ptypes": [ "Journal Article" ], "first_author": "Ning Z", "dirs": [ "A" ] }, "42598243": { "pmid": "42598243", "title": "Long-Term Stabilization of Non-Bovine Milk Exosomes Using Lyophilization: Toward Oral Delivery of PROTAC Therapeutics.", "abstract": "The clinical translation of exosome-based therapeutics is limited by challenges in long-term storage, stability, and scalability. We developed and optimized a lyophilization protocol to stabilize camel and donkey milk-derived exosomes for oral delivery of ARV-110, a proteolysis-targeting chimera (PROTAC). Using 5% trehalose, 5% mannitol, and 2.5% glycine as cryoprotectants, lyophilization preserved exosome integrity, protein content, and expression of key markers (CD63, CD81, TSG101, Alix) during storage at -80°C. Reconstituted exosomes, maintained particle size and zeta potential comparable to fresh preparations. In vitro, ARV-110-loaded lyophilized exosomes showed enhanced cytotoxicity in LLC cells (p < 0.001) and increased transepithelial transport in MDCK assays (p < 0.05). In rats, ARV-110 delivered via lyophilized exosomes achieved a 1.77-fold higher Cmax and 1.73-fold higher AUC0-24 compared to free drug, with no change in Tmax. Lyophilized exosomes retained over 90% of protein content after 8 weeks at -80°C, whereas non-lyophilized controls retained <25%. These findings demonstrate that lyophilized milk exosomes are a scalable, biocompatible platform for oral delivery of poorly bioavailable therapeutics like PROTACs, addressing major formulation and stability challenges and supporting their translational potential in drug delivery.", "journal": "Journal of drug delivery science and technology", "year": "2026", "doi": "10.1016/j.jddst.2025.107821", "ptypes": [ "Journal Article" ], "first_author": "Nathani A", "dirs": [ "A" ] }, "41100560": { "pmid": "41100560", "title": "Discovery of a Novel Sucrose 6F-Phosphate Phosphorylase and Its Application for Isomelezitose Production.", "abstract": "Rare sugars have gained attention as attractive alternatives to conventional sugars due to their potential health benefits and lower caloric value. However, their scarcity restricts widespread application, highlighting the need for efficient biosynthetic production strategies. In this study, a novel sucrose 6F-phosphate phosphorylase from Sphaerochaeta globosa strain Buddy was identified through sequence analysis within the glycoside hydrolase family GH13_18. Substrate screening revealed that this enzyme is capable of producing isomelezitose, a rare trisaccharide naturally found in honey, known for its osmoprotective and potential prebiotic properties. A proof-of-concept production at a 1 L scale, starting from cheap bulk sugars sucrose and isomaltulose, yielded 68 g of high-purity isomelezitose (>99%), which enabled a first evaluation of its properties as a food ingredient. Isomelezitose exhibited limited digestibility and a relative sweetness of 27% compared to sucrose. Moreover, its physicochemical properties were comparable to those of sucrose, including similar glass transition temperature, crystallinity, and flow/tribological behavior. To this end, isomelezitose might form an interesting opportunity as a sugar alternative in the food industry.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.5c07863", "ptypes": [ "Journal Article" ], "first_author": "Steynen M", "dirs": [ "A" ] }, "42403021": { "pmid": "42403021", "title": "Optimization of fermentation medium and preparation of a bacterial agent from Serratia marcescens G4.", "abstract": "Sclerotinia sclerotiorum causes severe yield losses in soybean. Overuse of chemical fungicides leads to pollution, residues, and resistance. Biological control offers a sustainable alternative. This study optimized fermentation of Serratia marcescens G4, developed microcapsules (G4Ms), and evaluated their efficacy against soybean sclerotiniosis. Under initial culture conditions, the OD600 of strain G4 reached 2.241 after 12 h. Sucrose and soybean peptone were identified as the most effective carbon and nitrogen sources, respectively. Response surface optimization determined the optimal medium as sucrose 13.21 g L-1, soybean peptone 10.33 g L-1, yeast extract 10.95 g L-1, and NaCl 10 g L-1, under which the predicted OD600 increased to 2.474 (10.4% improvement). The optimal culture conditions were 35 °C, pH 7.0, 3% (v/v) inoculum, 155 rpm, and 50 mL working volume. Microcapsules prepared with 80% maltodextrin (G4Ms) exhibited enhanced storage stability, stronger biofilm formation, and improved inhibitory activity compared to free-living G4 cells. In pot experiments, G4Ms achieved a control efficacy of 55.84% against soybean sclerotiniosis, and their application increased soil enzyme activities and microbial diversity. G4Ms is an effective, stable, and soil-friendly biocontrol agent against S. sclerotiorum-induced soybean sclerotiniosis, highlighting its potential as a sustainable alternative to chemical fungicides for integrated disease management. © 2026 Society of Chemical Industry.", "journal": "Pest management science", "year": "2026", "doi": "10.1002/ps.71092", "ptypes": [ "Journal Article" ], "first_author": "Gao A", "dirs": [ "A" ] }, "42288032": { "pmid": "42288032", "title": "An in-silico and experimental dual-screening strategy for robust lyoprotection of restriction endonucleases.", "abstract": "An in-silico and experimental dual-screening lyophilization strategy was developed for long-term ambient storage of two model restriction endonucleases (REase) of XbaI (oxidation-resistant) and XhoI (oxidation-sensitive). After molecular docking and experimental screening of lyoprotectants and their combinations, a trehalose-povidone-arginine ternary lyoprotectant \"combo\" was finally developed. The lyophilized formulations were primarily evaluated by four critical quality attributes (CQAs), including lyocake appearance, reconstitution rate, relative enzymatic activity (REA) and short-term storage stability. Four ternary lyophilized formulation candidates were further evaluated by thermogravimetric analysis (TGA) for the residual moisture contents (<2%). Based on the results of four CQAs and TGA, the optimized formulation was finally determined, including trehalose (15%), arginine (5%) and povidone (5%). Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) results indicated that REase lyocakes formed a co-amorphous glassy matrix. Scanning electron microscopy (SEM) images showed REase lyocakes with intact, loose and porous structure. Following six months of long-term stability testing under controlled conditions (25 °C, 60% RH), the lyophilized REase products still maintained comparable DNA cleavage efficiency to that of freshly prepared samples. This study presents a unique in-silico and experimental dual-screening strategy for developing cold-chain-free solid-state enzyme reagents.", "journal": "Cryobiology", "year": "2026", "doi": "10.1016/j.cryobiol.2026.105669", "ptypes": [ "Journal Article" ], "first_author": "Zhang J", "dirs": [ "A" ] }, "42255582": { "pmid": "42255582", "title": "Integrated Optothermal Paperfluidic Platform for Single-Step, Model-Driven CRISPR-Cas12a Diagnostics of Mycobacterium tuberculosis.", "abstract": "CRISPR-Cas12a molecular diagnostics offer high sensitivity and specificity for pathogen detection, yet their translation to point-of-care (POC) settings is often hindered by complex reagent handling, precise thermal requirements, and reliance on bulky laboratory equipment. In this study, we present an integrated opto-thermal paperfluidic platform engineered for the autonomous, single-step CRISPR-Cas12a detection of Mycobacterium tuberculosis (MTB). The platform's architecture was optimized by comparing laser and thermal transfer printing, identifying the latter as the superior method for creating robust, leak-proof hydrophobic barriers while preserving the structural integrity of the cellulose substrate. To achieve autonomous operation, we developed a hybrid mathematical modelincorporating boundary resistance and thermal evaporationto design an \"intrinsic timer\" that aligns capillary flow front progression with the 15 min CRISPR reaction kinetics. Thermal regulation and signal readout are managed by a custom-engineered, 3D-printed hardware module that provides closed-loop 39 °C incubation and high-contrast fluorescence imaging. By utilizing a red-shifted ROX-quencher reporter probe to overcome paper autofluorescence and sucrose-based lyoprotection for reagent stabilization, the platform achieved a limit of detection of 0.0335 ng/μL with prestored reagents. Our integrated detection system requires only a single sample-loading step and provides results in 15 min, offering a practical, low-cost solution for decentralized tuberculosis surveillance in resource-limited environments.", "journal": "ACS omega", "year": "2026", "doi": "10.1021/acsomega.6c00845", "ptypes": [ "Journal Article" ], "first_author": "Thepbut A", "dirs": [ "A" ] }, "42100725": { "pmid": "42100725", "title": "Drying parameters and aging modulate protective properties of vitrified trehalose.", "abstract": "Storage of biological materials underpins medical, research, and biotechnological applications. Although cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, offers an alternative by stabilizing biomolecules in vitrified matrices that limit molecular motion and degradation. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying conditions, environment, storage duration, and biomolecule type. However, systematic links between these factors and stability remain poorly defined. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in trehalose-based vitrified systems. DNA remained stable across all conditions, independent of trehalose or drying parameters, reflecting intrinsic resistance to desiccation damage. RNA exhibited moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured glassy properties. In contrast, enzymes were highly sensitive to drying in the absence of trehalose and strongly protected under conditions that promoted favorable vitrified properties. Short-term enzyme protection (30 min) positively correlated with higher glass transition temperature (Tg). However, during prolonged dry storage, higher Tg was inversely correlated with enzyme stability and instead tracked detrimental physical aging of the vitrified matrix. These findings demonstrate that optimal glass properties depend on both biomolecule class and timescale, providing a framework for rationally designing room-temperature preservation strategies.", "journal": "BBA advances", "year": "2026", "doi": "10.1016/j.bbadva.2026.100188", "ptypes": [ "Journal Article" ], "first_author": "Kumara UGVSS", "dirs": [ "A" ] }, "41648458": { "pmid": "41648458", "title": "Drying parameters and aging modulate protective properties of vitrified trehalose.", "abstract": "Storage of biological materials is essential for medical, research, and biotechnological applications. While cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, provides an alternative by stabilizing biomolecules in vitrified (\"glass-like\") matrices that limit molecular motion which if not reduced can lead to breakdown and loss of integrity. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying methods, environmental conditions, storage duration, and the type of preserved molecule. Systematic studies linking these factors to short and long-term stability remain limited. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in vitrified trehalose systems. DNA remained stable under all conditions, independent of trehalose or drying parameters, reflecting its intrinsic resistance to desiccation-induced damage. RNA showed moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured vitrified properties. In contrast, enzymes were highly sensitive to drying without trehalose and were strongly protected under conditions that promoted favorable vitrified properties. Enzyme protection after 30 min correlated with high glass transition temperature. However, during prolonged drying, increased glass transition temperature was inversely correlated with enzyme protection and was a better indicator of detrimental physical aging of the vitrified system. These findings present the first insight into how drying methods, environmental conditions, and storage duration shape vitrified properties and stability. They guide optimization of room-temperature preservation.", "journal": "bioRxiv : the preprint server for biology", "year": "2026", "doi": "10.64898/2026.01.16.700019", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Kumara U", "dirs": [ "A" ] }, "40761077": { "pmid": "40761077", "title": "Sucrose Monolaurate as a Stabilizer for Lactate Oxidase Electrodes at Low pH: A Structural Analysis Based on Grazing Incidence Small-Angle X-ray Scattering.", "abstract": "Sugars and sugar surfactants can increase the storage stability of enzyme electrodes. In this study, the feasibility of using sugar surfactants as stabilizers for enzyme electrode operation under acidic conditions was investigated along with their stabilizing mechanism. Lactate oxidase (LOx)-sucrose monolaurate-modified electrodes maintained ∼80% of their activity at pH 5.0, compared with ∼50% activity retention without a stabilizer. To elucidate the stabilizing mechanism, the structure of sucrose monolaurate with and without LOx on common electrode materials was analyzed using grazing incidence small-angle X-ray scattering (GI-SAXS). The results revealed that LOx was embedded in hexagonal arrangements and lamellar structures comprising sucrose monolaurate. Encapsulation protected the microenvironment of the enzyme against pH changes, without hindering its access to the substrate and mediator. This study confirms the high potential utility of GI-SAXS as a powerful tool for elucidating the structure-derived mechanisms of enzyme-electrode modifications.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.5c02857", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Shitanda I", "dirs": [ "A" ] }, "40498081": { "pmid": "40498081", "title": "Relaxation Processes in Freeze-Dried Monoclonal Antibody Formulations─The Role of Sucrose Concentration.", "abstract": "Sucrose is the most common stabilizer used in freeze-dried protein formulations. We have investigated, using several methods, the effect of monoclonal antibody (mAb) to sucrose weight ratio on the thermal, relaxation, and water sorption behavior of freeze-dried formulations. The influence of the sucrose content on the miscibility and retention of the native structure of mAb was also investigated. With decreasing mAb-to-sucrose weight ratio, the following effects were observed. Differential scanning calorimetry revealed a progressive decrease in the glass transition temperature of the formulation, while, based on dielectric spectroscopy, the α-relaxation time decreased, whereas both the β- and γ-relaxation times increased. The 1H T1 relaxation time of the antibody, determined by solid-state nuclear magnetic resonance spectroscopy, followed the same trend as the β-relaxation time. Finally, infrared spectroscopy indicated that the optimal retention of the native-like secondary structure of the antibody was achieved at a 4:1 mAb-to-sucrose weight ratio. At mAb-to-sucrose weight ratios of 1:1 and lower, there was no evidence of phase separation in the 20-50 nm scale. Taken together, the results provide new insights into the solid-state behavior of the antibody-sucrose system.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00378", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kumar NSK", "dirs": [ "A" ] }, "39571769": { "pmid": "39571769", "title": "Effects of buffers on spray-freeze-dried/lyophilized high concentration protein formulations.", "abstract": "Solid-state protein formulations are known to exhibit enhanced storage stability compared to their liquid dosage form counterparts. pH is one of the factors affecting the stability of protein formulations. The pH of protein formulations in the solution could be influenced by the buffer used, directly impacting their solid-state stability. During lyophilization, buffer components may interact with other formulation components present in the protein formulations, causing a pH shift. This study aimed to investigate the effects of phosphate buffer and amino acid buffers (such as histidine and/or arginine) on the physical properties and accelerated storage stability of spray freeze-dried or lyophilized protein formulations. A model protein, bovine serum albumin (BSA), was used to prepare high-concentration protein formulations. The formulations consisted of BSA, trehalose, and mannitol in an 80:15:5 ratio (w/w), respectively. Various buffers were utilized in the preparation of protein formulations, and the resultant solid formulations underwent screening via accelerated stability study using size exclusion chromatography (SEC). The combination of phosphate and arginine buffers resulted in increased monomer loss in the accelerated storage stability study. Additional characterizations, including solid-state Fourier transform infrared spectroscopy (ssFTIR) and powder X-ray diffraction (PXRD), were conducted. While these analyses did not definitively elucidate the mechanism behind the observed instability, their outcomes provide valuable insights for further investigation, highlighting the need for future research in this area.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2024.124974", "ptypes": [ "Journal Article" ], "first_author": "Patil CD", "dirs": [ "A" ] }, "40289875": { "pmid": "40289875", "title": "Carbohydrate-Based Polyester and Amino Acid Polyester Photocrosslinker and Their Resin Formulation for 3D Printing Applications.", "abstract": "Fully bio-based polyester was designed and synthesized using the carbohydrate-based diol 2,4:3,5-di-O-methylene-D-mannitol (Manx) and dimethyl ester of 2,3:4,5-di-O-methylene-galactaric acid (Galx). Photocurable resin formulations were prepared by incorporating up to 15 wt% of the carbohydrate polyester into hydroxyl ethyl methacrylate (HEMA) along with polyacrylamide crosslinker derived from L-glutamic acid. Complex 3D structures with good shape fidelity could be 3D printed using these novel polyester resin formulations. The incorporation of the carbohydrate polyester improved the glass transition temperature of the 3D-printed objects. Enzymatic erosion studies conducted using esterase enzyme revealed a higher degradation rate for the 3D-printed films containing the carbohydrate polyester. The hydrolytic degradation analysis conducted in both acidic and basic environments revealed that the 3D-printed polymer network exhibits stability and resilience in acidic conditions, while it undergoes complete degradation in basic conditions. This finding underscores the possibility of tailoring degradation processes under regulated circumstances.", "journal": "Chemistry, an Asian journal", "year": "2025", "doi": "10.1002/asia.202401474", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kamble GN", "dirs": [ "A" ] }, "41429994": { "pmid": "41429994", "title": "Polymers as Stabilizing Excipients for Spray-Dried Protein Formulations.", "abstract": "Drying is widely used to enhance the storage stability of biologic drug products which are susceptible to degradation in aqueous solutions. Compared to conventional freeze-drying, spray drying offers continuous, high-throughput manufacturing. Stabilizing excipients are critical for protecting proteins from stresses during drying and storage. This study evaluated the potential of polysaccharide- and protein-lysate-based polymeric excipients as alternatives to commonly used stabilizers such as trehalose and mannitol, using bovine serum albumin (BSA) as a model protein. Spray-dried BSA formulations were prepared with (2-hydroxypropyl)-β-cyclodextrin (HPβCD), hydrolyzed gelatin, dextran 20 kDa, or sodium carboxymethyl cellulose (NaCMC) polymers, either alone or in combination with trehalose or mannitol. Protein stability was assessed by monitoring monomer loss under stressed storage (40°C, 3 months). Crystallinity and changes in the secondary structure were analyzed using powder X-ray diffraction (PXRD) and solid-state Fourier transform infrared spectroscopy (ssFTIR), respectively. Particle size and size distribution, surface morphology and reconstitution time were also evaluated. Spray-dried BSA formulations containing HPβCD or hydrolyzed gelatin, either alone or with sugars, exhibited lower monomer loss than the trehalose- or mannitol-only formulations. In contrast, formulations with Dextran 20 kDa and NaCMC showed poor stability. PXRD revealed progressive sodium chloride crystallization during storage. ssFTIR detected secondary structure changes in the BSA over 3 months. The spray-dried powders with polysaccharides generally showed longer reconstitution times than those with polymers. HPβCD and hydrolyzed gelatin improved the physical stability of spray-dried BSA compared to sugar excipients, which highlights their potential use as stabilizing additives.", "journal": "Pharmaceutical research", "year": "2026", "doi": "10.1007/s11095-025-03996-z", "ptypes": [ "Journal Article" ], "first_author": "Patil CD", "dirs": [ "A" ] }, "41238664": { "pmid": "41238664", "title": "Comparative study of rebaudioside A and rebaudioside Aα: physicochemical properties and in vivo metabolism.", "abstract": "Stevioside (ST) and rebaudioside A (Reb A) are 250 times sweeter than sucrose, but their bitterness and low water solubility limit their application. Rebaudioside Aα (Reb Aα) was synthesized using dextransucrase. Reb Aα exhibited 80-fold greater water solubility and lower thermal mass loss than Reb A and had melting point >180 °C. Reb Aα was stable within pH range 1.4-10 and exhibited excellent storage stability in aqueous solution. In vitro digestion revealed that Reb Aα was hydrolyzed into ST and glucose. In vivo, Reb Aα administration did not affect body weight gain, glucose tolerance, energy homeostasis, or liver histology. Also, in the Reb Aα group, inflammatory crown-like structures were not observed in white adipose tissue and uncoupling protein 1 was significantly upregulated in brown adipose tissue. Gut microbiota analysis revealed no significant differences in microbial composition between the control and Reb A or Reb Aα groups.", "journal": "NPJ science of food", "year": "2025", "doi": "10.1038/s41538-025-00585-7", "ptypes": [ "Journal Article" ], "first_author": "Kim HJ", "dirs": [ "A" ] }, "39943706": { "pmid": "39943706", "title": "Modulating the Physical Form of Mannitol Crystallizing in Frozen Solutions: The Role of Cosolute and Processing.", "abstract": "Mannitol is widely employed as a bulking agent in lyophilized formulations. Our goal was to evaluate the role of noncrystallizing cosolutes in inhibiting mannitol crystallization and preventing the formation of mannitol hemihydrate (MHH) in frozen solutions. The individual influence of two common stabilizers (sucrose and trehalose) and three model proteins (lysozyme, bovine serum albumin, and immunoglobulin G) on the crystallization behavior of mannitol was investigated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). Sugars exerted a more pronounced crystallization inhibitory effect than proteins. In the presence of sugars, mannitol predominantly crystallized as MHH while the proteins facilitated the crystallization of δ-mannitol. Annealing the frozen solutions at -25 °C favored MHH crystallization. A higher annealing temperature of -10 °C accelerated mannitol crystallization and promoted the formation of the anhydrous δ-polymorph. The crystallization inhibitory effect of proteins was surmounted with annealing, while at a high sugar concentration, a substantial fraction of mannitol was retained amorphous even after annealing.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.4c01481", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Zeng C", "dirs": [ "A" ] }, "42372673": { "pmid": "42372673", "title": "Enhanced stability of RPA-CRISPR-Cas12a system for respiratory pathogen detection using Trehalose-Carboxymethyl Chitosan Lyoprotectant.", "abstract": "Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection. A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis. This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance. The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.", "journal": "Diagnostic microbiology and infectious disease", "year": "2026", "doi": "10.1016/j.diagmicrobio.2026.117523", "ptypes": [ "Journal Article" ], "first_author": "Yang H", "dirs": [ "A" ] }, "42336051": { "pmid": "42336051", "title": "Optimized composite cryoprotectants enhance survival and membrane integrity of Bifidobacterium breve BX-18 during lyophilization.", "abstract": "Bifidobacterium breve BX-18 is a probiotic strain with documented health benefits, yet lyophilization compromises its viability. We hypothesized that optimizing a composite cryoprotectant would enhance survival by preserving membrane integrity and reducing oxidative stress. Single-factor screening identified skim milk, trehalose, sodium acetate, and taurine as effective protectants. Response surface methodology (Box-Behnken design) determined the optimal formulation: trehalose (10.66%), sodium acetate (0.98%), taurine (0.42%), and skim milk (9.75%), achieving 71.01% survival versus <5% in unprotected controls. Mechanistic analyses revealed the composite protectant preserved membrane integrity (70.42% intact cells vs. 28.32% control), maintained membrane fluidity (generalized polarization 0.026 ± 0.011 vs. 0.047 ± 0.009 postlyophilization), and retained enzyme activities (Na+-K+-ATPase decreased 26.3% vs. 76.4% in controls, Ca2+/Mg2+-ATPase decreased 38.4% vs. 62.9%), and β-galactosidase activity remained higher (10.02 ± 0.15 U/mL vs. 2.62 ± 0.13 U/mL). Intracellular reactive oxygen species accumulation was significantly attenuated, and intracellular malondialdehyde content was significantly reduced, indicating alleviated lipid peroxidation in the protected group. Storage stability assessment further demonstrated that lyophilized B. breve BX-18 prepared with the optimal composite cryoprotectant maintained significantly higher viable counts during 21 d of refrigerated storage (4°C) in fermented milk, meeting the viable cell requirements for probiotic functional foods. These findings demonstrate that strategic cryoprotectant combinations preserve B. breve BX-18 viability through multilevel cellular protection, supporting the development of stable probiotic ingredients for functional dairy applications.", "journal": "Journal of dairy science", "year": "2026", "doi": "10.3168/jds.2026-28608", "ptypes": [ "Journal Article" ], "first_author": "Qi W", "dirs": [ "A" ] }, "39547422": { "pmid": "39547422", "title": "Preclinical development of lyophilized self-replicating RNA vaccines for COVID-19 and malaria with improved long-term thermostability.", "abstract": "Messenger RNA (mRNA) vaccines against COVID-19 have demonstrated high efficacy and rapid deployment capability to target emerging infectious diseases. However, the need for ultra-low temperature storage made the distribution of LNP/mRNA vaccines to regions with limited resources impractical. This study explores the use of lyophilization to enhance the stability of self-replicating mRNA (repRNA) vaccines, allowing for their storage at non-freezing temperatures such as 2-8 °C or room temperature (25 °C). We lyophilized repRNA molecules complexed to a novel cationic emulsion delivery system, LION™, with different sugar-based lyoprotectants to identify candidates that provided the best vaccine integrity and effectiveness after being thermally stressed. For screening, we used repRNA encoding the reporter protein secreted embryonic alkaline phosphatase (SEAP) and for proof-of-concept, we used repRNA vaccines encoding SARS-CoV-2 full-length spike (WA-1 isolate) or full-length surface protein circumsporozoite (CS) of Plasmodium yoelii (Py). We found that lyophilization of LION/repRNA with sucrose provided the best colloidal stability, preserved in vitro expression, and induced equivalent antigen-specific antibody responses in mice compared to freshly prepared liquid LION/repRNA. Furthermore, lyophilized vaccines were stable for at least one week at 25 °C and at least one year at 2-8 °C. The cumulative analysis of stability-determining physicochemical data, in vitro potency, and in vivo immunogenicity in mice enabled the selection of a lead lyophilized composition containing 10 % w/v sucrose as the lyoprotectant. The data presented here provide a foundation for the clinical evaluation of next-generation thermostable repRNA vaccines that will enable more equitable vaccine access globally.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2024.11.023", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Gulati GK", "dirs": [ "A" ] }, "40433913": { "pmid": "40433913", "title": "Enhancing the Viability of Spray-Dried Limosilactobacillus fermentum Using Quercetin-Integrated Encapsulation: Physicochemical, Metabolic, and Transcriptomic Insights.", "abstract": "The application of spray drying for the production of probiotic microcapsules offers many attractive advantages, yet now there are concerns regarding probiotic survivability. This study explores the impact of quercetin (Que) in a whey protein isolate (WPI) and trehalose (TR) encapsulation matrix to improve probiotic survival during spray drying. Results showed that probiotic survival increased by 4.95-fold (p < 0.05) with Que supplementation than the probiotics capsulated using WPI and TR. Physicochemical characteristics analysis indicated that adding Que resulted in slight changes in the moisture and water activity of probiotic microcapsules and improved the gastrointestinal digestion resistance and storage stability. The particle size of the spray-dried microcapsules varied from 9.84 to 11.17 µm. Cell membrane analysis demonstrated that the probiotics encapsulated with the WPI-Que-TR complex exhibited higher integrity and fluidity than WPI-TR-coated probiotics. Moreover, introducing Que reduced the ratio of saturated to unsaturated fatty acids and increased the pyruvate kinase activity of probiotics, contributing to the maintenance of cell activity. Transcriptomic results suggested that Que upregulated genes associated with fatty acid synthesis and energy supply while downregulating certain genes involved in amino acid biosynthesis, enabling probiotics to exist better in harsh conditions. Therefore, those results indicated that the co-microencapsulation of probiotics and hydrophobic active substance-Que-was realized, and the mechanism by which Que affects probiotic activity during spray drying has been revealed, which provides a scientific foundation for co-encapsulating probiotics with hydrophobic actives. PRACTICAL APPLICATION: In a rapidly growing market, the demand for dry probiotics has surged, highlighting the need for mass production. Spray drying, with its low energy costs and sustainable process, is a promising method for microencapsulating bacteria in protective substrates, enhancing their resistance during storage, processing, and digestion. Que can improve the survival rate of probiotics during spray drying, offering a novel approach for incorporating flavonoids in the creation of probiotic microcapsules with activity and stability.", "journal": "Journal of food science", "year": "2025", "doi": "10.1111/1750-3841.70293", "ptypes": [ "Journal Article" ], "first_author": "Hu L", "dirs": [ "A" ] }, "42826779": { "pmid": "42826779", "title": "Antifreeze polysaccharides as natural cryoprotectants for frozen meat and seafood products: Mechanisms, evaluation methods, and future prospects.", "abstract": "Freezing is widely employed to preserve meat and seafood products, yet ice recrystallization during frozen storage and thawing disrupts muscle ultrastructure, denatures myofibrillar proteins, and elevates drip loss. Conventional carbohydrate cryoprotectants, such as sucrose, sorbitol, and trehalose, stabilize myofibrillar proteins by modulating water activity, glass transition behavior, and hydrogen-bonding networks. Natural polysaccharides have emerged as promising alternatives that deliver cryoprotection while offering lower sweetness and reduced caloric contribution compared with conventional carbohydrate-based cryoprotectants. This review examines recent advances in applying these polysaccharides as cryoprotectants in comminuted matrices and intact muscle tissues and delineates four interrelated mechanisms: (1) Polysaccharide hydroxyl groups form hydrogen bonds with polar side chains of myofibrillar proteins, sterically limiting unfolding and disulfide-mediated aggregation induced by freeze-thaw cycling. (2) Extensive polysaccharide-water hydrogen bonding disrupts the tetrahedral network of water hydrogen bonds, reduces the fraction of freezable water, and elevates the energy barrier for ice nucleation. (3) Polysaccharide segments adsorb onto ice crystal surfaces, introducing local curvature that inhibits crystal growth through the Gibbs-Thomson effect. (4) Extended high molecular weight chains with an appropriate hydrophilic-hydrophobic balance restrict interfacial water migration, thereby inhibiting recrystallization. The efficacy of an antifreeze polysaccharide depends on its molecular weight, chain rigidity, degree of branching, hydrophilic-hydrophobic balance, and aggregation state. Current evaluation methodologies are compared, including direct and indirect ice crystal visualization, molecular dynamics simulations, thermal hysteresis analysis, and spectroscopic probing of hydrogen bonding. Unresolved questions concerning structure-activity relationships, polysaccharide-protein co-network architecture, and simulation tools suitable for complex food matrices are discussed.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.154700", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ma Y", "dirs": [ "A" ] }, "42504521": { "pmid": "42504521", "title": "Relaxation Processes in Freeze-Dried Monoclonal Antibody-Sucrose Formulations: The Role of Water Content.", "abstract": "Residual water in freeze-dried protein formulations plays a critical role in molecular relaxation behavior. In a previous study (Kumar et al., Mol Pharmaceutics 2025, 22, 4125-4136), we examined the impact of sucrose concentration on native structure retention in monoclonal antibody (mAb)-sucrose systems. The present work investigates how residual water content affects relaxation dynamics in this formulation system. Water affinity was first assessed via sorption isotherms across a range of water activities. There was an increase in residual water content as a function of mAb content in freeze-dried formulations. mAb-sucrose interactions were evident from detailed analyses of sorption data. There was a progressive decrease in the glass transition temperature with increasing water content. Dielectric spectroscopy was then employed to characterize the β- and γ-relaxation processes in the glassy state. While the β-relaxation time decreased with increasing water content, there was no significant effect on the corresponding activation energy. The effect of water content on the γ-relaxation was complex, with an increase in activation energies as a function of water content. In light of the influence of mobility on both physical and chemical stability, these results underscore the importance of controlling water content in optimizing the stability of freeze-dried mAb formulations.", "journal": "Molecular pharmaceutics", "year": "2026", "doi": "10.1021/acs.molpharmaceut.6c00350", "ptypes": [ "Journal Article" ], "first_author": "Kumar NSK", "dirs": [ "A" ] }, "41289169": { "pmid": "41289169", "title": "Exposure humidity and time control of lyophilization and enzyme engineering for stable isothermal nucleic acid detection reagents.", "abstract": "Isothermal nucleic-acid testing is ideal for on-site diagnostics, but large-scale lyophilization of reagents is hindered by three bottlenecks: costly in-situ capping of vials or PCR tubes, activity loss during non-in-situ transfer/sealing caused by ambient humidity and exposure time, and wild-type T7 RNA polymerase inactivation due to lyophilization-induced structural changes. We overcame these obstacles by optimizing the freeze-drying protocol, screening for robust enzyme variants, and refining the protectant recipe. Sealing at 15% RH prior to stoppering proved optimal; a lyophilization-tolerant T7 RNA polymerase mutant termed V5 was isolated; and a blend of 3%(w/v) PEG8000 plus 0.5%(w/v) mannitol markedly enhanced stability. The resulting cake contains 3%(w/w) residual moisture, reconstitutes in <1 s, and retains full activity after 13 months at 4 °C and for approximately 2 months at 25 °C. These specifications enable short-term, cold-chain-independent field deployment of isothermal nucleic-acid detection reagents.", "journal": "Preparative biochemistry & biotechnology", "year": "2026", "doi": "10.1080/10826068.2025.2593613", "ptypes": [ "Journal Article" ], "first_author": "Qin H", "dirs": [ "A" ] }, "42314049": { "pmid": "42314049", "title": "Mapping a Ternary Carbohydrate Design Space for Stable and Dispersible Protein Dry Powders.", "abstract": "Optimizing inhalable protein powders requires simultaneously preserving protein structure and minimizing interparticle cohesion, yet the mechanistic links between excipient composition, drying physics, surface energetics, and aerosol performance remain poorly defined. Here, a constrained ternary mixture design of inulin, trehalose, and mannitol was used to systematically interrogate composition-structure-function relationships in spray-dried lysozyme formulations. Protein secondary structure, glass transition temperature, humidity-dependent surface basicity (via inverse gas chromatography), and aerosol dispersibility (quantified by Wasserstein distance under controlled dispersion conditions) were measured and analyzed using linear and quadratic Scheffé mixture models. Carbohydrate composition strongly governed intermolecular β-sheet content and surface basicity, with trehalose positively associated with β-sheet formation and inulin promoting retention of surface basicity under humidity exposure. Most notably, the relative humidity at which surface basicity reached its minimum exhibited a strong inverse monotonic relationship with dispersibility (Spearman ρ = -0.86, p = 0.013), indicating that early moisture-induced surface reorganization corresponds to greater deviation from complete dispersion under inhalation conditions. These findings support a two-stage mechanistic framework in which drying-stage viscosity evolution defines a finite mobility window for compositional redistribution prior to vitrification, while postdrying surface relaxation dynamics govern susceptibility to moisture-mediated cohesion. Carbohydrate composition thus encodes both protein structural outcomes and aerosol performance through coupled drying- and relaxation-stage processes, providing a mechanistic foundation for rational design of inhalable biologic formulations.", "journal": "Molecular pharmaceutics", "year": "2026", "doi": "10.1021/acs.molpharmaceut.6c00277", "ptypes": [ "Journal Article" ], "first_author": "Xia G", "dirs": [ "A" ] }, "40558738": { "pmid": "40558738", "title": "Drying of Functional Hydrogels: Development of a Workflow for Bioreactor-Integrated Freeze-Drying of Protein-Coated Alginate Microcarriers for iPS Cell-Based Screenings.", "abstract": "Protein-coated ultra-high viscosity (UHV)-alginate hydrogels are essential to mimic the physiological in vivo environment of humans in several in vitro applications. This work presents an optimized bioreactor-integrated freeze-drying process for MatrigelTM-coated UHV-alginate microcarriers in the context of human induced pluripotent stem cell (hiPSC) expansion. The impact of freeze-drying on the UHV-alginate microcarriers using trehalose 100 mg/mL in 0.9% NaCl as a lyoprotective agent, as well as the stem cell response using hiPSCs, was analyzed using microscopy-based screenings. First observations of the process showed that the integrity of the cake was preserved in the samples with a maximum vapor exchanging rate. Following rehydration, the UHV-alginate microcarriers retained their original morphology. Upon the addition of Poloxamer 188, stickiness and bubble formation were reduced. The expansion of hiPSCs in a suspension bioreactor resulted in a 5-7-fold increase in total cell count, yielding at least 1.3 × 107 cells with viability exceeding 80% after seven days of cultivation. In flow cytometry analysis, the pluripotency factors OCT3/4 and SSEA4 resulted in positive signals in over 98% of cells, while the differentiation factor SSEA1 was positive in fewer than 10% of cells. Supported by preceding in silico predictions of drying time, this study presents, for the first time, basic steps toward a \"ready-to-use\" bioreactor-integrated freeze-drying process for UHV-alginate microcarriers in the iPSC context.", "journal": "Gels (Basel, Switzerland)", "year": "2025", "doi": "10.3390/gels11060439", "ptypes": [ "Journal Article" ], "first_author": "Balsters JM", "dirs": [ "A" ] }, "40987543": { "pmid": "40987543", "title": "\"Bigger, the Better?\"─The Influence of Sugar Size and Residual Moisture on Protein Stability and Accessibility in Lyophilizates.", "abstract": "Lyophilization is a key technology to improve the long-term stability of protein drug products, traditionally using the disaccharides sucrose and trehalose for cryo- and lyoprotection. Monosaccharides are less favored due to the low glass transition temperature and Maillard reaction potential. Additionally, trisaccharides and tetrasaccharides typically do not play significant roles, as they are often not approved for parenteral use and have been associated with lower protein stabilization. Key stability parameters include the preserved protein structure, solid-state accessibility, and monomer content. This study explores the long-term monomer retention of human serum albumin (HSA) in lyophilizates at 2-8, 25, and 40 °C by investigating the effect of a series of mono- to tetrasaccharides based on glucose (glucose, maltose, maltotriose, and maltotetraose) as well as glucose maltose and glucose maltotriose mixtures. We varied the residual moisture (RM) content (1, 1.5, and 2%) postlyophilization to understand the effects of water replacement, vitrification, and matrix mobility on protein stability. The molar ratios of maltose to HSA were set at 360:1 and 180:1 to investigate the impact of the sugar concentration at overall low sugar ratios. Solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX MS) was performed on a QDa benchtop mass spectrometer to evaluate protein accessibility and structural preservation using RMs of 1% D2O, 2% D2O, and 1% D2O + 1% H2O. The larger the sugar, the lower its stabilizing potential and the higher the protein accessibility, indicating insufficient water replacement. Increasing the RM from 1 to 1.5 and 2% enhanced stability, highlighting the superiority of residual water molecules, which was especially the case for the tri- and tetrasaccharides. Mixtures of small and large sugars showed stabilization benefits in maintaining the monomer content and structural preservation, indicating a good balance of water replacement and vitrification. Overall, the ssHDX MS findings of samples with headspace-spiked D2O did correspond with monomer retention, indicating that it could be a valuable tool for characterization and understanding the stabilizing capacity of lyophilized formulations. Our findings highlight the importance of RM control for optimal stability as well as the importance of the sugar size on lyoprotection based on water replacement and the potential of sugar mixtures to optimize the stability of lyophilized proteins.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00596", "ptypes": [ "Journal Article" ], "first_author": "Lo Presti K", "dirs": [ "A" ] }, "40022358": { "pmid": "40022358", "title": "Improving the physical stability of Lactobacillus plantarum LP90 during storage by mixing carboxymethylated dextran-whey protein conjugates and small-molecule sugars.", "abstract": "To explore the effect of small-molecule sugars on the physical stability of microcapsules and enhance probiotic preservation, carboxymethylated dextran and whey protein conjugate (WP-CD5d) by Maillard reaction were used as the wall material. Trehalose, lactose, and sucrose were used as lyoprotectants to encapsulate Lactobacillus plantarum LP90 (LP90) during storage, forming microcapsules labeled as WP-CD5dH, WP-CD5dR, and WP-CD5dZ, respectively. WP-CD5dH and WP-CD5dR exhibited the highest viability after freeze-drying, with survival rates of 97.8 % ± 0.9 % and 98.5 % ± 0.5 % respectively. LP90 microcapsules exhibited strong resistance to simulated gastrointestinal fluid over 5 h. The Guggenheim-Anderson-de Boer (GAB) and Gordon-Taylor model showed that WP-CD5dH had the best reduction in water plasticization (k was 4.0). 1H NMR spectra confirmed that WP-CD5dH exhibited the lowest molecular mobility. Furthermore, the storage experiment showed that WP-CD5dH provided the best protection for LP90 at 11 % RH, with a K value of 0.021. These results suggest that the probiotic mechanism of small-molecule sugars involves forming intermolecular forces with WP-CD5d, which helps to maintain the glassy state.", "journal": "Food research international (Ottawa, Ont.)", "year": "2025", "doi": "10.1016/j.foodres.2025.115834", "ptypes": [ "Journal Article" ], "first_author": "Li W", "dirs": [ "A" ] }, "40790703": { "pmid": "40790703", "title": "Beyond the water activity and Tg: water usability can govern xerophilic fungal and bacterial proliferation in sucrose/whey protein isolate solid matrices.", "abstract": "High-solid food matrices are often unstable; how water behaves in them dictates molecule mobility and microbial proliferation. Here, we employ lyophilized sucrose/whey protein isolate (WPI) mixtures as a model system to link water sorption isotherms, glass-transition temperatures (Tg), structural relaxation dynamics, and relaxation times to microbial growth of Aspergillus flavus and Bacillus subtilis. Although the sorption isotherms, Tg, and relaxation processes of the studied matrices were affected by aw and WPI, the microbial growth showed a high dependence on water mobility rather than aw (0.75-0.92 aw at 30 °C). Therefore, this study used water usability (Uw), a thermodynamic parameter (ranging from 0 to 1), where higher values indicate greater amounts of freely mobile water. Moreover, Uw quantifies the full plasticizing effect of bound and free water and the kinetic immobilization imposed by protein networks. As Uw declines with increasing WPI content, extensive hydrogen-bonding and microphase domains sequester water into tightly bound shells, reducing free volume and slowing segmental motions; this reduction in Uw predicts slower microbial growth behavior more reliably than aw or Tg alone. By uniting thermodynamic and dynamic facets of water behavior in a single parameter, Uw offers a quantitative framework to anticipate and control nutrient transport, textural stability, and microbial safety in complex, non-equilibrium solid foods, with broad implications for mitigating spoilage and ensuring product quality under shifting climatic conditions.", "journal": "Food research international (Ottawa, Ont.)", "year": "2025", "doi": "10.1016/j.foodres.2025.116919", "ptypes": [ "Journal Article" ], "first_author": "Cui T", "dirs": [ "A" ] }, "40107554": { "pmid": "40107554", "title": "Covalently immobilized β-galactosidase onto a novel alginate/lemon peel carrier: Catalytic, kinetic, stability studies, and its application in the production of whey high-protein beverage.", "abstract": "β-Galactosidase (βG) was immobilized on novel alginate/lemon peel (Alg/LP) beads, achieving an immobilization yield of 84.7 % and an efficiency of 96.6 %. Both free and immobilized enzymes exhibited optimal activity at 50 °C and pH 5. The immobilized enzyme (Alg/LP/βG) demonstrated significantly improved thermal stability and broader pH tolerance compared to its free form. Immobilization notably altered enzyme kinetics, reducing Km by 57 % and Vmax by 13.4 %. Alg/LP/βG displayed excellent storage stability at 4 °C, retaining 91.3 % activity after 21 days and 79.6 % activity after 35 days. It also maintained 100 % activity after 11 cycles and 75.4 % activity after 15 operational cycles. The enzyme's application was explored in producing high-protein beverages from 13 % whey protein concentrate, enhancing sweetness by hydrolyzing lactose into glucose and galactose, which were further sweetened with 7 % sucrose. Adding lemon peel powder (LPP) improved the beverage's flavor, antioxidant properties, total phenolic content, and viscosity while keeping its chemical composition low. Sensory evaluation identified the beverage with 0.4 % LPP as the most preferred. This study highlights the potential of Alg/LP beads as an effective support matrix for β-galactosidase, offering enhanced stability, reusability, and applicability, particularly for enzymatic processes in the food industry.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.142222", "ptypes": [ "Journal Article" ], "first_author": "Abdella MAA", "dirs": [ "A" ] }, "41794351": { "pmid": "41794351", "title": "Hydroxypropyl-β-cyclodextrin as a multifunctional excipient for enhancing stability and aerosol performance of spray-dried protein formulations.", "abstract": "Protein-based drugs are precise therapeutic options for various refractory lung diseases, significantly improving patient prognosis and quality of life. In this study, a comparative spray-drying approach was employed to prepare inhalable dry-powder formulations of bovine serum albumin (BSA), a surface-active model protein, using established pharmaceutical excipients, including 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), trehalose (Tre), leucine (Leu), and a Tre-Leu combination. The relative performance of HP-β-CD was systematically benchmarked against Tre, Leu, and dual-excipient systems under identical formulation and processing conditions, with respect to protein protection and aerodynamic properties. After 3 months of storage under accelerated stability condition (40 ± 2 °C and 20 ± 5% relative humidity), the protein monomer content in the HP-β-CD-based formulations was marginally lower than that observed in the Tre- and dual-excipient-containing dry powders but still demonstrated effective protein protection. Moreover, the aerosol performance of HP-β-CD formulations surpassed that of other excipients, indicating its superior potential for pulmonary delivery of protein molecules. Overall, this comparative study highlights the potential of HP-β-CD as an alternative excipient for protein-based aerosol dry-powder formulations.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.126729", "ptypes": [ "Journal Article" ], "first_author": "Yang Y", "dirs": [ "A" ] }, "40280484": { "pmid": "40280484", "title": "Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg\" and Tg' in frozen sucrose/water mixtures.", "abstract": "High-resolution synchrotron X-ray powder diffraction has been utilized to detect mechanical stresses in frozen solutions, via analysis of the profiles of the diffraction peaks of ice. Increase in the width of the peaks (peaks broadening) reflects disruption of the crystal lattice, with contributions including a decrease in the crystallite size and an increase in microstrain. Frozen sucrose solutions (5 and 10 % w/v) were frozen at 100 K and then heated and annealed at -45 °C (228 K) and -15 °C (258 K), i.e. between the two apparent glass transition events in the freeze-concentrated solutions (Tg\" and Tg'), and above the Tg', respectively. A decrease in microstrain and an increase in ice crystallite size were observed during annealing at -15 °C (above the Tg'), which is consistent with Ostwald ripening of ice crystals. Unexpectedly, and for the first time, an opposite trend was observed during annealing at the lower temperature of -45 °C, between Tg\" and Tg'. To the best of our knowledge, this is the first report of an increased strain in the crystalline ice domains and of simultaneous size reduction during annealing of frozen aqueous solutions. Considering that the interaction with ice crystals may result in protein destabilization, the size and microstrain of ice crystallites can serve as markers of the freeze/thaw stresses on biopharmaceuticals. A practical implication is that a prolonged hold between Tg\" and Tg' may increase stress imposed on protein molecules, i.e. a lower temperature is not always better for preserving biopolymers when freezing their aqueous solutions.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2025.103800", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kostyuchenko A", "dirs": [ "A" ] }, "42398871": { "pmid": "42398871", "title": "Biological control evaluation of bacteriophage freeze-dried powder against Aeromonas hydrophila infection, based on whey protein/trehalose.", "abstract": "Aeromonas hydrophila, as an important zoonotic conditional pathogen, can cause various diseases such as bacterial septicemia in fish, as well as acute gastroenteritis in humans. Bacteriophages, due to their significant advantage in combating antibiotic-resistant bacteria, have become a research hotspot for novel biological control tools. This study focused on A. hydrophila phage 003s1, which maintained high activity under a wide range of conditions from pH 3 to 11 and at temperatures from 30 to 60°C. After 60 min of ultraviolet irradiation, 63.97% of the original activity remained. To develop a novel antimicrobial agent against A. hydrophila, the phage 003s1 was loaded into different protective agents and subjected to freeze-drying. It was found that using 1% whey protein (WPI) as the protective agent resulted in the optimal viability of the phage after freeze-drying. When stored at 4°C for 6 months, the titer decreased by only 0.86 log10 PFU/mL. In the zebrafish embryo infection model, after treatment with phage powder at a MOI of 0.1, the survival rate of embryos infected with the minimum lethal dose of A. hydrophila significantly increased to 56.67%. In summary, the phage powder developed in this study not only exhibits excellent storage stability but also demonstrates significant protective effects in the zebrafish infection model, providing an innovative antimicrobial agent for the biological control of A. hydrophila.", "journal": "Microbial pathogenesis", "year": "2026", "doi": "10.1016/j.micpath.2026.108675", "ptypes": [ "Journal Article" ], "first_author": "Yang Y", "dirs": [ "A" ] }, "42144900": { "pmid": "42144900", "title": "Immobilization of an Invertase Produced by a Bacterium Isolated from the Peach Palm Fruit (Bactris gasipaes): Influence of the Support Material and Biochemical Characterization.", "abstract": "This study evaluated the immobilization of an invertase from Bacillus tequilensis, isolated from the fruit of the peach palm, on silica gel (SG) and polyhydroxybutyrate (PHB), aiming at its application in sucrose hydrolysis for invert sugar production. Immobilization was performed at 30 °C and pH 5.5 using glutaraldehyde-functionalized supports. Immobilization yield (IY), recovered activity (RA), kinetic parameters, and pH, thermal, storage, and operational stabilities were evaluated. IY values reached 72% (SG) and 77% (PHB), with RA of 35% and 67%, respectively. Both immobilized derivatives exhibited improved storage stability compared to the soluble enzyme. The SG-immobilized invertase demonstrated enhanced thermostability and higher fructose production and retained over 50% of its activity after three reuse cycles. In contrast, the PHB derivative showed greater substrate affinity. These results demonstrate that the B. tequilensis invertase immobilized on SG is a promising and stable biocatalyst derived from the Amazonian biodiversity, suitable for sustainable invert sugar production.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.6c02898", "ptypes": [ "Journal Article" ], "first_author": "Saraiva LS", "dirs": [ "A" ] }, "39910784": { "pmid": "39910784", "title": "Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi.", "abstract": "There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active 4 weeks after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.4c02029", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Pascual-Garrigos A", "dirs": [ "A" ] }, "41068553": { "pmid": "41068553", "title": "An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection.", "abstract": "Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.5c07919", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Wang YM", "dirs": [ "A" ] }, "41284053": { "pmid": "41284053", "title": "The impact of lyophilization media on fertilization potential of buffalo spermatozoa.", "abstract": "The current investigation aimed to exhibit the impact of lyophilization media of frozen spermatozoa on the fertility potential of buffalo spermatozoa as indicated by comet assay and ICSI. Semen specimens were centrifugated at 700 × g for 20 min using percoll gradient (45-90%), double washed in Tyrode's albumen lactate pyruvate (TALP) and diluted in the lyophilization media (media 1, 2, 3 and 4), correspondingly. Cooling of the diluted sperm cells in vapor of liquid nitrogen. Frozen samples were instantly put into the lyophilizer (-55 °C, pressure 0.001 Mbar). After 24 h of lyophilization, the semen specimens were kept for three months at 4 °C. Frozen-dried semen was re-hydrated at room temperature in of milli-Q water(100 µL). Comet assay results of the frozen-dried semen exhibited that the TCM medium exhibited the lowest % of DNA deterioration [6.17] and the superior % of embryonic developmental rate,while Tris-EDTA medium exhibited the highest % of DNA deterioration [13.09]. The lowest successful % of ICSI exhibited upon using EGTA and EDTA media. It could be concluded that ICSI of frozen-dried spermatozoa upon using TCM medium provides the highest % of embryonic expansion. Also, Tris-EGTA and Tris-EDTA media exhibited the lowest successful percent.", "journal": "Cell and tissue banking", "year": "2025", "doi": "10.1007/s10561-025-10200-4", "ptypes": [ "Journal Article" ], "first_author": "El-Sheshtawy RI", "dirs": [ "A" ] }, "42485833": { "pmid": "42485833", "title": "Cold-chain-free CRISPR diagnostics enabled by magnesium-tannic acid nanoencapsulation of the complete detection system.", "abstract": "Cold-chain dependency remains the major barrier to the widespread deployment of CRISPR-based diagnostics, particularly in resource-limited settings, where the thermal instability of protein and nucleic acid reagents necessitates refrigerated storage and transport from manufacture to point of use. Here we report a one-step aqueous nanoencapsulation strategy that overcomes this limitation using magnesium-tannic acid metal-phenolic nanoparticles (Mg2⁺-TA MPNs). The platform simultaneously co-encapsulates all functional components of a CRISPR-LbCas12a detection system, including the nuclease, guide RNA, and ssDNA reporter, within a single protective matrix. Magnesium ions serve as a structural coordinator of the metal-phenolic nanoparticles while maintaining biochemical compatibility with the released CRISPR system. Physicochemical characterization confirmed successful particle assembly and efficient cargo incorporation. Functional studies demonstrated that encapsulated CRISPR reagents retained greater than 70% of diagnostic activity after 8 days at 50 °C, whereas non-encapsulated controls were completely inactivated within 24 h under identical conditions. Released formulations also remained fully compatible with both fluorescence- and lateral flow-based detection formats. This approach requires no lyophilization, no specialized equipment, and no cold chain at any stage of production or storage, thereby offering a scalable and readily deployable route to thermally stable molecular diagnostics.", "journal": "Colloids and surfaces. B, Biointerfaces", "year": "2026", "doi": "10.1016/j.colsurfb.2026.115989", "ptypes": [ "Journal Article" ], "first_author": "J MA", "dirs": [ "A" ] }, "41461127": { "pmid": "41461127", "title": "A cold-chain free loop-mediated isothermal amplification test for detection of human malaria in point-of-care settings.", "abstract": "Malaria diagnosis in low-resource settings demands robust and cold-chain free molecular tests. To overcome infrastructure limitations of conventional loop mediated isothermal amplification (LAMP), we developed a refrigeration-independent assay for Plasmodium knowlesi detection. By using sugar-based enzyme preservation approach, sucrose shows most effectively to maintain Bst DNA polymerase activity during room-temperature storage, outperforming equivalent concentrations of glucose and galactose. The sucrose-preserved LAMP reagent demonstrated consistent performance for 12 days. This optimized assay achieved analytical sensitivity (10 copies/μL) and maintained 100 % specificity against Plasmodium species and human DNA. The chemical preserving method provided a cost-effective alternative to lyophilization, eliminating dependence on specialized equipment while achieving comparable short-term stability. The 12-day room-temperature stability suits typical field deployments, making the assay ideal for malaria surveillance and use in remote clinics.", "journal": "Diagnostic microbiology and infectious disease", "year": "2026", "doi": "10.1016/j.diagmicrobio.2025.117237", "ptypes": [ "Journal Article" ], "first_author": "Rosli NN", "dirs": [ "A" ] }, "42763167": { "pmid": "42763167", "title": "Rapid and sensitive on-site detection of PBoV G3 using an EsCas13d-based platform.", "abstract": "Rapid, sensitive, and user-friendly nucleic acid diagnostic methods are crucial for controlling the spread of PBoV G3. In this study, we developed SENTRY (Single-step Enzymatic Nucleic acid Targeted Reporter Yield), a pathogen detection platform designed for on-site deployment in resource-limited settings. Combined with GENE (General Extraction-free Nucleic acid Exposure reagent), a room-temperature lysis protocol, SENTRY can release viral nucleic acids within 5 min. The addition of accessory proteins enhances the sensitivity of the one-step SENTRY platform and shortens the reaction time. SENTRY provides results within 30 min, with clinical sample detection rates matching those of qPCR, and shows no cross-reactivity with non-target viruses. The system operates efficiently at body temperature (∼37 °C) without requiring temperature control devices. Lyophilized reagents make SENTRY robust to transport conditions and extend shelf life. Overall, these features make SENTRY highly suitable for POCT outside conventional laboratory environments.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.346108", "ptypes": [ "Journal Article" ], "first_author": "Fu PF", "dirs": [ "A" ] }, "41478037": { "pmid": "41478037", "title": "Extended strand-mediated target unmasking synergized with lyophilized hairpin probes for one-step viral RNA detection.", "abstract": "This method pioneers a synergistic strategy combining 'extended-strand unmasking with freeze-drying pre-packaging'. By designing unequal-length double-strand replacement strands that competitively bind to regions flanking the target site, it forces the unfolding of hairpin/cloverleaf conformations, thereby physically exposing concealed targets. Concurrently, a five-step lyophilization process (pre-folding-anchoring-sealing-encapsulation-freezing) employs graphene oxide (GO) π-π stacking to suppress >90 % of DNA breathing effects. Combining 0.02 % (w/v) low-melting-point agarose achieves in situ encapsulation and probe conformation fixation, forming microchannels for water molecules. The final lyophilized product forms a porous solid matrix (integrable into nitrocellulose test strips or 2-mm diameter lyophilized spheres), enabling stable probe storage at ambient temperature. This technology overcomes the 'impossible triangle' challenge in molecular diagnostics: achieving high room-temperature stability (>6 months, compared to <7 days for traditional liquid-phase CHA), ultra-high sensitivity (0.01 pM), and a simple, rapid testing process. This solution provides a \"sample-in, result-out\" point-of-care testing approach for home health management (no cold chain required), primary care clinics (no equipment dependency), and pandemic emergency response.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2025.118350", "ptypes": [ "Journal Article" ], "first_author": "Wang J", "dirs": [ "A" ] }, "41624074": { "pmid": "41624074", "title": "A thermo-responsive sol-to-gel phase transition hydrogel for sustained delivery of mesenchymal stem cell-derived exosomes.", "abstract": "Mesenchymal stem cells-derived exosomes (MSC-Exo) have significant therapeutic potential in regenerative medicine but face challenges such as rapid systemic clearance and poor retention at target sites. In this study, a thermo-responsive sol-to-gel phase transition hydrogel based on sodium alginate (SA), cellulose nanofibrils (CNF) and poloxamer 407 (P407) was prepared for sustained delivery of MSC-Exo. The MSC-Exo loaded CNF/SA hydrogel was prepared, which was freeze-dried and then dispersed in the P407 solution at room temperature. The mixture of CNF/SA/MSC-Exo and P407 could undergo thermo-responsive sol-to-gel phase transition due to the thermosensitive property of P407, and thus the CNF/SA/MSC-Exo was encapsulated in the P407 gel at physiological temperature (37 °C). Owing to the high degradation ratio of P404 gel and swelling ratio of CNF/SA hydrogel at physiological pH (∼7.4), sustained delivery of MSC-Exo from the gel was achieved with a high cumulative release of 80 % after 120 h. Cytotoxicity assay showed that the CNF/SA/P407 gel has excellent biocompatibility. These findings provide a feasible strategy to achieve the sustained delivery of Exo for regeneration therapy, giving valuable references for future research.", "journal": "Regenerative therapy", "year": "2026", "doi": "10.1016/j.reth.2026.101073", "ptypes": [ "Journal Article" ], "first_author": "Liu L", "dirs": [ "A" ] }, "42599083": { "pmid": "42599083", "title": "A rapid, sensitive, and field-deployable RAA-EsCas13d platform for detection of porcine adenovirus type 3.", "abstract": "Rapid, sensitive, and RADIANT-deployable nucleic acid diagnostics are essential for the prevention and control of porcine adenovirus type 3 (PAdV-3). In this study, we developed RADIANT (RAA-Cas13d DIagnostic plATform for extractioN-free Testing), an extraction-free platform capable of delivering accurate and portable detection in resource-limited settings. By systematically optimizing the reaction buffer composition, recombinase-aided amplification (RAA), T7 transcription, and CRISPR/EsCas13d-mediated cleavage were integrated into a one-pot reaction, reducing the total time-to-result to within 30 minutes. A simplified nucleic acid release step eliminated the need for laboratory-based extraction or complex heating, enhancing the accessibility of the assay. Incorporation of lyophilized reagents minimized cold-chain requirements and simplified assay preparation, enabling cost-effective storage and transport. The platform demonstrated high sensitivity in detecting PAdV-3 and offered two straightforward readout options, fluorescence under 470 nm blue/UV light and lateral flow assay (LFA), facilitating flexible interpretation under diverse RADIANT conditions. Validation with 56 clinical samples showed 100% concordance with quantitative PCR, confirming RADIANT as a rapid, user-friendly, and reliable on-site diagnostic tool for PAdV-3 detection.IMPORTANCEPorcine adenovirus type 3 (PAdV-3) is an important swine pathogen for which rapid, practical, and field-compatible diagnostic tools are still lacking. Here, we developed RADIANT, a CRISPR/EsCas13d-based platform that expands PAdV-3 detection beyond conventional laboratory workflows and supports accessible molecular testing in resource-limited settings. Its simple operation and adaptable visual readouts enhance its suitability for on-site use without compromising reliability. This study provides a valuable diagnostic approach for PAdV-3 surveillance and disease control and supports the broader application of portable CRISPR-based technologies in veterinary diagnostics.", "journal": "Microbiology spectrum", "year": "2026", "doi": "10.1128/spectrum.01291-26", "ptypes": [ "Journal Article" ], "first_author": "Li Y-y", "dirs": [ "A" ] }, "41947518": { "pmid": "41947518", "title": "An All-in-One Microfluidic Platform for POCT-Ready Nucleic Acid Extraction from Heterogeneous Clinical Samples.", "abstract": "In point-of-care (POC) molecular diagnostics, inhibitors from liquid residue in a microfluidic chip fundamentally limit the performance of automated nucleic acid extraction (NAE). In response to this challenge, a multifaceted strategy has been developed. We engineered a novel U-shaped microchannel via injection molding, which inherently reduces liquid retention by 2-fold over conventional square channels. Critically, we identified that surfactants, while essential for lysis, were a major source of undesired liquid retention; a simple water-rinse stage was developed to counteract this effect, further decreasing the residue by 5-fold. Process efficiency was radically enhanced by integrating ultrasonic excitation, achieving both a sample-to-pure-NA time of just 5 min and NA recovery rates in microchips comparable to conventional extraction methods. These physical, chemical, and process-level innovations were consolidated into the universal automated nucleic acid extraction (UNEX) system. Featuring lyophilized, shelf-stable reagents for cold-chain independence, the UNEX system is a truly automated, sample-in-to-result-out platform. When benchmarked against traditional methods using various clinical samples, UNEX exhibited exceptional precision (CV < 2%) and quantitative accuracy (R2 > 0.99). Our work provides a robust, field-deployable NAE solution, proving that a holistic approach (combining optimized channel geometry with intelligent fluidic protocols and energy-assisted elution) is key to unlocking the full potential of LOC technology in diagnostics.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c07787", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Chen Z", "dirs": [ "A" ] }, "42562533": { "pmid": "42562533", "title": "Field-deployable, lyophilized cell-free biosensor for robust and selective detection of inorganic arsenic in complex seaweed matrices.", "abstract": "Current on-site arsenic monitoring is hindered by cold-chain dependence and biosafety risks. We developed a field-deployable cell-free protein synthesis (CFPS) biosensor for the rapid, selective detection of toxic inorganic arsenic in seaweed matrices (Sargassum fusiforme and Porphyra sp.). An ArsR-regulated genetic circuit enables effective discrimination between inorganic and organic arsenic species. A tailored lyophilization strategy eliminated the need for ultra-low temperature storage, retaining ∼70% activity over four weeks at room temperature. Notably, freeze-drying serendipitously suppressed leaky expression, reducing background noise by 8-fold compared to liquid controls. Validated through spike-and-recovery tests in real seaweed extracts, this cold-chain-free, highly sensitive platform represents a significant advancement for decentralized food safety monitoring and environmental screening.", "journal": "Food research international (Ottawa, Ont.)", "year": "2026", "doi": "10.1016/j.foodres.2026.119766", "ptypes": [ "Journal Article" ], "first_author": "Jeong DS", "dirs": [ "A", "D" ] }, "42823406": { "pmid": "42823406", "title": "A fully integrated sample-to-answer diagnostic platform enables rapid quantification of post-transplant infection at the point-of-care.", "abstract": "Opportunistic infections after organ transplantation require frequent monitoring over years. However, current diagnostic tests are inaccessible to patients in resource-limited settings. Here, we present a microfluidic-enhanced nucleic acid test for on-site reporting (MONITOR), a fully integrated, sample-in-answer-out platform for point-of-care (POC) detection of opportunistic infections. MONITOR employs a gene-specific DNAzyme that, upon integration with recombinase polymerase amplification, enables CRISPR-like trans-cleavage signal amplification with single-molecule sensitivity. It further incorporates a rapid, extraction-free protocol for processing the urine sample, lyophilized assay reagents for cold-chain-free storage and transportation, and a 3D-printed microfluidic cartridge for self-contained, contamination-free fluid handling. A portable real-time fluorescence reader and smartphone application are further developed to enable on-site quantification. Clinical validation showed complete concordance with qPCR across the tested cohorts and a field trial identified high-risk patients with systemic BK polyomavirus infection. MONITOR fulfills ideal criteria for POC testing, offering a practical solution for decentralized, quantitative viral monitoring in low-resource settings.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-77054-6", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Yin P", "dirs": [ "A", "D" ] }, "42696600": { "pmid": "42696600", "title": "Amplification of RNA for identification of Zika and HCV in whole blood.", "abstract": "Direct RNA amplification from whole blood is fundamentally limited by rapid enzymatic degradation and inhibitory matrix effects. Here, we present a blood drying protocol that enables sensitive and robust RNA detection without the need for extraction, purification, or cold-chain logistics. Using whole blood, the platform achieves high detection sensitivity, down to 10 copies per microliter for Zika virus and 1 international unit per microliter for hepatitis C virus (HCV). We further demonstrate that the protocol can be scaled to larger blood volumes and achieve single-copy sensitivity without any sample loss. This is accomplished through thermal treatments of the sample combined with a primer-limited reverse transcription step, which together stabilize RNA within a dried blood matrix and permit spatially resolved enzymatic amplification. The system supports multiplexed detection from a single sample, enabling simultaneous identification of multiple targets. Separately, we introduce a concept wherein the very few copies of the preserved RNA within the matrix can be accessed repeatedly for molecular analysis. Furthermore, we demonstrated the detection of Zika and HCV using a portable fluorometer for point-of-care (POC) uses. With lyophilized reagents and minimal instrumentation such as a heater and an inexpensive portable fluorometer, this platform enables robust, reusable, and field-deployable diagnostics, advancing toward truly accessible on-site RNA testing in urgent care or low-resource settings from whole blood.", "journal": "Science advances", "year": "2026", "doi": "10.1126/sciadv.aeb6129", "ptypes": [ "Journal Article" ], "first_author": "Lim J", "dirs": [ "A" ] }, "42648816": { "pmid": "42648816", "title": "A lyophilized CHA-lateral flow platform for point-of-care detection of miR-106b-5p from L1CAM-Immunocaptured small extracellular vesicles in Alzheimer's disease.", "abstract": "The early, non-invasive diagnosis of Alzheimer's disease (AD) remains a significant clinical challenge. MicroRNAs carried by L1CAM-immunocaptured small extracellular vesicles (sEVs) in blood, particularly miR-106b-5p, have emerged as promising candidate biomarkers for AD. However, their detection requires methods that are sensitive, specific, stable, and suitable for point-of-care (POC) applications. Catalytic hairpin assembly (CHA) combined with lateral flow immunoassay (LFIA) offers a potential POC solution, but its practical application is limited by high background leakage caused by \"DNA breathing\" and poor probe stability. Herein, we propose an integrated POC platform that combines immunomagnetic enrichment of L1CAM-positive sEVs with a novel \"Fold-Adsorb-Block-Immobilize-Lyophilize\" (FABIL) process for CHA probe stabilization. The FABIL process utilizes magnetic graphene oxide (MGO) to adsorb and physically separate hairpin probes (H1 and H2), thereby suppressing nonspecific hybridization and generating lyophilized CHA microspheres with room-temperature stability. When integrated into a lateral flow test strip, the FABIL-NE-miR platform achieves a visual detection limit of 10 fM and a liquid-phase limit of detection of 1.14 fM, overcoming the traditional trade-off between low background and high sensitivity. Clinical validation using 100 serum samples, including 32 AD cases and 68 non-AD controls, demonstrated that FABIL-NE-miR outperformed traditional low-background CHA systems, with an area under the ROC curve (AUC) of 0.9563, a sensitivity of 90.63%, and a specificity of 94.12%. Overall, the FABIL-NE-miR platform enables rapid visual detection of low-abundance AD-associated miRNAs from L1CAM-immunocaptured sEV fractions. By addressing key challenges such as background leakage and probe instability, this study provides a practical tool for AD screening and supports the feasibility of the FABIL strategy as a universal platform for next-generation nucleic acid diagnostics.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345938", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Xu R", "dirs": [ "A" ] }, "42522199": { "pmid": "42522199", "title": "A portable, quick-visualization platform for field detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) in banana.", "abstract": "Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungal pathogen that causes Fusarium wilt of banana (FWB), leading to multiple outbreaks worldwide and resulting in severe economic losses. In the absence of any effective fungicides or soil disinfectants for this disease, rapid in-field detection of this pathogen is essential for early intervention and containment. However, current in-field detection methods still rely on instruments, making the process time-consuming and complex. Herein, we developed a portable and ultra-fast detection assay for Foc Tropical Race 4 (TR4), the most destructive Foc lineage, based on recombinase polymerase amplification (RPA) with lateral-flow dipstick (LFD) readout. We identified a TR4-specific DNA fragment as the diagnostic target and prepared crude templates using a room-temperature lysis buffer, eliminating the need for DNA purification. All RPA components, except template primer-probe mix, and magnesium acetate, were lyophilized in a single eppendorf tube to simplify the workflow and avoid step-by-step reagent addition, allowing the reaction can be conducted in the field by hand or portable metal bath. The developed RPA-LFD system offers a high specificity toward Foc TR4, showing no cross-reactivity with other Foc strains, and achieved a limit of detection of 10 fg/μL in the field. This sensitivity enables the detection of infections in asymptomatic plants at early stages. This platform provides a convenient, ultra-fast and user-friendly tool for on-site Foc TR4 surveillance and supports timely management decisions to curb the spread of FWB.", "journal": "Plant disease", "year": "2026", "doi": "10.1094/PDIS-05-26-0998-RE", "ptypes": [ "Journal Article" ], "first_author": "Bi F", "dirs": [ "A" ] }, "39827305": { "pmid": "39827305", "title": "A lyophilised formulation of chimpanzee adenovirus vector for long-term stability outside the deep-freeze cold chain.", "abstract": "The adenovirus-vaccine platform has come to prominence with the COVID-19 vaccination campaigns. The objective of this study was to validate a formulation that was suitable for lyophilisation and long-term storage at 5 (2-8) °C. Vaccine stability was assessed up to five years at 5 °C using a lyophilised formulation of the chimpanzee-adenovirus vector ChAd155 encoding a respiratory syncytial virus (RSV) antigen. Vaccine potency was assessed by functional infectivity assay. Other assessments of vaccine stability included those for capsid integrity, particle content, and DNA release. Vaccine efficacy and safety were assessed after two years in a murine model of RSV challenge and a rabbit toxicology model, respectively. Here, we show that the potency loss from lyophilisation was 0.12 log10. The potency loss over five years at 5 °C was estimated at 0.21 log10 (95%CI 0.10-0.30). This coincides with a 25% increase in the ratio of non-infectious particles/infectious particles. After two years of storage at 5 °C, (i) the loss of infectivity was 0.17 log10; (ii) the vaccine remained immunogenic and effective at clearing RSV from the lungs in a mouse-challenge model; and (iii) the vaccine was not associated with any adverse safety signal in a rabbit toxicology model. The 5-year stability of the lyophilised adenovirus-vector vaccine is within our acceptable limit ( < 0.3 log10 decrease). Its formulation process is amenable to manufacturing scale-up and should help in providing adenovirus-based vaccines where the cold chain is problematic, such as in low-income countries, and in pre-epidemic stockpiling.", "journal": "Communications medicine", "year": "2025", "doi": "10.1038/s43856-025-00740-x", "ptypes": [ "Journal Article" ], "first_author": "Mathot F", "dirs": [ "A" ] }, "42542071": { "pmid": "42542071", "title": "Development and validation of an agglutination test using in vitro-cultivated Trypanosoma evansi antigens for the diagnosis of surra in cattle.", "abstract": "Trypanosomosis is an important disease in livestock caused by Trypanosoma evansi infections. The CATT/T. evansi is recommended by WOAH for trypanosomosis screening, but its accessibility in Thailand and Southeast Asia remains limited by cold chain transportation and high costs. This study aimed to develop and validate an agglutination test using in vitro-cultivated T. evansi Thai strain antigens. The test was optimized using serum dilutions ranging from 1:2-1:64. Diagnostic performance was evaluated against the commercial CATT/T. evansi test and indirect ELISA. Cross-reactivity was assessed using bovine serum positive for Anaplasma spp., Babesia bigemina, Babesia bovis, Theileria spp., and Leishmania spp. Lyophilized antigen stability was evaluated for 12 months. The developed test showed a sensitivity of 87.07% and a specificity of 96.67% (PPV: 97.12%; NPV: 85.29%) versus CATT/T. evansi, with almost perfect agreement (κ = 0.83). It showed 77.95% sensitivity, 82.86% specificity (PPV: 80.49%; NPV: 80.56%), and substantial agreement (κ = 0.61) against indirect ELISA. No cross-reactivity was observed with five hemoparasites, and lyophilized antigen remained stable for ≥ 12 months at 4-8°C. The newly developed T. evansi agglutination test was applied to 851 cattle serum samples collected from 15 provinces across four regions of Thailand, revealing a seroprevalence of 50.29% (95% CI: 46.94-53.64). This study showed that the newly developed T. evansi agglutination test provides a rapid, field-adaptable alternative for trypanosomosis surveillance.", "journal": "Veterinary parasitology", "year": "2026", "doi": "10.1016/j.vetpar.2026.110876", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "Mutchimadilok T", "dirs": [ "A" ] }, "41448438": { "pmid": "41448438", "title": "Lyophilized chloroplasts as molecular capsules: A scalable platform for stable and functional human α-lactalbumin production.", "abstract": "The growing world population and the increasing demand for proteins present significant challenges to food security. Advances in synthetic biology have enabled the development of transplastomic plants engineered to produce milk proteins, offering a promising solution for the large-scale, economical production of alternative protein sources. This study demonstrated the extended stability of human α-lactalbumin (hLA) in freeze-dried transplastomic tobacco chloroplasts. hLA was expressed at high levels, constituting 39.1 % of the total soluble protein in chloroplasts (66.5 mg/g isolated chloroplasts). Western blot analysis of lyophilized tobacco chloroplasts stored at room temperature for up to six months indicated that the hLA protein remained stable, soluble, and properly folded over extended periods. The freeze-drying process of isolated chloroplasts resulted in a 34-fold increase in hLA protein concentrations. Importantly, the biological functionality of purified hLA from lyophilized chloroplasts was confirmed through an in vitro lactose synthesis assay, where recombinant hLA successfully activated galactosyltransferase, leading to efficient lactose production at level comparable to commercial standard. Lyophilized chloroplasts, free from nicotine and bacterial contamination, are suitable candidates for the oral administration of the dietary protein alpha-lactalbumin. These findings suggest that recombinant protein production in transplastomic plants could serve as a viable food source and alternative to conventional sources. Lyophilization further reduces expenses and streamlines downstream processing, purification, and storage. These methods facilitate relevant formulation practices for these compounds to meet the demand-oriented requirements. Future research should prioritize translation of this technology to edible plant hosts to enable direct human consumption and undertake comprehensive in vivo and clinical studies to validate safety, bioavailability, and efficacy.", "journal": "Journal of biotechnology", "year": "2026", "doi": "10.1016/j.jbiotec.2025.12.016", "ptypes": [ "Journal Article" ], "first_author": "Ehsasatvatan M", "dirs": [ "A" ] }, "42581111": { "pmid": "42581111", "title": "A point-of-care testing platform for pathogen nucleic acid analysis utilizing paper-based extraction and portable qPCR.", "abstract": "Rapid and reliable diagnostic methods are crucial for controlling animal disease outbreaks and reducing mortality, especially when effective drugs or vaccines are unavailable. While real-time quantitative polymerase chain reaction (qPCR) remains the gold standard for nucleic acid detection due to its superior sensitivity and specificity, conventional workflows are inherently cumbersome. Traditional extraction and amplification processes typically require 1.5 to 3 h, with further delays incurred by transporting samples to centralized laboratories. To overcome these limitations, this study introduces a point-of-care testing platform for on-site pathogen diagnostics. This system synergistically combines a centrifuge-free, glass fiber (GF) paper-based nucleic acid extraction card, room-temperature-stable lyophilized qPCR reagents, and a portable qPCR instrument. By eliminating the dependency on bulky benchtop equipment and cold-chain logistics, this platform streamlines lysis, extraction, and amplification, delivering sample-to-answer results within 45 min. The optimized system demonstrated excellent analytical sensitivity, achieving limits of detection (LoDs) of 10 copies/μL for the N gene of porcine epidemic diarrhea virus (PEDV) and 50 copies/μL for the PirA gene of Vibrio parahaemolyticus AHPND (VPAHPND). Furthermore, clinical validation using complex field samples yielded a 100% diagnostic concordance rate with standard laboratory equipment. This study highlights the potential of the proposed system for early diagnosis and monitoring of animal diseases, providing a promising solution for rapid on-site screening in resource-constrained settings.", "journal": "Analytical and bioanalytical chemistry", "year": "2026", "doi": "10.1007/s00216-026-06716-7", "ptypes": [ "Journal Article" ], "first_author": "Li J", "dirs": [ "A" ] }, "41621718": { "pmid": "41621718", "title": "Rapid colorimetric detection of Citrus tristeza virus combining portable sample preparation and reverse transcription-loop mediated isothermal amplification.", "abstract": "This article details combined portable sample preparation and colorimetric detection using loop-mediated isothermal amplification (RT-LAMP) of Citrus tristeza virus (CTV) from citrus leaves. The sample preparation employs an OmniLyse micro-homogenizer and cellulose paper disks to extract and isolate total nucleic acids in <15 min. Primer and dimethyl sulfoxide (DMSO) concentrations were optimized to minimize RT-LAMP assay reaction times and maximize the delay in the appearance of false positives due to non-specific amplification, respectively. The CTV primers were assessed against a panel of 24 CTV-positive isolates and 6 non-CTV pathogen isolates. To adapt the protocol for cold-chain-free field deployment, a lyophilized RT-LAMP reagent mix was developed and its rehydration solution was optimized to minimize false positives. In a greenhouse setting, the micro-homogenizer successfully extracted and isolated nucleic acids from CTV-positive trees, followed by the lyophilized RT-LAMP assay positively detecting the pathogen within 35 min. This study establishes the feasibility of quick and portable CTV detection without access to laboratory equipment, paving the way for larger-scale field studies, comprehensive validation of assay performance, and potential extension to other plant pathogens.", "journal": "SLAS technology", "year": "2026", "doi": "10.1016/j.slast.2026.100398", "ptypes": [ "Journal Article" ], "first_author": "Liu CW", "dirs": [ "A", "C" ] }, "42618126": { "pmid": "42618126", "title": "Field-deployable RspCas13d platform for rapid and extraction-free detection of giant panda- and canine-derived Canine Distemper virus.", "abstract": "Canine distemper virus (CDV) is a highly contagious RNA virus that causes severe disease in domestic dogs and diverse wildlife species, including endangered giant pandas. Current CDV diagnosis mainly relies on laboratory-based RT-qPCR, which requires nucleic acid extraction, trained personnel, and temperature-controlled instruments, limiting its use in field surveillance and resource-limited settings. Although CRISPR-based diagnostics offer promising alternatives, chemically defined extraction-free workflows compatible with one-pot Cas13d detection remain limited. This study addresses the need for a rapid, visual, extraction-free, and low-infrastructure method for CDV detection. We developed CLEAR-VISION, an integrated CRISPR diagnostic platform combining CLEAR (Chemical Lysis for Extraction-free Access to RNA) with VISION (Visual Isothermal Single-tube Integrated One-pot Nucleic acid detection). VISION integrates RPA amplification, T7 transcription, and RspCas13d-based detection into a single-tube reaction supported by a chemically defined buffer. CLEAR enabled rapid RNA release at room temperature without extraction kits or heating and was compatible with downstream one-pot detection. The PAM- and PFS-independent property of RspCas13d allowed flexible target selection, while lyophilized reagents improved storage and transportation convenience. CLEAR-VISION enabled CDV detection within 30 min and provided dual visual readouts, including fluorescence and lateral flow assays. Clinical evaluation in the current sample set showed consistent results with RT-qPCR for samples from giant pandas, stray dogs, and pet dogs. The assay also maintained stable performance at physiological temperature (37 °C), reducing reliance on temperature-controlled equipment. CLEAR-VISION provides a chemically defined, extraction-free, and low-infrastructure CRISPR diagnostic workflow for rapid CDV detection. Its novelty lies in integrating room-temperature chemical lysis with single-tube RPA-T7-RspCas13d detection, lyophilized reagents, and dual visual readouts. This platform supports the potential application of CRISPR-based diagnostics for on-site CDV surveillance in domestic animals and wildlife.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345960", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Tao Q", "dirs": [ "A" ] }, "39806456": { "pmid": "39806456", "title": "Stabilizing milk-derived extracellular vesicles (mEVs) through lyophilization: a novel trehalose and tryptophan formulation for maintaining structure and Bioactivity during long-term storage.", "abstract": "Extracellular vesicles (EVs) are widely investigated for their implications in cell-cell signaling, immune modulation, disease pathogenesis, cancer, regenerative medicine, and as a potential drug delivery vector. However, maintaining integrity and bioactivity of EVs between Good Manufacturing Practice separation/filtration and end-user application remains a consistent bottleneck towards commercialization. Milk-derived extracellular vesicles (mEVs), separated from bovine milk, could provide a relatively low-cost, scalable platform for large-scale mEV production; however, the reliance on cold supply chain for storage remains a logistical and financial burden for biologics that are unstable at room temperature. Herein, we aim to characterize and engineer a freeze-dried, mEV formulation that can be stored at room temperature without sacrificing structure/bioactivity and can be reconstituted before delivery. In addition to undertaking established mEV assays of structure and function on our preparations, we introduce a novel, efficient, high throughput assay of mEV bioactivity based on Electric Cell Substrate Impedance Sensing (ECIS) in Human dermal fibroblast monolayers. By adding appropriate excipients, such as trehalose and tryptophan, we describe a protective formulation that preserves mEV bioactivity during long-term, room temperature storage. Our identification of the efficacy of tryptophan as a novel additive to mEV lyophilization solutions could represent a significant advancement in stabilizing small extracellular vesicles outside of cold storage conditions.", "journal": "Journal of biological engineering", "year": "2025", "doi": "10.1186/s13036-024-00470-z", "ptypes": [ "Journal Article" ], "first_author": "Dogan AB", "dirs": [ "A" ] }, "41644546": { "pmid": "41644546", "title": "Paper-based fluorescent assay for blood typing and antibody titer determination using long-term ambient-stored bioengineered RBCs.", "abstract": "Methods for accurate, rapid blood typing and antibody titration at the point of care are constrained by cold-chain dependent red blood cell (RBC) reagents, time-consuming serial dilutions, and subjective visual interpretation. To overcome these limitations, here we develop a method based on bioengineered red-blood cells. We engineer hemoglobin-depleted RBC membranes that preserve ABO surface antigens, label them with fluorescent nanoparticles, and lyophilize them, demonstrating at least 2 years of stability at room-temperature. We capture hemagglutination on a dual-layer paper pad and quantify it by fluorescence in approximately 8 minutes, eliminating the need for serial dilution. We conduct a registered clinical trial (ChiCTR2300078864) and benchmark performance against the gel microcolumn assay. In a clinical cohort (n = 641), ABO typing achieves 100% concordance. In antibody-titration samples, quantitative titers agree with the gel microcolumn assay (coincidence 80.75%, κ = 0.8623). In summary, we develop a cold-chain-independent, handheld paper-based assay that enables simultaneous ABO grouping and quantitative antibody titration with clinical-grade performance, supporting scalable manufacturing, cross-site standardization, and transfusion decision-making in emergency and resource-limited settings.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-69213-6", "ptypes": [ "Journal Article" ], "first_author": "Li Y", "dirs": [ "A", "D" ] }, "41460219": { "pmid": "41460219", "title": "Rapid, Sensitive, and Low-Cost EsCas13d and RPA-Based Assay Platform for Rapid and Accurate Identification of SVA.", "abstract": "Rapid, sensitive, and user-friendly nucleic acid diagnostics are critical for controlling outbreaks of Seneca Valley virus (SVA). Here, we present Field-deployable Isothermal Enzymatic Lysis Detection (FIELD), an RNA detection platform for resource-limited settings that integrates rapid detection, operational simplicity, and minimal equipment requirements. FIELD employs a room-temperature lysis protocol to release viral nucleic acids within 2 min, followed by a single-step RPA-CRISPR/Cas13d reaction completed in 30 min. FIELD detects SVA RNA at 8 copies/μL using a plate reader and at 50 copies/μL via visual readout within 35 min, with no cross-reactivity against nontarget viruses. FIELD remains functional at both body (∼37 °C) and room (∼25 °C) temperatures, eliminating the need for temperature-controlled devices. Lyophilized FIELD reagents retain full activity for 1 week at room temperature and remain functional after long-distance transport and storage at 4 °C for up to two months, supporting field deployment. In a clinical evaluation of 88 field samples, FIELD achieved 100% sensitivity and specificity relative to RT-qPCR. Collectively, these features render FIELD well suited for point-of-care testing (POCT) beyond conventional laboratory settings.", "journal": "ACS infectious diseases", "year": "2026", "doi": "10.1021/acsinfecdis.5c00889", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhao L", "dirs": [ "A" ] }, "42761875": { "pmid": "42761875", "title": "A lyophilization-compatible colorimetric LAMP assay for cold chain-free, extraction-free and rapid post-enrichment detection of E. coli.", "abstract": "Developing reliable dry-format colorimetric LAMP assays remains challenging because most existing systems depend on pH-sensitive dyes e.g. Hydroxy Naphthol Blue and Phenol Red. These dyes lose performance after lyophilization or when exposed to extraction-free sample buffers, as they require low-buffer conditions to generate accurate color transitions. Lyophilization disrupts pH stability, and common lysis additives further interfere with signal clarity. Consequently, creating a robust, field-ready colorimetric LAMP platform has been constrained. Here, we developed a Mn-PAPS Colorimetric LAMP assay, a metal ion-based colorimetric system that is inherently pH-independent and fully compatible with freeze-drying. Using Escherichia coli as a model organism, this study demonstrates the feasibility, and diagnostic performance of the lyophilized colorimetric LAMP platform. The Mn-PAPS Colorimetric LAMP platform demonstrated sensitivity in a single-digit CFU range and maintained clear visual contrast with a high analytical accuracy across 69 spiked and unspiked reference samples. Incorporation of daughter primers reduced incubation time from 120 to 45 minutes. Two lyophilized formats demonstrated room-temperature stability, supporting field deployment. • Resolves the long-standing limitation of pH-sensitive colorimetric LAMP through a metal-ion dye system. • Demonstrates single-digit CFU sensitivity and high analytical accuracy using a micropipette-free workflow. • Provides stable performance under ambient conditions through lyophilization-compatible reagent formats.", "journal": "MethodsX", "year": "2026", "doi": "10.1016/j.mex.2026.104138", "ptypes": [ "Journal Article" ], "first_author": "Jaroenram W", "dirs": [ "A" ] }, "41750756": { "pmid": "41750756", "title": "Development and Evaluation of a Lyophilized Plasma-Based Internal Quality Control for Human Immunodeficiency Virus Rapid Diagnostic Tests.", "abstract": "Background/Objectives: Rapid diagnostic tests (RDTs) for human immunodeficiency virus (HIV) are widely used, but most kits lack standardized internal quality control (IQC) materials. In this study, we aimed to develop and evaluate a plasma-based IQC compatible with five HIV RDT brands and with proven long-term stability. Methods: Control samples at three reactivity levels were tested with five HIV RDT kits in lyophilized and liquid forms. Lyophilized samples were produced with and without trehalose, whereas liquid samples were prepared with and without StabilZyme™ SELECT Stabilizer (Stabilizer). Accelerated stability testing was performed at 37 °C and 45 °C for 28 days, and the most stable formulation was selected for long-term storage at 4 ± 2 °C and 25 ± 5 °C. Stability was assessed based on test-line visibility and signal intensity. Signal-intensity trends were analyzed using simple linear regression with a t-test on the slope; samples were considered stable when no significant trend was detected (p > 0.05). Results: Reactivity measured using the Elecsys HIV combi PT assay yielded cutoff index (COI) values of 772.65 (1:8) for the strong-positive control and 269.95 (1:25) for the weak-positive control. Trehalose-containing lyophilized samples maintained reactivity under accelerated testing at 37 and 45 °C and for 6 months at 4 ± 2 °C and 25 ± 5 °C, with no significant change in signal intensity (p > 0.05). Conclusions: The plasma-based IQC materials were compatible with all five HIV RDTs, and trehalose-stabilized lyophilized plasma showed high stability, supporting transport and storage without strict cold-chain requirements.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2026", "doi": "10.3390/diagnostics16040608", "ptypes": [ "Journal Article" ], "first_author": "Jomjunyoung S", "dirs": [ "A" ] }, "40590553": { "pmid": "40590553", "title": "Validation and multi-site deployment of a lyophilized qRT-PCR reagent for the molecular diagnosis of avian influenza and rabies in Sub-Saharan African regions.", "abstract": "Molecular methods are widely accepted as gold-standard techniques for the laboratory diagnosis of most human and animal pathogens. However, most molecular protocols rely on reagents that need to be transported and stored at a freezing temperature, a requirement that might affect their reliability in areas where the cold chain cannot be guaranteed. Over the years, several lyophilized molecular products have been marketed to circumvent this issue. We therefore evaluated the feasibility of replacing liquid reagents with freeze-dried formulations for the molecular diagnosis of avian influenza (AIV) and rabies (RABV) viruses, two priority zoonotic pathogens widely spread in Sub-Saharan Africa. Among six different commercial freeze-dried kits, we selected one reagent due to its easy-to-use features, single-reaction format, and preliminary performance assessment. Through a more in-depth evaluation, we determined its analytical and diagnostic performance and formulation stability and obtained results comparable to those of standard liquid master mixes. However, for the detection of divergent lyssaviruses, the lyophilized reagent's sensitivity was affected by suboptimal complementarity between the oligonucleotides and target sequences. Finally, a multi-site evaluation in four veterinary diagnostic laboratories located in Sub-Saharan Africa demonstrated the successful deployment of AIV and RABV assays utilizing the freeze-dried reagent, which can interchangeably replace liquid qRT-PCR/RT-PCR kits. Altogether, our results indicate that the investigated lyophilized master mix represents a valid alternative to liquid reagents for the molecular diagnosis of avian influenza and rabies and has the potential for broader applications to other relevant infectious diseases upon proper validation.IMPORTANCEMolecular diagnostic protocols rely on reagents that need to be transported and stored at freezing temperatures. Meeting this requirement can be challenging in areas where the maintenance of the cold chain is not guaranteed, such as in Sub-Saharan Africa. Our study aimed to assess the feasibility of using lyophilized reagents as a replacement for liquid reagents in the molecular diagnosis of two widespread zoonotic pathogens in Sub-Saharan Africa, namely, avian influenza and rabies. To accomplish this, we selected a commercially available lyophilized reagent based on its format and performance characteristics. We conducted a laboratory validation to assess the use of the lyophilized reagent throughout the entire diagnostic process. We also conducted a reproducibility test involving African laboratories as potential end-users. Our findings confirm that the lyophilized reagent can replace traditional liquid reagents to diagnose rabies and avian influenza and suggest its possible use for a wider range of infectious diseases after undergoing appropriate validation.", "journal": "Journal of clinical microbiology", "year": "2025", "doi": "10.1128/jcm.00080-25", "ptypes": [ "Journal Article", "Validation Study", "Research Support, Non-U.S. Gov't" ], "first_author": "Drzewnioková P", "dirs": [ "A" ] }, "42009546": { "pmid": "42009546", "title": "[A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and CRISPR/EsCas13d].", "abstract": "The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats-UV light (470 nm) and lateral flow assay (LFA)-providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/EsCas13d-based portable visual platform for rapid on-site diagnostics.", "journal": "Sheng wu gong cheng xue bao = Chinese journal of biotechnology", "year": "2026", "doi": "10.13345/j.cjb.250568", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "Xu T", "dirs": [ "A" ] }, "42251880": { "pmid": "42251880", "title": "A one-pot RPA-T7/crDNA-CRISPR/Cas13a assay for portable and ultrasensitive detection of Helicobacter pylori and clarithromycin resistance mutations.", "abstract": "The high global prevalence of Helicobacter pylori (H. pylori) infection and rising clarithromycin resistance demand rapid, sensitive, point-of-care diagnostic tools. Although CRISPR/Cas systems have revolutionized nucleic acid detection, challenges such as unstable pre-synthesized crRNA and multi-step procedures limit their practical application. Here, we report a one-pot assay, termed RT-CRISPR (RPA-T7/crDNA-mediated CRISPR), that integrates recombinase polymerase amplification (RPA) with T7 transcription and CRISPR/Cas13a detection in a single closed tube. A key innovation is the use of a T7 promoter primers-crDNA hybrid chain (T7/crDNA) as a template for in situ synthesis of crRNA by T7 RNA polymerase, eliminating exogenous labile crRNA. The assay detects the H. pylori 16S rRNA gene at 1 copy/μL and the A2143G clarithromycin resistance mutation at 5 copies/μL, with no cross-reactivity against seven other gastrointestinal pathogens. It reliably detects the A2143G mutation at a ratio as low as 0.5% in mixed DNA (total 1× 104 copies/μL), highlighting its ability to identify low-abundance resistance mutations in heterogeneous samples. The entire assay, from DNA extraction to result, completes within 40 min using a portable fluorescence detector. Clinical validation with 103 samples showed 100% concordance with qPCR. The reagents can be lyophilized into stable pellets, maintaining performance after two months at room temperature. RT-CRISPR offers a simple, rapid, and robust platform for molecular diagnosis of H. pylori and drug resistance, holding promise for point-of-care testing and large-scale screening.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.152949", "ptypes": [ "Journal Article" ], "first_author": "Han Y", "dirs": [ "A" ] }, "41953445": { "pmid": "41953445", "title": "A streamlined integrated system integrating lysate release, freeze-dried reagents for multiplex polymerase chain reaction, and intelligent analysis for TORCHes pathogen identification.", "abstract": "Current Toxoplasma gondii (TOX), Epstein-Barr virus (EBV), rubella virus, human cytomegalovirus (HCMV), and herpes simplex virus types I and II (HSV I and II) (TORCHes) diagnostic methods are limited by challenges, such as multi-step workflows and cold-chain dependency. This study aimed to develop an integrated \"sample-in-result-out\" system combining rapid nucleic acid release, room-temperature-stable multiplex polymerase chain reaction (PCR), and automated interpretation to improve diagnostic accuracy. We utilized a self-prepared lysis buffer for fast nucleic acid extraction from diverse sample types. Primers, probes, and reaction components for six TORCHes pathogens were fabricated into freeze-dried microspheres via vacuum freeze-drying, enabling single-tube multicolor melting curve analysis (MMCA). The companion software, TORCHes-MCAv1.0, was used to automate pathogen typing and result reporting. Evaluation metrics encompassed sensitivity, specificity, precision, stability, and anti-interference ability. The system was validated using 210 clinical samples compared to commercial single-target qPCR, with Sanger sequencing resolving any discrepancies. Nucleic acid extraction and release were completed within 1 min. Freeze-dried microspheres demonstrated exceptional repeatability, with T m intra- and inter-batch CVs of 0.03-0.18%. Limits of detection (LoD) were 200 copies/mL for HSV-I/II, HCMV, and TOX, and 500 copies/mL for EBV and RV. Specificity analysis showed no cross-reactivity with non-target pathogens. Stability testing confirmed the microspheres could be stored stably at room temperature for 1 year. Clinical validation showed near-perfect agreement with qPCR (Kappa = 0.965-1.000), with all seven discrepant results confirmed correct via sequencing. The TORCHes-MCAv1.0 software achieved 100% concordance with manual interpretation. We developed a robust, streamlined integrated TORCHes detection system. Its simplicity, stability, and high-throughput nature make it a valuable complementary tool for confirming active infection and differentiating pathogen subtypes in complex clinical scenarios.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1788209", "ptypes": [ "Journal Article" ], "first_author": "Fu M", "dirs": [ "A" ] }, "42321793": { "pmid": "42321793", "title": "Comparative analysis of laboratory-based and portable qPCR platforms: CFX, MIC, and Biomeme Franklin for African swine fever and avian influenza detection.", "abstract": "Rapid diagnosis is critical for the early detection and control of transboundary animal diseases such as African Swine Fever (ASF) and Avian Influenza (AI), particularly because it enables timely containment measures at the farm level. In low-resource settings where sample transport, cold chain maintenance, and access to centralized laboratories are challenging, portable qPCR systems offer a clear operational advantage by enabling on-site or near-site testing thus reducing diagnostic turnaround and response time. This study evaluated three qPCR platforms for detecting African Swine Fever Virus (ASFV) and Avian Influenza Virus (AIV): two laboratory-based systems, CFX96 Touch (Bio-Rad) and MIC (BioMolecular Systems), and the portable qPCR platform Franklin by Biomemex. Seventy-two ASF virus samples representing genotypes I, II, IX, XIV, XXIII, and a genotype I/II recombinant from eleven countries were tested on two laboratory platforms (CFX96, MIC) and one rapid platform (Franklin) alongside 50 AI virus samples (H5N1, H9N2) from seven countries. Both laboratory platforms achieved consistent results. Franklin required protocol optimization when the WOAH-recommended protocol produced predominantly false-negative results, but adapted amplification conditions restored performance. Limits of detection per reaction calculated by probit analysis were: CFX96 21.28 (14.85-68.67), MIC 32.76 (21.12-96.11), Franklin with lyophilized reagents 4.12 (2.71-14.97), and Franklin with adapted protocol 6.85 (4.33-20.11). After adaptation, all platforms exhibited high diagnostic sensitivity and specificity for ASFV detection. For the AIV detection protocol, false negative results were observed on MIC and Franklin platforms compared to the CFX96. The laboratory platforms tested in this study (CFX96 and MIC) provided robust high-throughput capabilities, while the portable system (Franklin) offered superior sensitivity but required protocol adaptation of established WOAH-recommended procedures.", "journal": "BMC veterinary research", "year": "2026", "doi": "10.1186/s12917-026-05606-5", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "Auer A", "dirs": [ "A" ] }, "42014536": { "pmid": "42014536", "title": "Optimization of automated radiosynthesis method for [18F]AlF-NOTA-Octreotide and PET/CT imaging in neuroendocrine neoplasms.", "abstract": "Neuroendocrine neoplasms (NENs) represent heterogeneous tumors with increasing occurrence rate. Somatostatin receptor (SSTR) imaging using 68Ga-labeled analogs is crucial but limited by the relatively short half-life of this radionuclide along with its reliance on generator supply. To overcome these limitations, this study aimed to establish an automated synthesis platform compliant with Good Manufacturing Practice (GMP) standards for the preparation of a novel SSTR-targeting probe, [18F]AlF-NOTA-octreotide. Its diagnostic efficacy in neuroendocrine neoplasms was evaluated through comparison with existing 68Ga-labeled and [18F]FDG probes. The fully automated-synthesis of [18F]AlF-NOTA-Octreotide was completed within 37 minutes, with a specific activity of 56 ± 5 GBq/μmol and a radiochemical yield (RCY) of 42 ± 1.5%. The RCP was > 95% and remained stable for 8 hours at room temperature. The [68Ga]Ga-NOTA-Octreotide kit demonstrated an RCP of 97.5 ± 0.3%, stability ≥94% for 4 hours, and long-term stability ≥ 96% over 6 months. In vivo imaging in mice showed comparable tumor uptake for both probes at 30 minutes post-injection (5.6 %ID/cc). However, [18F]AlF-NOTA-Octreotide exhibited significantly lower background activity in blood, muscle, and bone. The tumor uptake of [18F]AlF-NOTA-Octreotide remained high (5.92 ± 0.10 %ID/cc) at 2 hours post-injection, superior to that of the 68Ga-labeled counterpart (5.05 ± 0.68 %ID/cc). At the patient level, [18F]AlF-NOTA-Octreotide detected 41 (87.2%) among 47 lesions, whereas [18F]FDG detected only 13 (27.7%). For G1-G2 grade lesions, the SUVmax was significantly higher with [18F]AlF-NOTA-Octreotide compared to [18F]FDG (14.9 ± 13.8 to 4.8 ± 4.4, p < 0.001). [18F]AlF-NOTA-Octreotide demonstrated superior detection of primary tumors, lymph node metastases, hepatic, osseous, and peritoneal metastases compared to FDG. One case of pulmonary atypical carcinoid (G3) was FDG-positive but Octreotide-negative, illustrating the complementary nature of the two imaging modalities. A robust, GMP-compatible automated synthesis process for [18F]AlF-NOTA-Octreotide and a lyophilized kit for [68Ga]Ga-NOTA-Octreotide were successfully established, both exhibiting high RCP, high specific activity, and excellent stability. [18F]AlF-NOTA-Octreotide demonstrated significantly higher lesion detection rates and uptake intensity than [18F]FDG in SSTR2-positive NENs with lower background activity. The complementary profiles of these probes enable personalized, precise imaging based on tumor biology. This work provides substantial technical and data support for clinical translation.", "journal": "EJNMMI radiopharmacy and chemistry", "year": "2026", "doi": "10.1186/s41181-026-00447-x", "ptypes": [ "Journal Article" ], "first_author": "Wan X", "dirs": [ "A" ] }, "41757367": { "pmid": "41757367", "title": "Rapid and Visual Detection of Muscovy Duck Reovirus Using a Reverse Transcription Recombinase-Aided Amplification Assay in a Lyophilized Format for Point-of-Care Applications.", "abstract": "Muscovy duck reovirus (MDRV) causes substantial economic losses in the waterfowl industry, necessitating rapid and field-deployable diagnostic tools. In this study, we developed, for the first time, a lyophilized reagent-based reverse transcription recombinase-aided amplification (RT-RAA) assay targeting the σC gene of MDRV for point-of-care testing (POCT). The assay was optimized to operate at 39°C with a probe concentration of 100 nM. It achieved a detection limit of 1.03 × 101 copies/μL within 20 min, which is comparable to RT-quantitative polymerase chain reaction (qPCR) and superior to existing methods. It exhibited high specificity for MDRV with no cross-reactivity against other common waterfowl pathogens. Particularly, the assay was successfully lyophilized into a ready-to-use format, representing the first reported RT-RAA-based method for MDRV detection in such a format. The lyophilized reagents retained the key analytical performance characteristics without cold-chain dependance. When evaluated on 42 clinical samples, the lyophilized RT-RAA demonstrated 95.8% diagnostic sensitivity and 100% specificity compared to RT-qPCR, with almost perfect agreement (κ = 0.952). A visual readout using a portable blue light imager further enabled naked-eye interpretation without instrumentation. This robust, rapid, and field-deployable RT-RAA assay provides a practical solution for on-site MDRV surveillance, particularly in low-resource settings.", "journal": "Transboundary and emerging diseases", "year": "2026", "doi": "10.1155/tbed/5924181", "ptypes": [ "Journal Article" ], "first_author": "Chen X", "dirs": [ "A" ] }, "42150651": { "pmid": "42150651", "title": "Lyophilization of a viral vaccine: Strategies for surrogate use in analytical characterization of cake properties and considerations on virus infectivity.", "abstract": "Lyophilization has the potential to increase the stability of viral vectors and reduce the dependence on an uninterrupted cold chain. However, freezing and drying stress can inactivate viruses, resulting in reduced infectivity. Development of virus formulations presents additional challenges due to biosafety level restrictions that increase the complexity of sample handling. Particularly, when working with virus-containing lyophilized powders, the characterization of lyophilized cake properties by Karl-Fischer titration, differential scanning calorimetry, and X-ray-powder diffraction requires stringent safety precautions. Our feasibility study aimed to evaluate the use of the virus-free formulation buffers as surrogates for the analytical characterization of lyophilized cake properties, based on the hypothesis that the impact from the low mass fraction of the virus is negligible. We used the ChAdOx1 nCoV-19 vaccine as model viral vector; we formulated it with selected excipient combinations and applied different freezing protocols, including shelf-ramped freezing, quench freezing, and annealing for lyophilization to generate both amorphous and (partially) crystalline cake morphologies. Our results indicate that virus-free formulation buffers can be used as convenient surrogates for the evaluation of cake appearance, residual moisture content and crystallinity. In addition, we show that the extent of preservation of viral infectivity after lyophilization analyzed by TCID50 assay depends on both the formulation and the freezing protocol.", "journal": "Journal of pharmaceutical sciences", "year": "2026", "doi": "10.1016/j.xphs.2026.104336", "ptypes": [ "Journal Article" ], "first_author": "Arsiccio A", "dirs": [ "A" ] }, "40335035": { "pmid": "40335035", "title": "Dehydration-Driven Glass Formation in Aqueous Carbonates.", "abstract": "Amorphous carbonates, in their liquid and solid (glassy) forms, have been identified to play important roles in biomineralization, volcanism, and deep element cycling. Anhydrous amorphous calcium and calcium-magnesium carbonate (ACC and ACMC05, respectively) are structural glasses that exhibit a glass transition upon being heated. We report a significant effect of the water content on glass formation. The results yield a parametrization enabling prediction of the stability of their liquid and solid amorphous phases as a function of temperature and water content. These results, obtained through novel fast differential scanning calorimetry, demonstrate that hydrous ACC and ACMC05 do indeed exhibit the behavior of structural glasses and that dehydration of these materials by lyophilization is a route that can be used to isothermally cross the glass transition. This work presents a viable process for a significantly wider range of geo- and biomaterials. Dehydration-controlled formation of glassy ACC therefore constitutes the missing link in the transformation from supersaturated aqueous solutions through an intermediate amorphous glassy state to crystalline CaCO3 polymorphs. These results yield direct implications for the mechanistic interpretation of geological processes and biomineralization.", "journal": "The journal of physical chemistry letters", "year": "2025", "doi": "10.1021/acs.jpclett.5c00551", "ptypes": [ "Journal Article" ], "first_author": "Bissbort T", "dirs": [ "A" ] }, "40704748": { "pmid": "40704748", "title": "Water Mobility and Clustering in Lyophilized Human Serum Albumin.", "abstract": "Water plays a critical role in the stability of lyophilized protein therapeutics by stabilizing protein structures while also contributing to degradation processes like deamidation. Previous studies suggest a critical water threshold where degradation rates change, possibly due to shifts in water distribution between bound and free forms. However, the use of human serum albumin (HSA) as a model to understand the role of water clustering in protein stability has not been systematically explored. Our study examines water distribution, mobility, and clustering in lyophilized HSA using dynamic vapor sorption (DVS), solid-state NMR (ssNMR) spectroscopy, and molecular dynamics (MD) simulations. The water sorption isotherm follows Type II behavior, with Brunauer-Emmett-Teller (BET) analysis indicating a monolayer water content of 6 wt %. MD simulations also show water clusters forming at a similar threshold (∼5.5%). ssNMR analysis reveals increased water mobility between 7.4 and 11.5% water content, marking the transition from bound to free water. Our findings suggest that there is a critical water threshold for lyophilized HSA at 5-6%, where initial clustering occurs, leading to free water with increased mobility (7.4-11.5%). This indicates progressive water interactions and dynamics. This alignment of experimental and modeling data provides a useful insight into the role of water distribution in protein stability in lyophiles. In addition, these results offer a basis for further studies to explore the relationship between water thresholds and protein degradation, with potential applications for optimizing the stability of lyophilized pharmaceutical products.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00317", "ptypes": [ "Journal Article" ], "first_author": "Chen Y", "dirs": [ "A" ] }, "39806922": { "pmid": "39806922", "title": "Freeze-dried plasma: Hemostasis and biophysical analyses for damage control resuscitation.", "abstract": "Effective hemorrhage protocols prioritize immediate hemostatic resuscitation to manage hemorrhagic shock. Prehospital resuscitation using blood products, such as whole blood or alternatively dried plasma in its absence, has the potential to improve outcomes in hemorrhagic shock patients. However, integrating blood products into prehospital care poses substantial logistical challenges due to issues with storage, transport, and administration in field environments. We utilized hemostatic assays and advanced biophysical techniques, such as calorimetry, infrared spectoscopy, dynamic light scattering, and biolayer interferometry, to compare the functional and structural properties of freeze-dried plasma (FDP; OctaplasLG Powder, Octapharma AB) with those of fresh plasma controls. Hemostatic characterization of FDP revealed that clot formation properties and coagulation parameters were largely comparable to fresh plasma controls, with some variations observed in Von Willebrand factor-ADAMTS13 axis and fibrinolysis. No change to moisture content of FDP (~1% water content) was observed after 6-month storage at ambient conditions. Biophysical analyses of FDP during transfusion demonstrated spontaneous exothermic mixing of FDP in plasma, a dilution effect from saline, as well as comparable stability to plasma controls. Quantification of ligand-binding affinities of platelet receptors activated GPIIbIIIa and GPIbα showed comparable binding properties to plasma controls. Our results show that FDP exhibits hemostatic functionality and protein stability on par with fresh plasma, as assessed by novel, highly sensitive techniques. FDP therefore represents a viable alternative to conventional plasma in damage control resuscitation, offering significant logistical and storage advantages for prehospital and remote applications, especially in scenarios where whole blood is unavailable.", "journal": "Transfusion", "year": "2025", "doi": "10.1111/trf.18124", "ptypes": [ "Journal Article" ], "first_author": "Shoara AA", "dirs": [ "A" ] }, "39861719": { "pmid": "39861719", "title": "Dual-Mechanism Gastroretentive Tablets with Encapsulated Gentian Root Extract.", "abstract": "Background/Objectives: This study aimed to develop gastroretentive tablets based on mucoadhesive-floating systems with encapsulated gentian (Gentiana lutea, Gentianaceae) root extract to overcome the low bioavailability and short elimination half-life of gentiopicroside, a dominant bioactive compound with systemic effect. The formulation also aimed to promote the local action of the extract in the stomach. Methods: Tablets were obtained by direct compression of sodium bicarbonate (7.5%) and solid lipid microparticles (92.5%), which were obtained with lyophilizing double emulsions. A quality by design (QbD) was employed to evaluate the impact of formulation factors and processing parameters on emulsion viscosity, powder characteristics (moisture content, encapsulation efficiency, flowability), and tablet characteristics (floating lag time, gentiopicroside release, and assessment of dispersibility during in vitro dissolution). Results: The trehalose content and high-shear-homogenization (HSH) time of primary emulsion were critical factors. Trehalose content positively influenced emulsion viscosity, moisture content, floating lag time, encapsulation efficiency, and the release rate of gentiopicroside. HSH time positively affected powder stability and negatively gentiopicroside release. The selected powder had a high gentiopicroside encapsulation efficiency (95.13%), optimal stability, and good flowability. The developed tablets exhibited adequate floating lag time (275 s), mucoadhesive properties, and gentiopicroside biphasic release (29.04% in 45 min; 67.95% in 6 h). Furthermore, the optimal tablet formulation remained stable for 18 months and was primarily digested by duodenal enzymes. Conclusions: Dual-mechanism gastroretentive tablets with encapsulated gentian root extract were successfully developed. The in vitro digestion study demonstrated that the optimal formulation effectively resisted gastric enzymes, ensuring the release of its contents in the small intestine, even in the case of premature gastric evacuation.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17010071", "ptypes": [ "Journal Article" ], "first_author": "Mudrić J", "dirs": [ "A" ] }, "41012579": { "pmid": "41012579", "title": "Peste des Petits Ruminants Vaccine: Criteria for Assessing Its Thermotolerance.", "abstract": "The Peste des Petits Ruminants (PPR) live attenuated vaccines, the PPR virus (PPRV) Nigeria 75/1 strain (lineage II) and PPRV India Sungry 96 strain (lineage IV), currently used for control and eradication programme are very efficient vaccines as they provide the host, sheep and goats, a lifelong immunity after a single minimum recommended dose of 102.5 TCID50/mL. Unfortunately, both live attenuated vaccines are thermolabile and their use requires maintaining the cold chain from the manufactory premises to the field as most PPR-infected regions are facing of hot climate, with poor infrastructure, and the maintenance of an effective cold chain remains a challenge. To address this challenge, efforts have focused on developing thermotolerant (ThT) PPR vaccines using different stabilisers and improving the freeze-drying process. This study aimed to define the criteria for the evaluation of the stability of ThT PPR vaccines. A total of 37 batches of freeze-dried PPR vaccines using the PPRV Nigeria 75/1 strain, including eight (8) and twenty-nine (29) vaccines labelled as ThT and conventional formulations, respectively, were tested to evaluate the stability at temperatures of 40 °C to simulate the field conditions in some hot climate regions. All the vaccine batches included in this study initially showed acceptable levels of residual moisture, below 3%, and titres above the minimum WOAH standard requirement of 102.5 TCID50/mL. Following the incubation at 40 °C, 56.7% and 46% of the 37 vaccine batches tested retained titres above 102.5 TCID50/mL on day 3 and day 5, respectively. These vaccines use stabilisers such as skimmed milk, lactalbumin-sucrose, trehalose and one unnamed product (which may be protected for patent). The mean of titre loss among the PPR vaccines maintaining titres above 102.5 TCID50/mL was 0.78 log10 at day 3 and 0.99 log10 at day 5, suggesting a significant early degradation during the first 3 days. Based on these data, it is proposed that thermotolerant PPR vaccines should maintain a minimum titre of 102.5 TCID50/mL for vaccine dose on day 5 post-incubation at 40 °C with a titre loss below 1 log10 per mL. Preliminary immunogenicity test results showed that the PPR ThT vaccine meeting this criterion could be used in the field without maintaining a cold chain for up to 3 weeks, offering a practical solution for vaccination in remote areas.", "journal": "Viruses", "year": "2025", "doi": "10.3390/v17091151", "ptypes": [ "Journal Article" ], "first_author": "Bodjo CS", "dirs": [ "A" ] }, "39809457": { "pmid": "39809457", "title": "Water Activity as an Indicator for Antibody Storage Stability in Lyophilized Formulations.", "abstract": "Lyophilization remains a key method for preserving sensitive biopharmaceuticals such as monoclonal antibodies. Traditionally, stabilization mechanisms have been explained by vitrification, which minimizes molecular mobility in the lyophilized cake, and water replacement, which restores molecular interactions disrupted by water removal. This study proposes a novel design strategy that combines water activity and glass-transition temperature as the main indicators to predict long-term stability in lyophilized formulations. The water activity, calculated as the product of water activity coefficient and (residual) water content, serves as a mutual indicator of molecular interactions and influence of residual water content in the lyophilizate. By predicting beneficial excipient combinations through activity coefficient calculations using the perturbed-chain statistical association fluid theory model and calculating Tg using the Gordon-Taylor equation, the study identifies favorable excipient systems, such as sucrose/ectoine mixtures, providing formulation windows that offer broad stability ranges. The approach was validated with stability studies, confirming that formulations within a water activity range of 0.025-0.25 exhibit high (long-term) stability. This work advances formulation development by integrating water-excipient interactions and residual moisture content into a predictive model, moving beyond traditional empirical methods and offering a robust pathway to the design of stable biopharmaceutical formulations. This makes it possible to achieve high/favorable water activities despite low residual moisture (thus, high glass-transition temperatures) with plausible excipient concentrations and combinations.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.4c01106", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zäh M", "dirs": [ "A" ] }, "40167576": { "pmid": "40167576", "title": "A QbD approach for optimizing the lyophilization parameters of cyclophosphamide monohydrate.", "abstract": "Cyclophosphamide, an active pharmaceutical ingredient (API), is accessible in monohydrate form and esteemed for its remarkable stability. Maintaining this monohydrate form post-lyophilization is essential for product stability. This research aims to optimize essential lyophilization parameters for an effective and robust lyophilization cycle using a Quality by Design (QbD) methodology. Initially, thermal analysis is performed to evaluate the thermal qualities of the product. The research defines critical process parameters (CPPs) and important quality attributes (CQAs), employing a systematic Quality by Design (QbD) methodology to establish the design space in accordance with the required Quality Target Product Profile (QTPP). The lyophilization parameters being examined are primary drying temperature, primary drying duration, and primary drying vacuum. Meticulous analysis of the results identifies an optimum formulation. The test product successfully preserves water content between 6 and 7%, hence confirming the existence of the monohydrate form, as verified by X-ray diffraction (XRD) examination. Furthermore, the product demonstrates minimal concentrations of TBA and ACN, maintains an intact cake structure, and achieves a rapid reconstitution time of less than 30 s. The implementation of Quality by Design (QbD) concepts to enhance lyophilization parameters offers significant insights for developing generic pharmaceutical lyophilization formulations. Ultimately, a reliable freeze-drying technique is developed and then requires validation on industrial-scale lyophilizers for commercial applications.", "journal": "Drug development and industrial pharmacy", "year": "2025", "doi": "10.1080/03639045.2025.2487615", "ptypes": [ "Journal Article" ], "first_author": "Ahammad SR", "dirs": [ "A" ] }, "42202083": { "pmid": "42202083", "title": "Freeze-dried kit formulation and physicochemical assessment of a daratumumab-based radiopharmaceutical.", "abstract": "Daratumumab is a fully human anti-CD38 monoclonal antibody with strong potential as a targeting vector for therapeutic radionuclides. This study aimed to develop a freeze-dried daratumumab immunoconjugate kit by selecting a suitable chelator (DOTA-NHS, p-SCN-Bn-DOTA, or p-SCN-Bn-1B4M-DTPA) for the 177Lu-labelling, optimising the freeze-drying formulation, and evaluating the physicochemical properties and purity profiles. Conjugation performed in carbonate buffer at elevated temperature enhanced chelator incorporation and supported the selection of daratumumab-p-SCN-Bn-DOTA as the most suitable candidate, achieving radiolabelling yield up to 99.8 % without additional purification. Among the evaluated freeze-dried formulations, a saline-based, buffer-free sucrose-mannitol formulation containing polysorbate 20 (S.F5) provided the most favourable characteristics, including minimal residual moisture and the highest monomer purity with non-detectable HMWS species under the applied SE-HPLC conditions. ATR-FTIR and Raman spectroscopy confirmed preservation of the antibody structural integrity after conjugation and freeze-drying. In an in vitro study using human serum, [177Lu]Lu-daratumumab-p-SCNBn-DOTA maintained higher radiochemical purity for 168 h than [177Lu]Lu-daratumumab-p-SCN-Bn-1B4MDTPA, indicating greater stability. These results support the feasibility of a ready-to-use freeze-dried daratumumab-p-SCN-Bn-DOTA kit for the 177Lu-labelling.", "journal": "Acta pharmaceutica (Zagreb, Croatia)", "year": "2026", "doi": "10.2478/acph-2026-0013", "ptypes": [ "Journal Article" ], "first_author": "Apostolova P", "dirs": [ "A" ] }, "41285208": { "pmid": "41285208", "title": "Recent advances in freeze-drying technologies for mRNA vaccines against infectious diseases.", "abstract": "Currently, the storage and transportation of mRNA vaccines typically rely on ultra-low temperature conditions. To improve their stability and extend shelf life, recent studies have been devoted to converting liquid formulations into solid forms using drying technology. Among them, freeze-drying (lyophilization) is an effective strategy that freezes samples and removes moisture through primary (sublimation) and secondary (desorption) drying stages, maximally preserving the structural integrity and biological activity of mRNA vaccines. The significant reduction in moisture content effectively inhibits the rate of hydrolysis of mRNA, which is considered the primary factor contributing to the instability of mRNA vaccines. However, the freeze-drying process itself and its accompanying stresses pose key challenges, involving many critical variables closely related to formulation composition, process parameters, and manufacturing environment. This paper systematically reviews the application of different freeze-drying technologies in mRNA vaccines and the optimization strategies of lyophilized mRNA vaccines, aiming to provide theoretical foundation and guidance for optimizing freeze-drying processes, enhancing vaccine stability and expanding their application scope.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2025.126426", "ptypes": [ "Journal Article", "Review" ], "first_author": "Lou J", "dirs": [ "A" ] }, "41306796": { "pmid": "41306796", "title": "Fourier Transform Near-Infrared Spectrometry Reveals Significant Inter-Lot Variability in Thyrotropin Alfa (Thyrogen®) Quality.", "abstract": "Thyrotropin alfa is a recombinant heterodimeric glycoprotein that serves as a highly purified form of human thyroid stimulating hormone (TSH). It is produced through recombinant DNA technology within a genetically modified Chinese hamster ovary (CHO) cell line. The molecule is structurally complex, consisting of two non-covalently linked subunits: an alpha subunit (92 amino acid residues) with two N-linked glycosylation sites, and a beta subunit (118 residues) containing one N-linked glycosylation site. The resulting amino acid sequence is identical to that of human pituitary TSH. Thyrotropin alfa is supplied as a sterile, non-pyrogenic, white to off-white lyophilized product, intended for intramuscular (IM) administration after reconstitution with Sterile Water for Injection, USP. Each vial of Thyrogen contains 1.1 mg thyrotropin alfa, 36 mg mannitol, 5.1 mg sodium phosphate, and 2.4 mg sodium chloride. The intralot analysis of the 12 vials sampled from lot EY0155 revealed significant differences in the drug product within the same manufacturing lot, as evidenced by both spectral plots and multivariate analysis. The visual differences were observed in the following wavenumber regions: 4000 to 5000 cm-1, 5150 cm-1, and the 6700 cm-1 regions. The differences seen at 5150 cm-1 are very likely a difference in moisture content. 25% of the vials were flagged as outliers, with vials appearing 3.3, 3.8, and 5.3 SDs from the center of the cluster of 12 vials. The interlot analysis of a spectral library containing 171 vials from 18 different lots of thyrotropin alfa demonstrated tremendous variability in the near-infrared absorption profiles across the samples. Obvious differences were observed in the spectra of the library at several wavenumber regions, including around 5900 and 6650 cm-1. The ratio between two peak sizes between 5900 and 5950 cm-1 was dramatically variable. Differences were also noted around 4250, 4400, and 4540 cm-1, and from 4700 to 4800 cm-1. Only one vial was an outlier, appearing 7.8 SDs away from the center of the library spectral cluster. Some literature suggests differences in the spectra of vials may reflect differences in the stability and efficacy of the drug.", "journal": "Contact in context", "year": "2025", "doi": "10.6084/m9.figshare.30403540", "ptypes": [ "Journal Article" ], "first_author": "Yoon U", "dirs": [ "A" ] }, "41752511": { "pmid": "41752511", "title": "Buffalo Milk: Alternative Use for Soap Preparation Enriched with Vegetables.", "abstract": "The surplus in the production of buffalo milk determines the possibility of finding alternative solutions for its use. Indeed, the utilization of milk in cosmetic formulations has been met with great approval by consumers, primarily due to its highly appreciated emollient characteristics. The aim of this research was to test an alternative use of buffalo milk in the production of artisanal solid soaps, using buffalo milk as raw material and Lavender, Thyme, and Grape pomace as sources of natural bioactive compounds. The analytical approach was focused on using vegetable materials in three forms: fresh, dried, and freeze-dried. For this purpose, the chemical features of both raw materials and artisanal soaps were determined in order to understand the feasibility of these productions. All formulated artisanal soaps revealed good chemical characteristics, such as a low moisture content, and got high scores in the sensory evaluation, with those with Lavender and Grape pomace being the most appreciated formulations. Furthermore, adding vegetable materials increased the bioactive molecules content, as demonstrated by the data obtained from total polyphenol content and antioxidant activity. Therefore, the addition of plants and vegetables to the formulation could represent an innovative production of natural soaps and be a further element for the market trends.", "journal": "Molecules (Basel, Switzerland)", "year": "2026", "doi": "10.3390/molecules31040734", "ptypes": [ "Journal Article" ], "first_author": "la Gatta B", "dirs": [ "A" ] }, "40764529": { "pmid": "40764529", "title": "Inhibitory efficacy, production dynamics, and characterization of postbiotics of lactic acid bacteria.", "abstract": "Antimicrobial resistance (AMR) poses a significant threat to human health and food safety. Lactic acid bacteria (LAB) produce bioactive compounds, known as postbiotics, that act as promising natural preservatives with broad-spectrum antimicrobial activity. This study aimed to evaluate the antimicrobial spectrum, production dynamics, and physicochemical properties of postbiotics derived from five LAB strains: Lactobacillus plantarum NBRC 3070, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus rhamnosus GG ATCC 53103, and Bifidobacterium animalis subsp. lactis ATCC 27673. The antimicrobial activity of these postbiotics was assessed against several Gram-positive and Gram-negative pathogens. A crude bacteriocin-like inhibitory substance (BLIS), a postbiotic component, was partially purified using ammonium sulfate purification and characterized enzymatically. Its molecular weight was estimated by SDS-PAGE. The results showed that postbiotics, particularly those from L. plantarum and L. acidophilus, exhibited strong antimicrobial activity. The inhibitory effect was most pronounced against Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus after a 16-h exposure. The postbiotics production peaked between 24 and 36 h of incubation, achieving 85.71-89.28% inhibition. These postbiotics remained stable at high temperatures (up to 121 °C), across a wide pH range (3-5 and 9-11), and under varying salt concentrations. Neutralized cell-free supernatants from L. plantarum, L. acidophilus, L. casei, and L. rhamnosus GG retained antimicrobial activity, and enzyme treatments confirmed the proteinaceous nature of the BLIS. SDS-PAGE revealed diffuse protein bands between < 3.3 and 6.5 kDa. Lyophilization enhanced the concentration and stability of antibacterial compounds by reducing water content. In addition to BLIS, LAB strains produced other antimicrobial metabolites, including lactic acid, acetic acid, hydrogen peroxide, fatty acids, and notably, oleic acid. These postbiotic components remained effective after one month of storage at 4 °C and 20 °C for one month. The novelty of this study lies in its comprehensive characterization of postbiotics from well-established LAB strains across multiple functional parameters. Overall, the findings suggest that these LAB-derived postbiotics are stable, effective, and hold potential as natural antimicrobial agents in food preservation.", "journal": "BMC microbiology", "year": "2025", "doi": "10.1186/s12866-025-04123-z", "ptypes": [ "Journal Article" ], "first_author": "Rahman MM", "dirs": [ "A" ] }, "41285550": { "pmid": "41285550", "title": "Variability Assessment Methods for Lyophilized Drug Product, A Case Study.", "abstract": "Lyophilization is a critical process, removing water and/or solvent through sublimation to ensure the stability and longevity of injectable drug products. The complexity of the lyophilization process, which involves multiple stages such as freezing, primary drying, and secondary drying, necessitates a robust approach to ensure product quality and consistency. Along with applying Quality by Design (QbD) principles in lyo process development, robust statistical and risk-based methodologies are essential for assessing process variability. This paper presents a risk-based and statistically sound sampling methodology for assessing variability in lyophilized drug products during Continued Process Verification (Stage 3a) of Process Validation. Sampling plans are strategically designed, ensuring representative data collection from lyophilizer shelves. By integrating variance analysis, capability indices, and probability, the methodology provides a comprehensive understanding of both intra-batch and inter-batch variability. The assessment enables estimation of future batch performance concerning critical quality attributes, including water content, assay, pH, and impurities. Identifying and controlling both intra-batch (within-shelf and between-shelf) and inter-batch variability ensures robustness. The study highlights the importance of statistically rigorous sampling plans, justification of the plans, and data analysis to ensure process control. A case study is presented, which demonstrates the application of the approach in a lyophilization process. The methodology supports regulatory compliance and enhances process understanding, enabling tighter process control and continuous improvement. The risk-based Stage 3a framework provides a structured method for post-commercialization variability assessment, bridging process design and continued verification.", "journal": "PDA journal of pharmaceutical science and technology", "year": "2026", "doi": "10.5731/pdajpst.2025-000046.1", "ptypes": [ "Journal Article" ], "first_author": "Pazhayattil AB", "dirs": [ "A" ] }, "41843699": { "pmid": "41843699", "title": "Hierarchically Oriented Carbon Nanotubes/Poly(vinyl Alcohol) Composite Conductive Hydrogels for Flexible Strain Sensors.", "abstract": "Hydrogels have been widely used in flexible electronics due to their water content similar to that of muscles and other biological tissues. However, it was a challenge to significantly improve mechanical robustness and electrical conductivity owing to their trade-off. To address this, a synergistic method of freeze casting and hot pressing was proposed to create a multiscale hierarchical structure in the hydrogel. In detail, the mixture of poly(vinyl alcohol) (PVA) and cellulose nanofibers (CNFs) with dispersed carbon nanotubes (CNTs) was freeze-cast and freeze-dried, then hot-pressed to enhance the crystallinity, and swollen to form an oriented PVA/CNT@CNF hydrogels. The resulting hydrogel (CNT:PVA mass ratio of 2:10) exhibited good comprehensive performances, with an electrical conductivity of 0.124 S m-1, a tensile strength of 6.22 MPa, and a crystallinity of 13.98 wt %. Notably, the fracture energy displayed pronounced anisotropy, reaching 16.41 kJ m-2 along the frozen direction, 2.47 times that in the perpendicular direction. Owing to its high conductivity and structural stability, the assembled flexible sensor can reliably monitor human motions (e.g., finger and wrist bending) and functions effectively as a touch-sensitive keypad for rapid and accurate human-machine interaction. This work offered a new strategy to prepare conductive hydrogels with high crystallinity for wearable sensing, health monitoring, and soft robotics.", "journal": "ACS applied materials & interfaces", "year": "2026", "doi": "10.1021/acsami.5c23930", "ptypes": [ "Journal Article" ], "first_author": "Wang L", "dirs": [ "A" ] }, "42830159": { "pmid": "42830159", "title": "Development of orodispersible wafer-based delayed-release omeprazole systems for pediatric compounding.", "abstract": "Omeprazole (OMZ) is one of the most frequently compounded active pharmaceutical ingredients for pediatric use. Despite the availability of licensed pediatric formulations in some countries, extemporaneous oral suspensions remain widely used in hospital pharmacies, although their limited chemical stability, short shelf life, and need for specific storage conditions may complicate routine use. This study aimed to develop a child-appropriate solid alternative based on a hybrid lipid-wafer platform combining gastro-resistant OMZ microparticles with an orodispersible matrix. Orodispersible wafers (ODWs), composed of pectin, κ-carrageenan, and maltodextrins, were produced by lyophilization, yielding highly porous matrices with low residual moisture (2.74%) and rapid oral disintegration. To protect OMZ during gastric exposure, two encapsulation strategies were investigated: calcium alginate-zein beads and lipid microparticles (LMPs). Morphology, particle size, drug loading, encapsulation efficiency, and solid state were characterized, and drug release was evaluated in gastrointestinal buffers and pediatric biorelevant media. Trimyristin-based LMPs showed the most favorable performance, limiting OMZ exposure to the acidic environment and providing controlled intestinal release consistent with Higuchi kinetics. Their incorporation into ODWs yielded a solid unit containing approximately 2.5 mg OMZ. Importantly, OMZ content remained unchanged for up to six months, with comparable release profiles at t0 and after storage, including at room temperature. Overall, the proposed hybrid lipid-wafer platform provides a stable, child-appropriate solid dosage form to OMZ delivery and represents a promising formulation strategy for further development in suitably equipped hospital pharmacy compounding settings.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127509", "ptypes": [ "Journal Article" ], "first_author": "De Renzis E", "dirs": [ "A" ] }, "42320735": { "pmid": "42320735", "title": "Designing inhalable amorphous solid dispersions to mitigate crystallization risks in atropisomer development.", "abstract": "The development of inhalation formulations for CHF-6523, a selective PI3Kδ inhibitor, presents significant hurdles due to atropisomerism and a strong tendency to form amorphous solids, complicating the access to a suitable crystalline form of the pharmaceutically active isomer. NMR experiments and DFT calculations revealed a near-equilibrium distribution of two atropisomers (RaS and SaS, ≈55:45 in ethanol-d6/D2O), and their low interconversion energy barriers of 86.5 and 87.1 kJ/mol. After a comprehensive salt screening, CHF-6523 monomaleate was the only crystalline solid obtained as a single diastereoisomer (RaS). Concerns regarding the suitability of the maleate counterion for pulmonary administration prompted the exploration of amorphous solid dispersions (ASDs) using CHF-6523 xinafoate, identified as the most promising amorphous salt candidate. Three manufacturing techniques - blending, spray-drying and freeze-drying - were evaluated to preserve the amorphous phase, improve powder handling and ensure dissolution and aerosolization performance. Spray-dried powder provided the most favorable physicochemical profile, yielding particles with dv90 below 5 µm, a residual moisture of ∼ 6%, with water uptake below 1% and superior resistance to physical and chemical changes over 180 days. In vitro dissolution studies in simulated lung fluid showed that spray-dried and freeze-dried ASDs reached concentrations above 950 µg/mL within the first hour, outperforming the crystalline monomaleate and the free base (∼700-800 µg/mL at 4 h). Preliminary aerosol characterization confirmed the superior respirable fraction of the spray-dried ASD (∼73%), compared to blend (∼66%) and freeze-dried formulations (∼37%). This work demonstrates how atropisomer-related conformational dynamics can hinder crystalline form identification while guiding the development of stable amorphous formulations for inhalation therapies.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127099", "ptypes": [ "Journal Article" ], "first_author": "Disisto P", "dirs": [ "A" ] }, "40509247": { "pmid": "40509247", "title": "Antioxidant Activity and Microbial Quality of Freeze-Dried, Lactic Acid Fermented Peach Products.", "abstract": "Lactic acid fermentation has emerged as a promising strategy to enhance the functional and health-promoting qualities of plant-based foods. This study evaluates the impact of lactic acid fermentation on the antioxidant capacity, microbial viability, and chemical stability of freeze-dried peaches, aiming to develop a functional food with probiotic potential. Two bacterial strains-Fructilactobacillus fructivorans (P_FF) and Lactiplantibacillus plantarum (P_LP)-were used to assess strain-dependent effects on microbial and bioactive compound profiles. Microbiological analyses included total viable count (TVC), fungal count (TFC), and total lactic acid bacteria (TCLAB). Chemical analyses comprised polyphenol, flavonoid, anthocyanin, carotenoid, sugar, and vitamin C content, as well as antioxidant activity (DPPH, ABTS, reducing power). Thermal and structural stability were examined via thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR). Fermentation significantly increased the counts of lactic acid bacteria, achieving 8.38 and 7.86 log CFU/g after freeze-drying, respectively. While total polyphenols slightly decreased (by 9.5% and 1.1% for L. plantarum and F. fructivorans, respectively), flavonoid content increased notably by 16.1% in F. fructivorans-fermented samples. Antioxidant activities, assessed by ABTS and DPPH assays, were largely maintained, although a reduction in reducing power was observed. Additionally, fermentation led to sucrose hydrolysis, resulting in higher glucose and fructose contents, and increased water content in the final products. Minor increases in total fungal counts were noted after freeze-drying but remained within acceptable limits. Overall, the combination of fermentation and freeze-drying processes preserved key antioxidant properties, enhanced microbial safety, and produced functional peach-based products with improved bioactivity and extended shelf life. These findings highlight the potential of fermented freeze-dried peaches as innovative, health-oriented alternatives to traditional fruit snacks.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules30112360", "ptypes": [ "Journal Article" ], "first_author": "Ossowski S", "dirs": [ "A" ] }, "41471331": { "pmid": "41471331", "title": "From Berries to Capsules: Technological and Quality Aspects of Juneberry Formulations.", "abstract": "Background:Amelanchier alnifolia (Juneberry) is a phenolic-rich species with potential for pharmaceutical applications. This study aimed to optimize ultrasound-assisted extraction (UAE) conditions for producing ethanolic extracts from differently processed Juneberry berries collected in Lithuania and to develop solid oral dosage forms based on the obtained extracts. Methods: Extracts were prepared using varying ethanol concentrations, temperatures, and extraction times from dried, frozen, and freeze-dried berries. Total phenolic content (TPC) and total flavonoid content (TFC) were determined spectrophotometrically. Antioxidant activity was evaluated by DPPH and ABTS assays. Phenolic profiles were quantified by high-performance liquid chromatography (HPLC), identifying five major compounds. Extracts were converted into powders using lactose monohydrate, microcrystalline cellulose, or magnesium aluminum metasilicate as carriers. Hard capsules were manufactured and evaluated according to European Pharmacopoeia (Ph. Eur.) requirements, including mass uniformity, moisture content, and disintegration time. Results: Freeze-dried berries yielded the highest TPC, TFC, and antioxidant activity across all extraction conditions. The most efficient extraction parameters for freeze-dried berries were identified as 50% ethanol, 50-55 °C, and 30 min. HPLC analysis confirmed the presence of chlorogenic and neochlorogenic acids, rutin, hyperoside, and isoquercitrin. Among the powdered systems, lactose monohydrate demonstrated favorable flowability and moisture characteristics. Conclusions: Freeze-dried Juneberry berries are a suitable raw material for producing phenolic-rich extracts with strong antioxidant activity. Lactose-based powder blends showed the best technological performance and were successfully formulated into hard capsules. These findings support the potential of Juneberry extracts for incorporation into standardized pharmaceutical dosage forms and provide a basis for future formulation and bioavailability studies.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ph18121841", "ptypes": [ "Journal Article" ], "first_author": "Pudžiuvelytė L", "dirs": [ "A" ] }, "40795611": { "pmid": "40795611", "title": "Combination of trehalose and inulin efficacy as lyoprotectant for the preservation of human monocytic cell line.", "abstract": "Traditional cell cryopreservation is expensive and logistically complex, and it necessitates a constant cold chain [44,57]. Lyophilization presents an alluring substitute, so long as the right lyoprotectants are applied to preserve cell integrity [23,40,44,52]. This study examined whether the human THP-1 mononuclear cell line's viability and function are maintained during the lyophilization process when trehalose and inulin are combined as a lyoprotectant. Cell survival, membrane integrity, metabolic activity, and functional responsiveness (differentiation and cytokine secretion) were assessed after THP-1 cells were processed in various lyophilization conditions (trehalose alone versus trehalose in combination with inulin). The reference cells were cryopreserved cells. Following rehydration, the vitality of cells lyophilized in a mixture containing trehalose and inulin was comparable to that of cryopreserved cells. After a recovery period in culture, the cell membranes regained their integrity, despite being temporarily permeable right after rehydration. Additionally, the inclusion of inulin helped to reduce the residual moisture content and increase the stability of the lyophilizates, while also preserving metabolic activities and the ability to differentiate (including IL-1β release after stimulus). Trehalose and inulin work well together as a lyoprotectant, lowering the stress that THP-1 cells experience during the lyophilization process. Therefore, this approach may be a practical and affordable substitute for traditional cryopreservation, maintaining the cells' stability and activity even after extended storage.", "journal": "Cryobiology", "year": "2025", "doi": "10.1016/j.cryobiol.2025.105287", "ptypes": [ "Journal Article" ], "first_author": "Meiss A", "dirs": [ "A" ] }, "39694272": { "pmid": "39694272", "title": "Recent advances in drying and development of solid formulations for stable mRNA and siRNA lipid nanoparticles.", "abstract": "Current RNA lipid nanoparticle (LNP) based products are typically liquid formulations that require ultra-cold storage temperatures for stability. To address this limitation, recent efforts have focused on enhancing stability and enabling room temperature storage by converting these formulations into solid forms through drying processes such as lyophilization, spray drying, and spray-freeze drying. Nevertheless, the drying process itself can influence the stability of RNA/LNP formulations. Therefore, understanding the factors that contribute to instability during drying is essential. The choice of drying technique for LNPs depends on factors such as the mode of delivery, lipid components, and desired final product characteristics. Additionally, the drying mechanism and associated stresses must also be carefully considered. Drying methods involve a range of process parameters related to formulation, process settings, and the manufacturing environment. It is essential to understand how these parameters influence the final solid-state products' attributes, including appearance, moisture content, flow properties, and reconstitution time, as these can significantly affect the physical and chemical stability of the formulation. This review focuses on various drying techniques and their impact on the stability of RNA/LNP-based systems.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2024.12.013", "ptypes": [ "Journal Article", "Review", "Research Support, N.I.H., Extramural" ], "first_author": "Arte KS", "dirs": [ "A" ] }, "42612840": { "pmid": "42612840", "title": "Reshaping root-associated microbiota to enhance medicinal quality: Application of optimized functional microbial inoculants in Salvia miltiorrhiza cultivation.", "abstract": "The application of functional microbial preparations is an effective means to improve medicinal plants quality. In this study, the preparation processes of various functional microbial inoculants had been optimized, and the effects of these inoculants on the quality and root-associated microecology of Salvia miltiorrhiza had been evaluated. By regulating the moisture content and particle size of fungal freeze-dried powder, the optimal process parameters for Trichoderma longibrachiatum (Tl), Alternaria tellustris (At), and Paraphoma radicina (Pr) were determined, and fungal inoculants with high storage activity at both normal and low temperature were produced. Microcapsules of Bacillus licheniformis (Bl) and Bacillus subtilis (Bs) were prepared by sodium alginate-CaCl2 dripping method. Two microcapsules showed encapsulation efficiencies above 90 % and maintained high viability after 6 months of storage. Pot experiments showed that five microbial inoculants boosted the quality of S. miltiorrhiza with the dose-dependent effects. Among them, T1 and At improved biomass and contents of tanshinone I, tanshinone IIA, and cryptotanshinone at low to medium application rates (0.15-0.3 g/1.5 Kg soil). Bs microcapsule also had a better effect. Variation partitioning, mantel tests, and structural equation modeling showed that the inoculants boosted the quality of S. miltiorrhiza by adjusting soil pH, enhancing soil nutrients and enzyme activities, and reshaping root-associated microbial community. These inoculants enriched taxa, including Trichoderma, Funneliformis, Sphingomonas, and Actinoplanes, and suppressed pathogenic fungi. These findings demonstrated that rationally designed microbial inoculants sustainably improve yield and bioactive components through targeted modulation of root-associated microbial networks, offering an eco-friendly strategy for high-quality medicinal plant cultivation.", "journal": "Bioresource technology", "year": "2026", "doi": "10.1016/j.biortech.2026.135671", "ptypes": [ "Journal Article" ], "first_author": "Li W", "dirs": [ "A" ] }, "42412156": { "pmid": "42412156", "title": "Integral Use of Chia Seeds in Microencapsulation Processes for the Development of Oil-Based Potential Functional Food Ingredients.", "abstract": "An integral use approach of chia seeds was applied through microencapsulation, combining chia oil as the core with seed-derived proteins and mucilage as wall components for the development of potential functional food ingredients. Oil-in-water emulsions were formulated and dried by spray-drying and freeze-drying to evaluate how their distinct drying mechanisms influence the physicochemical, structural, and functional properties of chia oil microparticles. Both drying methods successfully produced chia oil microparticles, yielding powders with low moisture content, high encapsulation efficiency (approximately 91%, w/w), and high protein content. However, the drying method influenced the structural and functional properties of the microparticles. Scanning electron micrographs revealed lamellar structures in freeze-dried samples and spherical particles in spray-dried ones. After reconstitution, emulsion particle size increased during storage, particularly in freeze-dried systems, whereas spray-dried microparticles showed superior dispersibility and instant properties. Regarding oxidative stability, freeze-dried microparticles provided greater protection against lipid oxidation, reaching a peroxide value of 2.90 meq/kg oil after 45 days of storage. Besides, the α-linolenic acid content remained stable in freeze-dried microparticles, whereas a significant decrease was observed in spray-dried samples over the same period. A preliminary sensory evaluation of a beverage fortified with freeze-dried microparticles indicated that 94% of panelists were able to identify the fortified sample, which showed significantly lower sensory acceptance for taste, color, and appearance than the non-fortified product. Overall, these findings demonstrate the feasibility of using seed-derived proteins and mucilage as wall materials for chia oil encapsulation and highlight the potential of freeze-dried microparticles as ingredients for functional beverages.", "journal": "Plant foods for human nutrition (Dordrecht, Netherlands)", "year": "2026", "doi": "10.1007/s11130-026-01543-8", "ptypes": [ "Journal Article" ], "first_author": "Copado CN", "dirs": [ "A" ] }, "41328745": { "pmid": "41328745", "title": "Lyophilization process design and development of human plasma derived Alpha 1 proteinase inhibitor.", "abstract": "Lyophilization is the preferred method to stabilize labile protein formulations in order to attain longer shelf life. Thus, the understanding of different parameters of lyophilization along with inherent properties of plasma protein formulations is very essential to achieve improved stability. The present study aims to optimize the lyophilization cycle of one of the plasma proteins, Alpha-1 proteinase inhibitor (A1PI) to obtain a homogeneous and consistent cake structure using different lyophilization cycle parameters: slow freezing, fast freezing, and fast freezing with annealing. Process analytical technology (PAT) tools like product probe temperature, Lyo Rx, LyoLogplus software were implemented for continuous lyophilization cycle monitoring, determination of freezing and eutectic points. Formulation parameters like fill volume and solid content were screened to study the impact on the product quality. Fast freezing with annealing demonstrated homogenized cake appearance, similar size crystals with more number of pores, optimum reconstitution time as well as moisture content. High fill volume proved to be beneficial for obtaining a homogenized cake structure. The product quality attributes demonstrated similarity with the market comparator. Desired quality attributes of A1PI freeze dried cake were achieved by optimizing lyophilization cycle parameters along with formulation parameters. The understanding of different concepts of lyophilization can be utilized for the optimization of lyophilization parameters of other plasma proteins.", "journal": "Drug development and industrial pharmacy", "year": "2026", "doi": "10.1080/03639045.2025.2596296", "ptypes": [ "Journal Article" ], "first_author": "Bardhan S", "dirs": [ "A" ] }, "41376018": { "pmid": "41376018", "title": "Evaluation of Quality and Storage Characteristics of Freeze-Dried Powdered Mycelium Sausages According to Packaging Methods.", "abstract": "With the increases in the global population, demand for meat, and income, plant-based meat alternatives using mushrooms, soybeans, and other ingredients are attracting increasing attention to address food security. Mushroom mycelia have a high moisture content and are prone to quality deterioration; therefore, interventions, such as freeze-drying and optimized packaging, are necessary to improve shelf life. Furthermore, controlling storage temperature is a key factor in preserving product quality. Therefore, in this study, Lentinula edodes (shiitake mushroom) mycelium sausage were stored under various packaging methods (aerobic packaging, vacuum packaging, and modified atmosphere packaging) and storage temperatures (0 and 4 °C). Quality and storability were evaluated at 0, 7, 15, 30, and 50 days using tests for proximate components, pH, storage loss, color, water activity (Aw), aerobic bacterial count, thiobarbituric acid reactive substances (TBARS), and volatile basic nitrogen (VBN) using an electronic nose and an electronic tongue. The vacuum-treated groups showed superior pH, CIE L*, CIE a*, Aw, TBARS, and VBN levels. At 0 °C, each treatment group exhibited significantly lower VBN levels on days 30 and 50 (p < 0.05). Overall, vacuum packaging and low storage temperatures are expected to be beneficial for maintaining the quality and storage characteristics of shiitake mushroom mycelium sausages.", "journal": "Foods (Basel, Switzerland)", "year": "2025", "doi": "10.3390/foods14234080", "ptypes": [ "Journal Article" ], "first_author": "Oh YN", "dirs": [ "A" ] }, "39945434": { "pmid": "39945434", "title": "Water's Dual Role as a Chemical Catalyst and Physical Stabilizer in Deamidation of Lyophilized Proteins Studied via Molecular Dynamics Simulations.", "abstract": "Water plays a critical role in chemical degradations, such as deamidation, in freeze-dried proteins. Two distinct patterns for deamidation in relation to water have been reported, that is a \"hockey stick\"-type behavior with a water-independent deamidation rate, followed by a sharp increase above a water content threshold, and an inverted bell-shaped profile. To understand the underlying mechanism, molecular dynamics simulations are employed to study the explicit water distributions around reactive sites for amorphous and crystalline insulin as well as amorphous IgG1. The simulated water distribution on the protein surface is first validated by successfully predicting water vapor sorption isotherms for both amorphous and crystalline insulin. The \"hockey stick\"-type behavior is explained by a water threshold level beyond which there are two (Asn-Gly sequence in IgG1) or three (Asn at the C-terminus in insulin) water molecules assisting the cyclization reactions. Regarding the inverted bell-shaped profile for amorphous IgG1, the initial decreases in deamidation rate with increasing water content at low water levels can be rationalized by a lower density and higher free volume of IgG1 at a lower water content. When the free volume exceeds a percolation threshold, the produced ammonia gas can easily diffuse away, lowering the back reaction rate and thus raising the overall reaction rate. The \"free volume\" mechanism can also be applied to the abnormal stability ranking orders of crystalline and amorphous insulin. The faster deamidation and dimerization rates in insulin crystals compared to amorphous insulin as reported by Pikal and Rigsbee are due to the lower density and higher free volume (above the percolation threshold) in crystalline insulin, assuming that dehydration of insulin crystals does not result in a major collapse of the crystal structure.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.4c01185", "ptypes": [ "Journal Article" ], "first_author": "Feng S", "dirs": [ "A" ] }, "41832752": { "pmid": "41832752", "title": "Stability of house dust mite raw materials: Impact of storage conditions and lyophilization.", "abstract": "Dermatophagoides pteronyssinus and D. farinae are major indoor allergens. Raw materials derived from these house dust mites (HDMs) are used to produce allergen extracts. The stability of these materials is influenced by storage form and conditions, which can affect protein integrity. To compare storage formats and conditions for maintaining physicochemical stability of HDM raw materials. Pure mite bodies (PMBs), prepared in fresh frozen and lyophilized forms, were stored at -80°C, -20°C, and 25°C. Over a 12-month period, samples were assessed for total protein and major allergens (Der p 1 and Der f 1) using ELISA. Protein integrity was analyzed by SDS-PAGE. Accelerated stability testing at 25°C for up to 14 days was also performed. Storage at -80°C best preserved total protein and Group 1 allergen content over 12 months. At -20°C, moderate declines were observed, with D. farinae showing greater reductions than D. pteronyssinus. Lyophilized samples were generally more stable than fresh-frozen ones at -20C°. Accelerated testing at 25°C caused marked losses over 14 days. Ultra-low temperature storage (-80°C) remains optimal for preserving physicochemical stability of HDM raw materials. Lyophilization with -20°C storage is a promising, context-dependent option; however, in the absence of residual-moisture and immunological data, these findings should be interpreted as preliminary and limited to raw-material handling.", "journal": "Asian Pacific journal of allergy and immunology", "year": "2026", "doi": "10.12932/AP-090425-2062", "ptypes": [ "Journal Article" ], "first_author": "Wangapai T", "dirs": [ "A" ] }, "41982638": { "pmid": "41982638", "title": "Development and Stepwise Optimization of Freeze-Dried Seasoning Cubes From Traditional Fish Sauce: A Postharvest Approach for Value-Added Aquatic Products.", "abstract": "This study focuses on the technological development of freeze-dried seasoning cubes from traditional Vietnamese fish sauce (60°N), aiming to improve postharvest stability and product versatility. Formulation trials were conducted by blending fish sauce with calamansi juice, garlic, chili, sugar, and maltodextrin to optimize flavor balance and structural integrity. Multistage freeze-drying (10°C-50°C) was applied to evaluate the effects of drying temperature on moisture removal, structural properties, sensory quality, and retention of bioactive compounds. The optimal formulation (1:1 dilution, 10% calamansi, 15% garlic, 10% chili, 20% sugar, and 25% maltodextrin) combined with a final drying temperature of 50°C produced compact, well-defined cube-shaped products (approximately 25 × 25 × 8 mm) with a stable porous internal matrix and a smooth, nonsticky surface. The optimized cubes achieved a final moisture content of 2.79%, a sensory acceptance score of 4.50 ± 0.18, and retained considerable levels of bioactive compounds, including 44.64% DPPH radical scavenging activity, 21.15 mg GAE/100 g total polyphenols, and 24.75 mg AA/100 g vitamin C. These findings demonstrate a practical postharvest strategy to transform traditional liquid fish sauce into convenient, value-added aquatic products suitable for broader culinary and export applications.", "journal": "International journal of food science", "year": "2026", "doi": "10.1155/ijfo/2389589", "ptypes": [ "Journal Article" ], "first_author": "Pham VT", "dirs": [ "A" ] }, "42817278": { "pmid": "42817278", "title": "α-Tocopherol-loaded freeze-dried liposomes and proliposomes: powder properties and thermal performance.", "abstract": "Dry lipid-based formulations can enhance the handling and preservation of lipophilic bioactives, though their efficacy depends heavily on formulation composition and processing conditions. This study compared α-tocopherol-loaded freeze-dried liposomes (FD_LPs) and proliposomes (PLPs) formulated with either mannitol (PLPs_MAN) or trehalose (PLPs_TRE) as carriers. All three formulations were evaluated for reconstitution characteristics, powder properties, and thermal performance after rehydration. Additionally, a four-week storage stability test was conducted at 4, 25, and 40 °C, restricted to FD_LPs and PLPs_MAN. Upon reconstitution, all formulations exhibited mean vesicle diameters ranging from 561.5 to 669.3 nm and polydispersity index (PDI) values of approximately 0.57-0.60, indicating relatively broad particle size distributions. Initial entrapment efficiencies (EE) were high, exceeding 93% for all samples. Among the formulations, PLPs_MAN exhibited the lowest moisture content. Both PLP formulations demonstrated lower hygroscopicity and superior flow indices compared to FD_LPs. Under thermal stress (90 °C, 12 h), PLPs_TRE showed the lowest TBARS response, whereas PLPs_MAN maintained the highest DPPH radical-scavenging activity. After four weeks at 40 °C, mean EE values were lower than their initial values for both tested formulations, reaching 73.7% for FD_LPs and 79.0% for PLPs_MAN. Overall, both proliposome formulations exhibited more favorable powder flow indices and lower hygroscopicity than FD_LPs, whereas their thermal responses depended on the endpoint examined. The four-week storage findings apply only to FD_LPs and PLPs_MAN and do not establish the storage performance of PLPs_TRE. Differences in carrier loading and processing limit attribution of the observed effects to carrier identity alone.", "journal": "Journal of liposome research", "year": "2026", "doi": "10.1080/08982104.2026.2740627", "ptypes": [ "Journal Article" ], "first_author": "Lee J", "dirs": [ "A" ] }, "40509314": { "pmid": "40509314", "title": "Tailoring Polymeric Scaffolds with Buddleja globosa Extract for Dual Antimicrobial and Biocompatible Wound Healing Applications.", "abstract": "Integrating traditional herbal extracts into modern biomaterials offers a promising route for advanced wound care. A standardized Buddleja globosa Hope extract (BG-126), recognized for its therapeutic value, was incorporated into polymeric scaffolds with variable composition to explore their potential in promoting wound healing and controlling infections. This work aimed to identify the polymeric composition of a scaffold with BG-126 that maximizes its compatibility and antimicrobial properties. Scaffolds were developed by lyophilization using a Box-Behnken design (BBD) with chitosan, hyaluronic acid, and gelatin content as study factors. Thirteen scaffold formulations were tested for their antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa, including biofilm forms, as well as for their biocompatibility with normal human fibroblasts. Structural and physical properties, such as the moisture content and swelling capacity, were evaluated. The best-performing scaffold was analyzed using Raman spectroscopy. The chitosan content was strongly associated with antimicrobial efficacy, while gelatin enhanced fibroblast compatibility (R2 ≥ 0.9). No correlations were identified between the polymeric content and biofilm inhibition or physical properties. BG-126-loaded scaffolds reduced planktonic and biofilm proliferation and improved fibroblast compatibility compared to the control scaffold (without BG-126). The results support the rational design of botanical-loaded scaffolds with targeted properties for wound healing.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules30112428", "ptypes": [ "Journal Article" ], "first_author": "Ceriani R", "dirs": [ "A" ] }, "41193929": { "pmid": "41193929", "title": "Improving the CQAs and Lyophilization Process Efficiency of Freeze-dried Monoclonal Antibody Drug Products Using Controlled Ice Nucleation Technology.", "abstract": "The objective of this research was to explore the application of controlled ice nucleation (CN) technology to enhance the critical quality attributes (CQAs) and manufacturing efficiency of freeze-dried monoclonal antibody (mAb) drug products. The study aimed to address challenges associated with conventional freeze-drying process, such as low supercooling temperatures and inhomogeneous ice nucleation temperatures, and to evaluate whether CN could improve product quality, and the overall efficiency of the lyophilization process. Various mAb drug products were manufactured using three different lyophilization methods during the freezing step, including conventional, annealing, and CN. The CQAs of the drug products were assessed, including residual moisture content, reconstitution time, product appearance, microscopic morphology, specific surface area, protein purity, charge variance, and sub-visible particulates. Comparative analyses were conducted to evaluate the impact of the CN on product quality and process efficiency. CN demonstrated significant improvements in product appearance, uniformity, reconstitution properties, and stability. It notably reduced the primary drying time, enhancing the overall efficiency of the freeze-drying process. Furthermore, the CN method produced lyophilized drug products with consistent cake appearance, improved quality, and enhanced stability, ensuring therapeutic efficacy and patient safety. CN technology represents a major advancement in pharmaceutical manufacturing. The implementation of CN has the potential to improve the CQAs of drug products, optimize the lyophilization process, reduce operational costs, and increase production throughput. These findings underscore the importance of adopting innovative technologies like CN to meet the growing demand for high-quality and efficient biopharmaceutical production.", "journal": "Pharmaceutical research", "year": "2025", "doi": "10.1007/s11095-025-03930-3", "ptypes": [ "Journal Article" ], "first_author": "Chen Y", "dirs": [ "A" ] }, "41087409": { "pmid": "41087409", "title": "Impact of maltodextrin and gum Arabic ratio on freeze dried microencapsulated extract of microgreen kangkong (Ipomoea reptans Poir).", "abstract": "Kangkong (Ipomoea reptans Poir) is widely consumed in Southeast Asia and possesses pivotal health benefits. Microgreen kangkong can be a potential alternative to meet nutritional requirements as it contains higher concentrations of bioactive compounds than mature plants. The study aimed to determine the effect of the ratio of maltodextrin and gum Arabic as encapsulants on the physicochemical characteristics of microgreen kangkong extract microcapsules. Encapsulation is applied to protect certain substances, including bioactive compounds, resulting in their good stability and bioavailability. Microgreen kangkong extract was encapsulated using the freeze-drying technique with varying concentration ratios between maltodextrin and gum arabic 60:40, 50:50, 40:60, and 20:80. The results showed that the increase in gum arabic concentration provided better chemical characteristics; however, this resulted in high water content. Extract with a concentration of maltodextrin and gum arabic 20:80 exhibited the foremost chemical characteristics than other treatments, with solubility, encapsulation efficiency, total phenol, total flavonoid, antioxidant, and anti-inflammatory activity of 97.35%, 86.85%, 82.70 mg GAE/10 g microcapsules, 43.42 mg QE/10 g microcapsules, 0.38 M/10 g microcapsules, and IC50 of 535.37 mg/L respectively. Based on the evaluation of both chemical and physical properties, the 20:80 maltodextrin to gum arabic ratio is considered as the optimal formulation due to its balance between bioactive retention and acceptable physical characteristics. This formulation demonstrates potential as a dry, bioactive-rich ingredient for functional food or nutraceutical applications and introduces microgreen kangkong as a novel source for microencapsulation matrices.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-19707-y", "ptypes": [ "Journal Article" ], "first_author": "Kurniati D", "dirs": [ "A" ] }, "40946920": { "pmid": "40946920", "title": "Comparison of several methods for preserving ETEC-S3 bacteriophage and its application to food.", "abstract": "Food contamination caused by pathogenic bacteria is commonly controlled using chemical preservatives and physical preservation, which may negatively impact food quality. Bacteriophages are viruses that specifically infect bacteria, it can be used as an alternative antimicrobial agents. For further applications, phage need to be preserved. Phages show varying sensitivities to different preservation conditions, hence it is important to develop reliable long-term phage preservation methods. The ETEC-S3 phage, isolated from soil from previous study, showed lytic activity against Enterotoxigenic Escherichia coli (ETEC) as its original host. However, its preservation methods had not been investigated. This study investigates the effects of lyophilization and low-temperature storage, using trehalose, sucrose, or glycerol as stabilizing agents, on the titer stability of ETEC-S3, and evaluates their application in reducing ETEC populations in various food matrices. Low-temperature storage at -80 °C with sucrose or glycerol, and lyophilization with sucrose, effectively maintained ETEC-S3 stability, with titer reductions of 0.04, 0.08, and 0.26 log units, respectively, after 24 weeks. Lower temperatures performed more effective in preserving phage titers compare to higher temperatures. Stabilizers provided protection, which helped maintain phage viability throughout storage. Furthermore, lyophilized ETEC-S3 with the sucrose addition still performed high lytic activity after being applied to artificially contaminated various food samples. We found that food samples which high moisture content allowed easier phage diffusion, which resulted in higher bacterial reductions. The highest bacterial reductions were observed in tofu at 28 °C (99.86 %), followed by pasteurized milk at the same temperature (94.37 %). These findings indicate the potential preservation condition of ETEC-S3 as a natural food preservative.", "journal": "Journal of microbiological methods", "year": "2025", "doi": "10.1016/j.mimet.2025.107257", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "Pranata NZ", "dirs": [ "A" ] }, "40066621": { "pmid": "40066621", "title": "Lyophilized monkeypox mRNA lipid nanoparticle vaccines with long-term stability and robust immune responses in mice.", "abstract": "The World Health Organization (WHO) has recently declared another global health emergency due to the rapidly spreading monkeypox (Mpox) outbreak in numerous African countries. To address the unmet need to contain the outbreak using the existing vaccines, this study developed a lyophilization process for an effective, scalable and affordable Mpox mRNA-LNP vaccine candidate to address the global health crisis. A comprehensive evaluation and optimization of the vaccine formulation (the type/concentration of cryoprotectants, the type/concentration of buffer system, as well as the mRNA concentration and reconstitution solvent) and the freeze-drying process parameters (freezing method, temperature, cooling rate and primary/secondary drying conditions) were conducted. The freeze-dried product exhibits a uniform appearance and a moisture content of less than 1%. Reconstitution of the lyophilized mRNA-LNP resulted in equivalent particle size/polydispersity index, encapsulation efficiency and mRNA integrity compared to that of freshly prepared mRNA-LNP. Furthermore, the lyophilization process can be scaled up 100-fold to 2000 vials/batch. Notably, the lyophilized mRNA-LNP demonstrated a storage stability of at least 12 months at 4°C, and at ambient temperature for a minimum of 8 h post-reconstitution, exhibiting minimal deterioration in product quality. The in vitro biological activity and in vivo immunogenicity of the lyophilized mRNA-LNP was comparable to that of the freshly prepared mRNA-LNP. These results provide a compelling rationale for the utilization of lyophilization technology in enhancing the accessibility of the Mpox mRNA vaccine in developing countries, a strategy that is crucial for containing the global epidemic of Mpox infection.", "journal": "Human vaccines & immunotherapeutics", "year": "2025", "doi": "10.1080/21645515.2025.2477384", "ptypes": [ "Journal Article" ], "first_author": "Wang B", "dirs": [ "A" ] }, "42798006": { "pmid": "42798006", "title": "Improving the Quality of Lyophilized Animal Vaccines: A Review of Optimization Strategies for Freeze-Drying Processes.", "abstract": "Vacuum freeze-drying, commonly referred to as lyophilization, is one of the primary processes in vaccine industrial manufacturing. It offers significant advantages, including a marked extension of the shelf-life of dried products, effective preservation of biological activity, and user convenience. However, in the production of animal freeze-dried vaccines, the diverse nature of antigens, the complexity of heat-stable protectant formulations, and variations in manufacturing equipment pose substantial challenges to process design for animal freeze-dried vaccines. Commercially available animal freeze-dried vaccines commonly suffer from antigen potency loss, insufficient product stability, low production efficiency and high manufacturing costs. Freeze-drying process optimization can markedly improve the quality of freeze-dried vaccines, with key evaluation indicators including vaccine stability, shelf-life, sample morphology, and residual moisture content. Focusing on multiple types of animal vaccines, this review summarizes research advances in improving the quality of animal freeze-dried vaccines from the perspectives of modifying material properties, enhancing freezing-stage regulation, increasing the specific surface area for drying, and optimizing heat conduction. Finally, future research strategies for animal freeze-dried vaccines are prospected. Although numerous approaches have been proposed to enhance the quality of animal freeze-dried vaccines, comprehensive considerations from multiple dimensions are still required to provide more holistic theoretical support for cost reduction and efficiency improvement in the production of animal freeze-dried vaccines.", "journal": "Veterinary sciences", "year": "2026", "doi": "10.3390/vetsci13090972", "ptypes": [ "Journal Article", "Review" ], "first_author": "Huang F", "dirs": [ "A" ] }, "42492644": { "pmid": "42492644", "title": "Lyophilized perfluorocarbon nanodroplets: toward scalable and stable acoustic contrast agents for ultrasound biomedical applications.", "abstract": "Perfluorocarbon nanodroplets are emerging ultrasound-responsive agents with strong potential for diagnostic imaging and therapeutic applications due to their ability to undergo acoustic droplet vaporization. However, their inherent physicochemical instability limits their long-term storage and clinical translation. Here, we report a scalable freeze-drying protocol for lipid-stabilized perfluorocarbon nanodroplets to ensure long-term stability without compromising NDs characteristics and acoustic performance. In this study, nanodroplets composed of perfluorobutane or perfluoropentane were produced by microfluidic nanoprecipitation and protected by cryoprotectants. The influence of cryoprotectant type on cake appearance, particle size, polydispersity index, chemical composition and vaporization behavior was systematically evaluated. Chemical integrity was assessed by gas chromatography for perfluorocarbon core and ethanol content and by Karl Fischer titration for residual water. Acoustic performance was determined by measuring ADV thresholds under focused ultrasound. Disaccharide-based cryoprotectants, particularly trehalose at 10 wt%, provided optimal protection, preserving nanodroplet size (200-250 nm), monodispersity (Polydispersity Index <0.2), and acoustic responsiveness after freeze-drying. In addition, quantitative analyses showed that the chemical composition of nanodroplets was maintained, with perfluorocarbon and ethanol contents remaining unchanged, while residual water content stayed below 2 %, indicating effective preservation of the freeze-dried product. Stability studies demonstrated preservation of particles, chemical and acoustic properties for at least six months at 4 °C and 25 °C. These results establish a robust strategy for stabilizing perfluorocarbon nanodroplets and support their practical deployment as ultrasound-responsive agents for biomedical applications.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127208", "ptypes": [ "Journal Article" ], "first_author": "Melich R", "dirs": [ "A", "D" ] }, "41074368": { "pmid": "41074368", "title": "Hygroscopicity and quality stability of freeze-dried restructured jujube influenced by the addition of biopolymer-rich chickpea and quinoa.", "abstract": "Freeze-dried restructured jujubes (FDRJ) are highly hygroscopic, resulting in structural collapse and color browning, which ultimately leads to quality degradation. This study aimed to evaluate the effects of incorporating chickpea and quinoa on the hygroscopicity reduction of FDRJ, identify the key matrix components responsible for this effect, and elucidate the underlying mechanisms. Results showed that the lowest hygroscopicity was observed at a total addition level of 35 % (w/w) of chickpea and quinoa, with a mass ratio of 2:1. Further comparison of hygroscopic behavior, glass transition temperature, and microstructure among AQC, freeze-dried restructured pure jujube (PJ), and FDRJs formulated with corn starch (ACS), soy protein isolate (ASPI), and a combination of both (ACSSPI) revealed that AQC had the lowest equilibrium moisture content (EMC), with protein-based and starched-based biopolymers contributing to EMC reduction. In addition, ACS, AQC, and ACSSPI exhibited higher glass transition temperatures of anhydrous samples (Tgs). Moreover, biopolymer addition resulted in a smaller and denser internal pore structure in FDRJ. Changes in texture, color properties, moisture content, and water activity of AQC during storage followed zero-order kinetics, and the shelf-life prediction model based on moisture content showed the highest predictive accuracy (relative errors of 3.21 %-9.47 %). These results will provide insight into the mechanisms underlying hygroscopicity reduction and references for improving the quality stability of FDRJ using biopolymer-rich raw materials.", "journal": "Food research international (Ottawa, Ont.)", "year": "2025", "doi": "10.1016/j.foodres.2025.117126", "ptypes": [ "Journal Article" ], "first_author": "Li M", "dirs": [ "A" ] }, "40473686": { "pmid": "40473686", "title": "Mathematical modeling of sorption isotherms and the thermodynamic properties of vacuum-dried and freeze-dried Barhi dates.", "abstract": "Studying the moisture sorption behavior of Barhi dates is key to enhancing their drying efficiency, packaging strategies, and storage conditions to ensure long-term stability and shelf-life. This research analyzed the sorption isotherms of vacuum-dried (VDBD) and freeze-dried Barhi dates (FDBD) via a dynamic vapor sorption apparatus across different temperatures and a wide range of water activity levels. Various mathematical models were evaluated to determine their accuracy in predicting sorption behavior, and the thermodynamic properties of sorption were examined in relation to the equilibrium moisture content (Xe). The results demonstrated that VDBD and FDBD followed Type III adsorption and Type V desorption isotherms. Adsorption increased with increasing water activity, whereas desorption displayed the opposite trend, with significant hysteresis between the two processes. The Peleg model provided a precise fit for both the adsorption and desorption isotherms across all the tested conditions. To maintain safe storage at the studied temperatures and 50% relative humidity, the recommended moisture content was 5% for VDBD and 9%. for FDBD. Additionally, the net isosteric heat of sorption (Qst), differential entropy (ΔS), and Gibbs free energy (ΔG) exhibited inverse correlations with Xe, increasing as the moisture content decreased during sorption. These thermodynamic parameters were effectively modeled as functions of Xe, indicating that both adsorption and desorption were nonspontaneous processes. This study provides critical insights for food processors to refine drying protocols and establish optimal storage conditions for VDBD and FDBD, ultimately preserving quality and preventing microbial contamination.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-01935-x", "ptypes": [ "Journal Article" ], "first_author": "Fikry M", "dirs": [ "A" ] }, "42437520": { "pmid": "42437520", "title": "Machine Learning-Based PAT Tool for Real-Time Residual Moisture Monitoring in Lyophilized Biopharmaceuticals Using NIR Spectroscopy.", "abstract": "There is a growing demand in the pharmaceutical industry to optimize analytical methodologies, reducing time and resource consumption while ensuring rapid process development and cost-efficient manufacturing. Indeed, Food and Drug Administration (FDA) introduced the Process Analytical Technology (PAT) as an initiative enabling manufacturers to measure and control in real-time processes based on the Critical Quality Attributes (CQAs) of the product. This study explores the implementation of PAT as a strategic approach in the lyophilization process design and optimization. In this sense, Residual Moisture (RM) was selected as a CQA to evaluate lyophilized biopharmaceutical products, due to its impact on stability. Traditionally, RM quantification is obtained by Karl-Fischer (KF) titration, a destructive technique. Recently, Near-Infrared Spectroscopy (NIRS) was proposed as a non-destructive alternative; however, its effectiveness is hindered by spectral complexity and non-linear moisture-spectra relationships. Here, PAT tool integrating NIRS with Machine Learning (ML) is developed to enable rapid, at-line RM assessment, handling the complexity of non-linear relationships between RM values and spectral features. Different ML models - Partial Least Squares (PLS), Support Vector Regression (SVR), XGBoost, and an ensemble PLS-SVR approach - were trained on placebo samples and validated on drug products. SVR outperformed PLS at lower moisture levels, while PLS was more accurate at higher RM ranges. The ensemble model combining both approaches improved predictive accuracy across the full RM range tested. External validation using literature-sourced datasets, including placebo and simulated drug products with different excipients, confirmed the model's robustness and adaptability. Furthermore, a minimal dataset augmentation strategy enhanced model specialization for specific formulations. The proposed NIRS-ML ensemble model offers a scalable, non-destructive solution for real-time RM monitoring, supporting pharmaceutical manufacturing within the PAT framework. Its adaptability across formulations highlights its potential as a versatile tool for lyophilized biopharmaceutical manufacturing pipelines.", "journal": "Biotechnology and bioengineering", "year": "2026", "doi": "10.1002/bit.70302", "ptypes": [ "Journal Article" ], "first_author": "Fiorenza S", "dirs": [ "A" ] }, "40284531": { "pmid": "40284531", "title": "Comparison of Drying Techniques to Produce Stable and Bioavailable Encapsulated ACE-2 Nanoparticles.", "abstract": "Background/Objectives: COVID-19 infection continues globally, with frequent emergence of unfamiliar SARS-CoV-2 variants acting to impair immunity. The competitive binding of SARS-CoV-2 spike proteins and angiotensin-converting enzyme 2 (ACE-2) can decrease the binding of the virus on native ACE-2 receptors on healthy human cells. It remains a practical approach to lessen viral spread. In this study, a method to encapsulate ACE-2 in the form of chitosan/tripolyphosphate cross-linked nanoparticles (NPs) was developed with emphasis placed on the best dehydration method to secure functional ACE-2 nanoparticles. Methods: Methods: Preparation conditions were assessed by varying pH (4.0-6.5) and the ratio between chitosan and ACE-2 mixing ratios (1:1, 1.5:1, 2:1, 2.5:1, and 3:1). The formulated NPs were then dehydrated using different approaches that included spray-drying (SD), freeze-drying (FD), and spray-freeze drying (SFD) and used varying mannitol concentrations (0, 1:1, and 5:1 of total weight). The mannitol was served as a cryoprotectant in this study. Results: The best formulation achieved used a pH 5.5 with a mixing chitosan-ACE-2 ratio of 2:1, where ACE-2-loaded NPs had an average particle size of 303.7 nm, polydispersity index (PDI) of 0.21, encapsulation efficiency (EE) of 98.4%, and ACE-2 loading content (LC) of 28.4%. After reconstitution, all SD samples had a relatively low yield rate, but the ACE-2 NPs dehydrated specifically using SFD required a lower amount of added mannitol (1:1 of its total weight) and produced a higher yield rate (p < 0.05) and similar PDI and EE values, along with relatively good particle size and LC. This formulation also produced a high ACE-2 release and uptake in differentiated Caco-2 cells, thus representing an effective ACE-2 encapsulation procedure for use with dry powders. Conclusions: This work showed that spray-freeze drying was the best method to dehydrate ACE-2 NPs, using less cryoprotectant to create a significant advantage in terms of greater loading capacity with lower additive requirements.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17040537", "ptypes": [ "Journal Article" ], "first_author": "Guo Y", "dirs": [ "A" ] }, "40941082": { "pmid": "40941082", "title": "Recent Advances and Future Perspectives on Heat and Mass Transfer Mechanisms Enhanced by Preformed Porous Media in Vacuum Freeze-Drying of Agricultural and Food Products.", "abstract": "Preformed porous media (PPM) technology has emerged as a transformative approach to enhance heat and mass transfer in vacuum freeze-drying (VFD) of agricultural and food products. This review systematically analyzes recent advances in PPM research, with particular focus on spray freeze-drying (SFD) as the dominant technique for precision pore architecture control. Empirical studies confirm PPM's efficacy: drying time reductions of 20-50% versus conventional VFD while improving product quality (e.g., 15% higher ginsenoside retention in ginseng, 90% enzyme activity preservation). Key innovations include gradient porous structures and multi-technology coupling strategies that fundamentally alter transfer mechanisms through: resistance mitigation via interconnected macropores (50-500 μm, 40-90% porosity), pseudo-convection effects enabling 30% faster vapor removal, and radiation enhancement boosting absorption by 40-60% and penetration depth 2-3 times. While inherent VFD limitations (e.g., low thermal conductivity) persist, we identify PPM-specific bottlenecks: precision regulation of pore structures (<5% size deviation), scalable fabrication of gradient architectures, synergy mechanisms in multi-field coupling (e.g., microwave-PPM interactions). The most promising advancements include 3D-printed gradient pores for customized transfer paths, intelligent monitoring-feedback systems, and multiscale modeling bridging pore-scale physics to macroscale kinetics. This review provides both a critical assessment of current progress and a forward-looking perspective to guide future research and industrial adoption of PPM-enhanced VFD.", "journal": "Foods (Basel, Switzerland)", "year": "2025", "doi": "10.3390/foods14172966", "ptypes": [ "Journal Article", "Review" ], "first_author": "Hu X", "dirs": [ "A" ] }, "42546983": { "pmid": "42546983", "title": "Engineering High-Performance Inhalable Monoclonal Antibody Powders via Spray Freeze Drying.", "abstract": "Spray freeze-drying (SFD) is a cutting-edge technique for producing dry powders, ideal for pulmonary drug delivery, owing to its ability to generate porous microparticles with low density and excellent aerodynamic properties. The SFD process, involving atomisation, freezing, and drying, is particularly suited for formulating biologics, e.g., monoclonal antibodies (mAbs), due to its low-temperature conditions. However, the method poses challenges, including the potential denaturation of mAbs due to shear, freezing, and dehydration stresses during processing. Here, we investigated how formulation modulates mAb stability and delivery performance during and after SFD processing. A model mAb was spray freeze-dried in the presence of selected stabilizing excipients, including sugars/polyols, amino acids, cyclodextrins, and surfactants. MAb chemical stability was assessed by size exclusion chromatography after the SFD process to study the strength of the formulations in each stage. The resulting inhalable mAb powders demonstrated favourable aerodynamic properties, rapid dissolution rates, and stability under various storage conditions. Notably, trehalose combined with either leucine or HPβCD effectively preserved mAb stability throughout the SFD process, ensuring high fine particle fractions (50-60%), and good physical stability of the powders. The optimised dry powder was successfully administered to rats via inhalation, as evidenced by lung deposition and systemic exposure, supporting the suitability of inhaled mAb-based therapeutics. This work elucidates key formulation-process interactions governing the pulmonary delivery of inhalable mAbs and demonstrates how rational excipient design enables the production of stable, inhalable biologic dry powders via SFD.", "journal": "European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V", "year": "2026", "doi": "10.1016/j.ejpb.2026.115202", "ptypes": [ "Journal Article" ], "first_author": "Pasero L", "dirs": [ "A" ] }, "42580499": { "pmid": "42580499", "title": "Development of inhalable dry powders with a high content of naked cholesterol-modified siRNA by spray freeze drying and spray drying techniques.", "abstract": "Inhaled dry powder formulations of naked small interfering RNA (siRNA), even those without a transfection reagent, are promising in clinical applications for respiratory diseases. We herein attempted to newly produce inhalable spray-freeze-dried (SFD) and spray-dried (SD) powders mainly composed of naked siRNA and leucine, an excipient, at a high siRNA content of 10 or 20% (w/w) in order to examine the effects of a cholesterol (Chol) modification to siRNA on their physical and biological properties. The Chol modification to siRNA improved the aerosol performance of the SD powder, but not the SFD powder, even though it did not significantly change the particle structures of these powders. Irrespective of the content of or Chol modification to siRNA, the SFD powder exhibited excellent aerosol performance with a high fine particle fraction of >64%. The structural and biological integrities of siRNA were almost completely maintained in the SFD and SD powders regardless of the content of or Chol modification to siRNA, while powders loading naked Chol-modified siRNA, unlike those loading naked non-modified siRNA, attained gene silencing against cultured cancer cells even without a transfection reagent. After their intratracheal administration to mice, the SFD and SD powders loading naked Chol-modified siRNA both achieved gene silencing against cancer cells metastasized in the lungs and did not significantly affect any of the following toxicity indices: body weight, the arterial oxygen saturation level, lung weight, or the number of nucleated cells and amounts of lactate dehydrogenase and tumor necrosis factor alpha in bronchoalveolar lavage fluid.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127308", "ptypes": [ "Journal Article" ], "first_author": "Okuda T", "dirs": [ "A" ] }, "40768573": { "pmid": "40768573", "title": "A streamlined CRISPR-based test for tuberculosis detection directly from sputum.", "abstract": "Mycobacterium tuberculosis (Mtb) is a major threat to global health, and there is an urgent need for affordable, simple tuberculosis (TB) diagnosis in underresourced areas. Here, we combine recombinase polymerase amplification with Cas13a and Cas12a detection to create two parallelized one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and a human DNA internal control. These assays are compatible with lateral flow and can be readily lyophilized. Our final assay showed a limit of detection of 69.0 CFU per milliliter for Mtb H37Rv and 80.5 CFU per milliliter for Mycobacterium bovis BCG in spiked sputum, with no cross-reactivity to diverse bacterial or fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (six of six) sensitivity and 100% (seven of seven) specificity compared to culture. SHINE-TB streamlines TB diagnosis from sample to answer by combining amplification and detection while being compatible with lateral flow and lyophilization.", "journal": "Science advances", "year": "2025", "doi": "10.1126/sciadv.adx2067", "ptypes": [ "Journal Article" ], "first_author": "Bell AG", "dirs": [ "A" ] }, "41201953": { "pmid": "41201953", "title": "Highly Sensitive Field Detection Technology for Anthrax Based on the CRISPR/Cas13a System.", "abstract": "In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.", "journal": "Microbial biotechnology", "year": "2025", "doi": "10.1111/1751-7915.70240", "ptypes": [ "Journal Article", "Evaluation Study", "Research Support, Non-U.S. Gov't" ], "first_author": "Fan J", "dirs": [ "A" ] }, "40054154": { "pmid": "40054154", "title": "Development of a single-tube RPA/CRISPR-cas12a detection platform for monkeypox virus.", "abstract": "Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks primarily occurring in West and Central Africa. The recent global MPXV outbreak underscores the urgent need for effective detection methods. Currently, qPCR is considered the gold standard for MPXV detection; however, it requires specialized personnel and costly equipment. This study introduces a CRISPR-Cas12a-based detection system targeting the MPXV A27L gene, achieving a detection limit as low as 10 aM. This system exhibits high specificity, with no cross-reactivity with other orthopoxviruses, and delivers results in under 40 min. To support point-of-care testing (POCT), we developed a lateral flow assay (LFA) strip for easy result visualization. The detection system was validated using six different clinical sample types, revealing that herpes fluid and saliva are the most suitable sources. The findings of this study align with qPCR results. Additionally, we lyophilized the RPA and CRISPR reagents to improve transport, storage, and field deployment. In conclusion, this study presents a reliable molecular diagnostic approach for early MPXV detection and point-of-care testing, contributing to epidemic prevention and control.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117221", "ptypes": [ "Journal Article" ], "first_author": "Liu S", "dirs": [ "A", "C" ] }, "42407157": { "pmid": "42407157", "title": "CRISPR-based diagnostics for ESKAPE drug-resistant bacteria: From proof-of-concept to point-of-care.", "abstract": "The ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, represent a major challenge in the antimicrobial resistance (AMR) crisis. These pathogens are progressively acquiring pan-resistance and spreading beyond traditional healthcare settings, yet conventional diagnostic methods remain ill-equipped for point-of-care (POC) deployment due to slow processing times and limited adaptability. CRISPR/Cas systems feature programmable target specificity and intrinsic signal amplification, enabling rapid and accurate nucleic acid detection. Despite these advantages, the translation of CRISPR-based assays for ESKAPE pathogens from proof-of-concept to practical POC tools faces major challenges. Current research is fragmented, and key trade-offs between sensitivity, multiplexing performance and operational simplicity have not been fully addressed. This review offers a critical assessment of the field, moving beyond a simple summary of existing studies to analyze how various CRISPR systems (Cas9, Cas12, Cas13, and Cas14) and amplification strategies address the demands of POC testing. We identify key barriers to clinical application, particularly sample preparation, multiplex detection, reagent stability, and discuss emerging solutions such as microfluidic integration, lyophilized reagents development, and artificial intelligence-driven data interpretation. By focusing on the central question of how to transition from benchtop research to bedside application, this review provides a strategic framework for advancing next-generation CRISPR diagnostics capable of rapid, precise, and real-time detection of drug-resistant ESKAPE pathogens in the fight against AMR.", "journal": "Talanta", "year": "2027", "doi": "10.1016/j.talanta.2026.130240", "ptypes": [ "Journal Article", "Review" ], "first_author": "Mu Y", "dirs": [ "A", "D" ] }, "40128193": { "pmid": "40128193", "title": "Portable molecular diagnostic platform for rapid point-of-care detection of mpox and other diseases.", "abstract": "The World Health Organization's designation of mpox as a public health emergency of international concern in August 2024 underscores the urgent need for effective diagnostic solutions to combat this escalating threat. The rapid global spread of clade II mpox, coupled with the sustained human-to-human transmission of the more virulent clade I mpox in the Democratic Republic of Congo, highlights a critical gap in point-of-care diagnostics for this emergent disease. In response, we developed Dragonfly, a portable molecular diagnostic platform for point-of-care use that integrates power-free nucleic acid extraction (<5 minutes) with lyophilised colourimetric LAMP chemistry. The platform demonstrated an analytical limit-of-detection of 100 genome copies per reaction for monkeypox virus, effectively distinguishing it from other orthopoxviruses, herpes simplex virus, and varicella-zoster virus. Clinical validation on 164 samples, including 51 mpox-positive cases, yielded 96.1% sensitivity and 100% specificity for orthopoxviruses, and 94.1% sensitivity and 100% specificity for monkeypox virus. Here, we present a rapid, accessible, and robust point-of-care diagnostic solution for mpox, suitable for both low- and high-resource settings, addressing the global resurgence of orthopoxviruses in the context of declining smallpox immunity.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-57647-3", "ptypes": [ "Journal Article" ], "first_author": "Cavuto ML", "dirs": [ "A" ] }, "42639630": { "pmid": "42639630", "title": "A field-deployable CRISPR-based biosensing platform for monitoring marine ecosystems.", "abstract": "Ocean ecosystems are undergoing accelerating disruption from human impacts such as climate change. Warming ocean temperatures drive pathogenic outbreaks, increase harmful algal blooms, and cause coral stress. These can have serious consequences for marine ecosystems, human health and the aquaculture industry, representing a critical One Health issue. Monitoring key marine species offers valuable insights, but current methods are resource-intensive, low-resolution, and unsuitable for frequent deployment. Here, we introduce a low-cost, field-deployable CRISPR biosensing platform for detecting marine organismal DNA and RNA. Harnessing the programmability of CRISPR diagnostics for environmental biosurveillance, we demonstrate versatility across three climate-linked indicators: Vibrio spp., Pseudo nitzschia spp., and heat-stressed corals. Portable 3D-printed processor and incubator devices enable direct processing of filter-captured samples with temperature control. Field-readiness is reinforced by lyophilized reagents, lateral-flow readouts, dropper-based handling, and a two-step multiplexed workflow, delivering results within one hour without laboratory instruments. Benchmarking with authentic pathogens and environmental seawater confirmed seawater tolerance and robust detection of 108 CFU/filter Vibrio pathogens, equivalent to 102 cp/µL for a 1L filtered sample. This decentralized platform reduces barriers to routine monitoring and can provide early warnings of ecosystem disturbances, whilst supporting One Health initiatives in the marine space.", "journal": "Nature sustainability", "year": "2026", "doi": "10.1038/s41893-025-01752-0", "ptypes": [ "Journal Article" ], "first_author": "Kim N", "dirs": [ "A" ] }, "41590284": { "pmid": "41590284", "title": "Integrated Colorimetric CRISPR/Cas12a Detection of Double-Stranded DNA on Microfluidic Paper-Based Analytical Devices.", "abstract": "Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a's trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV.", "journal": "Biosensors", "year": "2026", "doi": "10.3390/bios16010032", "ptypes": [ "Journal Article" ], "first_author": "Zhang Z", "dirs": [ "A" ] }, "41444344": { "pmid": "41444344", "title": "A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses.", "abstract": "In recent years, viral co-infections, particularly with respiratory viruses, have resulted in more complex symptoms, a greater disease burden, and increased challenges in clinical decision-making. These complexities underscore the urgent need for improved diagnostic tools in the managing acute respiratory infections. To address the limitations of conventional qPCR and current POCT methodologies, we developed a passively driven microfluidic chip capable of rapidly screening multiple respiratory viruses. This platform is particularly suited for the point-of-care diagnosis of viral co-infections. Our device integrates nucleic acid amplification and CRISPR-based detection within a single, passively operated system. By utilizing a rapid, 10-minute sample preparation protocol and a 35-minute on-chip assay, this platform enables the multiplex detection of influenza A/B, human parainfluenza virus, and SARS-CoV-2. The total assay time from sample to answer is approximately 45 min, with equipment requirements minimized to a heating block. The assay demonstrated a detection sensitivity of about 10 copies/µL for viral RNA in dilution series experiments. The sensitivity of the assay was 98.44% (95% CI: 91.6%-99.96%), and the specificity was 100% (95% CI: 79.4%-100%). The system combines CRISPR-Cas12a-mediated sensing with reverse transcription recombinase polymerase amplification (RPA) for highly specific nucleic acid detection. The chip design utilizes capillary action and gravity-driven flow for autonomous fluid control, while lyophilized reagent preloading ensures storage stability and minimizes user intervention.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-31658-y", "ptypes": [ "Journal Article" ], "first_author": "Li X", "dirs": [ "A", "D" ] }, "40337838": { "pmid": "40337838", "title": "Handheld RPA-based molecular POCT system for rapid, low-cost 8-plexed detection of respiratory pathogens at home.", "abstract": "During seasonal influenza or emerging respiratory outbreaks, rapid home-based multiplex molecular point-of-care testing (POCT) for respiratory pathogens is crucial for early diagnosis and intervention, particularly in vulnerable populations. However, existing POCT systems, primarily designed for clinical settings, are often too complex, costly, and reliant on trained operators, limiting their suitability for home use. To overcome these barriers, we introduce a microfluidic cartridge-based system leveraging recombinase polymerase amplification (RPA) for multiplexed detection of respiratory pathogens in home environments. The microfluidic cartridge is designed with three parallel channels-each integrating a lysis chamber, an RPA chamber preloaded with lyophilized reagents, and an air storage chamber. Each detection channel enables extraction-free, single-channel 3-plex RPA assays, and by combining three-channel parallel detection, the system achieves simultaneous identification of eight respiratory pathogens and one internal control in under 25 min. A novel pneumatic pressure pumping strategy ensures precise flow control through dynamic bladder compression, paired with microchannel hydraulic resistance matching to guarantee uniform volumetric distribution and synchronized flow across all channels. Furthermore, a dynamic mixing method promotes homogeneous mixing of RPA reagents with lysed samples via a bidirectional flow between the lysis and RPA chambers, enhancing assay reliability. Our microfluidic design enables significant miniaturization, yielding a compact, lightweight system (<1 kg) suitable for handheld or desktop use. Its low power consumption (3 W) and remarkable cost-effectiveness ($1.4 per test) enhance the system's practicality and accessibility for home settings. Validation with 356 nasopharyngeal swabs further confirms its robust performance, achieving high sensitivity (>97%) and specificity (>99%), ensuring reliable at-home diagnosis of respiratory co-infections without requiring professional operation.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d5lc00343a", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zai Y", "dirs": [ "A", "D" ] }, "42546384": { "pmid": "42546384", "title": "An integrated centrifugal microfluidic CRISPR-based diagnostics platform for multiplexed point-of-care testing of respiratory pathogens.", "abstract": "Rapid, accurate, and multiplexed point-of-care testing (POCT) of respiratory pathogens is critical for clinical triage and infection control. However, existing platforms frequently necessitate trade-offs between sensitivity, throughput, and operational complexity. To address this, we developed the iCARD (integrated Centrifugal Assay on a Rotating Disc) platform, a streamlined microfluidic molecular diagnostic system based on single-step CRISPR kinetics and centrifugal microfluidics. Comprising a polymethyl methacrylate disc pre-loaded with lyophilized CRISPR/Cas13a reagents and a self-developed portable fluorescence analyzer, the platform enables automated multiplexed detection following sample loading. Furthermore, a novel resin-based pretreatment method was engineered to efficiently purify and concentrate nucleic acids from throat swabs. The iCARD system facilitates the parallel screening of six respiratory pathogens across four independent samples within 40 min. Analytical validation confirmed single-copy sensitivity (limit of detection: 0.25-1.0 copies/μL) without cross-reactivity. Clinical validation using 94 retrospective patient throat swabs demonstrated exceptional diagnostic accuracy, achieving a sensitivity of 95.5% (64/67; 95% CI: 87.64%-98.47%) and a specificity of 100.0% (27/27; 95% CI: 87.51%-100.00%). These findings demonstrate that the iCARD platform serves as a robust, high-throughput, and accurate diagnostic tool for decentralized molecular screening and epidemiological surveillance.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.119083", "ptypes": [ "Journal Article" ], "first_author": "Li L", "dirs": [ "A" ] }, "40150790": { "pmid": "40150790", "title": "Multiple-Channel Funnel Adapted Sensing Tube (MFAST) for the Simple and Duplex Detection of Parasites.", "abstract": "Leishmaniasis poses a significant global health threat, infecting millions of people annually, particularly in tropical and subtropical regions. Timely and accurate detection of the Leishmania species is crucial for effective treatment and control of this debilitating disease. This study introduces the multi-channel funnel adapted sensing tube (MFAST) chip, a simple diagnostic tool designed for the rapid detection of Leishmania panamensis. MFAST is fabricated through 3D printing and sacrificial molding of acrylonitrile butadiene styrene (ABS) and the reagents are transported between reservoirs by gravity. We combine experiments and finite element analysis to facilitate smoother fluid flow, improving the overall performance of the device. Highly sensitive and specific RPA-CRISPR/Cas12a assay is utilized in the chip, achieving a detection limit as low as 1000 parasites/mL (detecting as few as 5 parasites per reaction). The multichannel design enables duplex detection, allowing for simultaneous identification of both L. braziliensis and L. panamensis through distinct channels. Furthermore, stability tests indicate that lyophilized reagents retain functionality for up to 15 days when stored at 4 °C, underscoring the potential of this chip for practical diagnostic applications in low-resource settings.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.4c03306", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, N.I.H., Extramural", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Peng R", "dirs": [ "A" ] }, "40034782": { "pmid": "40034782", "title": "A Streamlined Point-of-Care CRISPR Test for Tuberculosis Detection Directly from Sputum.", "abstract": "Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (105 to 102 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.", "journal": "medRxiv : the preprint server for health sciences", "year": "2025", "doi": "10.1101/2025.02.19.25322517", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Dunkley ORS", "dirs": [ "A" ] }, "39488143": { "pmid": "39488143", "title": "Towards the identification of body fluids using RT-LAMP isothermal amplification coupled with CRISPR-Cas12a.", "abstract": "While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative technique that enables detection of mRNA at a single temperature (usually 60-65℃) for 10-30 minutes and has comparable sensitivity to PCR. We describe the coupling of RT-LAMP amplification (60℃ for 30 minutes) with CRISPR-mediated fluorescent detection of the body fluid specific mRNA markers MMP3 (menstrual fluid), CYP2B7P (vaginal material), TNP1 (spermatozoa), KLK2 (semen), and MUC12 (rectal mucosa). Following temperature optimization and final selection of RT-LAMP-CRISPR assays, their specificity across circulatory blood, buccal, menstrual fluid, vaginal material, semen, and rectal mucosa was assessed. Most assays were specific for their intended target body fluid, although MMP3 and CYP2B7P were detected in some rectal mucosa samples, the latter of which has been observed previously in the literature. A preliminary sensitivity assessment in target fluids was determined by a dilution series over six logs of RNA input. A range of assay approaches were investigated to develop a protocol suitable for use in a forensic screening laboratory. This included the determination of fluorescent assay results by eye, use of lyophilised reagents, and RT-LAMP and CRISPR reactions undertaken in one-tube in a lower resource setting.", "journal": "Forensic science international. Genetics", "year": "2025", "doi": "10.1016/j.fsigen.2024.103167", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Lynch CRH", "dirs": [ "A" ] }, "42618136": { "pmid": "42618136", "title": "A lyophilized RPA-CRISPR/Cas13d one-pot platform for rapid detection of porcine circovirus type 3.", "abstract": "Rapid molecular diagnostics are essential for effective surveillance of infectious diseases in swine production systems. Here, we report SHARP (Single-step Hybrid RPA-CRISPR/EsCas13d Platform), a one-pot CRISPR-based platform for detection of porcine circovirus type 3 (PCV3). By integrating rapid nucleic acid release with CRISPR/Cas13d detection, a single-step one-pot detection system was established, enabling detection within 30 min under simplified reaction conditions. In the visual readout mode, the detection limit was 50 copies/μL, with no cross-reactivity observed against common swine viruses. After lyophilization and rehydration, the SHARP system showed consistent detection performance across 50 clinical samples, in agreement with qPCR results. These results indicate that SHARP provides a simplified workflow for rapid PCV3 detection, with potential for decentralized molecular surveillance in livestock production systems.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345832", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Shao LN", "dirs": [ "A" ] }, "39754554": { "pmid": "39754554", "title": "Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.", "abstract": "Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve \"sample-in-answer-out\" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.4c05026", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Hu F", "dirs": [ "A" ] }, "40832287": { "pmid": "40832287", "title": "A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection.", "abstract": "Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. During the COVID-19 pandemic, we developed Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based technology for nucleic acid detection. Here, we introduce microfluidic SHINE (mSHINE) which enables >100-plex nucleic acid detection using a highly portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized SHINE reagents and a microscopic stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. A subsequent sealing step allows for >100 SHINE reactions to proceed independently and in parallel. We demonstrate that mSHINE has equal sensitivity to SHINE, allowing for highly multiplexed pathogen detection in ≤ 1 hour. In addition, mSHINE can detect single-nucleotide variants, including mutations associated with drug susceptibility. mSHINE shifts the paradigm of laboratory-based multiplexed nucleic acid testing, greatly benefiting patients and public health.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.08.11.669785", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Kramer S", "dirs": [ "A" ] }, "40872937": { "pmid": "40872937", "title": "Freeze-Drying of mRNA-LNPs Vaccines: A Review.", "abstract": "The instability of mRNA vaccines presents significant challenges for their storage, transportation, and large-scale distribution, particularly in resource-limited countries. Recently, freeze-drying (lyophilization) has been considered as a promising approach for preserving mRNA vaccine efficacy. This formulation technique enhances the long-term stability of mRNA vaccines by converting them into a stable dry powder. The purpose of this review is to provide an overview of the current knowledge on the progress of freeze-drying techniques for mRNA vaccines, with emphasis on the associated challenges. This review highlights the factors influencing the stability of freeze-dried mRNA vaccines and provides a comprehensive overview of the formulation components, including excipients, buffers, and surfactants, as well as the process parameters and storage conditions that aim to improve stability and shelf-life. By providing these insights, this review supports the advancement of more robust, scalable, and efficient lyophilization protocols, ultimately addressing the stability limitations of mRNA vaccines and enhancing their global accessibility.", "journal": "Vaccines", "year": "2025", "doi": "10.3390/vaccines13080853", "ptypes": [ "Journal Article", "Review" ], "first_author": "Khan MDFH", "dirs": [ "A" ] }, "39875036": { "pmid": "39875036", "title": "Formulation screening of lyophilized mRNA-lipid nanoparticles.", "abstract": "Lipid nanoparticles (LNPs) have demonstrated their therapeutic potential as safe and effective drug delivery systems for nucleic acids during the COVID-19 pandemic. However, one of the main challenges during technical CMC (Chemistry, Manufacturing, and Controls) development is their long-term stability at temperatures of 2-8 °C or higher, which may be improved by the removal of water by lyophilization. In this study, we identified lyo-/cryo-protectants for freeze-dried mRNA-LNP formulations beyond conventional excipients such as sucrose and trehalose as Tg-modifiers using polyA as a surrogate. Hydroxypropyl-beta-cyclodextrin, Kollidon® 12 PF (PVP), and dextran 40 kDa were tested in combinations to best stabilize the mRNA-LNPs during the lyophilization process as well as during storage for up to 6 months at 2-8 °C, 25 °C/60 % r.h., and 40 °C/75 % r.h.. We also tested the formulation principle including protectants in- and outside of the LNPs. Formulations were assessed for size, PDI, encapsulation efficiency, and properties related to the lyophilized dosage form. While 10 % (w/V) sucrose formulations successfully stabilized LNPs during the lyophilization process, they were not suitable for storage at temperatures beyond 2-8 °C. The most promising formulations for storage at higher temperatures were identified as 9 % (w/V) trehalose + 1 % (w/V) PVP with only a small increase in size over 6 months at 25 °C maintaining PDI and encapsulation efficiency. Results were verified with eGFP-mRNA-LNPs and tested in cell culture experiments. This study may serve as guidance for formulation scientists to further optimize freeze-dried mRNA-LNP formulations and eventually eliminate the cold chain for mRNA-LNP products.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125272", "ptypes": [ "Journal Article" ], "first_author": "Ruppl A", "dirs": [ "A" ] }, "41331990": { "pmid": "41331990", "title": "Not so cold! Improving the thermostability of mRNA vaccines.", "abstract": "One of the biggest challenges in the mRNA-LNP vaccine field is product stabilization to overcome the logistical hurdles linked to the ultra-cold distribution chain associated with first-generation mRNA SARS-CoV-2 vaccines. Despite recent progress in the field, many R&D efforts remain focused on the development of mRNA-LNP vaccines that would be as stable as liquid formulations for storage at refrigerated or room temperatures. After an overview of the underlying mechanisms of mRNA-LNP instability, this review provides an update on the different approaches that are currently explored to improve mRNA-LNP thermostability, encompassing mRNA sequence optimization, nucleotide modification and mRNA-LNP design strategies as well as formulation process optimization. Alternative approaches for mRNA-LNP stabilization such as lyophilization, dual-vial formulations and the replacement of water with deep eutectic solvents in the mRNA-LNP process and products are also discussed. Achieving robust thermostability of mRNA vaccines will require a multifactorial optimization strategy, integrating advances in sequence engineering, novel formulation designs, buffer composition, excipient selection and manufacturing processes.", "journal": "Expert review of vaccines", "year": "2025", "doi": "10.1080/14760584.2025.2596674", "ptypes": [ "Journal Article", "Review" ], "first_author": "Haensler J", "dirs": [ "A" ] }, "39839540": { "pmid": "39839540", "title": "Characterization of Amorphous Ibrutinib Thermal Stability.", "abstract": "The choice of method for drug amorphization depends on various factors, including the physicochemical properties of the active pharmaceutical ingredients, the desired formulation, and scalability requirements. It is often important to consider a combination of methods or the use of excipients to further enhance the stability and performance of the amorphous drug. This study presents a comparison of techniques including melt quench, hot melt extrusion, solvent evaporation, ball milling, and lyophilization used for the preparation of amorphous ibrutinib. The amorphous material was thoroughly investigated using numerous techniques to examine changes in the physicochemical properties, stability, and degradation pathways of the drug product. During the examination, the temperature was discovered to be a key parameter for controlling the solubility and permeability of ibrutinib, which is influenced by the presence of the degradation product. We found that this degradation product could potentially polymerize and increase the molecular weight. The quantity, polymerization rate, and structure of the impurity can be regulated by the temperature variation during the amorphization processes. Additionally, the molecular weight of the degradation product was determined using Zimm plot analysis, which appeared for the first time in the literature for molecules of this category.", "journal": "Organic process research & development", "year": "2025", "doi": "10.1021/acs.oprd.4c00299", "ptypes": [ "Journal Article" ], "first_author": "Trunov D", "dirs": [ "A" ] }, "42350438": { "pmid": "42350438", "title": "Next-generation mRNA vaccines: strategies to overcome stability challenges.", "abstract": "Messenger RNA (mRNA) vaccines represent a paradigm shift in rapid vaccine development and pandemic preparedness, enabling unprecedented speed in antigen design and scalable, cell-free manufacturing. Despite proven clinical efficacy, widespread mRNA vaccine deployment remains critically constrained by fundamental molecular instability requiring ultra-cold chain storage. The chemical lability of the RNA phosphodiester backbone, 2'-hydroxyl-mediated transesterification, oxidative nucleobase damage, and lipid nanoparticle (LNP) structural vulnerabilities collectively limit long-term stability and the feasibility of global distribution. This review provides a mechanistic analysis of RNA hydrolysis, oxidative degradation, lipid peroxidation, PEG-lipid desorption, and freeze-thaw-induced nanoparticle aggregation, linking molecular instability to translational challenges in storage and supply chain logistics. We critically evaluate engineering strategies to enhance stability, including optimized LNP architectures, lyophilization with vitrifying excipients, antioxidant incorporation, lipid-polymer hybrid nanocarriers, hydrogel-based depots, self-amplifying mRNA (saRNA), and circular RNA (circRNA) constructs. Stability modeling, cold-chain risk analysis, and regulatory Chemistry, Manufacturing, and Controls (CMC) considerations are discussed to contextualize formulation robustness within real-world distribution systems. Advancing mRNA vaccines beyond ultra-cold dependence will require coordinated innovation in RNA engineering, delivery system optimization, and scalable manufacturing to enable thermostable, resilient, and globally deployable vaccine technologies.", "journal": "NPJ vaccines", "year": "2026", "doi": "10.1038/s41541-026-01502-z", "ptypes": [ "Journal Article" ], "first_author": "Algaissi A", "dirs": [ "A" ] }, "39617053": { "pmid": "39617053", "title": "Molecular mechanisms for stabilizing biologics in the solid state.", "abstract": "Protein drugs exhibit challenges of biophysical and biochemical instability due to their structural complexity and rich dynamics. Solid-state biologics aim to enhance stability by increasing molecular rigidity within the formulation matrix, representing a primary category of drug products alongside sterile liquid formulations. Understanding the molecular mechanisms behind the stabilization and destabilization of protein drugs, influenced by formulation composition and drying processes, provides scientific rationale for drug product design. This review aims to elaborate on the two primary models of water-to-sugar substitution and matrix vitrification, respectively, via thermodynamic and kinetic stabilization. It offers an up-to-date review of experimental investigations into these hypotheses, specifically elucidating protein structure and protein-excipient interactions at the molecular level, molecular dynamics across a broad range of motion regimes, and microscopic attributes such as protein-sugar and protein-salt miscibility and microenvironmental acidity, in relevant liquid, frozen, and solid states, using advanced biophysical techniques for solid-state analysis. Moreover, we discuss how these mechanistic understandings facilitate the investigation and prediction of critical stability behaviors and enables the design of solid biological drug products.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2024.11.017", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ling J", "dirs": [ "A" ] }, "40021008": { "pmid": "40021008", "title": "Strategies of formulation and lyophilization process for sodium chloride-mannitol-protein-based products.", "abstract": "Sodium chloride (NaCl) serves as a necessary stabilizer for some proteins, yet it presents an unfriendly eutectic melting temperature and can easily lead to collapse for lyophilized products. Mannitol, serving as bulking agent with property of high eutectic melting point, can provide good mechanical support for lyophilized cake to avoid collapse, therefore it is applied for lyophilized formulations containing NaCl. However, the combination of NaCl and mannitol in formulations often results in structural collapse in lyophilization process due to unproper ratio or lyophilization recipe. In this study, the strategy to improve cake appearance with formulations containing NaCl and mannitol has been explored. The suitable lyophilization formulation compositions and lyophilization process parameters have been proposed with acceptable protein stability. Furthermore, the possibility of mutual inhibition by NaCl and mannitol crystallization is revealed by result of XRD, in which various mannitol crystal forms are observed. It is speculated that protein concentration and lyophilization process parameters are main factors to affect mannitol crystal forms. Our studies not only provide insights into the crystallization behaviors in NaCl-mannitol-protein-based formulations, but also find the strategies to improve the appearance of their lyophilized biopharmaceuticals, facilitating their widespread applications in pharmaceutical development.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2025.01.007", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Lin C", "dirs": [ "A" ] }, "41155963": { "pmid": "41155963", "title": "Short Peptides as Excipients in Parenteral Protein Formulations: A Mini Review.", "abstract": "Biopharmaceutical medicines represent one of the most dynamic sectors of the pharmaceutical industry, with therapeutic proteins forming the largest and most important group. Their structural complexity and inherent sensitivity to chemical and physical stressors, however, continue to pose major challenges for formulation development and long-term stability. Short peptides have emerged as a promising yet underutilized class of excipients for protein-based drug products. Their modular architecture allows for precise tuning of physicochemical properties such as polarity, charge distribution, and hydrogen-bonding potential, thereby offering advantages over single amino acids. Experimental studies indicate that short peptides can serve multiple functions: stabilizers, antioxidants, viscosity-lowering agents, and as lyo/cryoprotectants or bulking agents in lyophilized formulations. Notably, the relatively small and chemically defined space of short peptides-approximately 400 possible dipeptides and 8000 tripeptides-makes them particularly amenable to systematic screening and computational modeling. This enables rational identification of candidates with tailored excipient functions. This review summarizes current knowledge on the use of short peptides as excipients in parenteral protein formulations, with a focus on their functional versatility and potential for rational design in future development.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17101328", "ptypes": [ "Journal Article", "Review" ], "first_author": "Migoń D", "dirs": [ "A" ] }, "41126008": { "pmid": "41126008", "title": "Developing Subcutaneous Formulations for Irisin as a Potential Therapeutic.", "abstract": "Irisin, a hormone secreted by skeletal muscle during physical exercise, shows promise for health benefits. However, limited research on its characterization and formulation development has impeded its pharmaceutical development. This study aimed to develop fit-for-purpose subcutaneous formulations for irisin. Irisin was expressed in E.coli and purified. The prepared irisin was characterized using orthogonal biophysical techniques, and its biological activity was assessed. Subcutaneous formulations were then developed by screening pH, excipients, and formats (lyophilized vs. aqueous), and the product stability profiles were investigated under various conditions. Irisin was successfully expressed in both soluble and refolded forms, and after purification, both forms displayed similar biophysical properties and induced adipocyte browning. Lyophilized formulations, incorporating phosphate buffer (pH 7), sucrose or trehalose, and polysorbate 80, preserved irisin's properties and bioactivity after lyophilization and could be rapidly reconstituted for subcutaneous administration. The formulations also remained stable at 40°C/75% RH for at least one month. These findings lay a foundation for advancing irisin-based therapies for obesity and related diseases.", "journal": "Pharmaceutical research", "year": "2025", "doi": "10.1007/s11095-025-03945-w", "ptypes": [ "Journal Article" ], "first_author": "Hu G", "dirs": [ "A" ] }, "39827914": { "pmid": "39827914", "title": "Formulation development and feasibility of AAV5 as a lyophilized drug product.", "abstract": "The majority of adeno-associated virus (AAV) gene therapies are currently developed as frozen formulations (e.g., ≤ - 60 °C) that are challenging to maintain and distribute world-wide. Lyophilization can allow for long-term refrigerated storage and improved shelf-life that lowers long-term cost. Here, we performed a lyophilization feasibility study to assess the ability of several different excipients to stabilize AAV5 during lyophilization and on storage stability. A range of biophysical techniques were used to assess capsid integrity on a molecular level including quantification of externalized DNA, capsid particle size, and capsid monomer percent area. Additionally, transmission electron microscopy was used for the first time to monitor the size and integrity of the capsids subjected to the lyophilization process, and the results supported other characterization methods used in this study. A formulation containing hydroxyectoine and trehalose stabilized capsid structure directly after lyophilization, as observed directly by 5.0 % of internally stained capsids (empty) and indirectly with 7.5 % external DNA. A recombinant human albumin and trehalose formulation stabilized capsid structure on stability as observed by improved external DNA and monomer profiles overtime. Adversely, mannitol crystallization negatively affected capsid structure. Our findings indicate that lyophilization is a viable option to frozen formulation for stabilizing AAV5 drug products.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2025.01.004", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Vargas SK", "dirs": [ "A" ] }, "39028537": { "pmid": "39028537", "title": "Optimization of lyoprotectant for recombinant human acidic fibroblast growth factor by response surface methodology.", "abstract": "Recombinant human acidic fibroblast growth factor (rh-aFGF) is a widely used biological product, but it is unstable and its biological activity is easy to decrease. In order to maintain the long-term stability and biological activity of rh-aFGF, based on the response surface method, the freeze-drying characterization and cell proliferation rate of rh-aFGF freeze-dried powder were evaluated by scoring and Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in this study. The optimal concentrations of trehalose, glycine and BSA were optimized, and the optimal formulation was verified by regression experiment. The results showed that trehalose, glycine and BSA had significant effects on the characterization of lyophilized rh-aFGF and cell proliferation. The optimal formulation of 5.7% trehalose, 2.04% glycine and 1.98%BSA combined with rh-aFGF could achieve the optimal freeze-dried characterization and biological activity. Using the best formulation to verify, the freeze-dried formability index of the freeze-dried powder was 23.35, and the rate of cell proliferation was 43.59%, which was close to the expected 23 and 41.69%. This study determined a freeze-dried formulation of rh-aFGF that meets the requirements of freeze-dried formalization integrity and maintains biological activity, providing reliable support for the subsequent development of related drugs.", "journal": "Preparative biochemistry & biotechnology", "year": "2025", "doi": "10.1080/10826068.2024.2378098", "ptypes": [ "Journal Article" ], "first_author": "Huang Q", "dirs": [ "A" ] }, "41118408": { "pmid": "41118408", "title": "Preparation, characterization and in vitro evaluation of atorvastatin nanosuspensions.", "abstract": "Poorly water-soluble drugs present a significant challenge for pharmaceutical development, particularly affecting the pharmacokinetics of orally administered drugs due to their poor dissolution. This study aimed to enhance the dissolution of a low water solubility drug atorvastatin using nanosuspension technology. Antisolvent technique was utilized to prepare atorvastatin calcium nanosuspensions. Different stabilizers were used including Cremophor, HPMC, pluronics (F127, F108, and F68), PEG400, PEG600, PEG 1k, PEG8k, PVA, PVP k30, PVP k10, PVP 44k, SLS, sodium alginate, Tween 20, and Tween 80. The prepared nanosuspensions were lyophilized using mannitol or trehalose as a lyoprotectant. Several optimum formulations were obtained. The selected best optimum formulation was 2% pluronic F127, 80 mg mannitol, and 2 min sonication time. It exhibited a mean particle size of 54.5 nm, a zeta potential of -0.809, and 0.141 PDI after reconstitution. The crystalline state of the nanoparticles was evaluated using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Fourier-transform infrared spectroscopy (FTIR) was used to assess interactions between the drug substance and the additives. In vitro drug release study was conducted for lyophilized nanosuspension in comparison to the innovator product Lipitor® in two different media, 0.05M potassium phosphate buffer pH 6.8 and 0.1N HCL. The XRD analyses indicated that the lyophilized nanosuspension was partially crystalline with some amorphization and this confirmed by DSC. FTIR results suggested that there was a physical interaction between atorvastatin and the additives. The polymer and lyoprotectant successfully preserved and coated the drug nanoparticles. The lyophilized nanosuspension exhibited superior dissolution in 0.1N HCl. However, the innovator was faster in 0.05 M phosphate buffer. In conclusion, successful preparation of lyophilized nanosuspension of atorvastatin calcium was achieved. The current findings revealed that excipient functionality in terms of stabilization, effect on solid state properties, and drug release were critical for development of stable and efficient nanosuspension.", "journal": "PloS one", "year": "2025", "doi": "10.1371/journal.pone.0335024", "ptypes": [ "Journal Article" ], "first_author": "Akour YA", "dirs": [ "A" ] }, "40881288": { "pmid": "40881288", "title": "Standardized freeze-dried FMT: is the ideal protectant out there?", "abstract": "Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infections. Freeze-drying offers a next-generation, more practical, and aesthetically acceptable FMT formulation that could facilitate standardized preparation methods. Viable preservation is a critical step in freeze-drying, yet no universal medium effectively protects both anaerobes and aerobes. This study aimed to evaluate different protectants compared to trehalose 5% (T5) after confirming its efficacy. A mix of inulin and glucosamine (IG5) and a High-antioxidant Matrix with trehalose (HM) were tested. Viability was assessed using colony-forming unit (CFU) enumeration and flow cytometry with a LIVE/DEAD™ staining method. T5 demonstrated satisfactory bacterial recovery after freeze-drying, with viability of 84 ± 28% for anaerobes and 59 ± 39% for Bifidobacterium (BIF), confirming its efficiency in our preparation facilities. While HM showed highest results (91 ± 7% for anaerobes, 121 ± 33% for BIF), it did not significantly outperform T5. IG5, however, resulted in a significant loss of bacteria, with only 16 ± 12% viability for anaerobes (p = 0.016) and 19 ± 9% for BIF (p = 0.031). HM and T5 both proved effective for freeze-dried FMT, with HM yielding the highest recovery but not significantly outperforming T5. Given its simplicity and consistent results, T5 may serve as a reliable standalone protectant or as a base for improved formulations. IG5 showed significant bacterial loss and is unsuitable. Further biological validation and stability data will guide the development of optimized freeze-dried oral FMT capsules.", "journal": "Frontiers in microbiology", "year": "2025", "doi": "10.3389/fmicb.2025.1618067", "ptypes": [ "Journal Article" ], "first_author": "Pourrat A", "dirs": [ "A" ] }, "42450048": { "pmid": "42450048", "title": "Intravenous Everolimus Formulation (Sapu003) for Clinical Trials.", "abstract": "Everolimus is approved for the treatment of advanced renal cell carcinoma after VEGF-targeted therapy, metastatic HR-positive/HER2-negative breast cancer in combination with exemestane, and other oncologic indications. However, an intravenous option has not been developed, largely due to its pronounced hydrophobicity and limited oral bioavailability of approximately 15-20%. In this study, we report the development of Sapu003, a novel intravenous Everolimus formulation enabled through the Deciparticle™ platform. A diverse library of mPEG-based block copolymers was evaluated for their ability to encapsulate Everolimus and self-assemble into stable nanoparticle structures. mPEG-Chol was ultimately selected based on its favorable biocompatibility characteristics. In addition to Everolimus, mPEG-Chol and related analogs demonstrated broad formulation compatibility with multiple hydrophobic therapeutics, including Sirolimus, Tacrolimus, Cyclosporine, as well as representative peptides and polyketides. Clinical manufacturing was conducted in a cGMP environment over a 7-day production cycle. Production was carried out under amber light using light-protective vials to reduce drug degradation. The bulk material was sterile-filtered, and subsequent fill/finish/lyophilization operations were performed under temperature-controlled conditions with high precision in fill accuracy (≥98%). After reconstitution, the final product yielding uniform Deciparticles™ that met predefined sterility and particle size criteria. Stability studies demonstrated that the formulation remained stable for at least one month at 5 °C and retained acceptable in-use stability for at least 24 h at room temperature. The process was successfully scaled beyond 10 g, supporting an ongoing Phase 1b open-label dose escalation clinical study of Sapu003 in combination with exemestane in patients with advanced mTOR-sensitive solid tumors (NCT07369505). In vivo evaluation demonstrated strong antitumor efficacy following intravenous administration (QW × 3), with tumor growth inhibition reaching 97-98% in the U-87MG glioblastoma xenograft model. No evidence of phlebitis was observed with repeated tail vein dosing. In this model, Sapu003 dosed weekly showed superior tumor suppression compared with oral Everolimus. Collectively, screening of a mPEG-block copolymer library identified mPEG-Chol as a lead excipient capable of consistently forming stable Deciparticles™ with sub-20 nm mean particle size. The resulting intravenous Everolimus formulation demonstrated scalable manufacturing, favorable stability, and potent antitumor activity in preclinical models, supporting further clinical evaluation of Sapu003 in advanced solid tumors.", "journal": "International journal of molecular sciences", "year": "2026", "doi": "10.3390/ijms27135775", "ptypes": [ "Journal Article" ], "first_author": "Min SH", "dirs": [ "A" ] }, "40430843": { "pmid": "40430843", "title": "A Review on the Stability Challenges of Advanced Biologic Therapeutics.", "abstract": "Advanced biotherapeutic systems such as gene therapy, mRNA lipid nanoparticles, antibody-drug conjugates, fusion proteins, and cell therapy have proven to be promising platforms for delivering targeted biologic therapeutics. Preserving the intrinsic stability of these advanced therapeutics is essential to maintain their innate structure, functionality, and shelf life. Nevertheless, various challenges and obstacles arise during formulation development and throughout the storage period due to their complex nature and sensitivity to various stress factors. Key stability concerns include physical degradation and chemical instability due to various factors such as fluctuations in pH and temperature, which results in conformational and colloidal instabilities of the biologics, adversely affecting their quality and therapeutic efficacy. This review emphasizes key stability issues associated with these advanced biotherapeutic systems and approaches to identify and overcome them. In gene therapy, the brittleness of viral vectors and gene encapsulation limits their stability, requiring the use of stabilizers, excipients, and lyophilization. Keeping cells viable throughout the whole cell therapy process, from culture to final formulation, is still a major difficulty. In mRNA therapeutics, stabilization strategies such as the optimization of mRNA nucleotides and lipid compositions are used to address the instability of both the mRNA and lipid nanoparticles. Monoclonal antibodies are colloidally and conformationally unstable. Hence, buffers and stabilizers are useful to maintain stability. Although fusion proteins and monoclonal antibodies share structural similarities, they show a similar pattern of instability. Antibody-drug conjugates possess issues with conjugation and linker stability. This review outlines the stability issues associated with advanced biotherapeutics and provides insights into the approaches to address these challenges.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17050550", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sarvepalli S", "dirs": [ "A" ] }, "41163552": { "pmid": "41163552", "title": "Monoclonal antibody formulations: a quantitative analysis of marketed products and patents.", "abstract": "Since the approval of OKT3® in 1986, monoclonal antibodies (mAbs) have become a cornerstone of modern therapeutics. However, their complex physicochemical properties pose challenges, particularly for high-concentration formulation and subcutaneous administration. Excipient selection is crucial for maintaining mAb stability and efficacy, yet existing studies often lack systematic, cross-source analyses. This study integrates data from marketed products and patents to investigate formulation trends and excipient preferences. Data were retrieved from the Drugs@FDA databases, CAS Formulations Database and Derwent Innovation as of December 31, 2024. Extracted information included target, indication, dosage form, route of administration, and formulation composition. The associations between formulation-related factors (e.g., antibody concentration, route of administration) and excipient selection were evaluated using proportion tests. A total of 6,119 patent records and 108 marketed mAb products (covering 388 patented and 141 marketed formulations) were analyzed. Proportion tests revealed significant associations between antibody concentration and the use of histidine (marketed p = 0.0017) and citric acid (marketed p = 0.0047). The route of administration also influenced excipient choice, notably for hyaluronidase (marketed p = 0.0167; patent p = 0.0056). In addition, lyophilized formulations accounted for a relatively small fraction of both marketed (14.18%) and patented (14.69%) products, with sucrose emerging as the predominant lyoprotectant. This study analyzed excipient usage in marketed and patent formulations by time, concentration, and administration route. High-concentration products more frequently included histidine, arginine, and hyaluronidase, while low-concentration ones used citric/phosphoric acid, trehalose, and NaCl. Intravenous formulations commonly used phosphate/citric buffers, while histidine, arginine, hyaluronidase, and methionine buffers were favored for subcutaneous administration. Lyophilized formulations consistently contained sucrose as the main excipient to mitigate freeze-drying stresses. Additionally, surfactants were essential across formulations to prevent surface-induced aggregation. Patent data could provide early indications of emerging formulation strategies, though further validation is needed to confirm their predictive value.", "journal": "mAbs", "year": "2025", "doi": "10.1080/19420862.2025.2580696", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Peng Y", "dirs": [ "A" ] }, "41780686": { "pmid": "41780686", "title": "A functional lyoprotectant platform enables storage-stable, mucus-penetrating siRNA delivery to the lung.", "abstract": "Respiratory diseases remain a major global health concern, where pathological mucus accumulation and chronic inflammation severely compromise lung function. RNA therapeutics have emerged as a transformative modality to address underlying molecular pathologies beyond the capabilities of small-molecule drugs. However, effective delivery of RNA therapeutics to the lungs remains hindered by significant challenges. The instability of lipid nanoparticles (LNPs) in liquid formulations compromises their storage and cold-chain transport, while the pathological mucus hypersecretion characteristic of chronic airway diseases impedes nanoparticle penetration and delivery efficacy. Herein, we propose a functional lyoprotectant strategy that bridges formulation stability and biological functionality within a single design. Specifically, N-acetylcysteine (NAC), a clinically used mucolytic, was incorporated into a sucrose-based lyoprotectant matrix as a functional additive, enabling lyophilization while introducing mucus-modulating capability. The lyophilized LNPs preserved physicochemical integrity, maintained siRNA encapsulation, and achieved efficient mucus penetration and gene silencing in vitro and in vivo. In murine models of mucus-hypersecretory lung disease, a single-dose administration achieved enhanced therapeutic outcomes through a sequential and dual-action complementary mechanism, including extracellular NAC-mediated mucolysis and intracellular RNAi-mediated inflammation suppression. This work pioneers the concept of a functional lyoprotectant, offering a generalizable platform for storage-stable and biologically active inhaled RNA therapeutics.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2026", "doi": "10.1016/j.jconrel.2026.114749", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Sun W", "dirs": [ "A" ] }, "41123384": { "pmid": "41123384", "title": "A High-Throughput Microbial Viability Assay to Evaluate Lyoprotectants for Live Biotherapeutic Products.", "abstract": "Many cell-based therapies, such as live biotherapeutic products (LBPs), require preservation of cell viability during manufacturing, transportation, and storage before being delivered to patients. Although lyophilization is a common strategy to improve storage stability, it can compromise cell viability, thus reducing therapeutic efficacy. To mitigate this challenge, protective excipients, known as lyoprotectants, are necessary to protect cells during lyophilization. While current research centers on a few disaccharides such as trehalose and sucrose, there is a growing need to investigate and screen more alternatives to broaden the range of lyoprotectants and tailor formulations to specific strains. However, conventional methods for cell formulation screening, such as colony-forming assay, despite being considered as the gold standard for evaluating microorganism viability, are labor-intensive and expensive. In this study, we report a high-throughput screening (HTS) method based on well-plates to evaluate lyoprotectants for a representative LBP, BioPYM, a genetically modified Saccharomyces boulardii targeting gastrointestinal diseases. In this assay, various BioPYM formulations were lyophilized on a metal 96-well plate, washed to remove lyoprotectants, and then diluted and inoculated into a nutritional medium for growth monitoring. A significant inverse relationship was observed between log10(cell viability) and growth lag time, with a Pearson's coefficient of -0.9862, suggesting that higher post-lyo viability can be reflected by shorter lag times during recovery growth. Comparable post-lyo viability was observed between well-plate-based lyophilization and bulk-material lyophilization. With this high-throughput method, we expect to boost the range of excipient choices beyond commonly used disaccharides to help cells withstand the stresses of lyophilization and more efficiently customize the protective formulations for cell-based therapeutics.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00557", "ptypes": [ "Journal Article" ], "first_author": "Cui H", "dirs": [ "A" ] }, "40436220": { "pmid": "40436220", "title": "A spray dried replicon vaccine platform for pandemic response.", "abstract": "The recent COVID-19 pandemic, as well as the threat of a global pandemic caused by H5N1 avian influenza virus, has highlighted the need for the development of thermostable vaccines that can be manufactured and distributed rapidly to combat the next global pandemic. To address this need, we previously developed a replicon vaccine platform that utilizes a nanostructured lipid carrier (NLC) to protect and efficiently deliver antigen-expressing replicon molecules in vivo. The replicon-NLC vaccine platform uses readily sourced components and can be rapidly manufactured at scale with the potential for stockpiling, thus enhancing pandemic preparedness. Spray drying is a promising method of vaccine desiccation with reduced costs and increased scale-up capabilities compared to lyophilization. As proof of concept, we demonstrate for the first time the successful spray drying of a replicon-NLC vaccine complex designed to protect against H5N1 avian influenza A virus to enhance its long-term thermostability while maintaining vaccine immunogenicity in an in vivo mouse model. Several glass-forming disaccharide excipients were screened for formulation and process compatibility under low-temperature spray drying conditions, and it was determined that a suitable shell-forming excipient, L-leucine, was necessary to prevent excessive accumulation of replicon-NLC vaccine complexes on the dry powder surface and a subsequent loss in process yield. The spray dried replicon-NLC vaccine powders were chemically stable for 1 month of storage at 40 °C. Immunogenicity of the spray dried drug product was also well maintained for at least 3 months of storage at 4 °C when administered intramuscularly into C57BL/6 mice as a reconstituted liquid. Finally, we demonstrate the ability to precisely control the aerodynamic particle size of the spray dried vaccine product to generate dry powders that are theoretically suitable for nasal or pulmonary delivery without reconstitution. This work establishes the feasibility of spray drying a thermostable replicon-NLC vaccine for rapid pandemic response.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125777", "ptypes": [ "Journal Article" ], "first_author": "McClary WD", "dirs": [ "A" ] }, "41901238": { "pmid": "41901238", "title": "Formulation Matters: The Overlooked Engine of Stability and Success in Antibody-Drug Conjugates.", "abstract": "Backgrounds: Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the cytotoxic potency of drugs, representing a significant class of targeted cancer therapeutics. Despite their clinical success, formulation-related instability, rather than biological inefficacy, is a major contributing factor to setbacks in ADC development. This review examines the biochemical, physicochemical, and formulation factors that contribute to ADC stability, with a focus on excipient selection, conjugation site heterogeneity, and linker-payload reactivity. Methods: This comprehensive review was based on a selection of peer-reviewed mechanistic, analytical, and manufacturability studies on ADC stability. Our goal was to highlight formulation strategies, degradation pathways, and solid-state stabilization principles that affect the pharmacokinetics and therapeutic efficacy of ADC. Results: Results demonstrate how formulation variability including buffer composition, excipient choice, ionic strength, and lyophilization can directly affect payload release, linker cleavage, kinetics, and antibody conformation. It has been demonstrated that techniques, such as lyophilization with glass-forming matrices and the addition of surfactants, enhance stability against hydrolysis, oxidation, and aggregation. Developments in analytical characterization, such as real-time kinetic modeling and multi-attribute techniques based on mass spectrometry, have made quantification of degradation and bioactivity losses more predictable in ADC formulations. The connection between chemical stability and formulation outcomes is being redefined by new techniques, such as model-informed optimization and AI-driven design. Conclusions: ADC formulation is now a key component of molecular stability, clinical reliability, and regulatory compliance rather than a secondary consideration. By guaranteeing long-term stability, better pharmacokinetics, and improved therapeutic indices across next-generation designs, these approaches have the potential to revolutionize ADC development.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2026", "doi": "10.3390/ph19030393", "ptypes": [ "Journal Article", "Review" ], "first_author": "Torres-Dias L", "dirs": [ "A" ] }, "42658878": { "pmid": "42658878", "title": "Development and validation of the 6-dye lyophilized fluorescent multiplex PCR system.", "abstract": "Aiming to address the issues associated with traditional liquid STR reagents-such as dependence on cold-chain storage, cumbersome operation, and unsuitability for on-site rapid detection-this study has successfully developed a six-color fluorescent lyophilized multiplex amplification reagent. The reagent includes 29 autosomal STR loci and 1 gender identification locus. By screening high-tolerance Taq polymerase, optimizing lyoprotectant formulations, and developing a lyophilization process based on thermodynamic analysis, stable ambient-temperature storage of key reagent components has been achieved. Performance validation demonstrates that the lyophilized reagent exhibits high sensitivity (100% detection at 0.125 ng DNA), remains stable for 60 days at room temperature, rehydrates rapidly (<1 second), and shows some tolerance to common inhibitors. Benefiting from the \"whole-volume loading\" advantage, its detection capability for trace samples surpasses that of traditional liquid reagents. In parallel comparison with similar liquid reagents, it achieves completely consistent locus detection rates (100%), fully meeting the requirements for individual identification. With simple operation and a ready-to-use, disposable design, this reagent effectively overcomes the reliance on cold-chain logistics. It provides robust technical and reagent support for rapid on-site forensic testing, emergency response, grassroots applications, and DNA database construction, demonstrating significant application prospects.", "journal": "PloS one", "year": "2026", "doi": "10.1371/journal.pone.0356258", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "Cui Y", "dirs": [ "A" ] }, "40514623": { "pmid": "40514623", "title": "Development, Characterization, and Molecular Dynamics Simulation of Andrographolide Nanosuspensions Utilizing Hummer Acoustic Resonance Technology.", "abstract": "Andrographolide (AG) is a diterpenoid lactone, widely recognized for its potent anti-inflammatory and immunomodulatory properties. However, AG's clinical applications are significantly limited due to its poor water solubility. The aim of this study was to rapidly develop an andrographolide nanosuspension (AG-NS) using Hummer Acoustic Resonance (HAR) technology to enhance AG's solubility. AG-NS was prepared using HAR technology for high-throughput screening of stabilizers. Quality risk assessment was performed to identify critical formulation and process variables influencing AG-NS. A Box-Behnken design (BBD) was applied to evaluate the effects of these critical variables on AG-NS. Following lyophilization, the redispersibility of AG-NS was evaluated, and physicochemical characterization was conducted to verify the absence of significant interactions between AG and the excipients. The optimized AG-NS formulation exhibited a Z-Ave of 183.96 ± 4.40 nm, a PDI of 0.151 ± 0.065, and a zeta potential of -42.85 ± 1.09 mV. MD simulations revealed the internal mechanisms of AG-NS stabilization. Lyophilized AG-NS demonstrated excellent redisperseability. In vitro dissolution studies showed that the lyophilized AG-NS had a significantly faster dissolution rate and higher cumulative dissolution (120 min) compared to AG crude powder and the physical mixture (PM). The optimized AG-NS demonstrated favorable physicochemical properties and enhanced dissolution performance. The lyophilized formulation exhibited excellent redispersibility upon reconstitution. HAR technology is an innovative and efficient approach for the rapid development and optimization of nanosuspension formulations.", "journal": "AAPS PharmSciTech", "year": "2025", "doi": "10.1208/s12249-025-03160-1", "ptypes": [ "Journal Article" ], "first_author": "Wang L", "dirs": [ "A" ] }, "41274344": { "pmid": "41274344", "title": "Dry powder inhalation of siRNA therapeutics: Emerging strategies for pulmonary gene silencing.", "abstract": "Small interfering RNA (siRNA) holds great promise for treating pulmonary diseases by enabling targeted gene silencing. However, effective delivery of siRNA to the lungs faces multiple challenges, including enzymatic degradation, mucus clearance, surfactant interactions, epithelial barriers, and macrophage clearance. Dry powder inhalation (DPI) has emerged as an attractive approach, offering direct, non-invasive administration to the respiratory tract, improved patient compliance, and enhanced siRNA stability. This review summarizes recent progress in siRNA DPI development, focusing on delivery systems and manufacturing techniques. Lipid-based, polymeric, hybrid, and peptide carriers have been widely investigated to encapsulate and protect siRNA, facilitate cellular uptake, and promote endosomal escape. Manufacturing approaches such as spray drying, spray freeze-drying, thin-film freeze-drying, and lyophilization are discussed in terms of their impact on particle properties, stability, and aerosol performance. Strategies to overcome pulmonary barriers, including surface modifications and excipient selection, are highlighted. Finally, current challenges and future perspectives for clinical translation are considered, emphasizing the need for optimized formulation design, safety evaluation, and scalable production. Together, these advances provide a foundation for the development of siRNA DPI therapies, offering new opportunities for the treatment of lung diseases.", "journal": "Journal of pharmaceutical sciences", "year": "2026", "doi": "10.1016/j.xphs.2025.104076", "ptypes": [ "Journal Article", "Review" ], "first_author": "Lu Y", "dirs": [ "A" ] }, "42431558": { "pmid": "42431558", "title": "Formulation and stability design spaces for a new yellow fever vaccine.", "abstract": "Yellow fever (YF) is a life-threatening disease that is entirely preventable through vaccination. The objective of this study was to formulate a freeze-dried presentation of a live-attenuated, Vero cell-based yellow fever vaccine (vYF) with improved manufacturability and enhanced shelf-life. Considering four critical quality attributes (i.e., glass transition temperatures and infectious titers at two time points), a comprehensive formulation screening was performed to identify an optimal combination of excipients, capable of protecting the virus during lyophilization while ensuring long-term stability of the vYF infectious titer. Overall, L-proline, urea, and poloxamer 407 demonstrated a stabilizing effect, and the application of an advanced kinetic model enabled prediction of a 60-month shelf-life at 5 °C for the formulated vaccine. Machine learning models were used to build both formulation and stability design spaces that defined global stabilizing regions within the explored concentration ranges of each excipient. This study demonstrates that the combination of systematic excipient screening and advanced analytics enables the selection of robust formulations with long-term stability within an accelerated development timeframe. The developed vaccine formulation has the potential for large-scale industrial production and can address substantial market demand in the coming years, representing a significant advancement in vYF technology.", "journal": "European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V", "year": "2026", "doi": "10.1016/j.ejpb.2026.115177", "ptypes": [ "Journal Article" ], "first_author": "Clénet D", "dirs": [ "A" ] }, "42543522": { "pmid": "42543522", "title": "Development and evaluation of rizatriptan benzoate orally disintegrating tablets for migraine treatment.", "abstract": "This study aimed to develop rizatriptan (RZT) benzoate orally disintegrating tablets (ODTs), used in migraine treatment, using various excipients and, in particular, super-disintegrants, through the direct compression method, and to compare them with a commercial product (Maxalt® RPD). Migraine is a common disease that affects quality of life. ODTs are the preferred dosage form due to their rapid effect and patient compliance. In this study, ODTs with rapid disintegration time, rapid release, and high porosity were developed using superdisintegrant for RZT. ODT formulations were prepared by direct compression method using superdisintegrants such as Ludiflash®, Ludipress®, and Pharmaburst™ along with other excipients. Disintegration time and in vitro dissolution tests were performed and compared with Maxalt® RPD. Furthermore, the porosity, water absorption rate, wetting time, and surface morphology of the tablets were also examined. The interactions between RZT and excipients were investigated using DSC and XRD studies. ODT formulations containing Ludiflash® and Pharmaburst™ were found to be the most suitable according to their disintegration time, dissolution profiles. It was observed from the dissolution profile of the commercial product that 100% of the drug was dissolved within 3 min. This is thought to be due to Maxalt® RPD being manufactured by lyophilization. A highly porous structure was clearly observed on the surface of ODTs. There was no interaction between RZT and excipients. As a result, ODT formulation of RZT, used in the treatment of migraine, has been successfully developed.", "journal": "Drug development and industrial pharmacy", "year": "2026", "doi": "10.1080/03639045.2026.2713612", "ptypes": [ "Journal Article" ], "first_author": "Çelebi N", "dirs": [ "A" ] }, "42110367": { "pmid": "42110367", "title": "Spray-freeze-drying aprepitant with hydroxypropyl cellulose increases nasal bioavailability.", "abstract": "The design of free-flowing lyophilized powders by spray-freeze-drying (SFD) enables novel therapeutic applications, including nasal administration. In this study, a non-aqueous SFD approach using dimethyl sulfoxide (DMSO) as the spray solvent was investigated. Low-viscosity hydroxypropyl cellulose grades (HPC-SSL and HPC-UL) were employed as excipients to produce rapidly dissolving, porous, amorphous aprepitant powders at different drug-to-polymer ratios. The resulting spherical particles contained amorphous aprepitant and exhibited sizes between 250 μm and 500 μm. In vitro dissolution at pH 7.0 showed rapid drug release, with maximum dissolution achieved after 3 min for aprepitant/HPC-UL at a 20/80 ratio, exceeding the corresponding physical mixture by 2- to 3-fold. Film-cast amorphous solid dispersions (ASDs) reached comparable drug concentrations but exhibited slower dissolution. Formulations containing HPC-SSL showed higher supersaturation levels but reduced dissolution rates. Increasing drug loading to a 40/60 ratio led to a marked delay in dissolution. Nasal deposition in vitro exceeded 90% of the administered dose, attributed to the combination of low particle density (<0.1 g/cm3) and sufficient mechanical stability. Following nasal administration in rats, t max ranged from 0.8 h to 4 h, C max from 26.9 ng/ml to 64.0 ng/ml, and AUClast from 125.4 ng·h/ml to 316.3 ng·h/ml, with no statistically significant differences between HPC-UL and HPC-SSL. All SFD formulations demonstrated significantly increased nasal bioavailability compared to ASD controls. Despite similar t max values, HPC-based SFD formulations enhanced aprepitant bioavailability by at least threefold. These findings underline the potential of SFD as a formulation platform for the nasal delivery of poorly water-soluble drugs.", "journal": "International journal of pharmaceutics: X", "year": "2026", "doi": "10.1016/j.ijpx.2026.100551", "ptypes": [ "Journal Article" ], "first_author": "Rautenberg A", "dirs": [ "A" ] }, "41773051": { "pmid": "41773051", "title": "Nanoparticulate protectants for the dry storage of protein therapeutics.", "abstract": "Protein therapeutics are a cornerstone of modern medicine, yet their inherent instability often requires expensive and energy-intensive cold-chain logistics to maintain efficacy. Dry-state formulations offer a viable strategy to enhance protein stability, but the associated drying processes, such as lyophilization and spray drying, can introduce stresses that damage protein molecules. While excipients are widely used to protect proteins during dehydration, conventional formulations often require protein-specific, iterative optimization. Further, protein stabilizing excipients can crystallize during storage, thereby reducing product quality, increase product viscosity beyond thresholds for injection, and raise safety concerns. Nanoparticulate excipients are receiving increasing attention to address these challenges. This article provides an overview of nanoparticle excipients showing promise for stabilizing proteins during drying processes. It is envisioned that nanoparticulates may advance toward universal excipients capable of stabilizing a variety of proteins without the need for cumbersome optimization while also providing robust biocompatibility.", "journal": "Nanomedicine (London, England)", "year": "2026", "doi": "10.1080/17435889.2026.2634992", "ptypes": [ "Journal Article", "Review", "Research Support, N.I.H., Extramural" ], "first_author": "Park J", "dirs": [ "A" ] }, "41599218": { "pmid": "41599218", "title": "Structure-Based Prediction of Molecular Interactions for Stabilizing Volatile Drugs.", "abstract": "Background/Objectives: The high volatility of volatile drugs significantly restricts their clinical applicability. Although excipients capable of strong interactions can reduce volatilization, conventional screening methods rely on empirical trial-and-error, resulting in low efficiency and high resource consumption. To address this limitation, this study introduces an artificial intelligence (AI)-driven strategy for screening drug-excipient interactions. Using d-borneol as a model drug, this approach aims to efficiently identify strongly interacting excipients and develop stable nano-formulations. Methods: High-throughput simulations were performed using the Protenix structure prediction model to evaluate interactions between d-borneol and 472 FDA-approved excipients. The top 50 candidate excipients were selected based on these simu-lations. Molecular docking and stability experiments were conducted to validate the predictions. Results: Molecular docking and stability experiments confirmed the consistency between predicted and experimental results, validating the model's reliability. Among the candidates, soybean phospholipid (PC) was identified as the optimal excipient. A lyophilized liposomal formulation prepared with PC significantly suppressed the volatilization of d-borneol and improved both thermal and storage stability. Mechanistic investigations indicated that d-borneol stably incorporates into the hydro-phobic region of phospholipids, enhancing membrane ordering via hydrophobic interactions without disturbing the polar headgroups. Conclusions: This study represents the first application of a structure prediction model to excipient screening for volatile drugs. It successfully addresses the stability challenges associated with d-borneol and offers a new paradigm for developing nano-formulations for volatile pharmaceuticals.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18010111", "ptypes": [ "Journal Article" ], "first_author": "Zhao Y", "dirs": [ "A" ] }, "41304926": { "pmid": "41304926", "title": "In Vitro and in Vivo Efficacy of Different Ointment Formulations Containing Centaurium erythraea Rafn. Aerial Extract.", "abstract": "Background:Centaurium erythraea Rafn. (C. erythraea) is a medicinal plant traditionally used in European folk medicine for the treatment of wounds, skin inflammations, and other dermatological conditions, in addition to its well-documented systemic antioxidant and anti-inflammatory effects. However, its topical applications remain insufficiently investigated, particularly using plant material collected from Romania. The purpose of this study was to prepare different ointment formulations containing C. erythraea Rafn. extract obtained from the aerial parts of the plant, using various excipients, and to evaluate their in vitro and in vivo efficacy. Methods: The phytochemical profile of C. erythraea extract was characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lyophilized extract was pre-dissolved in different solubilizing agents-Transcutol® P (diethylene glycol monoethyl ether), Capryol® 90 (propylene glycol monocaprylate), or a combination of both-and then incorporated into five ointment formulations. Texture analysis and an in vitro membrane diffusion study were performed. The antioxidant capacity of the formulations was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), and total phenolic content (TPC) assays. Anti-inflammatory activity was evaluated in vitro using tumor necrosis factor-alpha (TNF-α)-induced interleukin-1 beta (IL-1β) production in human keratinocyte (HaCaT) cells, and in vivo using a carrageenan-induced rat paw edema model. Results: LC-MS/MS identified 18 polyphenolic compounds, with hyperoside (3.78 ± 0.05 µg/mL), protocatechuic acid (1.13 ± 0.06 µg/mL), chlorogenic acid (1.07 ± 0.06 µg/mL), and quercetin (0.53 ± 0.03 µg/mL) as the principal constituents. The formulation containing both Transcutol® P and Capryol® 90 exhibited the most pronounced antioxidant activity (65% DPPH inhibition; 69.71 ± 0.83 mg gallic acid equivalent/mL) and significantly reduced IL-1β levels by 45.7% compared to the inflamed control. In vivo, this formulation showed comparable anti-edematous effects to a methylprednisolone ointment. Furthermore, it demonstrated the highest skin permeation efficiency, with a quercetin diffusion coefficient of 35.12 × 10-5 cm2/min. Conclusions: These findings highlight the therapeutic potential of C. erythraea extract from aerial parts in topical formulations and underscore the enhancing role of Transcutol® P and Capryol® 90 in improving both the pharmacodynamic and pharmacokinetic properties of bioactive compounds.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ph18111681", "ptypes": [ "Journal Article" ], "first_author": "Karetka AJ", "dirs": [ "A" ] }, "42413883": { "pmid": "42413883", "title": "High-concentration biologic formulations: challenges, strategies, and emerging technologies for subcutaneous delivery.", "abstract": "The growing demand for subcutaneous (SC) self-administration of biotherapeutics, driven by the rising prevalence of chronic diseases and healthcare cost-containment pressures, has accelerated the development of high-concentration formulations (HCFs). However, protein concentrations exceeding 100 mg/mL introduce significant challenges, including solubility limitations, high viscosity, aggregation propensity, injectability constraints, and manufacturing complexities. This review systematically examines the primary obstacles in HCF development and highlights recent advances in formulation and process technologies. Key strategies include: (i) excipient-based viscosity reducers; (ii) hyaluronidase-enabled large-volume SC delivery; (iii) lyophilization for decoupling manufacturing from final concentration; (iv) spray drying platforms including SnapShot™ and XeriJect®; (v) electrostatic spray drying (Elektroject™ Hypercon™); (vi) Microglassification™ dehydration technology; and (vii) protein crystallization (Crystalomics®). Collectively, these innovations are reshaping the landscape of high-concentration biologic drug products, enabling the transition from intravenous to subcutaneous administration for a broader range of therapeutics. This review serves as a strategic guide for formulation scientists and drug developers engaged in next-generation subcutaneous biologics.", "journal": "European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V", "year": "2026", "doi": "10.1016/j.ejpb.2026.115178", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ma J", "dirs": [ "A" ] }, "42357308": { "pmid": "42357308", "title": "Fabrication of Microneedle Patches by Suspension Casting of Drugs in Organic Solvents.", "abstract": "Background/Objectives: Drug administration by microneedle patch (MNP) offers advantages over conventional dosage forms as a painless, self-administered skin patch for parenteral delivery. Dissolvable MNPs are typically manufactured by casting an aqueous formulation containing dissolved active pharmaceutical ingredient (API) and excipients into a mold and allowing it to dry. This process can be detrimental to APIs that are sensitive to dissolution and drying during the casting process. Methods: This study presents a MNP fabrication process in which drug particles are suspended in an organic solvent carrier without being dissolved in the solvent. Results: We started with drug particles either as pure API or formulated with excipients to stabilize them. We then screened nine organic solvents, ranging from high (methanol) to low (toluene) polarity, to identify those that suspend the drug particles without dissolution or damage to the API. To guide formulation of stabilized drug particles, we generated a companion database of 16 common stabilizing excipients and measured their solubility in our panel of organic solvents to identify excipient-solvent combinations that did not lead to excipient dissolution. We generated a second database of 14 water-soluble polymers to serve as the microneedle matrix material and determined their solubility in our panel of solvents to identify solvents that enabled polymer dissolution. Using these data, we designed casting solutions that suspended particles of API (and excipients) in an organic solvent that dissolved a matrix polymer. Casting and drying these solutions on molds produced MNPs for delivery of three model compounds: lyophilized tetanus toxoid (i.e., a vaccine), methotrexate (i.e., a small molecule drug), and insulin (i.e., a biologic). Conclusions: We conclude that this fabrication method, guided by the excipient and polymer solubility databases, offers a novel method to produce MNPs by suspension casting of drugs in organic solvents.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18060692", "ptypes": [ "Journal Article" ], "first_author": "Lu CY", "dirs": [ "A" ] }, "41662999": { "pmid": "41662999", "title": "Lyophilized formulation development and characterization of stable glatiramer acetate/oligonucleotide polyplexes at clinically therapeutic strengths.", "abstract": "Glatiramer acetate (GA) electrostatically complexes with cytosine-guanine oligodeoxynucleotides (CpG ODN), forming ∼100 nm cationic nanoparticles that localize this potent immunostimulant to the injection site. We previously reported GA-CpG nanoparticles retained the anti-tumor activity of CpG while mitigating systemic immune-related adverse events. Nonetheless, nanoparticulate systems like polypeptide-oligonucleotide complexes pose challenges with reproducible production, in-use stability, and storage stability, which must be solved before translation for clinical use. In this study, we systematically investigated a microfluidic mixing process to define reproducible production of GA-CpG nanoparticles. Dynamic light scattering measurements revealed significantly smaller and more uniform nanoparticles for microfluidic processing versus traditional mixing via pipette. We screened a range of buffer systems to determine the pH and ion types that could maintain colloidal stability and CpG potency. Buffer screening tests indicated that amino acid buffers, particularly glutamic acid, better maintained particle consistency than commonly used parenteral buffers. Finally, formulations of potential lyoprotectants and GA-CpG nanoparticles were developed. Freeze-thaw and freeze-drying experiments were conducted to assess the effects of buffers and lyoprotectants. Formulations with 5% HP-β-CD or trehalose yielded mean particle sizes of less than 127 nm and retained even after storage for 6 months at 40 °C/75% relative humidity. The work on electrostatic complexes reported here may provide valuable guidance to formulators aiming to optimize polypeptide-based oligonucleotide polyplex products for in-use or long-term stability.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.126659", "ptypes": [ "Journal Article" ], "first_author": "Gong H", "dirs": [ "A", "D" ] }, "40008995": { "pmid": "40008995", "title": "Mini Review on the Lyophilization: A Basic Requirement for Formulation Development and Stability Modifier.", "abstract": "Freeze-drying is popular for producing pharmaceutical formulations with structurally complicated active components and drug delivery system carriers. It is the process of eliminating water from ice crystals through the sublimation mechanism. Some formulations may require drug-specific excipients such as stabilizers, buffers, and bulking agents to maintain the appearance and assure the long-term stability of the drug product. This approach is utilized for therapeutic compounds that are moisture sensitive, thermolabile, and degrade in the atmosphere. Freezing and primary and secondary drying are critical processes in the lyophilization process because they directly impact the end result. This approach is effective for producing a variety of dosage forms, including oral, inhalation, and parenteral. As a result, lyophilization may be an important method for improving the therapeutic efficacy and delivery of various dosage forms delivered via different routes. Additionally, lyophilization is used in pharmacological research to preserve biological samples, stabilize reference/standards, and increase the solubility and bioavailability of poorly soluble drugs. Thus, lyophilization is critical for maintaining the stability, efficacy, and safety of pharmaceutical products throughout their development and lifecycles. This article includes a broad overview of the lyophilization process, principle, excipients for lyophilized medicine compositions, and new lyophilization technologies as well as their applications in a variety of fields.", "journal": "Assay and drug development technologies", "year": "2025", "doi": "10.1089/adt.2024.122", "ptypes": [ "Journal Article", "Review" ], "first_author": "Joshi S", "dirs": [ "A" ] }, "41901217": { "pmid": "41901217", "title": "Ambient-Stable mRNA Medicines: Emerging Paradigms in Dry and Solid-State Formulation.", "abstract": "The medical field now uses mRNA therapeutics to deliver fast programmable treatment options through versatile vaccination platforms. The worldwide adoption of mRNA therapeutics faces a major obstacle because these molecules require extreme cold storage and transportation systems. mRNA stability establishes a fundamental scientific and industrial challenge which requires researchers to unite formulation design with process control and material engineering for cold-chain independence. Current knowledge about RNA hydrolysis and lipid oxidation and water-mediated degradation is combined with new methods for solid-state stabilization through lyophilization and spray-freeze-drying and thin-film technologies. Mechanism such as vitrification, water replacement and excipient RNA interactions are assessed to establish the fundamental chemical properties needed for extended product stability. Advanced mRNA development strategies are also examined, including self-amplifying and circular RNA structures and nano-glass and metal-organic frameworks and artificial intelligence-based predictive design for creating stable mRNA formulations at room temperature. This review examines manufacturing and regulatory and logistical obstacles which affect real-world implementation of mRNA therapeutics through assessments of production scale and product quality tests and packaging strength and tropical environment testing. The combination of research findings presents a path to develop mRNA medicines which maintains their effectiveness when stored at 25 °C or above, thus enabling worldwide access to RNA-based treatments. The development of mRNA into a durable therapeutic platform requires scientists to merge molecular research with process development and regulatory standardization.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2026", "doi": "10.3390/ph19030370", "ptypes": [ "Journal Article", "Review" ], "first_author": "El-Tanani M", "dirs": [ "A" ] }, "39861734": { "pmid": "39861734", "title": "Development and Characterization of Lyophilized Chondroitin Sulfate-Loaded Solid Lipid Nanoparticles: Encapsulation Efficiency and Stability.", "abstract": "This study explores the development and characterization of lyophilized chondroitin sulfate (CHON)-loaded solid lipid nanoparticles (SLN) as an innovative platform for advanced drug delivery. Background/Objectives: Solid lipid nanoparticles are increasingly recognized for their biocompatibility, their ability to encapsulate diverse compounds, their capacity to enhance drug stability, their bioavailability, and their therapeutic efficacy. Methods: CHON, a naturally occurring glycosaminoglycan with anti-inflammatory and regenerative properties, was integrated into SLN formulations using the hot microemulsion technique. Two formulations (SLN-1 and SLN-2) were produced and optimized by evaluating critical physicochemical properties such as particle size, zeta potential, encapsulation efficiency (EE%), and stability. The lyophilization process, with the addition of various cryoprotectants, revealed trehalose to be the most effective agent in maintaining nanoparticle integrity and functional properties. Results: Morphological analyses using transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the dimensions of the nanoscales and their structural uniformity. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) revealed minimal excipient interaction with CHON, ensuring formulation stability. Stability studies under different environmental conditions highlighted that SLN-2 is the most stable formulation, maintaining superior encapsulation efficiency (≥88%) and particle size consistency over time. Conclusions: These findings underscore the potential of CHON-loaded SLNs as promising candidates for targeted, sustained-release therapies in the treatment of inflammatory and degenerative diseases.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17010086", "ptypes": [ "Journal Article" ], "first_author": "Bustos Araya ME", "dirs": [ "A" ] }, "40750017": { "pmid": "40750017", "title": "Hydroxypropyl cellulose-based lyospheres accelerate dissolution of aprepitant combined with its supersaturation for oral delivery.", "abstract": "Spray freeze-drying (SFD) leads to a free-flowing lyophilized powder, providing new therapeutic options. In this work, non-aqueous SFD particles are produced with dimethyl sulfoxide as a spray solvent for obtaining amorphous solid dispersions (ASDs) for oral administration of a poorly water-soluble drug, aprepitant. The SFD process was conducted with the low viscosity hydroxypropyl cellulose grades HPC-SSL or HPC-UL to achieve rapidly dissolving aprepitant powder. Besides different HPC grades, varying solid contents (5 and 10 %) and drug-excipient ratios (20/80, 40/60 and 100/0) were explored and compared to film casted ASDs and analysed regarding particle morphology and characteristics, storage stability, in vitro dissolution and in vivo pharmacokinetic studies in rats. Median particle size of the powder formulations ranged between 300 and 500 µm, increasing HPC content led to amorphous formulations, that remained amorphous for 12 months of storage at 25 °C. Dissolution at pH 1.2 revealed supersaturation across all SFD samples with concentration values twofold and fourfold the saturation concentration for 40/60 and 20/80 drug-excipient ratios respectively. After oral administration to rats, tmax, cmax and AUC varied between 2.5-4 h, 1.04-1.24 µg/ml and 5.4-8.1 µg*h/ml respectively. Pharmacokinetic parameters were not affected by HPC grade, but indicate a possible relationship between a decrease of geometric particle density and an increase in bioavailability. Low viscosity HPC grades proved to be suitable excipients for the use in non-aqueous SFD to produce amorphous, rapidly dissolving, low density powders.", "journal": "European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences", "year": "2025", "doi": "10.1016/j.ejps.2025.107220", "ptypes": [ "Journal Article" ], "first_author": "Kožák J", "dirs": [ "A" ] }, "41166273": { "pmid": "41166273", "title": "Systems-Integrated Thermostable Vaccine Delivery: Converging Cold-Chain-Free Design, AI-Augmented Formulation, and Climate-Resilient Infrastructure.", "abstract": "Thermostable vaccine delivery represents a transformative step toward equitable and climate-resilient immunization systems. Unlike conventional vaccines requiring strict cold-chain storage at 2-8 °C, thermostable formulations retain efficacy at elevated ambient temperatures (up to 40-45 °C), enabling deployment in low-resource, remote, and climate-vulnerable regions. Recent advances in excipients, nanoencapsulation, stabilization matrices such as polymers, metal-organic frameworks (MOFs), liposomes, and drying techniques like lyophilization and spray drying have significantly enhanced heat tolerance and shelf life stability. Existing reviews primarily emphasize material innovations and biophysical stabilization strategies, often highlighting case studies and high-throughput screening approaches. However, critical gaps remain regarding systems-level integration of thermostable vaccine platforms. Few studies explore the convergence of digital formulation design, AI-driven development pipelines, geospatial modeling, lifecycle engineering, and carbon-neutral delivery strategies. Similarly, the integration of smart logistics, real-time monitoring, blockchain-based vaccine authentication, and the policy-technology interface required for deployment in conflict zones, postdisaster areas, and regions severely affected by climate change remains underrepresented. Addressing these gaps is essential to move beyond cold-chain elimination toward climate-smart and sustainable vaccine systems capable of meeting the health needs of underserved populations and addressing emerging global immunization challenges.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c01296", "ptypes": [ "Journal Article", "Review" ], "first_author": "Das S", "dirs": [ "A" ] }, "41165456": { "pmid": "41165456", "title": "RAS-GTP Inhibition Overcomes Acquired Resistance to KRASG12C Inhibitors Mediated by Oncogenic and Wild-Type RAS Activation in Non-Small Cell Lung Cancer.", "abstract": "Small-molecule KRASG12C(OFF) inhibitors that bind to the inactive GDP-bound state of KRAS have demonstrated efficacy in patients with KRASG12C-mutant tumors, yet responses tend to be transient because of emergence of on-treatment resistance. Recently, RAS(ON) G12C-selective inhibitors, which bind to the active GTP-bound state of RAS, were described, and elironrasib is undergoing evaluation in multiple clinical trials. In this study, we generated resistant cell lines and patient-derived xenograft models to KRASG12C(OFF) and RAS(ON) G12C-selective inhibitors and interrogated resistance mechanisms using a multiomics strategy consisting of phosphoproteomics, whole-exome sequencing, and RNA sequencing combined with functional testing using small-molecule and CRISPR screens and RAS(ON) inhibitors being evaluated in clinical trials. Two models reactivated RAS signaling, either via KRASG12C gene amplification or NRASG13R mutation, and were vulnerable to dual inhibition by RAS(ON) G12C-selective and RAS(ON) multiselective inhibitors, RMC-4998 and RMC-7977. Two models, which lacked any discernable genomic alteration, acquired resistance associated with increased receptor tyrosine kinase activity and downstream persistent RAS activity and were sensitive to RAS-GTP inhibition by RMC-7977. Finally, one model displayed epithelial-mesenchymal transition, loss of RAS dependence, and acquired reliance on cell-cycle kinases and proteins associated with DNA damage response. This work highlights KRASG12C-selective inhibitor resistant states that parallel and complement clinical findings and demonstrate that a large subset could be overcome with a RAS(ON) multi-selective inhibitor as a stand-alone agent or in combination with other therapies. Multi-omic characterization of resistance mechanisms to KRASG12C-selective inhibitors in non-small cell lung cancer provides insights that could inform precision medicine-based therapeutic approaches for improving the treatment of KRASG12C mutant tumors. See related article by Stern et al., p. 485.", "journal": "Cancer research", "year": "2026", "doi": "10.1158/0008-5472.CAN-25-0600", "ptypes": [ "Journal Article", "Research Support, U.S. Gov't, Non-P.H.S.", "Research Support, Non-U.S. Gov't" ], "first_author": "Solanki HS", "dirs": [ "B" ] }, "41040355": { "pmid": "41040355", "title": "Targeting RAD52 overcomes PARP inhibitor resistance in preclinical Brca2-deficient ovarian cancer model.", "abstract": "BRCA-mutated ovarian cancer commonly develops resistance to poly (ADP-ribose) polymerase (PARP) inhibitors. Here, we investigated the DNA repair protein RAD52 as a potential target to overcome resistance. In analysis of The Cancer Genome Atlas datasets and immunohistochemistry of tissue microarrays, elevated RAD52 expression correlated with poor overall survival in patients with high-grade serous ovarian cancers. We tested two PARP inhibitor-resistant Brca2-deficient mouse ovarian cancer models, ID8-OR and HGS2-OR. HGS2-OR cells had higher RAD52 expression than parental lines. Rad52 knockout or knockdown restored PARP inhibitor sensitivity in both models. In syngeneic mice, ID8-OR cells in which Rad52 was knocked out yielded lower tumor burden and longer overall survival than control cells. Rad52 depletion impaired single-strand annealing and homologous recombination and led to accumulation of DNA double-strand breaks after PARP inhibitor treatment. RNA sequencing demonstrated that PARP inhibitor treatment induced Polq expression in Brca2- and Rad52-deficient cells, suggesting a switch to microhomology-mediated end joining. Finally, the RAD52 inhibitor D-I03 synergized with a PARP inhibitor to reduce cell viability and tumor burden and prolong survival. Collectively, our findings establish RAD52 as a promising therapeutic target to overcome PARP inhibitor resistance in BRCA2-mutated ovarian cancer and offer mechanistic insights to inform future clinical strategies.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.09.24.678351", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Ota Y", "dirs": [ "B" ] }, "41525963": { "pmid": "41525963", "title": "MicroRNA-CRISPR biosensors for cancer diagnostics.", "abstract": "Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer and cardiovascular disorders. However, their low sequence length, low abundance, high sequence homology (including iso-miRs), and strong matrix and preanalytical effects in biofluids require highly sensitive and robust analytical technologies. CRISPR-Cas systems, particularly Cas12a, Cas12b, Cas13a, and Cas9, offer programmable nucleic acid recognition with high mismatch discrimination combined with collateral nuclease activity, enabling versatile signal amplification through fluorescence, electrochemical, electrochemiluminescent (ECL), photoelectrochemical (PEC), colorimetric, and lateral-flow readouts. This review critically evaluates the latest advances in CRISPR-based miRNA biosensors, emphasizing their analytical performance and translational potential in clinical diagnostics across plasma/serum, saliva, whole blood, and extracellular vesicle samples. The detection limits are typically within the femtomolar to attomolar range. The requirements for clinical translation are equally influenced by factors such as sample preparation, inhibitor tolerance, miRNA panel multiplexing, quantitative readout, and reagent stability. We compared CRISPR-based workflows with RT-qPCR and digital PCR and provided a roadmap for standardization and quality control, as well as the minimal analytical and clinical validation standards required for adopting CRISPR technology in clinical chemistry laboratories.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2026", "doi": "10.1016/j.cca.2026.120837", "ptypes": [ "Journal Article", "Review" ], "first_author": "Habib AH", "dirs": [ "B" ] }, "41480640": { "pmid": "41480640", "title": "Replication-associated base excision repair/single-strand break repair regulates PARG inhibitor response via the PRMT1/PRMT5/ATR axis.", "abstract": "Poly(ADP-ribose) polymerases 1 and 2 (PARP1/PARP2), and poly(ADP-ribose) glycohydrolase (PARG), modulate the level of poly(ADP-ribose) (PAR), a post-translational protein modification, in response to DNA damage or replication stress. Here, we find that replication-dependent and PARP1/PARP2-mediated PARylation recruits the base excision repair (BER)/single-strand break repair (SSBR) scaffold protein XRCC1 and the associated factors DNA polymerase β (POLB), aprataxin (APTX), and DNA ligase isoform 3 (LIG3). Further, these BER/SSBR proteins promote resistance to inhibitors of PARP1/PARP2 and PARG, as loss of these proteins sensitizes glioblastoma and ovarian cancer cells to each. In addition, depletion of these replication-associated BER/SSBR factors leads to enhanced PAR levels and PARG inhibitor-induced activation of the ATR/CHK1 S-phase checkpoint kinases. Both PARG inhibition and ATR inhibition lead to elevated ATM- and DNA-PK-dependent KAP1 phosphorylation. In turn, inhibition of either ATR or CHK1 enhances the cellular response to PARG inhibitors. Finally, inhibition of the ATR regulators PRMT1 or PRMT5 synergizes with PARG inhibition, implicating replication-associated BER/SSBR and PARylation in the activation of the PRMT1/PRMT5/ATR axis. This study highlights the role of BER/SSBR in protecting the cell during S-phase to suppress PARylation-induced checkpoint activation, which may suggest a potential intervention strategy for PARG inhibitor-resistant tumors.", "journal": "NAR cancer", "year": "2025", "doi": "10.1093/narcan/zcaf057", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "Ibrahim M", "dirs": [ "B" ] }, "41177494": { "pmid": "41177494", "title": "Engineering and enhancement of Bst DNA polymerase variants toward improved diagnostic applications: A review.", "abstract": "Bst DNA polymerase, the key enzyme in loop-mediated isothermal amplification (LAMP), is pivotal for point-of-care diagnostics due to its strand displacement activity. Aiming to optimize Bst DNA polymerase for faster, more accurate, and more robust nucleic acid detection in clinical diagnostics and various point-of-care testing scenarios, this review summarizes current research focused on engineering Bst DNA polymerase to enhance its functional properties. We systematically analyze key mutation sites within its structural domains, detailing how specific amino acid substitutions influence thermostability, strand displacement efficiency, fidelity, inhibitor tolerance, and nucleotide selectivity. Additionally, we explore advancements on fusion protein strategies aimed at enhancing various properties of Bst DNA polymerase, including DNA affinity, processivity, salt tolerance, thermostability, and resistance to inhibitors. We also discuss the challenges and future directions of domain-specific engineering and emphasize the potential of integrating artificial intelligence and high-throughput screening for rapid mutant development.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.148691", "ptypes": [ "Journal Article", "Review" ], "first_author": "Yang R", "dirs": [ "B", "C" ] }, "39838098": { "pmid": "39838098", "title": "Targeting BRCA1-deficient PARP inhibitor-resistant cells with nickases reveals nick resection as a cancer vulnerability.", "abstract": "Tumors lacking the BRCA1 and BRCA2 (BRCA) hereditary breast cancer genes display heightened sensitivity to anti-cancer treatments, such as inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). However, when resistance develops, treatments are lacking. Using CRISPR technology, we discovered that enhancing homologous recombination through increased DNA end resection in BRCA1-deficient cells by loss of the 53BP1-Shieldin complex-which is associated with resistance to PARP inhibitors-also heightens sensitivity to DNA nicks. The sensitivity is caused by hyper-resection of nicks into extensive single-stranded regions that trigger cell death. Based on these findings and that nicks limit tumor formation in mice, we propose nickases as a tool for personalized medicine. Moreover, our findings indicate that restricting nick expansion is a critical function of the 53BP1-Shieldin complex.", "journal": "Nature cancer", "year": "2025", "doi": "10.1038/s43018-024-00902-1", "ptypes": [ "Journal Article" ], "first_author": "Whalen JM", "dirs": [ "B" ] }, "41808580": { "pmid": "41808580", "title": "Targeted therapy for DNA damage response and homologous recombination repair defects: The Olaparib Combinations trial.", "abstract": "Mutations in genes encoding proteins involved the DNA damage response (DDR) occur in up to 20% of patients with cancer. It is unknown whether poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors, alone or in combination with ATR or AKT inhibitors, have histology-agnostic clinical efficacy in tumors with DDR mutations or PI3K/AKT pathway mutations, respectively. The Olaparib Combinations (OLAPCO) trial enrolled patients in treatment arms based on next-generation sequencing results. In cohorts 1 and 2, patients with tumors harboring DDR mutations received either the PARP inhibitor olaparib or olaparib and the ATR inhibitor ceralasertib. In cohort 3, patients with tumors with PI3K-AKT pathway alterations or ARID1A mutations received olaparib and capivasertib. The primary end point was overall response rate (ORR) at 16 weeks assessed by the Response Evaluation Criteria in Solid Tumors, version 1.1. Sixty-six patients were treated, including 26 on olaparib monotherapy, 24 on olaparib and ceralasertib, and 16 on olaparib and capivasertib. Among all patients treated, the ORR was 6.1% and the clinical benefit rate was 31.2% with a median duration of benefit (DoB) of 11 months. Among seven patients with platinum- and PARP inhibitor-resistant high-grade ovarian serous cancer in the olaparib and ceralasertib arm, one had a partial response and four had stable disease with a median DoB of 10 months. No unexpected toxicities were observed. The study failed to meet its primary end point of ORR. DDR and homologous recombination repair defects are not consistently actionable with olaparib as monotherapy or in combination with other targeted therapies in a histology-agnostic manner.", "journal": "Cancer", "year": "2026", "doi": "10.1002/cncr.70332", "ptypes": [ "Journal Article" ], "first_author": "Doroshow DB", "dirs": [ "B" ] }, "41735312": { "pmid": "41735312", "title": "Targeting de novo pyrimidine synthesis confers vulnerability to copper-mediated ATR inactivation in PARP inhibitor-resistant ovarian cancer.", "abstract": "Although poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) as monotherapy or in combination with other DNA-damaging agents exhibit promising clinical efficacy, the therapeutic responses are usually transient, with subsequent development of acquired resistance posing a significant challenge. Here, through a small-molecule compound screening, we identify elesclomol, a potent copper ionophore, which sensitizes BRCA-proficient ovarian cancer cells to PARPi by inhibiting activation of the ATR-CHK1 pathway. Mechanistically, we demonstrate that copper directly binds to ATRIP, a critical cofactor of ATR activation, disrupting the ATR-ATRIP interaction, further impairing ATR-mediated DNA damage repair signaling and potentiating PARPi sensitivity. Importantly, we reveal a secondary metabolic vulnerability in PARPi-resistant ovarian cancer associated with de novo pyrimidine synthesis, suggesting that targeting this pathway as an effective strategy to eradicate drug-adaptive residual tumors and resistant patient-derived xenograft models following ATR and PARP co-inhibition. These findings propose de novo pyrimidine synthesis as an adaptive metabolic vulnerability that can be therapeutically targeted to overcome PARPi resistance in BRCA-proficient ovarian cancer.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-70001-5", "ptypes": [ "Journal Article" ], "first_author": "Nan Y", "dirs": [ "B" ] }, "41168257": { "pmid": "41168257", "title": "RECQL1 as a potential therapeutic target for PARP inhibitor-resistant ovarian cancer.", "abstract": "Overcoming resistance to poly (ADP-ribose) polymerase (PARP) inhibitors is an urgent challenge for ovarian cancer treatment. Here, we investigated RECQL1, a RecQ helicase involved in homologous recombination repair, as a potential target for overcoming PARP inhibitor resistance in this disease. Patients with platinum-sensitive recurrent ovarian cancer treated with the PARP inhibitor olaparib between March 2018 and December 2021 were included. Immunohistochemistry assays were conducted to assess RECQL1 protein expression patterns in surgically resected ovarian cancer tissues. The effect of RECQL1 knockdown on olaparib resistance was evaluated in vitro using ovarian cancer cells transfected with an anti-RECQL1 siRNA. Among 44 patients, those with no response to olaparib had significantly higher RECQL1 expression levels compared with responders (P = 0.020). Kaplan-Meier curves showed significantly shorter overall survival in the high RECQL1 expression group than in the low expression group (median, 26.0 months vs. not reached; P = 0.027). Multivariate analysis revealed high RECQL1 expression to be a significant prognostic factor for shorter overall survival (P = 0.036). RECQL1 knockdown significantly enhanced the sensitivity to olaparib in two ovarian cancer cell lines. Overall, RECQL1 plays a critical role in PARP inhibitor resistance and is a promising therapeutic target to improve ovarian cancer patient outcomes.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-21844-3", "ptypes": [ "Journal Article" ], "first_author": "Yoshida H", "dirs": [ "B" ] }, "42095258": { "pmid": "42095258", "title": "Loss of IGF-1R impairs DNA-PKcs recruitment to chromatin leading to defective end-joining.", "abstract": "The insulin-like growth factor (IGF) axis regulates cancer cell proliferation, growth, invasion, and therapy resistance. Elevated expression of the type 1 IGF receptor (IGF-1R) is linked to radioresistance and biochemical recurrence in prostate cancer, yet the molecular mechanisms underlying IGF-1R-mediated DNA damage responses remain unclear. We investigated the role of IGF-1R in DNA double-strand break (DSB) repair by assessing chromatin recruitment of DNA repair proteins, repair pathway usage, and therapeutic sensitivity in cancer cell models with altered IGF-1R status. Loss of IGF-1R impaired DNA-dependent protein kinase catalytic subunit (DNA-PKcs) localisation to chromatin, resulting in defective non-homologous end-joining (NHEJ) and a compensatory reliance on alternative repair pathways, including microhomology-mediated end-joining (MMEJ). Modulating IGF-1R expression restored radiosensitivity in poly (ADP-ribose) polymerase (PARP) inhibitor-resistant breast cancer cells. IGF-1R inhibition compromises canonical DSB repair and re-sensitises resistant cancer cells to therapy, supporting its potential as a therapeutic strategy in homologous recombination-deficient tumours. Furthermore, IGF-1R mutant cancers may benefit from targeted inhibition of the MMEJ pathway.", "journal": "Molecular oncology", "year": "2026", "doi": "10.1002/1878-0261.70266", "ptypes": [ "Journal Article" ], "first_author": "Ellis MO", "dirs": [ "B" ] }, "41687989": { "pmid": "41687989", "title": "Circular permutation engineering improves amplification efficiency of Bst DNA polymerase.", "abstract": "Previously, a chimeric Bst DNA polymerase (HpStBL) with high inhibitor tolerance and reverse transcriptase activity, was constructed by introducing Hp47-Sto7d domain. However, the complex primer design and the possibility of nonspecific amplification in loop-mediated isothermal amplification (LAMP) partly restricted the broader application of HpStBL in nucleic acid detection. In order to explore the polymerase with better performance for LAMP and reverse-transcription LAMP (RT-LAMP), four CP-Bst DNA polymerase mutants were generated by circular permutation of Hp47-Sto7d domain of HpStBL. The results showed that circular permutation had impacts on the hydrophobicity of the protein by altering the amino acid sequence thereby affecting the affinity of the polymerase for DNA, and the mutants exhibited both DNA synthesis and reverse transcriptase activity. Mutant CP-G23 especially showed highest amplification performance, which could successfully amplify 100 copies/μL Astroviruses plasmid DNA. Based on the CP-G23, a simple and rapid nucleic acid detection method was developed with the recombinase from Thermus thermophilus, in which the system can successfully amplify 100 copies/μL Astroviruses plasmid DNA and 0.01 pg/μL SARS CoV-2 RNA in 45 min with one pair of inner primers. This method simplified the primer design and eliminated the need for reverse transcriptase, can be used for quantitative or qualitative DNA/RNA detection, and holds significant potential for application in clinical diagnostics.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.150825", "ptypes": [ "Journal Article" ], "first_author": "Xiang R", "dirs": [ "B", "C" ] }, "41745836": { "pmid": "41745836", "title": "Synergistic Cellular Toxicity from Inhibition of Poly(ADP-ribose) Glycohydrolase (PARG) and Ubiquitin-Specific Protease 1 (USP1).", "abstract": "Ubiquitin-specific protease 1 (USP1) is an emerging target for poly(ADP-ribose) polymerase 1 (PARP1) inhibitor-resistant and BRCA1/BRCA2 mutant tumors. USP1 is a deubiquitylating enzyme responsible for the removal of the mono-ubiquitin mark on FANCD2, PARP1, and the replication factor proliferating cell nuclear antigen (PCNA), among other proteins. USP1 facilitates proper PCNA-mediated polymerase switching from error-prone trans-lesion synthesis DNA polymerases to replicative DNA polymerases. Due to the critical role of USP1 in DNA synthesis and DNA repair, and the discovery that USP1 deubiquitylates PARP1, USP1 inhibitors (USP1i) were found to have a synthetic lethal relationship with PARP1 inhibitors (PARPi), suggesting a mechanistic link between poly(ADP-ribose) (PAR) dynamics and USP1-mediated ubiquitin hydrolysis. However, the relationship between USP1 inhibition and inhibitors of poly(ADP-ribose) glycohydrolase (PARGi), the primary enzyme responsible for PAR hydrolysis, has not been resolved. Using cell cytotoxicity, synergy, PCNA-ubiquitin, and PAR analyses, it is demonstrated herein that PARG inhibition, combined with USP1 inhibition, leads to increased levels of mono-ubiquitinated PCNA, decreased PAR accumulation, and synergistic cytotoxicity between ML323, a potent USP1i, and PDD00017273, a model PARGi. Future studies will focus on the mechanism that contributes to USP1/PARG synthetic lethality, the mechanism of cell death, and the impact of USP1 on PAR/ubiquitin dynamics and replication stress signaling.", "journal": "Toxics", "year": "2026", "doi": "10.3390/toxics14020162", "ptypes": [ "Journal Article" ], "first_author": "Leonard SM", "dirs": [ "B" ] }, "41950345": { "pmid": "41950345", "title": "A Droplet-Free and Oil-Free Microfluidics Based on Self-Pumping Hydrogel for Point-of-Care Multiplex Digital Nucleic Acid Detection.", "abstract": "Digital PCR (dPCR) enables rapid and accurate nucleic acid quantification and has been widely applied in clinical diagnostics, pathogen screening, environmental monitoring, and food safety. However, current dPCR platforms based on droplet microfluidic chip cannot been applied for in-field test and multiplexing, due to their complex fluidic manipulation. Here, we report a self-pumping hydrogel chip for multiplex digital nucleic acid detection that exploits the intrinsic water-absorbing behavior of hydrogel to drive autonomous fluid transport while simultaneously leveraging the cross-linking hydrogel network for digital amplification. This strategy eliminates external pumps and intricate microfabrication, allowing a fully passive operation with high sensitivity and single-copy quantitative accuracy. The ready-to-use hydrogel chip with pre-embedded reagents for isothermal loop-mediated isothermal amplification exhibits excellent storage stability and inhibitor tolerance. Furthermore, integration of four spatially separated detection zones allows simultaneous digital quantification of Vibrio parahemolyticus, Salmonella typhi, and Staphylococcus aureus in a single assay within 35 min on-site. This self-pumping hydrogel chip offers a simple, low-cost, and scalable platform that advances digital nucleic acid analysis toward portable and high-throughput point-of-care testing.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.6c00222", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Wang Y", "dirs": [ "B" ] }, "40261914": { "pmid": "40261914", "title": "Rational Design of UvsX Recombinase Variants for Enhanced Performance in Recombinase Polymerase Amplification Applications.", "abstract": "Homologous recombination is a vital biological process for DNA repair, genomic stability, and genetic diversity, driven by the RecA/Rad51 recombinase family. However, as a T4 bacteriophage recombinase homologous to RecA/Rad51, UvsX has limited in vitro performance during recombinase polymerase amplification (RPA) due to ATP utilization and DNA affinity. In this study, UvsX was rationally engineered to enhance these properties through homology modeling, virtual saturation mutations, and consensus mutation strategies. Targeted mutagenesis produced UvsX variants (E198N, E198R, E198K, and K35G) with a 16 ± 4% to 39 ± 6% improvement in RPA activity, while the double mutant K35G/E198R showed an increase of up to 43 ± 4%. Structural analysis revealed that the K35G/E198R mutation enlarged ATP-binding pockets and increased the positive surface potential of DNA-binding sites, resulting in a 12 ± 4% improvement in ATP utilization and more ADP and less AMP generated, a 10 ± 2% enhancement in DNA interaction compared to the wild-type, and better inhibitor tolerance. These findings establish a foundation for the rational optimization of recombinases in nucleic acid amplification and promote their potential for industrial RPA applications.", "journal": "Biochemistry", "year": "2025", "doi": "10.1021/acs.biochem.5c00098", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhang L", "dirs": [ "B" ] }, "40542116": { "pmid": "40542116", "title": "Polymerase theta expression is correlated with proliferative capacity but not with DNA repair deficiency status in solid tumors.", "abstract": "Polymerase theta (POLθ) inhibitors were developed to overcome resistance to PARP inhibitor treatment in homologous recombination (HR) deficient cancer. Biomarkers identifying PARP inhibitor-resistant cancer cases that specifically rely on POLθ activity for cancer cell survival will help to identify the sensitive patient population. From the TCGA RNAseq data, we determined POLθ expression levels, and from whole-exome and whole-genome sequencing data, we determined the number of POLθ-associated mutational signatures in solid tumors with various HR deficiency status. We found that POLθ expression levels did not differ significantly between HR-proficient and HR-deficient cancers. However, POLθ expression correlated strongly with proliferation-associated gene expression signatures and was predominantly observed in the S and G2/M phases of the cell cycle. POLθ-associated mutational signatures are correlated with POLθ expression levels only in BRCA2-deficient cancers. POLθ expression level and POLθ-associated mutational signatures may be indicative of POLθ inhibitor sensitivity in BRCA2-deficient tumors, but are unlikely to be informative in other cancers.", "journal": "NPJ precision oncology", "year": "2025", "doi": "10.1038/s41698-025-01000-w", "ptypes": [ "Journal Article" ], "first_author": "Sztupinszki Z", "dirs": [ "B" ] }, "42132973": { "pmid": "42132973", "title": "Developing a TaqMan loop-mediated isothermal amplification assay for Oryzias latipes detection.", "abstract": "Environmental DNA (eDNA) analysis requires highly sensitive and specific detection methods because target DNA is often present at low concentrations among diverse biological materials. Although LAMP is a rapid and robust amplification technique, conventional assays can generate nonspecific products. Incorporating TaqMan probes into LAMP reactions may enhance both specificity and sensitivity for ecological monitoring. We developed a TaqMan probe-based loop‑mediated isothermal amplification (TaqMan LAMP) assay to detect Oryzias latipes from eDNA. Under controlled laboratory conditions, the assay showed approximately tenfold higher analytical sensitivity than turbidimetric LAMP and detected template DNA at low copy numbers in triplicate tests; however, this lowest detected concentration represents a preliminary estimate rather than a statistically defined limit of detection. Using crudely extracted eDNA obtained via the suspended glass fiber method, both TaqMan LAMP and turbidimetric LAMP detected O. latipes. These results indicate that TaqMan LAMP can function with minimally processed eDNA, although the present field data do not demonstrate improved inhibitor tolerance or superior performance relative to turbidimetric LAMP. Specificity testing confirmed that fluorescence signals were observed only for O. latipes and not for the tested nontarget fish species. The TaqMan LAMP assay provides a sensitive and species‑specific approach for detecting O. latipes from environmental samples. Its compatibility with simple DNA extraction workflows highlights its potential for practical and rapid biomonitoring applications, although further field‑based evaluations will be required to clarify whether it offers performance advantages over existing LAMP methods.", "journal": "Molecular biology reports", "year": "2026", "doi": "10.1007/s11033-026-11946-0", "ptypes": [ "Journal Article" ], "first_author": "Fukuta S", "dirs": [ "B" ] }, "40308947": { "pmid": "40308947", "title": "Synergistic enhancement of PARP inhibition via small molecule UNI66-mediated suppression of BRD4-dependent transcription of RAD51 and CtIP.", "abstract": "Targeted therapy leveraging synthetic lethality in homologous recombination (HR)-defective tumors, particularly in BRCA-mutated tumors through poly(ADP-ribose) polymerase (PARP)-dependent repair inhibition, has shown success. However, the challenge lies in the ability of the tumors to reactivate HR via diverse mechanisms, leading to resistance against PARP-dependent repair inhibition. Addressing this issue, the down-regulation of HR activity has been explored as a potential strategy to overcome PARP inhibitor-resistant tumors. Yet, the intricate modulation of HR gene expression in mammalian cells is still not fully understood. In this study, we used a small molecule, UNI66, identified from high-throughput screening, to investigate regulatory mechanisms of HR. UNI66 was observed to induce synthetic lethality in PARP1-deficient cells and enhanced the sensitivity of multiple cancer cells to PARP inhibitors, suggesting a role in HR down-regulation. Mechanistically, UNI66 was found to interact with and inhibit BRD4 protein binding to the promoters of CtIP and RAD51 genes, resulting in the down-regulation of their transcription. This decrease in CtIP and RAD51 expression was associated with reduced HR activity, thereby increasing the sensitivity of tumors to PARP inhibitors. These findings indicate that BRD4-mediated transcriptional regulation of CtIP and RAD51 influences HR activity, which may have implications for overcoming resistance to PARP inhibitors.", "journal": "NAR cancer", "year": "2025", "doi": "10.1093/narcan/zcaf013", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Amarsanaa E", "dirs": [ "B" ] }, "42092962": { "pmid": "42092962", "title": "Natural diversity-guided catalytic-core chimerism engineers a rapid and inhibitor-tolerant reverse transcriptase.", "abstract": "Reverse transcriptases (RTs) are essential components of RNA-based molecular technologies, yet their practical performance is often constrained by trade-offs between catalytic speed, thermal stability, and tolerance to inhibitory substances. Conventional optimization via mutagenesis or directed evolution often improves individual traits but struggles to integrate multiple traits within a single enzyme. Here, we applied catalytic-core recombination, guided by natural sequence diversity and diagnostic performance criteria, to recover reverse transcriptase phenotypes that would be difficult to obtain by single-trait optimization alone. Starting from 1,028 sequences, 24 representative RT variants were used to generate a library of chimeras by swapping a critical 405-residue polymerase domain into a validated M-MuLV scaffold. We identified chRT V18 as the lead candidate, demonstrating consistent activity across a broad temperature range (40-70 °C). In addition, chRT V18 enabled efficient and linear cDNA synthesis in as little as 1 min at elevated temperatures, while maintaining strong resistance to clinically relevant inhibitors. Beyond RT-qPCR, chRT V18 supported high-temperature RT-LAMP at 69 °C, a regime typically incompatible with conventional RTs, enabling improved specificity and rapid amplification. When formulated into one-step RT-qPCR master mixes, chRT V18 achieved low limits of detection and full concordance with IVD-approved assays, while reducing reverse transcription time by 90%, enabling shorter time-to-result. Natural diversity-guided catalytic-core chimerism enabled the development of a rapid, inhibitor-tolerant reverse transcriptase with an unusual combination of broad temperature compatibility, minute-scale reverse transcription, and resilience to clinically relevant inhibitors. Beyond the properties of chRT V18 itself, the results support modular recombination of evolutionarily optimized domains as a practical engineering strategy for integrating multiple performance traits in complex enzymes.", "journal": "Journal of biological engineering", "year": "2026", "doi": "10.1186/s13036-026-00692-3", "ptypes": [ "Journal Article" ], "first_author": "Fonseca Costa I", "dirs": [ "B" ] }, "40029391": { "pmid": "40029391", "title": "Response to PARP inhibitor in EGFR-tyrosine kinase inhibitor-resistant metastatic lung adenocarcinoma with germline PALB2 mutation.", "abstract": "Tumors with homologous recombination deficiency (HRD) can benefit from treatment with poly ADP-ribose polymerase inhibitors (PARPi). However, the methods for identifying HRD vary and are controversial. Several DNA repair genes in the homologous recombination repair pathway may be linked to PARPi susceptibility, and studies are underway to identify biomarkers that can predict the response to PARPi. We present a case of EGFR-tyrosine kinase inhibitor-resistant metastatic lung adenocarcinoma with a germline PALB2 mutation that was treated with fluzoparib (an orally administered PARPi). The treatment achieved surprising results and lasted for more than 4.5 months. Our study provided evidence that metastatic lung adenocarcinoma with germline PALB2 could benefit from PARPi, which improves patient outcomes.", "journal": "Anti-cancer drugs", "year": "2025", "doi": "10.1097/CAD.0000000000001712", "ptypes": [ "Journal Article", "Case Reports", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhu C", "dirs": [ "B" ] }, "42177699": { "pmid": "42177699", "title": "Translational prioritization of strategies to overcome poly ADP-ribose polymerase inhibitor resistance in breast cancer based on systematic review of preclinical evidence.", "abstract": "Resistance to poly ADP-ribose polymerase (PARP) inhibitors has emerged as a major obstacle to durable clinical benefit in breast cancer. While multiple preclinical strategies have been proposed, there is a lack of translational frameworks that prioritize interventions based on mechanistic robustness, biomarker relevance, and strength of evidence. We performed a systematic and evidence-informed synthesis of 22 preclinical studies investigating therapeutic approaches to overcome PARP inhibitor resistance. Studies were evaluated across experimental models, mechanistic endpoints, and therapeutic outcomes. Risk of bias was assessed using the SYRCLE and ROBINS-I tools, and the consistency and certainty of evidence were appraised using the GRADE framework to support translational prioritization. Epigenetic targeting via EZH2 inhibition and immune microenvironment modulation through STING pathway activation consistently demonstrated high-quality evidence for tumor suppression, survival benefit, and immunologic activation in BRCA1-mutant models. In contrast, strategies aimed at restoring homologous recombination exhibited strong in vitro synergy but limited in vivo translatability, underscoring pharmacokinetic and model-dependent constraints. This review advances an evidence-based translational prioritization framework to guide the clinical development of combination strategies for PARP inhibitor-resistant breast cancer. We identify critical gaps in biomarker integration, standardized resistance modeling-particularly in BRCA wild-type disease-and dynamic monitoring of resistance evolution, and propose key directions for future preclinical and early-phase clinical research.", "journal": "Discover oncology", "year": "2026", "doi": "10.1007/s12672-026-05288-3", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shen K", "dirs": [ "B" ] }, "41467381": { "pmid": "41467381", "title": "Integrated Engineering of T4 gp32 Achieves Synergistic Improvements in Thermostability, DNA Binding, and Diagnostic Robustness for Enhanced Nucleic Acid Amplification.", "abstract": "Single-stranded DNA-binding protein gp32 is essential for DNA replication and widely used in isothermal amplification techniques such as recombinase polymerase amplification (RPA). However, its inherent thermal instability and reaction condition sensitivity restrict its broader applications. Here we develop the Fermentation Integrated Rational Engineering (FIRE-3S) strategy to enhance the functional properties of gp32 through molecular dynamics (MD) simulations, structure-guided fermentation tuning and stability-focused rational design. Starting from a disulfide-bonded mutant (V62C/T80C) that showed enhanced RPA performance but reduced thermostability, MD simulations revealed local structural disruption near the Zn2+-binding motif. Then, structure-guided fermentation tuning was applied, which restored structural integrity and enhanced DNA-binding affinity. Building on 2S-step, a computationally guided clique-based design pipeline rapidly identified a multisite mutant, gp32-M1, with elevated thermostability (ΔTm = +6.03 °C), robust ssDNA-binding, and strong inhibitor tolerance─maintaining >65% activity under 4% isopropanol or ethanol. This work establishes a systematic framework for engineering ssDNA-binding proteins with enhanced biostability and environmental resilience for molecular diagnostics.", "journal": "Biomacromolecules", "year": "2026", "doi": "10.1021/acs.biomac.5c02469", "ptypes": [ "Journal Article" ], "first_author": "Zhang L", "dirs": [ "B" ] }, "41635070": { "pmid": "41635070", "title": "Development of real-time PCR and droplet digital PCR assays for the simultaneous quantification of bacterial and human mitochondrial DNA for forensic analysis.", "abstract": "Forensic evidence recovered from crime scenes often contains a mixture of human and bacterial DNA. Although short tandem repeat (STR) profiling of genomic DNA (gDNA) is widely used for human identification, its effectiveness can be limited in cases involving highly degraded DNA. In such cases, human mitochondrial DNA (mtDNA) and microbiome analysis may serve as alternative methods. In this study, we developed a multiplex quantification assay targeting the bacterial 16S rRNA V7 region and the human mitochondrial NADH-dehydrogenase subunit 5 (ND5) gene. Quantification was performed using TaqMan-based real-time PCR (Human-Bacteria qPCR; HBQ) and droplet digital PCR (Human-Bacteria ddPCR; HBD). Optimal primer and probe concentrations were at 7 μM for the HBQ assay, and 5 μM bacterial primer set, 7 μM human mtDNA primer set, and 700 nM probes for the HBD assay. Sensitivity testing showed that the HBQ assay detected all DNA samples-except G147A-down to 20 fg, while the HBD assay detected both bacterial and human DNA at 20 fg, demonstrating higher analytical sensitivity than the real-time PCR method. Moreover, mock forensic samples were analyzed to confirm the assay applicability, and PCR inhibitor tolerance tests using humic acid and tannic acid were conducted to further validate their performance. Furthermore, the HBQ and HBD assays may be used in quality control processes for samples potentially affected by bacterial DNA or human mtDNA contamination and could also be applied to other fields such as food safety, environmental science, and biological research involving microbial DNA and human mtDNA.", "journal": "Journal of forensic sciences", "year": "2026", "doi": "10.1111/1556-4029.70276", "ptypes": [ "Journal Article" ], "first_author": "Lee JW", "dirs": [ "B" ] }, "41843372": { "pmid": "41843372", "title": "Research progress on direct PCR and its applications.", "abstract": "Direct Amplification Technology (Direct PCR) is derived from conventional PCR and eliminates the cumbersome DNA extraction process, thereby enhancing detection efficiency and accuracy. Combined with evolving PCR-derived techniques, it has found extensive applications across multiple fields. This article provides an overview of the development process, fundamental principles, basic procedures, and derivative technologies of direct PCR. It further details its applications in testing various samples including blood, microbial specimens, and cellular/tissue samples, offering foundational knowledge to support the popularization and further advancement of this technology.", "journal": "Analytical sciences : the international journal of the Japan Society for Analytical Chemistry", "year": "2026", "doi": "10.1007/s44211-026-00893-2", "ptypes": [ "Journal Article", "Review" ], "first_author": "Xu T", "dirs": [ "B" ] }, "39964178": { "pmid": "39964178", "title": "Rapid and sensitive isolation of Campylobacter jejuni using immunomagnetic separation from patient specimens exposed to oxygen.", "abstract": "This study describes a method for detecting Campylobacter jejuni in patient stools with subsequent isolation using antibody-magnetic beads in conjunction with selective culture and PCR. Monoclonal antibodies specific for the flagellin A and major outer membrane protein of C. jejuni were generated; two clones (1C7 and 4B2) were used to coat magnetic beads for immunomagnetic separation (IMS). C. jejuni strain NCTC11168 was recovered from human stool samples spiked with varying concentrations (101-105 CFU/mL) by Campylobacter (Campy)-IMS or a conventional culture-based method and plated on modified charcoal-cefoperazone-deoxycholate agar; the number of colonies was enumerated. The detection limits of Campy-IMS and conventional culture-based method with spiked stool samples were 102 and 104 CFU/mL, respectively. The sensitivity of IMS-PCR was 10-10,000-fold higher than that of direct PCR. The recovery rate of C. jejuni from spiked stools stored for 12 to 72 h decreased from 72.3 to 5.9% with Campy-IMS and from 48.5 to 0.1% with the conventional culture-based method. Importantly, of 20 PCR (+)/bacterial culture (-) samples that were diagnosed as probable cases according to general criteria, 95% (19/20) were confirmed positive by Campy-IMS. Thus, this study suggests a solution to overcome the problems caused by the inconsistency between probable and confirmed cases of Campylobacter infection. The isolation, cultivation, and maintenance of Campylobacter spp. are difficult because of the microaerophilic conditions and specific medium needed. Although selective media are useful for the initial isolation of Campylobacter, subsequent exposure of the sample to oxygen has a detrimental effect on the positive culture rate of Campylobacter, significantly lowering the isolation rate from patient samples. In this study, the detection limit was improved by combining immunomagnetic separation and PCR methods to quickly detect Campylobacter jejuni in clinical patient stool samples using antibody-magnetic beads. Therefore, this study is expected to improve confirmation of C. jejuni infection where diagnosis would previously fail with patient samples because of oxygen exposure, inappropriate diagnostic methods, and interference from other bacteria in the sample.", "journal": "Microbiology spectrum", "year": "2025", "doi": "10.1128/spectrum.01907-24", "ptypes": [ "Journal Article", "Evaluation Study", "Research Support, N.I.H., Extramural" ], "first_author": "Kim SY", "dirs": [ "B" ] }, "39953527": { "pmid": "39953527", "title": "Performance of cellulose-based card for direct genetic testing of spinal muscular atrophy.", "abstract": "Spinal muscular atrophy (SMA) is a devastating neuromuscular condition resulting from the loss of the survival motor neuron 1 (SMN1) gene. Precise genetic testing has become essential after the authorization of several potent medications. To achieve this objective, the use of dried blood spot (DBS) has assured convenient and extensive testing from a distance. Nevertheless, developing countries such as Indonesia sometimes lack access to standard filter papers like FTA or Guthrie cards for DBS processing. Here, we aim to develop a cellulose-based card as an alternative filter paper for DBS preparation suitable for the genetic testing of SMA including but not limited to a direct polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex allele-specific amplification (multi-ASA). An in-house paper was developed from a 180 gsm cellulose-based paper and was used for DBS preparation. The performance of dried blood spotted on the cellulose-based card (DBSc) was compared to pure genomic DNA (gDNA) isolate and dried blood spotted on FTA cards (DBSf) for genetic testing. The results of the genetic testing of our cellulose-based card were completely matched with those of gDNA and DBSf in both direct PCR-RFLP and Multi-ASA to separate SMN1 from SMN2. In addition, after three months of storing, the DBSc continued to exhibit a clear result, suggesting its high stability for DNA storage. Our cellulose-based card has the potential to be used for DBS carrier and for further genetic testing using PCR. Our findings can assist physicians in sending DBS samples from SMA suspicion cases to genetic testing centers, thereby preventing diagnosis delay or misdiagnosis.", "journal": "BMC biotechnology", "year": "2025", "doi": "10.1186/s12896-024-00938-2", "ptypes": [ "Journal Article" ], "first_author": "Wijaya YOS", "dirs": [ "B" ] }, "42801370": { "pmid": "42801370", "title": "Molecular identification and resistance-associated gene profiling of non-tuberculous mycobacteria isolated from bovine clinical samples.", "abstract": "Non-tuberculous mycobacteria (NTMs) are environmental opportunistic pathogens capable of producing tuberculosis-like lesions in bovines, potentially complicating the diagnosis and surveillance of bovine tuberculosis. Molecular data on resistance-associated genes in bovine NTMs from India remain limited. This study aimed to isolate and characterize NTMs from bovine clinical samples and to investigate selected resistance-associated genes at molecular level. A total of 193 samples comprising faeces (n = 101), nasal swabs (n = 21), trans-tracheal washes (n = 13), tissues (n = 30), and blood (n = 28) were analysed using direct PCR and culture-based isolation. Genus-level identification was performed using hsp65 PCR followed by species-specific PCR assays. Eighteen confirmed NTM isolates were screened for resistance-associated genes (rpoB, katG, rpsL, and gyrA) and selected amplicons were sequenced. Statistical analysis was conducted using chi-square or Fisher's exact test. Mycobacterium vaccae predominated among isolates (66.7%). The rpoB gene was detected in 88.9% of isolates and was mainly associated with M. vaccae. The katG (16.7%) and gyrA (5.6%) genes were detected exclusively in M. vaccae, while rpsL gene was not amplified in any isolate. Sequence analysis showed no mutations in rpoB or katG hotspots, but non-synonymous substitutions were detected in quinolone resistance-determining region of gyrA. Resistance-associated genes were predominantly detected in M. vaccae, with rpoB most frequent, followed by katG and gyrA. These findings indicate genotypic variation only and cannot confirm phenotypic resistance because susceptibility testing was not performed. Further, phenotypic and species-specific analyses are required to establish functional significance.", "journal": "Molecular biology reports", "year": "2026", "doi": "10.1007/s11033-026-12814-7", "ptypes": [ "Journal Article" ], "first_author": "Panigrahi B", "dirs": [ "B" ] }, "42255530": { "pmid": "42255530", "title": "Evaluation of XRCC4 and VEGF Gene Variants in Relation to Breast Cancer Risk in a Jordanian Cohort.", "abstract": "Breast cancer develops through complex interactions of genetic and environmental influences. XRCC4 is a critical DNA repair gene that maintains genomic stability and is implicated in carcinogenesis, while VEGF is a key regulator of angiogenesis that plays an essential role in tumor progression and recurrence. The present study sought to evaluate the contribution of XRCC4 (rs28360071) and VEGF (rs35569394) polymorphisms to breast cancer susceptibility in a Jordanian Arab cohort. This case-control study included 300 breast cancer patients and 300 healthy individuals. Peripheral blood samples were collected for genomic DNA extraction. XRCC4 (rs28360071) and VEGF (rs35569394) polymorphisms were genotyped using direct PCR, and their associations with breast cancer risk were analyzed using appropriate statistical methods. The XRCC4 (rs28360071) polymorphism exhibited significant differences in allelic and genotypic distributions between breast cancer cases and control individuals in both unadjusted (p = 0.01) and adjusted analyses (p = 0.005), with the DD genotype associated with reduced breast cancer risk. The findings suggest that XRCC4 (rs28360071) is associated with breast cancer susceptibility among Jordanian Arab women, whereas VEGF (rs35569394) does not appear to influence risk. These results indicate a potential role for XRCC4 variation in breast cancer susceptibility in this population; however, this finding should be cautiously interpreted, as further research is required to confirm the association.", "journal": "Cancer management and research", "year": "2026", "doi": "10.2147/CMAR.S595329", "ptypes": [ "Journal Article" ], "first_author": "Al-Eitan L", "dirs": [ "B" ] }, "42524532": { "pmid": "42524532", "title": "Genetic Variants in the MTHFR and WNK1 Genes and Their Contribution to Hypertension Susceptibility.", "abstract": "Hypertension (HTN) is a common and complex disorder influenced by multiple genetic and environmental factor, where the underlying mechanisms of its etiology remain incompletely understood although the identification of several contributing elements. This research sought to evaluate the possible relationship between genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR) and With-No-Lysine Kinase 1 (WNK1) genes and susceptibility to hypertension. Genomic DNA was extracted from blood samples collected from 220 individuals with hypertension and 220 normotensive controls. Genotyping of MTHFR (rs1801133 and rs1801131) and WNK1 (AluYb8) polymorphisms was performed using direct PCR and PCR-RFLP techniques. The resulting data were subjected to appropriate statistical analyses. A statistically significant association was identified between the rs1801131 polymorphism of the MTHFR gene and susceptibility to hypertension (p = 0.0006). This association remained significant under the codominant, dominant, and recessive genetic models, with all p-values < 0.016. Furthermore, the CC haplotype of the MTHFR gene showed a significant association with hypertension (OR = 2.02, p = 1e-04). These findings indicate that the MTHFR rs1801131 polymorphism is significantly associated with hypertension susceptibility and may represent a potential genetic marker. This highlights its relevance for future studies exploring genotype-driven risk assessment and personalized approaches to hypertension management.", "journal": "International journal of medical sciences", "year": "2026", "doi": "10.7150/ijms.133244", "ptypes": [ "Journal Article" ], "first_author": "Al-Eitan LN", "dirs": [ "B" ] }, "41549071": { "pmid": "41549071", "title": "Direct PCR-Based VNTR Analysis of TPO Intron 10 for Rapid Detection of Maternal Cell Contamination in Prenatal Diagnosis.", "abstract": "Maternal cell contamination (MCC) in fetal specimens poses a major risk for misdiagnosis in prenatal genetic testing. Standard variable number tandem repeat (VNTR) analysis of the thyroid peroxidase (TPO) gene intron 10 is informative for MCC detection but traditionally requires DNA extraction, limiting its use in resource-limited laboratories. This study aimed to develop and validate a direct PCR-based VNTR assay for MCC detection without DNA extraction. Leftover whole blood and amniotic fluid specimens from 90 families undergoing prenatal diagnosis for severe thalassemia were analyzed, with white blood cell and amniotic fluid pellets directly subjected to PCR amplification of the TPO intron 10 VNTR locus using a high-pH buffer to overcome PCR inhibitors. The direct PCR results were compared with standard DNA-based VNTR analysis in a blinded study, and VNTR heterozygosity at the TPO locus was also assessed in 148 unrelated Thai individuals to evaluate marker informativeness. Among 253 specimens, informative VNTR patterns were observed in 65 families (72.2%), with MCC ruled out in 64 families (71.1%) and detected in 1 family (1.1%). Uninformative VNTR patterns occurred in 25 families (27.8%) due to maternal homozygosity or similarity between maternal and fetal VNTRs. Direct PCR showed 100% concordance with standard DNA-based VNTR analysis. The heterozygosity rate of the TPO intron 10 VNTR was 87.8% among unrelated Thai individuals, supporting its suitability for routine MCC screening. The developed direct PCR protocol is a practical, rapid, and reliable tool for MCC detection, streamlining laboratory workflows and ensuring accurate prenatal diagnosis in low-resource settings.", "journal": "Clinical and translational science", "year": "2026", "doi": "10.1111/cts.70474", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Wichian P", "dirs": [ "B" ] }, "41117598": { "pmid": "41117598", "title": "Evaluation of a sample-to-answer real-time PCR assay for enterovirus detection in cerebrospinal fluid.", "abstract": "Enteroviruses frequently cause aseptic meningitis, necessitating differentiation from bacterial meningitis to avoid unnecessary antimicrobial treatment and improve patient management. Polymerase chain reaction (PCR) remains the gold standard for enterovirus detection in cerebrospinal fluid (CSF) due to its high sensitivity and rapid turnaround time. The discontinuation of the Cepheid Xpert EV assay has created a gap in diagnostics. This study aimed to optimize two laboratory-developed real-time PCR assays using Diasorin direct amplification disc (DAD) and universal disc (UD) methods compared to the previously standard Cepheid Xpert EV assay. A total of 87 clinical CSF specimens were tested to assess sensitivity, specificity, and overall performance. There was no significant difference between the performance of the three assays compared to standard of care results. The Xpert EV and DAD methods had a 79.6% positive agreement, while the UD method had an 86.4% positive agreement. All methods achieved 100% negative agreement. Specificity testing against non-enteroviral pathogens confirmed no cross-reactivity. The estimated limit of detection was 100 copies/mL for Xpert EV, 250 copies/mL for UD, and 1,000 copies/mL for DAD, though clinical sensitivity remained high. Blood contamination affected the DAD assay but not the UD method due to its RNA extraction step. This study demonstrates the potential of DAD and UD methods as viable alternatives for rapid enterovirus detection in CSF, essential for patient management following the discontinuation of the Xpert EV assay. Further validation and comparison are needed for broader clinical applications.IMPORTANCERapid and accurate detection of enteroviruses in cerebrospinal fluid is crucial for patient management in cases of aseptic meningitis, especially following the discontinuation of the Cepheid Xpert EV assay. This study evaluates two laboratory-developed real-time PCR assays utilizing Diasorin direct amplification disc and universal disc methods, demonstrating their potential as viable alternatives for enterovirus detection with high sensitivity and specificity.", "journal": "Journal of clinical microbiology", "year": "2025", "doi": "10.1128/jcm.00864-25", "ptypes": [ "Journal Article", "Evaluation Study", "Research Support, Non-U.S. Gov't" ], "first_author": "Perkins G", "dirs": [ "B" ] }, "41502675": { "pmid": "41502675", "title": "Innovative Rapid Detection System for Nondeletion-Type α‑Thalassemia via Whole-Blood Amplification and Microfluidic Technology.", "abstract": "Thalassemia represents a major global public health challenge. This study developed an integrated detection system combining direct PCR-(Dir-PCR) with reverse dot blot hybridization (RDB), enabling accurate and rapid diagnosis of nondeletion α-thalassemia (αWSα, αCSα, αQSα). The core innovation lies in the systematic optimization of the Dir-PCR-RDB methodology, establishing an efficient amplification and hybridization system capable of directly utilizing whole blood samples, thereby effectively bypassing conventional nucleic acid extraction steps. The optimized method tolerates approximately 24% (12 μL/50 μL) whole blood, achieves a sensitivity of 1.5 copies/μL, and demonstrates no cross-reactivity with other thalassemia variants. Clinical validation showed 100% concordance with sequencing results. Furthermore, by integrating this optimized system with microfluidic technology, we constructed a closed-tube \"sample-in, result-out\" platform that reduces total detection time to 122 min. This work not only provides an efficient and cost-effective solution for nondeletion α-thalassemia screening but also establishes a technological paradigm for rapid molecular diagnosis of other genetic variants and pathogens.", "journal": "ACS omega", "year": "2025", "doi": "10.1021/acsomega.5c08718", "ptypes": [ "Journal Article" ], "first_author": "Wei Y", "dirs": [ "B" ] }, "40261456": { "pmid": "40261456", "title": "Direct amplification and sequencing of enterovirus genomes from clinical and wastewater samples reveals uncommon virus types in Cameroon.", "abstract": "Knowledge about the genetic diversity of enteroviruses in sub-Saharan Africa remains biased by the fact that it has mainly been described through the characterization of specimens isolated in cell cultures. The aim of this study was to characterize enteroviruses that circulate among humans in Cameroon by focusing more specifically on respiratory enteroviruses and on enteroviruses that are non-cultivable or non-cytopathogenic in standard cell lines. For this purpose, RNA was extracted from nasopharyngeal swabs, stool suspensions, and sewage concentrates collected in Cameroon from January 2018 to December 2019, and the RNA extracts were subjected to molecular screening to detect enterovirus genomes, which were subsequently sequenced using Illumina technology. Overall, 81 strains of 32 virus types were identified. Most of the strains (61.7%) belonged to the species Enterovirus coxsackiepol, followed by members of the species Enterovirus betacoxsackie (21%), Enterovirus alphacoxsackie (14.8%), and Enterovirus deconjuncti (2.5%). Almost all of the strains were found in sewage concentrates (79%) and stool suspensions (18.5%). Only two EV-D68 strains were identified in nasopharyngeal swabs. Interestingly, several virus types that have rarely been reported were detected, including non-cultivable/non-cytopathogenic EV-C strains, among which was a new virus type assigned as EV-C119. These results highlight the fact that virus isolation and unbiased cell-culture-free approaches complement each other to provide a more complete view of the enterovirus landscape in humans.", "journal": "Archives of virology", "year": "2025", "doi": "10.1007/s00705-025-06296-4", "ptypes": [ "Journal Article" ], "first_author": "Modiyinji AF", "dirs": [ "B" ] }, "41560289": { "pmid": "41560289", "title": "Extraction-Free Scalable Sample Preparation for the Rapid Detection of HIV in Blood Plasma.", "abstract": "Nucleic acid amplification tests that are rapid and low-cost are needed for expanding access to point-of-care (POC) diagnostics for infectious diseases and viral load monitoring. Several companies have commercialized POC devices using rapid isothermal amplification; however, these tests use nasal swabs or saliva samples. Translating these tests to blood plasma is challenging due to clotting factors, heat- or pH-induced coagulation of human albumin, high concentrations of ribonucleases (RNases), and other sample matrix inhibitors. Crude, extraction-free sample preparation methods are promising alternatives that reduce the complexity of sample preparation. In this paper, we present an extraction-free sample preparation method for in-tube and paper-based reverse transcription recombinase polymerase amplification for the detection of HIV in blood plasma samples. We use a rapid heat treatment protocol with a reducing agent to inhibit RNases and lyse HIV virus, enabling RNA preservation into direct amplification without the need for complex RNA purification steps. We demonstrate sample treatment in 5 min and amplification within 20 min in reaction volumes ranging from 50 to 300 μL and show that large volume reactions achieve a limit of detection of 800 copies/mL (16 copies/reaction) for HIV-positive plasma in both paper and tube formats.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c05443", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Shimazu KN", "dirs": [ "B" ] }, "40632864": { "pmid": "40632864", "title": "High-throughput multiplexed gene and cell doping analysis through CRISPR-Cas12a system integrated with blood direct PCR.", "abstract": "Advancements in gene and cell therapies introduce \"gene and cell doping,\" requiring efficient and sensitive detection methods. Here, we report a high-throughput multiplexed gene and cell doping analysis (HiMDA) using CRISPR-Cas12a system integrated with blood direct polymerase chain reaction (PCR). Blood direct PCR enables simultaneous amplification of multiple exogenous genes directly from whole-blood samples. Coupled with sequence-specific DNA recognition and fluorescence reporter system, HiMDA achieves multiplexed, on-target detection of doping genes and cells. Our results demonstrate HiMDA's feasibility with only 5 microliters of blood required for the entire 90-minute process. HiMDA exhibits exceptional sensitivity, detecting as few as 2.5 copies of doping target genes from blood-four times more sensitive than current anti-doping standards-and identifying in vivo doping up to 10 days. These findings highlight HiMDA's robust high-throughput, multiplexed capabilities, satisfying the sensitivity and selectivity demands of anti-doping research. HiMDA offers a flexible solution to meet future doping detection challenges.", "journal": "Science advances", "year": "2025", "doi": "10.1126/sciadv.adv7234", "ptypes": [ "Journal Article" ], "first_author": "Yi JY", "dirs": [ "B" ] }, "41456244": { "pmid": "41456244", "title": "First molecular identification and genetic diversity of Giardia duodenalis and Blastocystis sp. in patients with multiple sclerosis in Iran.", "abstract": "Giardia duodenalis (G. duodenalis) and Blastocystis sp. are common intestinal protists with potential immunomodulatory effects. This study investigated their epidemiology, genetic diversity, and associated risk factors in patients with multiple sclerosis (MS) in western Iran, a population with immune dysregulation that may be vulnerable to opportunistic infections. From June 2022 to November 2024, a cross-sectional study was conducted on 116 MS patients attenting neurology clinics in Sanandaj, Iran. Stool samples were analyzed using nested PCR targeting the triosephosphate isomerase(tpi) gene for G. duodenalis and direct PCR targeting the small subunit ribosomal RNA (ssu rRNA) gene for Blastocystis sp. All PCR-positive samples were sequenced. Phylogenetic analysis was performed using the Maximum Likelihood method. Multivariate logistic regression was used to identify risk factors. The prevalence of G. duodenalis and Blastocystis sp. was 9.48% (11/116) and 12.06% (14/116), respectively. This prevalence is notable compared to some reports in the general Iranian population. Genotyping revealed G. duodenalis assemblages A (36.36%, 4/11) and B (63.64%, 7/11), and Blastocystis sp. subtype 1 (ST1) (28.6%, 4/14), ST2 (35.7%, 5/14), and ST3 (35.7%, 5/14). Multivariate analysis identified younger age (< 30 years; adjusted odds ratio [AOR] = 6.10, p = 0.007) and animal contact (AOR = 5.85, p = 0.030) as significant risk factors for G. duodenalis, while female sex (AOR = 8.31, p = 0.009) and animal contact (AOR = 4.79, p = 0.047) were significant for Blastocystis sp. Phylogenetic analysis confirmed the genetic diversity and suggested zoonotic potential of the identified isolates. This first molecular study of G. duodenalis and Blastocystis sp. in Iranian MS patients highlights a notable prevalence of these parasites. The significant association with animal contact and the identification of assemblages/subtypes with low host specificity underscore a potential zoonotic risk. These findings suggest that such infections may be underrecognized in MS populations and warrant consideration in clinical management, including potential screening and public health interventions to mitigate risk.", "journal": "Molecular biology reports", "year": "2025", "doi": "10.1007/s11033-025-11370-w", "ptypes": [ "Journal Article" ], "first_author": "Shafiee M", "dirs": [ "B" ] }, "40348313": { "pmid": "40348313", "title": "A sample-in- result -out microfluidic system for β-thalassemia diagnostics via direct whole blood PCR-reverse dot blot.", "abstract": "Existing thalassemia detection methods demand high - end labs and have complex procedures, leading to long testing cycles. This study aims to develop a convenient detection method integrated with a microfluidic platform for a sample - to - result process. First, optimal conditions for the whole blood direct PCR - reverse dot hybridization (dPCR - RDB) system were explored. Then, its performance was evaluated with clinical samples. Finally, the entire process was integrated into a palm - sized microfluidic chip for \"sample - in, result - out\" detection. A stable dPCR - RDB system was established. Clinical verification on 149 samples showed a 0.1 μl whole - blood minimum detection limit, 100 % specificity, and resistance to high triglyceride and bilirubin levels. It had 100 % positive and negative coincidence rates with traditional methods (kappa = 1). The microfluidic - integrated platform achieved \"sample - in, result - out\" with 0.5-1 μl blood in 130 min, sans a PCR lab. A \"sample - in, result - out\" microfluidic gene detection platform using whole blood as the template was successfully established.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2025", "doi": "10.1016/j.cca.2025.120348", "ptypes": [ "Journal Article" ], "first_author": "Ye C", "dirs": [ "B" ] }, "41310493": { "pmid": "41310493", "title": "Comparison of different methods for the detection of HLA-B* 57:01 allele in people living with HIV in Eastern Uttar Pradesh, India.", "abstract": "The gold standard for detecting HLA-B*57:01 allele linked to Abacavir hypersensitivity is sequence-based typing (SBT), which is labour-intensive, not readily available and needs special expertise. HCP5 gene polymorphism is a surrogate marker for HLA-B*57:01 allele. Therefore, we compared, nested PCR for HLA-B*57:01 allele, HCP5 PCR with SBT method to develop a simple and cost-effective method for detection of HLA-B*57:01 allele. Whole blood cells were collected from PLHIV and Buffy coat and DNA was isolated. Direct PCR for HLA-B*57 gene followed by nested PCR for the HLA-B* 57:01 allele and HCP5 gene PCR was done in all patients. The PCR results of both methods were confirmed by the SBT method. Among 366 samples, 25 (6.83%) were positive for HLA-B* 57:01 allele by nested PCR. HCP5 gene PCR was positive in 25 (6.83%) individuals who were also positive for the HLA-B*57:01 allele with nested PCR. A strong correlation was observed between the HLA-B*57:01 allele and HCP5 with negative and positive predictive values of 100% respectively in our population. Nested (two-step) and HCP5 gene PCR (single-step) are simple, rapid and cost-effective methods for HLA-B*57:01 allele detection and either can be adapted in the ART centre located in resource-poor settings.", "journal": "BMC infectious diseases", "year": "2025", "doi": "10.1186/s12879-025-11790-w", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "Rai TK", "dirs": [ "B" ] }, "40517580": { "pmid": "40517580", "title": "Molecular detection of antibiotic-resistant Campylobacter spp. among broiler and layer chickens in Egypt: An outlook from a One Health concept.", "abstract": "Campylobacter is a zoonotic pathogen that poses a public health concern, with poultry acting as the main reservoir of human infection. This study aimed to look into the prevalence and risk factors associated with antibiotic-resistant Campylobacter spp. found in chicken cloacal swabs using direct polymerase chain reaction (PCR) detection, highlighting their importance for public health. In this study, 108 pooled chicken cloacal swabs from 270 broilers and 270 layers were included. Following DNA extraction, the extracted DNA was examined for the presence of Campylobacter 16S rRNA, along with the mapA gene in Campylobacter jejuni (C. jejuni) and the ceuE gene in Campylobacter coli (C. coli). Next, the key antibiotic resistance genes (gyrA Thr-86-Ile mutation for C. jejuni, gyrA Thr-86-Ile mutation for C. coli, tetO, cmeB, and blaOXA-61) were looked for in the pools that tested positive for Campylobacter. The prevalence rate of C. jejuni and C. coli was 12 % and 1.9 %, respectively, with 0.9 % carrying both species. TetO was the most common antibiotic resistance gene, found in 75 % of both broilers and layers, followed by blaOXA-61, which was found in 50 % of broilers and 12.5 % of layers, while the gyrA Thr-86-Ile mutation and cmeB genes were found in broilers at rates of 37.5 % and 25 %, respectively. The phylogenetic analysis of the obtained Campylobacter 16S rRNA sequences harbouring two or more antibiotic resistance genes reveals a genetic relatedness to Campylobacter strains isolated from chickens, animals, and humans, highlighting their public health significance. In conclusion, direct PCR detection of antibiotic-resistant Campylobacter in chicken feces provides better insights into the prevalence of antibiotic-resistant Campylobacter spp. among chickens in Egypt.", "journal": "Research in veterinary science", "year": "2025", "doi": "10.1016/j.rvsc.2025.105762", "ptypes": [ "Journal Article" ], "first_author": "Shaheed I", "dirs": [ "B" ] }, "41489819": { "pmid": "41489819", "title": "QuickDraw: detecting HIV in whole blood using an integrated paper-based consumable that enables direct amplification of purified RNA from paper.", "abstract": "A definitive diagnosis of HIV typically requires a positive nucleic acid test. Limited access to these tests means that initiation of anti-HIV therapy is delayed or does not occur in a significant part of the world. While rapid antigen tests are more broadly available, these are insufficient for diagnosis on their own. To address the challenge of improving access to HIV testing, we have developed a passive, paper-based microfluidic sample preparation device we term the QuickDraw. We demonstrate that QuickDraw efficiently processes HIV-containing finger stick-quantities of whole blood to yield purified viral RNA. The output of the QuickDraw is then used as input for a colorimetric reverse transcriptase - loop-mediated isothermal amplification (RT-LAMP) assay. Coupled with sample preparation conducted with the QuickDraw, the assay demonstrated a limit of detection of 1000 copies per mL. QuickDraw simplifies viral nucleic acid sample preparation and detection by dramatically reducing the amount of equipment needed, suggesting it could be suitable for deployment in clinical and low-resource settings. By decentralizing nucleic acid testing, the QuickDraw platform has the potential to expand access to nucleic acid diagnostics in low-middle income countries (LMICs), while also supporting the UNAIDS goals for HIV detection, leading to wider access to treatment and reduced community transmission. It is also a significant step towards the goal of a simple-to-use nucleic acid-based HIV self-test.", "journal": "Lab on a chip", "year": "2026", "doi": "10.1039/d5lc00897b", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Evans AL", "dirs": [ "B" ] }, "41666443": { "pmid": "41666443", "title": "A Novel Method for the Rapid and Efficient Preparation of Polymerase Chain Reaction Template DNA from Filter Paper Blood Samples.", "abstract": "Filter paper blood samples (i.e., dry blood spots [DBSs]) have been widely used for pathogen detection, especially in malaria control and elimination. Dried blood spot-based polymerase chain reaction (PCR) testing has also been increasingly used for diagnosis and gene monitoring. The current method of DNA extraction from DBSs is limited in practical application because of its low efficiency, time-consuming procedure, and high cost. Accordingly, a hemolysis preservation (HP) method was developed and compared with the most commonly used Tween-Chelex method and spin-column method kit (QIAamp DNA Mini) under equivalent conditions by extracting template DNA from the same batch of Plasmodium falciparum density-gradient DBSs. The HP method yielded the highest DNA recovery volume (557.32 ng/µL of blood) and the lowest limit of detection (LOD; equivalent to 0.1 parasites/µL) from DBSs with the minimum blood volume (4-5 µL) using the simplest reagent. Additionally, it had the shortest time (∼50 minutes) and the lowest cost ($0.27 per sample). The LOD of the direct PCR method also reached a value of 0.11 parasites/µL. Therefore, the DBS PCR test conducted using the HP method can detect asymptomatic low-density Plasmodium infection and is a cost-effective option in resource-deficient areas.", "journal": "The American journal of tropical medicine and hygiene", "year": "2026", "doi": "10.4269/ajtmh.25-0115", "ptypes": [ "Journal Article" ], "first_author": "Yuting H", "dirs": [ "B" ] }, "42327579": { "pmid": "42327579", "title": "Assessing the Association Between Genetic Variants in ACE, SOD1, and PER3 and their Role in Breast Cancer Risk among Jordanian Women.", "abstract": "Genetic and environmental factors regulate many physiological processes in the human body, and alterations in these processes may contribute to the development of various diseases, including breast cancer (BC), which is considered the most prevalent cancer among women and a leading cause of cancer-related mortality in the Jordanian population. Genes such as ACE, SOD1 and PER3 play important roles in regulating essential biological functions. These genes are involved in key physiological pathways, including blood pressure regulation, oxidative stress response and circadian rhythm maintenance, and genetic variants within them may influence susceptibility to cancer. Therefore, this study investigates the association between polymorphisms in the ACE, SOD1 and PER3 genes and the risk of breast cancer, with the aim of evaluating how these genetic variations relate to breast cancer susceptibility and clinical outcomes in Jordanian women. Blood samples of 300 women diagnosed with breast cancer, along with 300 healthy participants, were collected, and DNA was extracted from them. Genetic variants in the ACE (rs1799752), SOD1 (rs36232792) and PER3 (rs57875989) genes are examined through employing direct PCR to amplify the target regions. The ACE (rs1799752) variant was observed to be associated with breast cancer susceptibility, with the I/I genotype increasing risk of breast cancer (OR = 5.138, 95% CI = 1.38-19.03, p = 0.014). No associations were observed for SOD1 (rs36232792) and PER3 (rs57875989) variants. The rs1799752 polymorphism is suggested to have the potential of serving as a biomarker for breast cancer susceptibility in Jordanian women, as it is associated with elevating the risk.", "journal": "Journal of Cancer", "year": "2026", "doi": "10.7150/jca.133243", "ptypes": [ "Journal Article" ], "first_author": "Al-Eitan LN", "dirs": [ "B" ] }, "41967609": { "pmid": "41967609", "title": "Recombinant Taq DNA polymerase.", "abstract": "The sudden COVID-19 pandemic highlights the importance of diagnostic assays in health security readiness. One persistent difficulty is the rapid molecular detection of pathogenic microorganisms and viruses. Taq DNA polymerase remains an essential component for COVID-19 molecular detection. Taq DNA polymerase exhibits a high level of thermostability, which results from tighter hydrophobic packing, increased salt bridges, shortened loops, and proline substitutions that reduce flexibility, enabling activity above 90 °C. In addition, engineered variants address limitations by improving fidelity, processivity, and stability. The enzyme´s thermostability has significantly increased the efficiency, automation, and specificity of PCR. Hot-start modifications using antibodies, aptamers, or chemical inhibitors further reduce non-specific amplification, strengthening its role in sensitive diagnostics. Although Taq polymerase is available from several commercial sources, it is crucial and urgent to produce enzymes recombinantly for use in research and diagnostic procedures. To satisfy the demands of industry, it is necessary to maximize and optimize its production. We provide an overview of the recombinant Taq DNA polymerase´s characteristics, structural features, engineering advances, production background and strategies, and purification history in this paper. Together, these insights underscore its central role in diagnostics, research, and biotechnology, as well as its continued relevance as a model enzyme for protein engineering.", "journal": "Journal of biotechnology", "year": "2026", "doi": "10.1016/j.jbiotec.2026.04.002", "ptypes": [ "Journal Article", "Review" ], "first_author": "Žigová K", "dirs": [ "B" ] }, "41221096": { "pmid": "41221096", "title": "Protein Engineering of Biosynthetic Enzymes Unlocks Libraries of Bioactive Tilimycin Analogs.", "abstract": "Natural products continue to inspire therapeutic innovation due to their structural complexity and biological potency. Pyrrolobenzodiazepines (PBDs), known for antitumor activity, function by covalently binding to guanine bases in DNA, a mechanism that is inherently species-agnostic. However, their potential as antibiotics remains underexplored, as modest antibiotic activity is commonly seen with this class of compounds. Here, we produce analogs of tilimycin and tilivalline, two PBDs produced by the gut microbe Klebsiella oxytoca. We mutated NpsA, the nonribosomal peptide synthetase (NRPS) pathway protein responsible for initiating biosynthesis through adenylation of 3-hydroxyanthranilic acid on the pathway to form tilimycin and tilivalline, to enhance promiscuity with substrate analogs. Using structure and informatic-guided mutagenesis, we developed a rapid screening method to identify compatible enzyme-building block combinations to generate a panel of tilimycin and tilivalline analogs. We identified compounds that possess the ability to inhibit DNA polymerase and that show growth inhibitor activity with a DNA-repair mutant of E. coli. This work demonstrates the feasibility of NRPS reprogramming to use biocatalytic approaches to access non-natural derivatives with antibiotic potential and highlights tilimycin analogs as candidates for gram-negative antibacterial development.", "journal": "ACS catalysis", "year": "2025", "doi": "10.1021/acscatal.5c05921", "ptypes": [ "Journal Article" ], "first_author": "MacDonald MR", "dirs": [ "B" ] }, "40897294": { "pmid": "40897294", "title": "Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d.", "abstract": "The DNA polymerase from Thermococcus kodakarensis KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from Sulfolobus tokodaii (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from Saccharolobus solfataricus) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.", "journal": "Protein expression and purification", "year": "2025", "doi": "10.1016/j.pep.2025.106809", "ptypes": [ "Journal Article" ], "first_author": "Chen L", "dirs": [ "B" ] }, "42197341": { "pmid": "42197341", "title": "Deep Learning-Guided Engineering of Bst DNA Polymerase Improves LAMP-Based Detection of Foodborne Pathogens.", "abstract": "Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid detection method, but its application is often limited by the suboptimal performance of wild-type Bacillus stearothermophilus (Bst) DNA polymerase. This study employed a combined deep learning and semi-rational design strategy to engineer Bst DNA polymerase. High-throughput screening identified the A0A150MFP3 sequence and the L105M mutation, which increased enzymatic activity by 32.92%. Fusion with the CL7 protein generated a CL7-Bst mutant with enhanced thermal stability and tolerance to common inhibitors, including 7% (v/v) ethanol, 0.18‱ (w/v) SDS, 80 mmol/L NaCl, and 0.8 mmol/L EDTA. Systematic optimization of the LAMP reaction system determined the optimal pH (9.0), enzyme concentration (0.20 U/μL), and temperature (64 °C). When applied to Escherichia coli O157:H7 detection, the CL7-Bst mutant achieved Tt values of 15.13 and 12.78 for crude and purified DNA, respectively, with a limit of detection of 1 × 103 CFU/mL. In summary, integrating deep learning with semi-rational design and fusion protein engineering yielded a high-performance DNA polymerase that facilitates rapid, sensitive, and field-deployable LAMP-based pathogen detection.", "journal": "Microorganisms", "year": "2026", "doi": "10.3390/microorganisms14050954", "ptypes": [ "Journal Article" ], "first_author": "Chen H", "dirs": [ "B", "C" ] }, "41273506": { "pmid": "41273506", "title": "Enantioseparation by Thin-Layer Chromatography.", "abstract": "Both high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) techniques should be considered complementary rather than competing, with TLC being used widely in organic synthesis and analytical research. Chiral TLC provides an easy technique for the resolution, isolation, and routine quality control of the enantiomeric purity of active pharmaceutical ingredients and a variety of other racemates, especially for fast and cost-effective analysis with simple equipment. The present chapter describes several approaches in chiral TLC for the separation of amino acids and basic drugs. Analytical approaches for direct enantioseparation include the use of (1) \"chiral additive in achiral stationary phase (CAASP),\" (2) \"chiral mobile-phase additive (CMPA),\" (3) \"premixing of chiral reagent with the enantiomeric mixture,\" and (4) \"ligand exchange approach\" with copper metal complex. Both pre-coated and custom-prepared TLC plates have been used. (-)-Quinine, (-)-erythromycin, L-(+)-tartaric acid, (S)-naproxen, bovine serum albumin (BSA), and L-amino acids were used as chiral reagents/selectors in different manners for the enantioseparations of (±)-ketamine, (RS)-ketorolac, certain β-adrenergic drugs, and DL-dansyl amino acids, as examples.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2026", "doi": "10.1007/978-1-0716-5023-3_2", "ptypes": [ "Journal Article" ], "first_author": "Bhushan R", "dirs": [ "B" ] }, "41008821": { "pmid": "41008821", "title": "Evaluation of Mithramycin in Combination with Chemotherapeutic Agents Against Ewing Sarcoma Cell Lines.", "abstract": "Ewing Sarcoma (ES) is a rare, malignant bone neoplasm that is primarily diagnosed in childhood and adolescence. The aggressive nature of this neoplasm requires the use of surgery, radiation and a rigorous chemotherapy regimen. Metastatic ES carries a poor prognosis, which necessitates the development of new therapeutic agents. Mithramycin was tested in targeted therapy due to its specific inhibitory effects on the EWS-FLI1 fusion protein which is present in >85% of ES tumors. We tested the combination of Mithramycin with chemotherapeutic agents vincristine (VCR) and Etoposide (Eto) for inducing higher cytotoxicity against ES cells, CHLA10 and TC205. Cardiomyocyte cell line, H9C2 was used to test the effect on non-malignant cells. Cell viability was measured using the CellTiter-Glo kit, and the combination index was evaluated to determine the type of combination response (antagonistic, additive, or synergistic). Apoptotic cells were measured post-treatment with vehicle (DMSO, control), monotherapy (mithramycin or etoposide), or combination therapy (mithramycin + etoposide) using BD LSRII flow cytometer and analyzed utilizing FlowJo software V8.0. The apoptotic protein marker c-PARP in both treatment and control groups was analyzed using Western blot analysis. The results showed higher cytotoxicity for combination treatment when compared to individual agents, and the combination index confirmed the response as synergistic. H9C2 cells did not demonstrate significant decreases in cell viability when treated with combination therapy, highlighting the specificity of the treatment toward its target tissue. Flow cytometry confirmed the underlying mechanism as upregulation of apoptosis which is further supported by an increase in effector caspases 3/7 and elevated expression of c-PARP. These in vitro assays using ES cells provide preliminary evidence for the benefit of chemotherapy and mithramycin combination.", "journal": "Cancers", "year": "2025", "doi": "10.3390/cancers17182977", "ptypes": [ "Journal Article" ], "first_author": "Lambring CB", "dirs": [ "B" ] }, "39963238": { "pmid": "39963238", "title": "Bioprinted hydrogels in bone regeneration: a bibliometric analysis.", "abstract": "The application of bioprinted hydrogels in the field of bone regeneration is garnering increasing attention. The objective of this study is to provide a comprehensive overview of the current research status, hotspots and research directions in this field through bibliometric methods, and to predict the development trend of this field. A search was conducted on 27 December 2024, for papers published on the Web of Science from 2010 to 2025. We used the bibliometrix package in the software program R to analyze the retrieved data and VOSviewer and CiteSpace to visualize hotspots and research trends in bioprinted hydrogels for bone regeneration. We identified and reviewed 684 articles published in this field between 2010 and 2025. A total of 811 institutions and 1,166 researchers from 41 countries/regions contributed to these publications. Among them, China led in terms of the number of articles published, single-country publications (SCP), and multi-country publications (MCP). Our bibliometric-based visualization analysis revealed that the mechanical properties and osteogenic differentiation capacity of biomaterials have been a focal research topic over the past decade, while emerging research has also concentrated on the in vitro fabrication of stem cells for bone regeneration and osteogenic differentiation, particularly the precise application of in situ stem cell-loaded bioprinted organoids. This study provides an in-depth analysis of the research trajectory in the application of bioprinted hydrogels for bone regeneration. The number of research papers in this field is increasing annually, and the main research hotspots include bone regeneration, 3D printing, scaffolds, and hydrogels. Future research directions may focus on gelatin, additive manufacturing, and growth factors. Additionally, international collaboration is essential to enhance the effectiveness of bioprinted hydrogels in bone regeneration applications.", "journal": "Frontiers in pharmacology", "year": "2025", "doi": "10.3389/fphar.2025.1532629", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhang H", "dirs": [ "B" ] }, "41893728": { "pmid": "41893728", "title": "Dimethyl Sulfoxide Enhances HLA Peptide Identification.", "abstract": "Background: Mass spectrometry (MS)-based immunopeptidomics has emerged as the gold standard for profiling HLA-bound peptides, yet detection remains challenging due to their non-tryptic nature, variable lengths, and lack of basic residues, which limit ionisation and fragmentation efficiency. Methods: To address these limitations, we investigated the impact of incorporating 5% dimethyl sulfoxide (DMSO) into LC-MS/MS mobile-phase buffers on immunopeptidomic workflows. Using B-lymphoblastoid cell lines expressing HLA class I and II alleles and elastase-digested HeLa lysates as a surrogate for non-tryptic peptides, we assessed peptide identification, ionisation efficiency, charge state distribution, and fragmentation quality. Results: DMSO significantly increased peptide identifications across all sample types, with gains of ~1.33 folds for HLA class I, ~1.55 folds for HLA class II, and ~1.24 folds for elastase digests. Improvements were systematic and reproducible, driven by enhanced electrospray ionisation, higher charge states, and superior MS2 spectral quality, evidenced by ~2-fold increase in b- and y-ion intensities. Importantly, DMSO did not introduce major sequence bias, preserving motif integrity and predicted binding characteristics. Conclusions: Overall, these findings establish DMSO as a robust additive for improving sensitivity and reliability in immunopeptidomics, particularly for low-input or clinically derived samples.", "journal": "Proteomes", "year": "2026", "doi": "10.3390/proteomes14010013", "ptypes": [ "Journal Article" ], "first_author": "Lim Kam Sian TCC", "dirs": [ "B" ] }, "39861249": { "pmid": "39861249", "title": "Novel Ultrafiltration Polyethersulfone Membranes Blended with Carrageenan.", "abstract": "The development of ultrafiltration (UF) polymeric membranes with high flux and enhanced antifouling properties bridges a critical gap in the polymeric membrane fabrication research field. In the present work, the preparation of novel PES membranes incorporated with carrageenan (CAR), which is a natural polymer derived from edible red seaweed, is reported for the first time. The PES/CAR membranes were prepared by using the nonsolvent-induced phase separation (NIPS) method at 0.1-4.0 wt.% CAR loadings in the casting solutions. The use of dimethylsulfoxide (DMSO), which is a bio-based and low-toxic solvent, is reported. Scanning electron microscopy, atomic force microscopy, water contact angle, porosity, and zeta potential measurements were used to evaluate the surface morphology, structure, pore size, hydrophilicity, and surface charge of the prepared membranes. The filtration performance of PES/CAR membranes was tested with bovine serum albumin (BSA) solutions. It was shown that CAR incorporation in the casting solutions notably increased hydrophilicity, porosity, pore size, surface charge, and fouling resistance of the prepared membranes compared with plain PES membranes due to the hydrophilic nature and pore-forming properties of CAR. The PES/CAR membranes showed a significant reduction in irreversible and total fouling during filtration of BSA solutions by 38% and 32%, respectively, an enhancement in the flux recovery ratio by 20-40%, and an improvement in mechanical properties by 1.5-fold when compared with plain PES membranes. The findings of the present study indicate that CAR can be used as a promising additive for the development of PES UF membranes with enhanced properties and performance for water treatment applications.", "journal": "Polymers", "year": "2025", "doi": "10.3390/polym17020176", "ptypes": [ "Journal Article" ], "first_author": "Al Marri SH", "dirs": [ "B" ] }, "41476862": { "pmid": "41476862", "title": "Keratin Additive for Cellular Adhesion in Transcutaneous Prosthetics.", "abstract": "The dermal barrier is widely considered the body's first line of defense against most foreign bodies, protecting it from both moisture loss and bacterial invasion. However, when the skin is ruptured for long-term medical interventions (e.g., transcutaneous prosthetics), it is difficult to restore and maintain this protective barrier. Although there are no direct, biological examples of true transcutaneous features in the human body, similar phenomena can be observed in phalangeal nails. This study aims to investigate keratin, the primary component of fingernails, in its hydrolyzed form as an additive to induce cell adhesion in two representative scaffold types. Electrospun fibers and chitosan-gelatin cryogels-two well-characterized scaffolds used in dermal tissue engineering-were selected for this study as a fibrous and macroporous foundation. Both electrospun fibers and cryogels were fabricated with a range of keratin additive concentrations (0, 1, 3, 5, 7, and 10 wt/wt% and wt/v% for electrospun fibers and cryogels, respectively) and tested for surface properties, mechanical strength, biocompatibility, and material behavior. Overall, it was determined that hydrolyzed keratin had a positive effect on cell adhesion and proliferation but that high quantities of the keratin resulted in adverse effects on the scaffold properties. With dermal applications in mind, this study found that 5 and 7 wt/wt% keratin electrospun fibers possessed required cell counts, surface energies, tensile strength, and contact angle, all with consistent reproducibility. For the cryogels, 3 and 5 wt/v% keratin had the best combined performance, maintained structural integrity through swelling and porosity, and displayed minimal loss in compressive strength. Therefore, hydrolyzed keratin represents a promising additive for bothelectrospun fibers and cryogels in tissue engineering applications.", "journal": "Journal of tissue engineering and regenerative medicine", "year": "2025", "doi": "10.1155/term/4337554", "ptypes": [ "Journal Article" ], "first_author": "Cagle AL", "dirs": [ "B" ] }, "41781465": { "pmid": "41781465", "title": "Paradoxical oncogenic effects of hepatic Brca1 through modulating Bhmt.", "abstract": "The well-established tumor suppressor, Breast Cancer Susceptibility Gene 1 (BRCA1), has been implicated in lipid metabolism and in paradoxically promoting hepatocellular carcinoma (HCC); yet its mechanistic role remains unknown. Here we show murine hepatic Brca1 loss protected against fatty liver, glucose intolerance and tumor development. Using single nuclear RNA sequencing, we found betaine-homocysteine S-methyltransferase (Bhmt) was suppressed with Brca1-deficiency. Concomitant knockdown of BRCA1 and BHMT in human hepatoma cells revealed additive accumulation of DNA double strand breaks and increased susceptibility to cell death. In our mouse model and human hepatoma cells, BHMT repression with BRCA1-deficiency was associated with downregulation of choline metabolism, an emerging hallmark of cancer. Metabolomics in hepatic Brca1-deficient mice further revealed related disruptions in choline metabolism. Together, our data show hepatic Brca1-deficiency protected against tumor formation by inducing cell death through exacerbating DNA damage and suppressing choline metabolism by inhibiting Bhmt expression, challenging the role of BRCA1 solely as a tumor suppressor.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-39581-6", "ptypes": [ "Journal Article" ], "first_author": "Ghebreselassie M", "dirs": [ "B" ] }, "42491242": { "pmid": "42491242", "title": "Reconstructing the glioblastoma microenvironment in heterotypic 3D spheroids: a multicellular model to study tumor-stromal crosstalk.", "abstract": "The complex interplay between tumor cells and the stromal components of the glioma microenvironment necessitates the development of sophisticated in vitro models capable of modelling key aspects of cellular interactions that occur beyond the limitations of conventional monocultures. The development and characterization of homo- and heterotypic 3D spheroid models incorporating CCF-STTG1 astrocytes, HMC3 microglia, and U87MG glioma cells was undertaken. The assessment of morphological, molecular, and functional properties was performed via flow cytometry, cytokine arrays, ECM analysis and invasion assays (Matrigel™/gelatin). Heterotypic spheroids have been observed to spontaneously self-assemble into a spatially polarized architecture, with microglia and glioma cells segregating into distinct compartments, a pattern suggestive of the cellular topology at the invasive front. The morphological, molecular, and functional properties of the generated 3D models recapitulated several established features associated with in vivo tumors, including growth, invasion, resistance to chemotherapy, and metabolic reprogramming alongside the expression of stemness markers, and key pro-invasive mediators (MMPs, SDF-1α, VEGF). Secretome profiling revealed a marked, non-additive upregulation of chemokines (IP-10, MIP-1α) and the emergence of novel correlations (HGF/SDF-1α, MCP-1/LIF), indicating potential modulation of paracrine networks involved in immune cell trafficking in the heterotypic setting. The initial formation of a rigid ECM matrix appears to be initiated by microglia, while the supply of fibronectin and laminin may be linked to astrocytes exhibiting some features of reactive gliosis, which could help organize invasion pathways. These heterotypic 3D spheroid models offer a stroma-enriched, reproducible platform for the analysis of stromal contributions to glioma progression and for exploratory preclinical evaluation of therapeutic strategies.", "journal": "Frontiers in bioengineering and biotechnology", "year": "2026", "doi": "10.3389/fbioe.2026.1852454", "ptypes": [ "Journal Article" ], "first_author": "Nikitina Y", "dirs": [ "B" ] }, "40785070": { "pmid": "40785070", "title": "Acryloyl Gelatin as Polymer Additive to Droplet Digital LAMP for DNA Quantification.", "abstract": "Absolute quantification of target nucleic acids is essential for life analysis and can be achieved through droplet digital microfluidic nucleic acid quantification (droplet digital NAQ). Recent advancements in this field have incorporated polymers into droplets to form microgel particles, which add new functionalities to the droplets and offer opportunities for tandem analysis. However, the range of materials available for microgel-based droplet digital NAQ remains limited. In this study, we introduce acryloyl gelatin (GelA) as an additive polymer for droplet digital NAQ. GelA is photopolymerizable, biocompatible, and biodegradable, making it an ideal candidate for droplet digital NAQ applications. Our findings demonstrate that GelA exhibits minimal thermal variation in viscosity, which is favorable for droplet microfluidic production and handling. Additionally, GelA does not significantly affect the efficiency of droplet digital loop-mediated amplification (droplet digital LAMP) under the optimized conditions. Upon the addition of GelA, the droplet digital LAMP assay for the detection of the N gene in SARS-CoV-2 can be realized within the range of 25 to 250,000 copies/test, and the amplification is completed within 30 min. Following droplet digital LAMP, GelA droplets can be transformed into either physical or chemical microgel particles. In the microgel form, the particles are stable through transfer cycles, can prevent leakage of amplification products, and can facilitate controlled release of the products for further applications. This work broadens the range of materials available for droplet-based digital nucleic acid detection, increases the versatility of droplet digital LAMP, and opens new possibilities for advancements in this field.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c02761", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Deng J", "dirs": [ "B" ] }, "42438064": { "pmid": "42438064", "title": "CO2-Responsive Dual-Charge-Switchable Membranes for Protein Foulant Cleaning.", "abstract": "Known CO2-responsive membranes exhibit single-charge-switching (neutral-cationic), which limits their ability to regulate electrostatic interactions with oppositely charged foulants in membrane cleaning. Herein, CO2-responsive poly(ether sulfone) (PES) ultrafiltration membranes with a dual charge-switchable surface are prepared by adding an amphoteric diblock copolymer additive to PES via the nonsolvent-induced phase separation (NIPS) method. By tuning the isoelectric point (IEP) of the copolymer additive to around 5, the membrane surface charge reversibly switches between positive and negative states under alternating CO2 bubbling, lowering pH to ∼4 (below IEP), and N2 bubbling, raising pH to ∼7 (above IEP). ATR-FTIR and water contact angle analyses indicate that increasing the copolymer content in the casting solution increases the amount of amphoteric chains on the membrane surface. Using bovine serum albumin (BSA) as a model foulant with a similar IEP, protein-fouled membranes are effectively cleaned with water when CO2 and N2 are alternately bubbled due to the constantly existing electrostatic repulsion between the membrane and protein. The gas-triggered cleaning efficiency is comparable to conventional acid/alkaline cleaning and remains stable over repeated fouling-cleaning cycles, underscoring the potential of this approach for environmentally friendly and chemical-free membrane cleaning.", "journal": "Macromolecular rapid communications", "year": "2026", "doi": "10.1002/marc.70357", "ptypes": [ "Journal Article" ], "first_author": "Zamanian N", "dirs": [ "B" ] }, "40006097": { "pmid": "40006097", "title": "Potential of Chitosan/Gelatin-Based Nanofibers in Delivering Drugs for the Management of Varied Complications: A Review.", "abstract": "Drug delivery systems have revolutionized traditional drug administration methods by addressing various challenges, such as enhancing drug solubility, prolonging effectiveness, minimizing adverse effects, and preserving potency. Nanotechnology-based drug delivery systems, particularly nanoparticles (NPs) and nanofibers (NFs), have emerged as promising solutions for biomedicine delivery. NFs, with their ability to mimic the porous and fibrous structures of biological tissues, have garnered significant interest in drug-delivering applications. Biopolymers such as gelatin (Ge) and chitosan (CH) have gained much more attention due to their biocompatibility, biodegradability, and versatility in biomedical applications. CH exhibits exceptional biocompatibility, anti-bacterial activity, and wound healing capabilities, whereas Ge provides good biocompatibility and cell adhesion properties. Ge/CH-based NFs stimulate cellular connections and facilitate tissue regeneration owing to their structural resemblance to the extracellular matrix. This review explores the additive methods of preparation, including electrospinning, force pinning, and template synthesis, focusing on electrospinning and the factors influencing the fiber structure. The properties of Ge and CH, their role in drug release, formulation strategies, and characterization techniques for electrospun fibers are discussed. Furthermore, this review addresses applications in delivering active moieties in the management of orthopedics and wound healing with regulatory considerations, along with challenges related to them. Thus, the review aims to provide a comprehensive overview of the potential of Ge/CH-based NFs for drug delivery and biomedical applications.", "journal": "Polymers", "year": "2025", "doi": "10.3390/polym17040435", "ptypes": [ "Journal Article", "Review" ], "first_author": "Mohite P", "dirs": [ "B" ] }, "39714890": { "pmid": "39714890", "title": "Stepwise Modulation of Bridged Single-Benzene-Based Fluorophores for Materials Science.", "abstract": "In recent years, researchers studying fluorogenic samples have steadily shifted from using large, expensive, poorly soluble fluorophores with complex synthetic sequences to smaller, simpler π scaffolds with low molecular weight. This research article presents an in-depth study of the photophysical properties of five bridged single-benzene-based fluorophores (SBBFs) investigated for their solution and solid-state emission (SSSE) properties. The compounds O4, N1O3, N2O2, N3O1, and N4 are derived from a central terephthalonitrile core and vary in the amount of oxygen and nitrogen bridging atoms. These minimalized emitters show full-color tunable emission properties and exhibit moderate-to-high photoluminescence quantum yield values reaching up to 0.78 in dimethyl sulfoxide (DMSO). In addition to demonstrating excellent compatibility in poly(methyl methacrylate) (PMMA) films and additive manufacturing using stereolithography (SLA), white light emission was achieved in both solution and 3D-printed materials by controlling the mixing ratio of the compounds. Employing density-functional theory (DFT), well-correlating theoretical absorption and emission wavelengths were calculated as average values of the different possible conformers. Furthermore, cellular internalization of the substances was accomplished using Pluronic® F-127 nanoparticles. Overall, this study emphasizes the remarkable properties of single-benzene-based emitters, showcasing their accessibility and potential applications in biomedical fields and materials science.", "journal": "Chemistry (Weinheim an der Bergstrasse, Germany)", "year": "2025", "doi": "10.1002/chem.202404263", "ptypes": [ "Journal Article" ], "first_author": "Huber A", "dirs": [ "B" ] }, "41240001": { "pmid": "41240001", "title": "Simulating the Transmural Mechanical Response of Functionally Graded Arterial Grafts.", "abstract": "With coronary artery disease remaining the leading cause of mortality worldwide, the design and manufacture of clinically viable synthetic coronary artery grafts remains a fundamental healthcare challenge. It is widely accepted that vascular mimicking materials (VMMs) should emulate the heterogeneous biomechanical and biological functions of the multilayered artery wall to ensure long-term patency postimplantation. However, few VMMs can adequately meet these complex design requirements. Poly(vinyl alcohol) (PVA)/gelatin cryogels are prospective VMMs due to their combined mechanical (PVA) and biointegrative (gelatin) features, but their development thus far has been limited to homogeneous constructs. The aim of this research is to assess the mechanical response of biomimetically designed multilayered grafts, simulated using Finite Element Analysis. The impact of a sinusoidal interface on circumferential stress distribution and graft compliance, was explored. Using qualitative insight from research on hydrogel based functionally graded biomaterials, and in the context of subzero extrusion additive manufacturing, rough (infinite) friction was used to model the contact between the layer. It was found that transmural stress patterns were continuously graded (phased) as a function of interface amplitude and frequency. In contrast to laminated models, which displayed a discontinuity in transmural stress between layers. This design methodology illustrates a novel approach to achieving functionally graded synthetic grafts through interface design.", "journal": "ACS applied bio materials", "year": "2025", "doi": "10.1021/acsabm.5c01506", "ptypes": [ "Journal Article" ], "first_author": "Fegan KL", "dirs": [ "B" ] }, "41235717": { "pmid": "41235717", "title": "A Mixing System for Uniform, Reproducible Viscous Bioinks Preparation.", "abstract": "Rationale: Extrusion 3D bioprinting is an additive manufacturing tissue engineering technique that uses cell-laden viscous biomaterials known as bioinks. Manually mixing cell suspensions into viscous biomaterials can be challenging due to the high viscosity ratio between the two fluids. Static mixers are an attractive approach as they can quickly and reproducibly mix two fluids, including those with a high viscosity ratio. However, static mixers intended for viscous applications have not been comprehensively investigated for bioink preparation. This work evaluates the mixing performance, shear stress, and cell viability using four different types of static mixers intended for high viscosity mixing. Methods: Three static mixers intended for mixing viscous solutions were designed based on the Sulzer SMX, Ross ISG, and serpentine mixers and fabricated using resin 3D printing. CELLMIXER, a Kenics-style static mixer commercially available through CELLINK, was used as a comparator. Two biomaterial inks based on PEGDA and methacrylated gelatin were used to characterize each mixer's performance. Shear stress was estimated via fluid dynamics simulations using shear-thinning attributes measured experimentally through rheology. Mixing effectiveness was evaluated using fluorescent beads, from which the most effective design was chosen for live cell mixing experiments. Viability of cell lines (A549 and NIH-3T3) and primary human lung fibroblasts was evaluated postmixing. A demonstration of extrusion bioprinting was performed using the mixed bioinks. Results: The SMX-style mixer provided the most uniform mixing and yielded the lowest simulated shear stresses among the designs investigated. A549, NIH-3T3, and primary human lung fibroblasts maintained viabilities above 96% postmixing using the SMX-style mixer with a more homogeneous cell distribution compared to the CELLMIXER. The bioprinting demonstration validated our mixing system for producing viable tissue constructs with evenly distributed cells. Conclusions: We present a simple, reproducible, and flexible system for mixing cells into viscous biomaterial inks. Our approach facilitates standardized fabrication of cell-laden tissue constructs to ensure consistency in the growing field of extrusion 3D bioprinting.", "journal": "ACS biomaterials science & engineering", "year": "2025", "doi": "10.1021/acsbiomaterials.5c01334", "ptypes": [ "Journal Article" ], "first_author": "Singer R", "dirs": [ "B" ] }, "40246286": { "pmid": "40246286", "title": "Do co-solvents used in exposure studies equally perturb the metabolic profile of Daphnia magna?", "abstract": "Dissolution methods such as co-solvents are used to solubilize insoluble compounds in exposure experiments. Several exposure studies have followed the guidelines from the Organization for Economic Co-operation and Development where co-solvents are applied at 0.01% v/v of the total exposure volume. Although no observable apical endpoint abnormalities were reported following these guidelines, little is known about the molecular-level impacts of co-solvents used in exposure studies. A targeted metabolomics approach using liquid chromatography coupled with triple quadrupole mass spectrometry was used to assess Daphnia magna responses to four commonly used co-solvents, including acetone (ACT), acetonitrile (ACN), methanol (MeOH), and dimethyl sulfoxide (DMSO), at three different levels (0.01%, 0.05%, and 0.1% v/v) over 48 hr. Based on the observed metabolic disruptions, exposure to MeOH and DMSO induced higher metabolic perturbations in amino acid levels and associated biochemical pathways in comparison to ACT and ACN exposures. However, as with mixtures, when co-solvents are combined with the pollutants under investigation, there is a possibility for additive, synergistic, or antagonistic interactions. Hence, to examine the possible impairments in co-solvent and pollutant mixtures, ACT and ACN applied at 0.01% v/v were chosen to be tested with phenanthridine (PN). Daphnia magna exposure to PN dissolved in ACT had less disruptions; in contrast to PN prepared in ACN, which triggered a higher degree of antagonism in the D. magna metabolic profile. Consequently, exposing D. magna to ACT applied at 0.01% v/v resulted in the lowest metabolic perturbation in both parts of this study, suggesting that it is the least disruptive co-solvent for molecular-level exposure studies involving D. magna.", "journal": "Environmental toxicology and chemistry", "year": "2025", "doi": "10.1093/etojnl/vgaf068", "ptypes": [ "Journal Article" ], "first_author": "Hajir S", "dirs": [ "B" ] }, "40825176": { "pmid": "40825176", "title": "Site-Selective Electrochemical Intramolecular C(sp2)-H Selenylation for Dibenzoselenophene Synthesis.", "abstract": "We report a robust and eco-conscious electrosynthetic protocol for efficiently constructing structurally diverse and biologically relevant dibenzoselenophenes. This transformation proceeds via electrochemical intramolecular C-H activation of bis(biaryl) diselenides, employing LiClO4 as a green electrolyte and tetrabutylammonium bromide as an additive, conducted in DMSO at 70 °C under a constant current of 10 mA using Pt/Pt electrodes. The newly developed method offers several notable advantages, including mild and energy-efficient conditions, transition-metal-free catalysis, short reaction times, high to excellent yields, broad substrate tolerance, scalability to gram-scale synthesis, operational simplicity, and environmental friendliness.", "journal": "The Journal of organic chemistry", "year": "2025", "doi": "10.1021/acs.joc.5c01600", "ptypes": [ "Journal Article" ], "first_author": "Karmakar I", "dirs": [ "B" ] }, "41490607": { "pmid": "41490607", "title": "Automated active learning to optimize hydrogel drug release profiles.", "abstract": "Hydrogels are widely used in drug delivery due to their biocompatibility and tunable release properties. However, optimizing hydrogel formulations to the desired release of therapeutics remains experimentally intensive. In this study, we developed an automated, high-throughput and machine learning (ML)-guided framework to efficiently optimize alginate formulations for drug delivery. Using a liquid handling robot, we initially prepared a diverse seed library of 120 alginate hydrogel formulations loaded with bovine serum albumin (BSA) and measured their release profiles. A Gaussian process regression (GPR) ML model was trained to predict cumulative release across time, enabling implicit modeling of release curves. Feature importance analysis using Shapley additive explanations (SHAP) identified time, alginate molecular weight, and concentration as dominant factors influencing release kinetics. Through Bayesian optimization and active learning, we iteratively selected and tested new formulations, progressively reaching a near zero-order release. Finally, the top-performing BSA-optimized formulations were directly applied to the sustained release of chondroitinase ABC single-enzyme nanoparticles (chABC-SENs), achieving near-zero-order release with no further optimizations. This study demonstrates a scalable, data-driven strategy for hydrogel formulation optimization and highlights the potential of ML to accelerate the development of controlled release systems for sensitive and valuable therapeutics.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2026", "doi": "10.1016/j.jconrel.2026.114602", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Cheong E", "dirs": [ "B" ] }, "42605884": { "pmid": "42605884", "title": "Electrospun PEO/gelatin nanofibers for pDNA and mRNA delivery.", "abstract": "Electrospun nanofiber mats are a versatile delivery system that can be applied locally at a target site, making them attractive for localized gene delivery. Although some studies reported electrospinning of nucleic acids, polyethylene oxide (PEO) was primarily used as a co-spinnable additive rather than as a standalone carrier in previous studies. In this study, PEO and gelatin nanofiber mats were prepared via blend and coaxial electrospinning to encapsulate pDNA and mRNA polyplexes prepared with a commercial transfection reagent. Notably, mRNA complexes were directly encapsulated within electrospun nanofibers through blend electrospinning, an approach not previously reported for mRNA delivery from fiber matrices. The results showed that PEO fiber mats exhibit higher encapsulation efficiency (∼60% for pDNA), transfection performance (∼75% of the free-complex level), and maintenance of polyplex integrity than gelatin, blend, and coaxial fibers for both types of nucleic acids. Stability studies over time have shown that encapsulation within electrospun fibers significantly increased the functional lifetimes of both polyplexes compared with non-electrospun (free) polyplexes. Free pDNA polyplexes lost ∼95% of their activity within 24 h, whereas PEO fiber-encapsulated pDNA retained most of its activity, losing only ∼35% over the same period. Cytotoxicity analysis showed that all fiber mats maintained cell viability above 70% at low-to-moderate electrospinning polymer amounts. It was thus shown that PEO electrospun nanofibers can serve as efficient standalone carriers for nucleic acid delivery, with polymer selection influencing encapsulation, transfection, and stability.", "journal": "Biomaterials science", "year": "2026", "doi": "10.1039/d6bm00687f", "ptypes": [ "Journal Article" ], "first_author": "Akgün B", "dirs": [ "B" ] }, "41971453": { "pmid": "41971453", "title": "Natural product-induced apoptosis in oral squamous cell carcinoma via targeting mitochondrial multifunctionality.", "abstract": "Mitochondria, as the regulatory center of cellular energy metabolism, can influence ATP synthesis, redox balance, and apoptotic pathways. Recent studies have confirmed that some natural products, due to their self-assembling properties and multi-target effects, can inhibit OSCC progression by regulating mitochondrial function, providing key lead compounds for the development of new OSCC therapies. We retrieved data from PubMed, Embase, Web of Science, and CNKI to classify and summarize the relationship between mitochondria and OSCC, as well as the regulatory mechanisms of natural products. Natural products exhibit unique advantages in inducing apoptosis in OSCC cells through multi-pathway regulation mechanisms targeting mitochondria. Various active components, such as betaine and Arglabin, induce oxidative stress by promoting ROS accumulation, disrupting mitochondrial redox balance, and thereby activating the intrinsic apoptotic pathway; extracts like cantharidin and berberine induce changes in mitochondrial membrane permeability by regulating the expression ratio of Bcl-2 family proteins, promoting the release of Cyt-c and activating the caspase cascade. It is noteworthy that combining radiotherapy drugs with natural products for OSCC treatment offers greater stability without additive side effects. Moreover, nanodelivery technology, through the design of smart responsive carriers, introduces a competitive release mechanism, achieving precise, on-demand drug release at tumor sites, providing important candidates for the development of new antitumor drugs. Natural products targeting mitochondria provide a new direction for the treatment of OSCC, and their potential for clinical translation is expected to overcome existing therapeutic challenges.", "journal": "Frontiers in oncology", "year": "2026", "doi": "10.3389/fonc.2026.1749700", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhou Q", "dirs": [ "B" ] }, "39679719": { "pmid": "39679719", "title": "Tailoring Gelatin Films: Functionality, Stability, and Beyond Biodegradability.", "abstract": "This study investigates the enhancement of biodegradable gelatin films through the incorporation of glycerol as a plasticizer, and citric acid and zinc oxide as cross-linkers. The results showed notable improvements in various properties, including solubility, swelling behavior, thickness, pH, biodegradability, and both mechanical and thermal characteristics. The films demonstrated complete water solubility and UV-visible light absorbance in the 280-480 nm range. Soil burial tests indicated gradual weight loss over 15 days, leading to complete degradation. Structural and thermal analyses via FTIR and TGA confirmed the films' integrity and stability. Additionally, the study highlighted the effectiveness of these modified films in adsorbing copper (II) ions from acidic solutions, showcasing their potential for environmental applications like heavy metal remediation. These findings emphasize the potential of tailored additive combinations to produce biodegradable films with enhanced properties and functionality.", "journal": "Biopolymers", "year": "2025", "doi": "10.1002/bip.23645", "ptypes": [ "Journal Article" ], "first_author": "Bhardwaj N", "dirs": [ "B" ] }, "41112526": { "pmid": "41112526", "title": "Data-Driven Optimization of Bioink Formulations for Extrusion-Based Bioprinting: A Predictive Modeling Approach.", "abstract": "The field of tissue engineering has significantly advanced with the development of extrusion-based bioprinting. This technique utilizes shear forces to generate filaments for fabricating intricate structures. The printability and structural integrity of bioprinted constructs rely heavily on the rheological properties of bioinks, particularly viscosity, which varies with the shear rate for non-Newtonian materials. Since the shear rate at the nozzle tip fluctuates during extrusion, it is essential to understand how bioink composition influences this behavior. This study investigates the rheological behavior of ALGEC bioinks, a novel formulation composed of ALginate, GElatin, and 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO)-oxidized nanofibrillated cellulose (TO-NFC). The bioinks were prepared with varying concentrations: alginate (0-5.25%), gelatin (0-5.25%), and TO-NFC (0-1.5%), with a maximum total solid content of 8%. Viscosity was conducted over shear rates ranging from 0.1 to 100 s-1, with 252 viscosity data points used 80% for training and 20% for validation. To predict viscosity, polynomial fit and interaction-based multiple regression models were developed. Experimental data were used to estimate viscosity based on bioink composition and shear rate, with the best-performing model achieving an R2 of 0.98 and an mean absolute error (MAE) of 0.12. These predictive models were further utilized to optimize ALGEC formulations to achieve targeted viscosity ranges. Constructs were bioprinted using a random and an optimized composition, demonstrating the effectiveness of model-driven bioink optimization. These findings enhance tissue engineering by improving bioink printability, leading to structurally stable bioprinted constructs for regenerative medicine applications.", "journal": "Journal of manufacturing science and engineering", "year": "2025", "doi": "10.1115/1.4069041", "ptypes": [ "Journal Article" ], "first_author": "Sarah R", "dirs": [ "B" ] }, "40328275": { "pmid": "40328275", "title": "Bioprinted M2 macrophage-derived extracellular vesicle mimics attenuate foreign body reaction and enhance vascularized tissue regeneration.", "abstract": "Foreign body reaction (FBR) and insufficient vascularization greatly hinder the integration of 3D-bioprinted tissue substitutes with host tissues. Previous studies have shown that these problems are exacerbated by the stiffness of the 3D-bioprinted constructions, which is highly associated with the abnormal polarization of macrophages. Therefore, we developed an engineering strategy using membrane extrusion to prepare macrophage-derived extracellular vesicle mimics (EVMs). The EVMs derived from M1 and M2 macrophages (M1-EVMs and M2-EVMs) were rich in functional proteins. In the 2D environment, M1-EVMs promoted the fibrotic phenotype of fibroblasts, vascularization, and the M1 polarization of macrophages. In contrast, M2-EVMs effectively avoided the fibrotic trend, showed stronger angiogenic capabilities, and prevented excessive M1 polarization, demonstrating their potential to inhibit FBR and promote neovascularization. After bioprinting the EVMs loaded by gelatin-alginate bioink, the basic physical properties of the bioink were not significantly affected, and the biological functions of EVMs remain stable, indicating their potential as bioink additives. In the subcutaneous implantation model, unlike the FBR-aggravating effects of M1-EVMs, 3D-bioprinted M2-EVMs successfully reduced the immune response, prevented fibrous capsule formation, and increased vascular density. When applied to skin wound treatment, 3D-bioprinted M2-EVMs not only inhibited inflammatory levels but also exhibited pleiotropic pro-regenerative effects, effectively promoting vascularization, re-epithelialization, and appendage regeneration. As an innovative additive for bioinks, M2-EVMs present a promising approach to enhance the survival of bioengineered tissues and can further serve as a targeted drug loading system, promoting the development of regenerative medicine and improving clinical outcomes.", "journal": "Biofabrication", "year": "2025", "doi": "10.1088/1758-5090/add49f", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhang C", "dirs": [ "B" ] }, "40559360": { "pmid": "40559360", "title": "Impact of Additives on Poly(acrylonitrile-butadiene-styrene) Membrane Formation Process Using Non-Solvent-Induced Phase Separation.", "abstract": "Poly(acrylonitrile-butadiene-styrene) (ABS) is a common polymer used in toys, automobile parts, and membranes. Membranes fabricated with this copolymer commonly employ toxic solvents and have a dense architecture, which may not work in all applications. This work investigates the synthesis of ABS membranes, using green solvents and the influence of additives on the phase inversion process during the non-solvent-induced phase separation. The addition of water-soluble additives, ethanol, and acetone is hypothesized to provide additional control over viscosity and volatility, and, consequently, impact the phase inversion process. Membranes were fabricated with PolarClean and with various additive concentrations and evaporation times. The resulting membranes were characterized using scanning electron microscopy (SEM) and a pycnometer to visualize the pore structure and obtain porosity information. Membrane performance, including water flux and bovine serum albumin rejection, was evaluated using dead-end cell filtration. Membranes fabricated using only PolarClean had fingerlike pore morphology and relatively low protein rejection. The addition of additives resulted in a change in pore architecture and rejection, which is hypothesized to be a result of additives' volatility, humidity, and destabilization of liquid-liquid separation. This study provides a more detailed understanding of the impact of additives on the resulting ABS membrane structure and performance, with a focus on safer solvents.", "journal": "Membranes", "year": "2025", "doi": "10.3390/membranes15060181", "ptypes": [ "Journal Article" ], "first_author": "Dhameri S", "dirs": [ "B" ] }, "42514016": { "pmid": "42514016", "title": "Nutritional Strategies to Mitigate Heat Stress in Cattle: A Narrative Review.", "abstract": "Heat stress is a growing concern in cattle production systems due to the increasing frequency and intensity of extreme weather events driven by climate change. This review synthesizes current knowledge on the multifaceted impacts of heat stress and focuses on nutritional strategies to mitigate its effects on ruminating cattle. A comprehensive literature search was conducted using PubMed, Scopus, Web of Science, and Google Scholar. Heat stress adversely affects cattle physiology, behavior, rumen function, and overall productivity, particularly in dairy animals with high metabolic activity. During heat stress episodes, changes in the microbial population have been reported; however, there is no clear consensus, as findings vary widely among studies depending on diet, feed intake, animal type and experimental design. This variability limits the ability to draw general conclusions regarding changes in the rumen microbiome driven by heat stress. In this context, dietary nutritional intervention strategies offer a practical and scalable approach to enhance thermotolerance and maintain performance under heat stress conditions. Key nutritional strategies include modifications in diet composition to reduce metabolic heat production, with some approaches carrying potential risks to animal health, e.g., increasing dietary energy density through concentrates while minimizing forage content. Supplementation with rumen-protected nutrients like amino acids, vitamins, and minerals can be used to support immune function, antioxidant capacity, and metabolic stability. Polyphenols and betaine contribute to oxidative stress reduction and gut integrity, while probiotics may be used to improve rumen fermentation and nutrient utilization. Sensor technologies, including rumen boluses and wearable devices, offer the potential to monitor physiological responses to heat stress in real time and offer opportunities for precision feeding and early intervention. Most published studies only cover short periods of heat stress, and there is a lack of in vitro models simulating rumen hyperthermia. In parallel, future research should therefore prioritize longitudinal, in vivo trials that integrate physiological, metabolic, and microbial responses to understand the long term and systemic effect of heat stress. In addition, controlled trials in commercial settings are necessary to prove the transferability of results to commercial herds. A multidisciplinary approach combining nutritional, environmental, and technological strategies is likely to play an important role in safeguarding cattle welfare and productivity in a warming climate.", "journal": "Microorganisms", "year": "2026", "doi": "10.3390/microorganisms14071511", "ptypes": [ "Journal Article", "Review" ], "first_author": "Dhakal R", "dirs": [ "B" ] }, "40715080": { "pmid": "40715080", "title": "Concavity-enhanced chiral self-assembly of anisotropic nanoparticles toward strong chiroptical activity.", "abstract": "Chiral plasmonic nanomaterials have attracted significant attention due to their fascinating chiroptical properties and promising applications including chiral sensing, asymmetric catalysis, biomedicine, and chiroptics. Self-assembly of plasmonic nanoparticles is promising for constructing chiroplasmonic nanomaterials, but it remains challenging to obtain homochiral assemblies with high optical asymmetry. Here, gold nanodumbbells featuring a concave morphology are employed as achiral building blocks for controllable self-assembly into stable homochiral assemblies exhibiting strong chiroptical activity. The formation of helically stacked side-by-side assemblies with right handedness is triggered by introducing bovine serum albumin as the chiral additive. Remarkably, an asymmetry factor as high as 0.23 is obtained for the chiral assemblies. It is revealed that the concavity of the nanodumbbells considerably enhances the chirality and stability of the assemblies. Furthermore, the chiral assemblies are utilized as hosts for achiral fluorescent species to generate circular polarization luminescence. This work may advance the structural design of building blocks for chiral assembly toward novel chiroplasmonic nanomaterials.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-62165-3", "ptypes": [ "Journal Article" ], "first_author": "Zhou J", "dirs": [ "B" ] }, "39892116": { "pmid": "39892116", "title": "DMSO enhanced one-pot HDA-CRISPR/Cas12a biosensor for ultrasensitive detection of Monkeypox virus.", "abstract": "We present a dimethyl sulfoxide (DMSO)-enhanced one-pot HDA-CRISPR/Cas12a biosensor for the ultrasensitive detection of the monkeypox virus (MPXV). The MPXV B6R gene was initially amplified using DMSO-enhanced helicase-dependent amplification (HDA) in the bottom of the reaction tubes. DMSO was employed to enhance the amplification efficiency of HDA. CRISPR/Cas12a reagents, pre-added to the caps of the reaction tubes, were subsequently combined with HDA products to generate fluorescence signals. This DMSO-enhanced HDA-CRISPR/Cas12a biosensor enables the detection of synthetic B6R DNA within 1 hour, with a detection limit of 9 aM and a dynamic range of 10 aM to 100 pM. Our work demonstrated that 5% DMSO can enhance the sensitivity of the HDA -CRISPR/Cas12a assay by four orders of magnitude. For clinical applications, this approach can detect as low as 0.4 copies/μL of MPXV pseudovirus. A DMSO-enhanced HDA-CRISPR/Cas12a lateral flow biosensor (LFB) was developed for MPXV point-of-care testing (POCT), achieving a LOD of 10 fM. This method exhibits high specificity in distinguishing the monkeypox virus from closely related orthopoxviruses, including variola, vaccinia, cowpox, ectromelia, and camelpox. The assay is rapid (sample-to-answer times less than 1 h), cost-effective, and compatible with both fluorescence detection and the LFB for visual readouts.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2025.127660", "ptypes": [ "Evaluation Study", "Journal Article" ], "first_author": "Yu L", "dirs": [ "B" ] }, "40857695": { "pmid": "40857695", "title": "Tailoring High-Performance Poly(m-phenylene isophthalamide) Ultrafiltration Membranes Using Green Amphiphilic Sugar Esters.", "abstract": "This study pioneers the development of high-performance poly(m-phenylene isophthalamide) (PMIA) ultrafiltration membranes through synergistic integration of nonsolvent-induced phase separation (NIPS) and surface segregation techniques, employing sugar esters (SE-15) as an eco-friendly additive. Comprehensive characterization techniques were systematically conducted to investigate the influence of SE-15 on the microstructure, surface property and perm-selectivity of the SE-15/PMIA membranes. Specially, the nanoscale X-ray microscopy (Nano-CT) technique was employed to delve deeply into pore structures, revealing the effect of adding SE-15 on the evolution of pore channels. The synergistic interplay between SE-15's solubilization effect in the casting solution and its phase segregation behavior, as further verified through molecular dynamics simulations, significantly enhanced membrane porosity and hydrophilicity. With an appropriate SE-15 concentration (0.2 wt %), the SE-15/PMIA membrane demonstrated increased pore size (from 43.7 ± 3.0 to 62.2 ± 1.9 nm), porosity (from 67.0 ± 1.8 to 87.7 ± 2.2%), and hydrophilicity (from 69.6 ± 1.0 to 60.3 ± 1.3°) as well as a remarkable 32% improvement in pure water flux (from 576.0 ± 24.2 to 762.6 ± 5.4 kg·m-2·h-1) while maintaining bovine serum albumin (BSA) rejection rate >95%. As a green, nontoxic additive for advanced membrane engineering, SE-15 exhibits unique benefits in constructing porous PMIA membranes, offering a promising alternative to conventional petrochemical additives in polymer membrane fabrication. The SE-15/PMIA membranes demonstrate great potential in water treatment, particularly drinking water purification, municipal wastewater reclamation, and seawater pretreatment that require simultaneous high permeation and eco-safety.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c14782", "ptypes": [ "Journal Article" ], "first_author": "Li LL", "dirs": [ "B" ] }, "41569305": { "pmid": "41569305", "title": "High-Performance Perovskite Solar Cells Enabled by the Guanidine Derivative.", "abstract": "The commercialization process of perovskite solar cells (PSCs) is hindered by their intrinsic defects and instability, which would trigger ion migration and efficiency degradation. Additive strategies have been widely investigated to enhance the performance of PSCs, while the limited solubility of some potential additives inhibits their further application. Herein, the guanidine derivative creatine is introduced into the perovskite precursor to decrease defects and minimize ion migration. Through theoretical and experimental measurements, we convince that the strong interaction between creatine and PbI2 incites the solubility increase of creatine in DMSO. Specifically, the C=O in carboxyl and imine group could effectively chelate with Pb2+, while the amine group tends to form a hydrogen bond with iodide ions. Through synergistic effects, the addition of creatine can not only tailor the crystallization process to decrease defects but also obstruct ion migration. As a result, the PSCs achieve a champion power conversion efficiency of 25.51%, featuring an extremely enhanced stability, retaining over 93.2% of its initial value after exposure under an ambient environment for 1000 h. This work highlights the application potential of the additives with limited solubility in enhancing the performance of PSCs.", "journal": "ACS applied materials & interfaces", "year": "2026", "doi": "10.1021/acsami.5c23109", "ptypes": [ "Journal Article" ], "first_author": "Wang J", "dirs": [ "B" ] }, "40614649": { "pmid": "40614649", "title": "Betaine promotes chicken growth through modulating gut microbiota and FXR-mediated activation of IGF genes.", "abstract": "Betaine is a growth-promoting additive used in both the animal production and microbial fermentation industries. The primary mechanisms by which betaine promotes animal growth are well known through its direct action on the host cells. However, it remains unclear whether betaine exerts its growth-promoting effects in chickens dependent on the gut microbiota. Here, we found that betaine promotes the growth of broiler chickens while enhancing the richness and diversity of the cecal microbiota and increasing beneficial bacteria, such as Lactobacillus, Limosilactobacillus, and Prevotella (P < 0.05). However, the growth-promoting effects of betaine were abolished in broilers treated with an antibiotic cocktail. Furthermore, fecal microbiota transplantation from betaine-supplemented chickens could recapitulate the promoting effect of betaine on body weight, breast muscle weight, and hepatic insulin-like growth factors (IGFs) synthesis (P < 0.05), indicating that the gut microbiota plays an indispensable role in betaine's growth-promoting action. Mechanistically, betaine promotes (P < 0.05) the synthesis of bile acids, and microbial function predictions suggest that betaine upregulates the biosynthetic pathways of primary and secondary bile acids. Notably, the expression of the bile acid receptor farnesoid X receptor (FXR) was upregulated (P < 0.05) in the liver, promoting FXR binding to the IGFs genes promoter regions and activating the transcription of IGFs gene. Taken together, our findings suggest that betaine promotes broiler growth via microbiota-dependent mechanisms, accompanied by FXR-mediated upregulation of IGFs gene expression in the liver, providing new perspectives and theoretical support for understanding the complexity of animal growth regulation.", "journal": "Poultry science", "year": "2025", "doi": "10.1016/j.psj.2025.105455", "ptypes": [ "Journal Article" ], "first_author": "Wang Y", "dirs": [ "B" ] }, "40967546": { "pmid": "40967546", "title": "Preparation and application of citric acid crosslinked gelatin-polyvinyl alcohol composite films for active food packaging.", "abstract": "This study employed citric acid (CA) to crosslink Gelatin (GA) and Polyvinyl alcohol (PVA), enhancing their surface morphology and film properties through esterification. Magnolol (Mag) was subsequently incorporated as a functional additive to develop composite film with antibacterial and antioxidant properties. The results demonstrated that the CA-crosslinked composite film exhibited a tensile strength of 9.67 MPa, an elongation at break of 277.07 %, and a water vapor transmission rate of 2.3 (10-13 kg m-1s-1 Pa-1). In addition, the composite film demonstrated effectiveness in cherry preservation by preventing moisture loss, inhibiting microbial growth, and improving shelf life. This study proposes a novel multifunctional design approach for biodegradable bio-based packaging materials, demonstrating the potential of natural bioactive compounds combined with crosslinking modification technology in the field of food packaging.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.147715", "ptypes": [ "Journal Article" ], "first_author": "Zhuang Z", "dirs": [ "B" ] }, "41425917": { "pmid": "41425917", "title": "Machine learning-based early screening of mild cognitive impairment using nutrition-related biomarkers and functional indicators.", "abstract": "Mild cognitive impairment (MCI), an early stage of cognitive decline preceding dementia, poses a growing public health concern, especially in aging populations. Early identification of individuals at risk is essential for implementing timely interventions to delay or prevent progression to dementia. Nutritional factors and related biomarkers have emerged as promising targets for developing convenient, scalable screening strategies, particularly in resource-limited rural settings. This study aimed to develop and validate a machine learning (ML) model that integrates diet-related metabolites, physical examination indicators, lifestyle behaviors, and sleep quality to predict MCI risk and to evaluate the biological and predictive relevance of trimethylamine N-oxide (TMAO) and its dietary precursors among older adults in rural China. Data were derived from a large-scale epidemiological survey in Fuxin County, Liaoning Province, including 907 participants, of whom 270 were classified as MCI based on the Montreal Cognitive Assessment-Basic. Seven ML models were trained and evaluated using accuracy, sensitivity, and the area under the receiver operating characteristic curve (AUC). The best model's predictors were interpreted using Shapley Additive Explanation (SHAP) values. The random forest model showed the bestperformance (AUC = 0.74, 95% CI: 0.677-0.801; sensitivity = 0.72). SHAP analysis identified age, choline, carnitine, betaine, TMAO, daily intake of fruit and vegetables, body mass index, hip circumference, and daytime dysfunction as key predictors. TMAO-related metabolites consistently contributed positive SHAP effects, suggesting biologically relevant links between dietary metabolism and early cognitive decline. This interpretable ML framework offers a feasible, sensitive, and biologically informed approach for early MCI screening and supports the integration of nutritional biomarkers into cognitive health surveillance.", "journal": "Frontiers in aging neuroscience", "year": "2025", "doi": "10.3389/fnagi.2025.1641690", "ptypes": [ "Journal Article" ], "first_author": "Yuan L", "dirs": [ "B" ] }, "41424702": { "pmid": "41424702", "title": "Dietary Betaine Improves Immune Function, Antioxidant Status, Immune-Related Signaling Molecules, and Disease Resistance in Fish Species.", "abstract": "Phytochemicals derived from plants have attracted attention as feed additives in aquaculture due to their natural bioactive properties. These compounds have a wide range of benefits such as improving fish growth, increasing feed efficiency, and enhancing immunity. As natural alternatives to synthetic chemicals and antibiotics, phytochemicals contribute to disease resistance, reducing oxidative stress, and promoting gut health in aquatic animals. Betaine, a natural compound with osmotic and metabolic functions, has become a suitable feed additive in aquaculture due to its potential benefits for fish health. The role of betaine in maintaining cellular osmotic balance and enhancing physiological processes such as protein synthesis has made it an effective dietary supplement for enhancing fish growth. Recent studies indicate that this compound not only supports growth performance but also plays a fundamental role in enhancing fish immunity. Betaine enhances the innate defense mechanisms of fish against pathogens by increasing the activity of immune cells, especially macrophages, regulating cytokine production, and reducing inflammation. In addition, betaine's antioxidant properties reduce oxidative stress and improve immune signaling, helping fish maintain optimal immune function even in stressful environments. This comprehensive review aims to investigate the multifaceted role of betaine in improving fish immunity and antioxidant responses and the mechanisms associated with these roles.", "journal": "Aquaculture nutrition", "year": "2025", "doi": "10.1155/anu/4596572", "ptypes": [ "Journal Article", "Review" ], "first_author": "Yousefi M", "dirs": [ "B" ] }, "39228062": { "pmid": "39228062", "title": "Dialysis treatment, in vitro, and anticoagulation activity of polysulfone-polyacrylamide based-blend membranes: an experimental study.", "abstract": "The majority of treatments are performed with polysulfone (PSf) membranes. The main issue of the PSf membrane is its lack of endothelial function, leading to various processes like platelet adhesion, protein adsorption, and thrombus formation when comes in contact with blood. The crucial aspect in the development of hemodialysis (HD) membrane materials is a biocompatibility factor. This study aims to improve the performance and biocompatibility of PSf membranes by utilizing polyethylene glycol (PEG) as a pore-forming agent and polyacrylamide (PAA) as a multifunctional modifying additive owing to its non-toxic, and biocompatible nature. The formulated HD membranes were characterized using Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM), and Water Contact Angle (WCA) measurements. The biocompatibility results showed that PSf-PAA membranes reduced the adsorption of bovine serum albumin (BSA) protein, hemolysis process, thrombus formation, and platelets adhesion with improved in vitro cytotoxicity results as well as anticoagulation performance. The protein separation results showed that PSf-PAA membranes were able to reject 90.1% and 92.8% of BSA protein. The membranes also showed better uremic waste clearance for urea (76.56% and 78.24%) and creatinine (73.71% and 79.13%) solutes, respectively. It is conceivable that these modern-age membranes may surpass conventional HD membranes regarding both efficiency and effectiveness.", "journal": "Journal of biomaterials science. Polymer edition", "year": "2025", "doi": "10.1080/09205063.2024.2398325", "ptypes": [ "Journal Article" ], "first_author": "Zaman SU", "dirs": [ "B" ] }, "39422298": { "pmid": "39422298", "title": "Removal of Residual Additive Enabling Perfect Crystallization of Photovoltaic Perovskites.", "abstract": "Achieving high-efficiency perovskite solar cells (PSCs) hinges on the precise control of the perovskite film crystallization process, often improved by the inclusion of additives. While dimethyl sulfoxide (DMSO) is traditionally used to manage this process, its removal from the films is problematic. In this work, methyl phenyl sulfoxide (MPSO) was employed instead of DMSO to slow the crystallization rate, as MPSO is more easily removed from the perovskite structure. The electron delocalization associated with the benzene ring in MPSO decreases the electron density around the oxygen atom in the sulfoxide group, thus reducing its interaction with PbI2. This strategy not only sustains the formation of a crystallization-slowing intermediate phase but also simplifies the elimination of the additive. Consequently, the optimized PSCs achieved a leading power conversion efficiency (PCE) of 25.95 % along with exceptional stability. This strategy provides a novel method for fine-tuning perovskite crystallization to enhance the overall performance of photovoltaic devices.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2025", "doi": "10.1002/anie.202416887", "ptypes": [ "Journal Article" ], "first_author": "Bian ZK", "dirs": [ "B" ] }, "40691222": { "pmid": "40691222", "title": "Substrate profiling of marine-derived thermotolerant cysteine protease reveals unique cleavage preferences for industrial applications.", "abstract": "Due to their industrial importance, new proteases are constantly being sourced from the marine environment. However, their substrate specificities remain insufficiently studied, restricting the evaluation of their potential applications. Here, we applied multiplex substrate profiling by mass spectrometry (MSP-MS) to globupain, a marine thermotolerant clostripain-like protease and show that it has a novel substrate specificity. Globupain is an endopeptidase with a preference for cleavage of substrates on the C-terminal side of norleucine (Nle), Leu, Asn, Arg and Lys. While it can hydrolyze gelatin and collagen, its reaction rate is lower than that of papain, a commercial cysteine protease. The precise knowledge of substrate specificity of globupain led to the discovery that the calpain inhibitors MG101 and leupeptin inactivate globupain activity with IC50 values of 23.79 and 138.7 nM, respectively. Further investigation of additive effects revealed that globupain activity was stimulated by Triton X-100 and Tween 40 at concentrations of up to 1%. Globupain exhibited tolerance to elevated DTT concentrations and retained most of its activity in the presence of Mg2+ or Mn2+ compared to its preferred cation, Ca2+. In conclusion, globupain is a novel clostripain-like cysteine protease with a distinct substrate cleavage profile and remarkable stability in the presence of various additives, highlighting its potential for industrial applications.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-11635-1", "ptypes": [ "Journal Article" ], "first_author": "Røyseth V", "dirs": [ "B" ] }, "40277659": { "pmid": "40277659", "title": "Hybrid Hydrogels Augmented via Additive Network Integration (HANI) for Meniscal Tissue Engineering Applications.", "abstract": "Orthopedic soft tissue injuries, such as those to the fibrocartilaginous meniscus in the knee, present a significant clinical challenge, impacting millions globally and often requiring surgical interventions that fail to fully restore mechanical function. Current bioengineered meniscal replacement options that incorporate synthetic and/or natural scaffolds have limitations in biomechanical performance and biological integration. This study introduces a novel scaffold fabrication approach, termed Hybrid Hydrogels Augmented via Additive Network Integration (HANI) with great potential for meniscal tissue engineering applications. HANI scaffolds combine cross-linked gelatin-based hydrogels with polycaprolactone (PCL) additive networks, created via Fused Deposition Modeling (FDM), to enhance mechanical strength and replicate the anisotropic properties of the meniscus. Custom Stereolithography (SLA)-printed molds ensure precise dimensional control and seamless incorporation of PCL networks within the hydrogel matrix. The mechanical evaluation of HANI scaffolds showed improvements in compressive stiffness, stress relaxation behavior, and load-bearing capacity, especially with circumferential and 3D PCL reinforcements, when compared to hydrogel scaffolds without additive networks. These findings highlight HANI's potential as a cost-effective, scalable, and tunable scaffold fabrication approach for meniscal tissue engineering applications.", "journal": "Gels (Basel, Switzerland)", "year": "2025", "doi": "10.3390/gels11040223", "ptypes": [ "Journal Article" ], "first_author": "El Kommos A", "dirs": [ "B" ] }, "41916393": { "pmid": "41916393", "title": "Bioresorbable fiber reinforced hydrogel scaffold for cartilage tissue regeneration.", "abstract": "Melt electrowriting (MEW) is an innovative additive manufacturing technology utilized for producing fibrous three-dimensional structures for tissue engineering and regenerative medicine applications. While polycaprolactone (PCL) is the predominant polymer employed in MEW, its limited elasticity and slow degradation rate restrict its effectiveness in tissue regeneration. This study investigates the printability of medical-grade poly(L-lactide-co-caprolactone) (PLCL), which has a faster degradation rate, resulting in MEW PLCL scaffolds characterized by uniform fiber diameter, regular layer stacking, and customizable pore sizes. At a pore size of 500μm, the PLCL scaffolds exhibit superior mechanical properties, with a tensile strength of 22.4 MPa and an elastic modulus of 72.6 MPa. Additionally, compared with PCL scaffolds of the same pore size, PLCL scaffolds promote sustained cell proliferation, along with the upregulation of SOX9 and Col II and the downregulation of MMP7. These results indicate enhanced chondrocyte proliferation and increased synthesis of cartilage-specific matrix. When comparing pore sizes of 300μm and 500μm, PLCL-500 scaffolds support continuous cell proliferation even at lower cell densities. In contrast, PLCL-300 scaffolds promote initial cell adhesion but demonstrate a decline in proliferation after day 3. This constraint downregulates anabolic markers (SOX9, collagen II, Aggrecan) and upregulates matrix-degrading enzyme MMP7, primarily caused by excessive cell packing and compromised nutrient diffusion within scaffolds. Furthermore, the gelatin methacryloyl (GelMA) hydrogel is reinforced by PLCL-500 scaffolds, optimizing their mechanical properties and bioactivity. The composite scaffold exhibits an elongation at break of 1000% and a tensile strength of 0.5 MPa. Importantly, cell viability remains above 90%, with chondrocytes maintaining a spherical phenotype and establishing networks by day 14, accompanied by increased sulfated glycosaminoglycan (sGAG) deposition. This research clarifies the relationships among scaffold dimension, component, mechanical properties, and chondrogenesis, offering a promising strategy for cartilage tissue engineering applications.", "journal": "Biofabrication", "year": "2026", "doi": "10.1088/1758-5090/ae59b5", "ptypes": [ "Journal Article" ], "first_author": "Ye Y", "dirs": [ "B" ] }, "41082953": { "pmid": "41082953", "title": "Gelatin methacryloyl - thiolated graphene oxide composites for soft tissue engineering.", "abstract": "Soft tissue engineering represents a transformative solution for regenerating damaged tissues, with the development of advanced hydrogels playing a crucial role in this process. Gelatin methacryloyl (GelMA) has emerged as a promising material due to its biocompatibility and ability to support cell growth; however, its mechanical limitations have driven the exploration of composite hydrogels, with thiolated graphene oxide (tGONS) standing out as a key additive. In this study, we present a novel strategy involving thiolated graphene oxide nanosheets (tGONS) as covalent crosslinkers within the GelMA hydrogel matrix, wherein thiol groups on tGONS react with GelMA's acrylate groups, enhancing crosslinking and strengthening the hydrogel network. The results demonstrate that incorporation of tGONS at optimized concentrations (≤0.5 mg/mL) leads to a substantial increase in Young's modulus and gel fraction, along with a marked enhancement in antioxidant activity, evidenced by up to 40 % DPPH and 60 % H2O2 scavenging in G1GTII formulations. Despite the increased stiffness, acceptable elongation and high cytocompatibility (>85 % cell viability) were maintained. Additionally, the composite hydrogels showed improved resistance to enzymatic degradation and reduced swelling, offering more precise control over physicochemical properties.These findings highlight the multifunctional advantages of GelMA-tGONS composite hydrogels, positioning them as adaptable and robust scaffolds suitable for applications in soft tissue engineering, including cartilage, dermal, and neural regeneration.", "journal": "Methods (San Diego, Calif.)", "year": "2025", "doi": "10.1016/j.ymeth.2025.10.003", "ptypes": [ "Journal Article" ], "first_author": "Shiravandi A", "dirs": [ "B" ] }, "40029031": { "pmid": "40029031", "title": "Novel Three-Dimensional Printed Simulators for Ultrasound-Guided Prenatal Diagnostic Procedures.", "abstract": "Amniocentesis and chorionic villous sampling (CVS) are essential ultrasound-guided diagnostic procedures in modern obstetrics, but their declining frequency poses challenges for clinician training. We present novel 3-dimensional (3D)-printed simulators designed to train practitioners in performing these procedures under ultrasound guidance. Our models combine 3D-printed components with medical gelatin to mimic the abdomen and pelvis of a pregnant patient. Customizable inserts enable realistic simulated needle targeting practice. This standardized, reproducible, and realistic training tool offers a valuable resource for improving ultrasound-guided procedural skills.", "journal": "Journal of ultrasound in medicine : official journal of the American Institute of Ultrasound in Medicine", "year": "2025", "doi": "10.1002/jum.16675", "ptypes": [ "Journal Article" ], "first_author": "Keller NA", "dirs": [ "B" ] }, "40671639": { "pmid": "40671639", "title": "Methacrylated Carrageenan/Gelatin Interpenetrating Network Microspheres Loaded with Targeted Drugs and combined with PD-L1 Inhibitors for Hepatocellular Carcinoma Treatment.", "abstract": "Hepatocellular carcinoma (HCC) is a highly fatal malignant tumor. The clinical outcomes of vascular interventional therapy for HCC based on existing drug-eluting microspheres remain unsatisfactory. This study adopted a unique fabrication method to synthesize methacrylated carrageenan/gelatin hydrogel embolic microspheres (MCGs). The interpenetrating network formed by these two biocompatible and absorbable natural macromolecules endows MCGs with outstanding mechanical properties and vascular conformity. MCGs could be delivered through microcatheters smoothly, and they are provided in various sizes to accommodate different clinical requirements. Their in vivo degradation behavior is controlled and predictable. Due to the strong anionic nature of carrageenan, MCGs can efficiently load and sustainably release Lenvatinib, achieving the precise delivery of this antiangiogenic targeted drug in the tumor region. Animal models reveal that the microspheres block blood vessels effectively and MCGs allow for high-dose Lenvatinib to have prolonged contact with HCC. We also propose an HCC treatment strategy by combining PD-L1 inhibitors with MCGs carrying Lenvatinib. This combination therapy is safe and effective, and the additive and synergistic effects showed impressive antitumor potency and great clinical application potential.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c12363", "ptypes": [ "Journal Article" ], "first_author": "Ma M", "dirs": [ "B" ] }, "40505891": { "pmid": "40505891", "title": "Bacteriophage lysin-MOFs nanomaterials for treating apical periodontitis.", "abstract": "Enterococcus Faecalis (E. faecalis) infection is a leading cause of refractory apical periodontitis (RAP), where persistent inflammation and bone resorption contribute to poor healing outcomes. Traditional therapies often fail to effectively eradicate E. faecalis. In this study, we develop a novel nanozyme, LysPd138@ZIF-8, by synthesizing a ZIF-8 framework and encapsulated a bacteriophage lysin specific to E. faecalis. This nanozyme exhibits excellent biocompatibility, targeted antibacterial activity, and sustained drug release. Antibacterial assays demonstrate superior efficacy in eliminating E. faecalis, including biofilm-related bacteria. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining confirm that LysPd138@ZIF-8 promotes osteogenesis, while Quantitative PCR (qPCR) analysis show upregulation of osteogenesis-related genes in osteoblasts. Ex vivo tooth experiments further validate its antibacterial efficacy against E. faecalis within complex dental structures. In a mouse model of apical periodontitis, treatment with LysPd138@ZIF-8 effectively clear E. faecalis from the pulp, reduce bone resorption as observed via CT imaging, and significantly inhibit periapical inflammation as evidenced by histological staining. In conclusion, LysPd138@ZIF-8 represents a promising therapeutic strategy for refractory apical periodontitis, combining potent antibacterial efficacy against E. faecalis with osteogenic properties for enhanced clinical management of this challenging condition.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.113949", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kuong M", "dirs": [ "B" ] }, "40998040": { "pmid": "40998040", "title": "Exosomal miR-133b-3p modulates TGF-β1/Treg immunomodulation to ameliorate osteoporosis.", "abstract": "Osteoporosis (OP) is influenced by dysregulated miRNAs, particularly during osteoblast differentiation. The precise mechanisms are still under debate. This study aimed to explore the impact of bone marrow mesenchymal stem cells (BMSCs)-derived exosomal miR-133b-3p on the TGF-β1/Treg-mediated immune pathway, offering insights into OP's pathogenesis and potential therapeutic targets. Bioinformatics analysis of GEO dataset (GSE64433) identified differentially expressed miRNAs in osteoporosis. Target genes were predicted using TargetScan, miRDB, miRTarBase, and miRWalk databases, followed by GO and KEGG pathway enrichment analyses. An OP rat model was constructed by ovariectomy (n = 36, randomly allocated into three groups: control, OP, and OP+exosomal miR-133b-3p, n = 12 per group). BMSCs were isolated at 12 weeks post-OVX.Flow cytometry was used to identify the surface markers of BMSCs, CD29, CD44, CD106, CD34, and CD45. Exosomes were isolated from passages 3-5 BMSCs using ExoQuick kit. Transmission electron microscopy and nanoparticle tracking analysis were used to observe the morphology and size distribution of exosomes, and the expression of exosomal protein markers CD9, CD63, and TSG101 was detected by Western blot. qRT-PCR was performed to detect miR-133b-3p and TGF-β1 expression in exosomes. Dual-luciferase reporter assay validated the direct interaction between miR-133b-3p and TGF-β1 3'-UTR. Dual-energy X-ray bone densitometry was used to detect bone mineral density (BMD) after 4 weeks of treatment with miR-133b-3p-enriched exosomes (200 μg weekly via tail vein injection). Micro-CT was used to analyze the BV/TV, Tb.N, Tb.Th, SMI, Ct.Th, BA/TA, and Tb.Sp. In vitro experiments using isolated CD4+ T cells were conducted to assess TGF-β1 expression and CD4 + CD25 + Foxp3+ Treg cell differentiation via Western blot, RT-PCR, and flow cytometry. Osteoclast marker enzymes TRAP, MMP-9, and Cathepsin K were identified using immunohistochemistry. Bioinformatics analysis revealed 27 differentially expressed miRNAs. Target prediction of miR-133b-3p identified 44 high-confidence genes, with TGF-β1 emerging as a key target. BMSCs expressing CD29, CD44, and CD106 (but not CD34 and CD45) were isolated from both control and OP rats. The identified exosomes were roughly spherical with a double-layered membrane, they had a size distribution of about 103.5 ± 8.2 nm and 105.8 ± 10.6 nm, respectively, and had a positive expression of CD9 CD63, and TSG101. qRT-PCR analysis revealed significantly decreased miR-133b-3p expression in OP group exosomes (P < 0.001). Dual-luciferase assay confirmed direct binding of miR-133b-3p to TGF-β1 3'-UTR. Treating OP rats with exosomal miR-133b-3p improved various bone metrics, increased BV/TV, Tb.N, Tb.Th, BMD, Ct.Th, and BA/TA, decreased Tb.Sp and SMI, and improved bone histopathological changes in rat bone tissue. It decreased osteoclast marker enzyme TRAP, MMP-9, and Cathepsin K expression (P < 0.001). In vitro experiments demonstrated that miR-133b-3p-enriched exosomes promoted TGF-β1 expression and CD4 + CD25 + Foxp3+ Treg cell differentiation, while miR-133b-3p inhibitor exosomes had opposite effects. Exosomal miR-133b-3p derived from BMSCs mitigates OP in rats, acting via the TGF-β1/Treg-mediated immune pathway, presenting a promising avenue for OP therapy.", "journal": "Bone", "year": "2025", "doi": "10.1016/j.bone.2025.117658", "ptypes": [ "Journal Article" ], "first_author": "Zhao Y", "dirs": [ "B" ] }, "40484643": { "pmid": "40484643", "title": "C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance.", "abstract": "Breast cancer is the most diagnosed malignancy and a leading cause of cancer-related deaths among women globally. Cuproptosis plays a significant role in tumor progression and therapeutic response. Increasing studies suggest that targeting cuproptosis presents a promising strategy for cancer therapy, such as through the development of copper nanoparticles as therapeutic agents. However, resistance to cuproptosis has emerged as a critical hallmark of cancer. Therefore, it is essential to further investigate the mechanisms underlying cuproptosis resistance to enhance its therapy effect. The relationship between breast cancer progression and the C5a/C5aR pathway or cuproptosis was determined by single-cell RNA sequencing analyses, RNA-sequence analyses, bioinformatic analyses, survival analyses and immunohistochemistry. The antitumor effects of CuS nanoparticles and C5a receptor antagonists (C5aRA) were assessed by in vitro and in vivo strategies including cell counting kit-8, colony formation assay, relative reactive oxygen species level assay, western blots, real-time quantitative PCR, immunohistochemistry, immunofluorescence assay, flow cytometry and the xenograft mice models. Complement system activation by CuS nanoparticles was tested by ELISA. Our results indicated that activation of the C5a/C5aR pathway contributes to cuproptosis resistance by upregulating ATP7B expression via the Wnt/β-catenin pathway. Consequently, combining CuS nanoparticles with lazer treatment and C5aRA markedly enhanced the antitumor efficacy of CuS nanoparticles by overcoming cuproptosis resistance, leading to a synergistic effect in cancer therapy that included cuproptosis-targeting therapy, immunotherapy, and photothermal therapy. This study reports, for the first time, proved C5a/C5aR pathway-mediated cuproptosis resistance in cancer cells, and combining CuS nanoparticles and C5aRA offers a superior and novel therapeutic strategy for cancer.", "journal": "Journal for immunotherapy of cancer", "year": "2025", "doi": "10.1136/jitc-2025-011472", "ptypes": [ "Journal Article" ], "first_author": "Yang H", "dirs": [ "B" ] }, "41463037": { "pmid": "41463037", "title": "Ultrasensitive Detection of Rare Mutations via Amplifying-Cleaving-Enriching in Acute Myeloid Leukemia.", "abstract": "Background: Detecting low-frequency mutations is crucial for predicting prognosis and monitoring minimal residual disease (MRD) in acute myeloid leukemia (AML). However, the presence of abundant wild-type sequences hinders the detection of rare mutant alleles. We present a highly sensitive method called ACE (Amplifying-Cleaving-Enriching) to selectively enrich mutant sequences. Methods: ACE includes three steps: (1) initial PCR amplification using biotin-labeled primers, (2) cleavage of wild-type sequences with a specific restriction enzyme, and (3) enrichment of undigested mutant alleles via streptavidin-labeled magnetic beads. Results: Using two rounds of ACE, we achieved over 80,000-fold enrichment of mutant sequences carrying FLT3-TKD, enabling the detection of mutant alleles at levels as low as 0.0001% in AML patient blood samples. Additionally, the ACE method can be adapted to nearly any driver mutation by introducing wild-type-specific restriction sites through PCR with mismatched primers, which has been validated in the IDH1 mutation. Furthermore, the ACE method can be flexibly integrated into conventional detection techniques including Sanger sequencing, quantitative real-time PCR, allele-specific PCR, and even with advanced techniques like droplet digital PCR. Conclusions: ACE significantly enhances the sensitivity of existing techniques for rare mutation detection and holds potential for broad clinical applications.", "journal": "Biomedicines", "year": "2025", "doi": "10.3390/biomedicines13123026", "ptypes": [ "Journal Article" ], "first_author": "Zhuang X", "dirs": [ "B" ] }, "40782765": { "pmid": "40782765", "title": "Capsaicin ameliorates cholestasis through modulation of the FXR-SHP and FXR-FGF15 gut-liver axis in mice.", "abstract": "Cholestasis, including primary sclerosing cholangitis (PSC), remains a challenging condition with limited treatment options. The gut-liver axis and FXR signaling pathways play critical roles in maintaining bile acid homeostasis and preventing liver injury. Capsaicin, a natural compound with anti-inflammatory properties, has the potential to modulate these pathways and offer therapeutic benefits for cholestasis. In this study, capsaicin-loaded nanoparticles were developed and evaluated in a PSC mouse model to investigate the therapeutic effects and underlying mechanisms of action. Capsaicin-loaded nanoparticles (CAP@NPs) were synthesized via a solvent evaporation method and thoroughly characterized. Using a DDC-induced primary sclerosing cholangitis (PSC) mouse model, we evaluated their therapeutic effects on liver injury through serum biochemistry and histopathology. TRPV1 expression in fibrotic liver tissue was assessed via qRT-PCR, immunohistochemistry, and immunofluorescence. Inflammation, immune responses, and fibrosis were analyzed using molecular, histological, and cytometric techniques. Effects on bile acid metabolism were investigated by profiling hepatic and ileal gene expression, while gut microbiota shifts and colon injury markers were examined to elucidate underlying mechanisms. Biosafety was confirmed by hematological and pathological assessments. Our findings revealed that administration of CAP@NPs attenuated liver injury, inflammation, fibrosis, and bile duct hyperplasia. Mechanistically, CAP@NPs activated hepatic FXR-SHP and ileal FXR-FGF15 pathways to suppress Cyp7A1 expression, modulated gut microbiota composition (increased Clostridia, decreased Bacteroidia), enhanced intestinal barrier integrity, and reduced endotoxin translocation. This study represents a novel approach to treating cholestasis through targeted modulation of bile acid metabolism and inflammatory pathways, offering hope for new therapeutic strategies in liver disease.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2025.157130", "ptypes": [ "Journal Article" ], "first_author": "Du H", "dirs": [ "B" ] }, "40204766": { "pmid": "40204766", "title": "Lateral flow immunoassay using plasmonic scattering.", "abstract": "The lateral flow immunoassay (LFIA) is one of the most successful sensing platforms for real-world point-of-care (POC) testing. However, achieving PCR-level sensitivity without compromising the inherent advantages of LFIA, such as rapid and robust operation, affordability, and naked-eye detection, has remained a primary challenge. In this study, a plasmonic scattering-utilising LFIA was proposed, created by transparentising a nitrocellulose membrane and placing a light-absorbing backing card under the membrane. This LFIA minimised the background signal from its matrix, leading to substantially enhanced sensitivity and enabling naked-eye detection of the plasmonic scattering signal from gold nanoparticles without optics. Our plasmonic scattering-utilising LFIA showed an approximately 2600-4400 times higher detection limit compared with that of commercial LFIAs in influenza A assays. In addition, it exhibited 90% sensitivity in clinical validation, approaching PCR-level sensitivity, while commercial LFIAs showed 23-30% sensitivity. The plasmonic scattering-utilising LFIA plays a ground-breaking role in POC diagnostics and significantly boosts follow-up research.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-58663-z", "ptypes": [ "Journal Article" ], "first_author": "Lee B", "dirs": [ "B" ] }, "41258876": { "pmid": "41258876", "title": "Ultrasensitive Nucleic Acid Testing: From Foundational Research to Clinical Translation.", "abstract": "The ability to detect nucleic acids at ever-lower detection limits is crucial in molecular science, enabling disease management to shift from reactive response to proactive prediction and personalized medicine. This review synthesizes the technological landscape, which is organized into three strategies. First, we examine amplification-dependent platforms, charting their evolution from the quantitative polymerase chain reaction (PCR) to the absolute quantification of digital PCR (dPCR) and the rapid point-of-care utility of isothermal methods. Second, we explore amplification-free paradigms, where technologies such as clustered regularly interspaced short-palindromic repeat (CRISPR)-based diagnostics and nanopore sequencing achieve sensitivity through signal generation mechanisms that directly interrogate native target molecules. Finally, we detail the emergence of nanotechnology-based biosensors, which employ the physicochemical properties of nanomaterials to transduce molecular recognition events into measurable optical or electronic signals. The clinical and scientific translation of these platforms is very useful, enabling noninvasive cancer monitoring via liquid biopsy, real-time infectious disease surveillance, and the reconstruction of past ecosystems from trace environmental DNA. However, realizing the full potential of these technologies is contingent on overcoming significant translational hurdles. Critical challenges include the standardization of preanalytical workflows to ensure sample integrity and the engineering of fully integrated, cost-effective \"sample-in, answer-out\" systems suitable for widespread deployment. The future trajectory of the field points toward the convergence of these molecular tools with microfluidics, automation, and artificial intelligence, thus promoting decentralized and predictive healthcare.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.5c15324", "ptypes": [ "Journal Article", "Review" ], "first_author": "Li B", "dirs": [ "B" ] }, "39841191": { "pmid": "39841191", "title": "Nanoparticle-assisted PCR: fundamentals, mechanisms, and forensic implications.", "abstract": "Polymerase Chain Reaction (PCR) has transformed forensic DNA analysis but is still limited when dealing with compromised trace or inhibitor-containing samples. Nanotechnology has been integrated into nanoPCR (nanoparticle-assisted PCR) to overcome these obstacles. Nanomaterials improve PCR sensitivity, selectivity, and efficiency. Examples of these materials are semiconductor quantum dots and metal nanoparticles. They enhance DNA binding to primers, stabilize enzymes, and function as effective heat conductors, making accurate amplification possible even with tainted samples. The developments in nanoPCR have potential uses in forensics, as they allow for the more sensitive analysis of smaller, polluted, or deteriorated samples. Nevertheless, there are methodological and ethical issues. To provide credible and legitimate forensic evidence, rigorous validation and standardization of NanoPCR techniques are vital. The article addresses the relevant ethical and methodological aspects in forensic casework while examining the integration of nanotechnology into PCR.", "journal": "International journal of legal medicine", "year": "2025", "doi": "10.1007/s00414-024-03402-0", "ptypes": [ "Journal Article", "Review" ], "first_author": "Vajpayee K", "dirs": [ "B" ] }, "41047757": { "pmid": "41047757", "title": "Rapid and accurate detection of crop viruses by nano-electrochemical sensors.", "abstract": "This review aims to critically examine the development, capabilities, and future prospects of nano-electrochemical sensors as a next-generation solution for the rapid and accurate detection of crop viruses. The motivation stems from the urgent need to overcome the shortcomings of conventional diagnostic tools-such as ELISA and PCR-which, while accurate, suffer from drawbacks in speed, portability, and adaptability to evolving viral threats. We first introduce the fundamental architecture and working principles of electrochemical biosensors, emphasizing key transduction mechanisms including amperometry, voltammetry, and electrochemical impedance spectroscopy. A major focus is placed on the role of nanomaterials-such as gold nanoparticles, carbon-based nanostructures, quantum dots, metal-organic frameworks (MOFs), and MXenes-in enhancing sensor performance through improved surface area, electron transfer, and bioreceptor immobilization. Real-world applications are highlighted through recent advances in detecting agriculturally important viruses like tobacco mosaic virus, plum pox virus, and Citrus tristeza virus, with emphasis on sensitivity, selectivity, and response time. The review also explores current limitations, such as sensor reproducibility, field stability, and biofouling, as well as emerging directions including multiplexed detection systems and integration with Internet of Things (IoT) networks and artificial intelligence (AI) platforms for real-time crop health monitoring. By synthesizing technological advances and outlining actionable research pathways, this review underscores the transformative potential of nano-electrochemical sensors in plant virology and precision agriculture.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d5ay01205h", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Xie A", "dirs": [ "B" ] }, "40136948": { "pmid": "40136948", "title": "Recent Advances in Electrochemical Biosensors for Neurodegenerative Disease Biomarkers.", "abstract": "Neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD), represent a growing global health challenge with overlapping biomarkers. Key biomarkers, including α-synucleins, amyloid-β, and Tau proteins, are critical for accurate detection but are often assessed using conventional methods like enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which are invasive, costly, and time-intensive. Electrochemical biosensors have emerged as promising tools for biomarker detection due to their high sensitivity, rapid response, and potential for miniaturization. The integration of nanomaterials has further enhanced their performance, improving sensitivity, specificity, and practical application. To this end, this review provides a comprehensive overview of recent advances in electrochemical biosensors for detecting neurodegenerative disease biomarkers, highlighting their strengths, limitations, and future opportunities. By addressing the challenges of early diagnosis, this work aims to stimulate interdisciplinary innovation and improve clinical outcomes for neurodegenerative disease patients.", "journal": "Biosensors", "year": "2025", "doi": "10.3390/bios15030151", "ptypes": [ "Journal Article", "Review" ], "first_author": "Bae M", "dirs": [ "B" ] }, "40370821": { "pmid": "40370821", "title": "A snapshot on molecular technologies for diagnosing FAdV infections.", "abstract": "Fowl adenoviruses (FAdV) are prevalent in chickens worldwide, responsible for several poultry diseases, including inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), and gizzard erosion (GE), which result in significant economic losses in the poultry industry. Consequently, detection and efficient identification of FAdV serotypes are becoming extremely urgent to monitor outbreaks and develop vaccination strategies. Conventional PCR (cPCR) tests, combined with Restriction Fragment Length Polymorphism (RFLP) or sequencing, were developed for FAdV diagnosis. Although these molecular tests have considerably improved the accuracy of FAdV diagnosis compared with conventional methods, certain drawbacks remain unresolved, including lack of sensitivity and post-PCR analysis. Subsequently, advanced molecular technologies such as real-time PCR (qPCR), Loop Isothermal Amplification (LAMP), Cross-Priming Amplification (CPA), Recombinase Polymerase Amplification (RPA), Digital Droplet Polymerase Chain Reaction (ddPCR), Dot Blot Assay Combined with cPCR, Nanoparticle-Assisted PCR (nano-PCR), PCR-Refractory Quantitative Amplification (ARMS-qPCR), CRISPR/Cas13a Technology, and High-Resolution Melting Curve (HRM), have been developed to improve FAdV diagnosis.", "journal": "Frontiers in veterinary science", "year": "2025", "doi": "10.3389/fvets.2025.1558257", "ptypes": [ "Journal Article", "Review" ], "first_author": "Kardoudi A", "dirs": [ "B", "C" ] }, "40371045": { "pmid": "40371045", "title": "Nanomaterial-Based Biosensors for the Detection of COVID-19.", "abstract": "The COVID-19 outbreak began in December 2019 and has affected people worldwide. It was declared a pandemic in 2020 by the World Health Organization. Developing rapid and reliable diagnostic techniques is crucial for identifying COVID-19 early and preventing the disease from becoming severe. In addition to conventional diagnostic techniques such as RT-PCR, computed tomography, serological assays, and sequencing methods, biosensors have become widely accepted for identifying and screening COVID-19 infection with high accuracy and sensitivity. Their low cost, high sensitivity, specificity, and portability make them ideal for diagnostics. The use of nanomaterials improves the performance of biosensors by increasing their sensitivities and limiting detection by several orders of magnitude. This manuscript briefly reviews the COVID-19 outbreak and its pathogenesis. Furthermore, it comprehensively discusses the currently available biosensors for SARS-CoV-2 detection, with a special emphasis on nanomaterials-based biosensors developed to detect this emerging virus and its variants efficiently.", "journal": "Indian journal of microbiology", "year": "2025", "doi": "10.1007/s12088-024-01336-0", "ptypes": [ "Journal Article" ], "first_author": "Tewari M", "dirs": [ "B" ] }, "41109398": { "pmid": "41109398", "title": "Telomerase biosensors: A promising approach in cancer diagnostics.", "abstract": "Telomerase, an enzyme responsible for maintaining telomere length, plays a pivotal role in cellular immortality and is frequently activated in cancer cells. The detection of telomerase activity has emerged as a critical biomarker for cancer diagnosis and prognosis. This paper explores the development and application of advanced biosensors designed for the sensitive and specific detection of telomerase activity. We discuss various biosensor platforms, including electrochemical, optical, and nanomaterial-based systems, highlighting their mechanisms, advantages, and limitations. In addition, we review conventional methods for telomerase detection, such as the Telomeric Repeat Amplification Protocol (TRAP), quantitative PCR (qPCR), and Southern blotting. While these methods have been widely used, they present several limitations, including low sensitivity, time-consuming procedures, and the requirement for specialized equipment and expertise. The integration of nanotechnology and molecular recognition elements enhances the sensitivity and specificity of biosensors, enabling early cancer detection and monitoring of therapeutic responses. Furthermore, we address the challenges in translating these technologies from the laboratory to clinical settings and propose future directions for research in telomerase biosensing. This innovative approach holds significant promise for improving cancer diagnostics and personalized treatment strategies.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2026", "doi": "10.1016/j.cca.2025.120668", "ptypes": [ "Journal Article", "Review" ], "first_author": "Khalifezadeh M", "dirs": [ "B" ] }, "40019712": { "pmid": "40019712", "title": "Glioblastoma- derived exosomes (GBM-Exo) regulate microglial M2 polarization via the RAC1/AKT/NRF2 pathway.", "abstract": "The impact of exosome-mediated communication between glioblastoma and microglia on the formation of an immunosuppressive microenvironment remains to be explored. Tumor-associated macrophages are more likely to adopt an M2-like phenotype within the immunosuppressive environment. Here, we investigate the molecular mechanisms by which glioblastoma-derived exosomes promote microglial M2 polarization through RAC1. The expression of RAC1 in GBM was collected from public databases. A C57BL/6 mouse glioma xenograft model was established using intracranial stereotactic injection. RAC1 expression was validated by qRT-PCR, Western blotting, and immunohistochemistry. Glioblastoma-derived exosomes were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy, and Western blotting for exosome markers, with the content of RAC1 being profiled. RAC1 and AKT inhibitors were used to co-treat microglia with exosomes. Microglial polarization under different treatment conditions was assessed by Western blotting and immunofluorescence. Our study reveals that RAC1 is aberrantly expressed in glioblastoma and is associated with macrophage immune infiltration. GBM-derived exosomes, carrying RAC1, promote the M2 polarization of microglia. In microglia treated with GBM-derived exosomes, inhibition of RAC1 activity suppressed AKT phosphorylation and NRF2 nuclear translocation, while reducing the expression of M2 phenotype markers. Notably, following AKT inhibition, the exosome-induced NRF2 nuclear translocation was also significantly suppressed, highlighting the critical role of RAC1-mediated AKT activation in NRF2 translocation and microglial M2 polarization. Our study demonstrates that RAC1-carrying GBM-exosomes promote M2 polarization of microglia, a process mediated through the RAC1/AKT/NRF2 pathway.", "journal": "Journal of neuro-oncology", "year": "2025", "doi": "10.1007/s11060-024-04934-6", "ptypes": [ "Journal Article" ], "first_author": "Wu Q", "dirs": [ "B" ] }, "39681230": { "pmid": "39681230", "title": "Recent advances in lateral flow assays for MicroRNA detection.", "abstract": "Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2025", "doi": "10.1016/j.cca.2024.120096", "ptypes": [ "Journal Article", "Review" ], "first_author": "Nafari NB", "dirs": [ "B" ] }, "41023640": { "pmid": "41023640", "title": "Novel drug-free cascaded nanoparticles induce tumor-specific ROS storms via multimodal synergistic anticancer therapy.", "abstract": "Reactive oxygen species (ROS), generated by sonosensitizers, play a pivotal role in tumor cell apoptosis during sonodynamic therapy (SDT), particularly for tumors located deep within tissues. Nevertheless, conventional sonosensitizers present limitations including inadequate ROS generation, insufficient tumor-specific accumulation, and associated adverse effects, significantly restricting their clinical applicability. To address these limitations, novel drug-free multifunctional nanoparticles (designated HGMP NPs) were synthesized. These NPs consist of mesoporous polydopamine (MPDA)-loaded protoporphyrin IX (PpIX), further surface-modified with glucose oxidase (GOx) and hyaluronic acid (HA), to achieve integrated photothermal, sonodynamic, and starvation-based tumor therapy. Upon exposure to near-infrared (NIR) irradiation (808 nm) combined with ultrasound (US), HGMP NPs exhibited pronounced synergistic anticancer effects. Specifically, the photothermal effect triggered by NIR irradiation effectively enhanced local oxygen supply within tumor sites, thus significantly augmenting ROS production and improving the therapeutic outcomes of SDT. Concurrently, GOx-mediated glucose depletion induced tumor starvation and produced hydrogen peroxide (H2O2), further exacerbating oxidative stress within the tumor microenvironment. Transcriptomic analysis revealed that ROS and TNF signaling pathways represented key mechanisms underlying tumor elimination by this multimodal synergistic strategy. Real-time PCR analysis and ELISA assays further validated activation of the TNF signaling pathway. Importantly, this study first confirmed the high biocompatibility and biosafety of HGMP NPs via serum metabolomics, demonstrating no detectable systemic metabolic perturbations. Collectively, the prepared HGMP NPs provide a rational paradigm for synergistic anticancer therapy. These findings highlight the potential of HGMP NPs as an exceptionally safe and effective nanoplatform for cancer treatment, offering valuable insights into future developments in cancer nanomedicine.", "journal": "Journal of nanobiotechnology", "year": "2025", "doi": "10.1186/s12951-025-03705-3", "ptypes": [ "Journal Article" ], "first_author": "Wen M", "dirs": [ "B" ] }, "40418370": { "pmid": "40418370", "title": "Polymer-Based Electrochemical Sensors for the Diagnosis of Neurodegenerative Diseases.", "abstract": "Acute and chronic neurodegenerative diseases (NDs), including multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease (PD), are characterized by neurodegeneration, which is the gradual malfunction and damage of neurons and axons in the central nervous system. Improved clinical diagnostic workups and the development and tracking of successful disease-modifying treatments are made possible by detecting appropriate neurodegenerative disease (ND) biomarkers. Important biomarkers, such as Tau proteins, amyloid-β, and α-synucleins, are essential for precise identification but are often evaluated using time-consuming, expensive, and traditional techniques like polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Due to its exceptional selectivity and sensitivity, electrochemical biosensing has become a popular, low-cost substitute for more conventional diagnostic methods. Nanoparticles in biosensors are particularly noteworthy because they improve electron transport and aid in immobilizing biorecognition components. Conducting polymers have shown great potential in the field of electrochemical sensing. Conducting polymers have shown great potential in electrochemical sensing. Additionally, research has shown that polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), and poly(thiophene) are often regarded as among the best conducting polymers for fabricating electrochemical sensors. Moreover, a hydrogel biosensor allows for the detection of many parameters simultaneously with real-time monitoring, allowing for more accurate and timely tracking of multiple indicators of a patient. Hydrogel nano(bio)composite sensors that use electrochemical transduction methods to detect analytes are also available. Hydrogel-based polymer sensors for early-stage neurodegenerative diagnosis are examined in this review in a novel way. Afterward, we reviewed electrochemical sensors developed for detecting biomarkers related to diseases, including multiple sclerosis, Alzheimer's, Parkinson's, and Huntington's. There have also been developments devised to enhance efficacy of electrochemical diagnostic tools to address their limitations. In this respect, we have also reviewed many polymers used in electrochemical diagnosis of neurological disorders. Finally, we have also evaluated the limits and prospects of clinical trials involving these electrochemical means of diagnoses.", "journal": "Cellular and molecular neurobiology", "year": "2025", "doi": "10.1007/s10571-025-01570-0", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhang Q", "dirs": [ "B" ] }, "41100567": { "pmid": "41100567", "title": "Plant-Derived Nanovesicle Enhanced Microribonucleic Acid (MicroRNA) Transfer from Nasal Cavity to the Brain.", "abstract": "The nasal-to-brain route for drug delivery is potentially useful for the treatment of brain disorders. Not only chemically modified molecules but also nanosized carrier vesicles, such as cell culture-derived exosomes, as small molecules, could be delivered via this route. The focus of the present study was to evaluate the potential of vesicular plant-derived nanoparticles (PDNPs), which have been identified as multivesicular bodies, for transporting molecules via the nasal-to-brain route. PDNPs were isolated from the edible parts of onion (Allium cepa), cherry tomato (Lycopersicon esculentum var. cerasiforme), Delaware grape (Vitis labrusca \"Delaware\"), and grapefruit (Citrus × paradisi) using commercial kits without ultracentrifugation. Recombinant exogenous microRNA (cel-miR-39) was encapsulated into PDNPs by electroporation. Cel-miR-39-incorporated PDNPs were administered to adult male C57BL/6J mice via intranasal instillation or intravenous injection, and then tissue samples were collected. Cel-miR-39 transportation efficiency was evaluated by quantitative RT-PCR. Intranasal instillation was found to be more effective than intravenous injection for microRNA delivery to the brain. The onion-derived nanoparticle was the most effective transporter of microRNA to the olfactory bulb and caudal brain. The transportation potential and kinetics of other molecules (therapeutic drugs) using onion-derived nanoparticles are of future interest.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00931", "ptypes": [ "Journal Article" ], "first_author": "Umezawa M", "dirs": [ "B" ] }, "41791433": { "pmid": "41791433", "title": "Biosensor-based diagnosis for infectious diseases: Nano-enabled revolution.", "abstract": "Infectious diseases (IDs) pose a significant global health threat, exacerbated by the rise of multidrug-resistant (MDR) and antimicrobial-resistant (AMR) pathogens. The conventional diagnostic methods, such as culture characteristics, microscopical examinations, and polymerase chain reaction (PCR), are reliable but often face challenges like long turnaround times, high costs, and demanding infrastructure and trained personnel. Alternatively, biosensor-based diagnostics have emerged as rapid, cost-effective, and sensitive options for detection of different pathogens. This review delves into the principles, structural components, and classifications of biosensors, emphasizing nano-enabled platforms tailored for diagnosis of IDs. Biosensors are typically comprising a biorecognition element, signal transducer, and data processor. These tools able to convert the molecular interactions into measurable signals through various modalities, including optical (fluorescence, surface plasmon resonance (SPR), chemiluminescence, colorimetric), electrochemical (amperometric, potentiometric, impedimetric), and mass-sensitive formats. The review systematically classifies biosensors based on transduction mechanisms (optical, electrochemical, mass-sensitive, and magnetic) and biorecognition factors, focusing on their advantages and disadvantages. Biosensors are further categorized by bioreceptor type, including aptamers, antibodies, enzymes, peptides, and whole cells, each offering distinct recognition mechanisms. The integration of nanomaterials (NMs), such as gold, silver, magnesium oxide, quantum dots (QDs), and carbon nanotubes (CNTs), enhances the nano-biosensors' sensitivity, miniaturization, and applicability. The review also highlights nano-biosensors that specifically detect the pathogens like Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa), underscoring their clinical relevance and low detection limits. Ultimately, the review discusses current limitations and future pathways for innovation, including multiplexed detection, artificial intelligence (AI) integration, and improved biosensor portability. By enabling early and precise identification of resistant pathogens, nano-biosensors represent a transformative advancement in diagnostic microbiology, particularly in resource-limited settings. Overall, this review demonstrates that nano-enabled biosensors provide rapid and cost-effective options to traditional diagnostic techniques, providing crucial insights for early detection of MDR pathogens and outbreak manipulation.", "journal": "Microbial pathogenesis", "year": "2026", "doi": "10.1016/j.micpath.2026.108410", "ptypes": [ "Journal Article", "Review" ], "first_author": "Elkady FM", "dirs": [ "B" ] }, "40571859": { "pmid": "40571859", "title": "Future perspectives of GMO detection in agriculture: strategies for electrochemical nucleic acid detection.", "abstract": "The uncontrolled distribution of genetically modified organisms (GMO)-based food and feed is an increasing global concern, primarily due to limited information about their potential harmful effects. The growing diversity and complexity of GMOs present significant challenges for their detection, traceability, and safety monitoring. Traditionally, GMOs are detected using molecular methods, among which PCR methods are the most explored and are considered the gold standard. However, isothermal nucleic acid amplification methods, though less explored, hold great potential, especially when integrated with biosensor platforms, enabling the development of highly efficient and versatile biosensing systems. This paper provides a comprehensive overview of the recent advances in biosensors utilizing methods of isothermal nucleic acid amplification, highlighting their current progress and future perspectives. We discuss molecular methods for GMO detection, focusing on reaction conditions, amplification efficiency, and compatibility with various detection modalities. Additionally, we investigate the integration of various nanomaterials into transducers, such as electrochemical platforms, together with the electrochemical techniques and detection mechanisms, aiming to outline their synergistic effects with molecular techniques to improve detection sensitivity and enable real-time monitoring. Furthermore, we discuss the applications of GMO biosensors across diverse fields, including food safety and environmental monitoring, while addressing existing challenges and potential strategies for improving the performance, robustness, and practicality of biosensing platforms. Overall, this review highlights the significant progress achieved in GMO biosensors and underscores their promising role in advancing diagnostic and monitoring capabilities.", "journal": "Mikrochimica acta", "year": "2025", "doi": "10.1007/s00604-025-07267-x", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Kuprešanin A", "dirs": [ "B" ] }, "40882729": { "pmid": "40882729", "title": "Anti-inflammatory and osteogenic nanofibrous scaffolds of bioactive glass/carboxymethyl chitosan-reinforced PCL short fibers for alveolar bone regeneration.", "abstract": "Repairing critical alveolar bone defects in inflammatory microenvironments is challenging. To address this, dual-functional nanofibrous scaffolds with anti-inflammatory and osteogenic properties were developed. These scaffolds incorporate polycaprolactone (PCL) short fibers and mesoporous bioactive glass nanoparticles (MBGNs), fabricated through electrospinning and sol-gel processes, respectively. 3D Fibrous scaffolds were achieved through sequential dispersion, homogenization, thermal self-assembly, and surface modification. During this process, gelatin facilitated the self-assembly of the scaffold, while carboxymethyl chitosan was used to integrate MBGN into the PCL scaffolds. Characterization confirmed a porous structure of scaffolds and enhanced mechanical and in vitro acellular properties after MBGN incorporation. Non-cytotoxicity was confirmed by CCK-8 and Live/Dead assays. Flow cytometry showed scaffold-induced macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory), supported by qRT-PCR and ELISA, which showed reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines. In co-culture with human bone marrow mesenchymal stem cells, the scaffolds promoted osteogenic differentiation and mineralization, as shown by ALP/ARS staining and increased expression of osteogenic markers (RUNX2, OCN). These findings highlight the dual immunomodulatory and osteogenic functions of the scaffolds, suggesting their potential as a promising strategy for inflammatory alveolar bone regeneration.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.147197", "ptypes": [ "Journal Article" ], "first_author": "Feng Z", "dirs": [ "B" ] }, "39680916": { "pmid": "39680916", "title": "Rotavirus-Inspired Nanointerface Engineered Biosensors for All-in-One Cancer Diagnosis.", "abstract": "Ultrasensitive and population-scale cancer screening technologies are critical to reducing cancer mortality. However, the current qRT-PCR falls short in high-throughput screening of multiple cancers. Here, a rotavirus-inspired multicancer diagnosis system (RMDS) is developed via nanointerface engineering. RMDS employs Y-shaped DNA (YDNA) probes to encircle the graphene quantum dots (GQDs) for nanointerface modification. The biotransduction mechanisms at the nanointerface are systematically investigated. RMDS greatly enhances the transduction efficiency of biological analytes by optimizing the probe density and configuration. RMDS realizes ultrasensitive detection of the lung cancer KARS G12D mutation with a limit of detection (LoD) of 5.7 aM and the breast cancer-related AKT2 gene (LoD: 3.0 aM). The multiple chambers enable simultaneous diagnosis of multiple cancers and determination of cancer progression. Clinical validation shows RMDS can be a practical solution, which could complement or replace qRT-PCR and become the next-generation all-in-one tool for large-scale population cancer screening.", "journal": "Nano letters", "year": "2025", "doi": "10.1021/acs.nanolett.4c05210", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhu Y", "dirs": [ "B" ] }, "41477104": { "pmid": "41477104", "title": "Comprehensive Review on Viral RNA Extraction Strategies for Enhanced Molecular Diagnostics.", "abstract": "RNA viruses, characterized by their RNA-based genomes, have garnered significant concern due to their role in numerous lethal pandemics and remain a significant global health threat. Their predominance among emerging pathogens underscores the need for rapid detection and molecular characterization. PCR has been proven to be an indispensable tool for monitoring the emergence and spread of RNA viruses, but its success requires efficient viral RNA isolation. Poor RNA recovery may lead to failed detection, limiting downstream applications such as genome sequencing and molecular epidemiology. This review provides an overview of major RNA extraction strategies, including TRIzol-based, spin column-based, and magnetic bead-based methods, as well as novel and automated platforms. We compared their advantages, limitations, and trade-offs, emphasizing compatibility with downstream processes such as PCR and next-generation sequencing. Challenges related to sample collection, transport, storage, and RNA integrity are discussed, particularly in resource-limited settings where cold-chain and biosafety infrastructure may be limited. Additionally, we discussed applications of extracted RNA for diagnostics, viral characterization, and public health interventions, as well as advances in point-of-care (POC) technologies that integrate extraction with detection. Future directions focus on developing robust, cost-effective, and scalable extraction methods that are adaptable to both high-throughput laboratories and portable POC systems, ensuring global preparedness against emerging RNA viruses.", "journal": "Interdisciplinary perspectives on infectious diseases", "year": "2025", "doi": "10.1155/ipid/5579320", "ptypes": [ "Journal Article", "Review" ], "first_author": "Made Artika I", "dirs": [ "B" ] }, "41121215": { "pmid": "41121215", "title": "MiR-877, an exosomal miRNA from mechanical stretch induced adipose derived stromal cells, enhances fracture healing in nonunion rats with type 2 diabetes mellitus.", "abstract": "Bone nonunion or delayed union is a serious complication in diabetic patients with fractures, urgently requiring novel therapeutic strategies. Exosomes derived from stromal cells are naturally occurring nanoparticles carrying bioactive molecules that mediate intercellular communication and play crucial roles in diabetic fracture repair. Importantly, mechanical stimuli can modulate the cargo composition of exosomes, influencing bone healing outcomes. Here, we investigate for the first time whether exosomes derived from mechanically stretched adipose-derived stromal cells (MS-ADSC-Exos) enhance fracture healing in a type 2 diabetes mellitus (T2DM) nonunion model, and elucidate their underlying mechanisms. Exosomes secreted by ADSCs subjected to different magnitudes of cyclic mechanical stretch (0%, 6%, 18%; designated NMS, LMS, and HMS-ADSC-Exos) were applied to rat bone marrow mesenchymal stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) in vitro. Osteogenic differentiation, proliferation, migration, and angiogenesis were evaluated by Alizarin Red S and ALP staining, tube formation, scratch, and migration assays, respectively. Western blotting and immunofluorescence assessed osteogenic marker expression. In vivo, MS-ADSC-Exos or PBS were locally injected into the fracture sites of diabetic rat femoral nonunion models for 3 consecutive days post-operation. Bone regeneration was evaluated by micro-CT and histological analyses at 4 weeks. miRNA profiles of MS-ADSC-Exos were characterized by RNA sequencing, bioinformatics, and qRT-PCR. Functional roles of miR-877 were further validated via mimic and inhibitor transfection assays. In this study, it is shown that exosomes secreted from ADSCs induced via lower mechanical stretch can enhance fracture healing through the promotion of osteogenesis and angiogenesis in a rat model of nonunion with T2DM. Our results suggested miR-877 was significantly upregulated in LMS-ADSC-Exos, and can be transferred into BMSCs and HUVECs, which promotes osteogenesis and angiogenesis in diabetic conditions. This study reveals a novel mechanobiological mechanism whereby mechanical stretch modulates exosomal miRNA content to potentiate fracture repair. Transplantation of LMS-ADSC-Exos accelerates bone regeneration via miR-877-mediated osteogenic and angiogenic pathways. These findings highlight the therapeutic potential of mechanically stimulated ADSC-derived exosomes as natural bioactive nanotherapeutics for diabetic fracture nonunion.", "journal": "Stem cell research & therapy", "year": "2025", "doi": "10.1186/s13287-025-04692-w", "ptypes": [ "Journal Article" ], "first_author": "Tian L", "dirs": [ "B" ] }, "39675069": { "pmid": "39675069", "title": "Innovative strategies for enhancing AuNP-based point-of-care diagnostics: Focus on coronavirus detection.", "abstract": "Highly pathogenic coronaviruses have consistently threatened humanity, encompassing SARS-CoV, MERS-CoV, SARS-CoV-2 and others. Swift detection and accurate diagnosis play a crucial role in promptly identifying high-risk populations, enabling timely intervention, and effectively breaking the transmission chain to reduce casualties. However, the diagnostic \"gold standard\" reverse transcription-polymerase chain reaction (RT-PCR) failed to meet the overwhelming demand during the pandemic due to insufficient equipment and trained personnel, impeding the effective control of viral spread. Undoubtedly, there is an urgent need for the development of convenient, rapid, and sensitive point-of-care (POC) diagnostic technology. Gold nanoparticles (AuNPs) satisfy the substantial market demand for biosensors owing to their exceptional optical properties and stability. In this comprehensive review, we summarize the potential advantages of AuNPs in visual solution colorimetry and lateral flow assays (LFAs) for the diagnosis of COVID-19. We delve into the techniques for enhancing LFA signals, with the goal of increasing both detection sensitivity and specificity. Furthermore, we include the application of smartphones for unbiased and objective interpretation of results. The examples presented in this review are anticipated to inspire researchers in designing AuNPs biosensors to address current and potential outbreaks of infectious diseases in the future.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2024.127362", "ptypes": [ "Journal Article", "Review" ], "first_author": "Lai Z", "dirs": [ "B" ] }, "41409042": { "pmid": "41409042", "title": "RGD-Modified Gold Nanoparticles Loaded with SAHA Remodel the Hypoxic Inflammatory Microenvironment via Inhibiting HIF-1α-VEGF Signaling to Enhance Radiosensitivity in NSCLC.", "abstract": "This research explored the effectiveness of RGD peptide-functionalized gold nanoparticles (AuNPs) loaded with the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) to enhance the radiosensitivity of non-small cell lung cancer (NSCLC) by suppressing hypoxia signaling, thereby mitigating oxidative stress and inflammatory responses. RGD-AuNPs-SAHA was synthesized via citrate reduction, thiol-gold bonding for RGD modification, and SAHA loading. Structural and chemical characteristics were assessed via dynamic light scattering (DLS), transmission electron microscopy (TEM), UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). Elemental distribution was mapped using TEM-EELS. Drug release behavior was evaluated under neutral and acidic conditions (pH 7.4 and 5.5). SAHA release kinetics were assessed at pH 7.4 and 5.5. Cellular uptake and biodistribution were evaluated in A549 cells and xenograft mice using fluorescence labeling and flow cytometry. Therapeutic efficacy was examined via tumor volume measurement, serum cytokine profiling (TNF-α, IL-6, IL-10), oxidative stress markers (SOD, CAT, MDA), and molecular analyses (IHC, IF, Western blot, RT-PCR). DNA damage and apoptosis were quantified using TUNEL and γ-H2AX staining. RGD-AuNPs-SAHA exhibited uniform size (~20 nm), high SAHA encapsulation (85.2%), and pH-responsive release (60% at pH 5.5 vs 35% at pH 7.4). XPS and EELS mapping further verified the formation of Au-S bonds between thiol-modified RGD and the AuNP surface. Quantitative analysis of surface-bound RGD peptides was performed using UV-Vis spectroscopy combined with the Levenberg-Marquardt algorithm. In vivo, RGD-AuNPs-SAHA reduced tumor volume by 60% and modulated inflammatory cytokines (↓TNF-α/IL-6, ↑IL-10). Oxidative stress markers improved significantly (SOD: 110 U/mL; CAT: 85 U/mL; MDA: ↓2 nmol/mL). Hypoxia signaling proteins HIF-1α and VEGF decreased by 50% and 40%, respectively, confirmed by Western blot and RT-PCR. Apoptosis and DNA damage markers increased by 70% (TUNEL) and 65% (γ-H2AX), demonstrating enhanced radiosensitization. RGD-AuNPs-SAHA effectively remodeled the hypoxic tumor microenvironment, attenuated oxidative stress, and suppressed pro-tumorigenic signaling, leading to significant apoptosis and DNA damage. These findings highlight its potential as a radiosensitizer for NSCLC, offering a promising strategy to improve radiation therapy outcomes.", "journal": "International journal of nanomedicine", "year": "2025", "doi": "10.2147/IJN.S531731", "ptypes": [ "Journal Article" ], "first_author": "Lin J", "dirs": [ "B" ] }, "40890109": { "pmid": "40890109", "title": "Towards ultra-sensitive and rapid near-source wastewater-based epidemiology.", "abstract": "Wastewater-based epidemiology is emerging as a powerful early-warning public health surveillance tool. However, gold-standard PCR necessitates transporting samples to laboratories, with significant reporting delays (24-72 h), prompting growing interest in rapid, near-source tests for resource-limited settings. Research has focused on gold nanoparticle dipsticks, but these typically lack sensitivity in wastewater. Herein, we explore two complementary nanomaterial based approaches, using SARS-CoV-2 as an exemplar: 1) visually-read carbon black dipsticks; 2) spin-enhanced fluorescent nanodiamond dipsticks, exploiting selective separation from background autofluorescence. The assay provides a 2-hour turnaround from sample preparation to result with minimal equipment and achieves a limit of detection down to 7 copies per assay. A pilot study with samples from the Welsh National WBE programme finds 80% sensitivity and 100% specificity for carbon black, and 100% sensitivity, specificity for nanodiamonds. A proof-of-concept lab-in-a-suitcase nanodiamond assay tests raw, unprocessed wastewater samples. These findings lay the foundations for near-source WBE early-warning quantum sensors in the environment.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-63192-w", "ptypes": [ "Journal Article" ], "first_author": "Huang D", "dirs": [ "B" ] }, "41486158": { "pmid": "41486158", "title": "Polydopamine-encapsulated modified Pulsatilla decoction: a strategy to enhance ulcerative colitis therapy.", "abstract": "In Traditional Chinese Medicine (TCM), ulcerative colitis (UC) is often categorized as \"protracted dysentery.\" Pulsatilla Decoction has been reported to exert therapeutic benefits in patients with protracted dysentery. To potentially improve therapeutic outcomes in ulcerative colitis, we prepared a modified Pulsatilla Decoction (MPD). Preliminary clinical observations have suggested that MPD may alleviate symptoms in UC patients. However, rectal enema of MPD is often limited by suboptimal patient compliance and relatively short colonic retention. These limitations underscore the need for new TCM formulations. In this study, we encapsulated MPD within polydopamine (PDA) nanoparticles to prolong colonic residence and evaluate therapeutic effects in a UC model. First, PDA@MPD was prepared by encapsulating MPD with PDA. Fourier transform infrared spectroscopy (FT-IR) and other analytical instruments characterized its structure, morphology, and particle size. Drug release property was evaluated by UV-vis spectrophotometry. Subsequently, MPD active components were labeled with Fluorescein isothiocyanate (FITC); PDA@FITC-MPD was prepared similarly and administered orally to mice. In vivo fluorescence imaging tracked retention time and location in the gastrointestinal tract. Finally, the UC model was induced with 3% DSS. After 7 days of PDA@MPD treatment, therapeutic efficacy was assessed via disease activity index (DAI), colon length, histopathology, Western blot, quantitative real-time PCR (qRT-PCR), and ELISA. MPD was encapsulated by PDA. Relative to MPD, PDA@MPD showed a prolonged colonic retention time and modest improvements in colonic damage scores and inflammatory markers in DSS-induced UC mice. These changes were associated with up-regulation of tight-junction proteins (Occludin, ZO-1) and down-regulation of pro-inflammatory cytokines (IL-1β, TNF-α) in the PDA@MPD group. This study indicates that PDA@MPD prolongs colonic retention and is associated with modest improvements in DSS-induced colonic damage and inflammation in mice. These findings may offer a proof-of-concept for exploring polydopamine-based delivery systems in TCM formulations.", "journal": "Chinese medicine", "year": "2026", "doi": "10.1186/s13020-025-01280-1", "ptypes": [ "Journal Article" ], "first_author": "Chen YJ", "dirs": [ "B" ] }, "41357851": { "pmid": "41357851", "title": "GSTP1-modified exosomes derived from ADMSCs relieve LPS-induced acute lung injury.", "abstract": "Acute lung injury (ALI) is a life-threatening critical condition with high mortality. Adipose mesenchymal stem cell (ADMSC)-derived exosomes hold great potential for clinical therapy and drug delivery. Given that glutathione S-transferase Pi 1 (GSTP1), a key enzyme in oxidative stress response, is highly expressed in lung tissue and has been implicated in mitigating inflammatory and oxidative damage. However, the research on GSTP1-mediated ADMSC-derived exosomes in ALI is still blank. ADMSCs were characterized using flow cytometry, oil red O, Alizarin Red S, and clone formation assays. Gene expression was detected by quantitative real-time PCR (qRT-PCR) and western blotting. Exosomes were isolated and characterized by nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM), and their uptake was measured adopting the 3,3'-dioctadecyloxacarbocyanine perchlorate (DIO) probe. In vitro and in vivo ALI models were established exploiting lipopolysaccharide (LPS). Moreover, the cell viability, cytotoxicity, inflammatory response, cell apoptosis, oxidative stress markers, mitochondrial membrane potential, and mitochondrial DNA (mtDNA) and adenosine 5'-triphosphate (ATP) levels, as well as hematoxylin-eosin (H&E) staining were analyzed to evaluate the effects of GSTP1-modified exosomes on ALI. ADMSCs were positive for CD90 and CD73 and negative for CD34 and CD45. Exosomes were successfully isolated from ADMSCs, and GSTP1-modified exosomes exhibited enhanced uptake by human type II alveolar epithelial cells (HPAEpiCs). Furthermore, GSTP1-modified ADMSC exosomes attenuated LPS-induced inflammatory response, oxidative stress, mitochondrial dysfunction, and apoptosis in HPAEpiCs. GSTP1-modified exosomes derived from ADMSCs ameliorate ALI, suggesting a novel therapeutic target for this condition. GSTP1 up-regulated ADMSC-derived exosomes inhibit inflammation, apoptosis, oxidative stress, and mitochondrial dysfunction in LPS-induced ALI of HPAEpiCs. The online version contains supplementary material available at 10.1007/s10616-025-00863-y.", "journal": "Cytotechnology", "year": "2026", "doi": "10.1007/s10616-025-00863-y", "ptypes": [ "Journal Article" ], "first_author": "Guan H", "dirs": [ "B" ] }, "41321900": { "pmid": "41321900", "title": "GLX351322-Loaded Nanoparticles Alleviate Chronic Stress-Induced Depressive Behaviors Through Inhibition of Ferroptosis and Oxidative Stress.", "abstract": "Depression is a widespread neuropsychiatric disorder with limited treatment efficacy and frequent adverse effects. Ferroptosis, an iron-dependent and oxidative stress (OS) - related form of regulated cell death, is emerging as a key pathogenic mechanism in neurological diseases, yet its role in depression remains largely unexplored. This study aimed to evaluate the antidepressant and neuroprotective potential of GLX351322 (GLX), a selective inhibitor of NADPH oxidase 4 (NOX4), by formulating it into a nanocarrier system to overcome its pharmacokinetic limitations. GLX was encapsulated into methoxy poly (ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL) nanoparticles (GLX-NPs) via a simple nanoprecipitation method. Including particle size, zeta (ζ) potential, morphology, drug loading (DL), encapsulation efficiency (EE), biodistribution, and release efficiency, were characterized. In vivo, the antidepressant effect was assessed using a chronic unpredictable mild stress (CUMS) mouse model, while in vitro, the safety profile was evaluated in CORT-induced HT22 cells. Fluorescence, Quantitative real-time PCR (qRT-PCR), and Western blot (WB) experiments were conducted to explore the underlying neuroprotective mechanisms. The average particle size of GLX-NPs was 43.58 ± 3.09 nm, with a ζ potential of approximately -12.13 ± 0.35 mV, a DL of 6.90%, and an EE of 88.79%. GLX-NPs increased the accumulation of the drug in brain tissues. In CUMS mice, GLX-NPs improved depressive-like behaviors and preserved hippocampal neuronal integrity. Mechanistically, GLX-NPs inhibited NOX4 expression, suppressed reactive oxygen species (ROS) production and lipid peroxidation, and activated the Nrf2/HO-1/GPX4 pathway to alleviate ferroptosis. Co-administration with ferroptosis inducers or Nrf2 inhibitors reversed these protective effects. GLX-NPs effectively alleviate depressive-like behaviors by inhibiting neuronal ferroptosis and OS via modulation of the NOX4/Nrf2/HO-1/GPX4 signaling pathway. This study supports the therapeutic potential of GLX-NPs as a novel nanomedicine targeting ferroptosis in the treatment of depression.", "journal": "International journal of nanomedicine", "year": "2025", "doi": "10.2147/IJN.S555165", "ptypes": [ "Journal Article" ], "first_author": "Wang T", "dirs": [ "B" ] }, "39724853": { "pmid": "39724853", "title": "The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.", "abstract": "Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2024.127457", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhao L", "dirs": [ "B" ] }, "41260492": { "pmid": "41260492", "title": "MiR-133a encapsulated in hyaluronic acid nanoparticles alleviates sepsis-induced cardiomyopathy by decreasing M1 macrophage polarization via the SOCS3/JAK/STAT3 pathway.", "abstract": "Sepsis-induced cardiomyopathy (SIC) is a reversible myocardial dysfunction driven by excessive inflammation, with M1 macrophage polarization as a major contributor. This study explored the regulatory role of the miR-133a/SOCS3 axis in macrophage polarization and myocardial injury and assessed a hyaluronic acid (HA)-based nanogel for targeted miR-133a delivery. RAW264.7 macrophages were stimulated with LPS and co-cultured with H9C2 cardiomyocytes, followed by transfection with miR-133a mimics/inhibitor or SOCS3 overexpression plasmids. The direct binding between miR-133a and SOCS3 was verified by dual-luciferase reporter assay. A cecal ligation and puncture (CLP) rat model was used to assess in vivo therapeutic effects after intravenous administration of free or HA-encapsulated miR-133a. Macrophage polarization was measured by flow cytometry and qRT-PCR; inflammatory cytokines by ELISA; protein expression (SOCS3, JAK/STAT3 pathway, iNOS, CD206) by Western blot and immunohistochemistry; apoptosis by TUNEL; and myocardial injury by H&E staining. MiR-133a was significantly downregulated in LPS-treated macrophages and in SIC blood samples, with levels inversely correlated to SOCS3. MiR-133a overexpression suppressed M1 polarization, cytokine production, and SOCS3 expression, while enhancing M2 polarization; these effects were reversed by SOCS3 overexpression. In co-culture, miR-133a reduced cardiomyocyte inflammation and apoptosis through SOCS3/JAK/STAT3 inhibition. In CLP rats, HA-miR-133a achieved greater myocardial accumulation than free mimics, attenuated histopathological damage, lowered cytokine levels, iNOS, and phosphorylated JAK3/STAT3, and increased CD206 expression. MiR-133a alleviates SIC by inhibiting SOCS3/JAK/STAT3 signaling and promoting M2 polarization. HA-based targeted delivery enhances stability, tissue uptake, and therapeutic efficacy, representing a promising SIC therapy.", "journal": "Archives of biochemistry and biophysics", "year": "2026", "doi": "10.1016/j.abb.2025.110673", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zheng C", "dirs": [ "B" ] }, "41447753": { "pmid": "41447753", "title": "Nanoparticle albumin-bound paclitaxel (nab-Paclitaxel): Bridging pharmacology and translational medicine in breast cancer.", "abstract": "Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) represents a significant advancement in breast cancer therapy by enhancing drug delivery and minimizing solvent-related toxicity. Utilizing albumin-mediated transcytosis, nab-paclitaxel achieves superior intratumoral accumulation while eliminating hypersensitivity reactions linked to solvent-based taxanes. Clinical trials in metastatic breast cancer have demonstrated higher response rates and improved tolerability compared with conventional paclitaxel, especially in HER2-negative and triple-negative subtypes. In neoadjuvant settings, nab-paclitaxel-based regimens yield higher pCR rates and exhibit synergistic efficacy when combined with carboplatin, anthracyclines, or immune checkpoint inhibitors. Safety analyses show a favorable profile with reduced myelosuppression but an increased risk of peripheral neuropathy in certain populations. Additionally, nab-paclitaxel has been effectively incorporated into immunotherapy regimens for TNBC and HER2-targeted strategies in HER2-positive disease. Pharmacokinetic studies reveal improved tissue distribution and higher unbound paclitaxel exposure compared to solvent-based formulations. Overall, nab-paclitaxel offers distinct therapeutic advantages in terms of efficacy, safety, and tumor targeting, representing a major step forward in overcoming resistance and optimizing breast cancer treatment.", "journal": "Current research in translational medicine", "year": "2026", "doi": "10.1016/j.retram.2025.103561", "ptypes": [ "Journal Article", "Review" ], "first_author": "Alkhathami AG", "dirs": [ "B" ] }, "41491958": { "pmid": "41491958", "title": "Circulating extracellular vesicle PTPRO methylation: an exploratory biomarker for minimally invasive diagnosis of early-stage lung adenocarcinoma.", "abstract": "Protein tyrosine phosphatase receptor-type O (PTPRO), a member of the PTP family, has garnered attention for its diagnostic and prognostic potential through the methylation of circulating tumor DNA (ctDNA). However, the utility of ctDNA has shown limited sensitivity and specificity, particularly in early-stage lung adenocarcinoma (LUAD). Given the enhanced stability of tumor-derived DNA in small extracellular vesicles (sEVs) from cancer cells, this research investigates the feasibility of using PTPRO methylation in saliva-derived sEVs as a non-invasive and easily accessible biomarker for the early detection of LUAD. To explore the relationship between PTPRO methylation and prognosis in early-stage LUAD, we conducted Kaplan-Meier survival analyses and assessed the methylation status of the PTPRO promoter using methylation-specific PCR (MSP) and q-MSP. Saliva samples were collected from 60 early-stage LUAD patients, 30 pneumonia patients, and 21 healthy controls, with isolation and characterization of salivary sEVs through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and immunoblotting. Kaplan-Meier analysis revealed hypermethylation of PTPRO was linked to poorer overall survival in early-stage LUAD patients. PTPRO methylation was detected in salivary sEVs of 73.3% of early-stage LUAD patients, compared to only 35% in plasma sEVs. Receiver operating characteristic (ROC) analysis confirmed that PTPRO methylation in salivary sEVs effectively distinguished early-stage LUAD patients from both pneumonia patients and healthy individuals. This suggests that PTPRO hypermethylation is associated with adverse prognosis in early-stage LUAD. The detection of PTPRO methylation in salivary sEVs demonstrates high sensitivity and specificity, indicating its potential as an epigenetic biomarker for the non-invasive diagnosis of early-stage LUAD.", "journal": "Cancer cell international", "year": "2026", "doi": "10.1186/s12935-025-04127-9", "ptypes": [ "Journal Article" ], "first_author": "Dong H", "dirs": [ "B" ] }, "40533488": { "pmid": "40533488", "title": "Nanomaterials in PCR: exploring light-to-heat conversion mechanisms and microfluidic integration.", "abstract": "As a popular process in molecular-based diagnostics, polymerase chain reaction (PCR) can be employed for amplifying small amounts of DNA/RNA from different sources such as tissue, cells, peripheral blood and so on. Thanks to the unique physicochemical characteristics of nanomaterials and their progress, researchers have been encouraged to employ them as suitable candidates to address the PCR optimization challenges for enhancing efficiency, yield, specificity, and sensitivity. In nanoparticle-assisted PCR (nanoPCR), different nanoparticles (NPs) such as carbon nanotubes (CNTs), graphene, quantum dots (QDs), and gold (Au) might be used. Among different nanoPCR assays, photothermal PCR has emerged as a technique leveraging the excellent light absorption and heat conversion capabilities of nanomaterials. In addition to presenting recent advances in nanoPCR, this review also delves into the specific use of nanomaterials for photothermal PCR, including their applications in microfluidics as one of the best platforms for miniaturization of diagnostic techniques. Different types of NPs used in PCR are comprehensively examined, and detailed charts and tables are provided that outline features such as optimal concentration and size. The appropriate choice of nanomaterials for enhancing light conversion to heat in PCR applications is discussed. Finally, the related challenges and future trends are explored.", "journal": "Microsystems & nanoengineering", "year": "2025", "doi": "10.1038/s41378-025-00898-3", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shamsian S", "dirs": [ "B" ] }, "40845433": { "pmid": "40845433", "title": "Search and detect pathogens using magnetic iron oxide nanoparticles.", "abstract": "Superparamagnetic iron oxide nanoparticle (SPION)-based labeling is transforming microbial diagnostics by enabling the rapid, highly specific, and sensitive detection of pathogens in complex clinical and environmental samples. This technology improves the separation, tracking, and identification of microorganisms, especially in critical conditions like sepsis or foodborne infections, where prompt intervention is crucial. Coating magnetic particles with antibodies, antibiotics, lectins, or synthetic peptides allows for highly specific binding to microbial surfaces. This enables the concentration and detection of viable pathogens with minimal interference from the surrounding matrix. Magnetic labeling not only improves isolation and serves as a powerful analytical interface. Techniques such as magnetic resonance imaging (MRI) or magnetic particle spectroscopy can exploit the magnetic signal for non-invasive pathogen tracking and phenotyping, including the detection of virulence factors. Additionally, magnetic particles enhance traditional assays, such as PCR or 16 S rRNA sequencing by improving target concentration and the signal-to-noise ratio. Hyperspectral imaging can further distinguish between viable from dead cells based on their spectral signatures. This integrated approach is particularly valuable in time-sensitive clinical settings. For instance, magnetic separation allows for the quicker identification of pathogens responsible for sepsis, a condition where current diagnostics often yield from delayed or nonspecific results. Emerging methods like COMPASS (Critical Offset Magnetic Particle SpectroScopy) offer real-time analysis of pathogenicity factors, aiding in more targeted and effective therapies. Ultimately, the convergence of magnetic nanotechnology, molecular diagnostics, and advanced imaging holds great promise for next-generation microbial diagnostics, offering faster turnaround times, improved specificity, and the ability to distinguish live pathogens from residual genetic material-a key advance for precision medicine.", "journal": "Biochemical and biophysical research communications", "year": "2025", "doi": "10.1016/j.bbrc.2025.152462", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Tietze R", "dirs": [ "B" ] }, "39806951": { "pmid": "39806951", "title": "Current Scenario in Associating Clinical COVID-19 Biomarkers for Developing Surveillance Platforms.", "abstract": "The novel coronavirus that caused the epidemic and pandemic resulting in the acute respiratory illness known as coronavirus disease 2019 (COVID-19) has plagued the world. This is unlike other coronavirus outbreaks that have occurred in the past, such as Middle East respiratory syndrome (MERS) or severe acute respiratory syndrome (SARS). COVID-19 has spread more quickly and posed special challenges due to the lack of appropriate treatments and vaccines. Real-time polymerase chain reaction (RTPCR) and rapid antibody tests (surveillance tests) are the two most used tests (confirmation tests). However, the latter takes hours to complete, and the former may produce false positives. Scientists have invested significant effort to create a COVID-19 diagnostic system that is both highly sensitive and reasonably priced. Early detection of COVID-19 is a major area of focus for sensing devices based on nanomaterials. This overview enhanced insights into potential coronavirus biomarkers and, compared to earlier studies, introduced new avenues. Further, it covers the development of COVID-19 diagnostic systems from an analytical point of view, including clinical markers and their subsequent applications with biosensors.", "journal": "Current medicinal chemistry", "year": "2025", "doi": "10.2174/0109298673331044241031100627", "ptypes": [ "Journal Article", "Review" ], "first_author": "Lakshmipriya T", "dirs": [ "B" ] }, "40304581": { "pmid": "40304581", "title": "miR-27b-3p modulates liver sinusoidal endothelium dedifferentiation in chronic liver disease.", "abstract": "During chronic liver diseases, LSECs undergo a dedifferentiation process contributing to the development of hepatic microvascular dysfunction. Although microRNAs (miRNAs) have been associated with chronic liver disease, their role as modulators of liver endothelial phenotype is mostly unknown. Therefore, the aim of this study was to analyze miRNAs as regulators of hepatic sinusoidal endothelial dysfunction in chronic liver disease to suggest novel and translatable therapeutic options for cirrhosis. Global expression of miRNAs was determined in primary LSECs from healthy and cirrhotic patients (alcohol abuse) and rats (CCl4 inhalation). LSECs were transfected with the mimetic or inhibitor of dysregulated miRNAs or with quantum dot nano-complexes containing miR-27b-3p or negative control, and endothelial phenotype was analyzed by RNA sequencing, quantitative PCR, and western blot. Endothelial or mesenchymal phenotypes were analyzed in LSEC by RNA sequencing, followed by pathway analyses and gene deconvolution. In all, 30 and 69 dysregulated miRNAs were identified in human and rat cirrhosis, respectively, of which 6 miRNAs were commonly dysregulated. Specific exogenous downregulation of miR-27b-3p was associated with the upregulation of target genes, suggesting a correlation between loss of miR-27b-3p and LSEC dedifferentiation. Finally, the expression of miR-27b-3p was efficiently and physiologically re-established in cirrhotic LSECs using nano-miR-27b-3p, leading to modulation of 1055 genes compared with the negative control, ultimately leading to inhibition of the endothelial-to-mesenchymal transition process observed in cirrhosis. Loss of miR-27b-3p expression contributes to LSECs dedifferentiation in cirrhosis. The use of nano-miR-27b-3p represents a new therapeutic option for hepatic diseases coursing with endothelial dysfunction.", "journal": "Hepatology communications", "year": "2025", "doi": "10.1097/HC9.0000000000000700", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Abad-Jordà L", "dirs": [ "B" ] }, "40698406": { "pmid": "40698406", "title": "Hydrogel beads for enhanced nucleic acid analysis in complex fluid matrices.", "abstract": "We present a functionalized hydrogel bead platform designed to capture cell-free DNA (cfDNA), which is released from tumor cells into the bloodstream, and the use of this cfDNA as a biomarker for cancer detection. Hydrogel beads with covalently incorporated probes (HBP) are generated via photo-cross-linking in a two-phase microfluidic system. The precursor solutions from which the beads are generated are comprised of a photoreactive copolymer, magnetic nanoparticles, Cy3-labelled oligonucleotides serving as a barcode for bead identification, and a specific probe designed to bind the target DNA. From these solutions, droplets are generated in the microfluidic system and photocross-linked through C, H-insertion cross-linking (CHic). As a demonstration case, the hydrogel beads are used to detect a mutation (c.1633G>A) in MCF-7 cells, which is known to promote breast cancer progression by activating oncogenic signalling pathways. The HBPs were successfully employed to detect a fluorescently labelled mutation sequence (92 bp) in phosphate-buffered saline (PBS), serum, and whole blood using a fluorescent reader, followed by amplification through polymerase chain reaction (PCR). The sensitivity of the cfDNA detection method described here demonstrates a detection limit as low as 0.36 ng mL-1, which is lower than any other detection limit in the literature so far. The sensitivity of the hydrogel bead platform can be further significantly increased. This is because beads containing the captured DNA can be subjected directly to PCR for the amplification of the target sequence, eliminating the need for any additional purification steps. The simplicity of production, modification, and functionalization of the hydrogel beads, coupled with their high sensitivity in detecting cfDNA, makes this platform a promising approach for diagnosing a range of diseases.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d5lc00447k", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Alpsoy L", "dirs": [ "B" ] }, "40444879": { "pmid": "40444879", "title": "Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain.", "abstract": "Bone cancer metastases are the third most common site of cancer spread after lungs and liver. This condition often causes severe pain that impairs patients' physical, psychological, and social well-being. We aimed to explore the potential therapeutic benefits of magnesium hydride (MgH2) on bone cancer pain (BCP). A BCP model was established in Wistar rats. Daily oral dosing of 0.5% w/w MgH2 was administered. Assessment included pain sensitivity, motor coordination, and emotional behaviors. Hippocampal samples underwent RNA sequencing, Western blotting, immunofluorescence, and quantitative RT-PCR. MgH2 markedly reduced mechanical hypersensitivity and depressive behaviors in rats with BCP. These effects were linked to suppression of the TRPM2-NLRP3 signaling axis in hippocampal microglia. Additionally, MgH2 served as an adjuvant to reduce opioid tolerance during fentanyl co-treatment, enabling lower opioid dosages. Collectively, MgH2 inhibited TRPM2 activation, microglial activation, oxidative stress, and NLRP3 inflammasome formation, which together reduced neuroinflammation and improved therapeutic outcomes. MgH2 nanoparticles may relieve BCP and comorbid depressive symptoms by inhibiting TRPM2-mediated NLRP3 inflammasome activation in hippocampal microglia.", "journal": "Molecular pain", "year": "2025", "doi": "10.1177/17448069251348770", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Xu H", "dirs": [ "B" ] }, "39487879": { "pmid": "39487879", "title": "Digital PCR in Virology: Current Applications and Future Perspectives.", "abstract": "Digital PCR (dPCR) has been used in the field of virology since its inception. Technological innovations in microfluidics more than a decade ago caused a sharp increase in its use. There is an emerging consensus that dPCR now outperforms quantitative PCR (qPCR) in the basic parameters such as precision, sensitivity, accuracy, repeatability and resistance to inhibitors. These strengths have led to several current applications in quantification, mutation detection and environmental DNA and RNA samples. In high throughput scenarios, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the cost and throughput still significantly hampered the adaption of dPCR. There is much unexplored potential within the multiplexing capabilities of dPCR. This will allow simultaneous multi-target quantification and can also partially alleviate the throughput and cost drawback. In this review, we discuss the strengths and weaknesses of dPCR with a focus on virology applications and we discuss future applications. Finally, we discuss recent evolutions of the technology in the form of real-time dPCR and digital high-resolution melting.", "journal": "Molecular diagnosis & therapy", "year": "2025", "doi": "10.1007/s40291-024-00751-9", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Gleerup D", "dirs": [ "B" ] }, "39774267": { "pmid": "39774267", "title": "An optimized eDNA protocol for fish tracking in estuarine environments.", "abstract": "Environmental DNA (eDNA) is revolutionizing how we investigate biodiversity in aquatic and terrestrial environments. It is increasingly used for detecting rare and invasive species, assessing biodiversity loss and monitoring fish communities, as it is considered a cost-effective and noninvasive approach. Some environments, however, can be challenging for eDNA analyses. Estuarine systems are highly productive, complex environments, but samples collected from these settings may exhibit PCR inhibition and a low fish read recovery. Here we present an approach for detecting fish in turbid, highly productive estuarine systems. The workflow includes bead-based extraction, inhibition removal, high fidelity and specificity DNA polymerase (Platinum SuperFi II) and multiplexing the universal MiFish primers. By applying this hybrid method to a variety of complex estuarine samples with known inhibition, we have more than doubled the number of recovered fish species while removing most of the off-target amplification.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-85176-y", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Baidouri FE", "dirs": [ "B" ] }, "41300777": { "pmid": "41300777", "title": "STR-Uggles: Overcoming Humic Acid Inhibition Using Combined STR & qPCR Kit Chemistries.", "abstract": "DNA profiling can fail, or produce poor results, when naturally occurring materials are present during the amplification step. This study demonstrates that simple modifications to the reaction setup can overcome this obstacle. PCR inhibition is caused by a range of compounds including haem, humic acid and dyes. Various strategies to overcome this inhibitory effect have been explored, such as improving extraction methods to remove these compounds, diluting samples to reduce inhibitor concentration, or using inhibitor-tolerant DNA polymerases. In this study, we evaluate whether modified setups can help mitigate the effects of humic acid, a common inhibitor that induces various inhibition mechanisms. We combined the GlobalFiler STR kit with Investigator Quantiplex Pro into a single reaction. Supplementing the amplification GlobalFiler with additional reagents creates altered amplification environments that use additional DNA polymerase and reaction buffer. The modified setups outperformed the standard GlobalFiler protocol, even at the highest concentration of humic acid tested. The STR reactions supplemented with qPCR reagents produced higher-quality profiles with improved allele amplification and an even peak balance, indicating that a dual-DNA polymerase system offers a more robust and inhibitor-tolerant environment for STR amplification. In addition to demonstrating the value of this combined approach, these data provide a comprehensive dataset characterising the impact of increasing humic acid concentrations on profile quality from an ideal DNA input. For PCR inhibitors with similar mechanisms this approach offers broader applicability in forensic casework and a promising step toward more reliable and robust profiling of inhibited samples.", "journal": "Genes", "year": "2025", "doi": "10.3390/genes16111326", "ptypes": [ "Journal Article" ], "first_author": "McDonald C", "dirs": [ "B" ] }, "40560994": { "pmid": "40560994", "title": "Contemporary eDNA methods complement conventional microscopy in zooplankton diet studies: Case study with American lobster postlarvae.", "abstract": "The diets of pelagic marine larvae are difficult to analyze due to their small size and even smaller prey. Furthermore, different methods may lead to alternative interpretations of trophic interactions. Conventionally, diet studies have relied primarily on visual identification of prey through dissection and microscopy. While microscopy has clear benefits, it can yield an incomplete assessment of diet since smaller and soft-bodied prey items are often difficult to identify. Here, we combined conventional microscopy and two contemporary environmental DNA (eDNA) methods: DNA metabarcode sequencing (metabarcoding) and real-time Polymerase Chain Reaction (rtPCR), comparing their advantages/disadvantages in a diet analysis of planktonic American lobster (Homarus americanus) postlarvae. This is the first application of these molecular techniques on the postlarval lobster diet. We also describe the testing and development of a novel blocking primer designed to inhibit the amplification of lobster DNA, enhancing prey amplification. This approach allowed finer-scale identification of a greater variety of prey than microscopy. The targeted rtPCR approach identified a specific prey taxon with high fidelity - but involves a priori decisions regarding the choice of target. Here, an rtPCR assay was developed to target Calanus finmarchicus, an abundant copepod species in the Gulf of Maine, suspected to be an important prey item of larval lobsters. Microscopy revealed broad prey categories and the importance of arthropod prey in the postlarval diet. Metabarcoding confirmed the importance of arthropod prey, while filling in the gaps with additional prey species. Finally, rtPCR was able to detect a significant level of predation on Calanus finmarchicus that neither of the other two approaches identified. The combination of methods provided a richer understanding of diet than any single method alone and future diet studies of a wide range of consumers would benefit from the application of a mixture of microscopy and molecular-based methodologies.", "journal": "PloS one", "year": "2025", "doi": "10.1371/journal.pone.0325889", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Ascher A", "dirs": [ "B" ] }, "41300260": { "pmid": "41300260", "title": "Environmental DNA Metabarcoding in Marine Ecosystems: Global Advances, Methodological Challenges, and Applications in the MENA Region.", "abstract": "Environmental DNA (eDNA) metabarcoding has transformed marine biodiversity monitoring by allowing non-invasive, cost-effective detection of species with high resolution across diverse marine habitats. A systematic literature search was conducted using Google Scholar, Scopus, and the Qatar University Library databases. Relevant peer-reviewed publications were screened and selected based on predefined inclusion criteria to ensure comprehensive coverage of studies. This review synthesizes advances in global and regional eDNA applications, emphasizing the Middle East and North Africa (MENA) region, which faces unique environmental extremes, high endemism, and significant data gaps. eDNA metabarcoding often outperforms traditional methods under comparable sampling effort to traditional surveys in detecting rare, cryptic, and invasive taxa, but technical challenges like incomplete reference databases, primer biases, PCR inhibitors, and inconsistent methodologies limit their effectiveness, particularly in understudied areas such as MENA. Recent developments, including multi-marker approaches, autonomous samplers, and next-generation sequencing, are enhancing detection precision and enabling broader, real-time monitoring. In the MENA region, early studies have revealed eDNA's potential for habitat distinction, biogeographic research, pollution assessment, and the early discovery of non-indigenous species, although progress is hindered by gaps in reference libraries, infrastructure, and regulation. This review underscores the urgent need for regional collaboration, standardized protocols, and capacity-building. By integrating eDNA with traditional methods and leveraging emerging technologies, the MENA region can establish itself as a leader in marine biomonitoring under extreme environmental conditions, providing actionable insights for conservation and sustainable management of its unique marine ecosystems.", "journal": "Biology", "year": "2025", "doi": "10.3390/biology14111467", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sawh SK", "dirs": [ "B" ] }, "41143891": { "pmid": "41143891", "title": "Development and validation of an MPS-based 513-Plex SNP identity panel for degraded forensic samples.", "abstract": "Degraded samples pose a challenge in routine forensic practice. The commonly used short tandem repeat markers are not optimally suitable for the analysis of degraded samples because of their structural complexity and locus length. By contrast, single nucleotide polymorphisms (SNPs), characterised by their single base mutation feature, enable the design of short amplification fragments, conferring an advantage in detecting mutations in degraded samples. Hence, our team has developed a multiplex amplification system for individual identification of degraded samples, encompassing 507 autosomal SNP loci, five Y-InDel loci, and one amelogenin sex determination locus. The amplification fragment lengths in this multiplex system range from 81 to 116 bp. The forensic applicability of this panel was validated through sequencing analysis of 201 samples. Among these were 30 degraded samples (simulated degraded samples: heat degradation and ultrasonic fragmentation; formalin-fixed, paraffin-embedded samples). The results indicated that the genotyping accuracy of all loci included in this panel remained at 100% for samples with 0.1 ng of DNA input and severe degradation. Sensitivity experiments revealed that at a DNA input of only 31.25 pg, the locus detection rate reached 100%, with genotype accuracy of 92.5%. Based on population data analysis, the total discrimination power of this system reached 1-5.513 × 10-143. Furthermore, we included forensic case samples encompassing semen, saliva, menstrual blood, 10-year-old bloodstain cards, and 12-year-old bloodstain cards in the validation studies. The results demonstrated 100% genotyping accuracy across all sample types. Additional, validation data confirmed the system's species specificity (Homo sapiens-specific) and tolerance to inhibitors including humic acid, heme, ethylene diamine tetraacetic acid and indigo (up to 200 µM). In conclusion, this system can serve as a novel tool for the analysis of degraded samples in forensic work.", "journal": "Human genetics", "year": "2025", "doi": "10.1007/s00439-025-02785-3", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "Shi L", "dirs": [ "B" ] }, "41313025": { "pmid": "41313025", "title": "An effective DNA extraction protocol optimized for tropical swamp peat samples.", "abstract": "Peatlands are waterlogged ecosystems that store large amounts of carbon, harbor highly specialized endemic biodiversity, and play a crucial role in climate regulation. However, the microbial communities of tropical peatlands, particularly in the Atlantic Forest, remain largely unexplored. Molecular tools are essential for characterizing these communities, yet conventional DNA extraction kits are often ineffective for peat soils, likely due to their high organic matter content, low pH, and the presence of PCR inhibitors. This study aimed to optimize a conventional soil DNA extraction protocol to enhance DNA yield and purity from tropical peat soils, ensuring more reliable molecular analyses. The optimized protocol resulted in a four-times increase in DNA concentration compared to the conventional protocol and improved DNA purity. Agarose gel electrophoresis confirmed higher DNA integrity and also revealed the presence of RNA in samples subjected to the optimized protocol, indicating its potential for co-extraction of DNA and RNA. Quantitative PCR (qPCR) analysis showed significantly higher abundances of bacterial and archaeal 16S rRNA genes, as well as the mcrA gene, in optimized compared to conventional protocol, with statistically significant differences across soil profiles and depths. Furthermore, the optimized protocol yielded a lower coefficient of variation among replicates, enhancing reproducibility and consistency in microbial quantification. These findings demonstrate that the optimized protocol effectively extracts high-quality DNA from tropical peat soils, addressing common challenges associated with commercial kits. Its implementation can support improved future research on microbial-driven biogeochemical processes in tropical peatlands, particularly in the context of greenhouse gas emissions and climate change.IMPORTANCETropical peatlands play a central role in carbon storage and greenhouse gas dynamics, yet their microbial communities remain largely unexplored due to analytical challenges. High organic matter content, low pH, and PCR inhibitors commonly limit the efficiency of molecular tools in these environments. This study presents an optimized DNA extraction protocol that improves yield, purity, and reproducibility across peat profiles. The protocol enhanced the detection of microbial marker genes, such as bacterial and archaeal 16S rRNA and mcrA, and showed potential for co-extraction of RNA, expanding possibilities for future multi-omic approaches. This methodological advance enables more accurate and consistent microbial analyses in tropical peat soils, contributing to a better understanding of microbial roles in biogeochemical cycles and climate-related processes.", "journal": "Microbiology spectrum", "year": "2026", "doi": "10.1128/spectrum.01373-25", "ptypes": [ "Journal Article" ], "first_author": "Gontijo JB", "dirs": [ "B" ] }, "42050949": { "pmid": "42050949", "title": "[Comparison of the Forensic Testing Performance of Six STR Kits].", "abstract": "To compare the technical performance indicators of six commonly used STR kits, and to evaluate their capabilities in forensic testing. The STR genotyping results of forensic DNA standard sample 9948 with different template quantities were analyzed using IdentifilerTM Plus, AGCU EX28CS, NHID® 25A, VerifilerTM Plus, VersaPlexTM 27PY, and FastDirectTM 25A. Differen-ces in detection sensitivity, allelic drop-out/in, allele peak height and heterozygote balance were eva-luated. Various PCR inhibitors, including humic acid, heme, indigo, calcium ions and ethylenediaminetetraacetic acid (EDTA), were added to the multiplex amplification systems to compare the maximum tolerance levels of the six kits to PCR inhibitors. The detection sensitivity of IdentifilerTM Plus, AGCU EX28CS and VersaPlexTM 27PY was as low as 100 pg, whereas the sensitivity of FastDirectTM 25A, NHID® 25A and VerifilerTM Plus reached 50 pg. At different template quantities, the number of STR loci detected by the five kits was greater than that of IdentifierTM Plus. When detecting templates of 50 pg or less, the total number of allelic drop-out/in observed with the IdentifilerTM Plus was lower than that observed with the other five kits. The allele peak heights detected by AGCU EX28CS at 200 and 100 pg template quantities and by VersaPlexTM 27 PY at 50 pg were significantly lower than those detected by IdentifilerTM Plus (P<0.05). Regarding resistance to inhibitors, NHID® 25A showed stronger tolerance to humic acid (≤250 ng/μL), heme (≤0.6 mmol/L) and calcium ions (≤1.4 mmol/L) than other kits. It was found that VersaPlexTM 27PY showed the strongest tolerance to indigo (≤0.048 mol/L); and FastDirectTM 25A showed the strongest tolerance to EDTA (≤1.0 mmol/L). For heme, AGCU EX28CS and FastDirectTM 25A showed lower tolerance than IdentifilerTM Plus, whereas the other three kits exhibited higher tolerance. For other inhibitors, all five kits demonstrated tolerance comparable to or higher than that of IdentifilerTM Plus. Compared with IdentifilerTM Plus, AGCU EX28CS, FastDirectTM 25A, NHID® 25A, VersaPlexTM 27PY and VerifilerTM Plus show no obvious overall disadvantage in detection sensitivity, allelic drop-out/in, allele peak height, heterozygote balance and inhibitor resistance and even demonstrate superior performance in certain technical indicators.", "journal": "Fa yi xue za zhi", "year": "2026", "doi": "10.12116/j.issn.1004-5619.2024.540105", "ptypes": [ "Journal Article", "Comparative Study", "English Abstract" ], "first_author": "Sun YH", "dirs": [ "B" ] }, "40957701": { "pmid": "40957701", "title": "[Validation and Forensic Application of a Domestic Human DNA Quantitative Detection Kit].", "abstract": "To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration, male DNA concentration in mixed male/female DNA samples, the degree of DNA degradation and inhibitor tolerance. Samples with different concentrations, different male/female ratios, different concentrations of inhibitors, and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR. This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases. This human DNA quantification kit can effectively detect human DNA as low as 0.001 65 ng/μL, and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000. Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone, the DNA concentration can still be accurately detected. The degradation index can effectively characterize the degradation degree of the sample. This kit has been successfully applied in forensic practice. This human DNA quantification kit is accurate and reliable in detection. It can accurately reflect the degradation of DNA and inhibitor tolerance. It has good performance in quantitative accuracy, determination of the male/female ratio in mixed samples, and inhibitor tolerance. It has application potential in forensic case examination.", "journal": "Fa yi xue za zhi", "year": "2025", "doi": "10.12116/j.issn.1004-5619.2023.531101", "ptypes": [ "Journal Article", "Validation Study", "English Abstract" ], "first_author": "Chen J", "dirs": [ "B" ] }, "41619578": { "pmid": "41619578", "title": "Forensic botany clues: Development of a novel Osmanthus fragrans STR multiplex system.", "abstract": "Comprehensive research on human DNA genetic markers shows that short tandem repeats (STRs) have found extensive applications in individual identification and kinship testing. Botanical STRs are less commonly employed in forensic science due to the distinctive structure of plant cells and the incomplete state of genome sequencing. This study developed a novel 12-plex system for the individual identification of Osmanthus fragrans (O. fragrans), a species widely distributed across the world's temperate zones, to furnish essential botanical evidence for forensic investigations at crime scenes. Initially, a method for extracting plant DNA from trace samples was improved, which utilized glass beads to break cell walls and yielded significantly higher DNA quantities compared to the traditional method. Subsequently, five novel highly polymorphic STR loci (GHSTR1, GHSTR2, GHSTR4, GHSTR5, and GHSTR6) with trinucleotide repeats were identified from the O. fragrans reference genome (GCA_019395295.1) and characterized (expected heterozygosity > 0.8, polymorphic information content > 0.8, and power of discrimination > 0.9). These loci were integrated with seven previously reported loci (OF5, OF7, OF8, OF9, OF13, OF19, and OSM063) to establish a multiplex system. Developmental validation was conducted in accordance with ISFG and SWGDAM guidelines. The system demonstrated substantial species specificity, high sensitivity (optimal input: 50-500 pg), robust tolerance to common PCR inhibitors (e.g., ≤500 µM hematin and ≤400 ng/µL humic acid), and reliable performance in mixture studies (detecting minor contributors at 1:19-19:1 ratios). PCR conditions were further optimized to 58°C annealing, 20 min elongation, and 28 cycles. The system showed high precision (standard deviation <0.1 bp), accuracy (size deviation within ±0.5 bp), and inter-laboratory concordance. Population analysis of 273 samples revealed the high power of the system effectiveness (combined power of discrimination = 1 - 1.0045 ×10-19 and combined probability of exclusion = 0.993111589879955) despite observed Hardy-Weinberg equilibrium deviations. Ultimately, the system established definitive links between botanical evidence from suspects/victims and primary crime scenes in two real-world cases. In conclusion, the findings of the present study demonstrate the applicability of a novel STR multiplex system for individual identification of O. fragrans trace samples in forensic science. Without these forensic botany clues, these cases may not have been resolved.", "journal": "Forensic science international. Genetics", "year": "2026", "doi": "10.1016/j.fsigen.2026.103440", "ptypes": [ "Journal Article" ], "first_author": "Liang K", "dirs": [ "B" ] }, "40468880": { "pmid": "40468880", "title": "Molecular Profiling and Precise Diagnosis of Pratylenchus penetrans Infestation in Soil: A qPCR-Based Molecular Approach.", "abstract": "Pratylenchus penetrans, an important soil pathogen, has been reported on various crops in the temperate regions of South Korea. In concern, there is an urgent need for a precise, species-specific quantitative polymerase chain reaction (qPCR) kit to detect and quantify root lesion nematodes for early pest management and to controls yield losses. The present study focuses on D2-D3 region, a known marker for molecular profiling of Pratylenchus sp. A primer set mined from the highly conserved D2-D3 region of P. penetrans was used in a SYBR green based qPCR assay. Initial examination identified P. penetrans from infested soil samples using morphological and phylogenetic analyses. The DPp7F12R primer set demonstrated significant specificity in identifying P. penetrants by both conventional polymerase chain reaction (PCR) and qPCR assays. Linear regression of serially diluted DNA from nematode and nematode inoculated soil revealed a limit of quantification of 2 picograms (r² = 0.984), while also highlighting the impact of soil inhibitors. The qPCR using the DNA from varying densities of P. penetrans inoculated in soil demonstrated a robust correlation (r² = 0.98), indicating the limit of detection down to single nematode. Primer specificity evaluation with field soil sample precisely detected only P. penetrants. Species-specific DPp7F12R facilitate the direct detection of P. penetrants from soil DNA in very shorter time. Reliability of PCR was confirmed using BLAST algorithm, which identified partial sequence of PCR amplicon (300 bp) as P. penetrants. Finally, PCR assay using DPp7F12R is crucial for early detection of P. penetrans infestations, helping improve the plant health.", "journal": "The plant pathology journal", "year": "2025", "doi": "10.5423/PPJ.OA.11.2024.0181", "ptypes": [ "Journal Article" ], "first_author": "Natesan K", "dirs": [ "B" ] }, "40004450": { "pmid": "40004450", "title": "Enhancement of the Precision ID Mitochondrial DNA Whole Genome System for Challenging Unidentified Human Remains.", "abstract": "The Precision ID mitochondrial (mt) DNA Whole Genome system is a fully automated massively parallel sequencing (MPS) solution for the whole mitochondrial genome. While extremely sensitive, the Precision ID system is susceptible to inhibitors and microbial DNA that are often co-extracted from human remains. DNA templates spiked with varying amounts of hematin, humic acid, and calcium, along with bones containing degraded and non-human DNA, were sequenced using the Precision ID system with and without the addition of bovine serum albumin (BSA). BSA added to the initial PCR reaction successfully improved the robustness of the Precision ID system while not negatively impacting the sequencing success of uninhibited samples. The success of BSA is inhibitor-concentration dependent and is effective for templates containing at least 50 ng/μL humic acid, 50 μM hematin, and 1500 μM calcium ions. Furthermore, the presence of microbial DNA in addition to an inhibitor, results in non-specific adaptor ligation to the non-human DNA; BSA can alleviate the inhibition, allowing the human mtDNA to be amplified and sequenced. The addition of BSA to the Precision ID mtDNA system can yield successful sequencing results from challenging case samples that would otherwise fail.", "journal": "Genes", "year": "2025", "doi": "10.3390/genes16020119", "ptypes": [ "Journal Article" ], "first_author": "Canale LC", "dirs": [ "B" ] }, "42838440": { "pmid": "42838440", "title": "Simultaneous detection of RNA and DNA from pathogens with hyperplex PCR.", "abstract": "Rapid and reliable identification of pathogens is of great importance in healthcare settings and in the control of agonistic and antagonistic disease outbreaks. Broad-range methods for molecular detection of parasites, bacteria and viruses in samples with unknown contents are thus highly desirable. Hyperplex PCR (hpPCR) is a recently invented analysis technique that allows for parallel detection of up to 100 DNA and RNA targets. We present the development and evaluation of a proof-of-concept 16-plex hpPCR method targeting RNA viruses, bacteria and a parasite to enable detection of various gastrointestinal pathogens relevant in food or waterborne disease outbreak scenarios. The panel also includes highly pathogenic microbes such as Bacillus anthracis and Yersinia pestis. The hpPCR method showed high selectivity with detection of all expected targets. None of the tested bacteria (11), parasites (1) or viruses (4) gave false positive signals or cross-reactions for any other target in the panel. The limit of detection was comparable to single-plex qPCR/RT-qPCR assays targeting the same pathogens. HpPCR tolerated high amounts of humic acid, with somewhat better results for the bacteria compared to the viruses. The 16-plex hpPCR method may be supplemented with additional assays to further widen the group of targeted pathogens. A hpPCR method simultaneously targeting a multitude of microbes including bacteria, parasites and viruses may thus provide timely and selective detection and identification of pathogens in an outbreak situation.", "journal": "Analytical biochemistry", "year": "2026", "doi": "10.1016/j.ab.2026.116266", "ptypes": [ "Journal Article" ], "first_author": "Bergstrand S", "dirs": [ "B" ] }, "42154114": { "pmid": "42154114", "title": "DNA purity over yield: optimizing extraction and PCR in degraded Asian elephant (Elephas maximus) fecal samples.", "abstract": "Non-invasive genetic monitoring is essential for conserving the endangered Asian elephant (Elephas maximus). However, fecal DNA is highly susceptible to degradation and polymerase chain reaction (PCR) inhibition. Various DNA extraction techniques and optimized downstream PCR parameters were compared to establish a workflow that balances DNA quantity and quality. A modified phenol-chloroform-isoamyl alcohol (PCI) protocol was compared with a commercial extraction kit using 35 unpreserved, degraded fecal specimens to evaluate the trade-offs between DNA yield and purity. The PCI method produced significantly higher concentrations (> 500 ng/µL in some samples) but poor purity ratios (A260/A280 and A260/A230), resulting in complete PCR failure. In contrast, the kit yielded lower DNA amounts but superior purity (A260/A230 = 2.0-2.2), enabling a 57.1% (20/35) amplification success rate for the mitochondrial D-loop when combined with high-processivity KOD polymerase and optimized Mg2⁺ concentrations. Sanger sequencing confirmed E. maximus mitochondrial DNA in 15 specimens, whereas environmental eDNA was detected in two samples. These findings demonstrate that DNA purity, rather than yield, is the critical determinant of PCR success in degraded wildlife samples, underscoring the importance of inhibitor-removal technologies for the reliable and effective management of endangered megafauna.", "journal": "Genes & genomics", "year": "2026", "doi": "10.1007/s13258-026-01772-w", "ptypes": [ "Journal Article" ], "first_author": "Rerkdee S", "dirs": [ "B" ] }, "41017288": { "pmid": "41017288", "title": "Integrated Microfluidic System for Rapid 89-Plex Y-SNP Profiling: Development and Forensic Validation.", "abstract": "Current Y-chromosomal single-nucleotide polymorphism (Y-SNP) detection technologies in forensic genetics often rely on bulky equipment, complex procedures, and lack adaptability to field conditions. To address these limitations, we developed an 89-plex microfluidic Y-SNP system comprising a disposable DNA extraction/amplification chip and a capillary electrophoresis chip. Using in situ lyophilization, the reagents are stabilized for long-term storage. Integrated with a portable device, the system enables a fully automated \"sample-in-answer-out\" workflow and delivers complete 89-locus Y-SNP genotyping within 139 min. The system includes two panels-AIYSNP42 for global high-frequency haplogroups and AIYSNP47 for East Asian O-haplogroup subclades-designed on the basis of the International Society of Genetic Genealogy (ISOGG) phylogenetic tree for multi-level resolution. Validation showed a detection sensitivity of 2.5 ng of DNA, with a 93.6% genotyping success rate across 94 forensic samples. It maintained performance under environmental inhibitors (humic acid ≤ 100 ng/µL, hemin ≤ 300 µM, indigo ≤ 15 mM) and moderate UV-induced DNA degradation. The system demonstrated excellent reproducibility (coefficient of variation, CV < 0.5%) and reliably detected male DNA in mixtures (≥2% in male-female, ≥33% in male-male). This microfluidic system reduces the reliance on the need for conventional laboratory workflows and supports rapid, on-site Y-SNP analysis for pedigree tracing, ancestry inference, and mixture interpretation.", "journal": "Electrophoresis", "year": "2025", "doi": "10.1002/elps.70029", "ptypes": [ "Journal Article" ], "first_author": "Li Y", "dirs": [ "B", "D" ] }, "40765432": { "pmid": "40765432", "title": "A high-speed sequential liquid compartmentalization method for digital loop-mediated isothermal amplification in a microfluidic device.", "abstract": "Accurate and rapid quantification of nucleic acid targets is crucial for molecular diagnostics, particularly in resource-limited settings where simple and robust technologies are required. This study presents a high-throughput digital loop-mediated isothermal amplification (dLAMP) platform for the absolute quantification of nucleic acids in a sample, using a microfluidic device comprising ten thousand nanoliter-scale reaction microchambers. The polydimethylsiloxane (PDMS)-based device achieved complete liquid compartmentalization within 60 s in a single operation using an electronic pipette, without requiring surface modification, pre-degassing, pre-priming, or external pumping systems, which are typically necessary in conventional methods. The aqueous sample/reagent mixture was reliably compartmentalized using fluorinated oil, with 97% of the microchambers successfully filled to at least 80% of their designed volume, exhibiting excellent volumetric uniformity (CV = 0.07). Fluorescent LAMP assays targeting Salmonella and cannabis exhibited strong correlations between estimated and true DNA concentrations (R2 > 0.98), although quantification was consistently underestimated. Correction factors of 1000 and 10 000 were required for synthetic Salmonella and cannabis DNA, respectively, whereas only 10 were needed for cannabis seed-derived DNA, indicating these discrepancies were due to the intrinsic performance of the LAMP assays rather than device limitations. The dLAMP device also enabled the successful detection of cannabis seed DNA in the presence of 10 ng μL-1 humic acid, which inhibits amplification in conventional turbidity-based LAMP, demonstrating its robustness for point-of-care testing (POCT) applications. The distinctive compartmentalization strategy of the pipette-operated dLAMP platform enables high scalability without compromising operational simplicity, achieving high throughput, wide dynamic range, and accurate quantification.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d5lc00486a", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Honda R", "dirs": [ "B" ] }, "40948961": { "pmid": "40948961", "title": "Live culture-based qPCR screening of Taq DNA polymerase variants for resistance to PCR inhibitors.", "abstract": "We present a live culture PCR (LC-PCR) workflow that enables direct screening of randomly mutagenized Thermus aquaticus (Taq) and Klentaq1 DNA polymerase libraries without enzyme purification. Intact bacterial cells expressing individual variants serve as both enzyme source and DNA template in real-time PCR, allowing rapid selection for inhibitor resistance in a 96-well format. Screening ~14,000 clones in the presence of potent inhibitors (chocolate, black pepper) yielded two novel variants-Taq C-66 (E818V) and Klentaq1 H101 (K738R)-with superior resistance to diverse PCR inhibitors, including blood, humic acid, and plant extracts, compared to wild-type and previously known resistant mutants. Resistance persisted after purification, indicating intrinsic enzymatic tolerance. Structural mapping suggests these substitutions may enhance nucleotide binding or stabilize the polymerase-DNA complex, reducing susceptibility to inhibitor interference. LC-PCR reduces screening time, cost, and contamination risk, and is readily adaptable for evolving polymerases with enhanced speed, fidelity, or substrate versatility.", "journal": "Frontiers in bioengineering and biotechnology", "year": "2025", "doi": "10.3389/fbioe.2025.1624735", "ptypes": [ "Journal Article" ], "first_author": "Kermekchiev MB", "dirs": [ "B" ] }, "40416766": { "pmid": "40416766", "title": "DNA State Influences the Uptake and Persistence of Environmental DNA by Marine Sponge Natural Samplers.", "abstract": "Marine sponges as natural samplers of environmental DNA (eDNA) are receiving growing attention as an untapped source of biodiversity data. However, little is known about the state of DNA (e.g., cellular or extracellular) that is captured by these samples and how this compares to conventional aquatic eDNA samples. Here, we present an artificial spiking experiment where DNA in cellular and extracellular states was added into tanks containing two sponge species. Aquatic eDNA samples and sponge natural sampler DNA (nsDNA) samples were collected over 7 days and DNA from the two states was quantified in each sample using quantitative real-time PCR (qPCR). We found that there was a significant interaction between DNA state and sample type (eDNA and nsDNA), with lower detection and concentration of extracellular DNA, compared to cellular DNA, found in nsDNA samples. We also found that detection rate and concentration of DNA were significantly lower in nsDNA than in eDNA overall. During methodological testing, PCR inhibition was observed in both sponge species; this was prohibitive in one of the species. Further work to investigate the degree of PCR inhibition during nsDNA metabarcoding is important to understand its impact on the communities resolved using nsDNA methods. Synthesis and applications. We show that nsDNA may originate from a subset of the DNA present in environmental media, potentially providing a more stable picture of local communities. Natural samplers provide a promising option for hard-to-reach environments and for retrieving biodiversity data from archived samples; however, further work and optimization are required to understand what is and is not well represented by this sample type compared to widely applied aquatic eDNA approaches.", "journal": "Ecology and evolution", "year": "2025", "doi": "10.1002/ece3.70919", "ptypes": [ "Journal Article" ], "first_author": "Dowell R", "dirs": [ "B" ] }, "41098892": { "pmid": "41098892", "title": "Metabarcoding Close to Home: Songbird Nests as eDNA Aggregators for Trophic Ecology and Biodiversity Studies.", "abstract": "Environmental DNA (eDNA) sampling to monitor wildlife presence has mostly focused on water but increasingly includes soil, air, and creative biotic substrates like flowers and spiderwebs. Biotic substrates are unique in that they also provide insight into ecological interactions. Here we explore the ability of eDNA from songbird nests to reveal avian trophic ecology, such as nestling diet and nest predator identity, in addition to local insect biodiversity. Twenty-two nests comprising five New World sparrow species and two nonsparrow passerines were collected in a montane sagebrush steppe ecosystem shortly after confirmed nest predation events. A novel protocol was used to extract eDNA from whole nests, and each nest was sequenced twice-with and without a blocking oligonucleotide. The blocker was designed with alternating locked nucleic acids to specifically inhibit sparrow amplification and improve detection of rare species. A total of 126 species were detected, and the blocker proved highly effective, reducing sparrow reads ~100% with no discernable coblocking of nonsparrow passerines. Species richness in sparrow nests increased by 31% with the blocker when using a minimum read threshold of 10 copies. Most detected species were insects, including likely prey items and ectoparasites of nestling birds. Predators were detected in 36% of nests. We discuss the merits of this rich and unique data source and considerations for future implementation.", "journal": "Ecology and evolution", "year": "2025", "doi": "10.1002/ece3.72164", "ptypes": [ "Journal Article" ], "first_author": "Kronenberger JA", "dirs": [ "B" ] }, "40778187": { "pmid": "40778187", "title": "More than presence-absence; modelling (e)DNA concentration across time and space from qPCR survey data.", "abstract": "Environmental DNA (eDNA) surveys offer a revolutionary approach to species monitoring by detecting DNA traces left by organisms in environmental samples, such as water and soil. These surveys provide a cost-effective, non-invasive, and highly sensitive alternative to traditional methods that rely on direct observation of species, especially for protected or invasive species. Quantitative PCR (qPCR) is a technique used to amplify and quantify a targeted DNA molecule, making it a popular tool for monitoring focal species. Modelling of qPCR data has so far focused on inferring species presence/absence at surveyed sites. However, qPCR output is also informative regarding DNA concentration of the species in the sample, and hence, with the appropriate modelling approach, in the environment. In this paper, we introduce a modelling framework that infers DNA concentration at surveyed sites across time and space, and as a function of covariates, from qPCR output. Our approach accounts for contamination and inhibition in lab analyses, addressing biases particularly notable at low DNA concentrations, and for the inherent stochasticity in the corresponding data. Additionally, we incorporate heteroscedasticity in qPCR output, recognizing the increased variance of qPCR data at lower DNA concentrations. We validate our model through a simulation study, comparing its performance against models that ignore contamination/inhibition and variance heterogeneity. Further, we apply the model to three case studies involving aquatic and semi-aquatic species surveys in the UK. Our findings demonstrate improved accuracy and robustness in estimating DNA concentrations, offering a refined tool for ecological monitoring and conservation efforts. The online version contains supplementary material available at 10.1007/s42519-025-00477-9.", "journal": "Journal of statistical theory and practice", "year": "2025", "doi": "10.1007/s42519-025-00477-9", "ptypes": [ "Journal Article" ], "first_author": "Jones M", "dirs": [ "B" ] }, "41185695": { "pmid": "41185695", "title": "Optimizing target-to-total DNA ratio in eDNA studies: effects of sampling, preservation, and extraction methods on single-species detection.", "abstract": "There are many decisions to be made when sampling for environmental DNA (eDNA) analysis, whether using a targeted, single-species assay or community-based metabarcoding. Of the entire workflow from sampling water to bioinformatic analyses, the first steps in the process of collecting water, filtering it, and preserving the filter membranes represent major decision points upon which the success of downstream processes depend. Though many previous studies have compared water volume filtered, filter pore size, and preservation and extraction methods, the conclusions are often that they produce different results, but it is unclear which is the optimal approach for a given purpose. Here, rather than provide yet another methods comparison paper, we provide a framework for how to make informed decisions from a methods comparison and, importantly, how to combine data collected via different methodological choices. We investigate (1) the volume of water filtered and the filter pore size and (2) the preservation method and extraction method of samples with a specific lens on how these choices impact the detection of a single targeted species (Atlantic bottlenose dolphin, Tursiops truncatus, via quantitative PCR (qPCR)), although in principle these findings apply to single-species assays more generally. We find that larger pore size filters (5 µm vs. 1 µm) and larger volumes of water (3 L vs. 1 L) maximize the ratio of amplifiable target DNA to total DNA without compromising the absolute detection of target. We also find that maximizing total DNA yield during extraction (phenol chloroform vs. two commercial kits) does not always increase target detection likely due to the concentration of inhibitors and co-extraction of off-target DNA. We also comment on variation including technical and biological variability between replicates, finding that by homogenizing source water before filtering removes much of the biological variation. Finally, we present a statistical model that allows for inclusion of data from samples collected and processed in different ways, enabling researchers to change protocols or include data from other field sampling efforts, thereby opening up more possibilities to extend datasets and analyses.", "journal": "PeerJ", "year": "2025", "doi": "10.7717/peerj.20127", "ptypes": [ "Journal Article" ], "first_author": "Andruszkiewicz Allan E", "dirs": [ "B" ] }, "40359613": { "pmid": "40359613", "title": "Development of a multiplex recombinase amplification assay for the rapid and concurrent detection of human DNA and sex identification.", "abstract": "In forensic practice, it is essential to identify human DNA and determine the sex of individuals from biological samples collected at crime scenes. Currently, the common detection methods mainly focus on targeted DNA analysis based on PCR technology, which is time-consuming and relies on laboratory equipment. In recent years, recombinase polymerase amplification (RPA), as one of ubiquitous isothermal amplification technology, has gained popularity across various diagnostic fields due to its advantages of rapid processing and minimal temperature control requirements. This study has developed a multiplex RPA assay suitable for human and sex components identification. The assay has good sensitivity (as low as 25 pg) and strong tolerance to inhibitors (in the presence of 200 ng/μL humic acid, 400 ng/μL tannic acid, and 8000 ng/μL collagen). Furthermore, we combined the alkaline lysis and RPA detection to construct a rapid detection scheme, which can shorten detection time to half an hour. We also conducted a preliminary exploration of the visualization scheme for the constructed RPA assay. The above research demonstrates simultaneous and rapid detection of human and sex components, offering an accurate and sensitive detection scheme.", "journal": "Forensic science international. Genetics", "year": "2025", "doi": "10.1016/j.fsigen.2025.103300", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zheng Y", "dirs": [ "B" ] }, "41407971": { "pmid": "41407971", "title": "Development and validation of a STR and SNP multiplex detection system (88 STRs and 348 SNPs) using massively parallel sequencing.", "abstract": "Short tandem repeats (STRs) have long been the gold standard in forensic DNA analysis, while single nucleotide polymorphisms (SNPs) have increasingly emerged as valuable complementary markers. The advent of massively parallel sequencing (MPS) technology has significantly enhanced the detection resolution of both STRs and SNPs, providing novel approaches for forensic human identification. In this study, we developed and validated a comprehensive multiplex detection system utilizing MPS technology, which enabled the concurrent analyses of 88 STRs and 348 SNPs. Following the SWGDAM guidelines, validation studies were conducted to assess the forensic applicability of the system. Sensitivity study revealed that 100% STR profiles could be obtained from ≥ 0.125 ng, and 100% SNP profiles were achieved with ≥ 0.0625 ng. The panel showed high accuracy, achieving a 99.78% concordance rate with conventional capillary electrophoresis (CE) DNA profiles. The system exhibited robust performance in the presence of four common PCR inhibitors (including humic acid, melanin, indigo, and hematin). Additionally, 99.42% of STR genotypes and 100% of SNP genotypes were successfully obtained from degraded DNA shorter than 500 bp. Furthermore, across gender combinations in mixed samples, both STR and SNP markers performed robustly at 1:1 and 1:3 ratios. At extreme proportions, male-female mixtures (99:1) enable the detection of scarce minor contributors at Y-STR loci. For SNP loci, the ACR value serves as a key evaluative metric for distinguishing mixtures. ROC analysis of these SNP data yielded an AUC of 0.909. Population genetics analyses revealed that the cumulative power of discrimination (CPD) and cumulative probability of exclusion (CPE) both exceeded 0.999999, highlighting its extensive applicability and practical value in forensic genetics. This comprehensive validation study confirmed that the multiplex panel provided high sensitivity, accuracy, and reliability, representing a significant advancement in forensic DNA analysis methodology.", "journal": "International journal of legal medicine", "year": "2026", "doi": "10.1007/s00414-025-03682-0", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "Lu Y", "dirs": [ "B" ] }, "41223486": { "pmid": "41223486", "title": "Development and validation of a 55-Plex X-STR NGS panel for forensic casework: Population genetics, mutation analysis, and kinship discrimination.", "abstract": "X-chromosome short tandem repeats (X-STRs) are valuable genetic markers in forensic genetics, particularly for complex kinship analysis and the analysis of male mixtures, due to their unique inheritance pattern. However, the application of X-STRs has been limited by the relatively small number of loci included in commonly used capillary electrophoresis (CE)-based assays, and the lack of sequence-level polymorphism information provided by CE. This study addresses these limitations by developing and validating a novel next-generation sequencing (NGS) panel comprising 55 X-STR markers. The panel was constructed using multiplexed PCR capture and sequenced on Illumina platforms. We performed comprehensive developmental validation studies according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, assessing repeatability, concordance, sensitivity, resistance to PCR inhibitors, performance with degraded DNA, analysis of DNA mixtures, and species specificity. The panel demonstrated excellent performance across all validation parameters. A minimum input of 0.5 ng of DNA is recommended for reliable genotyping. The system showed robust resistance to humic acid (up to 7 ng/μL) and hematin (up to 30 μM). Accurate genotyping was maintained even with degraded DNA samples. For DNA mixtures, the minor contributor alleles could be reliably detected up to a 1:19 ratio (male-male) and 1:9 (female-male). Furthermore, we conducted a population genetics survey of 268 individuals (154 females and 114 males) from the Han Chinese population in Hebei Province, including a subset of 128 unrelated individuals. A total of 648 alleles were identified across the 55 loci. No significant deviations from Hardy-Weinberg equilibrium (HWE) or linkage equilibrium (LE) were observed after Bonferroni correction, except for three LD pairs. Population genetic parameters, including power of discrimination (DP) and power of exclusion (PE), were calculated. Furthermore, paternal and maternal mutation rates were estimated based on 51 father-mother-children pedigrees including 102 offspring. Finally, we evaluated the panel's ability to distinguish second-degree kinship relationships using both real-world samples (67 pairs) and simulated data (44,000,000 pairs). The results demonstrated the panel's enhanced discriminatory power compared to existing X-STR systems, achieving overall accuracies of 80.47 % to 93.20 % in simulated second-degree kinship tests. This 55-plex X-STR NGS panel provides a powerful new tool for forensic casework, particularly for complex kinship analysis, and contributes valuable population data for the Han Chinese population. The study also highlights the importance of considering sequence variation in X-STR analysis and provides insights into X-STR mutation rates.", "journal": "Forensic science international. Genetics", "year": "2026", "doi": "10.1016/j.fsigen.2025.103385", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "Ma G", "dirs": [ "B" ] }, "42473660": { "pmid": "42473660", "title": "Forensic validation and population genetic evaluation of an MPS-based integrated microhaplotype and Y-SNP/STR (MY) system.", "abstract": "Microhaplotypes (MHs) and Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and short tandem repeats (Y-STRs) are highly informative genetic markers that complement the limitations of conventional autosomal forensic markers. We evaluated an integrated MH and Y-SNP/STR (MY) system based on massively parallel sequencing (MPS), which incorporates 114 Y-SNPs, 45 Y-STRs, and 22 MHs. These 22 MHs were selected based on high effective number of alleles (mean global Ae > 8.3) and substantial ancestry informativeness (In > 0.185), suggesting their utility in mixture deconvolution and ancestry inference. In this study, we performed a comprehensive forensic validation of the MY system, demonstrating high sensitivity to 0.25 ng gDNA under the present experimental conditions, PCR inhibitor resistance (100 μM EDTA, 50 μM hemoglobin, 50 μM humic acid, and 25 μM indigo), species specificity (successfully distinguishing humans from 10 non-human species), repeatability (100%), and concordance (100% for 240 Y-STR alleles). In addition, the global forensic efficacy and ancestry inference of the 22 MHs were evaluated through forensic parameters and population genetic analyses, including principal component analysis (PCA), multidimensional scaling (MDS), F ST , phylogenetic tree, and STRUCTURE, based on data from 26 populations in the 1000 Genomes Project (1KGP) and a Guangdong Han (GDH) cohort. The results indicate that the integrated MH and Y-SNP/Y-STR panel provides complementary information for forensic mixture analysis, paternal-lineage characterization, and biogeographic ancestry inference, while further population-scale and inter-laboratory studies will be valuable for broader implementation.", "journal": "Biochemistry and biophysics reports", "year": "2026", "doi": "10.1016/j.bbrep.2026.102708", "ptypes": [ "Journal Article" ], "first_author": "Fan H", "dirs": [ "B" ] }, "41380605": { "pmid": "41380605", "title": "Optimizing viable bacteria detection in ballast tank sediments: Addressing PCR inhibitors and relic DNA.", "abstract": "The significant ecological risks posed by microbial communities in ballast water and sediments highlight the necessity for accurate species monitoring. However, the complex physicochemical conditions within ballast tanks limit the effectiveness of conventional environmental DNA (eDNA) metabarcoding approaches. This study focuses on two key challenges in monitoring viable microorganisms in ballast tank sediments: interference from relic DNA and PCR inhibition. For eDNA samples from the ballast tanks of three vessels, we paired PMA (propidium monoazide)-treated and untreated samples; each extract was either further processed to remove PCR inhibitors or left untreated. The resulting four sample groups were subjected to qPCR for 16S rRNA gene quantification, 16S rRNA amplicon sequencing, and comparative microbial diversity analyses to assess the impacts of these methodologies. PMA treatment successfully differentiated between intact and relic DNA, reducing total DNA and 16S rRNA gene copies by 9.45 % and 6.93 %, respectively. Subsequent inhibitor removal unmasked the true community abundance, yielding a 16-fold increase in 16S rRNA gene copies and a significant restructuring of the microbial composition. Notably, combining PMA treatment with inhibitor removal decreased 16S rRNA gene copies while increasing ASV richness, suggesting that inhibitors may impair PMA's ability to exclude relic DNA. Our findings emphasize the need to combine PCR inhibitor removal with PMA treatment for quantifying viable microorganisms; however, the optimal protocols for their co-processing require further refinement.", "journal": "Ecotoxicology and environmental safety", "year": "2026", "doi": "10.1016/j.ecoenv.2025.119534", "ptypes": [ "Journal Article" ], "first_author": "He H", "dirs": [ "B" ] }, "41313939": { "pmid": "41313939", "title": "Design of a 200-SNP panel for individual identification and evaluation of genotyping performance using isolated cells.", "abstract": "We developed a novel next-generation sequencing-based genotyping panel for 200 single nucleotide polymorphism (SNP) loci, and evaluated the genotyping performance when using small amounts of DNA. We analyzed 1144 previously characterized autosomal SNPs, and selected 200 informative SNPs, based on the allele coverage ratio (ACR) and amplicon size, to minimize inter-locus depth of coverage imbalances. Analysis of the 200 SNPs was performed using 1 ng of DNA from 66 Japanese individuals. The average ACR of most of the SNP loci ranged from 0.85 to 0.95. We also detected 200-SNP genotypes using diluted DNA samples (500, 250, 125, 60, 30, and 15 pg/reaction), and found high ACRs and concordance from less than 100 pg of DNA: the concordances were as high as 95.7 % and 88.0 %, respectively, when 60 and 30 pg of DNA were used. Our method also showed higher inhibitory tolerance than other methods when humic acid was added to the initial amplification reaction for SNP genotyping. When genotyping in the presence of 10 ng/µL humic acid, no SNP profiles were detected, but profiles with more than 99 % concordance were recovered on addition of bovine serum albumin. The genotyping performance was also evaluated using small groups of cells (10, 5, 4, 3, and 2 cells/reaction) isolated by micromanipulation with micro-tweezers. High concordance was identified on analysis of three cells (96.1 %). The use of isolated cells allowed accurate determination of the sensitivity, indicating that our method is highly sensitive and can provide informative SNP profiles for individual identification.", "journal": "Legal medicine (Tokyo, Japan)", "year": "2026", "doi": "10.1016/j.legalmed.2025.102759", "ptypes": [ "Journal Article" ], "first_author": "Namba R", "dirs": [ "B" ] }, "40534218": { "pmid": "40534218", "title": "Passive Sampling of Urban Runoff Solids to Capture Environmental DNA for the Detection of Rodents in Urban Spaces.", "abstract": "In urban environments, runoff may contain environmental DNA (eDNA) from nuisance animals, but its potential has not been investigated. Conventional eDNA methods, such as water sampling, may not be applicable to the detection of nuisance animals in urban areas due to the scarcity of target eDNA attributable to the sporadic contact of the animals with water sources, and the limited volume of samples collected may lead to false negative results. Given these limitations, this study aimed to develop a passive sampling method for the collection of runoff solids for the detection of rodents (mouse and rat) in urban areas using eDNA. The developed sampling method successfully collected 0.3-16.7 g dry weight of runoff solids per sampling event. Collected runoff samples contained 8.0-58.1 μg total DNA/g dry weight sample and were characterized by the presence of PCR inhibitors. Following the removal of these inhibitors, mouse eDNA was detected (albeit below the limit of quantification) using a designed species-specific assay. The results of this study demonstrate that runoff solids are a viable eDNA source for the detection of animals in urban environments. However, further work is required to improve the quantification of target DNA. Nevertheless, the developed methodology may provide new and important information on the ecology of animals in urban spaces that can be incorporated into the management of nuisance animals in urban areas. SUMMARY: A passive sampling scheme was successfully designed and captured 0.3-16.7 g dry weight of urban runoff solids in two sites in an urban space. Collected solids contain 8.0-58.1 μg/g of eDNA, which also contain PCR inhibitors which must be removed prior to qPCR. Mouse DNA was detected in six out of 19 samples, but the detections were below the determined quantification limit. This new passive sampling scheme can be applied for terrestrial animal detection in urban spaces to potentially track hidden animals.", "journal": "Water environment research : a research publication of the Water Environment Federation", "year": "2025", "doi": "10.1002/wer.70107", "ptypes": [ "Journal Article" ], "first_author": "Jolejole ME", "dirs": [ "B" ] }, "40825261": { "pmid": "40825261", "title": "Surveying marine biodiversity using eDNA metabarcoding of seawater and sediment in a high Arctic fjord during the polar night (Kongsfjorden, Svalbard).", "abstract": "The marine ecosystem of Kongsfjorden (KF), Svalbard, is directly affected by ongoing climate change in the Arctic. Shifts in species composition and distributions are already underway as a result of the transition from Arctic to Atlantic conditions in the fjord. The polar night is a period of challenging conditions and is historically understudied, and thus our understanding of the biodiversity of major eukaryotic groups is particularly limited at this time of year. Here we aimed to provide a comprehensive snapshot of eukaryotic biodiversity present in KF during the polar night using environmental DNA (eDNA) metabarcoding of the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Additionally, we sought to establish a baseline dataset for gelatinous zooplankton (GZP) during this period by combining eDNA with net sampling. Lastly, we tested for the impact of PCR-inhibition on the recovery of eDNA from turbid fjord waters. We successfully generated species lists for a large component of the pelagic community known to inhabit KF, benthic and hyperbenthic species typical to the area, as well as a number of new detections. We recovered taxa from major functional groups in the fjord, including macroalgae, phytoplankton, zooplankton, large vertebrates and benthic invertebrates. Finally, we recovered a richer polar night GZP community than previously detected with morphology-based methods, including other seasons. This study demonstrates the versatility of eDNA metabarcoding for marine biodiversity surveys in challenging environmental conditions and provides a baseline for future polar night eDNA-based biodiversity monitoring in KF.", "journal": "Marine environmental research", "year": "2025", "doi": "10.1016/j.marenvres.2025.107443", "ptypes": [ "Journal Article" ], "first_author": "Murray A", "dirs": [ "B" ] }, "42874529": { "pmid": "42874529", "title": "Simultaneous identification of saliva and semen by DNA methylation analysis using quantitative PCR and droplet digital PCR.", "abstract": "DNA methylation analysis is crucial in forensic science for age estimation, body fluid identification, and differentiating monozygotic twins. While massively parallel sequencing (MPS) and single-base extension (SBE) are standard, they are often time-consuming and costly. This study evaluated the feasibility of using quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to identify saliva and semen. DNA was bisulfite-converted using an EZ DNA Methylation-Gold Kit and two body-fluid-specific cytosine-phosphate-guanine (CpG) markers (cg09652652-2d for saliva and cg17610929 for semen) were analyzed using a QX600 droplet digital PCR system. This assay showed high sensitivity for samples with concentrations greater than 5 copies/μL. In contrast, qPCR yielded Ct values above 30 for the same samples. To assess specificity, blood, menstrual blood, vaginal secretion, and nasal secretion samples were tested for cross-reactivity with saliva and semen. The saliva-specific marker was amplified in saliva samples and slight amplification was also observed in nasal secretion samples. The semen-specific marker was detected only in semen samples. ddPCR effectively identified saliva in mixtures up to a 1:50 ratio and semen up to a 200:1 ratio. Moreover, ddPCR demonstrated greater tolerance to PCR inhibition by humic acid compared to qPCR. The DNA degradation test showed no significant difference between degraded and non-degraded saliva samples (paired t-test, p > 0.05). In contrast, a statistically significant difference was observed in semen. These findings demonstrate that ddPCR has the potential for application in forensic science.", "journal": "Legal medicine (Tokyo, Japan)", "year": "2026", "doi": "10.1016/j.legalmed.2026.103037", "ptypes": [ "Journal Article" ], "first_author": "Lee SE", "dirs": [ "B" ] }, "42234169": { "pmid": "42234169", "title": "Occurrence and Spatial Distribution of Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes in Surface and Groundwater of the Kelani River Basin, Sri Lanka.", "abstract": "Antimicrobial resistance (AMR) in aquatic environments represents a critical global public health threat, yet comprehensive environmental surveillance data remain scarce in low- and middle-income countries. The Kelani River Basin, supplying approximately 80% of Colombo's potable water and Sri Lanka's most industrially impacted waterway, lacks a basin-wide characterization of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) across spatial and seasonal gradients. This study provides the first comprehensive basin-wide assessment of ARB distribution, ARG occurrence, minimum inhibitory concentrations (MICs), and multidrug-resistant (MDR) pathogen diversity across surface water and groundwater during wet and dry seasons. A total of 71 water samples (surface water: 53; groundwater: 18) were screened against ten clinically relevant antibiotics using standard pour-plate and agar dilution methods. ARG profiling was conducted using environmental DNA (eDNA)-based PCR, and MDR isolates were characterized by sequencing of the 16S rRNA gene. The transitional zone was identified as the principal contamination hotspot, with ARB loads significantly elevated during the wet season (p < 0.05). Phenotypic resistance was highest against cloxacillin (20.4%), amoxicillin (14.8%), and cefuroxime (14.4%), while ciprofloxacin and tetracycline exhibited the lowest resistance rates. MIC profiling revealed that 41.9% of isolates tolerated concentrations up to 600 µg/mL, with MDR isolates exhibiting MICs ≥ 540 µg/mL. MAR indices ranged from 0.1 to 1.0, with high MAR (≥ 0.5) predominant in dry (59.9%) and wet (43.5%) seasons. ARG screening detected sul1 (93%), qnrB (66.2%), mefA (43.7%), and blaCTX-M (23.9%) basin-wide across both water compartments. 16S rRNA sequencing confirmed clinically significant pathogens, including Klebsiella pneumoniae, Pseudomonas fluorescens, and Proteus mirabilis among high-level MDR isolates. Water temperature, total phosphorus, and nitrogen compounds were significantly associated with ARB abundance spatially and seasonally (p < 0.05). These findings reveal the Kelani River Basin as a high-risk environmental AMR reservoir, while delivering a low-cost, replicable surveillance framework for resource-limited settings to support evidence-based water quality policy and Sri Lanka's National Action Plan for AMR.", "journal": "Current microbiology", "year": "2026", "doi": "10.1007/s00284-026-04915-0", "ptypes": [ "Journal Article" ], "first_author": "Kumara MAKMM", "dirs": [ "B" ] }, "42059966": { "pmid": "42059966", "title": "Development of a modified protocol for extraction of environmental DNA from water samples to assess the presence of fish species in wetland ecosystems.", "abstract": "Accurate fisheries assessment is challenged by limitations in traditional catch estimation methods, suggesting the need for advanced molecular tools such as environmental DNA (eDNA) analysis. This approach has transformed non-invasive biodiversity monitoring worldwide, yet standardized protocols remain underdeveloped in India, limiting practical application. The absence of validated, field-applicable eDNA extraction protocols optimized for complex aquatic environment like wetlands hinders reliable fish diversity assessment. This study validates a modified eDNA extraction protocol using mesocosm and field experiments. Water samples (300 mL) were filtered through glass fiber filters (1.5 μm), preserved in Longmire's and phosphate-buffered saline (PBS) lysis buffers, and processed using a modified phenol-chloroform isoamyl alcohol (PCI) extraction method. eDNA yield was quantified over 0, 10, and 20-day storage intervals and log transformed for analyses, eDNA degradation, and inhibitor modeling was performed. Amplification efficiency was compared for Ward barcode (COI) and Ac12S (12S rRNA) primers. The modified protocol outperformed the conventional protocol. PBS-preserved samples showed initial high yields (log10 mean ± SD, 3.06 ± 0.02 at Day 0) but degraded rapidly (λ = 0.102 day⁻1, half‑life = 6.8 days), falling to 2.18 ± 0.42 by Day 20. Longmire's buffer maintained statistically stable yields across storage (p > 0.05 for all pairwise comparisons), with no significant degradation. Ward barcode consistently amplified Longmire's preserved samples at 50 to 400 ng/μL; PBS samples showed no amplification. Ac12S showed limited amplification in both buffers. Application of the modified protocol in Harike wetland field samples confirmed its sensitivity and practical utility, detecting fish eDNA of Cyprinus carpio, Pethia conchonius, and Lepidocephalichthys thermalis. The findings suggest that buffer selection and standardized extraction are critical for reliable eDNA-based diversity surveys. The optimized workflow enhances flexibility for sampling in resource-limited areas, facilitating non-invasive surveys in protected wetlands.", "journal": "Environmental monitoring and assessment", "year": "2026", "doi": "10.1007/s10661-026-15375-1", "ptypes": [ "Journal Article" ], "first_author": "Saikia A", "dirs": [ "B" ] }, "40959136": { "pmid": "40959136", "title": "Rapid and accurate detection method for bluetongue virus based on CRISPR-Cas13a combined with RT-ERA.", "abstract": "Bluetongue virus (BTV), a vector-borne pathogen of domestic and wild ruminants, poses substantial global threats to livestock health and trade. Conventional detection methods, such as RT-qPCR, remain constrained by reliance on specialized equipment and prolonged turnaround times, limiting their utility in field settings. To overcome these challenges, we developed an integrated isothermal amplification-CRISPR detection platform-Reverse Transcription-Enzymatic Recombinase Amplification coupled with CRISPR-Cas13a (RT-ERA/CRISPR-Cas13a)-enabling rapid, sensitive, specific and visual pan-serotype detection of BTV. The assay demonstrated a sensitivity of 20 RNA copies/reaction within 55 min using three readout modalities: fluorescence values, visual fluorescence signals, and lateral flow test strips. Specificity evaluation revealed no cross-reactivity with 9 non-target pathogens, including epidemiologically significant viruses such as EHDV, AKAV, and CHUV. Clinical validation using 263 field samples demonstrated that RT-ERA/CRISPR-Cas13a achieved clinical sensitivities of 100%, 100%, and 96% with fluorescence values, fluorescence signals, and lateral flow strips, respectively, while maintaining 100% clinical specificity via all modalities. Field adaptation using Nucleic Acid Release Reagent (NARR) simplified crude sample processing, delivering 97% clinical sensitivity and 100% clinical specificity even in the presence of inhibitors from unpurified samples. This work represents the first CRISPR-Cas13a-based platform for pan-serotype BTV detection, combining portability, cost-efficiency, and detective accuracy suitable for point-of-care deployments. By bridging the gap between high laboratory sensitivity and practical field applicability, this system enables real-time BTV surveillance and facilitates timely outbreak containment in resource-constrained agricultural and veterinary settings.", "journal": "Frontiers in cellular and infection microbiology", "year": "2025", "doi": "10.3389/fcimb.2025.1621012", "ptypes": [ "Journal Article" ], "first_author": "Zhou D", "dirs": [ "B", "C" ] }, "40194910": { "pmid": "40194910", "title": "Rapid and Sensitive Digital Droplet PCR Assays for Detecting HPV16 DNA in Liquid Biopsies.", "abstract": "The combination of cell-free DNA (cfDNA) and digital droplet PCR (ddPCR) has significantly advanced the noninvasive screening, diagnosis, and monitoring of diseases, enabling highly sensitive and absolute quantification of target nucleic acids even in the presence of high background DNA. However, widespread adoption of ddPCR is hindered by higher costs, extended processing times, and the requirement for cfDNA purification, which adds expense and variability. To address these limitations, we developed two optimized ddPCR-based assays tailored for enhanced sensitivity, cost-efficiency, and ease of use. Our highly sensitive ddPCR assay for human papilloma virus (HPV)16 DNA detection in purified cfDNA from liquid biopsies from head and neck cancer patients significantly improved sensitivity by increasing cfDNA concentration by 8.5-fold, sample volume loading by 22-fold, and total cfDNA amount tested by 1200-fold without the need for restriction enzyme digestion. In parallel, we established a rapid ddPCR assay using unpurified cfDNA processed by heat treatment and centrifugation, achieving detection concordance rates of 55.6%, 66.7%, and 95.8% for plasma, serum, and surgical drain fluid (SDF), respectively, compared to purified cfDNA. Together, these complementary workflows, one optimized for unpurified cfDNA and the other for purified cfDNA, make ddPCR detection of specific targets in cfDNA more cost-effective, time-efficient, and standardizable across laboratories, paving the way for broader adoption in clinical diagnostics.", "journal": "Journal of medical virology", "year": "2025", "doi": "10.1002/jmv.70146", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, N.I.H., Extramural" ], "first_author": "Loo SK", "dirs": [ "B" ] }, "39721602": { "pmid": "39721602", "title": "Pathogen-Specific Electrochemical Real-Time LAMP Detection Using Universal Solid-Phase Probes on Carbon Electrodes.", "abstract": "Epidemic infections and spreading antibiotic resistance require diagnostic tests that can be rapidly adopted. To reduce the usually time-consuming adaptation of molecular diagnostic tests to changing targets, we propose the novel approach of a repurposable sensing electrode functionalization with a universal, target-independent oligonucleotide probe. In the liquid phase covering the electrode, the target sequence is amplified by MD LAMP (mediator-displacement loop-mediated isothermal amplification) releasing a generic methylene blue-labeled mediator, which specifically hybridizes to the solid-phase probe. To demonstrate the universality of the approach, two different pathogens, Staphylococcus aureus (crude lysate) and Treponema pallidum, are detected using the same solid-phase probe. The reactions reach a limit of detection of 1 × 103 and 4 × 102 copies per reaction within 30 min, respectively. The solid-phase probes carry a carboxymethyl aniline modification to form covalent C-C bonds on low-cost carbon electrodes. Maximum surface coverage and maximum hybridization signals are observed at grafting concentrations of ≥2 μM solid-phase probes. Successful detection of spiked target DNA in real swab samples and with three different commercial amplification buffers proved the broad applicability of this assay approach. The electrochemical MD LAMP is fast, compatible with dsDNA targets, and requires only minimal adaptation of an established amplification method. It is easily transferable to existing analytical electrochemical platforms, allowing the consumable to be synergistically used for different targets. The suggested approach of repurposable functionalized electrodes can also be considered to increase the preparedness for future epidemic or pandemic outbreaks as well as rapidly evolving resistance patterns or variants.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.4c02492", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Trotter M", "dirs": [ "B" ] }, "41611771": { "pmid": "41611771", "title": "Recombinase polymerase amplification-based rapid detection of Oryctes rhinoceros nudivirus (OrNV) infection in coconut rhinoceros beetles.", "abstract": "The coconut rhinoceros beetle (CRB) is an important pest of palms in the Asia/Pacific regions. A natural pathogen of CRB, Oryctes rhinoceros nudivirus (OrNV) has successfully been introduced into virus-free, invasive CRB populations in many Pacific islands leading to pest suppression. While PCR is currently used for virus monitoring, a more portable, simplified in-field method of virus detection would be helpful. Here, we developed a Recombinase Polymerase Amplification (RPA)-based assay to detect the partial gp54 gene of OrNV (OrNV-RPA) using fluorescence-based chemistry. The assay has a high degree of specificity with no cross-reactivity to the genomic DNA of various insects, soil, and bacterial species commonly found in the environment. Additionally, the assay has a high level of sensitivity with the ability to detect approximately 1610 copies of the gp54 gene (15 min reaction). OrNV-RPA assay results showed high consistency with the current standard PCR-based assay, whether using purified DNA (95.4%) or crude lysate (93.5%) from CRB midgut tissue, validating the ability of the OrNV-RPA assay to detect virus-free and virus-infected CRB specimens. The newly developed RPA-based assay provides an option for rapid in-field detection of OrNV to help improve CRB management in the Pacific Islands and other regions.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-025-34759-w", "ptypes": [ "Journal Article" ], "first_author": "Gupta SK", "dirs": [ "B" ] }, "39660908": { "pmid": "39660908", "title": "Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry.", "abstract": "Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world's most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.", "journal": "Microbiology spectrum", "year": "2025", "doi": "10.1128/spectrum.00784-24", "ptypes": [ "Journal Article", "Validation Study", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Dobhal S", "dirs": [ "B" ] }, "40674927": { "pmid": "40674927", "title": "Rapid and simple lateral flow detection for ADH1B and ALDH2 genotyping: PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay.", "abstract": "Single nucleotide polymorphisms (SNPs) in alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) significantly impact ethanol metabolism and alcohol-related health risks. Rapid and simple genotyping methods for these genes are crucial for clinical diagnostics and point-of-care testing (POCT), but existing techniques often require complex procedures or specialized equipment. We developed and validated a novel genotyping method, termed a PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay. The PRALL assay integrates: (i) template preparation from buccal swab, (ii) duplex PCR amplification using biotinylated primers incorporating a BslI recognition site, (iii) simultaneous BslI restriction digestion and allele-specific ligation of hapten-labeled adapters in a single tube, and (iv) visual detection by lateral flow assay (LFA). The method was successfully validated using model templates and human genomic DNA. The complete PRALL assay, from crude lysate preparation to visual result, was performed in approximately 1 h. The assay demonstrated high sensitivity (limit of detection: ∼100 pg genomic DNA, corresponding to ∼15 allele copies) and achieved 100 % concordance with Sanger genotyping for both SNPs across 25 human samples. The unique integration of streamlined enzymatic steps with an instrument-free visual LFA readout overcomes key limitations of previous methods. This makes PRALL a promising and practical tool for facilitating personalized alcohol risk assessment and enhancing clinical diagnostics, demonstrating strong potential for deployment in various settings, including those closer to the point of care.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2025.128588", "ptypes": [ "Journal Article" ], "first_author": "Shirasu N", "dirs": [ "B" ] }, "41718579": { "pmid": "41718579", "title": "Field-Compatible Detection of Spiroplasma citri Associated with Citrus Stubborn Disease Using CRISPR-Cas12a and Crude Sample Preparation.", "abstract": "Citrus stubborn disease (CSD), caused by Spiroplasma citri, presents a significant risk to citrus production, resulting in considerable yield losses when infections remain undetected. Accurate and timely diagnosis is crucial for effective disease management. However, existing nucleic acid-based methods, such as PCR and quantitative PCR (qPCR), require laboratory equipment and are not easily applicable in the field. This study developed a CRISPR-Cas12a-based DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) assay for the rapid, sensitive, and specific detection of S. citri, targeting the spiralin gene. An optimized recombinase polymerase amplification (RPA) primer pair and CRISPR-RNA (crRNA) were utilized for sequence-specific activation of Cas12a, enabling cleavage of fluorescent and lateral flow-compatible reporters. The assay demonstrated a detection limit of 1 attomolar (aM) (around 1.8 genome copies) using a fluorescence plate reader and 10 aM using blue-light visualization and a lateral flow assay (LFA). Specificity testing revealed discrimination against other phytopathogenic spiroplasmas, including S. kunkelii and S. melliferum. Validation in the plate-reader format with DNA extracted from symptomatic citrus samples showed 100% consistency with qPCR results. A 10-min NaOH-Tris crude extraction protocol was also assessed, facilitating straightforward and equipment-free sample preparation. Relative to DETECTR assays with kit-extracted samples, crude extracts preserved full diagnostic sensitivity in fluorescence assays and achieved 70% accuracy in LFA and visual formats in a subset of the same samples. These findings establish a dependable, portable, and highly sensitive diagnostic approach for S. citri, providing a practical tool for on-site detection and enhanced management of citrus stubborn disease.", "journal": "Plant disease", "year": "2026", "doi": "10.1094/PDIS-08-25-1699-RE", "ptypes": [ "Journal Article" ], "first_author": "Shih J", "dirs": [ "B" ] }, "39415058": { "pmid": "39415058", "title": "Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.", "abstract": "Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.", "journal": "Nature biotechnology", "year": "2025", "doi": "10.1038/s41587-024-02437-3", "ptypes": [ "Journal Article" ], "first_author": "Chen K", "dirs": [ "C" ] }, "41560317": { "pmid": "41560317", "title": "Sanger Sequencing of Xeno-Nucleic Acid.", "abstract": "Xeno-nucleic acids (XNAs) offer biostable genetic polymers for biotechnology and information technology, but are incompatible with prevailing DNA sequencing methodologies. Here we report a Sanger sequencing approach tailored for XNA, enabling direct readout of XNA strands up to 50 bases. This new method relies on a Bst DNA polymerase mutant, which not only recognizes XNA as a template but also efficiently incorporates dideoxyribonucleoside triphosphate (ddNTP) substrates, facilitating sequencing of XNA by synthesis of complementary DNA. The Bst F710Y mutant exhibits at least two orders of magnitude higher activity in catalyzing ddNTP incorporation. Our method demonstrates accurate sequence determination of diverse XNA strands of distinct backbone chemistries such as TNA and FANA. Lastly, we showcase proof-of-concept automated XNA sequencing on a genetic analyzer instrument, by leveraging Bst mutant's ability to stochastically incorporate BigDye-labeled ddNTP substrates in a single reaction. This work establishes a direct sequencing platform for XNA employing a chain termination strategy, and lays a foundation for future de novo identification of functional XNA and development of XNA-based data storage system.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2026", "doi": "10.1002/anie.202516617", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Wang Y", "dirs": [ "C" ] }, "41080446": { "pmid": "41080446", "title": "Assessing Temperature-Dependent DNA Cleavage by CRISPR-Cas9.", "abstract": "The RNA-guided CRISPR-Cas9 endonuclease has been a transformative tool for laboratory biochemistry with huge potential as a precision therapeutic. This tool site-specifically cleaves double-stranded DNA following the recognition of a unique protospacer-adjacent motif (PAM). Activation of the protein-nucleic acid Cas complex has also been widely recognized to feature an allosteric mechanism dependent on structural remodeling and interdomain crosstalk. Biophysical methods have probed the impact of allosteric perturbations on cleavage and specificity of Cas9, with the aim of engineering enhanced Cas effectors. These studies include Cas9 from thermophilic organisms that edit at higher temperatures and are active in human plasma. Validation of biophysical insights has necessitated the quantitation of DNA cleavage in vitro and, subsequently, the adaptation of established protocols to encompass temperature-dependent function that is evident in extremophilic Cas systems, such as Cas9 from Geobacillus stearothermophilus and the mesophilic SpCas9. This protocol is advantageous for probing functional temperature ranges of DNA cleavage that can theoretically be applied to any Cas-RNP system. Key features • Builds upon the original Cas9 cleavage assays reported by Jinek et al. [1] to include the active temperature range of thermophiles. • Validated for assessing the cleavage activity of both mesophilic and thermophilic Cas9 systems. • Allows for qualitative and quantitative assessment of DNA cleavage across multiple physiological regimes. • Can be adapted to assess cleavage at multiple genomic loci or with different PAM requirements. • Assay can be completed in a single day.", "journal": "Bio-protocol", "year": "2025", "doi": "10.21769/BioProtoc.5463", "ptypes": [ "Journal Article" ], "first_author": "Knight AL", "dirs": [ "C" ] }, "41288510": { "pmid": "41288510", "title": "Engineering and Assay of Enzymes for Expanded Genetic Alphabet Replication.", "abstract": "Use of noncanonical nucleotides for nucleic acid replication provides a critical way to understand how and why natural nucleotides were chosen by evolution and provides potential applications in biotechnology, diagnostics, therapeutics, and DNA computation. However, monitoring noncanonical nucleotide incorporation can be difficult, especially for high-throughput applications. Therefore, we have utilized a 6-letter genetic alphabet (G, A, T, C, Z, and P) for toehold-mediated DNA strand exchange assays. Toehold hybridization and branch migration were found to proceed readily through the formation of multiple consecutive non-natural base pairs between hydrogen bonding synthetic nucleotides dZ (6-amino-5-nitro-2(1H)-pyridone) and dP (2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one). Unlike other mismatches, a dZ:dG mis-pair was minimally disruptive to strand exchange. Overall, however, positional and numerary effects on mismatch tolerance could be leveraged to distinguish both dP and dZ mismatches. The success of the assay allowed a rapid assessment of the semisynthetic DNA replication by Escherichia coli DNA polymerases I, II, and IIIα and the identification of E. coli and Geobacillus stearothermophilus DNA Pol I mutants with improved fidelity. Given the difficulty of adapting organisms for expanded genetic alphabets, the ability to rapidly proof sequences and enzymes in vitro should lead to new parts and circuits that can be modularly introduced to improve the incorporation of more and more different noncanonical nucleotides.", "journal": "Biochemistry", "year": "2025", "doi": "10.1021/acs.biochem.5c00512", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "Bhadra S", "dirs": [ "C" ] }, "40563163": { "pmid": "40563163", "title": "Allosteric regulation of L-lactate dehydrogenase: Beyond effector-mediated tetramerization.", "abstract": "The activity of allosterically regulated enzyme is modulated through structural changes induced by effectors. L-lactate dehydrogenase from Geobacillus stearothermophilus (GsLDH) catalyzes the reversible conversion between pyruvate and lactate using NAD(H), and its activity is known to be activated by fructose 1,6-bisphosphate (FBP). However, the molecular basis of this regulation has not been explored using molecular dynamics (MD) simulations. In this study, we integrated MD simulations with biochemical assays to investigate the impact of FBP on GsLDH structure and function. MD revealed that FBP stabilizes the tetrameric form, reduces residue flexibility, and enhances pyruvate interactions with active site residues, despite a 23 Å distance between binding sites. Using MDavocado, we identified three P-axis-related dimer interface regions critical for stability and structural integrity. Microcalorimetry titration revealed that NADH binding (Kd = 1.2 ± 0.3 μM) occurs only in the presence of FBP, indicating an enthalpy-driven interaction involving a hydrogen-bond network. Single amino acid replacement, Gln189Leu, maintains tetrameric structure without FBP and enhances the substrate inhibition effect. However, this mutant fails to trigger the allosteric transition toward a conformation with higher affinity for the substrate, resulting in a high K0.5 value (2.3 ± 0.2 mM) and a low kcat/K0.5 (32 ± 4 s-1 mM-1), comparable to that of the WT without FBP. These findings suggest that oligomerization alone does not confer allosteric responsiveness, emphasizing the essential role of specific interactions in allosteric regulation. Collectively, our results advance the molecular understanding of FBP as a key allosteric effector to stabilize quaternary structure and improve enzyme activity.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70206", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Cai H", "dirs": [ "C" ] }, "40143462": { "pmid": "40143462", "title": "Cell-Free Reaction System for ATP Regeneration from d-Fructose.", "abstract": "Adenosine triphosphate (ATP)-dependent in vitro bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the in situ generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from Bifidobacterium adolescentis, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from Geobacillus stearothermophilus and a glycerol kinase from Cellulomonas sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C6 ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from Pseudomonas stutzeri. In the complete system, the ATP yield increased to 2.53 mol molfructose-1 with a maximum productivity of 7.2 mM h-1.", "journal": "ACS synthetic biology", "year": "2025", "doi": "10.1021/acssynbio.4c00877", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kraußer F", "dirs": [ "C" ] }, "40491232": { "pmid": "40491232", "title": "Revisiting D-Acylases for D-Amino Acid Production.", "abstract": "N-Acyl-D-amino acid deacylases (EC 3.5.1.81, also known as D-acylases) have been studied for decades for their utility in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity. In conjunction with an N-succinyl-amino acid racemase (NSAR), they impulse the dynamic kinetic resolution (DKR) of different NAAs until the corresponding enantiomerically pure D-amino acids. Besides the clear interest in this enzyme cascade, the application of D-acylase/NSAR tandems has been only briefly described outside the industrial field. In this work, we revisit D-acylases for the DKR of NAAs, reporting the characterisation of two new recombinant D-acylases belonging to Bordetella petrii and Klebsiella pneumoniae. The enzymes were successfully coupled with the recombinant NSAR from Geobacillus stearothermophilus for the biosynthesis of D-methionine or D-aminobutyric acid. We also carried out the structural characterisation of the D-acylase from Klebsiella pneumoniae (KleDacyl), providing the second experimental 3-D structure of a member of this family of enzymes. The structural model shows a highly dynamic character of this amidohydrolase superfamily member, supplying a snapshot of an open conformation of the enzyme most likely preceding substrate entrance into the catalytic cleft. Our results confirm for the first time the importance of an α/β mobile domain in the substrate specificity of D-acylases (region 282-341 in KleDacyl), opening up new strategies for structural-based protein engineering strategies.", "journal": "Microbial biotechnology", "year": "2025", "doi": "10.1111/1751-7915.70179", "ptypes": [ "Journal Article" ], "first_author": "Martínez-Rodríguez S", "dirs": [ "C" ] }, "39660873": { "pmid": "39660873", "title": "Assembly and engineering of BioBricks to develop an efficient NADH regeneration system.", "abstract": "The cofactor regeneration system plays a crucial role in redox biocatalysis for organic synthesis and the pharmaceutical industry. The alcohol dehydrogenase (ADH)-based regeneration system offers a promising solution for the in situ regeneration of NAD(P)H. However, its widespread use is hindered by low activity and poor expression of ADH in Escherichia coli. Herein, the BioBricks (promoter, ribosome binding site [RBS], functional gene, and terminator) were assembled and engineered to constitute an efficient NADH regeneration system. The semi-rational design was employed to enhance the catalytic efficiency of GstADH (an ADH from Geobacillus stearothermophilus), resulting in a beneficial GstADH variant with a 2.1-fold increase in catalytic efficiency. Furthermore, the RBS optimization was used to increase the expression of ADH genes, leading to the identification of an RBS with a 3.2-fold increased translation rate. Using this developed system, the NADH generating velocity reached more than 2 s-1 even toward 0.1 mM NAD+, indicating that it is the most promising NADH regeneration so far. Finally, the engineered system was utilized for the asymmetric biosynthesis of l-phosphinothricin (a chiral herbicide), with a high yield (>95%). The alcohol dehydrogenase (ADH)-based coenzyme regeneration system serves as a useful tool in redox biocatalysis. This system effectively replenishes NAD(P)H by utilizing isopropanol as a substrate, with the added advantage of easily separable acetone as a by-product. Previous studies focused on discovering new adh genes and engineering the ADH protein for higher catalytic efficiency, neglecting the optimization of other gene components. In this study, a remarkably efficient NADH regeneration system was developed using BioBricks assembly for system initialization. The ADH engineering was used to enhance catalytic efficiency, and RBS optimization for elevated ADH expression, which resulted in not only a 2.1-fold increase in catalytic efficiency but also a 3.2-fold increase in translation rate. Together, these improvements resulted in an overall 6.7-fold enhancement in performance. This system finds application in a wide range of NADH-dependent biocatalysis processes and is particularly advantageous for the biosynthesis of fine chemicals.", "journal": "Applied and environmental microbiology", "year": "2025", "doi": "10.1128/aem.01041-24", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Cheng F", "dirs": [ "C" ] }, "40804834": { "pmid": "40804834", "title": "Genogroup-Specific Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Point-of-Care Detection of Norovirus.", "abstract": "Background/Objectives: Norovirus is a major cause of acute gastroenteritis worldwide. Considering its highly infectious and transmissible nature, rapid and accurate diagnostic tools are of utmost importance for the effective control of outbreaks in the context of point-of-care testing (POCT). In this study, we developed a genogroup-specific multiplex reverse transcriptase loop-mediated isothermal amplification assay to detect the human norovirus genogroups I and II (GI and GII, respectively). Methods: For the comprehensive detection of clinically relevant genotypes, two sets of primers were incorporated into the assays targeting the RdRp-VP1 junction: one against GI.1 and GI.3, and the other for GII.2 and GII.4. Following optimization of the reaction variables, we standardized the reaction conditions at 65 °C with 6 mM MgSO4, 1.4 mM dNTPs, 7.5 U WarmStart RTx Reverse Transcriptase, and Bst DNA polymerase at 8 U and 10 U for GI and GII, respectively. Amplification was monitored in real-time using a thermocycler platform to ensure precise quantification and detection. Finally, the assay was evaluated through portable isothermal detection device to test its feasibility in on-site settings. Results: Both assays detected the template down to 102-103 copies per reaction and showed high target selectivity, yielding no non-specific amplification across 39 enteric pathogens. These assays enabled prompt detection of GI within 10-12 min and of GII within 12-17 min after the reaction was initiated. Onsite validation reveals all template detection below 15 min, demonstrating its potential feasibility in point-of-care applications. Including the sample preparation time, test results were obtained in less than 1 h. Conclusions: This method is a rapid, reliable, and scalable solution for detecting human norovirus in POCT settings for both clinical diagnosis and public health surveillance.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2025", "doi": "10.3390/diagnostics15151868", "ptypes": [ "Journal Article" ], "first_author": "Ansari WK", "dirs": [ "C" ] }, "42341607": { "pmid": "42341607", "title": "Beyond the protein lattice: bacterial S-layer glycans - from structure to functional frontier.", "abstract": "S-layers were long regarded as elegant but functionally inert molecular armor. The discovery, three decades ago, that the S-layer of Geobacillus stearothermophilus carries covalently attached glycans overturned this view and inaugurated a new chapter in bacterial cell biology. Since then, the field has expanded in taxonomic scope and mechanistic depth. Complex O-linked glycans have been characterized on bacterial S-layers across Gram-positive Firmicutes, including Paenibacillus alvei, commensal lactobacilli, and the nosocomial pathogen Clostridioides difficile, while the periodontal pathogen Tannerella forsythia has emerged as a landmark organism whose dual-species S-layer bears an O-linked decasaccharide containing terminal non-mammalian nonulosonic acid - a modification directly implicated in evasion of immune surveillance. What has emerged is a picture of remarkable functional versatility. S-layer glycans govern cell morphology, sporulation, epithelial adhesion, biofilm dynamics, and host immune recognition through pattern-recognition receptors, including DC-SIGN and Mincle. In terms of chemical complexity, they deploy an extraordinary repertoire of rare, taxonomically restricted monosaccharides - among them bacillosamine, pseudaminic acid, and 2,3-cyclophosphoryl-rhamnose - assembled through linkages exceeding the structural diversity of any eukaryotic glycan class. This modularity opens compelling avenues for glycosylation engineering: rewiring transferase specificity or swapping biosynthetic modules could tailor glycan composition for epitope display, self-assembling glyconanostructures, or targeted therapeutic carbohydrate delivery. Cryo-electron tomography and intact glycopeptide mass spectrometry are resolving glycan positioning at near-atomic resolution, while AlphaFold3 is modeling glycan-protein contacts driving lattice stabilization. S-layer glycans represent compelling targets for next-generation antimicrobials, mucosal vaccines, and precision bacteriocins. Their full biological and biotechnological repertoire remains to be uncovered.", "journal": "Current opinion in microbiology", "year": "2026", "doi": "10.1016/j.mib.2026.102784", "ptypes": [ "Journal Article", "Review" ], "first_author": "Schäffer C", "dirs": [ "C" ] }, "40243305": { "pmid": "40243305", "title": "Markerless mutagenesis enables isoleucine biosynthesis solely from threonine in Methanothermobacter marburgensis.", "abstract": "The archaeal model microorganism Methanothermobacter marburgensis has been studied for methane production for decades. However, genetic modifications are required to harness M. marburgensis for the generation of novel archaeal cell factories for industrial-scale production of commodity and high-value chemicals. Only the development of tools for genetic engineering opens up this possibility. Here, we present the establishment of the first markerless mutagenesis system for genetic modification of M. marburgensis. This system allows the recycling of positive selection markers and enables multiple sequential gene deletions or integrations. As a demonstration, we clarified the postulated isoleucine biosynthesis pathway directly from pyruvate via citramalate synthase (CimA). In doing so, we identified a putative CimA in M. marburgensis and deleted the CimA coding gene, resulting in auxotrophy for isoleucine. The complementation of cimA initiated through constitutive expression led to prototrophic growth similar to the wild type, demonstrating that cimA is essential for pyruvate-derived isoleucine biosynthesis in M. marburgensis. As it has been shown vice versa in Escherichia coli before, we were able to complement isoleucine biosynthesis with the integration of a synthetic isoleucine biosynthesis pathway from threonine for the first time in a methanogenic archaeon. This was achieved via genome integration of the characterized thermostable threonine deaminase from Geobacillus stearothermophilus. The successful integration of an alternative pathway for isoleucine production paves the road for future application of multi-gene biosynthetic pathways to overproduce industrially relevant chemicals. The autotrophic, hydrogenotrophic methanogen Methanothermobacter marburgensis is one of the best-studied model organisms in the field of thermophilic archaea. The fact that M. marburgensis shows robust growth and scalability in bioreactor systems makes it a highly suitable candidate for industrial-scale bioprocesses. Additionally, the reported study provides the tools for genetic engineering that enable sequential genome modification in M. marburgensis. Scalable bioreactor cultivation, the ability to genetically engineer, and the recent discovery of natural amino acid secretion in M. marburgensis set the cornerstone for the generation of the first cell factories in archaeal biotechnology to economically produce carbon dioxide-derived commodity and high-value chemicals at industrial scale.", "journal": "Microbiology spectrum", "year": "2025", "doi": "10.1128/spectrum.03068-24", "ptypes": [ "Journal Article" ], "first_author": "Klein M", "dirs": [ "C" ] }, "41028576": { "pmid": "41028576", "title": "One-Step RT-LAMP: A Method for Closed-Tube Colorimetric Detection of RNA Viruses in Plants.", "abstract": "A good virus indexing platform is essential for early detection of plant viruses and for providing virus free planting materials. Majority of the plant viruses are RNA viruses and the available detection methods include ELISA and RT-PCR. The ELISA is time consuming, and RT-PCR needs post PCR sample handling, predisposing to sample cross contamination. The LAMP (loop-mediated isothermal amplification) is an auto-cycling strand displacement isothermal DNA synthesis catalyzed by a strand displacing DNA polymerase like the Bst DNA polymerase. The RT-LAMP (reverse transcription LAMP) is a specific, highly sensitive, and relatively fast diagnostic method for RNA viruses. In one-step RT-LAMP, the total RNA extracted from symptomatic samples can be directly amplified under isothermal conditions by incorporating a reverse transcriptase and an RNase inhibitor in the LAMP assay. The inclusion of hydroxy naphthol blue dye in the RT-LAMP reaction mixture facilitates closed-tube visual detection of positive samples by a color change from violet to sky blue. Molecular fidelity of the LAMP amplicons can be assessed through restriction profiling.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2026", "doi": "10.1007/978-1-0716-4746-2_5", "ptypes": [ "Journal Article" ], "first_author": "Nair S", "dirs": [ "C" ] }, "40976505": { "pmid": "40976505", "title": "Development of a thermophilic l-arabinose-inducible system in Acetivibrio thermocellus (Clostridium thermocellum).", "abstract": "Inducible genetic operation systems constitute essential tools in microbial synthetic biology and metabolic engineering. However, inducible systems in non-model microbes, particularly thermophiles, are rarely reported. Acetivibrio thermocellus (previously termed Clostridium thermocellum), a representative strain of thermophilic non-model microbes, currently serves as a promising chassis organism in biorefinery. Although various genetic tools are available for A. thermocellus, superior thermophilic inducible systems are in high demand. In this study, we developed a thermostable l-arabinose-inducible system (ThermoARAi) in A. thermocellus by utilizing the inducible promoter PabnE and repressor AraR from Geobacillus stearothermophilus T-6. Through systematic promoter engineering and optimization of induction conditions using a thermostable β-glucuronidase as reporter, the system exhibited dynamic range improvement from a 5.4-fold induction to a 175-fold induction with negligible leakage. Furthermore, the ThermoARAi system was appropriate for use in metabolic engineering, as validated by its applications in whole-cell saccharification of cellulosic substrates and degradation of amorphous polyethylene terephthalate films. The ThermoARAi system significantly expands the genetic toolkit for precise gene expression modulation, metabolic engineering, and biotechnological applications in A. thermocellus. Importantly, this approach may also serve as a foundation for developing genetic tools in other Clostridia that play key roles in diverse ecosystems, including the gut.", "journal": "Metabolic engineering", "year": "2026", "doi": "10.1016/j.ymben.2025.09.008", "ptypes": [ "Journal Article" ], "first_author": "Liu F", "dirs": [ "C" ] }, "42196245": { "pmid": "42196245", "title": "Bst DNA Polymerase: Structure, Properties and Engineering Strategies in LAMP.", "abstract": "Bst DNA polymerase is a biotechnologically modified thermostable enzyme from the thermophilic Gram-positive bacterium Geobacillus stearothermophilus. The unique structure of Bst DNA polymerase determines its thermal stability, ability to replace a DNA strand and specificity. The high specificity of Bst DNA polymerase ensures the efficiency, sensitivity, and high rate of loop-mediated isothermal amplification (LAMP), which is widely used in vitro biotechnology. The review reveals the structural and functional features of the enzyme, its application in LAMP and methods of improvement of thermal stability (including directed evolution, site-directed mutagenesis, fusion constructs, and chemical modifications). The terminal transferase activity and ab initio synthesis are discussed regarding problems of Bst DNA polymerase and the ways to eliminate them. The questions of introducing modified nucleotides and primers to expand the diagnostic capabilities of LAMP are also discussed. Modern advances in Bst DNA polymerase engineering pave the way for the creation of reliable, thermostable, and highly specific test systems suitable for widespread diagnostic applications.", "journal": "International journal of molecular sciences", "year": "2026", "doi": "10.3390/ijms27104261", "ptypes": [ "Journal Article", "Review" ], "first_author": "Tikhonova E", "dirs": [ "C", "D" ] }, "40398777": { "pmid": "40398777", "title": "Point mutations enhance catalytic efficiency of Geobacillus stearothermophilus α-glucosidase: A biochemical characterization study.", "abstract": "α-Glucosidases are important enzymes with a broad spectrum of industrial applications. However, traditional protein engineering often struggles to improve activity without compromising stability and usually demands extensive screening. Here, we targeted the α-glucosidase (AG) from Geobacillus stearothermophilus (Gst), using in silico analyses and literature precedent to select point mutations N61H and N258P. We cloned the mutated gstAG gene into the pET-28a(+) vector and expressed in Escherichia coli. After expressing and purifying both wild-type and mutant enzymes, we performed detailed biochemical assays. Both mutants maintained GstAG's optimum temperature (60 °C) and pH (6.5). However, each displayed enhanced catalytic efficiency: N61H lowered the Michaelis constant (Kₘ) by 1.5-fold and raised the turnover number (kcat) by 1.7-fold relative to the wild type. These results offer a blueprint for engineering α-glucosidases with improved performance, unlocking new commercial and biotechnological applications.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.144379", "ptypes": [ "Journal Article" ], "first_author": "Hekim S", "dirs": [ "C" ] }, "41982909": { "pmid": "41982909", "title": "Screening of molecular elements and improvement of heat resistance in a thermophilic bacterium.", "abstract": "Engineering microorganisms to withstand extreme temperatures (>80 °C) remains a critical challenge in industrial biotechnology owing to limited genetic tools and poor mechanistic understanding of microbial thermoadaptation. We aimed to develop a novel Geobacillus stearothermophilus strain with remarkable thermal resilience through an integrated approach combining adaptive laboratory evolution and rational genetic engineering. Progressive thermal adaptation (70-80 °C) followed by genome reduction generated a mutant (SL-1-80) with enhanced stability at 80 °C. Subsequent combinatorial overexpression of eight heat-associated genes (murD, cysM, grpE, groES, hsp33, hslO, hrcA, clpE) synergistically extended its survival to 85 °C. Genomic and transcriptomic analyses revealed a triple mechanism: (1) strategic deletion of transposable elements (IS5377/IS4/IS110) reduced genomic instability, (2) co-activation of chaperone systems (GroES-GrpE) and redox homeostasis enzymes (HslO-Hsp33) enhanced protein folding and oxidative stress resistance, and (3) metabolic plasticity (BglG and HTH-domain transcriptional repressor), motility optimization (FliY), and transcriptional reprogramming (Sigma-D, DUF47-family chaperone and HTH-domain transcriptional repressor) facilitated nutrient acquisition and motility-based environmental navigation under stress. Furthermore, we established the first high-efficiency electroporation protocol (104 transformants/µg DNA) for this genus, enabling ATP-enhanced heterologous protein expression under heat stress. This study provided a robust platform organism for high-temperature bioprocessing and a mechanistic blueprint for engineering microbial thermotolerance, addressing key limitations in applications such as microbial-enhanced oil recovery and industrial enzyme production.", "journal": "Engineering microbiology", "year": "2025", "doi": "10.1016/j.engmic.2025.100225", "ptypes": [ "Journal Article" ], "first_author": "Cui J", "dirs": [ "C" ] }, "39777423": { "pmid": "39777423", "title": "Rational Design of a Novel DNA Polymerase From Clostridium thermocellum to Improve LAMP Detection Performance.", "abstract": "Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection. Through bioinformatics analysis, the enzyme and the DNA-binding domain (DBD) from Pyrococcus abyssi were mutated for enhanced interaction between proteins and DNA. A chimeric mutant DBDE146K-S738R reaches the detection threshold 13 min earlier than wild-type Cth DNA polymerase in real-time LAMP detection with a template concentration of 1.58 × 105 fg/µL. It also showed the highest enzymatic activity at pH 9.0 and 65°C. The chimeric enzyme DBDE146K-S738R exhibits good thermal stability, capable of performing LAMP reactions after treatment at 73°C or 70°C for 8 h. Moreover, it maintains high activity even under the inhibitory conditions of 50 U/mL heparin, 1.6 mM EDTA, 200 mM NaCl, 10% ethanol, 1.2 M urea, or 0.8% phenol. Notably, it was able to detect 1.58 × 102 ag/µL of the genome and 1.03 CFU/mL of the colony in Salmonella typhimurium detection. The enzyme's performance is superior to commercial Bst 2.0 and comparable to commercial Bst 3.0. The results suggest that DBDE146K-S738R in LAMP exhibits great potential for molecular biological studies and clinical diagnostic analysis.", "journal": "Biotechnology journal", "year": "2025", "doi": "10.1002/biot.202400559", "ptypes": [ "Journal Article" ], "first_author": "Wang C", "dirs": [ "C" ] }, "41416715": { "pmid": "41416715", "title": "Synergistic strategies for high production of Geobacillus stearothermophilus α-amylase in Bacillus subtilis.", "abstract": "Although the α-amylase (AmyS) from Geobacillus stearothermophilus exhibits high thermostability, its low enzymatic activity (44.36 ± 2.02 U/mL) severely hinders industrial applications. Given its strong protein secretion capability and GRAS (generally recognized as safe) status, this study employed Bacillus subtilis as the host to enhance AmyS production. Through error-prone PCR and high-throughput screening, a triple mutant T151A/K178E/T458A (AmySM) was generated, showing a 17.67% increase in activity (51.34 ± 1.11 U/mL). AmySM activity was further increased by 29.32% to 65.23 ± 2.33 U/mL using the signal peptide SPykwD, selected from a comprehensive library for superior secretion efficiency. The promoter PgsiB enhanced activity by 37.61% to 89.54 ± 2.95 U/mL. Optimizing the ribosome binding site (RBS) resulted in an additional 48.83% increase in activity, yielding a final activity of 134.02 ± 3.54 U/mL, which corresponds to a 3.02-fold improvement over the initial strain WBSW (44.36 ± 2.02 U/mL). Ultimately, scale-up fermentation in a 5-L bioreactor yielded a maximum extracellular activity of 1244.17 ± 48.66 U/mL at 72 hours, a 2.84-fold increase over the control. This multi-level strategy provides a rational framework for high-efficiency AmySM production, paving the way for extracellular production of high-value proteins in the GRAS host B. subtilis.", "journal": "Journal of industrial microbiology & biotechnology", "year": "2026", "doi": "10.1093/jimb/kuaf036", "ptypes": [ "Journal Article" ], "first_author": "Rao D", "dirs": [ "C" ] }, "41161424": { "pmid": "41161424", "title": "Biochemical characterization and activity enhancement of a GH13 thermostable oligo-α-1,6-glucosidase from Geobacillus stearothermophilus.", "abstract": "Oligo-α-1,6-glucosidase (OGL) catalyzes the hydrolysis of α-1,6-glycosidic linkages in oligosaccharides and plays a vital role in the production of high-glucose syrup, isomaltooligosaccharides (IMOs), and bioethanol. Industrial IMO production demands enzymes with both high thermostability and catalytic efficiency. Although thermophilic bacterial OGLs meet thermal requirements, their catalytic performance remains suboptimal. While random mutagenesis can identify beneficial mutations, traditional protein engineering often faces challenges such as stability trade-offs and extensive screening. Recently, computational tools like HotSpot Wizard and molecular docking have shown promise in semi-rational enzyme design. In this study, we aimed to enhance the catalytic activity of a thermostable OGL from Geobacillus stearothermophilus (Gst) via site-directed mutagenesis. Five point mutations (D59A, N61F, V101I, T257A, and P258N) were selected based on bioinformatics analysis and literature review. Wild-type and mutant enzymes were expressed in Escherichia coli BL21(DE3) and purified. All variants retained activity across a broad pH and temperature range, with optimal activity at 60 °C and pH 6.5. Compared to the wild-type enzyme, mutants D59A, V101I, T257A, and P258N exhibited 1.7- to 2.3-fold increases in catalytic efficiency (kcat/Km), with Vmax values increasing by 2.2- and 1.9-fold for D59A and P258N, respectively. Molecular docking with panose and isomaltose confirmed enhanced substrate binding, particularly for GstOGLD59A. These findings demonstrate the potential of rationally engineered GstOGL variants as efficient biocatalysts for industrial IMO production and underscore the value of integrating computational and experimental approaches in enzyme optimization.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.148568", "ptypes": [ "Journal Article" ], "first_author": "Hekim S", "dirs": [ "C" ] }, "41647422": { "pmid": "41647422", "title": "Nanobiocatalytic upgrading of heavy oil using Geobacillus stearothermophilus and alumina nanoparticles.", "abstract": "Challenges in heavy oil bioupgrading necessitate innovative approaches. Hence, we delved into the simultaneous application of a bacterium (Geobacillus stearothermophilus) and Al2O3 nanoparticles for heavy oil upgrading at a high temperature. We used the central composite design method within Design Expert software. The initial oil volume, upgrading time, and nanoparticle concentration were the main variables. Aromatic/aliphatic content and oil viscosity were the two independent responses. Results showed that nanoparticles could have accelerated bioupgrading. The zeta potential, FESEM and EDS confirmed the absorption of nano alumina on the bacterial cell due to the formed ion-dipole interaction between alumina and bacterial cells, which could have intensified the entry of heavy oil molecules into the bacterial cell. The best outcomes were observed during 11.86 days, with 26% v/v initial oil and 0.46% W nanoparticles, as indicated by FTIR spectroscopy, which showed a reduction in the aromatic/aliphatic index from 0.29613 to 0.07677 and a decrease in oil viscosity from 480 cp to 144 cp. Moreover, as determined by GC-MS, a remarkable 100% decrease in certain cyclo compounds and a considerable improvement in upgrading efficiencies for some contents such as cyclohexanes were observed. Also, a 14% decrease in asphaltene content was associated with a decrease in the solo use of bacteria. These findings highlight the synergistic efficacy of G. stearothermophilus and alumina in upgrading heavy oil.", "journal": "RSC advances", "year": "2026", "doi": "10.1039/d5ra07786a", "ptypes": [ "Journal Article" ], "first_author": "Maghzi A", "dirs": [ "C" ] }, "40589235": { "pmid": "40589235", "title": "Co-Expression of Chaperones for Improvement of Recombinant Geobacillus stearothermophilus Lipase Production in Pichia pastoris.", "abstract": "Pichia pastoris is among the most popular expression systems for recombinant protein production. The ability to secrete high titers of recombinant proteins is one of its primary advantages. Co-expression of folding-assisting factors is considered one of the strategies to improve protein production with this expression system. In this study, the effect of PDI, KAR2, HAC1, and ERO1 co-expression on Geobacillus stearothermophilus lipase production in P. pastoris was investigated. The chaperones were co-expressed under the regulation of the GAP promoter in clones with different lipase production levels (LPS#2 and LPS#8). Results showed that lipase-PDI co-expression clones have the highest activity. The effect of the other chaperones had varying effects for LPS#2 and LPS#8 clones. LPS#2-PDI and LPS#8-PDI were further analyzed to determine the effect of dimethyl sulphoxide (DMSO). Different concentrations (0.5%, 1%, and 2%) of DMSO were tested. The highest activity was obtained with approximately 1.5-fold activity in the LPS#8-PDI clone with 0.5% and 1% DMSO concentration levels. Comparison of the fermentation kinetic parameters revealed that the activity level of 56.54 U/mL provided with LPS#8 increased to 73.84 U/mL in the LPS#8-PDI clone with PDI co-expression, and when PDI co-expression was combined with DMSO conditions (0.5%), it reached 114.46 U/mL. It was observed that the productivity (U/g wet cell/g substrate/h) of the LPS#8 clone was increased 1.27-fold with LPS#8-PDI and 2.05-fold with LPS#8-PDI-DMSO conditions. In future studies, it is possible to achieve higher protein production levels by optimization at the fermenter scale.", "journal": "Biotechnology and applied biochemistry", "year": "2025", "doi": "10.1002/bab.70017", "ptypes": [ "Journal Article" ], "first_author": "Erden-Karaoglan F", "dirs": [ "C" ] }, "42526569": { "pmid": "42526569", "title": "Behind substrate specificity switch in lactate dehydrogenase: Active-site loop dynamics and epistatic enhancement.", "abstract": "Substrate specificity in lactate dehydrogenases (LDHs) controls metabolic flux and remains a central challenge in enzyme engineering, particularly when closely related substrates differ by only a single carboxylate group. Although Q102 (equivalent to Gln86 in Geobacillus stearothermophilus L-LDH) is recognized as a canonical substrate-specificity determinant, how this electrostatic switch cooperates with steric effects, conformational dynamics, and scaffold context to govern substrate recognition remains unclear. Here, we combined steady-state kinetics with molecular dynamics simulations to examine specificity switching from pyruvate to oxaloacetate in G. stearothermophilus L-LDH (GsLDH). The Q86R substitution enhanced oxaloacetate catalytic efficiency 34-fold (kcat/Km = 340 s-1 mM-1) through favorable electrostatic stabilization and increased substrate retention, but substantially reduced pyruvate activity. In contrast, I227V acted primarily through steric accommodation, broadening substrate acceptance by retaining substantial pyruvate activity (377 s-1 mM-1) while markedly increasing oxaloacetate activity (322 s-1 mM-1). The combined Q86R/I227V variant displayed positive epistasis and achieved the highest oxaloacetate efficiency among engineered LDH variants (738 s-1 mM-1), approaching that of native G. stearothermophilus malate dehydrogenase (GsMDH) (771 s-1 mM-1), indicating complementary contributions of electrostatic stabilization and steric optimization. Loop-state and productive-geometry analyses further showed that loop closure alone is permissive but insufficient for catalysis; productive turnover requires coupling between loop conformation, substrate retention, and hydride/proton-transfer geometry. Principal component analysis further revealed that the engineered mutations redistribute the conformational ensembles toward substrate-compatible productive states. Reciprocal mutations in GsMDH failed to restore efficient pyruvate catalysis, demonstrating that specificity switching is asymmetric and constrained by scaffold context. Collectively, these findings support an electrostatic-steric ensemble-selection model for LDH/MDH specificity and provide a mechanistic framework for engineering substrate specificity in Rossmann-fold dehydrogenases.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.153829", "ptypes": [ "Journal Article" ], "first_author": "Cai H", "dirs": [ "C" ] }, "42575356": { "pmid": "42575356", "title": "Bioinformatics-guided mutagenesis and biochemical characterization of the GDSL lipase GstLipC from Geobacillus stearothermophilus ATCC 7953.", "abstract": "GDSL lipases, members of the SGNH hydrolase superfamily, exhibit broad substrate specificity and catalytic adaptability, making them valuable model enzymes for investigating structure-function relationships under high-temperature conditions. Despite these features, residue-level determinants influencing the catalytic performance of thermophilic GDSL lipases remain insufficiently characterized. In this study, we investigated the effects of rationally selected amino acid substitutions on the catalytic properties of a thermophilic GDSL lipase using an in silico-guided strategy combined with experimental characterization methods. The gene encoding the GDSL lipase GstLipC from Geobacillus stearothermophilus ATCC 7953 was cloned into the pET28a(+) vector, and four point mutations (I184H, I184V, R196L, and R196F) were introduced based on structural modeling and molecular docking analyses. Wild-type and mutant enzymes were expressed and purified for biochemical and kinetic characterization. GstLipC, GstLipC_I184V, and GstLipC_R196F exhibited maximal activity at 90 °C, whereas GstLipC_I184H exhibited optimal activity at 100 °C. GstLipC_R196L displayed comparable activities at both temperatures. All variants showed optimal activity at pH 8 and high thermal stability. In the presence of 0.1 mM Triton™ X-100, the Km values of the mutant enzymes were lower than that of the wild type, suggesting altered substrate interactions, with GstLipC_R196F showing the lowest Km value. Overall, this study demonstrates that residue-level engineering can modulate the catalytic properties and thermal behavior of a thermophilic GDSL lipase, providing insights into the structure-function relationships of thermostable enzymes.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.154003", "ptypes": [ "Journal Article" ], "first_author": "Kaçiran A", "dirs": [ "C" ] }, "40387084": { "pmid": "40387084", "title": "Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9.", "abstract": "The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.", "journal": "eLife", "year": "2025", "doi": "10.7554/eLife.99275", "ptypes": [ "Journal Article" ], "first_author": "Belato HB", "dirs": [ "C" ] }, "38746279": { "pmid": "38746279", "title": "Structural and Dynamic Impacts of Single-atom Disruptions to Guide RNA Interactions within the Recognition Lobe of Geobacillus stearothermophilus Cas9.", "abstract": "The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2024.04.26.591382", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Belato HB", "dirs": [ "C" ] }, "41485486": { "pmid": "41485486", "title": "Structure-function analysis and exonuclease deletion yield an improved strand-displacing DNA polymerase from Aeribacillus pallidus for efficient recombinase polymerase amplification.", "abstract": "Isothermal nucleic-acid amplification relies on strand-displacing DNA polymerases that synthesize DNA while unwinding duplex templates. We previously identified two thermostable family-A polymerases, C1-Pol from Geobacillus zalihae and H1-Pol from Aeribacillus pallidus, which show a characteristic and reproducible trade-off between stability and activity. C1-Pol is more thermostable, whereas H1-Pol exhibits stronger strand-displacement activity and higher recombinase polymerase amplification (RPA) efficiency. Domain-swapping analysis indicated that a chimeric construct (C1-Pol#4), in which the C1-Pol 5'→3' exonuclease region was replaced with that of H1-Pol, enhanced strand-displacement and nucleotide-incorporation activities while maintaining parental-level stability, suggesting that this region modulates these important biochemical properties. However, C1-Pol#4 did not improve RPA efficiency. To clarify the functional contribution of this domain, we constructed Klenow-type deletion mutants lacking the 5'→3' exonuclease region. The H1-Pol large fragment (H1-PolLF) exhibited reduced thermostability but markedly enhanced polymerase and strand-displacement activities at 40 °C, the optimal temperature for RPA reactions. At this temperature, H1-PolLF showed ∼10-fold higher strand-displacement activity than Bst DNA polymerase 2.0 and 4.4-fold higher than full-length H1-Pol. H1-PolLF also accelerated RPA, producing equivalent amplicons in half the reaction time and improving detection sensitivity 100-fold (6 × 10³ → 6 × 10¹ copies) than parental H1-Pol. Coupling H1-PolLF with branched-chain polyamine (BCPA)-conjugated magnetic beads enabled reliable detection of ∼10² target molecules from 10 mL saline. These findings demonstrate that removing the 5'→3' exonuclease domain fine-tunes polymerase and strand-displacing functions, yielding an enzyme highly suited for rapid, ultrasensitive isothermal nucleic-acid detection when used in combination with BCPA beads.", "journal": "Journal of biotechnology", "year": "2026", "doi": "10.1016/j.jbiotec.2025.12.020", "ptypes": [ "Journal Article" ], "first_author": "Nishi K", "dirs": [ "C" ] }, "42781938": { "pmid": "42781938", "title": "An Integrated System for Geobacillus Protoplast Formation, Transfection, Recombinant Genomes Rebooting, Efficient Propagation, and Genomic DNA Isolation of the Thermophilic Bacteriophage TP-84.", "abstract": "Protoplast transformation has been an effective method for introducing plasmid DNA into Bacillus species; however, its application to deliver complete bacteriophage genomes has not been demonstrated previously. Here, we describe an optimized and reproducible method for forming Geobacillus stearothermophilus protoplasts and efficiently transfecting them with purified recombinant or wild-type genomic DNA from the thermophilic bacteriophage TP-84. Systematic optimization of key parameters, including bacterial cell density, lysozyme concentration, polyethylene glycol type and concentration, and DNA input, enabled robust phage genomic DNA uptake and subsequent production of infectious phage particles. In addition, we optimized and scaled up a single-layer solid-state bacteriophage propagation procedure for integration into the TP-84 genome-engineering workflow, enabling the recovery of high-titer lysates and sufficient genomic DNA for sequencing and subsequent genome manipulation from a single Petri dish. In our experimental system, solid-state propagation also provided greater stability of recombinant phage variants than propagation in liquid bacterial cultures. Together, these methods establish an integrated protoplast-based system for phage genomic DNA delivery, rebooting, propagation, and recovery, enabling controlled manipulation of bacteriophage genomes and facilitating the rapid generation and analysis of recombinant variants.", "journal": "MicrobiologyOpen", "year": "2026", "doi": "10.1002/mbo3.70421", "ptypes": [ "Journal Article" ], "first_author": "Sobolewski I", "dirs": [ "C" ] }, "42714150": { "pmid": "42714150", "title": "Development of a functional and disinfectable sewage vent high-efficiency particulate air filter system and its validation using vaporized hydrogen peroxide.", "abstract": "Airborne disinfection of high-containment facilities is mandatory for safe operations. However, sewage vent air-particularly in large-animal experimental units-can represent a critical gap and is typically not considered in the design of high-efficiency particulate air (HEPA) filter systems. Here, we present an engineering design enabling HEPA filtration of sewage vent air from primary biological containments, including large-animal facilities and necropsy suites. We additionally evaluated vaporized hydrogen peroxide (vH2O2) fumigation as an airborne disinfection approach. Disinfection efficacy was determined by quantitative carrier testing using commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus and compared with germ carriers coated with surrogate viruses, mycobacteria, and other spores. Temperature, relative humidity (RH), and vH2O2 concentration were monitored in real time to define process parameters and validate protocols targeting ≥4 log10 reduction. Inactivation efficacy differed substantially among challenge agents: CSC were least resistant to inactivation, whereas murine norovirus (MNV) on germ carriers was most challenging to inactivate. At medium-high vH2O2 concentrations (up to 520 parts per million [ppm]), increasing RH was the dominant driver of inactivation. Based on these data, we developed a biphasic disinfection cycle maintaining a saturated humidity environment with ~35% H2O2 for ≥10 h, achieving ≥4 log10 reduction of MNV infectivity and G. stearothermophilus spore viability on germ carriers. Overall, vH2O2 under high-to-saturated RH is an effective means to disinfect technical infrastructure in biological containment settings handling high-consequence pathogens.IMPORTANCEVent air from sewage systems in biological containment facilities where large animals are kept should be considered a contaminated waste stream. Therefore, effective high-efficiency particulate air (HEPA) filter systems that prevent mold or other moisture-related deterioration of the filters need to be installed. Airborne vaporized hydrogen peroxide (vH2O2) is an appropriate method to decontaminate these complex HEPA filter systems. High to saturated humidity is required for virucidal and sporicidal efficacy under medium to high concentration of vH2O2. Commercially available spore carriers are artificial in design, resulting in lower resistance to inactivation compared to custom germ carriers coated with non-enveloped viruses and thus should be avoided as read-out (validation means) by the biosafety community working with high-consequence pathogens in primary containments.", "journal": "Applied and environmental microbiology", "year": "2026", "doi": "10.1128/aem.01269-26", "ptypes": [ "Journal Article" ], "first_author": "Schinköthe J", "dirs": [ "C" ] }, "39681533": { "pmid": "39681533", "title": "Temperature-Adaptive Organic Scintillators for X-ray Radiography.", "abstract": "Organic phosphorescence or thermally activated delayed fluorescence (TADF) scintillators, while effective in utilizing triplet excitons, are sensitive to temperature changes, which can impact radioluminescence performance. In this study, we have developed a type of temperature-adaptive organic scintillator with phosphorescence and TADF dual emission. These scintillators can automatically switch modes with temperature changes, enabling efficient radioluminescence from 77 to 400 K. The highest photoluminescence quantum yield and light yield are 83.2% and 78,229 ± 562 photons MeV-1 excited by a UV lamp and X-ray, respectively. Their detection limit is 51 and 23 nGy·s-1 at room temperature and 77 K, respectively, which is lower than the standard dosage of 5.5 μGy s-1 for X-ray diagnostics. Moreover, given the high spatial resolution of 21.7 l p mm-1, we demonstrate their potential application in multiple-temperature X-ray radiography, offering promising new possibilities. This work opens a new route for developing organic scintillators to adapt to ambient temperature change and paves the way for their use in various temperature-sensitive radiography applications.", "journal": "Journal of the American Chemical Society", "year": "2025", "doi": "10.1021/jacs.4c12872", "ptypes": [ "Journal Article" ], "first_author": "Dong M", "dirs": [ "C" ] }, "39997825": { "pmid": "39997825", "title": "Si-HgTe Quantum Dot Visible-Infrared Photodetector.", "abstract": "Silicon photodetectors are well developed, with the advantage of their low cost and easy fabrication. However, due to the semiconductor band gap limitation, their detection wavelength is limited in the visible and near-infrared ranges. To broaden the detection wavelength, we stacked a mercury telluride (HgTe) colloidal quantum dot (CQD) photodiode and a silicon PIN photodiode in series. This detector shows response spectra ranging from visible to short-wave infrared (430 nm to 2800 nm) at room temperature. At zero bias, the total photocurrents are 112.5 μA and 1.24 μA, with a tungsten lamp and a blackbody serving as light sources, respectively. The response speed can reach 1.65 μs, with the calculated detectivities of the visible wavelength D* = 1.01 × 1011 Jones, and that of the short-wave infrared being D* = 2.66 × 1010 Jones at room temperature. At the same time, with a homemade trans-impedance amplifier (TIA) circuit, we demonstrate the device application for figuring out the amplified voltage of the VIS, SWIR, and the VIS-SWIR stacked layers.", "journal": "Nanomaterials (Basel, Switzerland)", "year": "2025", "doi": "10.3390/nano15040262", "ptypes": [ "Journal Article" ], "first_author": "Qian L", "dirs": [ "C" ] }, "41153241": { "pmid": "41153241", "title": "Rapid and Cost-Effective ABO Blood Genotyping Using a Freeze-Dried, Point-of-Care Ready Loop-Mediated Isothermal Amplification (LAMP) Assay.", "abstract": "Background: The accurate and rapid genotyping of ABO (chromosome 9q34.2) blood types is critical for clinical diagnostics and transfusion medicine, particularly in scenarios where serological methods yield uncertain results, such as in neonatal testing or with rare ABO subtypes. Methods: This study describes a loop-mediated isothermal amplification (LAMP)-based method for ABO genotyping that offers a faster and more cost-effective alternative to conventional PCR-based techniques. Results: The method targets four key single nucleotide polymorphisms (SNPs) at positions 261, 297, 703, and 930, allowing for the differentiation of common A, B, and O blood types, as well as the rare AB subtype B(A)01. The detection of the B(A)01 subtype is clinically important for preventing transfusion mismatches where serology may be inconclusive. Operating at a constant temperature, the assay can be completed in under an hour without the need for a thermocycler, offering significant time and cost benefits over qPCR. The method demonstrated high specificity, demonstrating detection down to 10 copies across all assays. When validated against a gold-standard method on clinical blood samples, the LAMP assay showed high accuracy (95% C value calculated via binomial exact method): 97.4% for type O, 98.7% for type A, 98.7% for type B, and 100% for the B(A)01 subtype. To enhance usability for point-of-care applications, freeze-dried reagents were developed that permit direct loading of lysed blood samples while maintaining high performance. Conclusions: This simplified and robust format positions the LAMP assay as a promising tool for rapid and reliable ABO genotyping in diverse clinical settings.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2025", "doi": "10.3390/diagnostics15202568", "ptypes": [ "Journal Article" ], "first_author": "Zhang J", "dirs": [ "C" ] }, "41666469": { "pmid": "41666469", "title": "Rapid Detection of Knowlesi Malaria Using Room-Temperature Stable and Ready-to-Use Colorimetric Loop-Mediated Isothermal Amplification Reagents.", "abstract": "A room temperature-stable and ready-to-use dried-format loop-mediated isothermal amplification (LAMP) diagnostic tool is proposed for detecting knowlesi malaria in the present study. Traditional LAMP assays require a cold chain to maintain the stability of their reagents during storage and transport. This requirement limits the use of LAMP-based methods in environments with limited infrastructure. Results can be obtained by directly observing color changes without the need for specialized instruments; positive samples appear yellow, whereas negative reactions remain pink. The developed dehydrated colorimetric LAMP was tested using 75 clinical blood samples collected from district and state hospitals in Malaysia. By using nested polymerase chain reaction-based assays as a reference method, dehydrated LAMP exhibited 100% sensitivity (95% CI: 92.1-100.0%) and 100% specificity (95% CI: 88.4-100.0%). The dehydrated LAMP reagents can be stored at room temperature for up to 15 days. This dehydrated LAMP assay offers an ideal diagnostic tool for resource-limited settings where knowlesi malaria is endemic.", "journal": "The American journal of tropical medicine and hygiene", "year": "2026", "doi": "10.4269/ajtmh.25-0201", "ptypes": [ "Journal Article" ], "first_author": "Lai MY", "dirs": [ "C" ] }, "42738108": { "pmid": "42738108", "title": "Photovoltaic Effect in the MnZn1-xCrxSb Weyl Semimetals.", "abstract": "The photovoltaic effect in the MnZn1-xCrxSb Weyl semimetals was studied in a wide spectral range. A correlation in the temperature behavior of the magnetization, photo-voltage, and photocurrent has been found and a sharp increase in the photocurrent below 140 K has been established. Increasing by 50% of the generation of photocurrent is accompanied grows of the Curie temperature by 3%. It is shown that, under the same sample illuminance, the photocurrent and photovoltage attain their maximum values under illumination with an incandescent lamp, rather than with a laser with a wavelength of 830 nm or a led lamp. The photocurrent and photovoltage are increased nonlinearly with increasing power of incident light and the maximum sensitivity is detected in the mid-IR range, increasing from 1.5 mA/W to 20 mA/W with the chromium doping concentration. As the chromium content in the MnZn1-xCrxSb semimetals grows, the drop in resistance changes to a rise under illumination. The possibility of magnetic field control of the photocurrent, photovoltage, and photoresistance with a maximum value at room temperature is demonstrated.", "journal": "Materials (Basel, Switzerland)", "year": "2026", "doi": "10.3390/ma19173670", "ptypes": [ "Journal Article" ], "first_author": "Aplesnin S", "dirs": [ "C" ] }, "40492321": { "pmid": "40492321", "title": "Point-of-care analysis for foodborne pathogens in food samples based on a fully enclosed microfluidic chip cartridge.", "abstract": "Foodborne pathogens endanger public health and rapid, sensitive, and accurate detection of them is vital. Portable, highly integrated detection devices have great application prospects in the screening and analysis of foodborne pathogens. In this study, a fully automated detection device based on a fully enclosed microfluidic chip cartridge was successfully designed. This device integrates multiple functions, including nucleic acid extraction, reagent preparation, LAMP reaction, and signal detection. By simply adding a sample, it can simultaneously detect four types of foodborne pathogens, making it advantageous for the analysis of complex samples and improving detection accuracy. Additionally, freeze-dried reagents are integrated into the fully enclosed microfluidic chip cartridge, which allows the reagents to be transported and stored at room temperature, greatly reducing the cost of detection. It has been successfully applied in actual samples contaminated with multiple foodborne pathogens and has excellent stability. The entire detection process can be completed in 45 minutes, with a sensitivity of approximately 500 CFU mL-1. Therefore, the automated microfluidic device would be adequate for point-of-care testing (POCT) with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d4lc00819g", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Tang D", "dirs": [ "C", "D" ] }, "41396898": { "pmid": "41396898", "title": "Joule-level, 20 ns Cr-Tm-Ho:YAG master oscillator power amplifier system.", "abstract": "We report on a compact, flash-lamp-pumped Cr-Tm-Ho:YAG (CTH:YAG) master oscillator power amplifier (MOPA) system operating at room temperature. The actively Q-switched oscillator produces 20 ns seed pulses at 2087 nm with an energy of 265 mJ. After a two-stage amplification, the system delivers a maximum output energy of 1.24 J, while maintaining the 20 ns pulse duration. To the best of our knowledge, this work represents the first demonstration of a joule-level, nanosecond CTH:YAG amplifier system, achieving the highest single-pulse energy yet reported from such a configuration. These results establish flash-lamp-pumped CTH:YAG as a viable architecture for developing high-energy, cost-effective 2 µm laser sources.", "journal": "Optics letters", "year": "2025", "doi": "10.1364/OL.577462", "ptypes": [ "Journal Article" ], "first_author": "Guo W", "dirs": [ "C" ] }, "40288444": { "pmid": "40288444", "title": "Room temperature storage and shipping of encapsulated synthetic RNAs as quality control materials for SARS-CoV-2 molecular diagnostic assays.", "abstract": "The Coronavirus pandemic unveiled the unprecedented need for diagnostic tests to rapidly detect the presence of pathogens in the population. Real-time RT-PCR and other nucleic acid amplification techniques are accurate and sensitive molecular techniques that necessitate quality control strategies and stable quality control materials. To meet this need, Twist Bioscience has developed and released synthetic RNA controls. However, RNA is an inherently unstable molecule needing cold storage, costly shipping, and resource-intensive logistics. Imagene provides a solution to this problem by encapsulating dehydrated RNA inside metallic capsules filled with anhydrous argon, allowing room temperature and eco-friendly storage and shipping. This technology initially developed for DNA storage has been successfully applied to RNA and other biospecimen and extensively validated through real time and accelerated aging. Here, RNA controls produced by Twist Bioscience were encapsulated in RNAshells and distributed to several laboratories that used them for COVID-19 detection tests by amplification. One RT-LAMP procedure, four different RT-PCR devices and 6 different PCR kits were used. The amplification targets were genes E, N; RdRp, Sarbeco-E and Orf1a/b. RNA retrieval was satisfactory, and the detection was reproducible. RNA stability was checked by real-time (3 years at room temperature) and accelerated aging (16 h at 90 °C, corresponding to approximately 10 years of storage at 25 °C, according to our previously published Arrhenius study for encapsulated RNA). The results were not significantly different from those for unaged capsules. This room temperature RNA stability allows the preparation and distribution of large strategic batches which can be stored for more than 10 years a long time and used for standardization processes between detection sites. Moreover, this provides the advantage of single-use and field usability across varying temperatures. Consequently, this type of encapsulated synthetic RNA, processed at room temperature, can be used as internal quality control materials for the SARS-Cov-2 virus as well as for detection of other RNA viruses.", "journal": "Journal of virological methods", "year": "2025", "doi": "10.1016/j.jviromet.2025.115169", "ptypes": [ "Journal Article" ], "first_author": "Colotte M", "dirs": [ "C" ] }, "40616652": { "pmid": "40616652", "title": "A Bromobenzoyl Pyridine Photosensitizer with Suppressed Phosphorescence for Efficient ROS-Based Photodynamic Therapy.", "abstract": "Organic room-temperature phosphorescent (RTP) and photodynamic therapy (PDT) both rely on triplet excitons generated through intersystem cross-system processes (ISC), but their energy dissipation pathways-the radiative decay of RTP and the reactive oxygen species (ROS) production of photodynamic therapy (PDT) are essentially competitive. Here, we report a photosensitizer based on bromobenzoylpyridine named 2N-Br, which achieves short-lifetime phosphorescence emission (lifetime = 0.018 ms, Φ≈0%), but generates a strong ROS. The molecular structure characterized by carbonyl and bromine atoms can effectively promote the efficient ISC process. Theoretical calculations show that it has a narrow ΔEST, while single crystal analysis shows that the dihedral angle (56.03°) and weak intermolecular interaction reduces the restricted molecular movement and enhances the non-radiative transition, which is conducive to the generation of oxygen-dependent ROS. When nanoparticles (2N-Br NPs) were fabricated, this material exhibited a strong PDT effect on 4T1 cells under 405 nm Xe lamp exposure. This work established a molecular strategy that links phosphorescence with the generation of ROS, demonstrating that the generation of strong ROS can coexist with ultra-low phosphorescence efficiency.", "journal": "Journal of fluorescence", "year": "2025", "doi": "10.1007/s10895-025-04427-3", "ptypes": [ "Journal Article" ], "first_author": "Wang S", "dirs": [ "C" ] }, "40310394": { "pmid": "40310394", "title": "Development of the MTB/IC LAMP-MS Assay for Rapid Detection of Mycobacterium tuberculosis.", "abstract": "Background: Tuberculosis (TB) remains a significant global health challenge, necessitating rapid and reliable diagnostic solutions. Current methods such as microscopy, culture, and PCR have limitations in resource-limited settings due to high costs and infrastructure requirements. Methods: In this study, we developed the MTB/IC LAMP-MS assay, a novel molecular diagnostic platform that integrates loop-mediated isothermal amplification (LAMP) technology with microscope (MS) detection. The assay enables TB diagnosis within 40 min, from nucleic acid extraction to result, requiring minimal equipment and simple operation for rapid and accurate detection. Results: Compared to the commercial AdvanSure TB/NTM real-time PCR kit, the MTB/IC LAMP-MS assay had a lower analytical sensitivity, with a limit of detection of 103 CFU/mL versus 102 CFU/mL for the commercial PCR kit, but showed a sensitivity of 93.3% and specificity of 100% in clinical tests. Additionally, the assay demonstrated minimal cross-reactivity with other respiratory pathogens, underscoring its robust specificity. In addition, the preloaded microchips demonstrated stable performance for up to 12 weeks at room temperature, supporting their field applicability. Conclusions: With its lower cost, simplified operation, and minimal infrastructure requirements, the MTB/IC LAMP-MS assay provides a practical and efficient solution for TB diagnosis in resource-limited settings.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2025", "doi": "10.3390/diagnostics15080996", "ptypes": [ "Journal Article" ], "first_author": "Jang WS", "dirs": [ "C" ] }, "41723155": { "pmid": "41723155", "title": "Formation of charge-polarized regions at dual single-atom sites for C-H bond activation in methane.", "abstract": "Direct conversion of CH4 into liquid oxygenates under mild conditions is of great significance but remains challenging due to the high dissociation energy of inert C-H bond. Here we report the fabrication of a dual atomic Fe and Pd catalyst with periodic macroporous structure (Fe1-Pd1 OMNC) toward the direct CH4 conversion at room temperature. Mechanism studies reveal that a charge polarization region (Oδ--Fe-Pdδ+) is formed in-situ on Fe-Pd atomic sites upon oxidant activation, wherein the electron-rich Oδ⁻ and electron-deficient Pdδ+ regions can respectively capture the Hδ⁺ and CH3δ⁻ in CH4 and lead to the activation of C-H bond. As a result, Fe1-Pd1 OMNC demonstrates attractive photothermal catalytic performance toward the selective oxidation of CH4 under Xe lamp irradiation, achieving the productivities of C1 oxygenates as high as 0.754 mmol h-1 and 0.035 mmol h-1 when using H2O2 or O2 as the oxidant, respectively.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-69822-1", "ptypes": [ "Journal Article" ], "first_author": "Chen D", "dirs": [ "C" ] }, "42128680": { "pmid": "42128680", "title": "[Study on the radiation intensity of new ultraviolet disinfection lamps at different ambient temperatures].", "abstract": "Objective: To understand the changes in radiation intensity of new ultraviolet disinfection lamps at different temperatures. Methods: Non-polar UV disinfection lamps, light-emitting diode (LED) UV disinfection lamps, 222 nm UV disinfection lamps, and general UV disinfection lamps were respectively placed at different ambient temperatures, including 20-25 ℃, 4 ℃, -20 ℃ and -40 ℃. At a vertical distance of 15 cm below the center of the UV disinfection lamp, the radiation intensity spectrum was recorded with the spectrometer every 5 minutes for a total of 30 minutes. Results: The radiation intensity of the non-polar UV disinfection lamp significantly decreased as the ambient temperature declined, with a reduction range of 10.65% to 77.58%. At room temperature, 4 ℃, and -20 ℃, the radiation intensity increased with the extension of the lighting time, with an increase of 28.66% to 85.41% compared to when the lights were turned on. However, at -40 ℃, it generally showed a downward trend, with a variation range of -8.06% to 24.53%. The radiation intensity of the LED UV disinfection lamp increased as the ambient temperature declined, with an increase range of 5.40% to 35.53%. At the four ambient temperatures, the radiation intensity decreased slightly with the extension of the lighting time, and the degree of reduction became smaller as the ambient temperature declined. The radiation intensity of the 222 nm UV disinfection lamp increased in low-temperature environments instead, with an increase range of 9.03% to 32.33%. At the four ambient temperatures, the radiation intensity remained relatively stable with the extension of the lighting time, with a variation range of only -2.87% to 8.27%. The radiation intensity of the general UV disinfection lamp decreased significantly as the ambient temperature declined, with a reduction range of 43.92% to 91.54%. At room temperature, 4 ℃, and -20 ℃, the radiation intensity increased significantly with the extension of the lighting time within the first 5 minutes and then stabilized. However, at -40 ℃, it generally showed a downward trend, with a variation range of -25.00% to 5.95%. Conclusion: New UV disinfection lamps vary in their radiation intensity characteristics. The radiation intensity of the non-polar UV disinfection lamp is greatly affected by low temperatures, but the radiation intensity of the LED UV disinfection lamp and the 222 nm UV disinfection lamp increases in low-temperature environments instead.", "journal": "Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]", "year": "2026", "doi": "10.3760/cma.j.cn112150-20251117-01089", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "Li HF", "dirs": [ "C" ] }, "42704152": { "pmid": "42704152", "title": "Zr-Oxo Cluster-Based Metal-Organic Framework for Selective C(sp3)-H Bond Photooxidation.", "abstract": "Selective aerobic oxidation of C(sp3)-H bonds to carbonyl compounds is an important but challenging transformation in green and sustainable catalysis, owing to the difficulty of achieving high chemoselectivity while suppressing overoxidation under mild conditions. Herein, we report HEBMU-1, a Zr-oxo cluster-based metal-organic framework photocatalyst, for the selective photooxidation of alkanes, arenes, and heteroarenes to aldehydes and ketones. Benefiting from its robust crystalline framework, photoactive organic linkers, and accessible Zr-oxo cluster sites, HEBMU-1 enables light harvesting, enhanced photoinduced charge separation, and molecular oxygen activation under xenon lamp irradiation at room temperature. This catalytic system exhibits broad substrate scope, good functional-group tolerance, high chemoselectivity, and effective suppression of overoxidation, affording the corresponding carbonyl products in good to high yields. The use of molecular oxygen as a green oxidant avoids stoichiometric chemical oxidants and harsh reaction conditions. In addition, HEBMU-1 can be readily recovered and reused over multiple cycles while maintaining its structural integrity. This work demonstrates the potential of Zr-oxo cluster-based MOF photocatalysts for selective aerobic C(sp3)-H oxidation and provides a green strategy for the synthesis of value-added carbonyl compounds.", "journal": "Inorganic chemistry", "year": "2026", "doi": "10.1021/acs.inorgchem.6c03239", "ptypes": [ "Journal Article" ], "first_author": "Gao G", "dirs": [ "C" ] }, "42322971": { "pmid": "42322971", "title": "Development of a chemical precipitated-LAMP assay for the diagnosis of knowlesi malaria.", "abstract": "A visualized air-dried precipitated loop-mediated isothermal amplification (ADP-LAMP) master mix, incorporating hydroxynapthol blue dye, was developed for the detection of Plasmodium knowlesi. The ADP-LAMP assay was designed to overcome the reliance of LAMP assays on cold-chain transportation for liquid reagents. This study analyzed 80 P. knowlesi and 40 non-P. knowlesi human blood samples collected from the district hospitals. Samples were confirmed by microscopy and nested PCR prior to ADP-LAMP method. The amplification buffer, enzyme and primers involved in ADP-LAMP were chemically precipitated followed by spin-drying into room-temperature stabled pellets. The ADP-LAMP assay generated result which could be visualized by naked eye without the use of specialized equipment and the limit of detection was found to be 10 copies/μL of P. knowlesi. The assay demonstrated high specificity, with no cross-reactivity against other human malaria species or human genomic DNA. When evaluated on clinical samples, the ADP-LAMP assay demonstrated 100% specificity and sensitivity with standard nested PCR and delivers results within 45 minutes at 65°C. This ADP-LAMP assay could be served as an alternative diagnostic tool in malaria endemic areas.", "journal": "Diagnostic microbiology and infectious disease", "year": "2026", "doi": "10.1016/j.diagmicrobio.2026.117518", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Lai MY", "dirs": [ "C" ] }, "41334030": { "pmid": "41334030", "title": "Use of freeze-dried umbilical cord amniotic membrane as a corneal patch graft.", "abstract": "The use of amniotic membranes for corneal perforation management has been extensively documented, demonstrating significant potential in ocular surface reconstruction. However, their application in emergency settings remains underexplored. This report presents a novel use of freeze-dried umbilical cord amniotic membrane as a corneal patch graft, highlighting its practical benefits in urgent clinical scenarios. A 64-year-old male presented to the emergency department with a painful, red eye, having a history of penetrating keratoplasty. Slit-lamp examination revealed a severe corneal abscess with a 3 mm-wide paracentral perforation. Emergency surgery was performed using a freeze-dried umbilical cord amniotic membrane (SclerFix, Amtrix®, TBF) with a \"mushroom plug and patch\" technique, combined with a fresh amniotic membrane overlay. Postoperative treatment included oral antibiotics, topical antifungal agents, and antibiotic eye drops. The fresh amniotic membrane dissolved within days, while the freeze-dried umbilical cord membrane facilitated rapid re-epithelialization, effectively sealing the perforation. At three months postoperatively, the cornea remained intact without recurrence or complications, and the patient reported complete pain resolution. This case demonstrates the successful use of freeze-dried umbilical cord amniotic membrane as a corneal patch graft, avoiding emergency keratoplasty and preserving ocular integrity. This technique represents a promising alternative in situations where corneal grafts are not readily available. Further studies are warranted to validate its efficacy.", "journal": "American journal of ophthalmology case reports", "year": "2025", "doi": "10.1016/j.ajoc.2025.102477", "ptypes": [ "Case Reports", "Journal Article" ], "first_author": "Bistour A", "dirs": [ "C" ] }, "41144386": { "pmid": "41144386", "title": "Battery-Powered Homeothermic Warming Pad for Maintaining Core Temperature in Mice and Rats.", "abstract": "Anesthesia decreases core body temperature and this seriously compromises the physiological status of an experimental animal. Importantly, hypothermia alters many aspects of neural function. When recording nervous system activity in anesthetized animals, the core temperature of the animal must be stabilized. Although commercial heating pads for small animals are readily available, they typically are expensive, somewhat bulky, and require power cords and a 120/240V source. Consequently, transporting an anesthetized animal from one location (e.g., surgical suite) to another (e.g., imaging rig) involves moving power cords. Moreover, commercial devices are not always compatible with custom stereotaxic frames, microscope stages, or holding boxes. This report describes a low-cost, battery-powered, temperature-controlled warming pad for mice and rats and documents its validity and utility. The device monitors an animal's core temperature continuously and automatically adjusts the heating pad to maintain normothermia. It can be constructed in under 30 min, the components are readily available, and the cost is less than $100. The device is portable, making it convenient for researchers who conduct procedures such as surgical preparations in one location and transport the anesthetized animal to another location for experimental recordings. The device can keep an anesthetized mouse normothermic ±0.7° for over 6 h without supplemental warmth (e.g., heat lamp), despite >15° differential between ambient room temperature and core body temperature. This report demonstrates how the warming pad can be used for in vivo imaging of neuronal activity for a prolonged period in mice.", "journal": "Journal of visualized experiments : JoVE", "year": "2025", "doi": "10.3791/69074", "ptypes": [ "Journal Article", "Video-Audio Media", "Research Support, N.I.H., Extramural" ], "first_author": "Fleites IR", "dirs": [ "C" ] }, "39939810": { "pmid": "39939810", "title": "Rapid on-site differentiation of two invasive whitefly cryptic species using LAMP.", "abstract": "Loop-mediated isothermal amplification (LAMP) is a promising technique for detecting pest species used on the spot due to its simplicity and rapidity. The whitefly is regarded as a species complex encompassing over 44 cryptic species. These species are morphologically indistinguishable but exhibit notable differences in their biological traits. Among them, Bemisia tabaci MEAM1 and MED are the most invasive and devastating members. Accurately and quickly distinguishing between these two invasive whiteflies in the field directly affects the implementation of control measures. However, it is often difficult to directly distinguish between the two when they occur together in the field and require extensive specialist knowledge or instrumentation. However, one of the limitations of the current LAMP is that the reaction mixture requires cold chain, which is not ideal for the detection on the spot. To promote its actual application of LAMP on the spot, the LAMP methods to differentiate B. tabaci MEAM1 and MED were developed in the laboratory, and Trialeurodes vaporariorum was used as a negative control. Then the effect of the preservation condition (room temperature and low temperature) of the reaction mixture on the efficiency of LAMP was compared. The study showed that the application of B. tabaci MED- and MEAM1- LAMP reaction mixture can differentiate B. tabaci MEAM1, B. tabaci MED, and T. vaporariorum after low-temperature preservation for more than 24 h. Finally, we validated the method with temperature-controlled hot-water cup in the field and proved its effectiveness when applied to the field. The results demonstrated that the low-temperature preservation of reaction mixture provides available technical support for the application of LAMP on the spot.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-89429-8", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Liu Y", "dirs": [ "C" ] }, "42679196": { "pmid": "42679196", "title": "Circularly Polarized Photoluminescence Obtained by Optical Spin Pumping in Achiral Lead-Halide Perovskites: Implications of Interface-Induced Rashba Splitting.", "abstract": "Spin-polarized electronic and optical properties in semiconductors are critical for potential spintronic device applications. However, such spin polarization is readily randomized by thermal fluctuations and generally observable only at low temperatures. By employing circularly polarized laser excitation for optical spin pumping, we detected circularly polarized emission from achiral two-dimensional/three-dimensional mixture films of (MBA)2PbBr4·(CsPbBr3)x (x = 0.5, 1, 1.5, 2, where x is the molar ratio). The circular polarization degree reaches 6%, exhibits a negative correlation with the x value, and is nearly insensitive to temperature variation. Moreover, these films display no circular dichroism absorption signals or circularly polarized luminescence upon excitation with a Xe lamp. We therefore conclude that strong Rashba-like splitting is enhanced in the interfacial regions around CsPbBr3 nanocrystals within the composite. Regulating the grain size may provide a new avenue for designing perovskite-based spintronic materials. More importantly, such spin polarization can be operating at room temperature.", "journal": "The journal of physical chemistry letters", "year": "2026", "doi": "10.1021/acs.jpclett.6c02095", "ptypes": [ "Journal Article" ], "first_author": "Yan P", "dirs": [ "C" ] }, "39710780": { "pmid": "39710780", "title": "An integrated and paper-based microfluidic system employing LAMP-CRISPR and equipped with a portable device for simultaneous detection of pathogens.", "abstract": "Point-of-care testing methods are essential for the large-scale diagnosis and monitoring of bacterial infections. This study introduces an integrated platform designed for the simultaneous detection of pathogenic bacteria. Users can simply inject samples into the system, which then conducts the entire procedure in a fully automated manner, eliminating the need for external power sources, all within 60 min or less. The innovative paper-based microfluidic system is capable of lysing bacteria and integrating loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12a system, achieving this with minimal reagent usage on a single piece of paper. The reaction reagents are pre-fabricated as freeze-dried powder on the paper, allowing for long-term storage. A portable and cost-effective detection device has been designed to provide stable temperature control and analyze fluorescent signals, complementing the paper-based microfluidic system. This compact device measures 150 × 150 × 100 mm, weighs less than 1.8 kg, has an average power consumption of under 15 W, and supports external power supply. The device utilizes non-contact QR codes for information transmission, ensuring functionality even in areas without Internet connectivity. This platform is capable of simultaneously detecting five typical pathogenic microorganisms, with a detection limit of 1 copy/μL. It boasts several advantages, including miniaturization, lightweight design, low power consumption, portability, affordability, rapid detection, and ease of operation, making it highly suitable for on-site detection.", "journal": "Analytical and bioanalytical chemistry", "year": "2025", "doi": "10.1007/s00216-024-05693-z", "ptypes": [ "Journal Article" ], "first_author": "Cui S", "dirs": [ "C", "D" ] }, "40862618": { "pmid": "40862618", "title": "Analytical evaluation of TriVerity, a rapid diagnostic and prognostic host gene expression test performed on the Myrna instrument using RT-LAMP.", "abstract": "We evaluated the analytical performance of the TriVerity test system, a benchtop system for rapid measurement and interpretation of host messenger RNAs (mRNAs) measured quantitatively from PAXgene Blood RNA specimens using the TriVerity cartridge on the Myrna instrument. In approximately 30 minutes, 3 scores are generated from 29 host mRNAs for the likelihood of a bacterial infection, a viral infection, and illness severity (7-day need for ICU-level care), each falling into 1 of 5 interpretation bands (very low, low, moderate, high, and very high). Reproducibility, precision, limit of detection, linearity, interference, and sample and cartridge stability were determined per Clinical Laboratory Standards Institute standards. An operator survey assessed TriVerity's ease of use. Reproducibility demonstrated SDs meeting the acceptance criteria of <5.5 score units. The lowest concentration at which 100% of replicates showed measurable amplification was 1 × 106 cp/mL of in vitro RNA transcripts; the limit of quantification and detection were equivalent to blood containing 500 leukocytes/µL. Interference was not observed for 17 potential interferents. The cartridge was stable at room temperature (RT) for 9 and 12 months using accelerated testing at 37°C. TriVerity results were equivalent using fresh and frozen PAXgene Blood RNA samples. All operators agreed or strongly agreed that the TriVerity test system was easy to use. In summary, the analytical performance presented here, along with diagnostic accuracy established in the SEPSIS-SHIELD trial, demonstrates that the TriVerity system is reliable and user friendly, assisting clinicians in managing patients with suspected acute infections and sepsis in acute care settings. The prompt diagnosis of acute infections and sepsis is critical for better patient outcomes. We introduce a groundbreaking messenger RNA-based test, the first of its kind, designed to diagnose the presence of infection and predict illness severity in adult patients with suspected acute infections or sepsis. Several key findings regarding the accuracy and robustness of the test system are presented, which will be relevant to laboratories or acute care settings implementing the test for patient care. Furthermore, these findings may assist clinical researchers in developing analytical trial protocols aimed at the combined evaluation of RNA-based multi-marker tests with both diagnostic and prognostic test characteristics.", "journal": "Journal of clinical microbiology", "year": "2025", "doi": "10.1128/jcm.00352-25", "ptypes": [ "Journal Article", "Evaluation Study", "Research Support, Non-U.S. Gov't" ], "first_author": "Figueiredo-Pereira C", "dirs": [ "C" ] }, "40342967": { "pmid": "40342967", "title": "A simple, inexpensive battery-powered homeothermic warming pad for mice and rats.", "abstract": "Anesthesia decreases core body temperature, and this seriously compromises the physiological status of an experimental animal. Hypothermia alters many aspects of neural function. When recording nervous system activity in anesthetized animals, their core temperature must be stabilized. This report describes an inexpensive, battery-powered, temperature-controlled warming pad for mice and rats and documents its validity and utility. The device is portable, making it convenient for researchers who conduct procedures such as surgical preparations in one location and transport the anesthetized animal to another location for experimental recordings. The device keeps anesthetized mice normothermic ±0.7° for over 6 hours without supplemental warmth (e.g., heat lamp), despite >15° differential between ambient room temperature and core body temperature. We demonstrate how the warming pad can be used for in vivo imaging of neuronal activity for a prolonged period in mice. Commercial heating pads for small animals are expensive, somewhat bulky, and require power cords and a 120/240V source. Transporting an anesthetized animal from one location (e.g. surgical suite) to another (e.g., imaging rig) involves moving power cords. Moreover, commercial devices are not always compatible with custom stereotaxic frames, microscope stages, or holding boxes. The device described here is small, inexpensive, battery-powered, and readily adaptable to experimental set ups. The homeothermic heating pad provides a simple method for maintaining the core temperature of anesthetized small animals. It can be constructed in under 30 minutes, the components are readily available, and the cost is less than $100. It is exceptionally useful for experiments on mice or rats.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.04.28.651075", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Fleites IR", "dirs": [ "C" ] }, "41543772": { "pmid": "41543772", "title": "Poly(lactic-co-glycolic acid) for reagent storage and controlled release in thermoplastic microfluidics.", "abstract": "The on-chip storage of dried reagents is an important technological challenge that must be addressed to improve the capabilities of microfluidic point-of-care (POC) chips. In this work, we investigate the use of poly (lactic-co-glycolic acid) (PLGA) as an encapsulant for the storage and controlled release of dried reagents integrated into disposable thermoplastic microfluidic chips. The PLGA layer allows multiple solid reagent deposits to remain isolated during sample introduction at room temperature and controllably released into the sample volume after heating the chip above a critical threshold temperature. Simple manual pipetting of a PLGA/ethyl acetate solution serves to form a protective PLGA shell encapsulating deposited reagents, with robust sealing between the PLGA and thermoplastic cyclic olefin polymer (COP) substrate preventing reagent leakage during sample introduction. When using a shell thickness below 20 μm to encapsulate nucleic acids as model reagents, over 90% of the deposits are retained following extended aqueous flow, while heating the chip above 40 °C leads to dramatic shrinkage of the PLGA, resulting in delamination of the encapsulating film and rapid reagent release. Using this approach, an on-chip loop-mediated isothermal amplification (LAMP) assay for the detection of methicillin-resistant Staphylococcus aureus (MRSA) is implemented using multiple encapsulated LAMP primer sets integrated directly into an array of on-chip wells. The PLGA encapsulation technique is shown to be a simple and effective method for reagent-integrated microfluidic device manufacturing, offering a new path towards true sample-in, answer-out point-of-care assays.", "journal": "Lab on a chip", "year": "2026", "doi": "10.1039/d5lc00632e", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Lee J", "dirs": [ "C" ] }, "39969709": { "pmid": "39969709", "title": "Copper metallurgical slag as a sustainable precursor of iron oxide photocatalysts to remove indigo carmine dye from water using the photo-Fenton process.", "abstract": "The synthesis of an efficient composite photo-Fenton nanocatalyst was investigated using acid leaching processes of copper slag (CS) with two types of citric acid (EC = edible and AR = analytical-grade reagent). For this purpose, citric acid at room temperature or at 40 °C was used to lixiviate the elements from CS, and then, the leachates were subjected to a thermal treatment (350 °C). The formation of maghemite (γ-Fe2O3) accompanied with calcite as the main mineralogical phase was observed by X-ray diffraction (XRD) studies. The development of nanospheres dispersed in the calcite matrix and the proportion of γ-Fe2O3 in the catalyst or composite material were revealed by scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS), respectively. The catalyst performance in the Fenton and photo-Fenton processes was investigated in the degradation of the indigo carmine dye (IC, 10 ppm), achieving an efficiency of 96.1 ± 2.1% in 60 min, using 5 mM of H2O2 (30% weight) and UV-light (λ = 365 nm). The activity of these materials was analyzed and compared as a function of the specific surface area and the % γ-Fe2O3 in the composites. The preparation of maghemite-rich nanocomposite using the CS leachate, generated with edible citric acid, is an excellent alternative to substitute materials produced with analytical grade reagents because in the removal of IC dye, it showed similar efficiency, kinetics, and stability, but with lesser requirements of lamp energy radiation and doses of photocatalyst and oxidant, with respect to data reported in the literature.", "journal": "Environmental science and pollution research international", "year": "2026", "doi": "10.1007/s11356-025-36072-5", "ptypes": [ "Journal Article" ], "first_author": "Valencia García K", "dirs": [ "C" ] }, "40447017": { "pmid": "40447017", "title": "Rapid detection of zoonotic malaria using room-temperature stable and ready-to-use colorimetric LAMP reagents.", "abstract": "Traditional loop-mediated isothermal amplification (LAMP) assays rely on a cold chain to preserve the stability of LAMP reagents during storage and transport. This requirement restricts the application of LAMP-based methods in resource-limited settings. To overcome this challenge, we developed a room-temperature stable, ready-to-use dried format of the LAMP diagnostic tool for detecting zoonotic malaria. In this study, we report the use of LAMP for detecting five zoonotic malaria species including Plasmodium fieldi, as there have been no previous publications on LAMP for fieldi to date. The reaction mixture was spin-dried in a vacuum container and stored with silica gel at room temperature before used. The detection limits were 1 copy/µL for P. cynomolgi, P. coatneyi, P. fieldi, and P. inui, while the detection limit for P. knowlesi was 10 copies/µL. Results can be visually observed through color changes, without the need for specialized equipment. Positive samples were indicated in yellow while negative reactions remained pink. The dehydrated LAMP reagents can be kept at room temperature for 15 days. Combination of the advantages of the raffinose, the dehydrated LAMP assay develop here is an ideal diagnostic tool in resource-limited settings where malaria is endemic.", "journal": "Acta tropica", "year": "2025", "doi": "10.1016/j.actatropica.2025.107678", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Lau YL", "dirs": [ "C" ] }, "42235064": { "pmid": "42235064", "title": "Ultrasensitive Portable LAMP-Powered Microfluidic Device for On-Site Rapid Forensic Species and Sex Identification.", "abstract": "Forensic investigations require the rapid and accurate identification of human DNA and sex. Although conventional PCR-based methods are reliable, they are limited by their speed, equipment requirements, and poor suitability for on-site analysis, warranting alternative approaches. Isothermal amplification─particularly, loop-mediated isothermal amplification (LAMP)─is a promising technique because of its speed, sensitivity, and minimal equipment requirements; however, its forensic application is constrained by primer design, extraction requirements, and reagent stability. In this study, we have developed a multiplex LAMP assay that targets the Cyt b and SRY genes for simultaneous human DNA detection and sex identification and further standardized the overall LAMP-based rapid detection workflow. The optimized assay showed a sensitivity of 25 pg, and reagents were formulated into lyophilized beads, which improved portability and long-term on-chip storage (≥9 months). The streamlined workflow integrated sample lysis and amplification and was completed in ≤35 min in a microfluidic device. This enabled rapid and user-friendly point-of-care testing (POCT). Robustness was confirmed by the reliable detection of trace male DNA in mixed samples, and 100% accuracy was confirmed via validation with diverse clinical body fluid specimens. Testing was performed with touch DNA from both simulated and real samples, and the accurate detection rate was 62.5%. Overall, the multiplex LAMP assay provides a sensitive, accurate, and portable solution for the rapid identification of human DNA and sex. The proposed device shows strong potential for forensic POCT application.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.6c02854", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zheng Y", "dirs": [ "C", "D" ] }, "41624191": { "pmid": "41624191", "title": "Non-volatile metabolomics analysis of heating withering method for processing white tea from Longjing 43 tea variety by UHPLC-Q-TOF/MS.", "abstract": "Withering is a crucial step in the production of white tea. However, the non-volatile metabolomic profile of white tea produced from the Longjing 43 cultivar using heated withering methods has not been fully elucidated. This study investigated the effect of a novel heated withering process on the taste quality and non-volatile metabolome of white tea. The application of heat significantly shortened the withering duration and enhanced the sensory quality of the tea. Specifically, white tea withered using hot air for 23 h, sunlight for 28 h, and fluorescent lamp for 31 h, all at 28 ± 1 °C, achieved higher taste sensory evaluation scores compared to traditional room-temperature withering at 20 ± 1 °C for 40 h. Non-targeted metabolomic analysis led to the identification of 1,372 metabolites across the different withering treatments. Relative to room-temperature withering, the heated withering processes-particularly hot air withering-resulted in higher concentrations of key taste-related compounds, including organic acids, amino acids, flavonoids, and catechins. Enrichment analysis of KEGG pathways indicated that the main metabolic pathways affected by heated withering were cysteine and methionine metabolism, glutathione metabolism, and isoquinoline alkaloid biosynthesis, with the most pronounced effects observed under hot air withering. In summary, hot air withering not only reduced the processing time but also improved the taste quality and markedly altered the profile of key metabolites in white tea.", "journal": "Frontiers in nutrition", "year": "2025", "doi": "10.3389/fnut.2025.1735183", "ptypes": [ "Journal Article" ], "first_author": "Cui H", "dirs": [ "C" ] }, "40185247": { "pmid": "40185247", "title": "Determination of limit of detection and relative limit of detection of Salmonella in raw pet food matrices using Salmonella bacteriological analytical manual methods.", "abstract": "Each year, there are multiple Salmonella recalls due to contaminated pet food products. Routine testing of finished products requires reliable, quick, and sensitive detection methods. In this study we comparatively evaluated the sensitivity of a culture-based reference method and two alternative methods, which are based on LAMP and PCR principles. The evaluation was performed in multiple trials using challenging pet food matrices, which were tested after various storage time intervals to better address the reliability of the methods. Raw freeze-dried treats, kibble, and patties were individually inoculated with Salmonella Typhimurium at fractional and high inoculation levels. Raw freeze-dried treats and kibble were inoculated at 0, 1, 3, 5, 7 and 10 CFU/25 g and raw patties were inoculated at 0, 1, 3, 5, 7, 10 and 30 CFU/25 g. For the culture method, the level of detection (LOD50) values were 0.9, 1.1, and 3.7 CFU/25 g for freeze-dried treats, kibble, and patties respectively. The calculated relative level of detection (RLOD) values were close to 1 CFU/25 g, indicating that LAMP and PCR methods have similar LOD50 values to the culture method. It was also confirmed that the alternative LAMP and PCR methods can provide reliable results within 24 h that match the reference culture method. Faster response times are invaluable when responding to potential pet food product contamination.", "journal": "Journal of microbiological methods", "year": "2025", "doi": "10.1016/j.mimet.2025.107116", "ptypes": [ "Journal Article", "Research Support, U.S. Gov't, Non-P.H.S.", "Research Support, N.I.H., Extramural" ], "first_author": "Kiener S", "dirs": [ "C" ] }, "40727280": { "pmid": "40727280", "title": "Mechanochemical synthesis of Sb3+-doped Cs2ZrCl6 double perovskites for excitation-wavelength-responsive trimodal luminescence and high-level anti-counterfeiting.", "abstract": "All-inorganic lead-free halide perovskites have shown great promise in the field of optoelectronics. However, the room-temperature, low-cost, and large-scale synthesis of such materials remains challenging. Here, we report a rapid mechanochemical synthesis strategy based on ethanol-assisted ball milling to successfully prepare Cs2ZrCl6 double perovskite with Sb3+ doping, which exhibits tricolor luminescence with excitation wavelength-dependent characteristics. The material emits blue (450 nm, [ZrCl6]2- self-trapped exciton emission), warm white (450/610 nm dual peaks), and red (610 nm, Sb3+ 3P1 → 1S0 transition) light under 254, 310, and 365 nm ultraviolet excitation, respectively, with a photoluminescence quantum yield (PLQY) stably exceeding 30%. When the Cs2ZrCl6:Sb3+ samples are fabricated into anti-counterfeiting patterns, they emit blue, warm white, and red light under 254, 310, and 365 nm ultraviolet lamp excitation, respectively, demonstrating excellent application prospects in the field of anti-counterfeiting encryption. This study provides a new idea for the development of efficient and environmentally friendly synthesis of multi-modal anti-counterfeiting materials.", "journal": "RSC advances", "year": "2025", "doi": "10.1039/d5ra03637b", "ptypes": [ "Journal Article" ], "first_author": "Chen Q", "dirs": [ "C" ] }, "40987432": { "pmid": "40987432", "title": "Molecular diagnostics for infectious disease.", "abstract": "Molecular diagnostic methods have been used in clinical and environmental settings for over three decades. Conventional approaches, which often involve long processing times and limited sensitivity, are gradually being replaced by molecular tests that offer improved analytical performance. Nucleic acid testing (NAT), particularly PCR-based methods and their variants such as quantitative PCR (qPCR) and reverse transcription PCR (RT-PCR), have demonstrated higher sensitivity and specificity than conventional assays. Recent developments in next-generation sequencing (NGS), including metagenomic and whole-genome sequencing, have also enhanced the identification of complex pathogens and resistance mechanisms. Point-of-care testing (POCT) is another advancement, supported by newly developed isothermal amplification (IA) techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), which allow rapid detection in decentralized settings. In addition, newer approaches such as droplet digital PCR (ddPCR) and CRISPR-based diagnostics are very promising due to their potential to provide accurate nucleic acid detection in low amounts of target. Aptamer-based sensors are also under extensive research and development for their application in diagnosis of infection. This review outlines both traditional and recent advanced molecular diagnostic tools, with a focus on their application to infectious disease detection.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2026", "doi": "10.1016/j.cca.2025.120619", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sayeli FG", "dirs": [ "C" ] }, "40780172": { "pmid": "40780172", "title": "A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT.", "abstract": "Mycoplasma pneumoniae (M. pneumoniae), a major human respiratory pathogen, necessitates the development of rapid point-of-care testing (POCT) platforms for clinical management. However, current two-step workflows suffer from operational complexity and aerosol contamination risks. This limitation stems from CRISPR-Cas12 mediated template degradation in single-reaction systems, which compromises amplification efficiency and detection sensitivity. Here, we combined RPA and CRISPR Cas12b by leveraging the difference in their optimal temperatures to construct a novel TRACER (Thermo-activated RPA Amplification for CRISPR-Cas12b Efficient Recognition) technology. Through precise temperature modulation, TRACER sequentially executes isothermal amplification and CRISPR-mediated detection while preventing premature template cleavage, thereby maintaining optimal reaction efficiency. The platform demonstrates exceptional analytical sensitivity with a detection limit of 1 copy/μL, representing a 100-fold improvement over conventional one-pot RPA-CRISPR-Cas12a systems. Clinical validation using 195 specimens revealed diagnostic performance metrics of 99.2 % sensitivity (119/120), 100.0 % specificity (75/75), and 99.5 % accuracy (194/195). This innovative combination of single-tube reaction, field-deployable instrumentation, and cost-effectiveness establishes TRACER as an ideal POCT solution for M. pneumoniae detection in diverse clinical settings.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117839", "ptypes": [ "Journal Article" ], "first_author": "Feng J", "dirs": [ "C" ] }, "40064782": { "pmid": "40064782", "title": "CRISPR-Cas-Based Diagnosis of Geminiviruses.", "abstract": "Successful disease management relies on rapid and accurate identification of the causal agent. Begomoviruses (family Geminiviridae) cause severe economic losses and pose a serious threat to sustainable agriculture. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection method utilizing the CRISPR/Cas12a and CRISPR/Cas13a system for begomoviruses used in our laboratory. CRISPR-based diagnostic tools are very precise and sensitive and visual readout-compatible reporters do not need expensive laboratory equipment such as qRT-PCR. The assay detects the targeted begomoviruses in infected plants with high sensitivity and specificity, and can provide highly sensitive and specific detection visual readouts using a simple, low-cost blue light lamp, ideal for on-site use.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2025", "doi": "10.1007/978-1-0716-4454-6_16", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Singhal T", "dirs": [ "C" ] }, "40760679": { "pmid": "40760679", "title": "CRISPR-based assays for the detection of BK virus and JC virus infections post-kidney transplantation.", "abstract": "Organ transplantation recipients encounter significant risks from acute or chronic infections that threaten graft survival. BK virus (BKV) and JC virus (JCV) are two prominent opportunistic infection viruses, and they may cause polyomavirus-associated nephropathy and graft kidney loss in patients who are in an immunosuppressed state after kidney transplantation. Hence, timely detection and sustained monitoring of the viral load are indispensable. However, the current diagnostic methods remain limited, and the development of new molecular detection technology is extremely urgent. The sequences and concentrations of clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), the concentration of Cas13a, and the primers for recombinase polymerase amplification (RPA) were optimized for BKV and JCV detection. Next, a novel microfluidic dual-droplet chip was designed and fabricated, and it was integrated with CRISPR (ddCRISPR) to simultaneously qualitatively detect BKV and JCV. Subsequently, the ddCRISPR assay was verified using clinical samples. Then, a lateral flow strip combined with CRISPR (LFCRISPR) was developed for the detection of BKV and JCV in resource-limited settings. A one-pot RPA-CRISPR reaction system was established and optimized for BKV and JCV detection. ddCRISPR can simultaneously and rapidly detect BKV and JCV with high sensitivity (10 copies/ml for BKV and 1 copy/ml for JCV), and provide absolute quantification, which is suitable for viral load detection and conducive to personalized and precise treatment for organ transplant recipients. LFCRISPR simplified the operational process through a simple visual readout, facilitating virus screening after organ transplantation. These platforms incorporate molecular testing into the transplantation treatment model, thereby reducing costs, prolonging the survival time of the graft, improving the clinical outcomes of postoperative management in kidney transplantation, and enhancing the patients' quality of life.", "journal": "Military Medical Research", "year": "2025", "doi": "10.1186/s40779-025-00632-0", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Liu Y", "dirs": [ "C" ] }, "41032977": { "pmid": "41032977", "title": "A 3D-printed, self-driven microfluidic sensor chip for point-of-care testing (POCT) of norovirus.", "abstract": "Early diagnosis of norovirus (NV) is critical for effective prevention and control of outbreaks. Clustered regularly interspaced short palindromic repeats (CRISPR)-coupled isothermal amplification has been widely employed for the highly sensitive and specific detection of nucleic acids; however, optimizing the compatibility among multi-enzyme reaction systems remains a challenge. In this study, a compartmentalized, self-driven, 3D-printed microfluidic sensor device was developed for cascade isothermal amplification and CRISPR-mediated multistep reactions. This chip integrated the sample pretreatment process with glucose biosensing technology. By leveraging the digital quantification capability of a personal glucose meter (PGM) as an endpoint readout, the system detected NV nucleic acids with high sensitivity and specificity. A series of target RNA concentrations (0.1-10,000 fM) were quantified using the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR sensing method developed in this study. The relationship was linear between the logarithm of the NV target RNA concentration (log C) and the corresponding fluorescence intensity. The RT-RPA-CRISPR assay was further engineered into a microfluidic chip-based point-of-care testing (POCT) system, and the limit of detection of NV was about 60 copies. This integrated approach facilitates field-deployable diagnostics of viral and bacterial pathogens.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.118048", "ptypes": [ "Journal Article" ], "first_author": "Wang W", "dirs": [ "C" ] }, "41932456": { "pmid": "41932456", "title": "Design principles for LAMP-CRISPR molecular diagnostics.", "abstract": "Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.", "journal": "Methods (San Diego, Calif.)", "year": "2026", "doi": "10.1016/j.ymeth.2026.03.014", "ptypes": [ "Journal Article", "Review" ], "first_author": "Silva CS", "dirs": [ "C" ] }, "39244837": { "pmid": "39244837", "title": "An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR/Cas12a.", "abstract": "Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2024.116740", "ptypes": [ "Journal Article" ], "first_author": "Fu J", "dirs": [ "C" ] }, "40349998": { "pmid": "40349998", "title": "Emerging insights of Staphylococcus spp. in human mastitis.", "abstract": "Human mastitis represents a prevalent and intricate condition that significantly challenges breastfeeding women, often exacerbated by pathogenic bacteria such as Staphylococcus aureus. A deep understanding of the interplay between human mastitis, the breast milk microbiome, and causative agents is imperative. This understanding must focus on the bacterium's virulence and resistance genes, which critically influence the severity and persistence of mastitis. Current methods for detecting these genes, including Polymerase Chain Reaction (PCR), 16S rRNA gene sequencing, shotgun metagenomic sequencing, multiplex PCR, whole genome sequencing (WGS), loop-mediated isothermal amplification (LAMP), CRISPR-based assays, and microarray technology, are vital in elucidating bacterial pathogenicity and resistance profiles. However, advanced attention is required to refine diagnostic techniques, enabling earlier detection and more effective therapeutic approaches for human mastitis. The involvement of Staphylococcus aureus in human infection should be a prime focus, especially in women's health, which deals directly with neonates. Essential virulence genes in Staphylococcus species are instrumental in infection mechanisms and antibiotic resistance, serving as potential targets for personalized treatments. Thus, this review focuses on Staphylococcusaureus-induced mastitis, examining its virulence factors and detection techniques to advance diagnostic and therapeutic strategies.", "journal": "Microbial pathogenesis", "year": "2025", "doi": "10.1016/j.micpath.2025.107685", "ptypes": [ "Journal Article", "Review" ], "first_author": "Patel M", "dirs": [ "C" ] }, "40938680": { "pmid": "40938680", "title": "CRISPR/Cas-based detection strategies for tumor biomarker detection.", "abstract": "Tumor biomarkers, such as nucleic acids, proteins, extracellular vesicles (EVs) and circulating tumor cells (CTCs), can provide valuable information for tumor risk assessment, diagnosis, prognosis and recurrence monitoring. Currently, polymerase chain reaction (PCR)-based approaches and enzyme linked immunosorbent assay (ELISA) are typically used for detecting tumor biomarkers in clinics. However, PCR-based methods have limits in sensitivity and detection channels. Besides, ELISA suffers from cumbersome operation and limited sensitivity. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are adopted for developing novel detection strategies due to the characteristics of high sensitivity, high specificity, simple operability and flexible programmability. Besides, CRISPR/Cas systems are amenable to combination with isothermal amplification techniques, primarily attributed to their compatibility and stability. Moreover, the combination of different CRISPR/Cas systems enables multiplex target detection. Therefore, CRISPR/Cas-based detection strategies have emerged as highly promising approaches for the sensitive, specific and multiplex detection of tumor biomarkers. In this review, we at first introduced the classification and working mechanisms of CRISPR/Cas systems. And then, we comprehensively summarized recently developed CRISPR/Cas-based detection strategies for tumor biomarkers. Besides, we reviewed detection strategies based on CRISPR/Cas systems for multiplex tumor biomarker detection. Furthermore, the challenges and prospects of existing CRISPR/Cas-based detection strategies were thoroughly discussed.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d5ay01150g", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Yang K", "dirs": [ "C" ] }, "42138264": { "pmid": "42138264", "title": "Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis.", "abstract": "Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c03226", "ptypes": [ "Journal Article" ], "first_author": "Gao M", "dirs": [ "C" ] }, "41192497": { "pmid": "41192497", "title": "Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe.", "abstract": "CRISPR-based nucleic acid detection technologies have revolutionized infectious disease detection and environmental monitoring by leveraging RNA-DNA complementarity to enable rapid, precise, and cost-effective detection of targets. However, achieving multitarget detection in one tube still presents challenges that necessitate further research. Here, we develop a nucleic acid detection module based on the CRISPR-Cas12i system. Importantly, we find that Cas12i and AapCas12b exhibit opposite trans-cleavage preferences for asymmetrically phosphorothioate-modified single-strand DNA probes, enabling the development of an effective dual-target nucleic acid detection platform by combining these two Cas12 nucleases in one tube. Moreover, this dual-target detection platform exhibits high specificity and sensitivity in genotyping the nucleic acid targets of human papillomavirus (HPV) 16 and HPV18, as well as Influenza A virus (FluA) and Respiratory syncytial virus. Notably, combined with loop-mediated isothermal amplification, this platform achieves high detection rates for clinical samples (18/18 FluA and 18/18 GAPDH internal reference detection rate). Taken together, these results can broaden the application of CRISPR-based Cas12 proteins for multi-target nucleic acid detection in one tube.", "journal": "Journal of genetics and genomics = Yi chuan xue bao", "year": "2026", "doi": "10.1016/j.jgg.2025.10.007", "ptypes": [ "Journal Article" ], "first_author": "Wang X", "dirs": [ "C" ] }, "40680677": { "pmid": "40680677", "title": "CRISPR-based one-pot detection: A game-changer in nucleic acid analysis.", "abstract": "The CRISPR/Cas system, originally developed as a gene-editing tool, has rapidly emerged as a powerful platform for nucleic acid detection due to its remarkable specificity, programmability, and robust trans-cleavage activity. While conventional CRISPR-based assays typically require separate amplification and detection steps, introducing complexity, manual intervention, and contamination risks. Recent innovations have led to the development of one-pot detection strategies that integrate target amplification with Cas-mediated signal generation within a single reaction vessel. These streamlined systems not only reduce handling steps and turnaround time but also enhance sensitivity and enable multiplexed detection of low-abundance nucleic acid targets. As such, one-pot CRISPR diagnostics are poised to transform point-of-care testing, pathogen surveillance, and clinical diagnostics. In this review, we summarize the latest advances in one-pot CRISPR-based detection technologies, evaluate their current limitations, and discuss strategies for optimization. Future efforts should focus on refining reaction kinetics, improving reagent stability, reducing costs, and expanding the repertoire of detectable targets. Furthermore, integration with portable platforms, AI-assisted signal interpretation, and the discovery of novel Cas effectors will be pivotal in realizing the full potential of CRISPR-based one-pot systems in diverse real-world applications.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117786", "ptypes": [ "Journal Article", "Review" ], "first_author": "Li X", "dirs": [ "C" ] }, "41223903": { "pmid": "41223903", "title": "Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay.", "abstract": "Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel. This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi. A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature. The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability. The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.", "journal": "Journal of equine veterinary science", "year": "2025", "doi": "10.1016/j.jevs.2025.105732", "ptypes": [ "Journal Article" ], "first_author": "Alsultan A", "dirs": [ "C" ] }, "41161575": { "pmid": "41161575", "title": "CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.", "abstract": "Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.", "journal": "Molecular and biochemical parasitology", "year": "2025", "doi": "10.1016/j.molbiopara.2025.111708", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Verma R", "dirs": [ "C" ] }, "40593148": { "pmid": "40593148", "title": "Recent advances in CRISPR-based single-nucleotide fidelity diagnostics.", "abstract": "Accurate point-of-care (PoC) detection of single nucleotide variants (SNVs) can support rapid and cost-effective clinical decision-making in tasks such as diagnosing pathogenic genetic variants, identifying pathogen resistance, or tracing viral lineage differentiation. Traditional nucleic acid diagnostics involving PCR and sequencing lack PoC applicability. CRISPR-based diagnostics (CRISPRdx) offer the necessary operational simplicity and ability to integrate specific nucleic acid sequence detection with isothermal amplification. However, achieving single-nucleotide fidelity is not self-evident and often requires empirical optimization. This Review explores recent strategics aimed at refining CRISPRdx specificity for SNV detection including various ways of tactical guide RNA (gRNA) design, fine-tuned effector selection, and improved reaction conditions. While the approaches described here are functional and can be occasionally combined, they often require optimizations to support specific clinical aims. Looking ahead, leveraging computational and AI tools for gRNA design, and harnessing newly discovered CRISPR systems, will broaden applicability and improve precision detection of CRISPRdx in diverse clinical settings.", "journal": "Communications medicine", "year": "2025", "doi": "10.1038/s43856-025-00933-4", "ptypes": [ "Journal Article", "Review" ], "first_author": "Kohabir KAV", "dirs": [ "C" ] }, "40664141": { "pmid": "40664141", "title": "Single-tube Lambda exonuclease-mediated LbuCas13a detect of ssDNA for single-nucleotide polymorphisms genotyping.", "abstract": "Single-nucleotide polymorphisms (SNPs) are critically important in genetics, disease association studies, and personalized medicine because they affect gene function, phenotypes, and drug response. Traditional methods like sequencing and PCR are time-consuming and often lack robust single-base resolution. Recent studies have shown that the Cas13a enzyme from Leptotrichia buccalis (LbuCas13a) can recognize single-stranded DNA (ssDNA) with exceptional specificity. Building on this, we have developed a rapid, isothermal, one-tube SNP genotyping platform (termed SNP-Specific Enzyme-based Nucleic acid Sensor for Efficient detection SNP-SENSE) that integrates recombinase polymerase amplification (RPA), Lambda exonuclease treatment, and LbuCas13a detection. In this assay, RPA amplifies the target allele CYP2C19∗2 (rs4244285, c.681G > A) CYP2C19∗3 (rs4986893, c.636G > A) and CYP2C19∗17 (rs12248560, c.806C > T), Lambda exonuclease converts the phosphorylated RPA product into ssDNA, and LbuCas13a-crRNA complexes cleave a fluorescent RNA reporter only in the presence of the perfectly matched target. This workflow operates at a constant temperature (∼39 °C) and completes in about 30 min. The method achieves single-nucleotide specificity, accurately distinguishing wild-type, heterozygous, and mutant genotypes. In tests with human whole blood samples, SNP-SENSE genotyped CYP2C19 alleles with 100 % concordance to Sanger sequencing. This single-pot platform offers a fast and precise tool for point-of-care genetic testing, supporting personalized treatment strategies and advancing precision medicine.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117760", "ptypes": [ "Journal Article" ], "first_author": "Luo S", "dirs": [ "C" ] }, "39890862": { "pmid": "39890862", "title": "Room temperature CRISPR diagnostics for low-resource settings.", "abstract": "Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-86373-5", "ptypes": [ "Journal Article" ], "first_author": "Nalefski EA", "dirs": [ "C" ] }, "39706401": { "pmid": "39706401", "title": "CRISPR-Cas12a2-based rapid and sensitive detection system for target nucleic acid.", "abstract": "Infectious diseases are extremely important public health issues, where the design of effective, rapid, and convenient detection platforms is critical. In this study, we coupled SuCas12a2, a novel Cas12 family RNA-targeting nuclease, with conventional PCR and recombinase polymerase amplification (RPA), respectively, to develop novel detection approaches, named PCR-SuCas12a2 and RPA-SuCas12a2. SuCas12a2 possesses collateral cleavage activity and cuts the additional single-stranded RNA (ssRNA) added to the reaction system once the ternary complex RNA-SuCas12a2-CRISPR RNA (crRNA) is formed. SuCas12a2 is specifically activated, where the cleaved fluorescent-labeled probes release fluorescent signals, with the strength of the fluorescent signal being proportional to the concentration of nucleic acids specifically bound to crRNA. Simultaneous transcription and SuCas12a2 detection can be performed in a single tube by introducing the T7 promoter sequence into the forward primer. Entamoeba histolytica (E. histolytica) and Mycoplasma pneumoniae (M. pneumoniae) were used as proof specimens to evaluate the performance of the platform. PCR-SuCas12a2 has excellent capabilities, including high specificity with no cross-reactivity from other species and ultra-sensitivity that achieves a detection of one copy per reaction for E. histolytica and M. pneumoniae. However, the sensitivity of the RPA-SuCas12a2 assay was 102 copies per reaction, which was inferior to PCR-SuCas12a2. Clinical samples were obtained from suspected infection patients of E. histolytica and M. pneumoniae, and used to evaluate the systems demonstrated 100 % specificity. The technique shows robust performance and suggests great potential for point-of-care testing of other pathogens to facilitate effective management and control of the spread of diseases.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2024.138996", "ptypes": [ "Journal Article" ], "first_author": "Yu H", "dirs": [ "C" ] }, "40687858": { "pmid": "40687858", "title": "Progress in the application of isothermal amplification technology in the diagnosis of infectious diseases.", "abstract": "Rapid detection of infectious diseases is critical for global public health prevention and control. However, the use of traditional molecular diagnostic methods, including PCR, has been limited because of their cumbersome procedures, complex equipment requirements, operation at different temperatures, and the level of expertise required for operation. Isothermal amplification technology (IAT) provides a rapid, sensitive, specific, simple and less costly method for diagnosing infectious diseases, which has led to revolutionary breakthroughs in molecular diagnostics. This paper summarizes recent progress in IAT technology, which focuses on the principles and applications of core technologies such as NASBA, LAMP, RPA, and RAA. In addition, the combination of IATs with the CRISPR/Cas system, which further revolutionizes nucleic acid detection technology, is explored in this review.", "journal": "Frontiers in microbiology", "year": "2025", "doi": "10.3389/fmicb.2025.1601644", "ptypes": [ "Journal Article", "Review" ], "first_author": "Bi Q", "dirs": [ "C" ] }, "40884799": { "pmid": "40884799", "title": "Engineered crRNA Drives RPA-T7-CRISPR/Cas14a Cascade for Ultrasensitive Detection of ctDNA PIK3CA H1047R.", "abstract": "The early detection of circulating tumor DNA (ctDNA) at mutant allele frequencies below 0.1% remains a critical challenge, significantly impeding therapeutic decision-making. To address this limitation, TIDE-Cas14a-an innovative CRISPR/Cas14a-based duplex detection system is developed that integrates recombinase polymerase amplification (RPA) with T7 exonuclease-mediated strand displacement. By strategically engineering crRNAs with synthetic mismatches, the platform achieves single-nucleotide resolution, enabling specific discrimination of the PIK3CA H1047R (c.3140A>G) variant from other mutant subtypes and wild-type sequences at a detection limit of 0.01% with attomolar sensitivity. The system leverages T7 exonuclease's 5'→3' digestion to convert RPA amplicons into single-stranded targets, thereby activating Cas14a without requiring thermal cycling. Furthermore, clinical validation using 32 breast cancer patient samples demonstrated that TIDE-Cas14a achieves 100% sensitivity and specificity, comparable to droplet digital PCR. When deployed on a low-cost digital microfluidic chip, the assay completes ctDNA profiling within 60 min at 37 °C, effectively bridging the gap between complex laboratory testing and point-of-care diagnostics. The work repurposes the CRISPR/Cas system's inherent specificity constraints as a precision oncology tool, establishing a scalable platform for early cancer detection and therapeutic monitoring.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2025", "doi": "10.1002/advs.202507126", "ptypes": [ "Journal Article" ], "first_author": "Yu Y", "dirs": [ "C" ] }, "40250151": { "pmid": "40250151", "title": "Point-of-need species identification using non-PCR DNA-based approaches to combat wildlife crime.", "abstract": "Wildlife crime, defined as any unlawful exploitation and trade of wildlife, is a lucrative illegal global industry, along with narcotics and weapons trafficking. It encompasses the harvest, transport, exchange, and end use of wildlife or wildlife-derived products. Regulated internationally by the Convention on the International Trade in Endangered Species of Flora and Fauna (CITES, 1973), wildlife crime is primarily detected using morphological or DNA sequencing methods. However, there is a growing demand for rapid, portable, and cost-effective screening tools to bypass time-consuming workflows and specialist laboratory equipment. Point-of-need testing, particularly at wildlife hotspots like international borders, offers a promising solution for the swift detection of illegal activities. Isothermal amplification methods such as loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and recombinase polymerase amplification (RPA), are favoured for their low resource needs compared to traditional PCR. These methods can be combined with target detection methods such as clustered regularly interspaced short palindromic repeats (CRISPR) and aptamers to enhance sensitivity. Integrating these methods with others, such as lateral flow assays (LFA) and microfluidic devices, simplifies sample preparation and visualisation. Already established in disease diagnosis and food safety, these innovations in genetic testing provide rapid, on-site detection. When applied to wildlife crime, they can serve as tools to complement traditional PCR and sequencing methods. This review explores how non-PCR based approaches could offer faster, simpler, and more cost-effective solutions to combat wildlife crime.", "journal": "Forensic science international. Genetics", "year": "2025", "doi": "10.1016/j.fsigen.2025.103278", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Yugovich O", "dirs": [ "C" ] }, "39725131": { "pmid": "39725131", "title": "CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.", "abstract": "Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.", "journal": "Clinica chimica acta; international journal of clinical chemistry", "year": "2025", "doi": "10.1016/j.cca.2024.120101", "ptypes": [ "Journal Article" ], "first_author": "Compiro P", "dirs": [ "C" ] }, "41435708": { "pmid": "41435708", "title": "RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio parahaemolyticus.", "abstract": "Vibrio parahaemolyticus is a major cause of seafood-associated gastroenteritis and aquatic animal diseases, posing persistent threats to public health and aquaculture. Rapid and accurate on-site quantitative detection is essential for risk assessment and early intervention. Although qPCR and digital PCR provide reliable quantification, their reliance on complex instrumentation limits field deployment. RPA-CRISPR-based isothermal assays offer a low-equipment alternative; however, existing approaches lack robust methodological strategies to achieve standard-curve-based quantitative reliability under field-deployable conditions. Here, we propose a methodological framework that enables in-run calibration and variance control for quantitative isothermal amplification by integrating reaction-volume locking and simultaneous standard-curve generation, implemented here within a closed centrifugal microfluidic system. A multi-unit microfluidic platform preloaded with gradient concentration standard plasmids allows concurrent construction of standard curves and sample analysis in a single run, reducing the impact of environmental and batch-to-batch variability. Signal generation was achieved using a one-pot RPA-CRISPR/Cas12a assay, in which balanced amplification and cleavage kinetics were obtained by screening crRNAs targeting suboptimal PAM sites and optimizing reaction conditions. The platform achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 100-104 copies/μL (R2 > 0.96), with performance comparable to qPCR (AUC = 0.984), and acceptable intra- and inter-assay variability under the tested conditions, with relative standard deviations of 2.63-6.07 %, at a cost of approximately $3.30 per test. Validation using spiked and real seafood samples demonstrated reliable on-site quantification. This work establishes a transferable quantitative methodology for RPA-CRISPR-based isothermal assays, advancing field-deployable pathogen detection in aquaculture and food safety.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2025.118327", "ptypes": [ "Journal Article" ], "first_author": "Fang J", "dirs": [ "C" ] }, "41754561": { "pmid": "41754561", "title": "CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.", "abstract": "Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.", "journal": "Viruses", "year": "2026", "doi": "10.3390/v18020218", "ptypes": [ "Journal Article", "Review" ], "first_author": "Wupori K", "dirs": [ "C" ] }, "41686849": { "pmid": "41686849", "title": "LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis.", "abstract": "Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the multicopy 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP). LAMP-coupled CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the L. (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, nor with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% (95% CI: 80.1-97.0%) for kDNA and 72.7% (95% CI: 59.0-83.9%) for 18S rDNA, both with 100% (95% CI: 90-100%) specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load. These new proof-of-concept LAMP-CRISPR assays, combining high sensitivity, multiple Leishmania species detection capability, and a portable lateral flow readout, hold promise as next-generation molecular tools to improve leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control.", "journal": "PLoS neglected tropical diseases", "year": "2026", "doi": "10.1371/journal.pntd.0013456", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Dueñas E", "dirs": [ "C" ] }, "42098301": { "pmid": "42098301", "title": "Ultrasensitive Detection and Its Potential Applications in the Diagnosis of Brucellosis.", "abstract": "Brucellosis is a major zoonotic disease caused by Brucella spp., notably B. melitensis, B. abortus, B. canis, and B. suis. It continues to impose a significant public health and economic burden in endemic regions, including parts of Asia, Africa, the Middle East, and Latin America. The clinical presentation of the disease is often nonspecific, and early, accurate diagnosis is further complicated by the susceptibility of conventional serological assays to cross-reactivity. This narrative review covers literature published between 2000 and 2025 regarding the detection of Brucella, indexed in PubMed, Web of Science, and China National Knowledge Infrastructure (CNKI). The review focuses on the limitations of traditional diagnostic methods and the performance characteristics and latest developments of emerging ultrasensitive detection technologies, including nucleic acid amplification (LAMP, RPA, SRCA), droplet digital PCR, CRISPR-Cas-based integrated systems, nanomaterial-enhanced assays, and protein/antigen-based biosensors. Their potential for integration into the One Health framework for zoonotic disease surveillance is also discussed.", "journal": "Current microbiology", "year": "2026", "doi": "10.1007/s00284-026-04941-y", "ptypes": [ "Journal Article", "Review" ], "first_author": "He L", "dirs": [ "C" ] }, "40871409": { "pmid": "40871409", "title": "Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic.", "abstract": "The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), catalyzed unprecedented innovation in molecular diagnostics to address critical gaps in rapid pathogen detection. Over the past five years, CRISPR-based systems, isothermal amplification techniques, and portable biosensors have emerged as transformative tools for nucleic acid detection, offering improvements in speed, sensitivity, and point-of-care applicability compared to conventional PCR. While numerous reviews have cataloged the technical specifications of these platforms, a critical gap remains in understanding the strategic and economic hurdles to their real-world implementation. This review provides a forward-looking analysis of the feasibility, scalability, and economic benefits of integrating these next-generation technologies into future pandemic-response pipelines. We synthesize advances in coronavirus-specific diagnostic platforms and attempt to highlight the need for their implementation as a cost-saving measure during surges in clinical demand. We evaluate the feasibility of translating these technologies-particularly CRISPR-Cas integration with recombinase polymerase amplification (RPA)-into robust first-line diagnostic pipelines for novel viral threats. By analyzing the evolution of diagnostic strategies during the COVID-19 era, we aim to provide strategic insights and new directions for developing and deploying effective detection platforms to better confront future viral pandemics.", "journal": "Microorganisms", "year": "2025", "doi": "10.3390/microorganisms13081905", "ptypes": [ "Journal Article", "Review" ], "first_author": "Papaneri A", "dirs": [ "C" ] }, "39287550": { "pmid": "39287550", "title": "Precise pathogen quantification by CRISPR-Cas: a sweet but tough nut to crack.", "abstract": "Pathogen detection is increasingly applied in medical diagnosis, food processing and safety, and environmental monitoring. Rapid, sensitive, and accurate pathogen quantification is the most critical prerequisite for assessing protocols and preventing risks. Among various methods evolved, those based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) have been developed as important pathogen detection strategies due to their distinct advantages of rapid target recognition, programmability, ultra-specificity, and potential for scalability of point-of-care testing (POCT). However, arguments and concerns on the quantitative capability of CRISPR-based strategies are ongoing. Herein, we systematically overview CRISPR-based pathogen quantification strategies according to the principles, properties, and application scenarios. Notably, we review future challenges and perspectives to address the of precise pathogen quantification by CRISPR-Cas. We hope the insights presented in this review will benefit development of CRISPR-based pathogen detection methods.", "journal": "Critical reviews in microbiology", "year": "2025", "doi": "10.1080/1040841X.2024.2404041", "ptypes": [ "Journal Article", "Review" ], "first_author": "Yao Z", "dirs": [ "C" ] }, "41292075": { "pmid": "41292075", "title": "Guanine-Quadruplex-Engineered crRNA Enables Light-Activated CRISPR/Cas12a System for Robust One-Pot Viral Assay.", "abstract": "Conventional one-pot detection platforms integrating CRISPR/Cas12a with isothermal amplification significantly streamline the nucleic acid detection workflow, while minimizing the risk of aerosol contamination. However, the intrinsic cleavage activity of the CRISPR/Cas12a system can substantially interfere with the nucleic acid amplification efficiency, ultimately compromising detection sensitivity. Herein, we develop a light-activated CRISPR/Cas12a system by engineering the crRNA with a guanine-quadruplex (G4) motif at its 3'-terminal, achieving precise regulation of Cas12a activity via photoswitching G4 structure formation. Through coupling with a recombinase polymerase amplification (RPA) reaction, we establish a one-pot detection platform that demonstrates superior detection performance compared to traditional Cas12a-based one-pot systems. The detection sensitivity has been improved by 2 orders of magnitude, reaching a level of 1 copy/μL. Notably, the platform demonstrated comparable sensitivity and specificity to PCR, the gold standard method, in detecting clinical samples, such as Epstein-Barr virus (EBV) and Influenza A virus (IAV), making it a promising technology for clinical diagnostics.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c04848", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Du J", "dirs": [ "C" ] }, "39923676": { "pmid": "39923676", "title": "LbCas12a-based DNA POCT facilitates fast genotyping on farm.", "abstract": "Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) detection system is now widely used for nucleic acid detection and disease diagnosis. However, there are still fewer detections for single nucleotide polymorphisms (SNPs) and limited diversified detection systems for pathogen and SNP sites detection, which greatly limits their applications. Obviously, the development of a more diversified and convenient suite of detection tools is essential to unlock the full potential of CRISPR/Cas12a technology and to expand its applications across a wider range of scenarios. We have successfully developed an integrated CRISPR/Cas12a assay system. This system introduces crRNA during protein expression, reducing the number of steps and reaction time by adding only a fluorescent reporter gene and target DNA during subsequent detection. It enables on-site visualization of the assay in combination with a Recombinase polymerase amplification (RPA) reaction. Combined with the RPA reaction, we are able to rapidly detect African swine fever virus (ASFV) pathogens with high specificity. The system also enables genotyping of the SNP site of the porcine prolificacy-associated estrogen receptor (ESR) gene and the sheep prolificacy-associated Fecundity booroola (FecB) gene. Visualization is possible up to a final concentration of 3 nM, and effective differentiation of low concentrations within the concentration range of the assay. The integrated CRISPR/Cas12a assay system we developed has a robust design that ensures high-fidelity genotyping and pathogen detection are no longer restricted to the lab, allowing for rapid field analysis, which is crucial for timely interventions in agricultural and clinical settings. In addition, it has the advantages of low cost, easy operation and visualization of results.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2025.127672", "ptypes": [ "Journal Article" ], "first_author": "Li X", "dirs": [ "C" ] }, "40527291": { "pmid": "40527291", "title": "Enhancement of CRISPR-Cas12a system through universal circular RNA design.", "abstract": "Precise control of Cas12a activity is crucial to address incompatibility in isothermal amplification-CRISPR-Cas12a one-pot nucleic acid detection. We developed a light-triggerable circular RNA system for dynamic LbCas12a regulation. By employing circular CRISPR guide RNA (crRNA) or a split circular universal direct repeat region with a replaceable spacer, we resolved the incompatibility between isothermal amplification and CRISPR detection. This system demonstrated robust performance in detecting trace nucleic acids in clinical samples. Furthermore, DNA modifications on circular crRNA enabled CRISPR-Cas12a regulation via base excision repair (BER) enzymes, offering potential for BER enzyme detection and modulation of LbCas12a cleavage activity by BER enzymes. This programmable strategy holds promise for selective gene editing in cells with elevated BER enzyme expression, such as uracil DNA glycosylase (UDG) in colon cancer cells. The circular RNA-assisted approach represents a resource-efficient method with significant potential for medical diagnostics and future clinical gene therapy applications.", "journal": "Cell reports methods", "year": "2025", "doi": "10.1016/j.crmeth.2025.101076", "ptypes": [ "Journal Article" ], "first_author": "Wang J", "dirs": [ "C" ] }, "39522277": { "pmid": "39522277", "title": "TtAgo-coupled-multiplex-digtal-RPA-CRISPR/Cas12a (TCMDC) for EGFR mutations detection.", "abstract": "Epidermal Growth Factor Receptor (EGFR) is an important target for the early evaluation, treatment, and postoperative follow-up in non-small cell lung cancer (NSCLC). Current detection technologies suffer from extended detection time and high rate of false positive amplification. Therefore, the development of rapid, highly sensitive and specific detection methods is of great significance for improving the diagnosis and treatment of lung cancer. In this study, we proposed a fast and sensitive detection method termed Thermus thermophilus Argonaute (Ttago)-Coupled-Multiplex-digital-recombinase polymerase amplification (RPA)-Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (TCMDC) detection method, integrating EGFR mutation template enrichment. Based on the cleavage principle of TtAgo, the wild type (WT) template was enriched under the action of double-guide DNA. Two CRISPR RNAs, not restricted by protospacer adjacent motif (PAM) sites, were introduced to target EGFR genes. By combining RPA with CRISPR-Cas12a, we established a single-pot, ultra-sensitive (1 copy, 0.1 %), and visually detectable method for EGFR detection. We further verified the feasibility of this approach using clinical serum samples from lung cancer patients, achieving rapid (within 1 h) and visual detection of EGFR, thereby presenting a promising clinical tool for the detection of lung cancer.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2024.127162", "ptypes": [ "Journal Article" ], "first_author": "Zhuang J", "dirs": [ "C" ] }, "40017424": { "pmid": "40017424", "title": "Deactivated Cas9-Engineered Magnetic Micromotors toward a Point-of-Care Digital Viral RNA Assay.", "abstract": "Digital nucleic acid assays, known for their high sensitivity and specificity, typically rely on fluorescent readouts and expensive and complex nanowell manufacturing, which constrain their broader use in point-of-care (POC) application. Here, we introduce an alternative digital molecular diagnostics, termed dCRISTOR, by seamlessly integrating deactivated Cas9 (dCas9)-engineered micromotors, extraction-free loop-mediated isothermal amplification (LAMP), low-cost bright field microscopy, and deep learning-enabled image processing. The micromotor, composed of a polystyrene sphere attached to a magnetic bead, incorporates a dCas9 ribonucleoprotein complex. The presence of human immunodeficiency virus-1 (HIV-1) RNA in a sample results in the formation of large-sized amplicons that can be specifically captured by the micromotors, reducing their velocity induced by an external magnetic field. The micromotor is propelled by an external magnetic field, which eliminates the need for chemical fuels, reducing system complexity, and allowing for precise control over micromotor movement, enhancing accuracy and reliability. A convolutional neural network classification-based multiobject tracking algorithm, CNN-MOT, accurately measures the change in micromotor motion, facilitating the binary digital assay format (\"1\" or \"0\") for simplified result interpretation without user bias. Incorporating an extraction-free LAMP assay streamlines the dCRISTOR workflow, enabling qualitative HIV-1 detection in spiked plasma (n = 21) that demonstrates 100% sensitivity and specificity and achieves a limit of detection (LOD) of 0.96 copies/μL. The assay also achieved 100% correlation with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in clinical patient samples (n = 9). The dCRISTOR assay, a label-free digital nucleic acid testing system that eliminates the need for fluorescence readouts, absorbance measurements, or expensive manufacturing processes, represents a substantial advancement in digital viral RNA diagnostics.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.4c14913", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, N.I.H., Extramural" ], "first_author": "Song Y", "dirs": [ "C" ] }, "39397560": { "pmid": "39397560", "title": "CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.", "abstract": "Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.", "journal": "Foodborne pathogens and disease", "year": "2025", "doi": "10.1089/fpd.2024.0078", "ptypes": [ "Journal Article" ], "first_author": "Gou S", "dirs": [ "C" ] }, "40717612": { "pmid": "40717612", "title": "CRISPR-Cas based platforms for RNA detection: fundamentals and applications.", "abstract": "The detection of RNA biomarkers is crucial for diagnosing many urgent diseases such as infections and cancer. Conventional RNA detection techniques such as RT-PCR, LAMP, and microarrays are effective, but often face limitations in terms of speed, sensitivity, and equipment demands. In recent years, CRISPR/Cas systems have emerged as versatile platforms for RNA detection, which offer high specificity, programmability, and adaptability across a wide range of diagnostic applications. This review first categorizes different CRISPR-based RNA detection systems according to the CRISPR effectors employed, including Cas13, Cas12, Cas14, Cas9, and newly characterized enzymes such as Cas7-11 and Cas10, detailing their mechanisms of target recognition, cleavage activity, and signal generation. The CRISPR detection platforms are coupled with or without pre-amplification steps to meet the different sensitivity needs. Preamplification-based systems integrate CRISPR with methods like RT-PCR and isothermal amplification to enhance sensitivity. In parallel, preamplification-free strategies, such as split-crRNA or split-activator systems, are gaining attention for their balanced assay performance and simplicity, which are especially attractive for point-of-care (POC) settings. Then, the diagnostic applications of these technologies are explored across two major domains: infectious disease detection and cancer biomarker identification via miRNAs, demonstrating the clinical potential of CRISPR-based RNA detection platforms. In addition, we explore ongoing challenges such as improving sensitivity in amplification-free formats, and developing field-deployable, cost-effective systems. The review concludes by outlining emerging trends and future directions in CRISPR-based RNA diagnostics, emphasizing their transformative potential in clinical settings.", "journal": "Chemical communications (Cambridge, England)", "year": "2025", "doi": "10.1039/d5cc03257a", "ptypes": [ "Journal Article", "Review" ], "first_author": "Bagi M", "dirs": [ "C" ] }, "40256781": { "pmid": "40256781", "title": "Rapid molecular detection of Senecavirus A based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a.", "abstract": "Senecavirus A (SVA), an emerging vesicular pathogen, is responsible for porcine idiopathic vesicular disease (PIVD). This disease is closely associated with porcine vesicular disease and acute neonatal piglet mortality, presenting a substantial threat to the global swine industry. At present, the absence of effective drugs or vaccines for treating the disease makes accurate diagnosis of SVA of paramount importance for the effective prevention and control of the disease. In this study, we combined reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein12a (CRISPR/Cas12a) using a dual-labelled fluorescence quencher or fluorescent biotin single-stranded DNA reporter molecule to develop two rapid, reliable, and portable visual SVA assays: RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB. The two methods exhibited comparable detection limits, with 9.6 copies/μL achieved in 40 and 45 minutes, respectively. They did not cross-react with non-target nucleic acids extracted from other related viruses and showed high specificity for SVA RNA detection. Furthermore, the methods demonstrated satisfactory performance in detecting 69 porcine adventitious samples, with no significant differences from that of quantitative reverse transcription polymerase chain reaction (RT-qPCR). In summary, the RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB methods developed are promising for early detection and routine surveillance of porcine SVA in resource-poor areas.", "journal": "Frontiers in bioengineering and biotechnology", "year": "2025", "doi": "10.3389/fbioe.2025.1451125", "ptypes": [ "Journal Article" ], "first_author": "Jiang C", "dirs": [ "C" ] }, "41440302": { "pmid": "41440302", "title": "Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay.", "abstract": "Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.", "journal": "Biosensors", "year": "2025", "doi": "10.3390/bios15120821", "ptypes": [ "Journal Article" ], "first_author": "Chen J", "dirs": [ "C" ] }, "39900222": { "pmid": "39900222", "title": "Recombinase polymerase amplification technology for point-of-care diagnosis of neglected tropical diseases.", "abstract": "Recombinase Polymerase Amplification (RPA) technology significantly advances the diagnostics of neglected tropical diseases (NTDs), providing rapid, isothermal, and minimally preparative testing ideally suited for under-resourced countries. This review critically assesses the current applications, limitations, and potential of RPA for detecting a broad spectrum of NTD pathogens, including viruses, bacteria, helminths, and fungi. The ability of RPA to operate under constant temperature conditions without the need for complex thermal cycling facilitates rapid pathogen detection within minutes, enhancing its utility for decentralized point-of-care testing in remote and underserved regions. RPA, however, faces limitations, including the labor-intensive and costly validation of primer design, especially for multiplex assays, and a susceptibility to nonspecific amplification. These challenges highlight the need for continuous refinement to ensure reliable and consistent performance across diverse environmental conditions. Despite these constraints, the scalability of RPA assays and their compatibility with portable detection platforms make them well-suited for deployment in field settings without access to traditional laboratory infrastructure. This review emphasizes the transformative potential of RPA in NTD diagnostics, enhancing accessibility, precision, and timeliness of interventions, ultimately contributing to improved global public health outcomes.", "journal": "International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases", "year": "2025", "doi": "10.1016/j.ijid.2025.107831", "ptypes": [ "Journal Article", "Review" ], "first_author": "Hueso L", "dirs": [ "C" ] }, "41500045": { "pmid": "41500045", "title": "A droplet digital LAMP-based lab-on-a-disc system for multiplex allele-specific detection of tumor-derived DNA mutations.", "abstract": "The absence of accurate, rapid, fully automated, and cost-effective detection methods has hindered the clinical translation of tumor-derived DNA mutations as cancer biomarkers. Here, we develop a sample-to-answer platform, termed multiplexed droplet digital loop-mediated isothermal amplification-based lab-on-a-disc (mddLAMP-LoD), for rapid and allele-specific detection of cancer-associated mutations. The system seamlessly combines automated microfluidics, an improved allele-specific LAMP assay with mismatch-engineered primer design for single-nucleotide discrimination, and artificial intelligence-assisted droplet imaging to enable precise mutation quantification. Full automation is realized through a combination of microfluidic passive valves, a gelatin-based thermosensitive active valve, a static magnetic field-driven on-chip nucleic acid extraction module, and polysaccharide additives that preserve water-in-oil droplet stability for over 1.5 h under thermal amplification. The mddLAMP-LoD platform demonstrates superior performance in detecting multiplex PIK3CA point mutations, achieving detection limits at the attomolar level or as low as100 cells, with a linear range of 0-6362 copies/μL. When coupled with deep learning, it exhibits high sensitivity, specificity and reproducibility, showing strong concordance with commercial kits in the analyses of breast cancer tissue and plasma samples. Using a 0.1 % mutation threshold, PIK3CA mutation frequency in adjacent tissue effectively predicts breast cancer prognosis, while serum PIK3CA profiles correlate with cancer staging and track disease progression. We validated that the mddLAMP-LoD system provides rapid, ultrasensitive mutation profiling suited for intraoperative molecular diagnosis and point-of-care liquid biopsy, representing a significant advance toward accessible precision oncology.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2025.118367", "ptypes": [ "Journal Article" ], "first_author": "Lu H", "dirs": [ "C" ] }, "42661818": { "pmid": "42661818", "title": "Recombinase polymerase amplification: transforming rapid detection of infectious diseases.", "abstract": "Pathogens are the agents responsible for these infections, and they use various mechanisms to invade and evade host defenses, emphasizing the importance of early detection. Molecular techniques such as polymerase chain reaction (PCR) have become the gold standard for detecting viral and bacterial pathogens due to their high sensitivity, specificity, and well-established validation. However, PCR requires thermal cycling equipment, a stable power supply, and trained personnel, which may limit its accessibility in resource-constrained settings. Isothermal amplification techniques are promising alternatives to PCR by providing simplicity, speed, and portability. Among these, recombinase polymerase amplification is particularly notable for its versatility, rapidity, and efficiency in amplifying nucleic acids at constant temperatures. This review discusses pathogen invasion mechanisms and recent advances in molecular diagnostics, with particular emphasis on RPA. The principles of RPA are compared with PCR, highlighting key advantages such as rapid amplification (typically within 20 min), low-temperature operation, minimal instrumentation, and compatibility with point-of-care (POC) platforms. At the same time, limitations of RPA, including primer design complexity, potential non-specific amplification, and current cost considerations, are addressed. RPA represents a versatile and promising tool that complements existing molecular methods and holds significant potential for expanding early infectious disease detection, particularly in decentralized and low-resource environments. By enabling timely and accessible diagnostics, RPA contributes substantially to improved disease surveillance, control, and patient outcomes. Traditional detection methodologies, such as culturing, PCR, and isothermal amplification techniques, with a focus on RPA. Due to its simple procedure, short detection time, high sensitivity and specificity, and suitability for point-of-care testing in remote areas, RPA can be the best alternative for the detection of infectious diseases.", "journal": "Virusdisease", "year": "2026", "doi": "10.1007/s13337-026-00963-z", "ptypes": [ "Journal Article", "Review" ], "first_author": "Khan S", "dirs": [ "C" ] }, "41090382": { "pmid": "41090382", "title": "Designing a locally produced DENV-1 nucleic acid diagnostic for low resource regions with endemic disease.", "abstract": "Using recombinant techniques, to support local production and availability of active diagnostic ingedients (ADIs) in regions with endemic or neglected disease, a reverse transcriptase (RT) fusion construct is reported (Δ3R5-TGP-RT), having the same modular architecture (R5-fluorescent protein-functional enzyme) as reported previously by us. In tandem with the R5-mCherry-Bst this RT construct completes the enzyme set required for RT-LAMP mediated detection of Dengue. The introduction of the thermophilic green fluorescence protein TGP, allowed a heat purification step at 95 °C to be introduced into the workflow, simplifying purification. A 3 terminal amino acid (SSK) truncation of the naturally occurring silaffin R5 binding peptide (Δ3R5) enabled improved protein purification and loading on silica and good storage stabilisation (≥3 months). L-arginine triggered elution of enzyme from silica, immediately prior to use, enabled a soluble enzyme to be delivered directly into the RT-LAMP reactions, producing a clear solution and compatibility with turbidity detection of PPi released complexes that form insoluble Mg2P2O7. A unique primer set was designed for the detection of Dengue Serotype 1 RNA, using a primer set (CAB 3), targeting the RNA-dependent RNA polymerase (RdRp), part of the NS5 region. This was chosen to minimise primer dimer formation, thereby reducing false positives. Amplification was achieved with high sensitivity (10 copies), with turbidity analysis able to distinguish positive and negative reactions. The approach developed has the potential to widen the application of LAMP diagnostics for neglected diseases such as Dengue by removing the availability of biological reagents as a limiting factor.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d5ay00889a", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Roberts A", "dirs": [ "C" ] }, "42094518": { "pmid": "42094518", "title": "Suppressing Primer-Driven Nonspecific Amplification in LAMP Using TrueLAMP.", "abstract": "Loop-mediated isothermal amplification (LAMP) is widely used for rapid nucleic acid detection but is limited by primer-driven nonspecific amplification that produces false positives. We developed TrueLAMP, a colorimetric LAMP formulation containing an inhibitor that suppresses nonspecific amplification while preserving target-dependent reactions. Using SARS-CoV-2 as a model, TrueLAMP was evaluated across multiple primer sets. TrueLAMP eliminated amplification in no-template controls while maintaining robust target amplification, achieving 100% specificity across all no-template controls and, using the Lamb primer set as a representative benchmark, a limit of detection of 250 copies per reaction with 95% detection probability. Stable endpoint colorimetric signals were maintained over extended incubation periods, enabling flexible readout without time-critical monitoring. TrueLAMP is compatible with readily available equipment, including convection ovens and temperature-controlled smart mugs, supporting simplified and decentralized molecular testing workflows.", "journal": "bioRxiv : the preprint server for biology", "year": "2026", "doi": "10.1101/2025.10.23.684253", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Chen Z", "dirs": [ "C" ] }, "42549884": { "pmid": "42549884", "title": "Advancement of LAMP technologies for rapid and accurate diagnosis of infectious diseases.", "abstract": "SUMMARYLoop-mediated isothermal amplification (LAMP) is a type of isothermal nucleic acid amplification test (NAAT). Compared to more traditional NAATs, like polymerase chain reaction (PCR), LAMP is run at one consistent temperature, utilizes four to six primers, and uses Bst polymerase. Some advantages that are provided by these differences are that LAMP has lower equipment requirements, resiliency to common PCR inhibitors, and a rapid time to result. Unfortunately, some disadvantages are also present due to these differences, such as more challenging primer design, reduced multiplexing capability, and difficulty with quantitation. Due to its unique characteristics, LAMP has proven to be an attractive option for molecular diagnostics in resource-limited, point-of-care, and near-patient testing settings. However, its adoption in clinical settings remains limited compared to its widespread use in research. In this review, we cover various detection methods within LAMP, recent advancements including extraction, multiplexing, and quantification, as well as key clinical applications of LAMP from a syndromic perspective. We also explore the commercial potential of LAMP and how it can be leveraged in resource-constrained settings and clinical laboratories, especially where early, affordable, and rapid diagnosis is essential.", "journal": "Clinical microbiology reviews", "year": "2026", "doi": "10.1128/cmr.00079-24", "ptypes": [ "Journal Article", "Review" ], "first_author": "Chaffee R", "dirs": [ "C" ] }, "40886110": { "pmid": "40886110", "title": "Technical aspects of loop-mediated isothermal amplification (LAMP) assay in cancer.", "abstract": "Cancer is a significant health problem worldwide, emphasizing the need for new diagnostic techniques. Among various molecular techniques, loop-mediated isothermal amplification (LAMP) has gained growing interest due to its rapidity, sensitivity, and cost-effectiveness. This review discusses the use of LAMP to target genes as a biomarker for cancer detection. We performed an extensive literature search to identify relevant oncology studies on LAMP. We highlighted its working principles, advantages over conventional diagnostic methods, and potential limitations in clinical settings. LAMP has been reported to be an effective molecular diagnostic technique with tremendous improvements in speed, sensitivity, and affordability. Its potential as a diagnostic tool for cancer detection can provide a viable alternative to conventional diagnostic methods, particularly in low-resource environments. However, challenges such as primer design complexity, possibility of false-positive signals, and standardization issues present hindrances to clinical application. Further research and development are required for further refinement and integration into routine diagnostics. Furthermore, integrating LAMP with other molecular technologies and new platforms may render it increasingly useful in the clinical setting. Continuing research in this field is important for establishing the overall efficacy of LAMP in oncology.", "journal": "Expert review of molecular diagnostics", "year": "2025", "doi": "10.1080/14737159.2025.2552820", "ptypes": [ "Journal Article", "Review" ], "first_author": "Asefi N", "dirs": [ "C" ] }, "40487219": { "pmid": "40487219", "title": "A comprehensive guide to loop-mediated isothermal amplification, an emerging diagnostic tool for plant pathogenic fungi.", "abstract": "The detection and identification of plant pathogenic fungi are crucial for effective plant protection measures. In the past two decades, loop-mediated isothermal amplification (LAMP) has emerged as a simple and cost-efficient tool for plant disease diagnosis, overcoming many drawbacks of traditional and PCR-based methods. LAMP relies on efficient DNA synthesis at a constant temperature, eliminating the need for thermocycling equipment. It is typically more robust, specific, and sensitive than PCR. This literature review summarizes LAMP primer design, reaction protocol development, sensitivity and specificity testing, and result detection methods. We provide examples of how LAMP's advantages are exploited in disease diagnosis and survey its diverse applications in plant pathogenic fungi research. These applications include the detection, identification, and monitoring of plant pathogenic fungi; the replacement of culture-based methods; the detection of genetic regions associated with functional changes; and the detection of single nucleotide polymorphisms. A comprehensive list of available assays is also provided. Despite its shortcomings-including difficulties with primer design, risks of cross-contamination, and the potential for false positives-LAMP holds significant potential to gain widespread recognition and popularity in the study of plant pathogenic fungi.", "journal": "Frontiers in plant science", "year": "2025", "doi": "10.3389/fpls.2025.1568657", "ptypes": [ "Journal Article", "Review" ], "first_author": "Németh MZ", "dirs": [ "C" ] }, "42202397": { "pmid": "42202397", "title": "Universal primer-based RPA combined with parallel CRISPR/Cas12a decoding for rapid multi-species meat authentication.", "abstract": "Rapid meat authentication is critical for food safety. Here, we report a two-step assay employing universal primer-based RPA amplification followed by species-specific CRISPR/Cas12a detection for the identification of 11 common meat species. A single universal primer pair enables broad amplification, while specific crRNAs allow parallel single-target CRISPR reactions. The assay takes approximately 40 min at constant temperature, costing ∼$4 per test with dual fluorescence and lateral flow strips. High specificity without cross-reactivity was observed, and detection limits ranged from 100 to 104 copies/μL. In binary meat mixtures, the fluorescence assay achieved adulteration detection limits of 0.05-0.5% (w/w), while the lateral flow format showed 0.05-5% (w/w) depending on the species. The method was verified using commercially processed products. This laboratory-validated strategy simplifies primer design and provides a promising platform for the qualitative screening of multiple meat targets, while further validation is required to assess its field robustness.", "journal": "Food chemistry", "year": "2026", "doi": "10.1016/j.foodchem.2026.148905", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Han S", "dirs": [ "C" ] }, "40204470": { "pmid": "40204470", "title": "Development of primers for loop-mediated isothermal amplification for Mycoplasma canadense detection.", "abstract": "Bovine mycoplasma mastitis is highly transmittable and hard to treat by chemotherapy. It causes severe economic loss and is considered a major problem for milk production. Mycoplasma canadense is one of the causative agents of mycoplasma mastitis. A primer set to detect M. canadense was developed based on single nucleotide polymorphism-specific loop-mediated isothermal amplification. Using this primer set, 10 fg M. canadense DNA corresponding to the DNA amount of ~13 cells was detected within 40 min. Cross-reactivity with other bovine Mycoplasma spp., Acholeplasma laidlawii, and mastitis-related bacteria was not observed when ≤1 pg DNA was applied. These results would provide a basis for validating future experiments with spiked-milk and field samples for the development of rapid detection of M. canadense.", "journal": "The Journal of veterinary medical science", "year": "2025", "doi": "10.1292/jvms.24-0303", "ptypes": [ "Journal Article" ], "first_author": "Kusumoto A", "dirs": [ "C" ] }, "42804951": { "pmid": "42804951", "title": "One-step MCDA-CRISPR/Cas12b platform for point-of-care testing of Shewanella algae.", "abstract": "Shewanella algae (S. algae) is an emerging marine zoonotic pathogen capable of causing various infections with a high mortality rate. Existing detection methods for S. algae tend to be nonspecific, complicated, and time-consuming. Here, we developed a CRISPR-based detection method, named MCTOS (Multiple Cross Displacement Amplification CRISPR/Cas12b-based Testing in One-Step), for S. algae detection. Through pan-genome analysis of 193 S. algae genomes against 103 other Shewanella genomes, we identified a S. algae-specific gene designated SA, as the detection target. MCTOS combines MCDA for target pre-amplification and CRISPR/Cas12b-based cleavage for signal readout in one-pot at a constant temperature of 61 °C for 45 min. Including a 15 min rapid nucleic acid extraction step, the total assay time is 60 min with real-time fluorescence for results visualization, or 65 min with an additional 5 min lateral flow biosensor readout. The limit of detection was 10 copies, and no cross-reactivity was observed against 14 common non-S. algae templates. In evaluations of 50 isolates and 47 samples, MCTOS showed 100% (27/27) specificity and sensitivities of 98.6% (69/70) and 95.7% (67/70) for the real-time fluorescence instrument and lateral flow biosensor, respectively. Compared with recent one-pot CRISPR methods and the loop-mediated isothermal amplification method for S. algae detection, MCTOS simplifies primer design and achieves higher sensitivity. In summary, MCTOS is a simple, rapid, convenient, highly sensitive, and specific method for pathogen detection that can serve as a point-of-care testing tool for clinical diagnosis and epidemiological surveillance, and is especially suited for basic, field, and clinical laboratories.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2026.130672", "ptypes": [ "Journal Article" ], "first_author": "Wang X", "dirs": [ "C" ] }, "40256705": { "pmid": "40256705", "title": "Development of a loop-mediated isothermal amplification assay for detecting Porphyromonas endodontalis.", "abstract": "Loop-mediated isothermal amplification (LAMP) may be used in the future to detect infecting microorganisms. LAMP assays exist for the endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum, but not yet for Porphyromonas endodontalis. To develop a LAMP assay for detecting P. endodontalis. It was an in vitro benchtop study. The National Center for Biotechnology Information GenBank Basic Local Alignment Search Tool was used to identify a segment of the dipeptidyl peptidase 11 (DPP11) gene unique to P. endodontalis. A primer design tool was used to generate six primers required for developing the LAMP assay. WarmStart Colorimetric LAMP 2X Master Mix was used to evaluate the LAMP assay, using purified P. endodontalis DNA as a control. Statistical parameters for sensitivity and specificity. The assay was performed in triplicate on pure DNA from P. endodontalis and P. gingivalis and on the DNA that was extracted from P. endodontalis, P. gingivalis, F. nucleatum, and E. faecalis cells and diluted two-fold from 1/2 to 1/256. Assays for the diluted samples were performed in triplicate, and the contingency tables indicated the LAMP assay to be 82% sensitive and 90% specific for P. endodontalis. LAMP assay could be a highly sensitive and specific chairside detection method for P. endodontalis.", "journal": "Journal of conservative dentistry and endodontics", "year": "2025", "doi": "10.4103/JCDE.JCDE_859_24", "ptypes": [ "Journal Article" ], "first_author": "Whitaker EJ", "dirs": [ "C" ] }, "41877674": { "pmid": "41877674", "title": "Development and Application of a LAMP Assay for Detecting E198A-Type MBC-Resistant Clarireedia monteithiana.", "abstract": "The E198A (GAG→GCG) mutation at codon 198 of the β-tubulin gene confers high levels of resistance to methyl benzimidazole carbamate (MBC) fungicides in Clarireedia monteithiana worldwide. Early detection of E198A-type resistant populations is therefore essential for effective dollar spot management. In this study, we developed a rapid loop-mediated isothermal amplification (LAMP) assay for detecting MBC-resistant C. monteithiana. Optimal amplification occurred at 64°C for 60 min, and positive reactions were readily identified by a distinct purple-to-sky-blue color change using hydroxynaphthol blue as the indicator. The selected primer set specifically distinguished E198A isolates from wild-type isolates, F200Y mutants, and closely related fungal species. The assay exhibited a detection limit of 2 × 104 copies, reflecting a 100-fold higher sensitivity than conventional PCR. In host inoculation assays, the LAMP method achieved an accuracy of 95.83 to 96.78% across two experimental groups under near-field conditions, whereas PCR produced successful amplification for only 124 of 140 samples (88.57%). Collectively, these results demonstrate that the developed LAMP assay provides a rapid, sensitive, and robust tool for monitoring E198A-type MBC-resistant isolates and offers substantial value for the early detection and management of dollar spot disease.", "journal": "Plant disease", "year": "2026", "doi": "10.1094/PDIS-01-26-0214-RE", "ptypes": [ "Journal Article" ], "first_author": "Huang J", "dirs": [ "C" ] }, "40292363": { "pmid": "40292363", "title": "In silico prediction of loop-mediated isothermal amplification using a generalized linear model.", "abstract": "Loop-mediated isothermal amplification (LAMP), a DNA amplification technique under isothermal conditions, provides the important benefits of high sensitivity, specificity, rapidity, and simplicity. Maximizing LAMP features necessitates the design of a complex LAMP primer set (LPS) consisting of four primers for six regions of a given target DNA. Furthermore, the LPS of a given target DNA is designed with LPS design support software such as Primer Explorer. However, even if the design is completed, we still must do many in vitro experiments and evaluations. Consequently, designing LPS often fails to achieve high performance, including efficient amplification. For this study, we examined in silico LAMP: a generalized linear model to predict DNA amplification from LPS. Using logistic regression with elastic net regularization, we identified factors that strongly affect LPS design. These factors, combined with domain knowledge for LPS design, led to the creation of LAMP kernel variables that are highly essential for high LAMP reaction. In silico LAMP, constructed using logistic regression with LAMP kernel variables, allows classification and performance prediction of LPS with an area under the curve of 0.86. These results suggest that a high LAMP reaction can be predicted using LAMP kernel variables and generalized linear regression model. Moreover, an LPS with high performance can be constructed without in vitro experimentation.", "journal": "Synthetic biology (Oxford, England)", "year": "2025", "doi": "10.1093/synbio/ysaf007", "ptypes": [ "Journal Article" ], "first_author": "Taguchi K", "dirs": [ "C" ] }, "39945656": { "pmid": "39945656", "title": "Development of a Highly Multiplexed RT-LAMP Assay for Coverage of Genetic Sequence Diversity.", "abstract": "Nucleic acid amplification tests (NAATs) can achieve high accuracy for diagnosing infectious diseases by targeting conserved genetic sequences specific to the target organism. Isothermal NAATs, such as reverse-transcription loop mediated isothermal amplification (RT-LAMP), simplify instrumentation requirements, facilitating point-of-care testing. However, sequence variation due to genetic variability can cause false negative results. Single-pot multiplex testing can improve sequence coverage, but RT-LAMP is complicated by requiring many primers for even a single assay, which can lead to nonspecific amplification. We implemented a process that leveraged manual primer design to develop a highly multiplexed RT-LAMP assay (Chain LAMP) targeting 7 adjacent genomic target regions of HIV, one of the most diverse clinically relevant pathogens. This process departed from standards for RT-LAMP design, including the omission of bumper primers whose activity was replaced by cooperative neighboring assays. The Chain LAMP is, to our knowledge, the highest order single-pot multiplexed RT-LAMP assay published. The assay has an analytical limit of detection of 25 copies of RNA/reaction without detectable nonspecific amplification, translating to 1000 copies of HIV/mL of plasma from a fingerstick sample, aligning with WHO standards for HIV viral load monitoring. When evaluated using 24 clinical RNA samples representative of global HIV diversity, Chain LAMP demonstrated robust coverage of sequence diversity, amplifying all samples with minimal sensitivity variation. We performed mechanistic analysis with Nanopore sequencing, identifying liftoff of multiple assay regions for each sample, indicating many initiation loci. The high level of multiplexing in the Chain LAMP effectively increases the coverage of HIV sequence diversity.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.4c05583", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Gilligan-Steinberg SD", "dirs": [ "C" ] }, "41097019": { "pmid": "41097019", "title": "CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.", "abstract": "Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 104 copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms26199754", "ptypes": [ "Journal Article" ], "first_author": "Kapitonova MA", "dirs": [ "C" ] }, "41018873": { "pmid": "41018873", "title": "Design, Optimization, and Analytical Performance Evaluation of LAMP-Based Rapid Detection Assay for Severe Fever with Thrombocytopenia Syndrome Virus.", "abstract": "Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), a tick-borne pathogen, presents a growing public health threat in East Asia. Although conventional RT-PCR methods are effective for detection, they are limited by the need for specialized equipment and time-consuming procedures. This study aimed to develop and evaluate a rapid, sensitive, and field-deployable diagnostic method for SFTSV using Loop-Mediated Isothermal Amplification (LAMP). Twelve sets of LAMP primers were designed to target the RNA-dependent RNA polymerase (L segment) of the SFTSV genome. These primers were screened through stepwise colorimetric LAMP assays to identify the optimal set. The sensitivity and specificity of the selected primer set were evaluated using serial dilutions of SFTSV RNA and a panel of control pathogens. The selected LAMP primer set demonstrated high amplification efficiency, successfully detecting as little as 10 attograms (ag) of SFTSV RNA. Moreover, the primer set showed no cross-reactivity with non-SFTSV samples, confirming its high specificity. The developed LAMP assay provides a rapid and reliable method for SFTSV detection, with potential for use in field settings. This diagnostic tool could enhance early detection and improve outbreak response for SFTSV infections.", "journal": "Journal of pharmacopuncture", "year": "2025", "doi": "10.3831/KPI.2025.28.3.240", "ptypes": [ "Journal Article" ], "first_author": "Na HH", "dirs": [ "C" ] }, "41593500": { "pmid": "41593500", "title": "Construction of an RPA-CRISPR visualization system for rapid detection of Burkholderia mallei.", "abstract": "BACKGROUND: Glanders is a chronic zoonosis caused by Burkholderia mallei (BM). The main sources of infection are infected horses, mules, donkeys and other single-hoofed equine animals. There is currently no commercial vaccine for glanders, so the mortality rate of animals with this disease is extremely high. The prevalence of the disease is due mainly to improper border trade quarantine, so early diagnosis of the disease is crucial for epidemic prevention and control. RESULTS: In this study, a one-step isothermal amplification detection method based on RPA and CRISPR was developed for BM targets. Through sgRNA design and verification and RPA primer design and screening, BM-sgRNA-3 and BM-RPA-F2/R1 were selected for the construction of the test system. The constructed RPA-CRISPR detection system has a detection limit of 1E1 copies/T and good specificity. On this basis, one-step visualization and test strip detection methods were further established. The results showed that the RPA-CRISPR one-step visualization process can achieve stable detection at a concentration of 1E1 copies/T, and the sensitivity of the RPA-CRISPR test strip detection system can reach 1E2 copies/T. CONCLUSIONS: This study established a one-step RPA–CRISPR assay specifically targeting Burkholderia mallei and translated it into both fluorescence-based and lateral flow formats. The assay demonstrated high analytical sensitivity and specificity, and its simple workflow and visual readout make it suitable for routine diagnosis and on-site screening of glanders.", "journal": "BMC microbiology", "year": "2026", "doi": "10.1186/s12866-026-04752-y", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Zhang Y", "dirs": [ "C" ] }, "42524368": { "pmid": "42524368", "title": "DISCO-LAMP: a novel discontinuous LAMP assay for isothermal antigen detection.", "abstract": "Proximity ligation assay (PLA) is a highly sensitive protein detection method in which DNA probes conjugated to affinity reagents are brought into proximity upon binding the same target molecule, thereby enabling greatly accelerated ligation of the DNA strands. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel discontinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent DNA fragment is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the full-length DNA while maintaining minimal background amplification at 10 nM of each individual DNA fragment. When functionalized with specific affinity reagents, DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM as well as both Wuhan-1 and Omicron spike protein using a newly engineered Designed Ankyrin Repeat Protein (DARPin) DS16 targeting a conserved epitope on the SARS-CoV-2 spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based technology that is readily adaptable for sensing diverse targets via PLA.", "journal": "Sensors & diagnostics", "year": "2026", "doi": "10.1039/d6sd00073h", "ptypes": [ "Journal Article" ], "first_author": "Thomas BM", "dirs": [ "C" ] }, "41577412": { "pmid": "41577412", "title": "Recombinase-aided amplification technology: A comprehensive review of innovations and multi-technology integration for rapid infectious disease diagnosis.", "abstract": "Recombinase-Aided Amplification (RAA), as an emerging isothermal nucleic acid amplification technique, demonstrates significant potential in infectious disease diagnostics due to its advantages of rapidity, high efficiency, and reduced reliance on complex instrumentation. Amid frequent global outbreaks of infectious diseases, the demand for rapid and accurate diagnostic technologies has intensified. RAA, leveraging its unique amplification mechanism and application flexibility, has garnered increasing research interest. Compared to other amplification techniques, RAA exhibits shorter reaction times and higher sensitivity, enabling the detection of diverse pathogens including viruses, bacteria, fungi, and parasites. Furthermore, the integration of RAA with techniques such as CRISPR and microfluidics provides innovative strategies for point-of-care testing. However, current RAA implementation faces industrial bottlenecks such as low primer design tolerance, nonspecific amplification due to sample matrix interference, and lack of standardized frameworks. This review aims to summarize the fundamental principles of RAA technology, its current applications in infectious disease diagnosis, synergistic innovations with complementary methods, and future development trajectories, thereby offering theoretical insights for the optimization and advancement of rapid diagnostic technologies.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2025.344932", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Xie M", "dirs": [ "C" ] }, "42055489": { "pmid": "42055489", "title": "Methylation-sensitive LAMP (MS-LAMP): assay design, readouts, and clinical translation in microbiology.", "abstract": "Loop-mediated isothermal amplification (LAMP) is widely used when rapid nucleic acid detection with minimal instrumentation is required. This review focuses on methylation-sensitive LAMP (MS-LAMP), a narrower and still underdeveloped extension in which methylation status is converted into an amplification outcome. We concentrate on microbiology-relevant applications and discuss the two principal gating routes currently used: bisulfite conversion followed by methylation-discriminating primer design, and methylation-sensitive restriction enzyme digestion before amplification. Rather than reviewing LAMP broadly, we examine how methylation gating reshapes assay design, readout selection, upstream quality control, and result interpretation. We also distinguish direct MS-LAMP evidence from the larger supporting literature on microbial methylomes and general LAMP engineering. Current evidence suggests that the main translational bottlenecks lie upstream of amplification, particularly in template damage during bisulfite treatment, digestion specificity, control design, and performance in complex clinical matrices. We conclude that MS-LAMP remains promising but methodologically immature for routine microbiology, and that progress will depend on robust locus selection, process controls, predefined thresholds, and validation across clinically relevant specimen types.", "journal": "Journal of microbiological methods", "year": "2026", "doi": "10.1016/j.mimet.2026.107520", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sahu TK", "dirs": [ "C" ] }, "40427583": { "pmid": "40427583", "title": "Modeling a Standard Loop-Mediated Isothermal Amplification Reaction and Its Modification Involving Additional Inner Primers.", "abstract": "Loop-mediated isothermal amplification (LAMP) was developed a quarter of a century ago, but it is still not exactly clear how this reaction proceeds. Only a few articles have focused on the kinetics of LAMP and the types of products formed. In this work, 10 types were identified and named. A basic dumbbell structure, Z6_dmb(1), consists of six zones and triggers the LAMP cycle. Due to self-priming, Z6_dmb(1) transforms into hairpin structure Z9_hp(1) and then into linearized strand Z9_li(1), carrying also strand Z6_dmb(2). Through similar transformations, it again generates strand Z6_dmb(1), completing the first LAMP cycle and starting a new one. The next stage of the exponential phase starts from two Z15_hp hairpin structures generated in the LAMP cycle, which next turn into Z15_li → Z27_hp → Z27_li → Z51_hp → and so forth. Modeling of a new type of the reaction, namely, pseudo-hemi-nested LAMP (phn-LAMP), was carried out. phn-LAMP involves three inner primers: two forward (FIP and extraFIP) and one backward inner primer, or vice versa. phn-LAMP has an advantage over LAMP involving loop or stem primers and over MIP-LAMP (multiple inner primers).", "journal": "Biomolecules", "year": "2025", "doi": "10.3390/biom15050690", "ptypes": [ "Journal Article" ], "first_author": "Akhmetzianova LU", "dirs": [ "C" ] }, "40905700": { "pmid": "40905700", "title": "A Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Salmonella Ser. Typhimurium in Egg Products.", "abstract": "As a leading cause of foodborne illness worldwide, detection of Salmonella enterica subsp. enterica serovar Typhimurium is essential for food safety and public health. This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella ser. Typhimurium in egg products. The primer set targeting the open reading frame STM3845 of Salmonella ser. Typhimurium was designed using PrimerExplorer v.4. The LAMP assay was optimized by adjusting reagent concentrations, reaction temperature, and incubation time, achieving the highest amplification/fluorescence in a 25.0 µL reaction at 65°C for 30 min. Results indicated that the newly designed assay could successfully detect and differentiate Salmonella ser. Typhimurium from other Salmonella serotypes and non-Salmonella bacterial pathogens except for Salmonella serotypes Montevideo, Michigan, and Senftenberg after testing 73 Salmonella ser. Typhimurium, 100 non-Typhimurium Salmonella, and 35 non-Salmonella bacterial pathogens of pure cultures. The LAMP assay was further compared with a commercial real-time PCR and FDA BAM culture method by testing pure culture and 200 inoculated (1-5 CFU/25g) egg and egg product samples, and proved to be comparable to the FDA BAM culture method; it also demonstrated 100 times more sensitivity than the real-time PCR assay in pure culture testing, with a detection limit of 0.56 log CFU/mL. The newly developed LAMP assay offers a rapid, specific, and sensitive method for detecting Salmonella ser. Typhimurium in egg products. Its simplicity, speed, and sensitivity position it as a powerful tool for routine monitoring, outbreak investigation, and on-site testing in the food industry. Developed a LAMP assay for specific detection Salmonella ser. Typhimurium in egg products. The newly developed LAMP assay was 100 times more sensitive than the real-time PCR assay. Our new LAMP assay was validated with hundreds of pure isolates and food samples.", "journal": "Journal of AOAC International", "year": "2025", "doi": "10.1093/jaoacint/qsaf078", "ptypes": [ "Journal Article" ], "first_author": "Hu L", "dirs": [ "C" ] }, "41490282": { "pmid": "41490282", "title": "Genome mining algorithm for identifying identical repeat sequences to enhance DNA-based diagnostic assays.", "abstract": "A pair of primers that can bind at multiple loci across the genome and randomly amplify multiple copies increases the analytical sensitivity of the currently used diagnostic assays. We developed a novel genome mining algorithm to identify short identical repeat sequences (IRSs) dispersed across the genome. The genome mining algorithm for IRS identification can be accessed from the GitHub portal (https://github.com/BPaul-bioinfoLAB/IRS-Finder). Using this algorithm, we have identified the IRS from five pathogens, namely, human gammaherpesvirus, vaccinia virus, Mycobacterium tuberculosis, Plasmodium falciparum, and Phytophthora palmivora. In silico PCR revealed that these IRSs can amplify multiple nonhomologous regions of variable amplicon sizes via three priming combinations. We further performed a polymerase chain reaction (PCR) assay with an IRS pair identified in M. tuberculosis. Interestingly, the PCR with single IRS amplified multiple nonhomologous copies and even more variable-sized copies in pair. These results indicate that the IRS-based diagnostic assays can detect pathogens in case of low-concentration DNA during disease progression. The genome mining algorithm can be used as a translation technology platform for developing highly sensitive varieties of PCR, microarray, loop-mediated isothermal amplification, and fluorescence in situ hybridization-based diagnostic assays.", "journal": "Genome", "year": "2026", "doi": "10.1139/gen-2025-0027", "ptypes": [ "Journal Article" ], "first_author": "Rajeswari K", "dirs": [ "C" ] }, "41581303": { "pmid": "41581303", "title": "Restriction Endonuclease-Assisted Recombinase Polymerase Amplification: Application towards the Detection of ctDNA.", "abstract": "Circulating tumor DNA (ctDNA) represents an invaluable liquid biomarker with significant applications in early cancer screening, therapeutic efficacy evaluation, and recurrence monitoring. However, ctDNA detection faces some challenges: low abundance in plasma cell-free DNA (cfDNA), difficulty in distinguishing mutant from wild-type signals using conventional methods, and loss of short fragments due to length heterogeneity. To address these biological challenges, we developed Restriction Endonuclease-assisted Recombinase Polymerase Amplification (REA-RPA). Its core advantage is the continuous, specific degradation of wild-type sequences during amplification, which reduces false positive risks by minimizing non-specific signals and enhances detection sensitivity via mutant target enrichment. Based on REA-RPA, we developed a one-pot real-time fluorescent strategy with genotyping probes to suppress residual wild-type signals. We also extended REA-RPA to a primerless system, utilizing wild-type digestion products as endogenous primers. This approach overcomes conventional primer design constraints and detects ctDNA fragments of varying lengths. Using the APC c.4012C > T mutation as a proof-of-concept, our results demonstrated that the one-pot real-time fluorescent detection strategy can identify mutations abundance as low as 0.01 %, while the primerless genotype switching strategy is capable of detecting mutations at a 0.1 % abundance. These findings highlight the exceptional sensitivity and accuracy of REA-RPA, and underscore its translational potential in clinical applications such as early cancer detection and treatment monitoring.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2026.129445", "ptypes": [ "Journal Article" ], "first_author": "Song Z", "dirs": [ "C" ] }, "42401475": { "pmid": "42401475", "title": "A dual-modal RPA-CRISPR/Cas12a biosensor for rapid and ultrasensitive detection of Staphylococcus aureus in bloodstream infections.", "abstract": "Bloodstream infections (BSIs) caused by Staphylococcus aureus (S. aureus) require rapid and accurate diagnosis to guide effective antimicrobial therapy and improve patient outcomes. However, current diagnostic methods often struggle to balance speed, sensitivity, portability, and cost. After systematic optimization of reaction parameters (including primer design, reaction temperature and time, buffer composition, and probe concentration), the fluorescence assay demonstrated high specificity and completed detection within 35 min. Under optimized conditions (1.0 μM HS-ssDNA-MB and 10 min CRISPR incubation), the electrochemical sensor achieved a detection limit of 138 copies/mL, with a wide linear dynamic range from 4.37 × 100 to 105copies/μL, and showed high specificity against non-target pathogens. Furthermore, the platform demonstrated reliable performance in spiked artificial blood samples, with recovery rates ranging from 100.7% to 107.3%. This dual-modal recombinase polymerase amplification (RPA)-CRISPR/Cas12a biosensor combines the rapid amplification capability of isothermal methods with the high specificity of CRISPR-based detection. This platform provides both a high-throughput fluorescence detection mode and a portable, low-cost electrochemical detection mode, offering an efficient and flexible solution for the rapid point-of-care diagnosis and large-scale screening of BSIs caused by S. aureus, with promising potential for clinical translation.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345800", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Luo G", "dirs": [ "C" ] }, "42887808": { "pmid": "42887808", "title": "Development and Evaluation of an RPA-CRISPR/Cas12a Molecular Assay for Rapid Diagnosis of Theileria annulata Infection in Bovines.", "abstract": "Tropical theileriosis, caused by Theileria annulata, continues to be the most significant tick-borne disease affecting cattle, particularly in endemic regions where it leads to significant production losses. Prompt and reliable diagnosis is critically important for timely treatment and disease control; however, conventional diagnostic methods have important limitations. Microscopic examination lacks sensitivity, particularly in carrier animals, while PCR requires specialized laboratory infrastructure. To overcome these limitations, a fluorescence-based RPA-CRISPR/Cas12a assay targeting the multicopy mitochondrial cytb gene was developed for rapid detection of T. annulata. Five RPA primer pairs were designed and evaluated, and primer set-5, generating a 324-bp amplicon, exhibited the best amplification performance. A CRISPR RNA (crRNA) was designed within the selected amplicon to enable specific Cas12a-mediated target recognition. The assay was completed within 50 min and demonstrated no cross-reactivity with DNA from Theileria orientalis, Babesia bigemina, Babesia bovis, Anaplasma marginale, and bovine host DNA, confirming excellent analytical specificity. Sensitivity analysis using recombinant plasmid DNA demonstrated a detection limit of approximately ten copies per reaction. The field evaluation of the assay was done by testing 206 bovine field samples. showed that the developed assay identified 163 positive samples, exceeding the detection rates obtained by PCR (151 positives) and microscopy (116 positives). On comparison with PCR, the assay achieved 100% diagnostic sensitivity, 78.18% specificity, 92.64% positive predictive value, 100% negative predictive value, with an overall agreement of 94.17%. These findings indicate that the RPA-CRISPR/Cas12a platform is a rapid, sensitive, and accurate molecular diagnostic approach with strong potential for field application and point-of-care diagnosis of bovine tropical theileriosis, particularly in resource-constrained settings.", "journal": "Acta tropica", "year": "2026", "doi": "10.1016/j.actatropica.2026.108371", "ptypes": [ "Journal Article" ], "first_author": "Mondal A", "dirs": [ "C" ] }, "41557543": { "pmid": "41557543", "title": "Development and evaluation of loop-mediated isothermal amplification (lamp) for rapid detection of campylobacter jejuni.", "abstract": "To develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Campylobacter jejuni in stool samples associated with acute diarrheal disease (ADD) and Guillain-Barré syndrome (GBS) for implementation in regional laboratories and primary health care centers. Four sets of LAMP primers for C. jejuni were designed and initially evaluated with DNA from C. jejuni, Campylobacter coli, Salmonella Infantis, and Escherichia coli extracted using a commercial kit. The best-performing primer set was selected for assay optimization, determination of the limit of detection, and analytical validation with DNA from the aforementioned strains. Subsequently, clinical validation was performed using stool samples from patients with suspected ADD due to C. jejuni, applying the same standardized conditions used for the strain DNA samples. The sensitivity and specificity of the test were determined using real-time PCR (qPCR) as the standard. The LAMP assay for the detection of C. jejuni in stool samples using a primer set for the cdtC region showed a sensitivity of 97% (CI: 81.49-99.83%), a specificity of 93% (CI: 75.79-98.80%), a positive predictive value of 94% (CI: 77.78-98.91%), and a negative predictive value of 96% (CI: 79.76-99.81%). Furthermore, a strong agreement was demonstrated between this LAMP assay and qPCR (k=0.9). The LAMP assay based on the cdtC gene is a reliable and rapid method, with high sensitivity and specificity comparable to qPCR for the identification of C. jejuni associated with ADD and GBS, especially in low-resource areas, due to its low cost and ease of implementation.", "journal": "Revista peruana de medicina experimental y salud publica", "year": "2025", "doi": "10.17843/rpmesp.2025.423.14501", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Caro-Castro J", "dirs": [ "C" ] }, "42507508": { "pmid": "42507508", "title": "Advanced next-generation vaccines for dengue and other flaviviral infections.", "abstract": "Notifiable flaviviral pathogens, particularly dengue, continue to pose significant health and economic challenges worldwide, especially in tropical regions where these infections remain endemic. In the Philippines, the risk remains high following the 2019 withdrawal of the Dengvaxia vaccine, with no alternative immunization currently available locally. Recent interest in nucleic acid-based immunogens highlights the need for scalable, non-infectious vaccine production platforms; however, generating high-dose DNA constructs capable of inducing effective immune responses without live-virus propagation remains challenging. Our work from 2018 to 2024 addressed this need through the development of computationally designed, cell-free, PCR-amplifiable DNA constructs using algorithmic overlap extension PCR (OE-PCR) and an improved technique, asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). Subsequent integration of machine learning, including recurrent neural network-based amplification prediction and LAMP primer design, further improved synthesis efficiency and expanded applications for surveillance and diagnostics. The AESOE-guided OE-PCR platform enabled the assembly of synthetic flaviviral DNA fragments of at least 1 kilobase in length from overlapping oligonucleotides, supporting scalable production of synthetic immunogens without the need for infectious viral materials. These live-virus-free strategies may enhance biosafety, reduce development timelines, and improve accessibility for laboratories in resource-limited settings, potentially supporting next-generation vaccine development for dengue and other flaviviral diseases.", "journal": "Journal of vector borne diseases", "year": "2026", "doi": "10.4103/jvbd.jvbd_319_25", "ptypes": [ "Journal Article" ], "first_author": "Camer GA", "dirs": [ "C" ] }, "42861960": { "pmid": "42861960", "title": "Five ways to find a parasite: comparison of five molecular assays for the detection of Strongyloides spp.", "abstract": "Strongyloidiasis, caused by nematodes of the genus Strongyloides, is a neglected tropical disease with significant public health implications. Accurate diagnostics are essential for disease surveillance and control, yet traditional stool-based and serological methods have limitations in sensitivity and specificity. Molecular assays offer an alternative diagnostic approach. In this study, we compared four probe-based real-time PCR (qPCR) assays and one loop-mediated isothermal amplification (LAMP) assay using human fecal DNA samples from Gabon and Papua New Guinea with both Strongyloides stercoralis and Strongyloides fuelleborni infections. Our results revealed discordance between assays, with no single primer set identifying all positive samples across all assays. One assay was identified as specific for S. stercoralis only, while other assays amplify other Strongyloides spp. This may impact the specificity of molecular diagnostics in epidemiological surveys, particularly with increasing identifications of S. fuelleborni infections in humans from Asia and New Guinea. Of the assays examined, four were probe-based qPCR assays and a LAMP assay. On sequencing positive amplicons, enteric bacterial sequences were identified for two assays. However, subsequent testing and BLAST results identified that including a probe, as the assays were designed, would increase specificity, so that the bacterial sequences would not be amplified. These findings provide a context for the interpretation of results from various Strongyloides molecular assays. Strongyloidiasis is an underdiagnosed neglected parasitic infection that poses a growing challenge for public health surveillance and control, particularly in regions where multiple Strongyloides species exist and clinical case detection is challenging. Molecular assays are increasingly used as reference diagnostic tools, yet their performance and species specificity have not been systematically evaluated across diverse endemic settings. In this study, we tested the hypothesis that commonly used molecular assays differ in their ability to accurately and consistently detect and discriminate Strongyloides stercoralis and Strongyloides fuelleborni in human fecal samples. By comparing five molecular assays using samples from Gabon and Papua New Guinea, we demonstrate discordance between tests, including species-dependent detection biases. These findings provide evidence that molecular diagnostics may misrepresent species distribution. Our results highlight the necessity for awareness of target species specificity and for regionally validated molecular tools to ensure the reliable interpretation of assay results.", "journal": "Journal of clinical microbiology", "year": "2026", "doi": "10.1128/jcm.00137-26", "ptypes": [ "Journal Article" ], "first_author": "Haidamak J", "dirs": [ "C" ] }, "41763755": { "pmid": "41763755", "title": "Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.", "abstract": "Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.", "journal": "Food research international (Ottawa, Ont.)", "year": "2026", "doi": "10.1016/j.foodres.2026.118370", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ma C", "dirs": [ "C" ] }, "40226432": { "pmid": "40226432", "title": "Development of a Novel Nested-RT-LAMP Assay for the Rapid and Accurate Coronavirus Disease-2019 Diagnosis.", "abstract": "Background and Aims: Coronavirus disease 2019 (COVID-19), an emerging life-threatening viral disease, has rapidly spread worldwide, exerting a detrimental impact on public health. We aimed to devise an innovative platform based on the loop-mediated isothermal amplification (LAMP) method, having priorities over real-time PCR (RT-PCR) in terms of sensitivity, specificity, and low running costs. Methods: To develop a novel assay, a new primer set plus four primer sets were designed targeting the N gene of the COVID-19 agent, resulting in the sensitivity reinforcement. The limit of detection (LOD) of the developed approach was determined and compared to those of the standard RT-LAMP and RT-PCR. Two hundred confirmed positive and negative samples initially tested by RT-PCR were recruited to assess the nested-RT-LAMP assay. Furthermore, for the one-step nested-RT-LAMP assay, positive samples were tested directly without the need for RNA extraction. Results: The LOD of nested-RT-LAMP, LAMP, and RT-PCR were 5, 15, and 15 copies/μL, respectively. The findings of the investigation illustrated 100% sensitivity and 98% specificity for both LAMP assays. Moreover, respectively, 94% and 97% sensitivity and specificity were determined regarding the one-step nested-RT-LAMP assay. Conclusion: We offered a novel approach with more sensitivity compared to RT-PCR and common RT-LAMP, not only being a simple, accurate, cost-effective alternative diagnostic tool for RT-PCR but also being able to detect asymptomatic or mildly symptomatic patients more accurately in 2 h by naked eyes.", "journal": "The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale", "year": "2025", "doi": "10.1155/cjid/3343309", "ptypes": [ "Journal Article" ], "first_author": "Mirzaei H", "dirs": [ "C" ] }, "41069987": { "pmid": "41069987", "title": "Comprehensive web-based platform for advanced PCR design, genotyping, synthetic biology, molecular diagnostics, and sequence analysis.", "abstract": "We present a comprehensive web-based platform that integrates a suite of molecular biology tools tailored for PCR-based applications. Key features include custom multiplex tiling PCR panel design for amplicon sequencing, loop-mediated isothermal amplification (LAMP), allele-specific PCR genotyping assay development, Gibson assembly, oligonucleotide analysis and design, and the de novo identification, classification, and visualization of repetitive sequences. The platform supports a wide range of PCR primer design tasks, including standard, multiplex, reverse, long-range, quantitative fluorescence (TaqMan and MGB probe), and bisulfite PCR. In silico PCR analysis ensures primer and probe specificity across diverse applications such as gene discovery via homology analysis, molecular diagnostics, DNA profiling, and repeat sequence identification. The multiplex tiling PCR tool is optimized for both next-generation and third-generation sequencing technologies. LAMP primer design includes support for loop primers that target eight distinct regions of the DNA template. The allele-specific PCR module enables genotyping of single nucleotide polymorphisms, insertions/deletions, multi-nucleotide variants, and haplotypes at specific loci. Additionally, the platform facilitates genome-wide identification, masking, and clustering of repetitive elements, including interspersed repeats and low-complexity sequences such as simple sequence repeats and telomeres. All tools are freely accessible at https://primerdigital.com/tools/.", "journal": "Molecular therapy. Nucleic acids", "year": "2025", "doi": "10.1016/j.omtn.2025.102716", "ptypes": [ "Journal Article" ], "first_author": "Kalendar R", "dirs": [ "C" ] }, "40129576": { "pmid": "40129576", "title": "Establishment and application of a loop-mediated isothermal amplification method based on MetAP2 gene for the detection of Nosema bombycis in silkworms (Bombyx mori).", "abstract": "Pebrine, caused by Nosema bombycis, is a devastating disease of silkworms that causes huge economic losses to the sericulture farmers. Although pebrine is a quarantine disease, currently the development of effective molecular diagnostic or therapeutic tools for its management is still a lagging hotspot in sericulture research. In the present study, a highly specific, sensitive, and field-applicable LAMP assay targeting MetAp2 gene was developed. LM1 primer set produced better results, with fluorescence (amplification) signals appearing in ~50 min. The reaction temperature of 60.9°C and outer primer to inner primer ratio of 1:8 were found to be optimal, with the shortest amplification time and strongest fluorescence intensity. The LAMP assay showed high specificity for the DNA of Nosema bombycis spores, as the templates of other common microorganisms of silkworms showed no amplification. The LAMP assay detected pMD-19T-met positive plasmid at the lowest concentration of 103 copies, with a detection time of ~80 min. The practicality test showed that the LAMP assay can detect Nosema bombycis spore DNA at the lowest concentration of 10-3 ng/μL. At concentration of 1.2 ng/μL, the real-time fluorescence signals appeared in ~60 min. The LAMP assay detected Nosema bombycis at all life stages of untreated silkworms. In fumagillin treated silkworms, no real-time fluorescence amplification was observed at 90 h and later, indicating the reliability of LAMP in detecting Nosema bombycis, and effectiveness of fumagillin, to some degree, in treating pebrine infection. The developed LAMP assay holds good promise for its application as a specific and field-applicable tool for the detection/control of pebrine in the field settings.", "journal": "Frontiers in veterinary science", "year": "2025", "doi": "10.3389/fvets.2025.1549224", "ptypes": [ "Journal Article" ], "first_author": "Qazi IH", "dirs": [ "C" ] }, "40777388": { "pmid": "40777388", "title": "A Rapid Multiplex LAMP Assay for Point-of-Care Detection of CT, NG, TV, and Fluoroquinolone Resistance in NG.", "abstract": "Rapid point-of-care (POC) diagnostics are essential tools for improving timely treatment and reducing the transmission of sexually transmitted infections (STIs). The STI NG Plus Assay is a rapid multiplex LAMP (loop-mediated isothermal amplification) NAAT (nucleic acid amplification test) capable of simultaneously detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and fluoroquinolone resistance-associated mutations in NG ( gyrA S91F). In this study, we assessed the STI NG Plus assay primer design and analytical sensitivity. Using a bioinformatically optimized primer design pipeline and empirical screening, the assay demonstrated high inclusivity and specificity, with no cross-reactivity to 48 urogenital organisms or the human genome. Analytical sensitivity testing showed reliable detection of all targets in both lysis buffer and clinical matrix. Limits of detection were lower than those of an existing FDA-cleared test. The assay's robustness, speed, and sensitivity support its potential for decentralized STI testing with integrated antimicrobial resistance profiling.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.07.24.666671", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Dong S", "dirs": [ "C" ] }, "40711424": { "pmid": "40711424", "title": "PAM-free activation of CRISPR/Cas12a via semi-nested asymmetric RPA: highly specific detection of HPV16 dsDNA.", "abstract": "Early and accurate detection of HPV16 nucleic acids is therefore critical for the effective screening, diagnosis, and prevention of cervical cancer. Although CRISPR/Cas12a-based molecular diagnostics offer a rapid and sensitive approach for HPV16 detection, their application to double-stranded DNA (dsDNA) targets remains constrained by two major limitations: the strict requirement for a protospacer adjacent motif (PAM) site and the insufficient specificity of current amplification strategies, which can lead to off-target amplification and false-positive results. To address these challenges, we developed a semi-nested asymmetric recombinase polymerase amplification (SNA-RPA) method combined with CRISPR/Cas12a for the detection of HPV16 dsDNA. This strategy employs a semi-nested primer design to significantly enhance target sequence specificity during amplification, while asymmetric primer ratios promote the efficient generation of single-stranded DNA (ssDNA) that directly activates Cas12a without the need for a PAM site. Using this approach, we achieved rapid and highly specific detection of HPV16 dsDNA, with a limit of detection as low as 18 aM. Beyond achieving PAM-free detection, our method also substantially improves amplification fidelity, offering a promising solution for precise and reliable HPV diagnostics and cervical cancer screening.", "journal": "The Analyst", "year": "2025", "doi": "10.1039/d5an00491h", "ptypes": [ "Journal Article" ], "first_author": "Zou X", "dirs": [ "C" ] }, "42164265": { "pmid": "42164265", "title": "Rapid identification of Lepiota brunneoincarnata in China with loop-mediated isothermal amplification.", "abstract": "Many edible fungi and poisonous mushrooms share similar morphologic characteristics, making them difficult to distinguish with the naked eye. Lepiota brunneoincarnata is a major lethal toxic mushroom widely distributed in China and responsible for frequent poisoning incidents across the country. The present study developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the translation elongation factor 1-a (tef1) sequence of L. Brunneoincarnata. A set of specific LAMP primers were designed for the specific detection of L. brunneoincarnata, and the assay showed no amplification from 63 non-target fungal specimens. In this experiment, the limit of detection for DNA concentration was 100 pg/mL, and the limit of detection for spore concentration was 10 spores/mL. The method also successfully detected L. brunneoincarnata in boiled and digested mixed mushroom samples, and the whole detection procedure can be completed within 60 minutes at 63 °C. The study is suitable for rapid identification of L. brunneoincarnata in China, and the method has potential for further optimization toward field application.", "journal": "Frontiers in cellular and infection microbiology", "year": "2026", "doi": "10.3389/fcimb.2026.1809128", "ptypes": [ "Journal Article" ], "first_author": "Fu D", "dirs": [ "C" ] }, "41161594": { "pmid": "41161594", "title": "Molecular Detection of Salmonella.", "abstract": "The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, the development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing, etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.", "journal": "Journal of food protection", "year": "2025", "doi": "10.1016/j.jfp.2025.100659", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Fu H", "dirs": [ "C" ] }, "40627810": { "pmid": "40627810", "title": "Deep Learning-Enhanced Hand-Driven Spatial Encoding Microfluidics for Multiplexed Molecular Testing at Home.", "abstract": "The frequent global outbreaks of viral infectious diseases have significantly heightened the urgent demand for molecular testing at home. However, the labor-intensive sample preparation and nucleic acid amplification steps, along with the complexity and bulkiness of detection equipment, have limited the large-scale application of molecular testing at home. Here, we propose artificial intelligence-enhanced hand-driven microfluidic system (MACRO) based on RPA and CRISPR technologies for home diagnosis of multiple types of infectious diseases. Leveraging a multidimensional space hourglass structure design, precise spatiotemporal control of fluids can be achieved simply by flipping the chip. Through dual chemical reactions, the system eliminates the need for nucleic acid extraction and purification, simplifying sample preparation and obviating the reliance on heating equipment. The MACRO achieves attomolar sensitivity within 60 min from sample input to result, and 100% specificity for 27 HPV subtypes. Clinical validation using 140 cervical swab specimens demonstrated 98.57% accuracy with 100% specificity. Further, we validated MACRO through multiplex detection of three clinically critical respiratory pathogens (SARS-CoV-2, Influenza A, and Influenza B) in 70 samples, achieving 100% diagnostic concordance. To circumvent subjective errors and enable real-time data collection, we further developed a mobile health platform based on the YoLov8 image recognition algorithm to ensure rapid and precise result output. With the performance of cost-effectiveness ($1.34 per target), and independence from instrument support, MACRO provides a comprehensive solution for molecular testing at home, offering significant implications for enhancing early warning systems for major epidemics and improving public health emergency response capabilities.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.5c04309", "ptypes": [ "Journal Article" ], "first_author": "Zhang Y", "dirs": [ "C" ] }, "41155464": { "pmid": "41155464", "title": "Measuring the Invisible: Microbial Diagnostics for Periodontitis-A Narrative Review.", "abstract": "Periodontitis is a biofilm-driven inflammatory disease in which conventional indices (probing depth, clinical attachment level, and radiographs) quantify tissue destruction without capturing the biology of infection. In this review, we synthesized microbiological diagnostics, from chairside tools to omics. We outline sampling strategies and emphasize the quantitative monitoring of bacterial load. Enzymatic assays (e.g., N-benzoyl-DL-arginine-2-naphthylamide hydrolysis assay test) measure functional activity at the point of care. Immunological methods include rapid immunochromatography for Porphyromonas gingivalis and enzyme-linked immunosorbent assay for the high-throughput measurement of bacterial antigens. Molecular platforms encompass quantitative polymerase chain reaction (qPCR) (TaqMan, SYBR, multiplex panels; propidium monoazide quantitative-qPCR for viable cells), checkerboard DNA-DNA hybridization for semi-quantitative community profiling, loop-mediated isothermal amplification (LAMP)/molecular beacon-LAMP for portable isothermal detection, and microarrays. Complementary modalities such as fluorescent in situ hybridization, next-generation sequencing, and Fourier transform infrared spectroscopy provide spatial, ecological, and biochemical resolutions. We discuss the limitations of current approaches, including sampling bias, presence-activity discordance, semi-quantitation, method biases, limited strain/function resolution, low-biomass artifacts, and lack of validated cutoffs. To address these challenges, we propose a pragmatic hybrid strategy: site-specific quantitative panels combined with activity and host-response markers interpreted alongside clinical metrics under standardized quality assurance/quality control. Priorities include outcome-linked thresholds, strain-aware/functional panels, robust point-of-care chemistry, and harmonized protocols to enable personalized periodontal care.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms262010172", "ptypes": [ "Journal Article", "Review" ], "first_author": "Usui M", "dirs": [ "C" ] }, "41241835": { "pmid": "41241835", "title": "Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.", "abstract": "In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c04524", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhang J", "dirs": [ "C" ] }, "40875162": { "pmid": "40875162", "title": "Advances in diagnosis of diseases causing diarrhea in newborn calves.", "abstract": "Diarrhea in newborn calves is a serious global health problem. It poses challenges for animal industry, veterinarians and researchers due to the rapid onset of dehydration. Mixed infections make treatment complicated, and many young calves suffer high rates of illness and death from this condition. Numerous enteropathogens are associated with diarrhea in newborn calves, encompassing viruses, bacteria, parasites, and protozoa. Their occurrence differs by region, yet the most prevalent infections include E. coli, Salmonella species, Clostridium perfringens, Clostridium difficile, Rotavirus, Coronavirus, Cryptosporidium, Toxocara, Giardia and Eimeria. This review outlines the diagnostic techniques for diseases that lead to diarrhea in newborn calves. Diagnosis is based on clinical manifestations; however, the laboratory identification of etiological items is the only valid way for detecting the illness's aetiology and initiating treatment protocols. Classic methods such as bacterial culturing, fecal flotation, direct microscopy, and virus isolation help us understand pathogens better. Immunological assays like ELISA and immunochromatography are fast, accurate, affordable, and useful for on-farm detection. They help identify specific antigens or antibodies efficiently. Molecular methods including PCR (standard, multiplex, real time and digital), LAMP assays, DNA microarrays and whole-genome sequencing allow highly accurate and sensitive detection. They can identify pathogens effectively, even at very low levels. Nanotechnology-based assays introduce a novel level of sensitivity and specificity, often yielding quick results with minimal sample volumes. In conclusion, accurate and rapid diagnosis using advanced techniques is critical for managing and preventing diseases that lead to diarrhea in newborn calves.", "journal": "Veterinary research communications", "year": "2025", "doi": "10.1007/s11259-025-10855-0", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sedky D", "dirs": [ "C" ] }, "42104518": { "pmid": "42104518", "title": "A rapid multiplex platform for simultaneous detection of chikungunya virus, dengue virus, and dengue serotyping based on isothermal amplification and lateral flow dipsticks.", "abstract": "The expeditious and precise diagnosis of dengue virus (DENV) and chikungunya virus (CHIKV) is paramount for effective patient management and the control of outbreaks. In this study, a duplex reverse transcription multi-enzyme isothermal amplification (RT-MIRA) assay was established for the simultaneous detection of DENV and CHIKV, followed by a nested RT-MIRA assay for DENV serotyping (DENV-1 to -4). Specific primers and probes targeting the DENV 3'-UTR, CHIKV E1 gene, and four DENV serotypes were designed. The duplex RT-MIRA and nested DENV RT-MIRA serotyping reaction systems were optimized at 39 °C with portable fluorescence or lateral flow dipstick readouts. For methodological validation, specificity was evaluated against 35 related pathogens, and the 95% limit of detection (LOD95) was determined via probit regression. For clinical validation, serum samples from 236 suspected patients were tested, benchmarking against RT-qPCR and serology. Statistical analyses included the Wilson score method for calculating 95% confidence intervals (CIs) and Cohen's kappa (κ). For external verification, 12 CHIKV-positive clinical samples and 5 artificially simulated co-infection samples were retrospectively analyzed to validate assay accuracy. The duplex RT-MIRA assay exhibited no cross-reactivity with other pathogens. The LOD95 values were 13.47 copies/μl for DENV and 10.49 copies/μl for CHIKV. Clinical validation demonstrated sensitivities of 96.15% (95% CI: 89.28%-98.67%) for DENV and 88.89% (95% CI: 67.20%-96.90%) for CHIKV. Specificity was 100% (95% CI: 92.87%-100%) for both. Agreement with RT-qPCR was strong for DENV (κ = 0.96) and CHIKV (κ = 0.92). The nested RT-MIRA serotyping assay showed high sensitivity (LOD95: 1.6-18.7 copies/μl) without cross-reactivity, accurately differentiating 75 DENV-positive samples into 71 DENV-1 and 4 DENV-2. In the external verification, the assay accurately detected 10 CHIKV mono-infections and 2 CHIKV/DENV co-infections, and distinguished four DENV serotypes in simulated matrices. A rapid and sensitive integrated method has been developed that combines duplex RT-MIRA for detecting DENV and CHIKV, and nested RT-MIRA for serotyping DENV. The simplicity and speed of the amplification and detection steps demonstrate this platform's potential for use in point-of-care testing and surveillance in areas with limited resources, particularly when used alongside portable extraction methods.", "journal": "Infectious diseases of poverty", "year": "2026", "doi": "10.1186/s40249-026-01450-9", "ptypes": [ "Journal Article" ], "first_author": "Liu G", "dirs": [ "C" ] }, "40304466": { "pmid": "40304466", "title": "Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography.", "abstract": "Although human tuberculosis (TB) caused by Mycobacterium bovis is clinically, pathologically, and radiologically indistinguishable from Mycobacterium tuberculosis-caused TB, M. bovis is innately resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. The rapid differentiation of these two biovars is therefore of high clinical and epidemiologic importance. Most current molecular tools in resource-limited settings identify mycobacteria only to the M. tuberculosis species (MTB) level. In this study, we report a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography for the rapid and easy differential detection of M. bovis and M. tuberculosis. The assay was optimized and validated using 143 isolates comprising six MTB reference strains, 50 M. bovis isolates, 58 M. tuberculosis isolates, 24 non-tuberculous mycobacterial (NTM) strains, and five other respiratory pathogens. The multiplex LAMP correctly detected MTB and distinguished between M. tuberculosis and M. bovis simultaneously with sensitivities of 500 fg and 1 pg DNA, respectively, within 60 min, and the results were visualized by dipstick chromatography within 10 min. The assay was specific in that no major respiratory pathogens tested, including NTM strains, were positive. The multiplex dipstick LAMP assay is therefore a useful and accurate low-cost method for the differential identification of M. bovis and M. tuberculosis, especially in endemic areas where bovine and human TB coexist. The distinction between M. bovis and M. tuberculosis can also aid in monitoring the spread of M. bovis to humans and allow for correct treatment, which will ultimately contribute to TB control in both humans and animals. Human tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. Most available molecular tools cannot distinguish the two biovars. This study addresses this gap by introducing a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography that can simultaneously and differentially detect M. bovis and M. tuberculosis within 60 min. The LAMP method does not require sophisticated high-cost equipment and can be easily implemented in resource-limited settings. Our LAMP facilitates rapid and accurate tuberculosis diagnosis, enabling appropriate therapeutic agents to be selected in areas where bovine and human tuberculosis coexist. It can also screen for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking.", "journal": "Microbiology spectrum", "year": "2025", "doi": "10.1128/spectrum.02421-24", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Akapelwa ML", "dirs": [ "C" ] }, "40748632": { "pmid": "40748632", "title": "Development of a CRISPR/Cas-Based Detection Platform for Tracking Decreased Susceptibility to Cephalosporins in Neisseria gonorrheae.", "abstract": "Gonorrhea has become an escalating public health issue due to the rapid emergence of antimicrobial resistance (AMR). Developing efficient and accurate detection of resistant strains is urgently needed for their management and treatment. We have developed the Multiplex Integrated RPA-CRISPR/Cas12a detection Assay (MIRCA) for simultaneous detection of Neisseria gonorrheae (Ng) and mutations with decreased susceptibility to cephalosporins. MIRCA enables multiplex detection of Ng and single-nucleotide polymorphisms in resistance-associated genes within 40 min, with high specificity and sensitivity (10-20 copies/reaction). Clinical evaluation showed 100% concordance with qPCR for Ng identification and Sanger sequencing for FC428 strain tracking. For predicting decreased-susceptibility strains with A501 mutations, MIRCA achieved 98.33% agreement with Sanger sequencing. Simulated tests demonstrated 100% consistency between MIRCA results in centrifuge tubes and microfluidic chips. This robust and cost-effective approach addresses current challenges in AMR surveillance. Its integration with microfluidic chip offers an affordable and user-friendly diagnostic solution, making it highly valuable for timely infectious disease diagnosis and resistance monitoring. It also holds significant potential for point-of-care testing in resource-limited areas.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c01400", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhao Z", "dirs": [ "C" ] }, "41365539": { "pmid": "41365539", "title": "Portable RT-LAMP Platform for Rapid Point-of-Care Detection for SFTSV.", "abstract": "Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) is a tick-borne pathogen associated with high mortality, particularly among the elderly, and poses a significant public health threat in East Asia's rural areas. Improved diagnostic approaches are crucial for effective disease management in resource-limited settings. In this study, we developed a portable Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) platform for rapid and accurate SFTSV detection. Primers targeting the conserved L gene were designed using 158 sequences collected between 2019 and 2023, and a multiplex assay incorporating the β-actin gene as an internal control to verify amplification efficiency in alignment with clinical performance requirements. We assessed the assay's sensitivity and specificity, ultimately establishing the RT-LAMP platform in a portable, clinical-grade device. The RT-LAMP assay achieved a detection limit of 500 RNA copies per reaction and provided results within 20 min. It demonstrated high specificity without cross-reactivity with other pathogens. Performance comparisons with commercial qRT-PCR kits using clinical specimens confirmed the RT-LAMP platform's reliability. The newly developed RT-LAMP platform is a rapid, accurate, and cost-effective diagnostic tool for SFTSV with strong potential for point-of-care testing (POCT). Its portability and ease of use make it ideal for application in resource-constrained environments, thereby enhancing SFTSV surveillance and improving public health outcomes in affected regions.", "journal": "Journal of microbiology and biotechnology", "year": "2025", "doi": "10.4014/jmb.2509.09027", "ptypes": [ "Journal Article" ], "first_author": "Lee GC", "dirs": [ "C" ] }, "41976544": { "pmid": "41976544", "title": "Advances of Digital Detection for Foodborne Pathogens.", "abstract": "The implementation of stringent regulatory policies for foodborne pathogens necessitates ultra-sensitive analytical methods. Digital detection, characterized by absolute quantification and tolerance to complex matrices, serves as a robust approach for food safety monitoring. This review summarizes recent advances in digital detection for foodborne pathogens, including nucleic acid amplification-based platforms such as droplet digital PCR and digital isothermal amplification, as well as emerging preamplification-free approaches based on enzyme-mediated signal conversion, functional nanomaterials, and microfluidic devices. We also profile the applications of digital detection technologies for achieving highly specific and accurate detection of foodborne pathogens and discuss their capabilities in viable bacteria quantification, antimicrobial resistance analysis, and multiplex detection. We finally discuss emerging trends, including partition-free digital detection and artificial intelligence-assisted analysis. These advances are expected to promote the development of intelligent and data-driven food safety surveillance strategies.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15071250", "ptypes": [ "Journal Article", "Review" ], "first_author": "He R", "dirs": [ "C" ] }, "42114484": { "pmid": "42114484", "title": "PCR-free cascaded CRISPR-Cas13a colorimetric platform (CLAMP) for non-invasive miRNA-based allergen-specific subtyping of allergic rhinitis.", "abstract": "Accurate identification of causative allergens is critical for personalized management of allergic rhinitis (AR), yet current methods often fail to distinguish clinical relevance from sensitization, particularly in polysensitized patients. To address this, we developed CLAMP (Cascaded CRISPR-Cas13a LAMP Colorimetric Assay), a platform that leverages a single Cas13a/crRNA complex to initiate a simplified CRISPR cascade amplification, enabling one-pot two-step detection without tube opening after sample addition. The assay employs a colorimetric readout based on CIELAB ΔE, allowing quantitative biomarker analysis with minimal equipment, requiring only a simple heat source. CLAMP achieves a detection limit of 1.15 × 10-15 M with excellent specificity and a turnaround time under 60 min. In preclinical validation using nasal lavage fluid from 200 allergen-specific AR mouse models, CLAMP enabled allergen-based AR classification via multiplex miRNA detection, achieving 91.5% accuracy in distinguishing major allergen subtypes. This low-cost, non-invasive platform holds strong potential for rapid allergen classification and future translation toward personalized immunotherapy in AR.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.118773", "ptypes": [ "Journal Article" ], "first_author": "Wan Y", "dirs": [ "C" ] }, "39913278": { "pmid": "39913278", "title": "Multiplex recombinase polymerase amplification (RPA) assay for carrier detection and prenatal diagnosis of α0-thalassemia (SEA and THAI deletions).", "abstract": "Hemoglobin Bart's hydrops fetalis is the most severe form of α-thalassemia, caused by homozygous α0-thalassemia, which is highly prevalent in Southeast Asian countries. Simple and rapid identification of α0-thalassemia carrier and prenatal diagnosis of Hemoglobin Bart's hydrops is essential in the region. We have developed a multiplex RPA assay for simple detection of α0-thalassemia (SEA and THAI deletions) and tested it in carrier detection (n = 125) and prenatal diagnosis of Hb Bart's hydrops fetalis syndrome (n = 30). The sensitivity, specificity, positive predictive value, and negative predictive value for detecting α0-thalassemia carriers were 100% for both SEA and THAI deletions. The assay correctly identified 42 carriers of α0-thalassemia (SEA deletion), 4 carriers of α0-thalassemia (THAI deletion), and 79 non-carriers. For prenatal diagnosis, the results of RPA revealed a 100% concordance (30/30) with the conventional gap-PCR analysis. The multiplex RPA assay is a rapid, reliable, and cost-effective method for diagnosing α0-thalassemia-related disorders in routine clinical settings. This assay has the potential to significantly contribute to the prevention and control of Hb Bart's hydrops fetalis in the region.", "journal": "Scandinavian journal of clinical and laboratory investigation", "year": "2025", "doi": "10.1080/00365513.2025.2463071", "ptypes": [ "Journal Article" ], "first_author": "Sitthisophonkamjorn Y", "dirs": [ "C" ] }, "40704969": { "pmid": "40704969", "title": "Rapid, multiplex, one-pot CRISPR/Dx system for detecting cancer fusion genes.", "abstract": "Targeted therapies directed at fusion genes have proven remarkably effective against cancers. Therefore, the rapid and reliable identification of cancer fusion genes can guide subsequent therapeutic treatment and predict prognosis. By integrating the RT-RPA and CRISPR/Cas12a approaches, we developed a one-pot CRISPR/Dx system for the rapid and multiplex detection of cancer fusion genes. A tube with unique assemblies was created using 3D printing technology to realize this application. As proof of principle, we demonstrated the feasibility of the one-pot CRISPR/Dx system in detecting lung cancer by targeting ROS1 fusions. The performance of the one-pot CRISPR/Dx detection system was comparable to a two-tube-based testing platform. When tested with synthetic RNA fusions, both approaches efficiently detected all 14 ROS1 fusions with an LOD in the range of 5-10 copies per μL, without generating a background signal, even in the presence of a large excess of wild-type RNA. The total reaction time for both approaches was 30 minutes. Notably, the one-pot CRISPR/Dx detection system minimized the operation steps and aerosol contamination without compromising detection sensitivity and specificity. Furthermore, its diagnostic power was validated using clinical samples. Thus, we successfully developed a rapid, multiplex, one-pot CRISPR/Dx detection system for detecting 14 clinically relevant ROS1 fusions with high sensitivity and specificity. It is also cost-effective and simple to operate, thereby realizing the ultimate goal of establishing CRISPR/Dx as the paragon of cancer diagnostics for home self-testing and point-of-care testing.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d5ay00783f", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Li J", "dirs": [ "C" ] }, "41754406": { "pmid": "41754406", "title": "Challenges and Solutions in pgRNA Measurement: Toward Improved Monitoring of Hepatitis B Therapy.", "abstract": "Hepatitis B virus (HBV) pregenomic RNA (pgRNA), transcribed directly from nuclear covalently closed circular DNA (cccDNA), is an essential component in viral replication. The synthesis and encapsidation of pgRNA depend significantly on the transcriptional activity of cccDNA, making serum pgRNA a recently recognized non-invasive biomarker for evaluating cccDNA activity. However, its clinical application is limited by factors including preanalytical variables, methodological inconsistencies in detection, and a lack of standardization in quantification. This review provides an overview of the biological origins of pgRNA and its critical role in the HBV replication cycle, highlighting the stability challenges encountered during the collection, processing, and storage of plasma/serum samples. Furthermore, it analyzes recent significant advancements in pgRNA detection technologies, encompassing modified reverse transcription quantitative polymerase chain reaction (RT-qPCR), nucleocapsid-captured methodologies, automated testing platforms, multiplex digital PCR, isothermal amplification, and clustered regularly interspaced short palindromic repeats-based assays. A comparison of these technologies revealed that discrepancies in pgRNA quantification arise primarily from variations in sample processing and measurement systems, rather than from inherent biological limitations. Therefore, establishing standardized sample handling procedures, harmonized detection methods, and unified measurement systems is imperative before pgRNA can be reliably applied to monitor treatment, guide cessation decisions, or evaluate cure in chronic hepatitis B.", "journal": "Pathogens (Basel, Switzerland)", "year": "2026", "doi": "10.3390/pathogens15020153", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhu Z", "dirs": [ "C" ] }, "42155550": { "pmid": "42155550", "title": "Advances in the detection of deadly free-living amoebae (FLA).", "abstract": "Free-living amoebae (FLA), including Naegleria fowleri, Acanthamoeba castellanii, Balamuthia mandrillaris, and Sappinia pedata, are ubiquitous protozoa capable of causing severe infections such as primary amoebic meningoencephalitis (PAM), granulomatous amoebic encephalitis (GAE), and Acanthamoeba keratitis (AK). Early diagnosis remains challenging due to disease rarity, nonspecific clinical presentation, and limited access to specialized laboratory methods. Rapid and accurate detection is critical for patient management and public health response, particularly amid changing environmental exposures. This review summarizes current diagnostic approaches in clinical and environmental contexts, including specimen handling, microscopy, culture, immunohistochemistry, antigen detection, and molecular methods such as conventional PCR, real-time PCR, multiplex qPCR, LAMP, and metagenomic next-generation sequencing. Environmental surveillance, biomarker discovery, quality assurance, and standardized protocols are also discussed. By evaluating strengths and limitations of available tools, this review highlights diagnostic gaps and future priorities to enhance sensitivity, turnaround time, and global accessibility.", "journal": "Diagnostic microbiology and infectious disease", "year": "2026", "doi": "10.1016/j.diagmicrobio.2026.117465", "ptypes": [ "Journal Article", "Review" ], "first_author": "Pandit S", "dirs": [ "C" ] }, "39927596": { "pmid": "39927596", "title": "A multiplex point-of-care test for discriminatory inference of SARS-CoV-2 Omicron lineages using reverse transcription loop-mediated isothermal amplification and lateral flow detection.", "abstract": "The ongoing increase in genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages remains a significant global challenge, mainly due to the incompatibility of genetic sequencing with the technological and economic capabilities of most countries worldwide. Additionally, the continued circulation and emergence of numerous lineages of the Omicron variant with some specific mutations represent a challenge in developing straightforward tests for their discrimination. In this study, we developed a multiplex assay based on reverse transcription loop-mediated isothermal amplification with lateral flow assay detection (RT-LAMP-LFA). This assay aims to enhance the accessibility of genomic surveillance and to deliver more precise epidemiological data to support public health decision-making. To demonstrate its utility, we used the assay as a proof of concept for discriminating between the SARS-CoV-2 Omicron lineages BA.1 and BA.2. When comparing the results of the new assay with the gold standard method of genetic sequencing in a panel of clinical samples, the multiplex RT-LAMP-LFA demonstrated excellent diagnostic performance. For BA.1 lineage detection, the assay achieved 100% sensitivity, 100% specificity, and 100% accuracy, while for BA.2, it showed 100% sensitivity, 95% specificity, and 96% accuracy. The overall simplicity of the method combined with advantages such as short analysis time (40 min), low cost (∼$15 per test) and adaptability to the point-of-care format make the multiplex RT-LAMP-LFA assay an important screening tool for inferring SARS-CoV-2 Omicron lineage positive samples, thus alleviating the high demand for sequencing and expanding genomic surveillance, even in remote locations.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d4ay01798f", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Estrela PFN", "dirs": [ "C" ] }, "42261770": { "pmid": "42261770", "title": "Machine Learning-Enhanced Ultrasensitive Immuno-CRISPR Array Facilitates Early Diagnosis of Alzheimer's Disease by Detecting Multiple Plasma Biomarkers.", "abstract": "Early and accurate diagnosis of Alzheimer's disease (AD) remains a significant challenge due to the multifactorial and dynamic nature of its pathology. Although plasma-based biomarkers such as amyloid-β (Aβ) and phosphorylated tau (p-tau) have shown promise as diagnostic indicators, current single-biomarker detection techniques lack the requisite sensitivity and specificity for early-stage diagnosis. Here, we present the development of an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) capable of concurrently detecting six core AD biomarkers, including Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404. By integrating antibody pair-based multiplex recombinase polymerase amplification (RPA) with spatially encoded CRISPR-Cas12a detection, the UCMDA achieves a detection limit of 1 fg/mL, which is 10 000-fold more sensitive than conventional ELISA. Clinical validation in a cohort of 155 plasma samples demonstrated that logistic regression (LR)-based integration of the six biomarkers significantly enhanced diagnostic performance, with the multi-biomarker model substantially outperforming single-biomarker approaches in diagnosing AD-MCI and AD. This platform offers a scalable, cost-effective, and minimally invasive strategy for early detection and disease monitoring. This work highlights the potential of CRISPR-based multiplex protein detection technologies combined with machine learning-assisted analysis to enhance the precision of diagnosing neurodegenerative disorders.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2026", "doi": "10.1002/advs.75983", "ptypes": [ "Journal Article" ], "first_author": "Zhang L", "dirs": [ "C" ] }, "42239534": { "pmid": "42239534", "title": "Simultaneous molecular detection of Mycobacterium tuberculosis and multidrug resistance using CRISPR-AaCas12b-based nucleic acid assay.", "abstract": "To address the unmet need for rapid, accurate diagnosis of Mycobacterium tuberculosis (MTB) and multidrug-resistant tuberculosis (MDR-TB), we developed and validated a clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas)-based diagnostic assay. A multiplex-recombinase polymerase amplification (RPA) coupled CRISPR-Alicyclobacillus acidiphilus Cas12b (AaCas12b) assay was established for simultaneous detection of MTB by targeting the specific insertion sequence IS6110 and the two most common drug resistance mutations, rpoB 1349C>T for rifampicin resistance and katG 944G>C for isoniazid resistance. The assay supported dual-readout signal detection using both a fluorescent platform and lateral flow chromatography (LFC). Its diagnostic performance was evaluated in 48 clinical samples using WHO-recommended GeneXpert MTB/RIF, phenotypic drug susceptibility testing (pDST), and sequencing as reference standards. The multiplex-RPA CRISPR-AaCas12b assay showed a limit of detection (LoD) of 1.5 CFU/mL for MTB detection, with a sensitivity of 97.1% and a specificity of 100% using culture as the reference standard, and a total turnaround time of 30 min (20 min for RPA and 10 min for CRISPR cleavage). For MDR-TB-related mutations, the assay achieved a sensitivity of 94.1% and a specificity of 100% for rpoB 1349C>T, and 94.7% sensitivity and 93.1% specificity for katG 944G>C, using sequencing as the reference standard. Notably, the LFC-integrated assay maintained comparable diagnostic accuracy with a total turnaround time of 35 min (20 min for RPA, 5 min for CRISPR cleavage, and 10 min for lateral flow strip reading). The established multiplex-RPA CRISPR-AaCas12b assay enables simple, accurate, and sensitive detection of MTB and common mutations associated with MDR-TB. With a rapid, simplified workflow and low resource requirements, this approach holds considerable potential for point-of-care testing in resource-limited settings, thus facilitating improved surveillance and control of TB and drug-resistant TB.", "journal": "Frontiers in cellular and infection microbiology", "year": "2026", "doi": "10.3389/fcimb.2026.1844184", "ptypes": [ "Journal Article" ], "first_author": "Hu Y", "dirs": [ "C" ] }, "41135356": { "pmid": "41135356", "title": "A multiplex RPA-CRISPR/Cas12a platform for rapid and accurate toxinotyping of Clostridium perfringens.", "abstract": "Clostridium perfringens (C. perfringens) is a leading cause of foodborne disease worldwide, requiring rapid and accurate toxinotyping for effective outbreak control and surveillance. Herein, we developed C. perfringens-multiplex RPA-CRISPR/Cas12a, an integrated detection platform combing multiplex Recombinase Polymerase Amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 12a (CRISPR/Cas12a)-mediated detection for comprehensive toxinotyping. The system simultaneously identifies six key toxin genes (cpa, cpb, etx, iap, cpe, netB) in two reaction tubes, enabling discrimination of all seven C. perfringens toxinotypes (A-G). The C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform exhibited exceptional analytical performance, achieving a detection limit of ≤10 copies/μL for across all targets while maintaining absolute specificity against the human genomic DNA and 5 common foodborne pathogens. In validation testing with 12 naturally contaminated food samples, the C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform demonstrated superior performance to commercial qPCR kits, accurately identifying eight Type A (cpa-gene-positive) and four Type F (cpa-gene and cpe-gene co-positive) strains. When coupled with a portable detection device, the platform completed the entire diagnostic workflow within 50 min while maintaining laboratory-level accuracy under field conditions. The rapid, cost-effective, and equipment-free system is particularly suited for decentralized toxin surveillance in resource-limited settings. By integrating high sensitivity, multiplex capability, and field applicability, this system significantly advances Point-of-care Testing (POCT) capabilities for food safety monitoring, supporting global food safety initiatives.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2025.128998", "ptypes": [ "Journal Article" ], "first_author": "Wang W", "dirs": [ "C" ] }, "42031469": { "pmid": "42031469", "title": "Challenges and advancements in the diagnosis of central nervous system tuberculosis.", "abstract": "Central nervous system (CNS) tuberculosis is one of the most severe forms of extrapulmonary TB, often leading to substantial morbidity and mortality owing to its complex clinical presentation and frequent diagnostic delays. Despite global TB control efforts, CNS TB continues to present unique difficulties owing to its nonspecific clinical manifestations, the paucibacillary nature of cerebrospinal fluid (CSF), and the limitations of conventional diagnostic methods. The diagnostic dilemma is further compounded in resource-limited settings where advanced molecular tools and neuroimaging may not be readily available. Timely and accurate diagnosis remains a cornerstone in reducing mortality and neurological sequelae. Clinically, CNS TB can present as tuberculous meningitis (TBM), tuberculoma, abscess, or spinal TB, each with overlapping features with other infectious and non-infectious conditions. This spectrum makes early clinical diagnosis particularly elusive. Traditional diagnostic methods such as CSF smear for acid-fast bacilli (AFB), culture, and routine biochemistry often fail to provide definitive answers. AFB smear sensitivity is dismal (less than 10 %), and culture, although considered a reference standard, takes weeks, during which the disease may progress rapidly. In recent years, advances in molecular diagnostics have brought hope. Nucleic acid amplification tests (NAATs), including GeneXpert MTB/RIF and the more sensitive GeneXpert Ultra, have enhanced detection compared to smear microscopy; however, their diagnostic yield in cerebrospinal fluid (CSF) remains suboptimal due to the typically low bacillary burden in CNS tuberculosis. WHO's 2021 Module 3 guidelines emphasize that despite these advances, NAAT sensitivity for CNS TB remains suboptimal and cannot replace clinical judgment and imaging. CSF Xpert positivity in TBM varies between 50 and 70 %, with a negative result not excluding disease. LAMP (loop-mediated isothermal amplification) and multiplex PCR assays are also emerging but lack standardization for CNS specimens. Neuroimaging techniques like MRI, MR spectroscopy, and diffusion tensor imaging (DTI) play an essential adjunctive role in diagnosis, particularly when CSF results are inconclusive. However, findings are often non-specific and require integration with clinical and laboratory data. Advanced imaging and artificial intelligence (AI)-based radiomic analysis are being explored for pattern recognition in neuro-TB, showing promise but are not yet widely applicable in clinical practice. Biomarkers like adenosine deaminase (ADA) along with interferon gamma, neopterin, and IP-10 in CSF have been studied, but none have yet replaced microbiological confirmation. Furthermore, diagnosis in specific populations-such as extremes of age, people living with HIV, and elderly individuals-presents additional complexities due to atypical presentations and altered immune responses. This review comprehensively discusses the multifaceted challenges in diagnosing CNS TB and the emerging diagnostic tools and strategies. It evaluates the sensitivity, specificity, and utility of conventional and advanced modalities and highlights evidence from WHO guidelines and recent literature. Addressing diagnostic barriers and integrating novel technologies into practice, particularly in high-burden countries, is essential to improving outcomes.", "journal": "The Indian journal of tuberculosis", "year": "2026", "doi": "10.1016/j.ijtb.2025.08.011", "ptypes": [ "Journal Article", "Review" ], "first_author": "Garg R", "dirs": [ "C" ] }, "41810315": { "pmid": "41810315", "title": "Research Progress on Point-of-Care Testing Technology for Mycoplasma Pneumonia.", "abstract": "Mycoplasma pneumoniae (MP) is a significant respiratory pathogen in children, often causing refractory and severe pneumonia. Sensitive, rapid, and portable diagnostic tools are crucial for guiding clinical management. Although traditional methods like culture, ELISA, and PCR are widely used, they suffer from drawbacks such as lengthy turnaround times, complex procedures, or reliance on laboratory equipment. Consequently, Point-of-care testing (POCT) technologies, valued for their speed, portability, and ease of use, have emerged as a key research focus for MP diagnosis. This review systematically summarizes advancements in POCT platforms, covering: (1) immunological methods; (2) molecular biology methods; and (3) biosensor technologies. Their sensitivity, specificity, and clinical performance are comparatively analyzed. Colloidal gold immunochromatography delivers results within 15 minutes but exhibits limited sensitivity. Molecular methods like LAMP and CRISPR-coupled systems achieve single-copy detection limits via isothermal amplification and gene editing, with processing times under 1 hour. Biosensors enable high-sensitivity automated detection through integrated signal amplification and microfluidics. Despite these advantages, POCT development faces challenges including cost-sensitivity tradeoffs, standardization barriers, and sample matrix interference. Future directions encompass multi-modal detection, AI-assisted interpretation, multiplex pathogen screening, and dynamic drug resistance gene monitoring. These innovations will expand POCT device deployment in primary care and home settings. This will ultimately improve effectiveness in controlling respiratory infections.", "journal": "International journal of general medicine", "year": "2026", "doi": "10.2147/IJGM.S584824", "ptypes": [ "Journal Article", "Review" ], "first_author": "Guo Z", "dirs": [ "C" ] }, "40047062": { "pmid": "40047062", "title": "FEN1-Aided RPA (FARPA) Coupled with Autosampling Microfluidic Chip Enables Highly Multiplexed On-Site Pathogen Screening.", "abstract": "A simple, rapid, low-cost, and multiplex detection platform is crucial for the diagnosis of infectious diseases, especially for on-site pathogen screening. However, current methods are difficult to satisfy the requirements for minimal instrument and multiplexed point-of-care testing (POCT). Herein, we propose a versatile and easy-to-use platform (FARPA-chip) by combining multiplex FARPA with an autosampling microfluidic chip. A pair of universal recombinase polymerase amplification (RPA) primers introduced during double-stranded cDNA (ds-cDNA) preparation are employed to amplify multiple targets, followed by amplicon-decoding with the chip, indicating no bias in amplifying different targets due to the universal RPA primers. FARPA-chip exhibits that as low as 10 copies of each target RNA in the starting sample can be sensitively detected by 12-plex detection of vector-borne viruses within 45 min and no cross-talk is observed between different targets. The feasibility of this platform is confirmed by designing a 9-plex FARPA-chip to detect 6 kinds of clinically common respiratory viruses from 16 clinical samples of nasopharyngeal swabs, and the results are completely consistent with RT-qPCR. Furthermore, by integrating quick extraction reagent, the turnaround time can be significantly decreased to <50 min, highlighting that our FARPA-chip enables a cost-effective on-site pathogen screening with a relatively high level of multiplexing. Depending on the number of chambers in the chip, the current design is theoretically capable of detecting up to 24 different pathogens, which should fulfill most clinical purposes. We believe that the proposed platform could provide an effective way for a series of healthcare-related applications in resource-limited settings.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.4c07015", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Ma Y", "dirs": [ "C" ] }, "40159755": { "pmid": "40159755", "title": "Rapid Universal Detection of High-Risk and Low-Abundance Microbial Contaminations in CAR-T Cell Therapy.", "abstract": "Live microbial contamination poses high risks to cell and gene therapies, threatening manufacturing processes and patient safety. Rapid, sensitive detection of live microbes in complex environments, such as CAR-T cell cultures, remains an urgent need. Here, an innovative sample-to-result workflow is introduced using digital loop-mediated isothermal amplification (dLAMP), enhanced by Electrostatic Microfiltration (EM)-based enrichment, for rapid sterility testing. By rationally designing primers targeting 16S and 18S rRNA, dLAMP assay enables both universal detection (covering >80% of known species) and strain-specific identification of bacterial and fungal contaminants in CAR-T cell spent medium and final products, directly from microorganism lysates. Enhanced by EM-based enrichment of low-abundance live microbes, the workflow achieves unparalleled sensitivity and speed, detecting contamination levels as low as 1 CFU/mL in complex CAR-T cell cultures within 6 h. Compared to qPCR and 14-day compendial methods, the approach demonstrates superior accuracy and significantly faster turnaround times. This workflow holds transformative potential for real-time monitoring in cell therapy manufacturing and rapid safety assessments of CAR-T cell products prior to patient infusion. Beyond cell therapy, the method is broadly applicable to infectious disease diagnostics, biomanufacturing monitoring, food safety, and environmental surveillance.", "journal": "Small methods", "year": "2025", "doi": "10.1002/smtd.202500253", "ptypes": [ "Journal Article" ], "first_author": "Wu X", "dirs": [ "C" ] }, "41965302": { "pmid": "41965302", "title": "A power-free sample-loading microfluidic chip integrated with a colorimetric loop-mediated isothermal amplification and a chromatic analysis via image processing for multiplex detection of parasitic diseases.", "abstract": "The gold standard diagnostic methods of gastrointestinal helminth parasites are the identification based on morphological characters of adult worms and the separation of the egg stage from host feces using the flotation or sedimentation techniques, both of which are time-consuming and require technical skills and parasitic knowledge. In our study, we develop a power-free sample-loading microfluidic chip integrated with a colorimetric loop-mediated isothermal amplification (LAMP) and a chromatic analysis for multiplex detection of parasitic diseases. The microfluidic chip can detect five parasitic groups simultaneously. The results can be interpreted with the naked eye or by chromatic analysis via image processing. The analytical specificity of this assay is specific to R. echinobothrida, R. tetragona, R. cesticillus, A. galli, and the Echinostomatidae group, and had minimum DNA detectable concentrations ranging from 4 ng to 40 pg/chip. The performance of the developed assay on fecal samples revealed a strong agreement with the gold standard method (Cohen's kappa value = 0.9); McNemar's test indicated no significant differences between both assays (p ‍> ‍0.05). Consequently, the proposed assay has potential to apply the detection of a wide range of pathogens and infectious diseases.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345419", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Panich W", "dirs": [ "C" ] }, "42515080": { "pmid": "42515080", "title": "Nucleic Acid Amplification Tests for Candida Species Identification: A Systematic Review of Diagnostic Performance.", "abstract": "Rapid and accurate identification of Candida species is critical for guiding antifungal therapy, especially with the emergence of intrinsically resistant pathogens. However, diagnostics using culture-based methods remain slow and labor-intensive, limiting timely treatment decisions. This systematic review evaluated the diagnostic performance and clinical applicability of nucleic acid amplification tests (NAATs) for Candida species identification using a PubMed search completed on 23 June 2025. A total of 888 records were screened, 333 full-text articles were assessed, and 158 studies were included based on criteria including comparison with standard diagnostic methods, diagnostic performance reporting, and involvement of clinical samples. PCR-based approaches were the most widely used, including conventional, nested, multiplex, real-time, and droplet digital PCR. Isothermal methods such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were increasingly represented. Common molecular targets included the ITS and 18S/28S rDNA regions. The risk of bias assessment was completed with the QUADAS-2 tool. Diagnostic performance varied depending on assay design, specimen type, gene target, and reference method. Excellent specificity and low limits of detection were achieved, especially with isothermal platforms offering the shortest turnaround times and greatest potential for point-of-care implementation. Multiplex assays were particularly advantageous for detecting mixed-species samples, while highly specific assays were optimal for distinguishing clinically important species such as Candidozyma auris, Nakaseomyces glabratus, and Pichia kudriavzevii. Overall, NAATs represent a promising diagnostic tool for Candida species identification, but broader clinical adoption will require improved standardization, validation across diverse patient populations, and clearer interpretation of fungal burden in the context of colonization versus infection.", "journal": "Pathogens (Basel, Switzerland)", "year": "2026", "doi": "10.3390/pathogens15070753", "ptypes": [ "Journal Article", "Systematic Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Czajka KM", "dirs": [ "C" ] }, "41249661": { "pmid": "41249661", "title": "A direct multiplex isothermal amplification-reverse dot blot hybridization system for β-thalassemia diagnosis.", "abstract": "Molecular diagnostic methods largely rely on expensive equipment and complex operations, which makes it difficult to achieve rapid and low-cost detection. In recent years, the increasing demand for point-of-care testing has driven the rapid development of isothermal amplification techniques, due to their simplicity and low equipment requirements. However, complex nucleic acid extraction steps are still required before most isothermal amplification. In this study, we propose a nucleic acid extraction-free gene detection method: direct multiplex Recombinase Aided Amplification combined with reverse dot blot hybridization (dmRAA-RDB). This method can detect multiple targets simultaneously and offers advantages such as high sensitivity, high specificity, low cost, no need for expensive instruments, and visual detection. By pre-treating whole blood samples with sodium hydroxide solution, the samples can be directly used for isothermal amplification and combined with commercial reverse dot blot (RDB) technology to rapidly detect 17 types of β-thalassemia mutations. The experimental results demonstrated that the pre-treated whole blood samples allowed for the simultaneous, stable, and efficient enrichment of all three target fragments via direct multiplex isothermal amplification. This single-tube triple amplification strategy demonstrates significant innovation in the field of isothermal amplification. Moreover, the workflow is shortened by nearly half, and a side-by-side comparison of 60 clinical samples showed 100 % agreement with the commercial PCR-RDB kit. The dmRAA-RDB method offers a highly promising, innovative solution for large-scale, rapid, and low-cost β -thalassemia screening in primary-care and resource-limited settings, and opens a new avenue for detecting other mutant genes.", "journal": "Annals of hematology", "year": "2025", "doi": "10.1007/s00277-025-06711-5", "ptypes": [ "Journal Article" ], "first_author": "Ye C", "dirs": [ "C" ] }, "41330665": { "pmid": "41330665", "title": "A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.", "abstract": "Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2025.344830", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Bao Y", "dirs": [ "C" ] }, "41224160": { "pmid": "41224160", "title": "A critical review of diagnostic strategies for bacterial pathogens in opium poppy (Papaver somniferum) cultivation.", "abstract": "Opium poppy (Papaver somniferum) is a pharmaceutically and economically strategic crop in which bacterial diseases cause measurable yield and quality losses. Yet the evidence base for diagnostics in poppy is fragmented, taxonomically inconsistent, and constrained by the crop's narcotic status. This review synthesizes historical records and updates taxonomy for priority taxa-most notably seed-borne Xanthomonas campestris pv. papavericola and soft-rot agents within Pectobacterium spp.-and compares conventional, molecular, immunological, point-of-care, and non-invasive optical/spectral approaches with a focus on fitness-for-purpose in poppy matrices (seed, capsule, vegetative tissues). We propose a unified, three-stage diagnostic workflow-screening (multiplex qPCR/LAMP with internal controls and viability-aware PMA/PMAxx-qPCR), target-specific confirmation (amplicon/targeted sequencing or ddPCR), and omics-level validation & QA-explicitly aligned with internationally recognized standards. The review integrates analytical performance with operational and economic considerations (throughput, matrix inhibition, LoB/LoD/LoQ reporting, measurement uncertainty) and maps diagnostic outputs into Integrated Disease Management (IDM) via threshold-based decision rules and precision-agriculture surveillance (UAV/HSI-assisted hotspot detection followed by molecular confirmation). We identify critical evidence gaps-poppy-specific inclusivity/exclusivity panels, inter-laboratory ring tests, validated molecular markers and primers, and real-time seed-health surveillance-and outline a phased regulatory pathway suited to narcotic-status constraints (license-bound sampling, chain-of-custody, and cross-border equivalence). Collectively, this roadmap translates contemporary diagnostic technologies into implementable, accreditation-ready practice for P. somniferum, enabling earlier, more reliable detection and directly informing sustainable IDM.", "journal": "Microbial pathogenesis", "year": "2026", "doi": "10.1016/j.micpath.2025.108167", "ptypes": [ "Journal Article", "Review" ], "first_author": "Erarslan G", "dirs": [ "C" ] }, "40070703": { "pmid": "40070703", "title": "Rapid Point-of-care Testing to Inform Intrapartum Treatment of Group B Streptococcus-Colonized Women in Uganda.", "abstract": "Maternal Group B Streptococcus (GBS) rectovaginal colonization is an important risk factor for invasive disease in neonates, yet availability of culture-based methods for detection is limited in low-resource settings. We evaluated the diagnostic performance of the HiberGene (HG) GBS loop-mediated isothermal amplification (LAMP) assay for the rapid detection of GBS in rectal/vaginal swabs collected from women in Uganda. This work forms a part of the PROGRESS GBS study. In phase 1, 1294 rectal and vaginal swabs were collected from pregnant women and inoculated in enrichment (Lim) broth, which was then tested using the HG GBS LAMP assay (sip gene target) and culture on chromogenic agar. In phase 2, 166 swabs from nonpregnant women were tested directly (without the enrichment step). For samples with discordant results, an additional method of testing against multiplex real-time polymerase chain reaction assay was used. Overall, the HG GBS LAMP assay detected more GBS-positive samples (31.3%; 452/1445) than culture-based methods (13.3%; 192/1445). Multiplex polymerase chain reaction-tested results were concordant with LAMP results in 96.3% of cases. The sensitivity and specificity of the LAMP assay, after adjusting for the tiebreaker results of discordant samples, were 94.4% (95% confidence interval, 86.2-99.4) and 99.0% (95% confidence interval, 94.3-100), respectively. The results of this study demonstrate high sensitivity and specificity of the HG GBS LAMP assay for the detection of GBS rectovaginal colonization in our setting. Given its rapid turnaround time, the HG GBS LAMP assay could appropriately be used to screen women for GBS rectovaginal colonization during labor to enable provision of intrapartum antibiotic prophylaxis.", "journal": "Open forum infectious diseases", "year": "2024", "doi": "10.1093/ofid/ofae605", "ptypes": [ "Journal Article" ], "first_author": "Nsimire Sendagala J", "dirs": [ "C" ] }, "39810344": { "pmid": "39810344", "title": "Multiplex Digital Nucleic Acid Analysis by a LAMP-Argonaute Coupling Assay via a Parallel Droplet Fusion SlipChip.", "abstract": "Multiplex digital nucleic acid analysis (NAA) allows the precise quantification of multiple target nucleic acids with single-molecule sensitivity, making it highly appealing for life science research and clinical diagnostics. Nucleic acid-guided endonucleases, such as CRISPR, have demonstrated great potential in digital NAA. However, performing multiplex digital NAA with an endonuclease remains challenging. The thermophilic Argonaute protein (Ago) enables specific targeting of multiple sequences by a single enzyme, exhibiting superior potential in multiplex detection. Here, we developed a multiplex digital NAA by coupling nucleic acid amplification and Ago-specific detection using parallel droplet fusion facilitated by a SlipChip. The SlipChip can generate a series of droplets to perform multiplex digital loop-mediated isothermal amplification (LAMP), followed by a series of droplets containing Ago reagents for parallel mixing and reactions, resulting in three distinct digital fluorescence signals (FAM, ROX, and Cy5) corresponding to each specific target sequence. We performed viral load analysis of respiratory viruses, including influenza A, influenza B, and SARS-CoV-2, within 60 min. In addition, we used this digital LAMP-Ago assay to analyze viral loads in 34 clinical samples. The system provides a multiplex digital NAA capable of precise nucleic acid quantification with high sensitivity and specificity.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.4c05145", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Luo Y", "dirs": [ "C" ] }, "42210595": { "pmid": "42210595", "title": "Development of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay and on-site rapid detection evaluation for Rodentibacter heylii and Rodentibacter pneumotropicus.", "abstract": "Rodentibacter pneumotropicus and Rodentibacter heylii are important exclusion pathogens in specific pathogen-free (SPF) laboratory animals. However, routine detection methods such as traditional bacterial culture remain labor-intensive and time-consuming. The loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay provides a self-contained format for nucleic acid testing that combines speed, sensitivity, and specificity while minimizing aerosol contamination, making it well-suited for point-of-need applications. This study aimed to develop a streamlined, on-site detection platform capable of rapidly identifying both pathogens with high analytical accuracy, operational simplicity, and suitability for routine laboratory surveillance. A LAMP-LFD system was established for the detection of the two pathogens using primers designed against the conserved 16S-23S rRNA ITS region. The assay's specificity, sensitivity, repeatability, and limit of detection (LoD) were evaluated. The assay achieved detection limits of 1 × 10-4 ng/μL for R. pneumotropicus and 1 × 10-5 ng/μL for R. heylii, demonstrating significantly higher sensitivity than PCR. No cross-reactivity was observed with 15 non-target bacteria. Among 28 infected samples, the method detected 19 positives, compared with 21 by multiplex PCR and 23 by qPCR. In 842 laboratory animal samples, culture-based, qPCR, and LAMP-LFD detected 2, 41, and 37 positive samples, respectively. The LoDs were 102 and 101 CFU/mL in fecal samples and 101 and 100 CFU/mL in throat swab samples for R. pneumotropicus and R. heylii, respectively. This method provides an approach for monitoring the health status of laboratory mice by enabling the detection of these two target pathogens.", "journal": "Animal models and experimental medicine", "year": "2026", "doi": "10.1002/ame2.70214", "ptypes": [ "Journal Article" ], "first_author": "Yan H", "dirs": [ "C" ] }, "40683387": { "pmid": "40683387", "title": "A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid colorimetric detection of pepper mild mottle virus (PMMoV).", "abstract": "Pepper mild mottle virus (Tobamovirus capsici, PMMoV) is a plant virus in the genus Tobamovirus that infects peppers and other members of the family Solanaceae. The virus is transmitted mechanically, poses a significant threat to crops globally, and is one of the most abundant viruses found in human feces and wastewater. Two colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect PMMoV, one targeting the RNA-dependent RNA-polymerase (PMMoV_RdRp) and the other targeting the coat protein (PMMoV_CP). Synthetic gBlock positive controls were used to determine the detection limit of each assay. PMMoV_RdRp detected PMMoV at concentrations greater than or equal to 100 copies/µL, the same sensitivity as the published RT-qPCR assay for this gene. In contrast, the detection limit of the PMMoV_CP RT-LAMP assay was an order of magnitude greater. Both assays were specific to PMMoV and did not amplify plant host tissue or related tobamoviruses. Since these RT-LAMP assays do not require specialized laboratory equipment and yield positive results within 20-30 min, they are advantageous for point-of-use testing. Overall, the RT-LAMP assays described here are sensitive, specific, and more rapid than existing methods for PMMoV detection and quantification and thus have potential widespread applications for agriculture, wastewater treatment assessment, recreational water quality testing, and food safety.", "journal": "Journal of virological methods", "year": "2025", "doi": "10.1016/j.jviromet.2025.115225", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Gross AJ", "dirs": [ "C" ] }, "40285984": { "pmid": "40285984", "title": "Pan-serotype reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting 2B-NSP coding region for colorimetric detection of foot-and-mouth disease virus in clinical samples.", "abstract": "Foot-and-mouth disease (FMD) is a highly contagious viral disease of even-toed animals. Rapid, early, and accurate diagnosis of the disease is important for the swift control of FMD. Although PCR-based assays are being used routinely for the effective diagnosis of FMD, these assays require sophisticated equipment, dedicated manpower, and complex procedures for the detection of amplified viral-genome. Colorimetric isothermal amplification assay with a sharp contrast in colour changes for the positive amplification of viral-genome would be qualified for quick and simple diagnosis of FMDV in both laboratory and field. Here, we report the development and evaluation of FMDV 2B-NSP coding region-based colorimetric RT-LAMP assay for pan-serotypic detection of viral-genome. Addition of 1 mg/ml of bovine serum albumin (BSA) as an additive, could reduce the detection time of the RT-LAMP assay from 60 to 30 min/reaction. Analytical sensitivity test showed that the RT-LAMP assay can detect up to 1000 copies of FMDV genome/reaction, simultaneously, the assay was found specific for the detection of FMDV genome as revealed on testing with serotypes O, A and Asia1 circulating in India during the last two decades. In addition, analysis of 312 clinical samples from various field outbreaks of FMDV in the country showed that RT-LAMP assay exhibited a sensitivity of 96.07%, and a specificity of 100% with an overall accuracy of 97.12%. Therefore, owing to the naked eye distinct visualization of amplified product (pink to yellow colour change), the RT-LAMP assay may facilitate rapid screening of FMD-suspected clinical samples without the use of hazardous DNA-binding dyes and simultaneously prevents aerosolization of amplified product, and subsequent carry over contamination in the diagnostic laboratory.", "journal": "Virus genes", "year": "2025", "doi": "10.1007/s11262-025-02158-y", "ptypes": [ "Journal Article" ], "first_author": "Biswal JK", "dirs": [ "C" ] }, "40290764": { "pmid": "40290764", "title": "Development of rapid and simple detection of bean common mosaic virus (BCMV) in mung beans (Vigna radiata) using reverse transcription-loop mediated isothermal amplification (RT-LAMP).", "abstract": "Bean common mosaic virus (BCMV) is one of the most serious and devastating Potyvirus of leguminous crops. In mung bean (Vigna radiata), BCMV is an emerging virus causing enormous losses to the crop, thereby reducing the production and profitability of the crop. Being seed borne and aphid transmitted virus, it important to reduce the spread and prevent its transfer to new geographical locations using rapid, specific and sensitive detection techniques. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was devised to rapidly and specifically detect BCMV. Three pairs of specific primers were designed targeting the BCMV genome. To determine the ideal temperature, reactions were carried out across a temperature range of 45 °C to 70 °C, with intervals of 5 °C. The optimal temperature for the assay was determined to be 60 °C with a 30-min incubation period. Comparison between the RT-LAMP and conventional reverse transcription polymerase chain reaction (RT-PCR) revealed that former can detect the BCMV upto 10- 9 and was one hundred times more sensitive than later. It was also determined that RT-LAMP was specific only in detecting BCMV, with no cross-reactivity with other closely related non-target viruses [potato virus Y (PVY), bean common mosaic necrosis virus (BCMNV), clover yellow vein virus (ClYVV) and soybean mosaic virus (SMV)]. After incubating the reactions at constant temperature of (60 °C/30 min), a characteristic ladder like banding pattern was observed on agarose gel for positive samples. Colorimetric tests (SYBR Green I) were also performed to reduce the requirement of laboratory equipment for visualizing RT-LAMP results. The results developed by SYBR Green I were comparable to that of agarose gel and can be visualized with naked eye. The developed RT-LAMP assay enables rapid detection of BCMV at 60 °C within a time period of 30-min.", "journal": "Virusdisease", "year": "2025", "doi": "10.1007/s13337-025-00916-y", "ptypes": [ "Journal Article" ], "first_author": "Ullah P", "dirs": [ "C" ] }, "39623085": { "pmid": "39623085", "title": "Diagnostic potential of rhinovirus C using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).", "abstract": "Rhinoviruses (RVs), particularly RV-C, frequently cause acute respiratory infections and asthma exacerbations. However, there is a lack of routine detection methods. Thus, this study aims to develop a rapid molecular and differential diagnostic detection method for RV-C using the reverse transcription (RT) loop-mediated isothermal amplification (LAMP) approach. The RT-LAMP assay targeting the 5'UTR region of RV-C genome was optimized by varying the reaction temperature, magnesium sulphate, betaine concentrations, and reaction time. Compared with conventional RT-PCR with a sensitivity of 106 copies of RNA, RT-LAMP demonstrated a significant increase in efficiency and sensitivity with a quantifiable viral load of at least 101 copies of RNA by gel electrophoresis and colour change, and 104 copies of RNA for end-point detection with a turbidimeter for 40 min. The assay is also specific without amplifying RV-A16 and RV-B72 genomic RNA. In the proof-of-concept assay using 30 clinical respiratory samples with known etiological agents, it detected all RV-C isolates, of which its accuracy was confirmed by sequencing. The newly developed RT-LAMP assay demonstrated good analytical sensitivity and specificity toward RV-C. The assay provides an alternative for improved RV-C diagnosis.", "journal": "International microbiology : the official journal of the Spanish Society for Microbiology", "year": "2025", "doi": "10.1007/s10123-024-00621-8", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Wong YP", "dirs": [ "C" ] }, "40256951": { "pmid": "40256951", "title": "Reverse-Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Viral Detection - A Case Study.", "abstract": "An assay and protocol framework are provided for utilizing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) at the point-of-care for diagnosing and monitoring a hypothetical zoonotic viral outbreak in a resource-limited area. This manuscript utilizes a previously published decision tree algorithm to determine an appropriate molecular diagnostic point-of-care test that can effectively address the outbreak presented in the hypothetical case study. © 2025 Wiley Periodicals LLC.", "journal": "Current protocols", "year": "2025", "doi": "10.1002/cpz1.70127", "ptypes": [ "Journal Article" ], "first_author": "Hassman A", "dirs": [ "C" ] }, "40883771": { "pmid": "40883771", "title": "Colorimetric reverse transcription loop-mediated isothermal amplification assay for visual, sensitive, and specific detection of enterovirus G.", "abstract": "Enterovirus G (EV-G) is widely prevalent in pigs worldwide, and co-infections of EV-G and other diarrhea-causing pathogens have been reported in many countries, threatening the pig farming industry. There are some methods available for EV-G detection; however, the RT-LAMP method for detecting EV-G has not yet been developed. The aim of thisstudy was to establish a highly sensitive and visual RT-LAMP assay for the detection of EV-G. A colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the visual detection of porcine enterovirus G (EV-G). The assay can be completed in 45 min at 64 °C, and the results can be observed by the unaided eye using the colorimetric LAMP master mix. The limit of detection (LOD) of the RT-LAMP assay was 46.8 copies of EV-G RNA, which is comparable to that of the previously described real-time RT-PCR (qRT-PCR) method and is 100-fold more sensitive than that of conventional RT-PCR. The assay specifically amplified the RNA of EV-Gs, and there was no cross-amplification with other porcine pathogens. In the clinical evaluation, the Kappa coefficient of the established RT-LAMP assay and the previously reported qRT-PCR was was 0.862 (P < 0.01), and the nucleic acid of EV-G could be tested from the fecal samples of porcine at different infection stages. In this study, we successfully established a visual, sensitive, specific, rapid, and convenient RT-LAMP method for the detection of EV-G, which was successfully applied in the detection of EV-Gs in clinical samples.", "journal": "BMC veterinary research", "year": "2025", "doi": "10.1186/s12917-025-04970-y", "ptypes": [ "Journal Article" ], "first_author": "Li ZH", "dirs": [ "C" ] }, "41397899": { "pmid": "41397899", "title": "A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.", "abstract": "Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations. After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout. HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100 IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500 μL of plasma, genotype 1 RNA ≥100 IU/mL was detected within 40-45 minutes, while genotype 3 had an LLOD of 5000 IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection. Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45 minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.", "journal": "The Journal of infectious diseases", "year": "2026", "doi": "10.1093/infdis/jiaf609", "ptypes": [ "Journal Article" ], "first_author": "Chowdhury A", "dirs": [ "C" ] }, "40021320": { "pmid": "40021320", "title": "Two colorimetric LAMP systems for nucleic acid-based diagnostics.", "abstract": "This study introduces an advanced 8-well loop-mediated isothermal amplification (LAMP) system specifically designed for the automated colorimetric detection of SARS-CoV-2. Incorporating two distinct configurations having either three light-emitting diodes (LEDs) with varying emission wavelengths per well, paired with a photodiode detector, or utilizing white LED illumination with a red, green, and blue (RGB) sensor. The colorimetric LAMP aims to provide a more accessible and rapid diagnostic tool than traditional fluorescence methods due to the system's simplicity. We designed, assembled, and compared two colorimetric home-assembled LAMP systems, the first one based on three LEDs, each with a different color with a photodiode, and the second one having RGB and a white LED, with traditional fluorescence-based LAMP method performed on a commercial qPCR instrument. Results demonstrated that the colorimetric RT-LAMP assays achieved critical threshold time (CT), closely matching the CT value of fluorescence-based detection accomplished by the qPCR instrument. We performed the fundamental experiment employing an identical RNA copy number of 1,570copies·μL-1, getting the CT value of (16.70 ± 0.43) min (mean ± standard deviation from 23 measurements). Then, we also performed different RNA numbers of copies between the highest and lowest RNA contents of ≈ 157,000 copies·μL-1 and ≈ 1570 copies·μL-1, respectively, getting CT values from (13.30 ± 0.04) min to (13.75 ± 0.30) min and (17.04 ± 0.02) min to (17.26 ± 0.02) min, all (mean ± standard deviation from three measurements). The colorimetric systems demonstrated rapid response and precision across varied viral loads while keeping the system simple due to the colorimetric detection method. The LAMP system's rapid and precise detection capabilities underscore its potential as an effective tool for point-of-need diagnostics. It is crucial for timely responses in ongoing and future pandemic scenarios. This system enhances testing accessibility and provides a robust platform for potential adaptation to other pathogenic threats, making it a valuable asset in global health diagnostics.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.343752", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Sun A", "dirs": [ "C" ] }, "41086990": { "pmid": "41086990", "title": "WarmStart colorimetric reverse transcription-loop-mediated isothermal amplification assay for the visual, sensitive, and specific detection of epizootic haemorrhagic disease virus in China.", "abstract": "A pan-specific colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect Chinese epizootic haemorrhagic disease virus strains (EHDVs) inaugurally. The assay could be completed in 45 min at 64 °C, and could detect as few as 15 copies of EHDV RNA, the coincidence rates of the established RT-LAMP assay and previously reported qRT-PCR were more than 86.7 % in the clinical evaluation.", "journal": "Journal of microbiological methods", "year": "2025", "doi": "10.1016/j.mimet.2025.107292", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Li Z", "dirs": [ "C" ] }, "40640128": { "pmid": "40640128", "title": "Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection.", "abstract": "The COVID-19 pandemic evidenced the urgent need for rapid, accurate, and scalable diagnostic methods for emerging infectious diseases. Droplet digital reverse transcription LAMP (ddRT-LAMP) is a promising technique for pathogen detection and accurate quantification, as it overcomes traditional LAMP's limitations in viral load estimation through reaction partitioning and digital analysis. However, many parameters must be adjusted to avoid spurious results. This study evaluates the critical conditions for effective ddRT-LAMP quantification of the SARS-CoV-2 N gene in plasmid DNA, synthetic RNA, and nasopharyngeal swab samples. Using a polydimethylsiloxane (PDMS) microfluidic device, the RT-LAMP reaction mixture with a fluorescent dye was divided into thousands of droplets stabilized by a surfactant in fluorinated oil. After incubation, the droplets were injected into a PDMS chamber for fluorescent imaging to determine the proportion of positive droplets and quantify the samples based on the Poisson distribution. The results showed that primer design and master mix composition significantly impacted the amplification. The selection of GelGreen® as the fluorescent dye was crucial, as other dyes tested diffused into the oil phase. Optimal amplification occurred with 105 µm droplet diameter and 30-min incubation, achieving detection and quantification limits of 102 cp/µL. By addressing these operational challenges, ddRT-LAMP can become a more effective tool for viral detection and quantification in clinical diagnostics.", "journal": "Microsystems & nanoengineering", "year": "2025", "doi": "10.1038/s41378-025-00982-8", "ptypes": [ "Journal Article" ], "first_author": "Chávez-Ramos K", "dirs": [ "C" ] }, "41536658": { "pmid": "41536658", "title": "[Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Detection of Genogroup Ⅱ Norovirus].", "abstract": "To establish a rapid testing method for genogroup Ⅱ (GⅡ) norovirus based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The conserved region of norovirus was screened using the genomic data from the National Center for Biotechnology Information (NCBI) database, and specific primers were designed using the online software Primer Explorer V5. The testing system was established through optimization of experimental conditions, including reaction temperature, MgSO4 concentration, dNTP concentration, etc. The sensitivity and specificity of the method were evaluated. The optimized testing method was used to analyze clinical stool samples, and the results were compared with those of a commercially available kit. The established testing method exhibited high specificity, showing no cross-reactivity with adenovirus, human enterovirus, or rotavirus. The testing method also exhibited high sensitivity, with a detection limit of 51 copies/μL. The turnaround time was 45 min. For 34 stool samples from patients infected with norovirus, the testing results were highly consistent with those of the commercial kit, with a sensitivity of 100%, a specificity of 83.33%, and an area under the receiver operating characteristic (ROC) curve of 0.97 (95% CI: 0.92-1.00), indicating the high accuracy and reliability of the testing method. In this study, we successfully developed an RT-LAMP-based testing method for GⅡ norovirus, which exhibits excellent sensitivity, specificity, and practical performance in clinical sample testing, making it suitable for use in primary care laboratories and for rapid testing in field settings.", "journal": "Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition", "year": "2025", "doi": "10.12182/20251160201", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "Liu Y", "dirs": [ "C" ] }, "40768948": { "pmid": "40768948", "title": "Point-of-need one-pot multiplexed RT-LAMP test for detecting three common respiratory viruses in saliva.", "abstract": "Respiratory viral infections pose a significant global public health challenge, partly due to the difficulty in rapidly and accurately distinguishing between viruses with similar symptoms at the point of care, hindering timely and appropriate treatment and limiting effective infection control and prevention efforts. Here, we developed a multiplexed, non-invasive saliva-based, reverse transcription loop-mediated isothermal amplification (RT-LAMP) test that enables the simultaneous detection of three of the most common respiratory infections, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza (Flu), and respiratory syncytial virus (RSV), in a single reaction via specific probes and monitored in real-time by a machine-learning-enabled compact analyzer. Our results demonstrate that the multiplexed assay can effectively detect three target RNAs with high accuracy. Further, testing with spiked saliva samples showed strong agreement with reverse transcription polymerase chain reaction (RT-PCR) assay, with area under the curve (AUC) values of 0.82, 0.93, and 0.96 for RSV, Influenza, and SARS-CoV-2, respectively. By enabling the rapid detection of respiratory infections from easily collected saliva samples at the point of care, the device presented here offers a practical and efficient tool for improving outcomes and helping prevent the spread of contagious diseases.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117836", "ptypes": [ "Journal Article" ], "first_author": "Kshirsagar A", "dirs": [ "C" ] }, "40526856": { "pmid": "40526856", "title": "Enabling SARS-CoV-2 Wastewater Surveillance Using an Integrated Microfluidic Chip.", "abstract": "Wastewater-based epidemiology (WBE) has emerged as a critical public health tool, offering cost-effective and noninvasive surveillance of infectious diseases through wastewater testing. Despite its potential, WBE implementation in low-resource settings is hindered by infrastructure limitations, lack of technical expertise, and dependence on centralized laboratories. This study presents a novel concentration-extraction-identification device (CEID), a point-of-use platform that integrates nucleic acid concentration, extraction, and detection into an affordable and user-friendly chip. The CEID employs immiscible filtration assisted by surface tension (IFAST) for nucleic acid extraction and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for pathogen detection. Key parameters of the CEID were characterized and optimized, resulting in efficient nucleic acid extraction with minimal carry-over of inhibitory molecules. The performance was validated using SARS-CoV-2 spiked wastewater samples, achieving a detection limit of 113 Cp/mL. The CEID was further validated through a 6-week surveillance study, successfully detecting SARS-CoV-2 in wastewater from two locations with a high correlation to standard WBE methods and RT-qPCR. Costing approximately $11 per sample and requiring minimal hands-on time, the CEID demonstrates significant advantages in affordability, simplicity, and adaptability for rural and underserved regions. This technology offers a scalable solution to democratize WBE, enhancing global infectious disease surveillance and response, particularly in low- and middle-income countries.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c00831", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Dehghan Banadaki M", "dirs": [ "C" ] }, "40608622": { "pmid": "40608622", "title": "An alternative real-time fluorescence reverse transcription loop-mediated isothermal amplification assay for the rapid detection of SARS-CoV-2.", "abstract": "COVID-19, caused by SARS-CoV-2 virus infection, remains a public health concern in many countries. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and cost-effective alternative test for COVID-19 diagnosis. In this study, we developed and evaluated a real-time RT-LAMP (rRT-LAMP) assay coupled with a melting curve analysis to detect SARS-CoV-2. The reaction was carried out in a real-time thermal cycler at 63 °C for 45 min to amplify the region of SARS-CoV-2 orf8; real-time monitoring of amplification was performed by fluorescence detection. The performance was assessed by comparing it to a real-time reverse transcription-polymerase reaction (rRT-PCR) as a reference. The rRT-LAMP could detect as few as 15 copies of SARS-CoV-2 RNA per reaction. Positive results appeared within 30 min, while the melting-temperature analysis could verify the amplification specificity. No positive results from non-SARS-CoV-2 templates and no mis-amplification were observed. The comparative analysis using 262 RNA extracted from nasopharyngeal swab samples revealed the overall accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the rRT-LAMP at 88.55% (95% CI: 77.52-100%), 84.13% (95% CI: 71.56-98.27%), 100% (95% CI: 78.38-100%), 100% (95% CI: 85.06-100%), and 70.87% (95% CI: 55.55-89.11%), respectively. The greatest sensitivity was as high as 98-100% for specimens with threshold rRT-PCR cycle (Ct) values of less than 30 cycles. Overall, this rRT-LAMP showed good performance for the rapid detection of SARS-CoV-2. It is proposed as a potential method for real-time amplification detection, offering increased laboratory capacity for SARS-CoV-2 testing in a cost-effective and timely manner.", "journal": "Revista do Instituto de Medicina Tropical de Sao Paulo", "year": "2025", "doi": "10.1590/S1678-9946202567037", "ptypes": [ "Journal Article" ], "first_author": "Rudeeaneksin J", "dirs": [ "C" ] }, "39899502": { "pmid": "39899502", "title": "Surfaces environmental monitoring of SARS-CoV-2: Loop mediated isothermal amplification (LAMP) and droplet digital PCR (ddPCR) in comparison with standard Reverse-Transcription quantitative polymerase chain reaction (RT-qPCR) techniques.", "abstract": "The persistence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on substrates, and the impact of fomites on Coronavirus Disease 19 (COVID-19) transmission, is until now, widely discussed. Consequently, further investigations are required for a correct risk assessment in high-risk facilities such as hospitals, healthcare facilities (HCFs), and long-term care facilities (LTCFs). Therefore, appropriate surveillance and disinfection programs represent the best approach to guarantee the safety of these communities. This study proposes an environmental SARS-CoV-2 surfaces routine monitoring approach in HCF and communities' settings, to provide rapid and effective evaluation of surface hygienic conditions and the effectiveness of applied sanitization measures. Surfaces samples (n = 118) were collected using the SRK® kit (Copan Italia) from 2020 to 2023. Three molecular techniques were compared: Reverse Transcription Loop mediated isothermal AMPlification (RT-LAMP, Enbiotech), Reverse-Transcription quantitative polymerase chain reaction (RT-qPCR) (RT-qPCR, Seegene) and droplet digital PCR (ddPCR, Bio-Rad). For ddPCR, two RNA extraction methods were compared: TRIzol LS (Invitrogen) versus QIAmp Viral Mini kit (QIAGEN), showing how the latter is more suitable for surfaces. Regarding the quantitative ddPCR results, the ROC analysis allowed to reduce the manufacturer cut-off for droplets number (from 3 to 1) for the positive samples. Moreover, a new cut-off for the viral RNA copies' number/μL for each target (N1 and N2) on environmental monitoring was fixed at 2,82. The results obtained using the QIAmp kit, suggested that the N2 target is more stable in the environment and could be most suitable for the virus environmental detection. The percentage of positive samples was similar among the techniques (26% for RT-LAMP, 36% for ddPCR and 23% for RT-qPCR). Using RT-qPCR as reference method, a sensitivity (SE) of 30% for RT-LAMP and 41% for ddPCR was observed. By contrast, specificity (SP) was higher for RT-LAMP (75%) respect to ddPCR (66%). Comparing the faster RT-LAMP with the sensitive ddPCR the 26% and 74% of SE and SP for RT-LAMP, were reported. The low sensitivity for RT-LAMP and ddPCR could be explained with the use of clinical rather than environmental kits, other than the changing in the virus prevalence during the sampling campaign. Although the RT-LAMP requires improvements in term of SE and SP, this research presents an innovative environmental monitoring and prevention method for SARS-CoV-2, that could be extended to other pathogens that are under environmental surveillance.", "journal": "PloS one", "year": "2025", "doi": "10.1371/journal.pone.0317228", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "Spiteri S", "dirs": [ "C" ] }, "40573373": { "pmid": "40573373", "title": "Evaluation of RT-LAMP for SARS-CoV-2 Detection in Animal Feces.", "abstract": "The wide host range, potential lethality, and zoonotic potential of SARS-CoV-2 infection in animals highlights the need for additional surveillance strategies. We validated a commercial, pH-based, colorimetric RT-LAMP assay for the detection of SARS-CoV-2 RNA in animal feces. The comparator assay was rRT-PCR. The limit of detection of the RT-LAMP assay was 72 genome copies per reaction. RT-LAMP was highly specific for SARS-CoV-2 and did not detect other human or animal coronaviruses. RT-LAMP was robust, with valid results generated for incubation lengths of 30 to 45 min, incubation temperatures of 60 to 70 °C, and reaction volumes of 10 to 25 µL. The diagnostic sensitivity was 100% for clinical fecal samples with high viral loads (Ct ≤ 25), 97.4% for samples with moderate to high viral loads (Ct ≤ 33), and 62% overall (Ct ≤ 40). The diagnostic specificity was 97.9%. Blinded method testing organized by an independent laboratory confirmed the satisfactory reproducibility of the assay. To our knowledge, this study represents the first validation of RT-LAMP for SARS-CoV-2 detection in animals. RT-LAMP testing could detect SARS-CoV-2 infection more rapidly and at the point of care in animals with moderate to high viral loads, allowing for earlier implementation of control measures.", "journal": "Viruses", "year": "2025", "doi": "10.3390/v17060783", "ptypes": [ "Journal Article", "Evaluation Study", "Research Support, U.S. Gov't, P.H.S." ], "first_author": "Pepper A", "dirs": [ "C" ] }, "40302734": { "pmid": "40302734", "title": "Development of a Rapid Surveillance System for Ross River Virus in Mosquitoes Through Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).", "abstract": "The global rise in arboviral diseases can be attributed to the ongoing effects of climate change. Ross River virus (RRV) is an illustrative example of such diseases, with case reports in Australia experiencing a significant surge since 2020. RRV is transmitted to susceptible species, such as horses and humans, through multiple mosquito vectors, namely Culex annulirostris, Aedes camptorhynchus, and more recently Ae. notoscriptus. This disease is not only endemic to Australia but has caused outbreaks in surrounding countries such as Fiji and Papua New Guinea. Currently, there are no therapeutic regimes or vaccinations available for RRV, leaving public health warning systems and advice relying upon disease prediction and surveillance. Commonly utilised methods, such as predictive modelling, are experiencing challenges resulting from an increased mosquito presence and extreme weather patterns, often yielding inaccurate advice. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) provided a promising solution to mitigate these challenges and is now considered the gold standard in many Australian states. However, this method must be performed in a laboratory setting and requires expensive machinery, thus rendering it inadequate for resource-poor or rural communities. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) serves as a simple and field-deployable substitute with comparable sensitivities and specificity to RT-qPCR, whilst possessing the ability to provide rapid results within 20 min. This paper describes a novel RRV RT-LAMP assay that can detect RRV in as little as one mosquito, with a limit of detection of 1 × 10-7 ng/µl (~620 copies/µl) and a clinical sensitivity of 84%. Through the addition of tetramethylammonium chloride (TMAC), our assay achieved a 100% specificity and was able to detect RRV RNA as early as 2 min in crude field samples. The simplistic sampling method coupled with our RRV RT-LAMP assay can provide an in-field and low-cost alternative to current routine surveillance techniques.", "journal": "Transboundary and emerging diseases", "year": "2025", "doi": "10.1155/tbed/1772438", "ptypes": [ "Journal Article" ], "first_author": "Knox A", "dirs": [ "C" ] }, "40694257": { "pmid": "40694257", "title": "Development of RT-LAMP assay for variants of HCV in Pakistan.", "abstract": "Hepatitis C Virus (HCV) elimination requires improved diagnostic methods, particularly in regions with high screening demands like Pakistan. Rapid and accurate detection is crucial, yet commercially available RT-qPCR assays often have limitations in sensitivity and specificity, reducing their reliability across diverse populations. Additionally, RT-qPCR is time-consuming, costly, and requires skilled personnel. In contrast, Re-verse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) offers a robust, cost-effective alternative with superior sensitivity and specificity. In the present study, for the first time, we reported a novel RT-LAMP for HCV diagnosis in Pakistan. We designed and optimized an RT-LAMP assay using degenerate primers targeting the 5’ UTR of HCV, incorporating sequences from reference and emerging genotypes, including Pakistani isolates. Key assay parameters, including reaction time, volume, primer concentration, and temperature, were optimized. Triple confirmation of HCV clinical samples using different sets of primers for conventional PCR, commercial lab RT-qPCR and RT-LAMP provided a unique validation approach. It was demonstrated that RT-LAMP detected HCV at a limit of detection of 131 copies, outperforming commercial RT-qPCR by identifying false negatives (71%) and achieving 100% sensitivity and specificity, compared to 82% sensitivity of RT-qPCR. The assay’s processing time was 60 min, supporting large-scale diagnostics. BLAST analysis confirmed specificity, with no significant alignment to related Flaviviridae genomes. RT-LAMP represents a significant advancement in HCV detection, offering high sensitivity, specificity, and ease of use, making it ideal for effective prevention and control strategies.", "journal": "Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]", "year": "2025", "doi": "10.1007/s42770-025-01745-3", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Mumtaz Z", "dirs": [ "C" ] }, "42655647": { "pmid": "42655647", "title": "Single-Tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Red-Spotted Grouper Nervous Necrosis Virus in Fish Species.", "abstract": "Betanodavirus is a causative agent of viral nervous necrosis (VNN) and a major threat to marine and brackish-water aquaculture globally. This virus causes epizootic outbreaks with particularly high morbidity and mortality in larval and juvenile stages and causes significant economic losses in aquaculture. Here, we developed a rapid and highly sensitive single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) assay for the detection of red-spotted grouper nervous necrosis virus (RGNNV) genotype infection in fish tissue samples. Six primers targeting eight conserved regions of the RNA2 coat protein gene of RGNNV were designed with conservation regions across RGNNV genotypes. RT-LAMP assay was completed within 60 min at 65 °C using a single-tube format that combined reverse transcription and isothermal amplification, and results were directly visualized by the addition of SYBR Green I dye, producing a colour change from orange (negative) to green (positive), observable with the naked eye or under UV illumination. The developed RT-LAMP assay was able to detect five copies of RGNNV from infected samples, which was 20-fold more sensitive than conventional reverse transcription-PCR. The assay demonstrated diagnostic sensitivity and specificity in two fish hosts (Asian seabass and cobia) and showed no cross reactivity with other fish viruses such as tilapia lake virus and cyprinid herpesvirus-2. The developed assay is simple, cost-effective, specific, and enables rapid detection of RGNNV in fish tissues. This single-tube RT-LAMP assay can be applied to screening broodstock facilities, fingerlings, aquaculture farms, quarantine facilities, and juveniles before stocking in ponds or cages, helping prevent disease outbreaks and the transmission of RGNNV in aquaculture systems.", "journal": "Viruses", "year": "2026", "doi": "10.3390/v18080827", "ptypes": [ "Journal Article" ], "first_author": "Pradeep MA", "dirs": [ "C" ] }, "41113156": { "pmid": "41113156", "title": "A One-Pot RT-LAMP Diagnostic Assay for SARS-CoV‑2 from Saliva Samples.", "abstract": "Results of efforts to diagnose infections with SARS-CoV-2 using a sampling method that was less invasive than the nasopharyngeal swab led to the rapid adoption of anterior nasal swabs. Saliva was also shown to have potential as a sample matrix and, like anterior nasal swabs, could be obtained noninvasively (e.g., passive drool). However, due to its inherent complexity and heterogeneity across patient populations (e.g., presence of mucins and RNases), saliva was largely disregarded as point-of-care diagnostics were being developed and broadly implemented. For molecular diagnostic approaches (e.g., RT-PCR or RT-LAMP), these matrix effects from saliva could lead to undesirable false positives or false negatives. The opportunity to address these challenges by normalizing the performance of saliva could enable important applications of molecular tests, particularly at the point-of-care. Toward these goals, we developed a one-pot RT-LAMP assay for the colorimetric detection of SARS-CoV-2 from saliva samples. The assay is performed in five steps: (i) a patient collects a passive saliva sample, (ii) the sample is placed on a heat block for 10 min at 95 °C, (iii) the undiluted sample is added to the one-pot RT-LAMP assay, (iv) the RT-LAMP reaction tube is place on a heat block for 40 min at 65 °C, and, (v) immediately postamplification, the reaction tube is inverted to observe the colorimetric output. We demonstrated the clinical performance of our assay using a panel of 127 patient samples. Our assay had an overall accuracy of 98%, with a sensitivity of 88% and a specificity of 100%. These results indicate excellent diagnostic agreement with the gold standard, RT-PCR, and highlight the potential to improve the clinical utility of saliva for point-of-care (e.g., mobile clinics) testing of SARS-CoV-2 and other upper respiratory viruses and emerging pathogens.", "journal": "ACS measurement science au", "year": "2025", "doi": "10.1021/acsmeasuresciau.5c00063", "ptypes": [ "Journal Article" ], "first_author": "Mora AC", "dirs": [ "C" ] }, "41992849": { "pmid": "41992849", "title": "Hydrogel RT-LAMP Enables Rapid, Quantitative Assessment of Mycobacterium tuberculosis Immune Responses.", "abstract": "Tuberculosis (TB) remains a global health challenge, requiring rapid and accessible diagnostics to guide preventive therapy. C-X-C motif chemokine ligand 10 (CXCL10) mRNA is a promising biomarker of TB infection, yet current assays rely on long stimulation times and complex instrumentation. Here, we present a hydrogel-based reverse transcription loop-mediated isothermal amplification (HRT-LAMP) assay that partitions amplification reactions within a porous hydrogel matrix, enabling digital-like quantification of CXCL10 mRNA using only a heater and smartphone imaging. A hand-held 3D-printed imaging device was developed to capture fluorescence signals, which were subsequently quantified by a convolutional neural network (CNN). The assay demonstrated a detection limit of 10 copies per reaction with excellent linearity (101-105 copies/test, R2 = 0.998), supporting a dynamic range up to 105 copies/test via high-resolution microscopy and 10 to 104 copies/test using the portable smartphone platform. In 79 samples used to establish diagnostic thresholds, HRT-LAMP reached 93.6% sensitivity and 100% specificity. In an independent validation cohort of 91 samples, it achieved 91.2% agreement with clinical diagnosis with sensitivity 92.3%, specificity 89.7%, κ = 0.82, and area under the curve (AUC) = 0.918, outperforming interferon-γ release assays (IGRA) used in clinics. Moreover, accurate results were maintained with as little as 50 μL of blood (95.7% agreement). HRT-LAMP thus enables rapid (<4 h), sensitive, and field-deployable quantification of CXCL10 mRNA responses, offering a practical platform for TB diagnosis in resource-limited and pediatric settings.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c07282", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Wang J", "dirs": [ "C" ] }, "40135490": { "pmid": "40135490", "title": "CRISPR/Cas12-mediated detection of GI and GII Norovirus in different food samples.", "abstract": "Norovirus is one of the leading causes of infectious diarrhea, occurring in about 18% of diarrhea cases worldwide. Norovirus is characterized by a low infectious dose, rapid onset, and strong transmission capacity. Given the lack of specific drugs and vaccines, developing efficient and accurate detection technologies is of great significance to prevent and control the spread of diseases. This study combined the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology with the clustered regularly interspaced short palindromic repeats (CRISPR) technology to develop a sensitive and rapid detection method, which can reduce the reliance on temperature control and expensive real-time fluorescent polymerase chain reaction (PCR) devices. The RT-LAMP/CRISPR Cas12a method demonstrated good specificity and sensitivity, testing food samples of three different substrates with 100% positive qualitative accuracy. The detection sensitivity is 32.8 copies/reaction for Norovirus GI and 22.8 copies/reaction for Norovirus GII. This method helps to effectively identify food products contaminated with Norovirus, thereby preventing human infections and economic losses due to disease outbreaks.", "journal": "Journal of food science", "year": "2025", "doi": "10.1111/1750-3841.70160", "ptypes": [ "Journal Article" ], "first_author": "Gou S", "dirs": [ "C" ] }, "40236396": { "pmid": "40236396", "title": "ViraLite: An Ultracompact HIV Viral Load Self-Testing System with Internal Quality Control.", "abstract": "The availability of effective antiretroviral therapy has made HIV manageable, provided patients have consistent access to routine viral load (VL) testing. Nonetheless, access to frequent VL testing remains limited. There is a need for accessible, user-friendly testing systems that allow people living with HIV (PLHIV) to monitor their VL more frequently and empower self-management. Here, we developed ViraLite, a sample-to-answer, compact, accessible, and battery-powered system for HIV viral load monitoring. The system is built upon a probe-based RT-LAMP assay that allows for multiplexed detection and quantification. An internal quality control targeting the RNase P was incorporated to enhance the reliability of the results. A software-reconfigurable real-time sensing system empowered by machine learning and a smartphone-guided protocol was developed in tandem to analyze the multiplexed assay. We analyzed 45 clinically archived samples using ViraLite and benchmarked our results against qRT-PCR, which showed 21 positive and 23 negative samples. Using our process control, ViraLite first identified 17 inconclusive samples that would otherwise be classified as negative. Then, ViraLite classified 14 out of 15 HIV-positive samples (93.3%) and 13 out of 13 HIV-negative samples (100%). The incorporation of RNase P as a process control increased the sensitivity of ViraLite from 66.66% to 93.33%, while maintaining a high specificity (100%). To assess the acceptance of ViraLite among PLHIV, we recruited 480 participants from online and three clinical sites to complete a survey. Over 86% of participants indicated ViraLite had benefits in convenience and privacy, on the other hand 61% of participants indicated concerns with test accuracy. The integration of compact hardware, a reliable assay, and smartphone guidance provides an accurate, easy to use system for PLHIV to self-manage their viral load and update their prescriptions frequently.", "journal": "medRxiv : the preprint server for health sciences", "year": "2025", "doi": "10.1101/2025.04.01.25325036", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Politza AJ", "dirs": [ "C" ] }, "41275804": { "pmid": "41275804", "title": "High throughput influenza A virus detection by isothermal amplification in sequential-injection paper-based microfluidics.", "abstract": "The recognized impact of epidemics and pandemics caused by Influenza A virus highlights the need for rapid, sensitive, and affordable diagnostic methods. In this work, we propose a molecular detection strategy for Influenza A viruses that combines Electrochemical reverse transcription Loop-Mediated Isothermal Amplification (E-RT-LAMP) using methylene blue (MB) as a redox-active probe, with detection carried out on a sequential-injection paper-based microfluidics (μPAD). The high amplification efficiency of the LAMP technique, following specific target recognition, combined with the intercalation of MB into double-stranded DNA enabled label-free detection of the target sequence through current variation with μPAD. The microfluidic platform was based on the combination of a filter paper disc with 3D pen-templated electrodes, enabling low-cost, portable, and reproducible analysis. The μPAD system exhibited a limit of detection of 9.24 × 101 copies per μL, and following the amplification reaction, detection provided results within seconds (∼3 diagnoses per minute). When tested on a panel of sequenced clinical samples, the assay showed no cross-reactivity with other similar respiratory viruses and demonstrated 100 % accuracy relative to reverse transcription quantitative PCR (RT-qPCR). These results demonstrate the potential of this strategy for point-of-care (POC) diagnostics, offering a promising alternative to conventional laboratory-based molecular methods.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2025.118249", "ptypes": [ "Journal Article" ], "first_author": "de Lima LF", "dirs": [ "C" ] }, "42075686": { "pmid": "42075686", "title": "Detection of Dengue Virus Serotype 3 Using a Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay: Evaluation with Clinical Samples from Southeastern Mexico.", "abstract": "Dengue virus (DENV), an important mosquito-borne orthoflavivirus, represents a growing global threat due to its geographic expansion and recent outbreaks worldwide. In resource-limited endemic settings, the development of affordable diagnostic assays is needed. In this study, we developed and validated a colorimetric reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the detection of DENV type 3 (DENV-3) using 95 previously diagnosed clinical samples from Southeastern Mexico. Primers targeting the 3' untranslated region (3' UTR) of DENV-3 were designed, and assay conditions were standardized. The colorimetric RT-LAMP DENV-3 system achieved a preliminary limit of detection of 1 × 103 copies per reaction, with 90.7% sensitivity and 100% specificity. The colorimetric format enabled visual readout without specialized equipment, supporting its potential applicability in point-of-care and resource-limited settings. The developed colorimetric RT-LAMP detection for DENV-3 is intended as a rapid screening/triage tool that can trigger confirmatory testing or public-health actions.", "journal": "Pathogens (Basel, Switzerland)", "year": "2026", "doi": "10.3390/pathogens15040359", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "Pérez-Tepos P", "dirs": [ "C" ] }, "41802806": { "pmid": "41802806", "title": "Armored RNA technology as a clinical diagnostics tool for future pandemic preparedness.", "abstract": "The COVID-19 pandemic highlighted the critical role of reliable molecular diagnostics in outbreak response and the vulnerabilities of existing systems to delays and reagent instability. Armored RNA technology, which packages RNA within bacteriophage-derived capsids, offers a robust solution by combining nuclease resistance, safety, and versatility into a single platform. Armored RNA has become a trusted internal and external control for RT-qPCR and RT-LAMP, enabling accurate detection across a wide range of viral pathogens. Also, recent advances in alternative expression systems, such as plant-based and cell-free platforms, as well as the use of more stable scaffolds from bacteriophage Qβ, are enhancing yield, stability, and accessibility of armored RNA. Engineering innovations, including capsid polymorphism and optimized downstream purification, further improve efficiency and broaden possible applications. Looking ahead, armored RNA holds promise not only as a diagnostic standard but also as a delivery vehicle for vaccines and therapeutics. Encapsulation of self-amplifying RNA, small interfering RNA, or microRNA could open new pathways for rapid-response vaccines and targeted therapies, aligning this technology with the future of precision medicine. By uniting stability, scalability, and adaptability, armored RNA represents a critical component of global health preparedness, with the potential to strengthen diagnostic resilience and accelerate biomedical countermeasures in future pandemics.", "journal": "Journal of microbiology (Seoul, Korea)", "year": "2026", "doi": "10.71150/jm.2510016", "ptypes": [ "Journal Article", "Review" ], "first_author": "Tan JH", "dirs": [ "C" ] }, "42198723": { "pmid": "42198723", "title": "Development and Evaluation of Molecular Diagnostic Tests for SARS-CoV-2 at English NHS Sites Throughout the COVID-19 Pandemic.", "abstract": "The COVID-19 pandemic placed unprecedented pressure on diagnostic services worldwide. The first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the UK were confirmed on 31 January 2020, prompting National Health Service (NHS) laboratories to scale diagnostic procedures. The demand for testing rapidly exceeded historical norms for respiratory virus diagnostics, necessitating substantial government investment in consumables, assay development, and workforce expansion. This review presents a retrospective evaluation of SARS-CoV-2 diagnostic platforms deployed within the Norfolk and Norwich University Hospital (NNUH) trust and compares them with those implemented by other regional laboratories during the pandemic. It examines the molecular mechanisms, performance, scalability, and specificity of the multiple molecular testing approaches to optimise workflow based on the evolving technology. The integration of complementary platforms through a stratified testing strategy enabled high-throughput population screening while preserving diagnostic resolution for complex respiratory cases, substantially improving laboratory efficiency and resilience. The emerging diagnostic methodologies, RT-LAMP and CRISPR-based assays, are described, and we discuss their potential roles in future outbreaks. We critically evaluate the overall preparedness of UK health services for the COVID-19 pandemic and highlight key priorities for future pandemic preparedness at both local and national levels.", "journal": "Viruses", "year": "2026", "doi": "10.3390/v18050517", "ptypes": [ "Journal Article", "Review" ], "first_author": "Griffith LD", "dirs": [ "C" ] }, "41309229": { "pmid": "41309229", "title": "Development of an RT-LAMP-CRISPR/Cas12a assay for rapid and specific detection of Bandavirus dabieense.", "abstract": "Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.", "journal": "Journal of microbiology (Seoul, Korea)", "year": "2025", "doi": "10.71150/jm.2506013", "ptypes": [ "Journal Article" ], "first_author": "Song BS", "dirs": [ "C" ] }, "40494717": { "pmid": "40494717", "title": "Corrigendum to \"Rapid and sensitive detection of shrimp infectious myonecrosis virus using a reverse transcription loop-mediated isothermal amplification and visual colorogenic nanogold hybridization probe assay\" [J. Virol. Methods, Vol. 193 (2013) 542-547].", "abstract": "", "journal": "Journal of virological methods", "year": "2025", "doi": "10.1016/j.jviromet.2025.115199", "ptypes": [ "Published Erratum" ], "first_author": "Arunrut N", "dirs": [ "C" ] }, "40195165": { "pmid": "40195165", "title": "Porous GNPs assisted LAMP-CRISPR/Cas12a amperometric biosensor as a potential point of care testing system for SARS-CoV-2.", "abstract": "A simple and ultrasensitive amperometric biosensor is introduced which has the potential to be applied as a point of care test for SARS-CoV-2 monitoring. It was prepared by integrating the reverse transcription loop-mediated isothermal amplification (RT‑LAMP) and CRISPR/Cas12a nuclease activity on a modified gold screen-printed electrode (GSPE). The GSPE is modified with double-end thiolated oligonucleotide reporters conjugated to porous gold nanoparticles (PGNPs) and inserted into a homemade poly-methyl methacrylate cartridge. This biosensor was integrated with a low-cost electronic kit to make a platform with the potential to be applied as a point-of-care testing system. The PGNPs on the reporters create a dense, negatively charged barrier that repels the redox couple of [Fe(CN)6]3-/4- from the GSPE surface. Upon the addition of a real sample, followed by LAMP amplification and Cas12a nuclease activity on disposable GSPE, in the presence of SARS-CoV-2, the single-guide RNA binds to the target sequence and activates Cas12a. The activated Cas12a then cleaves the reporters, releasing the PGNPs. This removal of electrostatic hindrance allows the redox couple of [Fe(CN)6]3-/4- to approach the positively charged GSPE, enhancing the amperometric signal. This biosensor offers an outstanding detection limit of 143 zM (~ 86 copies/mL) and a linear response from 4.7 to 7062 aM for SARS-CoV-2 real samples. By using double-end thiolated reporters and porous GNPs, this novel testing system makes it possible to minimize the required sample volume and reagent costs.", "journal": "Mikrochimica acta", "year": "2025", "doi": "10.1007/s00604-025-07094-0", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Rad MB", "dirs": [ "C" ] }, "41571380": { "pmid": "41571380", "title": "Advances in miRNA detection.", "abstract": "MicroRNAs (miRNAs) are experiencing increased interest for their potential as biomarkers and therapeutic agents and targets. In this review, we discuss the various advances in miRNAs detection from sample collection to result interpretation. Emphasis is placed on the necessity for standardized protocols in both collection and analysis to ensure reproducibility and comparability of results across studies. The challenges associated with miRNA detection stem from their small size, low concentration in biofluids, and complexity of their various transport forms. Conventional methods for miRNA measurement, including RT-qPCR, digital PCR, microarrays, and sequencing, are detailed. Others, such as isothermal amplification, have recently been developed to improve sensitivity, specificity, and multiplexing capability. These new methods have contributed to the development of point-of-care testing for near patient miRNA quantification. Furthermore, the evolution of artificial intelligence will enable detailed and more comprehensive analysis of big data thus facilitating miRNA biomarker discovery. Artificial intelligence can also enable the integration of this data with other omics approaches such as transcriptomics and proteomics as well as clinical and imaging data for improved patient management as part of personalized medicine.", "journal": "Advances in clinical chemistry", "year": "2026", "doi": "10.1016/bs.acc.2025.10.010", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zendjabil M", "dirs": [ "C" ] }, "39843806": { "pmid": "39843806", "title": "Advances and Challenges in the Diagnosis of Leishmaniasis.", "abstract": "Leishmaniasis remains a significant public health challenge, particularly in endemic regions with limited resources. Traditional diagnostic methods, including microscopy, culture, and serology, though widely utilized, often suffer from limitations such as variable  sensitivity, time delays, and the need for specialized infrastructure. Some of these limitations have been addressed with the emergence of molecular diagnostic techniques. Quantitative PCR (q-PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) assays have improved  the diagnostic sensitivity and specificity, enabling species identification and detection of asymptomatic infections. Further, nanodiagnostics and portable sequencing technologies such as the MinION™, along with lab-on-chip platforms, are revolutionizing the diagnostic landscape of leishmaniasis by offering point-of-care (POC) options for remote settings and field-based diagnosis. This review provides an in-depth analysis of these cutting-edge advances, discusses their application in resource-constrained settings, and evaluates their potential to reshape the future of leishmaniasis diagnosis and management.", "journal": "Molecular diagnosis & therapy", "year": "2025", "doi": "10.1007/s40291-024-00762-6", "ptypes": [ "Journal Article", "Review" ], "first_author": "Mehrotra S", "dirs": [ "C" ] }, "40872741": { "pmid": "40872741", "title": "Advances in Epstein-Barr Virus Detection: From Traditional Methods to Modern Technologies.", "abstract": "The Epstein-Barr virus (EBV) is a prevalent virus linked to various diseases, including infectious mononucleosis (IM), nasopharyngeal carcinoma, and Hodgkin's lymphoma. Over the past few decades, EBV diagnostic strategies have evolved significantly-progressing from traditional serological assays and histopathology to more sensitive and specific molecular techniques such as nucleic acid amplification and high-throughput sequencing (HTS). While conventional methods remain valuable for their accessibility and established clinical use, they are often limited by sensitivity, speed, and multiplexing capability. In contrast, emerging technologies, including isothermal amplification, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics, multi-omics integration, and AI-assisted analysis, have demonstrated great promise in improving diagnostic accuracy, speed, and applicability in diverse clinical settings, including point-of-care testing (POCT). This review systematically explores the historical development of EBV diagnostic technologies, highlighting key milestones and future trends in precision medicine and global health readiness.", "journal": "Viruses", "year": "2025", "doi": "10.3390/v17081026", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sun Y", "dirs": [ "C" ] }, "41204872": { "pmid": "41204872", "title": "Integrative Advances in Dengue Virus Diagnostics: From Technological Innovation to Tiered Diagnostic Systems.", "abstract": "Dengue virus (DENV) represents an escalating global public health threat, with rising incidence and expanding geographic distribution. Early and accurate diagnosis remains the cornerstone of effective outbreak management in the absence of universally accessible vaccines or antiviral therapies. Conventional diagnostic methods including viral nucleic acid detection, antigen/antibody testing have achieved substantial progress but still face limitations in sensitivity, speed, and applicability within resource-limited settings. Recent technological innovations are reshaping the diagnostic landscape. Advances in isothermal amplification, CRISPR-based detection, and NGS have markedly enhanced the sensitivity, specificity, and rapidity of DENV detection. In addition, the incorporation of noninvasive samples such as saliva and urine, together with home-based and POCT systems, is expanding diagnostic accessibility. The WHO's REASSURED framework further provides essential guidance for developing affordable, rapid, and field-deployable diagnostic tools. This review summarizes current DENV diagnostic strategies, spanning nucleic acid and serological detection, and highlights recent innovations that integrate these approaches into tiered diagnostic systems. By combining technological advancement with clinical and public health applicability, these strategies hold great promise for strengthening early detection, improving outbreak surveillance, and supporting the global effort toward dengue elimination.", "journal": "Journal of medical virology", "year": "2025", "doi": "10.1002/jmv.70688", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shi Y", "dirs": [ "C" ] }, "40268631": { "pmid": "40268631", "title": "Diagnosing invasive fungal infections in the laboratory today: It's all good news?", "abstract": "Despite the advances in medical science, invasive fungal infections (IFI) remain a diagnostic challenge. The increasing prevalence of IFI, driven by immunosuppressive therapies, advances in intensive care and emerging pathogens, underscores the need for early and accurate diagnosis. This review evaluates current laboratory methods for the diagnosis of IFI, highlighting their strengths and limitations. Conventional techniques, including fungal culture, direct microscopy, and histopathology, remain the gold standard for the diagnosis of proven IFIs. These methods allow pathogen isolation, species identification and antifungal susceptibility testing. However, these techniques have limitations in terms of sensitivity and turnaround times. Although microscopy is a rapid technique, its sensitivity and species discrimination profile are limited. Modern serological assays, such as β-d-glucan and galactomannan detection, have improved the diagnostic accuracy of probable IFI cases. Integration of these assays with clinical and radiological findings, enables earlier intervention, although this is accompanied by an increased risk of false positives and necessitates careful clinical correlation. Molecular diagnostics, particularly polymerase chain reaction (PCR), allow rapid, species-specific identification directly from clinical samples. The advent of MALDI-TOF mass spectrometry has further improved diagnostic efficiency, particularly for yeast identification, although challenges remain for filamentous fungi. Innovative techniques, such as metagenomic sequencing, lateral-flow assays, and loop-mediated isothermal amplification, offer the potential for rapid and precise detection, even in resource-limited settings. The combination of conventional and innovative methods provides a comprehensive diagnostic framework. The continuous refinement of these tools, in conjunction with multidisciplinary collaboration, is imperative to improve the early diagnostic and targeted treatment of patients with IFI.", "journal": "Revista iberoamericana de micologia", "year": "2025", "doi": "10.1016/j.riam.2025.01.004", "ptypes": [ "Journal Article", "Review" ], "first_author": "Pemán J", "dirs": [ "C" ] }, "39834374": { "pmid": "39834374", "title": "The diagnostic accuracy of point-of-care nucleic acid-based isothermal amplification assays for scrub typhus: a systematic review and meta-analysis.", "abstract": "The diagnosis and detection of pathogens such as Rickettsia and Orientia is a cause of major concern among the public health community. Unavailability of rapid, cost-effective diagnostic assays contributes to delayed diagnosis and timely treatment. Using the methodology of systematic reviewing and meta-analysis, the study aimed to synthesize and compare the diagnostic performances of all the available isothermal assays for the detection of classical rickettsial diseases. Studies were retrieved from PubMed and Scopus, and selection and screening were conducted using pre-determined inclusion and exclusion criteria. Analysis was performed using Meta-DiSc 2.0 for the assessment and comparison of diagnostic performance of the isothermal assays. Overall, six studies were selected as a part of this systematic review. All the selected studies (n = 6) optimized LAMP as their index test to detect scrub typhus. The quality assessment of the selected studies revealed only (n = 1) study to be of poor quality with a QUADAS-2 score of (<2). Meta-analysis revealed the pooled sensitivity of LAMP to be 66% [95% CI (0.40-0.85)] with a pooled specificity of 94% [95% CI (0.81-0.98)]. LAMP was estimated with a positive likelihood ratio of 8.3 [95% CI (3.8-18.1)] and a negative likelihood ratio of 0.3 [95% CI (0.2-0.7)] with a false positivity rate of 0.07 [95% CI (0.02-0.2)]. The diagnostic odds ratio was reported to be 21.96 [95% CI (10.2-47.3)]. Due to severe heterogeneity in the body of evidence (I 2 = 0.77), a meta-regression was performed with certain covariates to explore the potential causes. A case-control design was found to exaggerate the sensitivity {0.84 [95% CI (0.5-0.9)]} and specificity {0.73 [95% CI (0.6-0.8)]}. The findings reveal subpar performance of LAMP for the detection of scrub typhus. Active research and development focused on optimization of novel molecular diagnosis that are efficient, rapid, and cost-effective shall foster timely diagnosis and aid in reduction of the overall burden of scrub typhus. A detailed protocol of this review is registered and available in Prospero at: https://www.crd.york.ac.uk/prospero/. (registration number CRD42024511706).", "journal": "Frontiers in microbiology", "year": "2024", "doi": "10.3389/fmicb.2024.1516921", "ptypes": [ "Journal Article", "Systematic Review" ], "first_author": "Dixit R", "dirs": [ "C" ] }, "42529880": { "pmid": "42529880", "title": "Emerging point-of-care technologies for bacterial pathogen detection.", "abstract": "Bacterial infections remain a significant threat to public health worldwide, driving an urgent need for rapid, accurate, and field-deployable diagnostic techniques. Point-of-care testing (POCT) has emerged as a transformative strategy, providing timely detection, operational simplicity, and portability. Recent studies have aimed at enhancing sensitivity, specificity, multiplexing capability, and automation through the integration of molecular diagnostics with microfluidics and lab-on-chip technologies, alongside the development of low-cost, portable devices equipped with smartphone-based readout and cloud connectivity for real-time surveillance in resource-limited settings. Nonetheless, evidence-based frameworks for selecting optimal detection targets-such as genomic sequences, conserved protein epitopes, or viable whole cells-and matching them to appropriate POCT modalities remain notably underrepresented in the literature. This review systematically summarizes recent advances in POCT strategies for bacterial detection, categorized according to three major types of detection targets, including cellular phenotypic characteristics, surface antigens, and nucleic acids. We discuss the principles, advantages, limitations, and representative applications of key POCT platforms, which include microscopy-based visualization, immunoassays, isothermal amplification, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems, and microfluidic biosensors. Critical challenges, such as sample pretreatment, detection sensitivity, and operational simplicity, have been partially addressed through recent innovations. Finally, we outline the main future research directions focused on the development of integrated, automated, and intelligent POCT systems for clinical deployment.", "journal": "Journal of Zhejiang University. Science. B", "year": "2026", "doi": "10.1631/jzus.B2500782", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhang W", "dirs": [ "C" ] }, "41885207": { "pmid": "41885207", "title": "Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection.", "abstract": "Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the cis-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate cis-cleavage while preserving robust trans-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT-RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100-1000-fold improved sensitivity and achieves >10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and cis-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.", "journal": "Nucleic acids research", "year": "2026", "doi": "10.1093/nar/gkag267", "ptypes": [ "Journal Article" ], "first_author": "Yin W", "dirs": [ "C" ] }, "40489859": { "pmid": "40489859", "title": "The application of microfluidic technology in allergen detection: A review.", "abstract": "The escalating prevalence of allergic diseases has intensified the need for rapid and precise allergen detection, prompting the exploration of microfluidic technology in this domain. This review delves into the utility of microfluidics in enhancing allergen detection, underscoring its high sensitivity, throughput, and miniaturization advantages. We survey the spectrum of microfluidic chip materials, from silicon wafers to paper-based substrates, and discuss various fabrication techniques. The integration of microfluidics with detection methods like enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, Western blotting, loop-mediated isothermal amplification (LAMP), and biosensors has transformed allergen diagnostics, offering more efficient, sensitive, and user-friendly platforms. These advancements facilitate point-of-care testing and hold significant potential for personalized medicine. Our review highlights the novel applications of microfluidics in allergen detection, contributing valuable insights for the development of future diagnostic tools and clinical research in allergy treatment. The innovative convergence of microfluidics with existing diagnostic methods presents a paradigm shift in the field of allergen detection, promising enhanced prevention and management of allergic diseases.", "journal": "Medicine", "year": "2025", "doi": "10.1097/MD.0000000000042645", "ptypes": [ "Journal Article", "Review" ], "first_author": "Luo D", "dirs": [ "C" ] }, "41017813": { "pmid": "41017813", "title": "Measles and rubella: From global health challenges to advancements in molecular diagnostics in the elimination era.", "abstract": "Measles and rubella are highly contagious viral infections with significant public health implications, particularly in low- and middle-income countries. Despite the availability of effective vaccines, these diseases continue to cause periodic outbreaks, contributing to substantial global morbidity, mortality, and economic burden. Immunization programs have drastically abridged disease incidence; however, gaps in vaccination coverage and surveillance systems deter complete elimination. The economic impact of outbreaks includes direct healthcare costs and indirect societal losses, emphasizing the need for robust disease control strategies. Accurate and timely diagnosis is pivotal to measles and rubella elimination efforts. Current diagnostic approaches range from conventional RT-PCR (including multiplex and real-time formats), ELISA, and plaque reduction neutralization test (PRNT), to emerging methods such as isothermal amplification loop-mediated isothermal amplification, recombinase polymerase amplification), CRISPR-Cas systems, next-generation sequencing (NGS), microfluidics, and lateral flow assays. Despite their sensitivity, many of these methods require complex infrastructure and skilled personnel, limiting their utility in field settings. To bridge diagnostic gaps, there is an urgent need for rapid, affordable, and field-deployable nucleic acid-based diagnostics that are simple to use with minimal training. Innovations like CRISPR-Cas and microfluidic platforms hold promise for decentralized testing and real-time surveillance, potentially transforming global measles and rubella elimination programs for the future.", "journal": "Molecular therapy. Nucleic acids", "year": "2025", "doi": "10.1016/j.omtn.2025.102698", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sharma S", "dirs": [ "C" ] }, "41002010": { "pmid": "41002010", "title": "A Field-Deployable RotEx-LAMP-LFA Platform for Molecular Triage of HPV-Driven Oncogenesis.", "abstract": "Persisting as a major global health disparity, HPV16/18-associated cervical carcinogenesis continues to disproportionately affect populations in resource-limited regions with compromised screening infrastructure. While molecular detection of E6/E7 oncogenic transcripts surpasses conventional cytology in clinical specificity, current nucleic acid amplification platforms remain impeded by technical complexity, prolonged turnaround times (>4 h), and substantial per-test costs (>$50) that hinder scale-up in low-income settings. Here, RotEx-LAMP-LFA is presented: an innovative point-of-care system integrating microfluidic nucleic acid extraction, rapid isothermal amplification (30 min at 63 °C), and combined lateral flow detection in a single disposable cartridge. Validated with 69 clinical specimens (19 histology-confirmed SCC, 14 HPV+ precancerous lesions, 15 HPV+ infections, and 21 healthy controls), this sample-to-answer platform achieved 94.74% sensitivity (18/19; 95% CI: 84.72-100%) for invasive carcinoma detection with 100% (36/36; 95% CI: 90.44-99.93%) specificity for non-cancer. In addition, a subset of samples is collected through paired self-sampling to validate consistency with clinician-collected specimens, indicating that this innovative platform shows promise for community-level molecular stratification of persistent and progressive HPV infections. It paves the way for accessible, patient-centered cervical cancer risk reporting, with the potential to reduce psychological burden on patients.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2025", "doi": "10.1002/advs.202509468", "ptypes": [ "Journal Article" ], "first_author": "Gao Y", "dirs": [ "C" ] }, "41966922": { "pmid": "41966922", "title": "A universal light-controlled highly sensitive one-pot CRISPR/Cas12a diagnostic based on structure-engineered crRNA.", "abstract": "Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has transformed molecular diagnostics through its speed and accuracy; however, one-pot formats are often limited by sensitivity and field suitability. Herein, we developed a universal light-controlled high-sensitivity one-pot CRISPR/Cas12a testing (ULTRAt) platform based on structure-engineered CRISPR RNA (crRNA) scaffolds. By incorporating photocaged 6-nitropiperonyloxymethyl groups into the crRNA stem-loop, Cas12a activity is transiently suppressed during isothermal amplification via structural modulation, enabling efficient target enrichment. Subsequent UV irradiation removes the protecting groups, restoring the native conformation and activating robust trans-cleavage. ULTRAt achieves a limit of detection of two copies of monkeypox virus per reaction with a 15-min time-to-result, representing a 100-fold sensitivity improvement over conventional assays. The platform further supports single-nucleotide polymorphism discrimination and human papillomavirus 16/18 genotyping. Analysis of 91 clinical samples demonstrates strong concordance between ULTRAt and reference qPCR and sequencing assays. Collectively, ULTRAt enables rapid, ultra-sensitive, and versatile one-pot detection, supporting near-patient diagnostics and genotyping.", "journal": "Trends in biotechnology", "year": "2026", "doi": "10.1016/j.tibtech.2026.03.018", "ptypes": [ "Journal Article" ], "first_author": "Cui J", "dirs": [ "C" ] }, "42277912": { "pmid": "42277912", "title": "CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.", "abstract": "The field of nucleic acid-based testing experienced a decade-long stagnation since the development of quantitative polymerase chain reaction (qPCR) in 1992 and isothermal amplification methods in the early 2000s. However, in 2016, the discovery of trans-nuclease activity in CRISPR-Cas systems revolutionized the molecular diagnostics for nucleic acids. A typical CRISPR diagnostic workflow comprises three phases: (1) target recognition through CRISPR RNA (crRNA)-guided hybridization; (2) signal transduction via trans-cleavage of engineered reporters (e.g., fluorophore-quencher oligonucleotides), and (3) signal readout using fluorescence, electrochemical, or colorimetric platforms. Emerging shortly prior to the COVID-19 pandemic, CRISPR diagnostics quickly gained prominence as a field-deployable alternative to qPCR due to its rapidity (< 1 h), minimal equipment requirements, and field adaptability. This technological paradigm underwent rigorous validation and refinement alongside the rapid evolution of SARS-CoV-2 detection, which facilitated its adaptation for cancer diagnosis. Recent advancements in sensitivity (attomolar-level detection) and specificity (single-nucleotide discrimination) have enabled transformative applications in cancer diagnostics, including: (1) identification of nucleic acid biomarkers, such as high-frequency somatic mutations, circulating nucleic acids and miRNAs; and (2) detection of non-nucleic acid biomarkers, including epigenetic aberrations, proteins, small molecules and metabolite biomarkers. This review chronicles the decadal evolution of CRISPR diagnostics, with particular emphasis on recent advancements of its application in cancer diagnosis. We critically evaluate persistent technical limitations, including PAM sequence restriction, suboptimal sensitivity and specificity, quantitative constraints, and unmet point-of-care testing (POCT) in complex biological matrices. Additionally, we discuss prospective solutions to address these challenges.", "journal": "Cell & bioscience", "year": "2026", "doi": "10.1186/s13578-026-01603-1", "ptypes": [ "Journal Article", "Review" ], "first_author": "Chang L", "dirs": [ "C" ] }, "41306592": { "pmid": "41306592", "title": "Evolving HPV diagnostics: current practice and future frontiers.", "abstract": "Human papillomavirus (HPV) infection serves as a primary causative agent of cervical cancer, highlighting the importance of early screening and detection in mitigating the incidence and mortality rates of HPV-related diseases. Over the past decades, HPV detection technologies have evolved considerably, transitioning from traditional methods to more advanced, patient-centered approaches. This review provides a comprehensive overview of both established and emerging HPV detection strategies, with a particular focus on their clinical applicability, technical advantages, and limitations. Conventional methods such as hybrid capture and PCR-based assays remain the backbone of clinical screening, offering robust sensitivity and specificity. However, their reliance on invasive sampling and centralized laboratory infrastructure limits accessibility and patient compliance, particularly in low-resource settings. To address these limitations, emerging technologies-including CRISPR/Cas systems, droplet digital PCR (ddPCR), next-generation sequencing (NGS), isothermal amplification techniques (IAT) and artificial intelligence (AI) combined with hpv screening offer enhanced accuracy, rapid turnaround, and the potential for point-of-care deployment. In parallel, innovations in sampling such as self-collected vaginal swabs and liquid biopsy using urine, blood, or extracellular vesicles are improving test acceptability and broadening screening coverage. By summarizing current progress and highlighting ongoing challenges, this review aims to guide the development of more precise, non-invasive, and scalable HPV detection strategies to reduce the global burden of HPV-related disease, support global prevention efforts, and guide public health policies.", "journal": "Frontiers in cellular and infection microbiology", "year": "2025", "doi": "10.3389/fcimb.2025.1681779", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shi L", "dirs": [ "C" ] }, "39670632": { "pmid": "39670632", "title": "Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.", "abstract": "CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.", "journal": "ACS synthetic biology", "year": "2025", "doi": "10.1021/acssynbio.4c00695", "ptypes": [ "Letter", "Research Support, Non-U.S. Gov't" ], "first_author": "Chen W", "dirs": [ "C" ] }, "41591771": { "pmid": "41591771", "title": "Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.", "abstract": "Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, requires ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms that fail to address the growing requirements of sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein, we present a CRISPR/Cas12a-mediated ALP detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow (exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U L-1 and a wide linear range (0.1-10 U L-1), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of the readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value of about 7 U L-1, offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor for clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, thus offering significant potential for early disease diagnosis and long-term therapeutic monitoring.", "journal": "Analytical methods : advancing methods and applications", "year": "2026", "doi": "10.1039/d5ay01195g", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Chen D", "dirs": [ "C" ] }, "40076597": { "pmid": "40076597", "title": "Point-of-Care Diagnostic Test for Rapid Detection of Infectious Laryngotracheitis Virus by Loop-Mediated Isothermal Amplification and Nanoprobes.", "abstract": "Infectious laryngotracheitis virus (ILTV), a DNA virus classified as Gallid alphaherpesvirus 1, causes a highly contagious respiratory disease in chickens, leading to significant economic losses and health risks for the poultry industry. The rapid detection of ILTV is essential to control its spread and prevent outbreaks. Traditional diagnostic methods like PCR are costly, require specialized personnel, and delay response efforts. To address this, we developed a point-of-care diagnostic test combining loop-mediated isothermal amplification (LAMP) with DNA nanoprobes on respiratory swabs. LAMP targets the ILTV-glycoprotein E (gE) gene, enabling rapid nucleic acid amplification at 65 °C without extraction, making it suitable for on-site detection. DNA nanoprobes provide a colorimetric readout visible to the naked eye. Gold nanoparticles drive this readout, as their red color, based on localized surface plasmon resonance, persists in the presence of ILTV DNA through DNA-DNA hybridization, ensuring reliable detection. The assay achieved 100% sensitivity and specificity for ILTV-gE, with a detection limit of 200 copies per reaction, allowing for the early identification of infections. The results are available within 45 min, enabling prompt measures to control ILTV spread. Cost-effective and user-friendly, this method enhances disease management and biosecurity in poultry farms.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms26051971", "ptypes": [ "Journal Article" ], "first_author": "Cea-Callejo P", "dirs": [ "C" ] }, "41081989": { "pmid": "41081989", "title": "Laboratory detection methods for Chlamydia trachomatis infection in children: a review.", "abstract": "Chlamydia trachomatis (CT) infections in children present distinct diagnostic challenges, ranging from perinatal conjunctivitis and pneumonia to trachoma and, in older children, sexually transmitted infections. This review systematically evaluates contemporary laboratory methods for detecting CT in children. The methods are categorized by their distinct targets, beginning with morphological identification via stains (e.g., Giemsa, immunofluorescence) with or without culture; followed by immunological detection of antigens or antibodies (e.g., ELISA); and finally, molecular analysis using NAATs for DNA or RT-PCR for RNA. Each technique is rigorously examined based on analytical sensitivity, specificity, turnaround time, and technical requirements, with particular emphasis on age-specific specimen selection, sampling logistics, and ethical-legal considerations-especially in cases of suspected abuse. Our analysis confirms that NAATs, particularly real-time PCR, are the first-line diagnostic choice for their superior sensitivity in detecting asymptomatic and low-bacterial-load infections in children. RNA-based detection is highlighted as the preferred method for treatment monitoring due to its ability to differentiate viable pathogens. In resource-limited settings, rapid antigen tests offer a practical screening solution, though require NAAT confirmation. The review also discusses emerging technologies, including isothermal amplification and biosensors, for point-of-care (POC) testing. By synthesizing current evidence with pediatric-specific guidelines from the CDC, WHO, and European health authorities, this review aims to establish an evidence-based framework to guide optimal test selection, improve early detection, and inform public health strategies to reduce the CT disease burden in children.", "journal": "European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology", "year": "2026", "doi": "10.1007/s10096-025-05306-2", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sun Y", "dirs": [ "C" ] }, "41347242": { "pmid": "41347242", "title": "Genomic evolution of enteric pathogens: mechanisms of pathogenicity and diagnostic innovations.", "abstract": "Genomic evolution serves as a pivotal driver of pathogenicity and host adaptation in intestinal pathogens. This review systematically dissects, from a phylogenetic perspective, the key genomic evolutionary mechanisms underpinning pathogenesis across five major classes of intestinal pathogens and their significance. Bacteria (e.g., Escherichia coli) acquire virulence- and antibiotic resistance-enhancing genes via horizontal gene transfer and genomic recombination, equipping them to disrupt the intestinal mucosal barrier and evade host immune defenses. Fungi (e.g., Candida albicans and Cryptococcus spp.) significantly augment their pathogenic potential through chromosomal rearrangements and dynamic expansions or losses within gene families. Parasites (e.g., Giardia lamblia) successfully evade host immune recognition and clearance through complex life cycles and stage-specific gene expression regulation. Viruses (e.g., rotaviruses and noroviruses) rapidly adapt to host cellular environments via genomic mutation and recombination, triggering acute gastroenteritis. Although prions primarily propagate via the nervous system, the pronounced cellular stress response they elicit in intestinal tissues suggests the gut may serve as a potential secondary transmission or amplification site. Collectively, these diverse evolutionary mechanisms confer unique colonization, survival, and competitive advantages upon distinct pathogen classes within the complex gut microenvironment. Employing Escherichia coli as a paradigm, systematic bioinformatic analysis of 335 key virulence factors revealed evolutionarily stable functional clusters (e.g., effector/toxin systems, 21.0%) with core contributions to pathogenicity. These conserved genomic signatures provide a robust foundation for developing novel high-precision diagnostics. For instance, CRISPR-based platforms achieve 100% clinical concordance in detecting the Shiga toxin gene (stx2), while loop-mediated isothermal amplification coupled with lateral flow assay (LAMP-LFA) enables rapid (< 40 min) and accurate detection of bla NDM - 1-mediated carbapenem resistance. The deep integration of multi-omics data (genomics, transcriptomics, proteomics, etc.) with artificial intelligence (AI) is substantially accelerating the discovery of novel biomarkers. Looking forward, innovative technologies such as real-time nanopore sequencing and nanomaterial-enhanced high-sensitivity biosensors hold promise for achieving rapid, broad-spectrum pathogen detection, thereby robustly supporting the World Health Organization (WHO)'s \"One Health\" strategic goals. In conclusion, the \"Genomic Evolution-Biomarker Discovery-Diagnostic Development\" integrated triad framework presented herein offers crucial insights and actionable pathways for advancing next-generation precision diagnostics and formulating effective global infection control strategies.", "journal": "Frontiers in microbiology", "year": "2025", "doi": "10.3389/fmicb.2025.1647437", "ptypes": [ "Journal Article", "Review" ], "first_author": "Li T", "dirs": [ "C" ] }, "41344285": { "pmid": "41344285", "title": "A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.", "abstract": "Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.", "journal": "Veterinary parasitology", "year": "2026", "doi": "10.1016/j.vetpar.2025.110662", "ptypes": [ "Journal Article", "Review" ], "first_author": "Dasanayaka BP", "dirs": [ "C" ] }, "39874920": { "pmid": "39874920", "title": "Lift-CM: An integrated lift-heater centrifugal microfluidic platform for point-of-care pathogen nucleic acid detection using isothermal amplification and CRISPR/Cas12a.", "abstract": "Pathogen nucleic acid detection technology based on isothermal amplification and CRISPR/Cas12a system offers advantages in terms of high sensitivity, high specificity, and rapidity. However, this method has not been widely applied because of its shortcomings in utilizing conventional instruments, which cannot satisfy the requirements for Point of Care Testing (POCT), such as integration, convenience, and miniaturization. In this study, we developed an integrated lift-heater centrifugal microfluidic platform (Lift-CM) to automate the processes of isothermal amplification and CRISPR/Cas12a detection. A spatially encoded centrifugal microfluidic disc (SEC-disc) was employed to physically separate the amplification and detection processes while expanding the number of targets. The design of the dual-temperature and lift-heating centrifugal mechanism of the Lift-CM platform ensures that there is no manual intervention during amplification and detection processes. A smartphone-based app enables the setting of key parameters and monitoring of the experimental process, presenting results through a generated report that includes real-time fluorescence curves. We analyzed the plasmids of the Crimean-Congo hemorrhagic fever virus, Ebola virus, and five influenza viruses using different amplification methods (RPA/LAMP) to demonstrate the good compatibility of the Lift-CM platform with various amplification schemes. In clinical validation, the detection of H3N2-positive samples was completed within 30 min, and the results were highly consistent with qPCR results. This portable and compact platform offers a novel alternative solution for both clinical and at-home pathogen nucleic acid detection in the future.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117178", "ptypes": [ "Journal Article" ], "first_author": "Shu T", "dirs": [ "C" ] }, "40070264": { "pmid": "40070264", "title": "Development of Vibrio parahaemolyticus sensitive and specific loop-mediated isothermal amplification combined with lateral flow device.", "abstract": "Vibrio parahaemolyticus a gram-negative, rod-shaped bacterium with salinophilic properties is found mainly in rivers, oceans, and coastal environments. With the expanding scale of aquaculture in coastal regions of China, the contamination of seafoods with Vibrio parahaemolyticus is becoming a significant cause of food poisoning with symptoms including gastroenteritis, wound infection and sepsis. Current methods for detecting this microorganism are unsuitable in the present context. We developed a rapid LAMP-LFD method-by combining the loop-mediated isothermal amplification technique (LAMP) and lateral flow device (LFD). The thermolabile hemolysin tlh gene of Vibrio parahaemolyticus was used as the target, and we designed five specific primers in its conserved region. The primers were used to carry LAMP reaction with biotin labelling, the products completed hybridisation with the FAM-labelled primers, and the hybridisation products were tested for results on LFD. The results showed that the LAMP-LFD method specifically detected Vibrio parahaemolyticus and was negative for proximate strains such as Vibrio vulnificus and other Vibrio pathogens as well as common pathogens such as Escherichia coli. The optimised reaction conditions for LAMP were 40 min at 60 °C, plus 5 min of probe hybridisation and 3-5 min of LFD color development. The lowest concentration of Vibrio parahaemolyticus pure culture bacterial fluid of 1.5×102 cfu/mL could be detected, and the pathogen could be detected from tissue samples with a contamination concentration of 0.75×103 cfu/mL. The method has higher specificity and sensitivity, and the pathogen can be detected within 1.5 h. The LAMP-LFD method for Vibrio parahaemolyticus established in this study has the advantages of convenient operation, low dependence on equipment, high sensitivity and rapid detection, all of make it ideally suited to the detection of Vibrio parahaemolyticus at the grass-roots level.", "journal": "Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia", "year": "2026", "doi": "10.5507/bp.2025.007", "ptypes": [ "Journal Article" ], "first_author": "Liang X", "dirs": [ "C" ] }, "42217635": { "pmid": "42217635", "title": "Advances in rapid microbiological testing for animal diseases: A review.", "abstract": "Animal pathogenic microorganisms destabilize livestock economies and jeopardize human health through zoonotic transmission and foodborne illness. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site or point-of-care (POC) monitoring required to prevent disease outbreaks and manage animal health effectively. By integrating latest research advances, this study reviews advances in rapid detection technologies for animal pathogens, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification (LAMP); recombinase polymerase amplification (RPA)). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification from clinical livestock samples and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption from days to less than one hour while challenges regarding sample preparation from complex clinical matrices such as whole blood, serum, tissue homogenates, and fecal samples remain. The future of animal health surveillance lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors for farm-level early warning. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for POC veterinary diagnostics, while qPCR/dPCR (dPCR)remain indispensable for quantitative regulatory applications such as disease certification and vaccine efficacy monitoring. We propose a structured technology selection framework to guide researchers and veterinary practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements for different livestock species and production systems.", "journal": "Journal of microbiological methods", "year": "2026", "doi": "10.1016/j.mimet.2026.107564", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhen Z", "dirs": [ "C" ] }, "39942047": { "pmid": "39942047", "title": "Super-Fast Detection of Bacillus cereus by Combining Cellulose Filter Paper-Based DNA Extraction, Multienzyme Isothermal Rapid Amplification, and Lateral Flow Dipstick (MIRA-LFD).", "abstract": "Bacillus cereus is a widespread foodborne pathogen that can cause food poisoning when present in food at certain levels. Ingesting contaminated food may lead to symptoms such as abdominal pain, diarrhea, and, in severe cases, life-threatening conditions. In this study, a simple and super-fast method for detecting B. cereus was developed, which combines cellulose filter paper-based DNA extraction, multienzyme isothermal rapid amplification (MIRA), and lateral flow dipstick (LFD) technology. Initially, PCR was adopted to evaluate the DNA extraction efficiency of the filter paper, followed by the optimization of the lysis formula and extraction conditions. With the above optimization, DNA that can be used for subsequent nucleic acid amplification can be obtained within 3 min. Then, the isothermal amplification of MIRA-LFD was established and optimized to evaluate the detection specificity and sensitivity. Finally, the developed method was applied to detect B. cereus in cooked rice samples. The results indicated that the entire amplification procedure of MIRA-LFD only takes 15 min at 39 °C. The whole super-fast detection system could be completed in less than 20 min, from DNA extraction to result interpretation, which achieved a detection limit of 12 fg/μL of DNA concentration, corresponding to approximately 115 CFU/mL in actual samples.", "journal": "Foods (Basel, Switzerland)", "year": "2025", "doi": "10.3390/foods14030454", "ptypes": [ "Journal Article" ], "first_author": "Yi S", "dirs": [ "C" ] }, "39831125": { "pmid": "39831125", "title": "Swift and portable detection of soybean mosaic virus SC7 through RNA extraction and loop-mediated isothermal amplification using lateral flow device.", "abstract": "The soybean mosaic disease-caused by the soybean mosaic virus (SMV)-significantly impacts soybean quality and yield. Among its various strains, SMV-SC7 is prevalent in China. Therefore, rapid and accurate diagnosis is deemed critical to mitigate the spread of SMV-SC7. In this study, a simple and rapid magnetic bead-based RNA extraction method was optimized. Furthermore, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay that requires no specialized equipment such as PCR Amplifier was proposed, employing a lateral flow device (LFD) for visual interpretation of SMV-SC7. The RT-LAMP-LFD approach facilitated specificity testing of SMV-SC7. Moreover, the limit of detection (LOD) of this method was as low as 10-5 ng (2.4 copies). The sensitivity of RT-LAMP-LFD was 10-fold higher than that of the colorimetric RT-LAMP method. In 194 field samples tested, the RT-LAMP-LFD detection of the SMV-SC7 had accuracy of 98.45% in comparison to RT-qPCR. In conclusion, the assay exhibited high specificity, sensitivity, and rapidity, enabling economical and portable detection of SMV-SC7 and providing technical support to identify SMV-SC7-infected soybeans.", "journal": "Frontiers in microbiology", "year": "2024", "doi": "10.3389/fmicb.2024.1478218", "ptypes": [ "Journal Article" ], "first_author": "Guo SX", "dirs": [ "C" ] }, "42519721": { "pmid": "42519721", "title": "Establishment of a loop-mediated isothermal amplification-lateral flow dipstick assay for the point-of-care testing of feline herpesvirus-1.", "abstract": "Feline herpesvirus-1 (FHV-1) is a major pathogen responsible for feline upper respiratory tract disease (URTD), with a global distribution that poses significant threats to both the health of domestic cat and the conservation of wild felids. Thus, the development of a rapid and specific diagnostic method is crucial for controlling FHV-1 infection. In this study, we established a visual detection assay for FHV-1 by combining loop-mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD). The assay utilized six of primers targeting the highly conserved TK gene of FHV-1. The LAMP amplification was performed using biotin-labeled H-FIP and 6-FAM-labeled H-LF, followed by visual detection using LFD. After optimizing the reaction conditions, the LAMP-LFD assay was established and systematically evaluated for its specificity, sensitivity, and repeatability. Furthermore, the performance of this assay was assessed using nasal swabs from 87 cats suspected with feline URTD. The optimal reaction conditions were determined as follows: 0.2 μM each of outer primers, 1.2 μM each of inner primers, and 0.6 μM each of loop primers, with amplification at 63 °C for 40 min. The assay showed no cross-reactivity with other pathogens caused feline URTD, demonstrating high specificity. The detection limit was 102 copies/μL for recombinant plasmid and 101.5 TCID50/mL for FHV-1 viral culture. Good repeatability was also confirmed. Clinical testing of 87 nasal swabs revealed positive rates of 49.43% (43/87). Compared to qPCR, the assay exhibited a specificity of 97.56%, sensitivity of 91.30%, and an overall agreement of 94.25%, with a kappa value of 0.89, indicating almost perfect concordance between the two methods. The LAMP-LFD assay developed in this study is highly sensitive, specific, simple, independent of specialized equipment and easy to perform with results visualized by the naked eye, making it well-suited for point-of-care testing of FHV-1 in primary veterinary hospitals and field setting.", "journal": "Frontiers in veterinary science", "year": "2026", "doi": "10.3389/fvets.2026.1863672", "ptypes": [ "Journal Article" ], "first_author": "Yi S", "dirs": [ "C" ] }, "41178145": { "pmid": "41178145", "title": "Enzyme-Programmable DNA-PEG Hydrogel Spatiotemporally Regulates Bone Regeneration Microenvironment.", "abstract": "Conventional hydrogel drug delivery systems are limited in recapitulating the natural spatiotemporal progression of bone regeneration due to their passive release mechanisms. Here, an enzyme-responsive deoxyribonucleic acid (DNA)-polyethylene glycol (PEG) hydrogel is developed to actively coordinate the sequential processes of angiogenesis, osteogenesis, and mineralization through rational material design. The hydrogel integrates matrix metalloproteinase (MMP)-cleavable peptide-crosslinked PEG networks conjugated with actin-stabilized vascular endothelial growth factor (VEGF)-binding DNA strands, enabling spatiotemporally controlled therapeutic release. Upon implantation, MMPs trigger the hydrogel degradation, releasing VEGF to induce angiogenesis while simultaneously promoting osteogenic differentiation. The actin-stabilized DNA framework maintains structural integrity during this stage, preventing premature phosphate release. Subsequent matrix remodeling liberates nuclease that catalyzes DNA to generate phosphate ions, which synergize with peptides in hydrogels and endogenous calcium to drive mineralization. Molecular dynamics simulations reveal the underlying mechanism of hydrogel-mediated mineralization, demonstrating enhanced calcium phosphate formation. Such temporally controlled cascade significantly improves vascular density, osteogenic marker expression, and mineral deposition compared to controls. This work establishes a bioresponsive platform that dynamically interacts with the biological microenvironment to orchestrate multi-phase bone regeneration, offering new possibilities for complex tissue repair.", "journal": "Advanced materials (Deerfield Beach, Fla.)", "year": "2026", "doi": "10.1002/adma.202514461", "ptypes": [ "Journal Article" ], "first_author": "Wu X", "dirs": [ "D" ] }, "39531216": { "pmid": "39531216", "title": "Alginate Formulation for Wound Healing Applications.", "abstract": "Significance: Alginate, sourced from seaweed, holds significant importance in industrial and biomedical domains due to its versatile properties. Its chemical composition, primarily comprising β-D-mannuronic acid and α-L-guluronic acid, governs its physical and biological attributes. This polysaccharide, extracted from brown algae and bacteria, offers diverse compositions impacting key factors such as molecular weight, flexibility, solubility, and stability. Recent Advances: Commercial extraction methods yield soluble sodium alginate essential for various biomedical applications. Extraction processes involve chemical treatments converting insoluble alginic acid salts into soluble forms. While biosynthesis pathways in bacteria and algae share similarities, differences in enzyme utilization and product characteristics are noted. Critical Issues: Despite its widespread applicability, challenges persist regarding alginate's stability, biodegradability, and bioactivity. Further understanding of its interactions in complex biological environments and the optimization of extraction and synthesis processes are imperative. Additionally, concerns regarding immune responses to alginate-based implants necessitate thorough investigation. Future Directions: Future research endeavors aim to enhance alginate's stability and bioactivity, facilitating its broader utilization in regenerative medicine and therapeutic interventions. Novel approaches focusing on tailored hydrogel formations, advanced drug delivery systems, and optimized cellular encapsulation techniques hold promise. Continued exploration of alginate's potential in tissue engineering and wound healing, alongside efforts to address critical issues, will drive advancements in biomedical applications.", "journal": "Advances in wound care", "year": "2025", "doi": "10.1089/wound.2024.0081", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "Alrata L", "dirs": [ "D" ] }, "40979561": { "pmid": "40979561", "title": "Self-Organized Vascularized Hepatic Organoids in Microcapsules for Liver Regeneration.", "abstract": "Hepatic organoids represent a promising strategy for liver regeneration; however, challenges remain in flexibly tailoring them to achieve complex architectures and vascularized structures for enhanced functionality. Here, we propose a novel system of self-organized vascularized hepatic organoids (SOVHOs) encapsulated in hydrogel microcapsules with biomimetic features for liver regeneration. The hydrogel shell microcapsules encapsulating human umbilical vein endothelial cells and hepatic organoids derived from human induced pluripotent stem cells were generated using microfluidic encapsulation technology. The hydrogel shell provides a 3-dimensional culture environment that supports the formation of uniform hepatocyte spheroids with vascular networks inside the microcapsules. The SOVHOs exhibit liver-specific functions, including drug metabolism, urea synthesis, and serum protein production. In rats with acute liver failure, we demonstrated that the SOVHOs markedly enhanced survival rate and normalize the inflammatory response after transplantation, indicating their remarkable repopulation capacity in facilitating injured liver recovery. Furthermore, SOVHOs remained viable in the host liver for at least 7 days post-implantation, exhibiting low immunogenicity and no detectable adverse effects in rats during this period. These results suggest that our SOVHOs are potentially valuable for clinical application in liver disease treatment.", "journal": "Research (Washington, D.C.)", "year": "2025", "doi": "10.34133/research.0898", "ptypes": [ "Journal Article" ], "first_author": "Zhi Y", "dirs": [ "D" ] }, "40779637": { "pmid": "40779637", "title": "A sonoelectric niche for noninvasive intervertebral disc regeneration via targeted cell cycle modulation.", "abstract": "Cell cycle regulation is pivotal for tissue regeneration yet remains challenging in degenerative microenvironments. We engineered a sonosensitive conductive hydrogel incorporating polypyrrole-encapsulated porphyrin derivatives {[Tetrakis (4-carboxyphenyl) porphyrin (TCPP)]@PPy} to regulate cell cycle dynamics. Upon ultrasound irradiation, TCPP@PPy generates free electrons, establishing a controlled microcurrent within degenerative tissues. This sonoelectric niche induces nucleus pulposus cell (NPC) membrane depolarization, activating calcium voltage-gated channels (Cav) to drive Ca2+ influx. Subsequent calcium- and calmodulin-dependent protein kinase I activation up-regulates cyclin-dependent kinases CDK1/CDK2, forming a sonoelectricity-ion-kinase axis that stimulates NPC proliferation and anabolism. Concurrently, ultrasound-responsive borate ester bonds in the hydrogel amplify reactive oxygen species scavenging, counteracting oxidative stress-induced NPC ferroptosis. In a goat model of intervertebral disc degeneration, ultrasound-guided hydrogel implantation alleviated degenerative progression by synergistically reactivating cell cycle progression and suppressing oxidative damage. This strategy demonstrates a noninvasive, dual-targeted approach to regulate degenerative microenvironments through spatiotemporal control of sonoelectric and biochemical cues, offering a translatable strategy for tissue regeneration therapies.", "journal": "Science advances", "year": "2025", "doi": "10.1126/sciadv.adu6860", "ptypes": [ "Journal Article" ], "first_author": "Zhang X", "dirs": [ "D" ] }, "41177463": { "pmid": "41177463", "title": "Hydrogel formulations for sustained-release of broadly neutralizing antibodies.", "abstract": "Sustained serum levels of broadly neutralizing antibodies (bnAbs) are crucial for effective passive immunization against infectious diseases as protection persists only while these bnAbs remain at adequate concentrations within the body. Current obstacles, such as poor pharmacokinetics (PK) and burdensome administration, must be overcome to make bnAbs a viable option for pre- and post-exposure prophylaxis. In this work, we explore how a polymer-nanoparticle (PNP) hydrogel depot technology can be engineered to prolong protein delivery and enable drug exposure on the order of weeks to months. In-vivo studies in mice and rats demonstrate extended protein release compared to bolus administration, and modeling efforts predict the impact of both the elimination half-life of the active pharmaceutical ingredient and hydrogel depot volume on overall pharmacokinetics. Moreover, flow cytometry characterization reveals that immune cell infiltration into the hydrogel depot can result in faster-than-expected release of antibody cargo on account of active transport via cellular uptake. We then demonstrate that co-formulation of antibodies with an anti-inflammatory agent reduces cellular infiltration and resulting active transport, further extending delivery and pharmacokinetics. Finally, multicompartmental modeling predicts the human PK profiles of clinically relevant HIV bnAbs delivered via subcutaneous hydrogel injection. These findings aid in the development of next generation hydrogel materials that stabilize and slowly release bnAbs for long-term pre-exposure immunoprophylaxis.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.114349", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "Jons CK", "dirs": [ "D" ] }, "40585975": { "pmid": "40585975", "title": "Bioinspired nano-micron hydrogel microspheres for periodontitis therapy through synergistic multi-targeted remodeling of microenvironment.", "abstract": "Background: Periodontitis is a prevalent oral inflammatory disease that leads to alveolar bone resorption and tooth loss. It is hard to control and prone to recurrence. Current treatments often fall short due to deep, tortuous periodontal pockets and antibiotic-resistant bacteria, such as Fusobacterium nucleatum (F. nucleatum). Notably, current clinical therapies fail to simultaneously fulfill three critical objectives: robust antimicrobial efficacy, potent anti-inflammatory activity, and effective periodontal regenerative capacity. Methods: Inspired by the structure of the lotus flower, nano/micron-combined hydrogel microspheres (PDA/BBR@Gel@BMP9-PDLSC), encapsulating polydopamine (PDA) nanoparticles carrying berberine (PDA/BBR) and BMP9-infected PDLSCs (BMP9-PDLSC), were developed. Results: Microspheres exhibited excellent biocompatibility and sustained drug release, along with significant antibacterial, anti-inflammatory, and bone tissue regenerative effects. In vivo studies confirmed their efficacy in treating calvaria defects and periodontitis with persistent F. nucleatum infection, showing superior new bone formation and anti-inflammatory effects without organ toxicity. Notably, our study demonstrated that the anti-inflammation and osteogenesis effects were due to the synergistic effects of BBR and BMP9 released by PDA/BBR@Gel@BMP9-PDLSC microspheres. RNA-Seq and Western blot analysis showed that BBR and BMP9 synergistically reduced inflammation and promoted bone formation by regulating key genes involved in TNF, TGF-β, and PPAR signaling pathways. Conclusions: Our study provides a novel approach for comprehensively treating periodontitis with antibacterial, anti-inflammatory, and bone tissue regenerative effects.", "journal": "Theranostics", "year": "2025", "doi": "10.7150/thno.112782", "ptypes": [ "Journal Article" ], "first_author": "Zhou S", "dirs": [ "D" ] }, "40677759": { "pmid": "40677759", "title": "Three-stage sequential targeted nasal drops for correcting abnormal mitochondrial division in microglia.", "abstract": "Abnormal mitochondrial division in microglia significantly impacts central nervous system (CNS) diseases. However, treating CNS diseases through microglial mitochondria presents several challenges: intracerebral delivery of drugs, microglial targeting, and mitochondrial regulation. Herein, a novel three-stage sequential targeted nasal drops delivery system that achieves precise drug delivery to the core of brain lesions through noninvasive nasal delivery, targeting microglia, and regulating mitochondria were developed. Firstly, dehydroepiandrosterone (DHEA), identified from clinical data and transcriptomic analyses as a key neurosteroid regulating mitochondrial fission, was selected. Secondly, surface-positively charged hydrogel microspheres were prepared to adhere to the nasal mucosa, thereby avoiding rapid clearance and achieving the first stage of nasal mucosa targeting. Subsequently, targeted liposomes carrying cytotoxic T lymphocyte-associated protein-4 were constructed and modified into microspheres, which released liposomes through the nasal cavity to enter the brain and bound to the activated microglial surface receptors CD80/86 accomplishing the second stage of cell targeting. In the third stage, the system released DHEA in response to the microenvironment, precisely regulating dynamin-related protein 1 involved in mitochondrial membrane remodeling, which inhibited abnormal mitochondrial division, stabilized mitochondrial morphology and function, inhibited microglial activation. This study demonstrated that three-stage sequential nasal drops efficiently traversed the nose-to-brain pathway via nasal mucosa in both murine (n = 200) and porcine (n = 16) models, while significantly ameliorating anesthesia/surgery-induced cognitive dysfunction in mice. Therefore, the three-stage sequential nasal drip is a promising method for the treatment of central nervous system diseases.", "journal": "Bioactive materials", "year": "2025", "doi": "10.1016/j.bioactmat.2025.06.029", "ptypes": [ "Journal Article" ], "first_author": "Fei M", "dirs": [ "D" ] }, "40384273": { "pmid": "40384273", "title": "Engineered Exosome-Loaded Silk Fibroin Composite Hydrogels Promote Tissue Repair in Spinal Cord Injury Via Immune Checkpoint Blockade.", "abstract": "Spinal cord injury (SCI) is a severe central nervous system disorder characterized by a high rate of disability and limited axonal regeneration. Excessive post-injury inflammation often leads to further neuronal damage. Immune checkpoint (IC) genes, which regulate immune cell activity, play a critical role in modulating post-injury inflammation and thus influence neural repair and functional recovery. In this study, analysis of the GEO database reveals that the IC gene T cell immunoglobulin and mucin domain-containing protein 3 (Tim3) is highly expressed in microglia following SCI, contributing to an exacerbated inflammatory response. To address this, an RNAi-Tim3-Exo@SF hydrogel system is designed to deliver siRNA-Tim3 via exosomes, thereby regulating Tim3 expression after injury. Furthermore, miRNA sequencing indicates that the engineered exosomes (RNAi-Tim3-Exo) encapsulated within the hydrogel have the potential to promote axonal regeneration and modulate the spinal cord microenvironment. Preclinical studies demonstrate that the RNAi-Tim3-Exo@SF hydrogel could stabilize microtubules, promote damaged axon regeneration, stimulate angiogenesis, modulate the inflammatory environment, and ultimately improve motor function in SCI mouse models. Mechanistically, these reparative effects may be associated with miR-155-5p contained within the RNAi-Tim3-Exo. By integrating bioinformatics, biomedical science, and tissue engineering, this study presents a novel hydrogel-based therapeutic strategy with significant potential for the treatment of SCI.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2025", "doi": "10.1002/smll.202412170", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Dong X", "dirs": [ "D" ] }, "41360394": { "pmid": "41360394", "title": "Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag.", "abstract": "Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components, such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labeling of various droplet components, which may affect phase separation behavior and are thus not able to probe biomolecules in their chemically native state. Here, we introduce a noninvasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both the dilute and condensed phases. We demonstrate the robust performance of this 1D 1H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.", "journal": "Journal of the American Chemical Society", "year": "2025", "doi": "10.1021/jacs.5c14476", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Pálfy G", "dirs": [ "D" ] }, "41287253": { "pmid": "41287253", "title": "LRRC15 Silencing Reprograms Costal Chondrocytes with Enhanced Anti-Hypertrophic Capacity for Functional Cartilage Regeneration.", "abstract": "Costal chondrocytes (CCs) are promising alternatives to articular chondrocytes (ACs) when selecting donor cells for cartilage tissue engineering. However, their clinical utility is hindered by an inherent tendency toward hypertrophic differentiation and subsequent calcification. RNA sequencing identifies leucine-rich repeat-containing protein 15 (LRRC15) as a novel regulator of hypertrophic differentiation in CCs. CCs in which LRRC15 is silenced (shLRRC15@CCs) significantly enhance cartilage matrix synthesis and suppress the expression of hypertrophic markers in vitro. Notably, shLRRC15@CCs pellets maintain a stable chondrogenic phenotype under IL-1β-induced inflammatory conditions and promote robust cartilage regeneration in a rat defect model. To enhance clinical translatability, shLRRC15@CCs are encapsulated within an injectable thermosensitive poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PLEL) hydrogel (PLEL@shLRRC15-CCs), enabling minimally invasive delivery and sustained cell retention at defect sites. Transcriptome profiling reveals the mechanism by which LRRC15 knockdown attenuates AP-1 transcriptional activity. Collectively, FOS expression is downregulated, thus disrupting assembly of the AP-1 complex. Collectively, these findings demonstrate that PLEL@shLRRC15-CCs constitute a synergistic cell-hydrogel therapy with two advantages: intrinsic anti-hypertrophic properties and an injectable gelation capacity. Both advantages support minimally invasive cartilage repair.", "journal": "Advanced healthcare materials", "year": "2026", "doi": "10.1002/adhm.202502326", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Lin ZH", "dirs": [ "D" ] }, "40501883": { "pmid": "40501883", "title": "Engineering Sustained-Release Broadly Neutralizing Antibody Formulations.", "abstract": "Sustained serum levels of broadly neutralizing antibodies (bnAbs) are crucial for effective passive immunization against infectious diseases as protection persists only while these bnAbs remain at adequate concentrations within the body. Current obstacles, such as poor pharmacokinetics (PK) and burdensome administration, must be overcome to make bnAbs a viable option for pre- and post-exposure prophylaxis. In this work, we explore how a polymer-nanoparticle (PNP) hydrogel depot technology can be engineered to prolong protein delivery. In-vivo studies in mice and rats demonstrate prolonged protein release, and modeling efforts predict the impact of both the elimination half-life of the active pharmaceutical ingredient and hydrogel depot volume on overall pharmacokinetics. Moreover, flow cytometry characterization reveals that immune cell infiltration into the hydrogel depot can result in faster-than-expected release of antibody cargo on account of active transport via cellular uptake. We then demonstrate that co-formulation of antibodies with an anti-inflammatory agent reduces cellular infiltration and resulting active transport, further extending delivery and pharmacokinetics. Finally, multicompartmental modeling predicts the human PK profiles of clinically relevant HIV bnAbs delivered via subcutaneous hydrogel injection. These findings aid in the development of next generation hydrogel materials that stabilize and slowly release bnAbs for long-term pre-exposure immunoprophylaxis.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.05.27.656504", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Jons CK", "dirs": [ "D" ] }, "39460406": { "pmid": "39460406", "title": "Aptamer-Encapsulated Cellular Nanoparticles for Neurotoxin Neutralization.", "abstract": "Aptamers are single-stranded oligonucleotides that fold into defined architectures for specific target binding. In this study, aptamers are selected that specifically bind to small-molecule neurotoxins and encapsulate them into cell membrane-coated nanoparticles (referred to as 'cellular nanoparticles' or 'CNPs') for effective neutralization of neurotoxins. Specifically, six different aptamers are selected that bind to saxitoxin (STX) or tetrodotoxin (TTX) and encapsulate them into metal-organic framework cores, which are then coated with neuronal cell membrane. The resulting CNPs exhibit high colloidal stability, minimal aptamer leakage, and effective protection of aptamer payloads against enzyme degradation. This detoxification platform combines membrane-enabled broad-spectrum neutralization with aptamer-based specific toxin binding, offering dual-modal neutralization mechanisms for efficient neurotoxin neutralization. The in vitro neutralization efficacy is demonstrated using a neuron osmotic swelling assay, a Na+ flux fluorescence assay, and a cytotoxicity assay. The in vivo neutralization efficacy is further validated using mouse models of STX and TTX intoxication in both therapeutic and preventative regimens. Overall, integrating aptamers with CNPs combines the strengths of both technologies, resulting in a robust solution for broad-spectrum toxin-neutralization applications.", "journal": "Advanced healthcare materials", "year": "2025", "doi": "10.1002/adhm.202403539", "ptypes": [ "Journal Article", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Kai M", "dirs": [ "D" ] }, "41538424": { "pmid": "41538424", "title": "Versatile DNA Hydrogel-Mediated Delivery of Ginsenoside-Encapsulated Small Extracellular Vesicles to Boost Diabetic Wound Repair.", "abstract": "Diabetic wound healing is often hindered by poor outcomes, prolonged recovery, and high recurrence. To address this, a new therapy approach was demonstrated in this study, in which ginsenoside (GS) molecules are incorporated into small extracellular vesicles (sEV) secreted by mesenchymal stem cells (MSCs), and the formed complexes are then anchored in DNA hydrogels via aptamer-CD63 affinity as \"GS/sEV@DNAgels\". Besides the tissue-restorative ability that sEVs inherit from MSCs, in GS/sEV@DNAgels, GS molecules provide a superior antimicrobial/anti-inflammatory environment at wound sites, while DNA hydrogels serve as wound dressings to ensure sustained release kinetics and enhanced skin penetration. An innovative ultrasonic stimulation was developed to promote the massive production of sEVs. By triggering multiple cellular responses that alter membrane fluidity, calcium levels, and relevant protein expression, our approach achieves a 57.7-fold increase in sEV yield. The synergistic effects of GS and sEVs enhance cell viability, migration, and angiogenesis, as well as local anti-inflammatory and antibacterial conditions during diabetic wound healing. The upregulation of miR-424/322 is confirmed as an essential mechanism of this GS/sEV@DNAgel system in accelerating skin restoration. Our work provides a new and promising strategy for diabetic tissue regeneration.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2026", "doi": "10.1002/advs.202522920", "ptypes": [ "Journal Article" ], "first_author": "Xing J", "dirs": [ "D" ] }, "42253785": { "pmid": "42253785", "title": "Enzyme-Integrated Hydrogels for Advanced Biological Applications.", "abstract": "Natural enzymes, with their precise structural organization and compartmentalization in metabolic pathways, exhibit remarkable catalytic efficiency, inspiring the design of enzyme-integrated hydrogels for advanced biocatalysis. In this review, we first systematically discuss commonly employed physical strategiesincluding self-assembly, electrostatic adsorption, and direct encapsulationfor constructing enzyme-integrated hydrogel systems. Subsequently, we elaborate on enzyme-mediated self-confined polymerization and cross-linking strategies, encompassing natural enzymes, enzyme cascades, nanozymes, and physical-biochemical coupling catalysis. When considering advanced biological applications, we highlight the diverse applications of enzyme-integrated hydrogels across various fields, including imaging, tumor therapy, tissue engineering, and other biomedical diagnostics and therapeutics. Lastly, we present critical perspectives on current research challenges and future opportunities for enzyme-integrated hydrogels, aiming to advance their development and expand their biomedical and clinical applications.", "journal": "Polymer science & technology (Washington, D.C.)", "year": "2026", "doi": "10.1021/polymscitech.5c00076", "ptypes": [ "Journal Article", "Review" ], "first_author": "Hu M", "dirs": [ "D" ] }, "41387105": { "pmid": "41387105", "title": "NIR-Activatable Domino Cascade Catalysis Nanozyme Reactor for Multi-Mechanism Synergistic Immunotherapy in Bladder Cancer.", "abstract": "Current intravesical therapies for bladder cancer after resection are limited by poor tissue penetration, off-target effects, and insufficient efficacy. To address these challenges, this study designs a thermo-responsive hydrogel (PNH) that encapsulates chitosan (CS)-coated Fe/Mn bimetallic nanozymes (FMCC) together with cholesterol oxidase (ChOx). FMCC displays multiple enzyme-mimicking activities, including peroxidase (POD), catalase (CAT), and glutathione oxidase (GSHox). ChOx amplifies this catalytic cascade, enhancing reactive oxygen species (ROS) production and inducing ferroptosis-mediated tumor cell death. The CS coating improves mucosal adhesion and tissue permeability, thereby facilitating intravesical delivery. Upon near-infrared (NIR) irradiation, FMCC generates heat that liquefies the hydrogel, enabling spatiotemporally controlled drug release and providing mild photothermal therapy (MPTT). This photothermal effect acts synergistically with ferroptosis induction and immune modulation, concurrently minimizing damage to normal tissues. In parallel, ChOx disrupts cholesterol-rich membrane rafts and promotes pro-inflammatory M1 macrophage polarization. Released Mn2+ ions further potentiate immune activation by stimulating the cGAS-STING pathway, driving IFN-β and IL-6 secretion, dendritic cell maturation, and T cell infiltration. Together, this nanozyme-hydrogel system integrates tissue penetration, metabolic disruption, and immune stimulation, representing a promising strategy for localized bladder cancer therapy.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2026", "doi": "10.1002/advs.202513913", "ptypes": [ "Journal Article" ], "first_author": "Jiang Y", "dirs": [ "D" ] }, "40977628": { "pmid": "40977628", "title": "Submucosal Hydrogel for Spring-Mediated Intestinal Lengthening.", "abstract": "Spring-mediated distraction enterogenesis has shown success in intestinal lengthening, with spring confinement achieved by external plication with sutures to reduce the lumen diameter at both ends of the intestinal segment. Endoscopic spring placement would minimize the morbidity associated with device insertion. This study investigates the use of submucosal injection of engineered hydrogel to temporarily confine a compressed spring within an intestinal segment. Engineered hydrogels were composed of hyaluronic acid (HA) alone or HA with elastin-like protein (HELP). To simulate endoscopic injection in six juvenile pigs, hydrogel was injected into the submucosa in everted jejunum, followed by the placement of a gelatin-encapsulated, compressed nitinol spring. The jejunum was then unfolded over the spring, and hydrogel was injected distally into the submucosa. Sutures were placed as fiducial markers. After 7 days on a liquid diet, the pigs were euthanized, and their intestinal segments were analyzed for lengthening and histological changes. The spring-containing jejunal segments expanded in all animals, lengthening to 132% in the HA group and 188% in the HELP group. HELP hydrogels exhibited slower biodegradation than HA-only hydrogels. Histological analysis showed increased crypt width and decreased crypt density in the spring-containing segments compared to controls. Hydrogel effectively provides temporary spring confinement within intestinal segments without adverse effects. The mechanical stimulation from the spring induces crypt fission, expanding the intestinal epithelium. These results support the feasibility of gel-enabled, spring-mediated distraction enterogenesis for intestinal lengthening.", "journal": "Journal of biomedical materials research. Part A", "year": "2025", "doi": "10.1002/jbm.a.37986", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "Salimi-Jazi F", "dirs": [ "D" ] }, "41190751": { "pmid": "41190751", "title": "Virus-Inspired mRNA Delivery Vehicle Enabled by a Multilayered Nucleic Acid Nanocapsule.", "abstract": "The chemical instability of mRNA complicates its future clinical application, including issues surrounding its storage, handling, and potential for delivery to patients. Herein, we describe a chemical formulation, inspired by the dynamic multidimensional structure of a virus, with the potential to address many mRNA delivery limitations by enhancing stability, effective cytosolic delivery, and enabling the ability to target mRNA expression to a specific cell receptor. It consists of a liposomal core, stabilized by a metal organic framework, encapsulated within our nucleic acid nanocapsule formulation, ultimately imparting an enzymatic trigger to gate mRNA release and expression in cells, much as a virus is degraded by proteolytic enzymes under acidic conditions in the endosome to release its genetic cargo. We refer to this formulation as a liposomal metal organic framework-nucleic acid nanocapsule, wherein we show how a multilayered delivery carrier can use both pH and enzyme-specific triggers to gate efficient mRNA delivery to the cytosol of cells in vitro, as well as in vivo. Our system can achieve sustained expression from mRNA when stored at room temperature (∼14 days), mRNA structural integrity after >100 days at -20 °C, and therapeutic efficacy in an avian influenza in vitro model. In vivo, we show the expression of therapeutically relevant proteins and changes in the distribution of fluorescent proteins within a tumor microenvironment, observing more homogeneous expression using aptamers. This work provides an approach to mRNA delivery, which extends its shelf life while providing a route to receptor-specific delivery.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.5c12871", "ptypes": [ "Journal Article" ], "first_author": "Pal S", "dirs": [ "D" ] }, "41088453": { "pmid": "41088453", "title": "Cell line-specific estrogen responses uncover functional sex differences in murine macrophages.", "abstract": "RAW 264.7 (male-derived) and J774A.1 (female-derived) cell lines are widely used in immunology research and are considered preferred models for studying signaling pathways, yet their responses to gonadal hormones remain poorly understood. Gonadal hormones, particularly estrogen, shape immune cell function and contribute to sex differences in disease outcomes, with macrophages playing a central role through their expression of intracellular estrogen receptors (ERs). Herein, we investigated ER expression and functional responses to 17β-estradiol (E2) in male-derived RAW 264.7 and female-derived J774A.1 macrophages, in 2D culture. Additionally, we looked at sex-matched and mismatched media conditions in a 3D hydrogel system. Our results reveal distinct phenotypic and functional differences between the cell lines, emphasizing the need for sex-aware approaches in immunological research and model design. RAW 264.7 and J774A.1 macrophages were cultured in basal media for 24 hours, then treated with varying concentrations of 17β-estradiol (5, 25, 100 nM), as well as hormone-free and control media. Post-treatment analyses included viability, estrogen receptor expression, phenotype skewing, matrix metalloprotease 9 (MMP9) activity, and phagocytosis. These macrophages were also used to condition sex-specific media environments and were encapsulated in a hydrogel network containing adhesive and cleavable sites. Encapsulated cells were then exposed to sex-matched or sex-mismatched conditioned media, and proliferation and MMP9 activity were assessed. Our results revealed distinct differences in estrogen receptor gene and protein expression, as well as in core macrophage functions such as proliferation, inflammation, matrix remodeling, and phenotype skewing. Additionally, the sex-derivation of the surrounding molecular environment affected macrophage behavior in a 3D hydrogel system. Female-derived macrophages were more sensitive in terms of proliferation to sex-mismatched environments, while male-derived macrophages exhibited altered enzyme activity when exposed to female-conditioned media. These findings underscore the importance of accounting for both the origin of immune cells as well as the hormonal and environmental context in which they are studied. Without these considerations, experimental models risk missing critical biological differences that shape immune responses and disease outcomes.", "journal": "Biology of sex differences", "year": "2025", "doi": "10.1186/s13293-025-00760-1", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "Veintimilla AM", "dirs": [ "D" ] }, "39933315": { "pmid": "39933315", "title": "Hierarchical protein nano-crystalline hydrogel with extracellular vesicles for ectopic lymphoid structure formation.", "abstract": "Among cancer therapies, immune checkpoint blockade (ICB) has emerged as a prominent approach, substantially enhancing anti-tumor immune responses. However, the efficacy of ICB is often limited in the absence of a pre-existing immune response within the tumor microenvironment. Here, we introduce a novel hierarchical protein hydrogel platform designed to facilitate the formation of artificial tertiary lymphoid structures (aTLS), thereby improving ICB efficacy. Through the integration of self-assembling ferritin protein nanocages, rec1-resilin protein, and CP05 peptide, our hierarchical hydrogels provide a structurally supportive and functionally adaptive scaffold capable of on-demand self-repair in response to mild thermal treatments. The effective encapsulation of extracellular vesicles (EVs) via the CP05 peptide ensures the formation of aTLS with germinal center-like structures within the hierarchical hydrogel. We demonstrate that, combined with ICB therapy, EV-loaded hierarchical hydrogels also induce the TLS within the tumor, markedly promoting immune responses against ICB-resistant tumor. This bioactive hydrogel platform offers a versatile tool for enhancing a broad range of immunotherapies, with potential applications extending beyond TLS to other frameworks that support complex tissue architectures.", "journal": "Biomaterials", "year": "2025", "doi": "10.1016/j.biomaterials.2025.123166", "ptypes": [ "Journal Article" ], "first_author": "Ahn W", "dirs": [ "D" ] }, "40128393": { "pmid": "40128393", "title": "Osteopontin attenuates the foreign-body response to silicone implants.", "abstract": "The inflammatory process resulting in the fibrotic encapsulation of implants has been well studied. However, how acellular dermal matrix (ADM) used in breast reconstruction elicits an attenuated foreign-body response (FBR) remains unclear. Here, by leveraging single-cell RNA-sequencing and proteomic data from pairs of fibrotically encapsulated specimens (bare silicone and silicone wrapped with ADM) collected from individuals undergoing breast reconstruction, we show that high levels of the extracellular-matrix protein osteopontin are associated with the use of ADM as a silicone wrapping. In mice with osteopontin knocked out, FBR attenuation by ADM-coated implants was abrogated. In wild-type mice, the sustained release of recombinant osteopontin from a hydrogel placed adjacent to a silicone implant attenuated the FBR in the absence of ADM. Our findings suggest strategies for the further minimization of the FBR.", "journal": "Nature biomedical engineering", "year": "2025", "doi": "10.1038/s41551-025-01361-4", "ptypes": [ "Journal Article" ], "first_author": "Griffin MF", "dirs": [ "D" ] }, "40487156": { "pmid": "40487156", "title": "Curcumin-encapsulated exosomes in bisphosphonate-modified hydrogel microspheres promote bone repair through macrophage polarization and DNA damage mitigation.", "abstract": "Repairing critical-sized bone defects represents a major challenge in clinical therapeutics due to inhibited osteogenic differentiation, harsh bone tissue microenvironment, and abnormal inflammatory response. Mesenchymal stem cell-derived exosomes (MSC-Exos) have demonstrated tremendous regenerative potential in tissue repair. However, the confined therapeutic efficacy, deficient targeting capability, and poor retention rate have rendered MSC-Exos-based cell-free therapies insufficient for clinical bone defect repair. This study prepared curcumin-loaded MSC-Exos (Cur@Exos) based on an endogenous drug delivery approach and encapsulated in bisphosphonate-modified GelMA hydrogel microspheres by microfluidics. The CE@BP-Gel microspheres demonstrated superior biocompatibility and were competent to accelerate biomineralization. The sustained-release Cur@Exos in the composite hydrogel microspheres actively regulated the polarization of RAW264.7 cells toward the regenerative M2 type and inhibited the osteoclastic activity, thereby creating an immune microenvironment suitable for osteogenesis. Meanwhile, the composite hydrogel microspheres can directly support the adhesion, proliferation and osteogenic differentiation of BMSCs and facilitate the migration and angiogenesis of HUVECs. In vivo experiments demonstrated that the CE@BP-Gel microspheres significantly accelerated the repair of critical-sized cranial bone defects in SD rats. The targets and mechanisms of action of CE@BP-Gel in bone immune regulation were investigated based on network pharmacology, molecular dynamics simulation and RNA sequencing. It was found that CE@BP-Gel mitigates DNA damage induced by ROS in inflammatory environments. The encapsulated curcumin enhances DNA damage repair by activating the TDP1 enzyme, consequently reducing the expression of inflammatory factors in macrophages. This study demonstrates a promising therapeutic strategy to design an exosome-based drug delivery system for bone defect repair.", "journal": "Materials today. Bio", "year": "2025", "doi": "10.1016/j.mtbio.2025.101874", "ptypes": [ "Journal Article" ], "first_author": "Si Y", "dirs": [ "D" ] }, "41395957": { "pmid": "41395957", "title": "Bioactive Multifunctional Hydrogel Scaffolds Remodel the Inflammatory Microenvironment and Osteogenic-Osteoclastic Homeostasis to Advance Osteoporotic Bone Defect Repair.", "abstract": "Bone defect repair in patients with osteoporosis remains a major challenge due to the imbalance in bone metabolism and the inflammatory microenvironment with high levels of reactive oxygen species. To address these issues, a three-dimensional-printed bioactive multifunctional hydrogel scaffold consisting of a polycaprolactone skeleton and a gelatin/hyaluronic acid framework encapsulated with MgMn-layered double hydroxide nanosheets and calcium alendronate nanowires is constructed to promote osteoporotic bone defect repair. The as-prepared hydrogel scaffold continuously scavenges excess reactive oxygen species and generates oxygen through the multiple enzyme mimicry catalytic (catalase, superoxide dismutase, laccase, and hydroxyl radical scavenging) activities, regulates macrophage phenotype, and decreases the expression of pro-inflammatory cytokines, thereby improving the inflammatory microenvironment and alleviating hypoxia. Concurrently, the sustained release of alendronate and Mg2+ ions from the hydrogel scaffold inhibits osteoclast activities, while the released bioactive ions (Mg2+, Ca2+, and Mn3+) enhance osteoblast activities and promote osteogenic differentiation, which synergistically restore the balance of osteogenic-osteoclastic homeostasis. Animal experimental results reveal that the hydrogel scaffold can reconstruct the osteoclast/osteoblast balance and significantly accelerate bone tissue defect repair in the osteoporotic rats through synergistic effects of functional components in the implanted scaffold. These findings demonstrate the clinical potential of the as-prepared hydrogel scaffold in addressing the multifaceted complications of the pathological microenvironment and in enhancing osteoporotic bone defect repair.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.5c15635", "ptypes": [ "Journal Article" ], "first_author": "Wang Z", "dirs": [ "D" ] }, "41200551": { "pmid": "41200551", "title": "Synthetic cells for phage therapy: a perspective.", "abstract": "A synthetic cell is a membrane-bound vesicle that encapsulates cell-free transcription/translation (TXTL) systems. It represents a transformative platform for advancing bacteriophage therapy. Building on experimental work that demonstrates (i) modular genome assembly, (ii) high-yield phage TXTL systems, and (iii) smart hydrogel encapsulation, we explore how synthetic cells can address major limitations in phage therapy. The promising advances include point-of-care phage manufacturing, logic-responsive antimicrobial biomaterials, and new chassis to dissect the dynamics of phage-host interactions. We also propose a roadmap for the deployment of synthetic cells as programmable and evolvable tools in the context of laboratory research and translational clinical adoption.", "journal": "Frontiers in cellular and infection microbiology", "year": "2025", "doi": "10.3389/fcimb.2025.1690404", "ptypes": [ "Journal Article", "Review" ], "first_author": "Kulkarni V", "dirs": [ "D" ] }, "41424863": { "pmid": "41424863", "title": "Verteporfin-loaded hydrogel targeting YAP-mediated MDSCs recruitment for the treatment of residual tumors after incomplete radiofrequency ablation.", "abstract": "Background: Hepatocellular carcinoma (HCC), the major form of primary liver cancer, contributes markedly to cancer-related mortality worldwide and remains a serious global health concern, particularly affecting individuals with underlying chronic liver disorders. In hepatocellular carcinoma, insufficient radiofrequency ablation (iRFA) has been reported to drive local tumor relapse and distant spread, possibly by aggravating the immunosuppressive features of the tumor microenvironment. The present work seeks to clarify the underlying pathways driving the development of an immunosuppressive milieu after RFA and to identify potential therapeutic approaches to counteract this process. Methods: An injectable hydrogel composed of quaternized chitosan (QCS) and tannic acid (TA) was constructed to encapsulate verteporfin (VP), a well-established photosensitizer that has been clinically applied for treating neovascular retinal disorders such as age-related macular disease. Beyond its ophthalmologic application, VP has recently been reported to display anti-tumor activity through inhibition of oncogenic regulators such as Yes-associated protein (YAP), indicating its potential utility in cancer therapy. This hydrogel formulation is designed to target residual tumor tissue post-RFA, providing localized delivery and sustained release of VP to enhance anti-tumor immune responses. Results: Our findings identified YAP activation as a critical mediator of immunosuppression in residual tumors following RFA. Pharmacological inhibition of YAP significantly reduced the infiltration of myeloid-derived suppressor cells (MDSCs) and effectively reversed the immunosuppressive microenvironment conditions. Furthermore, the QCS/TA hydrogel enabled sustained local release of VP, resulting in enhanced antitumor immune responses via MDSC suppression. When administered as an adjuvant therapy following suboptimal RFA, the hydrogel markedly inhibited the progression of residual tumors, highlighting its therapeutic potential in improving post-RFA outcomes. Conclusion: Collectively, our data suggest YAP pathway inhibition as a promising immunomodulatory strategy to complement RFA in HCC management. This work demonstrates that the QCS/TA hydrogel-based delivery system can remodel the tumor immune milieu to overcome immunosuppression and delay post-ablation tumor recurrence, supporting its potential as a translational drug delivery strategy.", "journal": "Theranostics", "year": "2026", "doi": "10.7150/thno.119377", "ptypes": [ "Journal Article" ], "first_author": "Chen J", "dirs": [ "D" ] }, "40489013": { "pmid": "40489013", "title": "De Novo Design of Integrin α5β1 Modulating Proteins to Enhance Biomaterial Properties.", "abstract": "Integrin α5β1 is crucial for cell attachment and migration in development and tissue regeneration, and α5β1 binding proteins can have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5β1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5β1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin (FN) and RGD peptides in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed FN- and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.", "journal": "Advanced materials (Deerfield Beach, Fla.)", "year": "2025", "doi": "10.1002/adma.202500872", "ptypes": [ "Journal Article" ], "first_author": "Wang X", "dirs": [ "D" ] }, "39938720": { "pmid": "39938720", "title": "Injectable hydrogel microspheres promoting inflammation modulation and nucleus pulposus-like differentiation for intervertebral disc regeneration.", "abstract": "Local inflammation modulation and stem cell therapy have attracted much attention in the treatment of intervertebral disc degeneration (IDD). However, severe oxidative stress and limited nucleus pulposus (NP)-like differentiation of stem cells largely impair biomaterial implantation's therapeutic efficacy. Due to their excellent performance in injectability and flowability, and minor compression to NP tissue, hydrogel microspheres have become an attractive carrier for IDD treatment. Herein, an injectable hydrogel microsphere consisting of Wnt5a-mimetic peptide Foxy5- and the antioxidative peptide-grafted gelatin methacryloyl matrix (GFA), was developed as a stem cell delivery system for IDD therapy. Being fabricated and encapsulating bone marrow-derived mesenchymal stem cells (BMSCs) using the microfluidic technology, GFA hydrogel microspheres ameliorate IDD by promoting inflammation inhibition, NP-like differentiation and extracellular matrix regeneration. They efficiently eliminated reactive oxygen species, and downregulated the inflammation level through the inhibition of interleukin-17B/nuclear factor-κB signaling pathway. Moreover, the NP-like differentiation of BMSCs was effectively stimulated by Foxy5 via the calcium/calmodulin dependent protein kinase kinase 2/protein kinase A/sex determining region Y box protein 9 signaling pathway, thereby leading to a rebalance between the generation and degradation of NP matrix. In vivo rat IDD model demonstrated that BMSC-loaded GFA hydrogel microspheres mitigated local inflammation, preserved disc height, and promoted intervertebral disc regeneration. In conclusion, this study introduces an BMSC-loaded injectable hydrogel microspheres as a promising therapy for regulating the microenvironment and alleviating the progression of IDD.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.02.016", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Chen Y", "dirs": [ "D" ] }, "40240370": { "pmid": "40240370", "title": "Hydrogels with programmed spatiotemporal mechanical cues for stem cell-assisted bone regeneration.", "abstract": "Hydrogels are extensively utilized in stem cell-based tissue regeneration, providing a supportive environment that facilitates cell survival, differentiation, and integration with surrounding tissues. However, designing hydrogels for regenerating hard tissues like bone presents significant challenges. Here, we introduce macroporous hydrogels with spatiotemporally programmed mechanical properties for stem cell-driven bone regeneration. Using liquid-liquid phase separation and interfacial supramolecular self-assembly of protein fibres, the macroporous structure of hydrogels provide ample space to prevent contact inhibition during proliferation. The rigid protein fibre-coated pore shell provides sustained mechanical cues for guiding osteodifferentiation and protecting against mechanical loads. Temporally, the hydrogel exhibits tunable degradation rates that can synchronize with new tissue deposition to some extent. By integrating localized mechanical heterogeneity, macroporous structures, surface chemistry, and regenerative degradability, we demonstrate the efficacy of these stem cell-encapsulated hydrogels in rabbit and porcine models. This marks a substantial advancement in tailoring the mechanical properties of hydrogels for stem cell-assisted tissue regeneration.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-59016-6", "ptypes": [ "Journal Article" ], "first_author": "Xue B", "dirs": [ "D" ] }, "41322143": { "pmid": "41322143", "title": "Spermidine-encapsulated chondroitin sulfate methacryloyl hydrogel delivery system for remodeling bone homeostasis in rheumatoid arthritis.", "abstract": "Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent synovial inflammation, progressive cartilage degradation, and osteoclast-mediated bone erosion. While current systemic therapies alleviate inflammation, they often fail to prevent structural joint damage or promote local tissue regeneration. Herein, we developed a chondroitin sulfate methacryloyl (ChSMA) hydrogel-based delivery system encapsulating spermidine (SPD), a naturally occurring polyamine with emerging anti-inflammatory, senescence-attenuating, and chondroprotective properties, to achieve localized and sustained treatment of RA-related joint destruction. Using an RA-mimicking co-culture model comprising RA patient-derived synovial organoids and chondrocytes, we demonstrated that ChSMA@SPD effectively attenuated chondrocyte apoptosis and suppressed the expression of pro-inflammatory cytokines and matrix metalloproteinases (MMPs). In addition, ChSMA@SPD significantly inhibited osteoclast differentiation in vitro using primary bone marrow-derived monocytes from mice by downregulating dendritic cell-specific transmembrane protein (DC-STAMP) expression. In vivo studies using the collagen-induced arthritis (CIA) mouse model further confirmed that intra-articular administration of ChSMA@SPD reduced arthritis severity, preserved cartilage integrity, and mitigated joint inflammation. Furthermore, network pharmacology and molecular docking analyses identified key signaling pathways and potential molecular targets of SPD, such as TGFB2, XIAP, MMP8, and PLA2G1B. Collectively, our findings highlight ChSMA@SPD as a dual-functional hydrogel platform that simultaneously protects cartilage and suppresses bone resorption, offering a promising localized therapeutic strategy for RA treatment.", "journal": "Materials today. Bio", "year": "2025", "doi": "10.1016/j.mtbio.2025.102531", "ptypes": [ "Journal Article" ], "first_author": "Wang X", "dirs": [ "D" ] }, "41696152": { "pmid": "41696152", "title": "Smart nebulized ROS-responsive hydrogel microspheres loaded with UC-MSCs-derived exosomes for the treatment of acute lung injury.", "abstract": "Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are rapidly progressing, highly fatal clinical syndromes characterized by acute and progressive hypoxic respiratory failure. These conditions result in significant morbidity and mortality, yet no specific treatments are currently available, with management relying largely on symptomatic and supportive care. The pathogenesis of ALI/ARDS is closely linked to the excessive production of reactive oxygen species (ROS) and an uncontrolled inflammatory response, which collectively drive disease progression and tissue damage. To address this, we have developed a novel ROS-responsive hydrogel microsphere encapsulating human umbilical mesenchymal stem cell-derived exosomes, designated Exo@TK@CaAlg hydrogel microspheres. The design incorporates thioketal bonds, endowing the microspheres with both effective ROS-scavenging ability and specific responsiveness to oxidative stress. Exo@TK@CaAlg microspheres exhibit a small particle size and excellent biocompatibility in vitro and in vivo. Their potent ROS scavenging capacity positions them as a promising therapeutic approach for alleviating ALI. In preclinical studies, these microspheres have been successfully delivered via nebulization to the lungs of ALI mice, where they play a critical role in mitigating oxidative stress and inflammation. The treatment enhances pulmonary capillary barrier integrity, reduces protein exudation, restores mitochondrial function, and promotes the transition of macrophages from a pro-inflammatory to an anti-inflammatory phenotype. These collective findings highlight the potential of Exo@TK@CaAlg hydrogel microspheres as a therapeutic strategy for ALI/ARDS.", "journal": "Materials today. Bio", "year": "2026", "doi": "10.1016/j.mtbio.2026.102868", "ptypes": [ "Journal Article" ], "first_author": "Liu C", "dirs": [ "D" ] }, "40292340": { "pmid": "40292340", "title": "Hydroxyapatite microspheres encapsulated within hybrid hydrogel promote skin regeneration through the activation of Calcium Signaling and Motor Protein pathway.", "abstract": "Hydroxyapatite (HAp), traditionally recognized for its efficacy in bone regeneration, has rarely been explored for skin regeneration applications. This investigation explored HAp microspheres with distinct physicochemical properties tailored away from conventional bone regeneration parameters, and the capacity promoting skin regeneration and mitigating the aging process were investigated when encapsulated in hyaluronate hydrogels. By benchmarking against well-established dermal fillers like PMMA and PLLA, it was revealed the specific attributes of HAp that were conducive to skin regeneration, providing initial insights into the underlying mechanism. HAp enhanced the fibroblast functionality by triggering minimal adaptive immune responses and enhancing the Calcium Signaling and Motor Protein Signaling pathways. This modulation supported the production of normal collagen fibers, essential for ECM maturation and skin structural integrity. The significant ECM regeneration and remodeling capabilities exhibited by the HAp-encapsulated hybrid hydrogels suggested promising application in facial rejuvenation procedures, potentially making a breakthrough in aesthetic and reconstructive surgery.", "journal": "Bioactive materials", "year": "2025", "doi": "10.1016/j.bioactmat.2025.04.002", "ptypes": [ "Journal Article" ], "first_author": "Liu S", "dirs": [ "D" ] }, "41727273": { "pmid": "41727273", "title": "ROS-responsive hydrogel patch orchestrating macrophage reprogramming and mitochondrial protection for post-MI repair.", "abstract": "Myocardial infarction (MI) leads to irreversible cardiomyocyte loss accompanied by oxidative stress, inflammation, and fibrotic remodeling, ultimately progressing to heart failure. Using network pharmacology analysis, salvianolic acid B (DB), a major bioactive component of Salvia miltiorrhiza, was identified as a promising therapeutic candidate for MI. Oral administration of DB showed therapeutic efficacy in functional, molecular, and histological assessments, but its clinical translation is limited by poor bioavailability. To overcome this limitation, we developed an injectable reactive oxygen species (ROS)-responsive hydrogel patch for localized MI repair. The hydrogel, composed of whey protein isolate methacrylate (WPI-MA) and o-nitrobenzyl alcohol modified hyaluronic acid (HA-NB), forms a biocompatible and adhesive network through dynamic covalent interactions. ROS-sensitive liposomes encapsulating DB were incorporated into the hydrogel, enabling localized and on-demand drug release in response to the oxidative microenvironment. In vitro studies confirmed that the hydrogel exhibited favorable mechanical strength, selective myocardial adhesiveness, and sustained antioxidant capacity. In a murine MI model, a single administration of the hydrogel patch markedly attenuated fibrosis, promoted angiogenesis, and restored cardiac function. This study establishes a rational design strategy that begins with network pharmacology-based drug discovery, proceeds through validation of therapeutic efficacy, and culminates in the construction of a responsive delivery system, providing a promising approach for localized and sustained post-infarction cardiac repair.", "journal": "Bioactive materials", "year": "2026", "doi": "10.1016/j.bioactmat.2026.01.015", "ptypes": [ "Journal Article" ], "first_author": "Li M", "dirs": [ "D" ] }, "40385972": { "pmid": "40385972", "title": "Microenvironment-responsive living hydrogel containing engineered probiotic for treatment of massive bone defects.", "abstract": "Self-activating and microenvironment-responsive biomaterials for tissue regeneration would address the escalating need for bone grafting, but remain challenging. The emergence of microbial living therapeutics offers vast potential in regenerative medicine, as genetically engineered probiotics possess efficient stimuli-responsiveness and tunable biological functions. Here, using elevated endogenous nitric oxide (NO) signals as a biological trigger in bone fracture injuries, a Living Responsive Regenerative Medicine (LRRM) strategy for in situ bone defect repair through real-time controlled release of bone morphogenetic protein-2 (BMP2) is proposed. The Escherichia coli Nissle 1917 (EcN) strain, genetically engineered to sense NO signals and correspondingly produce and secrete BMP2, was firstly encapsulated in gelatin methacryloyl (GelMA) microspheres and then embedded in a bulky hyaluronic acid methacryloyl (HAMA) hydrogel to form a living hydrogel device that circumvents immune attack and prevents bacterial leakage. In vivo multiple bone defect models demonstrated the efficacy of the living hydrogel in enhancing the maturation of bone callus, promoting neovascularization, and facilitating full-thickness bone union. Strategic incorporation of engineered probiotics and the bilayer-structured encapsulation system may emerge as an effective and microenvironment-responsive medicine approach for tissue regeneration.", "journal": "Bioactive materials", "year": "2025", "doi": "10.1016/j.bioactmat.2025.04.020", "ptypes": [ "Journal Article" ], "first_author": "Fang H", "dirs": [ "D" ] }, "42174624": { "pmid": "42174624", "title": "ROS-responsive hydrogel microspheres deliver shTMEM2-loaded liposomes to suppress pyroptosis and ameliorate intervertebral disc degeneration.", "abstract": "Low back pain (LBP) is a highly prevalent musculoskeletal disorder worldwide. Nucleus pulposus cell (NPC) pyroptosis contributes to intervertebral disc degeneration (IVDD), but the underlying mechanisms remain unclear. We identified TMEM2, a transmembrane protein not previously linked to IVDD, as markedly upregulated in degenerated NPCs by single‑cell RNA sequencing. TMEM2 expression correlated positively with IVDD severity and NPC pyroptosis, and its knockdown alleviated pyroptosis. Mechanistically, TMEM2 binds BAX to promote mPTP opening, leading to mtDNA release and activation of the STING-TBK1-NF‑κB pathway, ultimately triggering NLRP3 inflammasome‑mediated pyroptosis. To address the avascular nature of the disc, we developed an injectable ROS‑responsive hydrogel microsphere system encapsulating shTMEM2‑loaded liposomes (Lipo/shTMEM2@MS). This platform enables sustained release, on‑demand degradation, and single‑dose intradiscal administration. In a rat model, Lipo/shTMEM2@MS effectively ameliorated disc degeneration. Collectively, these findings identify TMEM2 as a key driver of NPC pyroptosis and establish a ROS‑responsive microsphere strategy as a promising translational approach for IVDD.", "journal": "Journal of nanobiotechnology", "year": "2026", "doi": "10.1186/s12951-026-04579-9", "ptypes": [ "Journal Article" ], "first_author": "Xu Z", "dirs": [ "D" ] }, "42439201": { "pmid": "42439201", "title": "Gold Nanoparticles-Embedded Mn/Zn-MOF with Enhanced Antioxidant Activities for Treating Atopic Dermatitis.", "abstract": "Atopic dermatitis (AD) is a skin disease characterized by impaired barrier function and immune dysregulation, in which oxidative stress imbalance plays a key pathogenic role. Current clinical therapies for AD treatment primarily target immune and inflammatory responses, but rarely address direct clearance of reactive oxygen species (ROS). To correct this imbalance, a multi-enzyme-mimetic nanocomposite, an Au@Mn/Zn bimetallic metal-organic framework (Au@Mn/Zn-MOF; AMZM) is constructed. Manganese sites in AMZM reversibly cycle among Mn(II/III/IV), thereby mimicking superoxide dismutase (SOD) and catalase (CAT) activities. Density functional theory calculations confirm that incorporated gold nanoparticles (AuNPs) optimize the electronic structure of Mn sites by stabilizing the key Mn(III) intermediate and lowering energy barriers, enabling efficient cascade scavenging of O2·- and H2O2. In cellular models, AMZM suppresses ROS, stabilizes mitochondrial function, and downregulates inflammatory cytokines. In a 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model, topical application of polyvinyl alcohol (PVA)-coated AMZM significantly alleviates skin lesions, restores barrier function, and reduces immune infiltration. Transcriptomic and mechanistic studies further indicate that AMZM exerts antioxidant and anti-inflammatory effects through direct ROS scavenging and activation of the FOXO3 pathway. These results support AMZM as a nanotherapeutic strategy for AD treatment.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.74586", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Dong L", "dirs": [ "D" ] }, "41288241": { "pmid": "41288241", "title": "Controlling Supramolecular Assembly through Peptide Chirality.", "abstract": "Peptide-based self-assembled hydrogels are promising materials for diverse applications due to their biocompatibility, tunable mechanical properties, and ability to form nanostructured networks via noncovalent interactions. One of the most extensively studied hydrogelators, fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF), rapidly self-assembles into a 3D hydrogel capable of encapsulating enzymes and proteins, making it an attractive candidate for drug delivery applications and the protection of oxygen-sensitive biomolecules. However, its fast gelation results in heterogeneous structures and low-density cavities, limiting its uniformity and complicating the handling. Chirality plays a critical role in peptide self-assembly, yet its impact on hydrogel functionality remains underexplored. Here, we investigate how chirality influences the self-assembly kinetics, morphology, and structural properties of all four enantiomeric forms of Fmoc-FF. Using a range of analytical techniques, we tracked the morphological transitions from monomers to supramolecular nanostructures. Hydrogels formed from homoenantiomers displayed greater rigidity and faster gelation, while heteroenantiomeric systems exhibited a slower, three-phase transition from turbid nanospheres to transparent fibrillary gels. This slower gelation may be advantageous for controlled encapsulation, allowing for homogeneous distribution of the cargo. Finally, all enantiomeric hydrogels effectively prevented oxygen diffusion through their nanofiber networks, allowing H2 production by the oxygen-sensitive enzyme hydrogenase, which was encapsulated within the hydrogels. These findings highlight the potential of enantiomeric design in developing peptide hydrogels for various applications, particularly the encapsulation of small molecules and large proteins as well as oxygen-sensitive processes.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c14913", "ptypes": [ "Journal Article" ], "first_author": "Chatterjee M", "dirs": [ "D" ] }, "40703078": { "pmid": "40703078", "title": "Hydrogel particle-based protein display enabled by particle-templated emulsification.", "abstract": "Protein display technology enables high-throughput screening and plays an important role in protein discovery and engineering. Conventional in vivo display methods face challenges such as inefficient gene transformation and complex cell proliferation dynamics, while in vitro display methods are often limited to affinity-based selection and suffer from expression bias due to homogeneous reaction conditions. Here, we present a hydrogel particle-based protein display method enabled by particle-templated emulsification. This approach uses functionalized polyacrylamide hydrogel particles as isolated microreactors, incorporating DNA primers for genotype immobilization and Ni-NTA groups for capturing histidine-tagged protein phenotypes. Displayed proteins are synthesized via cell-free protein expression within isolated droplets, overcoming the limitations of in vivo cell culture and enabling compartmentalized screening, which is challenging in conventional homogeneous in vitro systems. Using particle-templated emulsification, single hydrogel particles can be rapidly encapsulated with individual DNA templates into isolated water-in-oil droplets within 30 seconds, without the need for specialized instrumentation. Up to 109 particles can be emulsified in a standard 50 ml conical tube. Compared to conventional droplet microfluidics, particle-templated emulsification achieves higher single-particle encapsulation and improved one-to-one particle-DNA pairing efficiency, reducing reagent consumption and minimizing DNA library loss caused by improper pairing. Digital PCR and cell-free protein expression are sequentially performed within droplets, with both the amplified DNA and the expressed protein immobilized on the same particle, thereby establishing a stable genotype-phenotype linkage. This method eliminates the need for cell handling, enables compartmentalized functional screening, and provides a fast, scalable, and user-friendly workflow for protein display, offering strong potential in directed evolution and protein engineering.", "journal": "RSC advances", "year": "2025", "doi": "10.1039/d5ra03622d", "ptypes": [ "Journal Article" ], "first_author": "Wu H", "dirs": [ "D" ] }, "40761027": { "pmid": "40761027", "title": "Ce6-DNAzyme-Loaded Metal-Organic Framework Theranostic Agents for Boosting miRNA Imaging-Guided Photodynamic Therapy in Breast Cancer.", "abstract": "Breast cancer remains a leading cause of mortality among women globally, underscoring the critical need for effective theranostic strategies. MicroRNA-21 (miR-21) imaging-guided photodynamic therapy (PDT) has attracted significant attention in recent years due to its selectivity and sensitivity toward breast cancer. However, key challenges remain, particularly regarding the low abundance of miR-21 caused by low-quality imaging at the tumor site and the low efficiency of PDT. To address these issues, we developed theranostic Ce6-DNAzyme@ZIF-8@PEG nanoparticles (CDZP NPs) for breast cancer, which integrates dual-cycling signal amplification for miR-21 detection and enhanced PDT through GPX4-DNAzyme-mediated gene editing to inhibit reactive oxygen species (ROS) scavenging. The CDZP NPs are based on a dodecahedral metal-organic framework (MOF) ZIF-8, encapsulating a dual-cycling miR-21 imaging system and Ce6-DNAzyme therapeutic system via one-pot synthesis. CDZP NPs exhibit excellent biocompatibility, acid-responsive release behavior, and a high loading capacity. These properties enable the control release of Zn2+, Ce6, and dual-cycling signal magnification system for miR-21 detection and enhanced PDT. In vivo studies with tumor-bearing mice demonstrated that intravenous injection of CDZP NPs could effectively target tumors. The dual-cycling signal amplification system, comprising three hairpin probes (H1, H2, and H3), achieved a detection limit for miR-21 as low as 3.4 pM. Moreover, Zn2+-activated GPX4-DNAzyme significantly inhibited GPX4 protein expression, reducing ROS scavenging and further enhancing PDT efficiency with a high tumor inhibition rate of 72.3%. This proposed theranostic strategy holds promise for advancing precision theranostics in breast cancer treatment.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.5c09287", "ptypes": [ "Journal Article" ], "first_author": "Yang Z", "dirs": [ "D" ] }, "42379309": { "pmid": "42379309", "title": "Bio-recruiting hydrogel targeting mitochondrial homeostasis under neutrophil extracellular traps for diabetic wound repair.", "abstract": "Diabetic wound healing is a complex process that requires precise coordination among functional cells, with endothelial cells (ECs) playing a critical role in tissue vascularization. We begin by identifying neutrophil extracellular traps (NETs) as one of critical stressors that disrupts mitochondrial homeostasis in ECs. In addition, inadequate recruitment of ECs often leads to unsatisfactory regenerative outcomes. To address these issues, we developed a composite hydrogel formulation co-encapsulating C-X-C motif chemokine ligand 12 (CXCL12) mRNA-loaded exosomes to promote pro-regenerative endothelial cell homing, and leonurine (Leo) to regulate cellular functionalities, thereby preserving endothelial function essential for neovascularization. By pairing exosomal mRNA delivery with the MS2 coat protein (MCP)-MS2 tethering system for mRNA payload multiplication, we achieved sustained CXCL12 production and cascade-amplified recruitment of CXCR4-positive cells. Moreover, Leo released from the composite hydrogel protectively rescued mitochondrial dysfunction. Further, application of this hydrogel to full-thickness skin defects led to significantly improved wound regeneration in diabetic mice. In summary, this study establishes a therapeutic \"recruit-reinforce\" platform based on a dual-delivery hydrogel armed with CXCL12 mRNA-enriched exosomes and Leo, thereby precisely targeting mitochondrial homeostasis under NETs stress for efficient diabetic wound repair.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2026", "doi": "10.1016/j.jconrel.2026.115143", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Song J", "dirs": [ "D" ] }, "40528841": { "pmid": "40528841", "title": "Thermosensitive hydrogel composite with si-Cx43 nanoparticles and anti-VEGF agent for synergistic treatment of diabetic retinopathy.", "abstract": "Diabetic retinopathy (DR) is characterized by pathological angiogenesis, inflammation, and retinal neurodegeneration, leading to vision loss. Current therapies, such as anti-VEGF agents, face challenges of low bioavailability and frequent invasive injections. Connexin43 (Cx43), a gap junction protein, plays a key role in DR progression through its modulation of inflammation and vascular dysfunction. A thermosensitive hydrogel composite was developed to encapsulate siRNA targeting Cx43 (si-Cx43) nanoparticles (NPs) and anti-VEGF (Avastin). The hydrogel was characterized for gelation, injectability, and degradation. In vitro studies evaluated the cytotoxicity, anti-angiogenic effects, and permeability regulation in hyperglycemic retinal cells under hyperglycemic conditions. In vivo therapeutic efficacy was assessed in a diabetic retinopathy rat model. si-Cx43-NPs demonstrated high siRNA encapsulation efficiency and stability, effectively silencing Cx43 expression in retinal endothelial cells. The hydrogel exhibited excellent injectability, temperature-sensitive gelation, and controlled degradation. In vitro, si-Cx43-NPs@Avastin-hydrogel significantly suppressed VEGF expression, reduced angiogenesis, and restored cell permeability under hyperglycemic conditions. In vivo, the hydrogel composite reduced neovascularization, inflammation, and apoptosis, restoring retinal structure and function more effectively than either single-agent treatment alone. Biocompatibility studies confirmed minimal toxicity and favorable degradation. The si-Cx43-NPs@Avastin-hydrogel provides a synergistic and minimally invasive therapeutic strategy for DR by targeting angiogenesis, inflammation, and neuroprotection with sustained drug delivery.", "journal": "Materials today. Bio", "year": "2025", "doi": "10.1016/j.mtbio.2025.101917", "ptypes": [ "Journal Article" ], "first_author": "Wen L", "dirs": [ "D" ] }, "40521190": { "pmid": "40521190", "title": "Programmatically activated DNA hydrogel microcapsules for precision therapy in inflammatory bowel disease.", "abstract": "Rationale: DNA-based nanomedicines have shown significant therapeutic potential for various diseases; however, efficiently utilizing DNA nanomedicines without chemicals or small-molecule drugs is still a major challenge. Methods: In this study, we presented programmed activated DNA hydrogel microcapsules (HAMs) specifically designed for the treatment of inflammatory bowel disease (IBD). It was constructed by encapsulating a DNA hydrogel in sodium alginate microcapsules (AMs) shells. The DNA hydrogel is self-assembled from aptamer-functionalized tetrahedral DNA nanostructures (TDNs) with anti-inflammatory properties and a Y-shaped DNA scaffold in response to ATP. This design provides HAMs with characteristics of precisely targeted release and enhanced local concentrations, thus ensuring better therapeutic outcomes. Results: HAMs exhibited a multistage response to intestinal fluids, a characteristic positive charge at IBD lesions, a high concentration of ATP in the inflammatory microenvironment, and high expression of the membrane protein TLR4 on immune cells, thereby enabling precisely targeted therapy for IBD. Both in vivo and in vitro studies demonstrated that this system possessed precise targeting ability and excellent stability. In a dextran sodium sulfate-induced colitis model, we demonstrated that HAMs effectively alleviate IBD by reducing the production of inflammatory cytokines, restoring the intestinal barrier, and modulating the diversity of the gut microbiota. Furthermore, no significant long-term toxicity of HAMs was detected in the treated mice. Conclusions: This stable, specific, and highly biocompatible system of programmatically activated HAMs overcomes the challenges associated with developing pure DNA nanostructures for therapy and presents a promising approach for IBD treatment.", "journal": "Theranostics", "year": "2025", "doi": "10.7150/thno.111583", "ptypes": [ "Journal Article" ], "first_author": "Lu P", "dirs": [ "D" ] }, "41900927": { "pmid": "41900927", "title": "Ionic Liquid Biospheres.", "abstract": "Liquid is a fundamental requirement for life as we understand it, but whether that liquid has to be water is not known. We propose the hypothesis that ionic liquids (ILs) and deep eutectic solvents (DES) constitute a class of non-aqueous planetary liquids capable of persisting on a wide range of bodies where stable liquid water cannot exist. This hypothesis is motivated by key physical properties of ILs and DES. Many exhibit vapor pressures orders of magnitude lower than that of water and remain liquid across exceptionally wide temperature ranges, from cryogenic to well above terrestrial temperatures. These properties permit stable liquids to exist where liquid water would rapidly evaporate or freeze and outside of bulk phases as persistent microscale reservoirs-such as thin films and pore-filling droplets. In other words, ILs and DES can persist in environments without requiring oceans, thick atmospheres, or narrowly regulated climate conditions. We further hypothesize that ILs and DES could act as solvents for non-Earth-like life, based on their polar nature and the demonstrated stability and functionality of proteins and other biomolecules in ionic liquids. More speculatively, our hypothesis extends to the idea that ILs and DES could enable prebiotic chemistry by providing long-lived, protective liquid environments for complex organic molecules on bodies such as comets and asteroids, where liquid water is absent. Additionally, based on the occurrence of DES-like mixtures as protective intracellular liquids in desiccation-tolerant plants, we propose that ILs and DES might be solvents that life elsewhere purposefully evolves. We review protein and other biomolecule studies in ILs and DES and outline planetary environments in which ILs and DES might occur by discussing available anions and cations. We present strategies to advance the IL/DES solvent hypothesis using laboratory studies, computational chemistry, planetary missions, analysis of existing spectroscopic datasets, and modeling of liquid microniches and chemical survival on small bodies.", "journal": "Life (Basel, Switzerland)", "year": "2026", "doi": "10.3390/life16030408", "ptypes": [ "Journal Article" ], "first_author": "Seager S", "dirs": [ "D" ] }, "40975175": { "pmid": "40975175", "title": "An ionic liquid nanoemulsion transdermal delivery system for targeted melanoma therapy.", "abstract": "Melanoma is the deadliest form of skin malignancy. The existing transdermal treatment strategies are difficult to achieve effective concentration of drugs in deep-seated tumors, which seriously affects the effect of percutaneous treatment. The low efficacy of medications for treating melanoma and their percutaneous penetration into tumor tissues are the urgent problems to be solved in percutaneous treatment of skin malignancies. In this work, two dacarbazine/benzothiocycloheptane (DTIC/14) couplets were designed and synthesized, among which compound HIT-1 exhibited significantly cytotoxicity to B16-F10 (IC50 = 7.86 μM), and 10.3-fold stronger than DTIC. HIT-1 could significantly induce apoptosis and G0/G1 phase arrest of B16-F10 cells, showing a good anti-melanoma effect. Three self-assembled nanoemulsions (HIT-1/PM-MEs) were prepared on the basis of molecular simulation, among which HIT-1/PM-ME-1-1 had the best transdermal drug delivery effect. HIT-1/PM-ME-1-1 was not only 11.8-fold stronger than DTIC in terms of anti-B16-F10 activity, but also a good regulator of mRNA and protein expression of DNAJB1, HSPA1B, p53, Bcl-2 and Cleaved-caspase 3. Furthermore, the HIT-1/PM-ME-1-1 transdermal delivery system showed optimal antitumor effects and stimulated antitumor immune response. Altogether, these results strongly support that this transdermal drug delivery system provides a valuable new strategy for the treatment of cutaneous melanoma.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.114243", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Lin R", "dirs": [ "D" ] }, "40680945": { "pmid": "40680945", "title": "Effects of deep eutectic solvent on laccase activity and thermal stability.", "abstract": "Eighteen deep eutectic solvent (DES) systems were systematically evaluated to optimize laccase biocatalysis. Five hydrogen bond acceptors (HBAs) significantly enhanced both enzyme activity and thermal stability. The choline dihydrogen phosphate (ChDHP)-glycerol DES yielded the highest activity increase (198 %). Moreover, a 50 % betaine-sorbitol (BS) solution retained 92 % activity after 1 h at 70 °C-55-fold higher than buffer-addressing a key thermal stability challenge. Fluorescence spectroscopy (FS) and molecular docking revealed that DES solutions stabilized laccase's native structure by forming hydrogen bonds with catalytic center histidine (His) and phenylalanine (Phe) residues. BS-laccase pretreatment increased glucose conversion by 30 % in distillers' grains (DG) and 54 % in poplar wood (PW). These findings establish DES as a sustainable, low-cost co-solvent that boosts enzymatic efficiency, offering molecular insights into HBA-enzyme interactions to guide biocatalytic system design for circular bioeconomy.", "journal": "Bioresource technology", "year": "2025", "doi": "10.1016/j.biortech.2025.132992", "ptypes": [ "Journal Article" ], "first_author": "Yang L", "dirs": [ "D" ] }, "41515317": { "pmid": "41515317", "title": "Microalgae and Macroalgae as Advanced Sources of Tyrosinase Inhibitors.", "abstract": "Tyrosinase (EC 1.14.18.1) is the primary enzyme responsible for melanogenesis in mammals and enzymatic browning in food, creating a high demand for potent, safe inhibitors of this enzyme in the cosmetic, medical, and agricultural sectors. Conventional synthetic inhibitors often face limitations concerning their cytotoxicity and stability, necessitating the exploration of marine natural products (MNPs). Marine algae, comprising macroalgae (seaweeds) and microalgae (including cyanobacteria), represent an underexploited source of structurally diverse bioactives. Macroalgae, particularly brown species, yield complex phlorotannins, such as the non-competitive oligomer dieckol, which exhibits an IC50 of 2.16 µg/mL. Conversely, microalgae deliver high-potency, low-molecular-weight compounds, notably the synthesizable scytonemin monomer (ScyM) with an IC50 of 4.90 µM-significantly stronger than kojic acid. Mechanistic analysis, supported by molecular docking, reveals diverse modes of action, from the two-step slow binding of complex phlorotannins to the highly specific competitive binding of red algal bromophenols. Translational success requires the consistent application of green extraction techniques, such as Natural Deep Eutectic Solvents (NADESs), and advanced delivery systems, like Nanostructured Lipid Carriers (NLCs), to ensure the stability and bioavailability of these compounds for future cosmeceutical and medical applications.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules31010020", "ptypes": [ "Journal Article", "Review" ], "first_author": "Harasym J", "dirs": [ "D" ] }, "39688336": { "pmid": "39688336", "title": "Critical Role of Water beyond the Media to Maintain Protein Stability and Activity in Hydrated Deep Eutectic Solvent.", "abstract": "Hydrated deep eutectic solvents (DESs) are recognized for their potential in biocatalysis due to their tunability, biocompatibility, greenness, and ability to keep protein stable and active. However, the mechanisms governing enzyme stability and activity in DES remain poorly understood. Herein, using bromelain as the model enzyme and acetamide (0.5)/urea(0.3)/sorbitol(0.2) as the model DES, we provide experimental evidence that modulation of associated water plays a key role in dictating protein stability and activity in hydrated DES. Specifically, rigid associated water at higher DES concentrations (beyond 40% v/v) stabilizes bromelain through entropy but destabilizes it through enthalpy. On the other hand, flexible associated water dynamics at lower DES concentrations result in an opposite thermodynamic outcome. Importantly, the bulk water dynamics cannot explain the stability trend, which emphasizes the critical role of water near the protein surface. Strikingly, associated water dynamics also correlates strongly with bromelain's proteolytic activity. An increasing flexibility of the associated water dynamics leads to the enhancement of the activity. This is the first study to experimentally link associated water dynamics to enzyme behavior in hydrated DES, offering insights that could guide future developments in solvent engineering for enzyme catalysis.", "journal": "The journal of physical chemistry. B", "year": "2025", "doi": "10.1021/acs.jpcb.4c07039", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Khan T", "dirs": [ "D" ] }, "41079779": { "pmid": "41079779", "title": "Influence of ionic liquids on enzymatic asymmetric carboligations.", "abstract": "The asymmetric mixed carboligation of aldehydes catalyzed by thiamine diphosphate (ThDP)-dependent enzymes provides a sensitive system for monitoring changes in activity, chemo-, and enantioselectivity. While previous studies have shown that organic cosolvents influence these parameters, we now demonstrate that similar effects occur upon addition of water-miscible ionic liquids (ILs). In this study, six ThDP-dependent enzymes were analyzed in the presence of 14 ILs under comparable conditions to assess their influence on enzymatic carboligation reactions yielding 2-hydroxy ketones. ILs exerted a moderate to strong influence on activity and, more notably, altered enantioselectivity. (R)-selective reactions were generally stable upon IL addition, while (S)-selective reactions frequently showed reduced selectivity or even inversion to the (R)-enantiomer. The most significant change was observed for the ApPDC_E469G variant of pyruvate decarboxylase from Acetobacter pasteurianus, where the enantiomeric excess shifted from 86 % (S) to 60 % (R) in the presence of 9 % (w/v) Ammoeng 102. Control experiments indicated that this shift was primarily due to the Ammoeng cation rather than the anion. To explore the molecular basis of this phenomenon, all-atom molecular dynamics (MD) simulations were performed on wild-type ApPDC and the E469G variant in Ammoeng 101 and Ammoeng 102. The simulations revealed that hydrophobic and hydrophilic regions of the Ammoeng cations interact with the (S)-selective binding pocket, thereby favoring formation of the (R)-product. These results highlight the potential of solvent engineering for modulating enzyme selectivity and demonstrate that MD simulations can capture functionally relevant enzyme-solvent interactions at the atomic level.", "journal": "Computational and structural biotechnology journal", "year": "2025", "doi": "10.1016/j.csbj.2025.09.029", "ptypes": [ "Journal Article" ], "first_author": "Gerhards T", "dirs": [ "D" ] }, "42065023": { "pmid": "42065023", "title": "Cellulase enzymes for sustainable sugarcane waste management: a comprehensive review.", "abstract": "This review assesses the role of cellulase enzymes in the sustainable management of sugarcane waste, focusing on bagasse, trash, and press mud as key lignocellulosic residues. It synthesizes recent experimental and industrial research to evaluate cellulase sources, enzyme systems (endoglucanase, exoglucanase, and β-glucosidase), and pretreatment techniques that impact hydrolysis efficiency. The review underscores that physicochemical pretreatments, such as dilute acid, alkali, steam explosion, and ionic liquids, greatly enhance cellulose accessibility, achieving glucose yields of 70-90% under optimal conditions. Fungal cellulases, particularly from Trichoderma reesei and Aspergillus species, outperform bacterial systems in the saccharification of sugarcane biomass, especially when used in synergistic enzyme cocktails. Key findings also reveal that enzyme immobilization and genetic engineering improve thermal stability, reusability, and catalytic efficiency, thereby lowering the overall processing costs. Furthermore, life cycle and techno-economic assessments show that enzymatic valorization of sugarcane waste significantly reduces greenhouse gas emissions and supports the production of high-value products, including bioethanol, biogas, organic acids, and biopolymers. Overall, this review establishes cellulase-based bioconversion as a technically viable and environmentally sustainable method for large-scale sugarcane waste utilization, while identifying enzyme cost, pretreatment severity, and scale-up challenges as major barriers to industrial application.", "journal": "3 Biotech", "year": "2026", "doi": "10.1007/s13205-026-04783-2", "ptypes": [ "Journal Article", "Review" ], "first_author": "Sinha A", "dirs": [ "D" ] }, "40508473": { "pmid": "40508473", "title": "Advancements in the Research on the Preparation of Isoamyl Acetate Catalyzed by Immobilized Lipase.", "abstract": "This study aims to delve into the application potential of immobilized lipases in the catalytic synthesis of isoamyl acetate. Through a comparative analysis of various immobilization methods, including physical adsorption, encapsulation, covalent binding, and crosslinking, along with the utilization of nanomaterials, such as magnetic nanoparticles, mesoporous silica SBA-15, and covalent organic frameworks (COFs) as carriers, the study systematically evaluates their enhancing effects on lipase catalytic performance. Additionally, solvent engineering strategies, encompassing the introduction of organic solvents, supercritical fluids, ionic liquids, and deep eutectic solvents, are employed to intensify the enzymatic catalytic process. These approaches effectively improve mass transfer efficiency, activate enzyme molecules, and safeguard enzyme structural stability, thereby significantly elevating the synthesis efficiency and yield of isoamyl acetate. Consequently, this research provides solid scientific rationale and technical support for the industrial production of flavor ester compounds.", "journal": "Materials (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ma18112476", "ptypes": [ "Journal Article", "Review" ], "first_author": "Guan G", "dirs": [ "D" ] }, "40005090": { "pmid": "40005090", "title": "Designed Reactive Natural Deep Eutectic Solvents for Lipase-Catalyzed Esterification.", "abstract": "Natural deep eutectic solvents (NADESs) are a sustainable, green option for extraction and reaction media in biorefineries and various chemical and biotechnological applications. Particularly, enzymatic reactions profit from NADES applications, as these solvents help to maintain high substrate solubility while improving both enzyme stability and efficiency. Recent studies confirmed that NADESs can perform multiple functions simultaneously, as reaction media for biocatalytic conversions, but also as substrates and catalysts for reactions, fulfilling the role of a reactive solvent. This study reports the beneficial effect of designed reactive natural deep eutectic solvents (R-NADESs) on the esterification activity and thermal stability of free and immobilized lipases in the synthesis of polyol- and carbohydrate-based biosurfactants. We manufactured and characterized 16 binary and ternary R-NADES systems with choline chloride (ChCl) as the hydrogen bond acceptor (HBA) and carbohydrate polyols; mono-, di-, and oligosaccharides; urea (U); N-methyl urea (MU); and water as the hydrogen bond donors (HBDs), in different combinations and molar ratios, most of which are reported for the first time in this paper. We determined their physicochemical, thermal, and molecular properties, including among others viscosity, polarizability, and the number of hydrogen bonds, and we showed that these properties are controlled by composition, molar ratio, molecular properties, temperature, and water content. Many lipases, both native and immobilized, showed high stability and remarkable catalytic performance in R-NADESs during esterification reactions.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules30040778", "ptypes": [ "Journal Article" ], "first_author": "Buzatu AR", "dirs": [ "D" ] }, "41069152": { "pmid": "41069152", "title": "Low Concentration Ionic Liquid-Assisted Efficient Enzymatic Degradation of Xanthan.", "abstract": "One of the main obstacles to preparing oligoxanthan through the enzymatic degradation of xanthan gum (XG) is its dense structure. To achieve the large-scale production of oligoxanthan, it is necessary to perturbate the conformation of XG and increase enzyme accessibility. This research developed an efficient system for the enzymatic degradation of XG assisted by low concentrations of ionic liquids (ILs). Structural characterization results indicated that a low concentration of [EMIM]DEP (1%, v/v) not only loosens the structure of XG but also alters the secondary structure of enzyme, thereby dramatically increasing its hydrolytic activity and eventually enhancing the production of oligoxanthan. The results were further elucidated by molecular dynamics (MD) simulation. Under the hydrolysis conditions of 1.5 mg/mL Aspergillus niger cellulase (AnCel), 0.2 mg/mL XG, and 1% (v/v) [EMIM]DEP at 40°C and pH 5.0 for 20 min, XG can be hydrolyzed into a mixture with a glucose equivalent of 32.25%. The degree of polymerization (DP) of XG hydrolysates was 3-10. Moreover, the xanthan hydrolysates obtained using this system exhibit excellent antioxidant activity. This study proposed an effective method for the preparation of functional oligosaccharides, which is of significant importance for the valorization of XG.", "journal": "Biotechnology journal", "year": "2025", "doi": "10.1002/biot.70136", "ptypes": [ "Journal Article" ], "first_author": "Chen C", "dirs": [ "D" ] }, "40865227": { "pmid": "40865227", "title": "Impact of imidazolium ionic liquid on biochemistry of marine mussels Mytilus trossulus.", "abstract": "Ionic liquids (ILs) are increasingly used in industrial applications, raising concerns about their environmental fate and toxicity in marine ecosystems. However, their impact on marine organisms, particularly under different salinity conditions, remains poorly understood. This study investigates the bioaccumulation and biochemical effects of 1-decyl-3-methylimidazolium chloride ([IM1-10] Cl) in Mytilus trossulus under salinities of 8 and 16 PSU. Mussels were exposed to [IM1-10] Cl for 21 days, followed by a 21-day depuration period. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantified IL accumulation, while biochemical assays assessed oxidative stress, detoxification responses, and lipid metabolism alterations. The [IM1-10] Cl was significantly accumulated in mussel tissues (511 ± 163 - 1347 ± 204 μg kg-1 DW). The substantially higher accumulation was observed at 16 PSU, suggesting salinity-dependent bioavailability. During depuration, IL levels declined gradually, yet complete elimination was not achieved. Biomarker analysis showed increased oxidative stress and neurotoxicity. Carboxylesterases (CEs) were significantly modulated, suggesting detoxification responses. Fatty acid profiling revealed reductions in polyunsaturated fatty acids and altered enzyme activity (ELOVL1, ELOVL6 and SCD1), implying disruptions in membrane stability and energy metabolism. These biochemical effects persisted beyond the depuration period, indicating potential long-term physiological consequences.", "journal": "Journal of hazardous materials", "year": "2025", "doi": "10.1016/j.jhazmat.2025.139610", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Dołżonek J", "dirs": [ "D" ] }, "41061860": { "pmid": "41061860", "title": "Ultra-high concentration low-viscosity subcutaneous antibody formulations using ionic liquids.", "abstract": "Monoclonal antibodies (mAbs) are the single largest class of therapeutics used in various indications including oncology, immunology, and neurology. Given their high prevalence, the development of subcutaneously administrable mAb formulations via conventional needles is an urgent and unmet need. Subcutaneous mAb formulations need to meet several stringent requirements, including high concentrations to enable the delivery of therapeutic doses through small volumes, low viscosity to allow self-administration, and high shelf stability, all of which are inherently linked to protein-protein interactions, which have collectively limited mAb concentrations in current commercial formulations to ∼150 mg/ml. Here, we report the use of ionic liquids (ILs) to mitigate protein-protein interactions to formulate antibodies at an ultra-high concentration in excess of 200 mg/ml. In addition to solubilizing antibodies at such high concentrations, IgG-IL formulations maintained a viscosity below the injectable threshold of 20 cP, remained stable at even room temperature and 37 °C, and exhibited improved bioavailability compared to saline formulations upon subcutaneous administration.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.114295", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Ramesh A", "dirs": [ "D" ] }, "40221211": { "pmid": "40221211", "title": "Multi-nanozyme cascade system for boosting colorimetric sensing by selective etching bimetallic MOFs.", "abstract": "Rational engineering of multienzyme system architecture is essential for achieving high-performance multi-enzyme cascade catalysis in sensing applications. In this context, the initiation step of the cascade reaction pathway plays a pivotal role in enhancing catalytic efficiency. CoFe Prussian blue analogue (CoFePBA), a dual-metal organic framework, is an ideal template for multi-enzyme design, leveraging its diverse dual-metal ion functionalities and synergistic effects. Defect engineering approaches enable the fine-tuning of catalytic properties, optimizing electron transfer and promoting reaction intermediates. Therefore, the rational design of the multienzyme system structure is critical for efficient cascade catalysis. In this study, we utilized deep eutectic solvents (DES) to selectively induce Co defects in CoFePBA (CoFePBA-DES) under mild conditions, providing more active sites and enhancing the Co2+/Co3+ ratio, significantly boosting the initial step of the cascade reaction. This then triggers the three-enzyme cascade reaction system-oxidase (OXD), superoxide dismutase (SOD), and peroxidase (POD)-which facilitates the conversion of products from O2 to O2•- to endogenous H2O2, achieving a two-fold increase in its yield and subsequently to OH• in a sequential reaction, demonstrating excellent multi-enzyme cascade catalytic activity. Utilizing the inhibitory effect of glutathione (GSH) on multi-enzyme cascade catalytic activity, we designed a highly efficient and rapid colorimetric sensor for the sensitive detection of GSH, with a detection range of 0.5-160 μM and a detection limit of 0.15 μM. Compared to traditional etching techniques, DES-based methods offer superior selectivity, lower toxicity, and better structural preservation of the MOF framework, making them a promising tool for controlled defect engineering. By selectively creating defects, the initial steps of the cascade reaction are activated, resulting in a significant enhancement of catalytic activity. This approach provides a viable pathway for the preparation of high-performance dual-metal catalysts for cascade reaction catalysts and sensing applications.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.343976", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Lin Y", "dirs": [ "D" ] }, "41289825": { "pmid": "41289825", "title": "Advances in ultrasound-enhanced recovery of marine algal polysaccharides: toward sustainable bioprocessing.", "abstract": "The growing emphasis on environmental sustainability has intensified interest in the extraction and valorization of polysaccharides from marine algal species due to their non-toxic nature and notable bioactivities. Although various conventional extraction techniques have been employed, these often involve high energy consumption, prolonged processing times, limited selectivity, and the potential degradation of polysaccharide bioactivity. To address these limitations, a range of sustainable and cost-effective extraction methods has been developed, including enzyme-assisted extraction (EAE), supercritical fluid extraction (SFE), subcritical water extraction (SWE), microwave-assisted extraction (MAE), and ultrasound-assisted extraction (UAE). Among these, the UAE, either as a standalone method or integrated within a biorefinery framework, has emerged as a promising and green technology. It offers higher extraction efficiency with reduced time, energy, and cost requirements. Polysaccharides extracted via ultrasound (US) have demonstrated enhanced anticancer, bioactive, and rheological properties. Furthermore, integrating the UAE with green solvents, such as natural deep eutectic solvents (NaDESs), or complementary techniques, including MAE, hot water extraction (HWE), and EAE, has been shown to further improve yields by 2-6 % and enhance functional quality. This review highlights the potential of the UAE, both individually and in combination with other methods for the efficient recovery of algal polysaccharides. It also evaluates the influence of the UAE on the biofunctional properties of the extracted compounds, underscoring their applicability across diverse industrial sectors. Nonetheless, further research is warranted to optimize process parameters, ensure safety, enhance antioxidant activity (AOA), and improve purification and characterization, as well as better understand the structure-activity relationships of these valuable biopolymers.", "journal": "Ultrasonics sonochemistry", "year": "2025", "doi": "10.1016/j.ultsonch.2025.107683", "ptypes": [ "Journal Article", "Review" ], "first_author": "Naseem T", "dirs": [ "D" ] }, "41441790": { "pmid": "41441790", "title": "Eutectozymes as Soft Hybrid Materials for Advanced Biocatalysis.", "abstract": "The development of soft hybrid materials that combine structural integrity, biocompatibility, and catalytic function is a central challenge in advanced biocatalysis. Here, we introduce eutectozymes, enzyme-loaded eutectogels built from natural hydrophobic deep eutectic solvents (HES) and stabilized through a dual supramolecular polymeric network. This unique architecture not only preserves the conformational integrity of enzymes but also creates confined microcavities acting as protective microreactors, thereby enhancing their stability and substrate affinity. Eutectozymes exhibit remarkable resilience under harsh operational conditions, including high temperatures, extreme pH, and organic solvents, while maintaining high catalytic efficiency and reusability. Their versatility is further demonstrated in environmental remediation, achieving over 90% degradation of recalcitrant dyes, and in antimicrobial applications against resistant bacterial strains. By synergistically merging the stabilizing properties of HES with robust gel matrices, eutectozymes establish a transformative platform for heterogeneous biocatalysis, opening new avenues in biotechnology, bioelectronics, and environmental technologies.", "journal": "Advanced materials (Deerfield Beach, Fla.)", "year": "2026", "doi": "10.1002/adma.202517014", "ptypes": [ "Journal Article" ], "first_author": "Martinez Cartagena ME", "dirs": [ "D" ] }, "41304833": { "pmid": "41304833", "title": "Targeted Biocatalyst Design for Asymmetric Citalopram Conversion in a Membrane Reactor.", "abstract": "Objective: This study aimed to develop a stable and active biocatalytic system for enzyme immobilization, utilizing an electrospun support doped with a metal-organic framework (MOF) and supplemented with an ionic liquid as a lipase stabilizer and activity enhancer. Methodology: The system was applied for an efficient and enantioselective resolution of racemic citalopram. Key parameters, including MOF concentration, electrospinning and immobilization conditions, ionic liquid selection, and reaction time, were optimized to enhance biocatalyst performance. Results: The optimal immobilization time was determined to be 24 h, achieving 52% immobilization efficiency and 100% activity recovery. The resulting biocatalytic system HIGH PVC-MOF-lip-CA exhibited superior storage stability, retaining 80% of its initial activity, a 75% improvement over the free enzyme. In the resolution of citalopram, the system achieved 96% conversion of S-citalopram within 24 h, with the enantiomeric excess of 93% in favor of the S-ester over the R-ester. These findings demonstrate the system's potential for efficient and stereoselective biocatalytic applications.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17111497", "ptypes": [ "Journal Article" ], "first_author": "Degórska O", "dirs": [ "D" ] }, "41150263": { "pmid": "41150263", "title": "Fungal and Microalgal Chitin: Structural Differences, Functional Properties, and Biomedical Applications.", "abstract": "Chitin, one of the most abundant natural polysaccharides, has gained increasing attention for its structural diversity and potential in biomedicine, agriculture, food packaging, and advanced materials. Conventional chitin production from crustacean shell waste faces limitations, including seasonal availability, allergenic protein contamination, heavy metal residues, and environmentally harmful demineralization processes. Chitin from fungi and microalgae provides a sustainable and chemically versatile alternative. Fungal chitin, generally present in the α-polymorph, is embedded in a chitin-glucan-protein matrix that ensures high crystallinity, mechanical stability, and compatibility for biomedical applications. Microalgal β-chitin, particularly from diatoms, is secreted as high-aspect-ratio microrods and nanofibrils with parallel chain packing, providing enhanced reactivity and structural integrity that are highly attractive for functional materials. Recent progress in green extraction technologies, including enzymatic treatments, ionic liquids, and deep eutectic solvents, enables the recovery of chitin with reduced environmental burden while preserving its native morphology. By integrating sustainable sources with environmentally friendly processing methods, fungal and microalgal chitin offer unique structural polymorphs and tunable properties, positioning them as a promising alternative to crustacean-derived chitin.", "journal": "Polymers", "year": "2025", "doi": "10.3390/polym17202722", "ptypes": [ "Journal Article", "Review" ], "first_author": "Yin L", "dirs": [ "D" ] }, "39842147": { "pmid": "39842147", "title": "Deep Eutectic Solvents based green approach for bioactive molecules recovery from Spirulina.", "abstract": "Spirulina is a unicellular microalga, characterized by blue/green color, that has received significant attention for its interesting nutritional composition. Phenolic compounds and phycocyanin (PC) are responsible for the many biological activities of Spirulina. Spirulina phenolic compounds are usually extracted using organic solvents, while PC is extracted with water or phosphate buffer solution, obtaining an extract characterized by low stability and low purity. Deep Eutectic Solvents (DESs) are green solvents, widely applied for bioactive molecules extraction, composed by two or more molecules which act as hydrogen bond acceptor or hydrogen bond donor to create an eutectic mixture. A Natural Deep Eutectic Solvent (NADES), is a DES composed only by natural compounds. This work aimed to optimize a green extraction procedure based on NADES to obtain an extract rich in phenolic compounds and PC from Spirulina. The application of NADESs as extraction solvents is an important strategy for recovering phenolic compounds and to increase and stabilize the protein PC. The optimized extraction procedure involved to use a betaine-glucose NADES under the following conditions: matrix-to-solvent ratio of 1:20 (w/w), temperature of 50 °C and one hour as extraction time. The developed extraction procedure guaranteed to recover a phenolic compounds and PC quantity of 11.77 ± 1.23 mg GAE (gallic acid equivalents) and 27.56 ± 2.46 g per g of Spirulina powder respectively. PC in the NADES extract degraded more slowly than in an aqueous extract. The extract phenolic compounds profile was determined through high performance liquid chromatography coupled with mass spectrometry analysis.", "journal": "Journal of chromatography. A", "year": "2025", "doi": "10.1016/j.chroma.2025.465695", "ptypes": [ "Journal Article" ], "first_author": "Della Posta S", "dirs": [ "D" ] }, "40065704": { "pmid": "40065704", "title": "Ionic Liquid-Induced Modulation of Ubiquitin Stability: The Dominant Role of Hydrophobic Interactions.", "abstract": "Despite the widespread use of imidazolium-based ionic liquids (ILs) in biotechnology, pharmaceuticals, and green chemistry, their detailed interactions with proteins, particularly affecting structural stability, remain poorly understood. This study examines the effects of ILs on ubiquitin, a thermodynamically robust protein with a β-grasp structure. We found that IL-induced destabilization follows a consistent order with previous findings: [BMIM]+ > [BMPyr]+ > [EMIM]+ for cations and [BF4]- > [MeSO4]- > [Cl]- for anions. Through pH and ionic strength-dependent studies, we observed that hydrophobic interactions predominantly influence the stability of positively charged ubiquitin, with electrostatic interactions playing a secondary role. NMR studies identified residues impacted by [BMIM][BF4]; however, site-directed mutagenesis of these residues showed minimal changes in destabilization, suggesting a global effect. This led us to conduct a broader empirical analysis, incorporating solvent-accessible surface area evaluations, which confirmed that hydrophobic residues are the primary drivers of stability alterations in ubiquitin, with charged residues playing a minimal role. Additionally, single-molecule force spectroscopy results indicate that imidazolium ILs lower the unfolding barrier without altering the transition state structure, offering insights into protein folding dynamics. ILs appear to modulate the stability landscape of proteins by energetically and kinetically favoring the unfolded state over the folded state. These insights offer potential strategies for the selective tuning of protein stability, which could be exploited to modulate protein-protein or protein-substrate interactions in various applications of ILs.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.4c03886", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Shrivastava A", "dirs": [ "D" ] }, "39637317": { "pmid": "39637317", "title": "Stability and Fibrillation of Lysozyme in the Mixtures of Ionic Liquids with Varying Hydrophobicity.", "abstract": "Combinatorial effects of small molecules provide newer avenues to improve protein stability. The combined effect of two different classes of ILs on the stability and fibrillation propensity of lysozyme (Lyz) was investigated. Imidazolium-ILs (an aromatic moiety) with varying alkyl chains, methyl (MIC), butyl (BMIC) and hexyl (HMIC), and pyrrolidinium-IL (alicyclic moiety) with butyl substitution (BPyroBr) were chosen. The fibrillation was delayed by the addition of any of the IL. While added as a mixture with varying molar ratios, the presence of HMIC with MIC or BMIC at the ratio of 2:1 increased the fibrillation time synergistically by increasing lag time and reducing elongation rate. The protein stability was significantly reduced in these conditions compared to lower molar ratios of HMIC with MIC or BMIC. Molecular dynamics simulation studies indicated that upon adding Im-ILs water molecules were reduced around Lyz, whereas BPyroBr slightly increased the water around Lyz. Preferential interaction studies suggest that the preferential binding of HMIC with the protein was the most favored and it synergistically facilitated the preferential binding of MIC. Though BMIC was preferentially binding to the protein, it disfavoured the interaction of MIC. BMIC and BPyroBr had a competitive binding on the surface of Lyz. The results suggested that the mixture of ILs containing the longer alkyl chain destabilizes the protein and delays the fibril formation to a larger extent than the shorter alkyl chain ILs. Further, the effect of aromatic ILs could be greater than alicyclic ILs having the same alkyl chain length.", "journal": "Chemphyschem : a European journal of chemical physics and physical chemistry", "year": "2025", "doi": "10.1002/cphc.202400743", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kushwaha P", "dirs": [ "D" ] }, "40414447": { "pmid": "40414447", "title": "A systematic investigation of TMB substrate composition for signal enhancement in ELISA.", "abstract": "3,3',5,5'-Tetramethylbenzidine (TMB) remains one of the most widely utilized chromogenic substrates for horseradish peroxidase (HRP) in colorimetric immunoassays, including enzyme-linked immunosorbent assays (ELISA). Despite its introduction into ELISA workflows over four decades ago, limited research has been conducted to systematically optimize TMB substrate formulations. Recent advancements in the field have proposed innovative approaches to enhance HRP catalysis, such as the use of deep eutectic solvents and ionic liquids, alongside investigations into the chemical properties of TMB and its analogs to identify more efficient alternatives. However, the development of stable and high-performance TMB solutions for clinical diagnostics requires a comprehensive understanding of how formulation parameters influence signal intensity and stability. In this study, we address these gaps by conducting a systematic evaluation of key factors affecting TMB substrate performance, including buffer pH, composition and molarity, specific ion effects, incorporation of organic solvents, and the use of polymer stabilizers. Additionally, novel strategies for signal amplification, identified through an extensive review of literature and patents, were experimentally tested. Based on these findings, we developed an optimized TMB formulation comprising 0.2 mol/L sodium citrate buffer (pH 4.5), 5 % DMSO, 0.37 mmol/L CaCl2, 0.4 mmol/L 2-hydroxy-β-cyclodextrin, 0.8 mmol/L TMB, and 1.3 mmol/L H2O2 as final concentrations. The performance of this optimized formulation was evaluated in comparison to previously reported formulations from the literature.", "journal": "Analytical biochemistry", "year": "2025", "doi": "10.1016/j.ab.2025.115908", "ptypes": [ "Journal Article" ], "first_author": "Khramtsov P", "dirs": [ "D" ] }, "39611237": { "pmid": "39611237", "title": "An ionic liquid-based delivery system of small interfering RNA targeting Bcl-2 for melanoma therapy.", "abstract": "Melanoma, characterized by rapid tumour progression and a strong tendency to metastasize, poses significant challenges in clinical treatment. Given the vital role of B-cell lymphoma 2 (Bcl-2) protein overexpression in inhibiting apoptosis in tumour cells, the suppression of Bcl-2 has emerged as a promising anticancer therapy. Here, we have developed a straightforward and effective delivery system that combines small interfering RNA (siRNA) targeting Bcl-2 (siBcl-2) with ionic liquids (ILs) for treating melanoma. The unique properties of ILs including structural tunability, inherent charge, and chemical stability have garnered significant attention in the biomedical fields; however, their application in siRNA delivery remains nascent. Rather than the weak function of free siBcl-2, our delivery system (1-hexyl-3-methylimidazolium-siBcl-2, designated as C6-siBcl-2) demonstrated an outstanding capacity to improve the cellular uptake and lysosomal escape, resulting in robust apoptosis and cytotoxicity in melanoma cells. In addition to exhibiting superior gene silencing activity in vitro, such events were also evident in mice bearing melanoma tumours. In particular, this IL-based delivery system showed advantages in suppressing tumour growth, preventing metastasis, and enhancing the survival time of mice with melanoma tumours. Therefore, our study offered a novel and powerful nanoplatform that integrated ILs and RNA interference therapy, presenting new strategies for cancer treatment.", "journal": "Biomaterials science", "year": "2025", "doi": "10.1039/d4bm01159g", "ptypes": [ "Journal Article" ], "first_author": "Xing Y", "dirs": [ "D" ] }, "41046881": { "pmid": "41046881", "title": "Hydrated ionic liquids enhance stability and preserve functionality in transmembrane proteins.", "abstract": "Transmembrane proteins, especially α-helical transmembrane proteins, are critical cellular membrane components. They play a crucial role in various biological processes, such as molecular transport, signal transduction, and metabolism. Therefore, structural and functional studies are essential for the advancement of fields such as fundamental biology, medical science, drug discovery, biotechnology, and bioengineering. However, challenges such as insolubility and the preservation of native structure have hindered research progress. Various methods, including amphiphilic molecule development and the use of nanodiscs, have been adopted to address these challenges. However, a simple and effective method for the stable handling of membrane proteins is yet to be developed. This study introduces hydrated ionic liquids that directly and stably dissolve transmembrane proteins, including TehA and bacteriorhodopsin. Careful selection of the basic backbone and functional groups of the component cations, along with the kosmotropicity of the anions, revealed that these proteins can be dissolved while retaining their higher-order structure and functionality. The thermodynamic stability of these transmembrane proteins increased by over 20 °C. Bacteriorhodopsin, a light-driven H+ transporter, retained its functional capacity. Additionally, its resistance to laser irradiation significantly improved. This study highlights the crucial role of hydration state and specific ionic interactions in the preservation of transmembrane protein structure and functionality. This enhanced stability facilitates the physicochemical analyses of transmembrane protein structures and functions. Furthermore, it opens new avenues for drug discovery and transmembrane protein-based device development through more efficient screening techniques.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.148096", "ptypes": [ "Journal Article" ], "first_author": "Fujita K", "dirs": [ "D" ] }, "40769369": { "pmid": "40769369", "title": "Natural deep eutectic solvents (NADES)-based chitosan hydrogel for enhanced photoimmunomodulation treatment on various wounds.", "abstract": "Natural Deep Eutectic Solvents (NADES) have recently emerged as eco-friendly, biodegradable alternatives to conventional solvents, offering excellent solubility and low toxicity (10 μg/mL). In this study, we developed a novel NADES-chitosan-based hydrogel combined with photoimmunomodulation (PIM) to enhance healing of ulcer-related wounds. The hydrogel exhibited a nanostructured network with globular aggregates (20-50 nm) interacting with amine and alcohol groups, contributing to improved biocompatibility, biodegradability, and thermal stability (50-120 °C and 130-200 °C). Cytotoxicity and permeability assays confirmed the hydrogel's enhanced bioactivity and extraction efficiency. Following PIM treatment, significant wound healing effects were observed, including increased cell migration, improved mitochondrial membrane potential, reduced intracellular ROS, and differential protein expression in AGS and HT29 cells (p < 0.05 and p < 0.01, respectively). These findings highlight the novelty of using NADES as a gelation solvent in hydrogels for wound therapy, with strong translational potential in diabetic and gastric ulcer treatment and scalability in pharmaceutical, food, and nutraceutical industries. Chemical compounds: chitosan, polyvinyl chloride, bentonite, chlorine chloride, lactic acid and malic acid.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.146591", "ptypes": [ "Journal Article" ], "first_author": "Shanmugapriya K", "dirs": [ "D" ] }, "42255576": { "pmid": "42255576", "title": "Screening of Ionic Liquids and Deep Eutectic Solvents for Enhancing the Activity and Stability of Fungal Laccase: Empowering Kinetic Parameters and Long-Term Conservation.", "abstract": "Ionic liquids (ILs) and deep eutectic solvents (DESs) are currently of great interest due to their highly interesting physicochemical properties; they were studied throughout many chemical and biological approaches, and they were used intensively in biocatalysis and biomedicine.In the present study, ten synthesized ILs and four synthesized DESs have been used to study their effect on the laccase enzyme. Hence, ILs and DESs have been screened in a conventional reaction-buffered medium (25 °C and pH 7.0) and using ABTS and DMP as substrates to study the catalytic ability and the stability of the enzyme. From 14 newly synthesized materials, only three water-miscible ILs ([BMIM]-[Ac], [Chol]-[Ac], and [Chol]-[Prop]) and one DES ([Chol]-[Ac.Ox]) have shown interest in improving the enzyme activity and stability. Among them, [BMIM]-[Ac] has shown impressive empowering power for the laccase activity (almost a 20-fold enhancement versus control); however, it was not efficient to protect the enzyme against high temperature or long conservation. The two choline-based ILs, [Chol]-[Ac] and [Chol]-[Prop], have allowed the increase of laccase activity with a rate of 1.7-fold and 7.8-fold, respectively, and they were shown to have interesting properties to protect the enzyme against temperature and storage. Regarding the DES [Chol]-[Ac.Ox], it improved the laccase at about 8.9-fold, but this rate has not been stable along the experiment due to the high hydrophobic nature of the solution; it could be more useful when applied at high temperatures. The four screened compounds in general improved the activity of the enzyme, especially at pH 7.0, and showed a great sensitivity against temperature. By kinetic studies versus [BMIM]-[Ac], it was demonstrated that the IL can improve the laccase activity by increasing the catalytic markers (V m and K cat); however, a decrease of the affinity has been noted by K m decreasing, which testifies that the interaction of the enzyme with ILs can affect the proteinic structure in an aqueous medium.", "journal": "ACS omega", "year": "2026", "doi": "10.1021/acsomega.5c09946", "ptypes": [ "Journal Article" ], "first_author": "Haj Kacem S", "dirs": [ "D" ] }, "40333001": { "pmid": "40333001", "title": "Surfactant-free microemulsion as a fluid scaffold for the thermal stabilization of lysozyme.", "abstract": "Electrostatic forces supported by hydrogen-bonding (H-bonding) interactions in the presence of surfactants generally stabilize microemulsions. So, contrary to this common wisdom, developing a surfactant-free microemulsion (SFME) that is predominantly stabilized by weak but large number of H-bonding interactions would be remarkable. Herein, the formulation and characterization of an SFME comprising a hydrophobic ionic liquid (IL) and a deep eutectic solvent (DES) exhibiting high thermal stability are reported. The constituents of DES, namely, ethylene glycol (EG) and choline chloride (ChCl), served as polar and amphiphile components, respectively, and an IL, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, worked as a hydrophobic entity to form the SFME. The formation mechanism and high-temperature thermal stability of the SFME are discussed in terms of relative changes in the thickness of interfacial films, predominantly stabilizing the polar and non-polar pseudo-domains via alteration in H-bonding interactions, which is supported by computational studies. The sufficiently low interfacial energy in the SFME is exploited to thermally stabilize lysozyme (LYZ) in SFME, resulting in remarkable thermal stability of up to 150 °C, which is higher than that observed in buffer, as revealed by comparative enzyme activity at room temperature after heating. This study not only adds to the existing knowledge about the formation and stability of SFMEs but is also expected to prompt other researchers to design relatively greener IL or deep eutectic solvent (DES)-based SFMEs for various biological and other applications.", "journal": "Physical chemistry chemical physics : PCCP", "year": "2025", "doi": "10.1039/d5cp00678c", "ptypes": [ "Journal Article" ], "first_author": "Kaur M", "dirs": [ "D" ] }, "40105133": { "pmid": "40105133", "title": "Hydrogen-bond induced non-linear size dependence of lysozyme under the influence of aqueous glyceline.", "abstract": "Natural deep eutectic solvents (NADESs) are environmentally friendly green solvents and hold great promise in the pharmaceutical industry. The secondary structure of a protein, lysozyme, follows a non-monotonous behavior in aqueous glyceline (choline chloride + glycerol) as the wt. % of water is increased. However, it is unclear how the hydration affects the stability of the protein in a non-linear way. In this work, we have performed all-atom molecular dynamic simulations for 1 μs with the lysozyme protein in an aqueous glyceline deep eutectic solvent (DES) by varying the wt. % of water. The simulated radius of gyration, Rg, values can qualitatively reproduce the protein behavior such that the Rg increases initially with an increase in wt. % of water, reaches the peak at 40 wt. %, and then gradually decreases with dilution. Several other properties, including root mean square deviation, root-mean square fluctuation, secondary structure of the protein, and solvent accessible surface area, are examined to explore the NADES effect on the protein structure. Next, we analyze the hydrogen bond profile of intra-protein and among various interspecies, e.g., protein-DES, DES-DES, protein-water, and water-water. The variation in protein-protein hydrogen bonds with concentrations can qualitatively explain the non-linear conformational dependence of the protein. The radial distribution function analyses show various microscopic structures formed due to the DES and water interaction, which play a critical role in protein behavior. This study indicates that at lower wt. % of water, the protein is constrained in a strong hydrogen bond network formed by glycerol and water molecules, resulting in a lower Rg. As the wt. % of water increases, the protein-water interaction drives the protein to expand, reflecting an increasing Rg. At sufficiently higher wt. % of water, the DES constituent and the water molecules interact strongly with the protein, resulting in a decrease in Rg. Overall, the investigation offers a microscopic insight into the protein conformation in DES.", "journal": "The Journal of chemical physics", "year": "2025", "doi": "10.1063/5.0251283", "ptypes": [ "Journal Article" ], "first_author": "Sarkar ID", "dirs": [ "D" ] }, "40186427": { "pmid": "40186427", "title": "Enhancing stability and catalytic activity of urate oxidase using natural deep eutectic solvent: insights from experimental and computational approaches.", "abstract": "This study investigated the impact of natural deep eutectic solvents (DES) based on sucrose, fructose and glycerol on the recombinant uricase enzyme. The plasmid recombinant pET-28a+ containing uricase coding sequence was extracted from the DH5α strain and transferred into the BL21 expration strain. Subsequently, overnight culture, induction with IPTG, and purification of the recombinant uricase using Ni-NTA affinity chromatography methods were carried out. The effect of DES containing sucrose, fructose and glycerol was assessed and 5% DES concentration verified for subsequent experiment. Thermodynamic parameters were analyzed using thermal inactivation and intrinsic fluorescence methods at temperatures of 35, 45, 55, and 65 °C. The results demonstrated lengthened enzyme half-life by approximately 61 min, higher activation energy and Tm, indicating improved thermal stability compared to the free enzyme. Kinetic tests revealed a reduction in the km value from 0.16 mM in the free enzyme to 0.09 mM in the treated enzyme, suggesting enhanced substrate binding affinity. Moreover, the Kcat/Km ratio, reflecting enzyme specificity towards the substrate, was enhanced. In the molecular dynamics simulation section, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and solvent accessible surface area (SASA) were analyzed. Lower RMSD and RMSF values indicate that the structure is more stable in the presence of the eutectic solvent compared to the free enzyme.", "journal": "Journal of biomolecular structure & dynamics", "year": "2026", "doi": "10.1080/07391102.2025.2486444", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Soltani-Nezhad Z", "dirs": [ "D" ] }, "40160783": { "pmid": "40160783", "title": "Preparation of Temperature-Sensitive Molecularly Imprinted Cryogel for Specific Recognition of Proteins.", "abstract": "In order to maintain the stability of the structure of protein molecules and improve the recognition during the separation process, molecular imprinting technology is combined with freeze polymerization to synthesize molecular imprinting cryogels (MICs). This study uses bovine serum albumin (BSA) as a template protein, low critical cosolubility temperature (LCST)-type ionic liquids as temperature-sensitive functional monomers, imidazole ionic liquids, and acrylamides as auxiliary functional monomers to prepare MICs with specific recognition, temperature sensitivity, interpenetrating macroporous structure, and large specific surface area. The MICs prepared at freezing temperature have uniform macroporous structures and good mechanical properties, which is conducive to the improvement of the mass transfer and adsorption capacities. Due to the advantages, the MIC reaches the adsorption equilibrium within 125 min with a saturated adsorption capacity of 741.5 mg g-1 and an imprinting factor of 1.65. Their static and dynamic adsorption behaviors are more in line with the Langmuir model and the quasi-secondary kinetic model, respectively. In addition, the MIC has obvious temperature sensitivity, and the maximum adsorption amount is reached at 37 °C. The separation factor (relative to cytochrome c, bovine blood hemoglobin, and lysozyme) of the MICs for BSA is up to 1.39. Repeatability experiments reveal that the adsorption capacity of molecularly imprinted cryogels is retained at 87% after five adsorption-desorption cycles, indicating excellent recyclability and potential for practical application.", "journal": "ACS omega", "year": "2025", "doi": "10.1021/acsomega.4c11143", "ptypes": [ "Journal Article" ], "first_author": "Cheng D", "dirs": [ "D" ] }, "41187397": { "pmid": "41187397", "title": "Novel deep eutectic solvents promote enzyme-substrate molecular dynamic adaptation to enhance sucrose ester synthesis efficiency.", "abstract": "The eco-friendly synthesis of sucrose esters represents a critical technological advancement for the food and pharmaceutical sectors, where conventional chemical methods face persistent challenges including insufficient selectivity, economic inefficiency, and environmental concerns. To overcome these limitations, we developed an enzymatic synthesis platform utilizing a novel deep eutectic solvent (DES) system comprising cyclohexanone:lauric acid:pyridine, which facilitates the dynamic molecular adaptation between the enzyme and bulky substrates. Through systematic optimization, a remarkable sucrose ester yield of 98.21 % was achieved under the identified conditions (substrate molar ratio of 1:6, enzyme addition of 0.1 g, used cyclohexanone, lauric acid, and pyridine as the medium, reaction at 60 °C in 72 h). Density functional theory (DFT) analysis confirmed the formation of extensive hydrogen bonds between pyridine and lauric acid, validating the successful establishment of this novel DES. The molecular dynamics simulations (MD) elucidated that cyclohexanone and lauric acid-maintained enzyme structural stability and beneficial to the anchoring of substrates, while the introduction of pyridine moderately increases the flexibility of the enzyme's active site, optimizes the substrate-binding pocket size, and enhances dynamic adaptation with large substrates such as sucrose, achieving a balance between activity and stability. This study provides a new approach for the rational design of enzymatic catalytic systems.", "journal": "Bioorganic chemistry", "year": "2025", "doi": "10.1016/j.bioorg.2025.109169", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Chen Z", "dirs": [ "D" ] }, "40505917": { "pmid": "40505917", "title": "Immobilized lipase in the resolution of racemic atenolol - a possible key to sustainable pharmaceuticals synthesis.", "abstract": "This study focuses on the development of a biocatalytic system based on lipase from Pseudomonas fluorescens immobilized on a poly(acrylamide)- and poly(ionic liquid)-based hydrogel. It was established that temperature significantly impacts polymerization rate of the monomers used, leading to the application of 70 °C for 30 min for the most suitable hydrogel structure. Under these conditions, the polymerization of PIL and PAM reached 63.5 % and 90.9 %, respectively. The produced hydrogel with immobilized lipase was characterized by improved by around 30 % chemical and thermal stability as compared to native enzyme and provided a suitable environment for successful lipase deposition, proven by elution of <2 % of the enzyme from the support. What is more, it was established that a higher enzyme loading resulted in greater catalytic activity, providing over 30 % higher performance after 10 reusability cycles. High enzymatic activity and enantioselectivity demonstrated by the produced system was proved in dynamic kinetic resolution of atenolol reaching over 55 % reaction yield, that was over 40 % higher as free counterpart, and attaining 100 % enantiomeric excess for the final (S)-atenolol ester.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.145174", "ptypes": [ "Journal Article" ], "first_author": "Degórska O", "dirs": [ "D" ] }, "40882734": { "pmid": "40882734", "title": "Sustainable food protein extraction from biomass using deep eutectic solvents: A review.", "abstract": "The increasing universal demand for eco-friendly and value-added proteins requires novel and environmental friendly extraction methods. Deep eutectic solvents (DES) are gaining recognition as a promising class of green solvents because of their biodegradable nature, low toxicity, and tuneable physicochemical features. This review critically investigates the advances in DES as an environmental friendly substitute for protein extraction from various biomass sources, including plant, animal, microbial, and algal origins. Key topics discussed include the composition, varieties, and protein interactions, emphasizing structural and functional mechanisms like hydrogen bonding, electrostatic interactions, and van der Waals forces that contribute to protein structure preservation and functionality. The influence of physicochemical parameters-such as viscosity, polarity, melting point, and pH-on protein stability and extraction efficiency is thoroughly assessed. Refinement technologies, including ultrasonic-assisted and solid-liquid extractions, are assessed in conjunction with real world applications in valorizing agricultural and industrial by-products. The review discusses challenges as well, such as the high viscosity of some DES formulations, constraints in mass transfer, potential toxicity and obstacles in separation and scalability for industrial use. Additionally, it underscores the potential of DES-extracted proteins in food packaging and other bio-based applications. By incorporating structural analysis, sustainability considerations, and application-focused discussions, this review offers a new and detailed understanding to guide future research and industrial adoption of DES for sustainable protein extraction.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.147074", "ptypes": [ "Journal Article", "Review" ], "first_author": "Saleem F", "dirs": [ "D" ] }, "39740381": { "pmid": "39740381", "title": "Gingerols: Preparation, encapsulation, and bioactivities focusing gut microbiome modulation and attenuation of disease symptoms.", "abstract": "Gut dysbiosis, chronic diseases, and microbial recurrent infections concerns have driven the researchers to explore phytochemicals from medicinal and food homologous plants to modulate gut microbiota, mitigate diseases, and inhibit pathogens. Gingerols have attracted attention as therapeutic agents due to their diverse biological activities like gut microbiome regulation, gastro-protective, anti-inflammatory, anti-microbial, and anti-oxidative effects. This review aimed to summarize the gingerols health-promoting potential, specifically focusing on the regulation of gut microbiome, attenuation of disease symptoms, mechanisms of action, and signaling pathways involved. Research findings from experimental and clinical studies have been summarized regarding gingerols effects on the modulation of gut microbiome and its metabolites, and attenuation of disease symptoms. Gingerols are phenolic compounds characterized by a common 3-methoxy-4-hydroxyphenyl moiety in their chemical structures, and further divided into different gingerol types, including gingerols (major), shogaols, paradols, gingerdiols, gingerdiones, and zingerones (minor). Advanced extraction techniques (e.g., ionic liquid-based-, enzyme-assisted-, microwave-assisted-, pressurized liquid-, ultrasound-assisted-, and supercritical fluid extractions) were reported as optimal alternatives to conventional methods for gingerols extraction. Research studies reported that gingerols positively modulated the composition of gut microbiome that helped to combat disease symptoms (e.g., obesity by decreasing weight gain- (Lactobacillus reuteri and Lachnospiraceae) and increasing weight loss associated-bacteria (Akkermansia, Muribaculaceae, and Alloprevotella). Gingerols intervention also ameliorated ulcerative colitis by increasing relative abundance of the beneficial bacteria (Akkermansia, Lachnospiraceae NK4A136, and Muribaculaceae_norank), and decreasing pathogenic microorganisms (Bacteroides, Parabacteroides, and Desulfovibrio). Emerging delivery systems (e.g., microcapsules, nanoparticles, nanostructured lipid carriers, nanoemulsions, and nanoliposomes) can enhance the bioavailability and therapeutic efficacy of gingerols by preserving their inherent properties and addressing challenges of stability, solubility, and absorption. Gingerols are promising therapeutic agents to modulate gut microbiome (increase beneficial bacteria and inhibit pathogenic microbes), and attenuate chronic disease symptoms such as diabetes, colitis, obesity, oxidative stress, and cancer. Despite significant progress, challenges persist in transforming research findings into industrial applications, such as stability and solubility during processing and low bioavailability in the distal gut to impart desirable health benefits.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2024.156352", "ptypes": [ "Journal Article", "Review" ], "first_author": "Abdullah", "dirs": [ "D" ] }, "41791814": { "pmid": "41791814", "title": "DrPISA: Deep eutectic solvent-assisted reverse proteome-integrated solubility alteration for high-sensitivity drug target identification.", "abstract": "Thermal stability-based proteomic strategies, including thermal proteome profiling (TPP) and proteome integral solubility alteration (PISA), enable proteome-wide target identification but predominantly analyze soluble proteins, overlooking heat-induced aggregates that may contain informative targets. Conventional denaturants used for solubilization of these precipitates offer limited performance and compatibility. Here, we developed a deep eutectic solvent (DES)-assisted reverse PISA (DrPISA) strategy to enhance insoluble proteome accessibility and improve detection sensitivity for subtle thermal stability alterations. A systematic evaluation of 65 DES formulations identified DES-48 (l-proline:glycerol:water, 1:1:4) as an optimal solubilization reagent for heat-aggregated proteomes, delivering up to 71.7% more identified proteins than GuHCl and 23.5% more than urea, together with 80.6% fully cleaved peptides and excellent quantitative reproducibility. Integrating DES-48 into a reverse PISA workflow enabled sensitive detection of early-stage aggregation events not captured in soluble-focused assays. Across five reference compounds, DrPISA reproducibly recovered known targets and increased kinase coverage, identifying 45 kinases among 1142 quantified proteins, including marginal kinase responses in staurosporine-treated samples undetectable by conventional PISA. A simplified six-temperature dimethyl labeling workflow was applied, pooling samples from six discrete heat treatments prior to isotopic labeling and quantitative mass spectrometry, reducing reagent consumption and MS acquisition time by more than 50%. Application to celastrol identified LULL1 as a previously under-recognized interacting protein, illustrating the biological insights gained by expanding target coverage into aggregated fractions. DrPISA extends thermal profiling to insoluble fractions with improved performance over conventional workflows, offering a scalable approach for high-sensitivity target deconvolution. The introduction of DES-48 enables enhanced recovery and quantification of heat-aggregated proteins, broadening the analytical scope of chemical proteomics. These advances establish DrPISA as a practical and complementary strategy for expanding the discoverable drug-protein interaction landscape.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345234", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Yang L", "dirs": [ "D" ] }, "42588874": { "pmid": "42588874", "title": "Cultivar-Dependent Biosynthesis, Sustainable Recovery, and Industrial Applications of Tomato-Derived Carotenoids.", "abstract": "Tomato (Solanum lycopersicum L.) exhibits extensive cultivar diversity in fruit morphology, color, and bioactive compounds. These are closely associated with genetic background, ripening stage, and carotenoid metabolic regulation. Carotenoids, especially lycopene and β-carotene, are major determinants of tomato coloration and are also nutritionally and industrially important high-value-added antioxidants. Carotenoid profiles are regulated by changes in related biosynthetic pathways, including precursor supply, desaturation, isomerization, and cyclization. Key genes such as PSY1, CRTISO, LCY-B and LCY-E play central roles in carotenoid synthesis and accumulation, thereby determining color phenotypes. Mutations and genome-editing (CRISPR/Cas9) approaches can modify carotenoid metabolic flux and develop cultivars with improved nutritional traits. Furthermore, advanced and sustainable recovery strategies for carotenoids (ultrasound-, microwave-, enzyme-, high-pressure-, deep eutectic solvent-, and supercritical fluid-assisted extraction methods) have been developed for efficient pigment extraction. In this review, we compare the physiological and molecular characteristics of diverse cultivars and traits of advanced bioprocesses in terms of carotenoid extraction efficiency and solvent safety. Finally, we discuss various encapsulation techniques that improve product stability and storage performance in industrial applications. Taken together, we review practical strategies combining cultivar-based carotenoid production, sustainable recovery, and product stabilization.", "journal": "Plants (Basel, Switzerland)", "year": "2026", "doi": "10.3390/plants15152371", "ptypes": [ "Journal Article", "Review" ], "first_author": "Kim HS", "dirs": [ "D" ] }, "39788224": { "pmid": "39788224", "title": "Harnessing deep eutectic solvent for enhanced inulinase production from agricultural via submerged fermentation with Aspergillus niger.", "abstract": "This study aimed to enhance inulinase production from agricultural biomass pretreated with deep eutectic solvents (DES) using Aspergillus niger A42 (ATCC 204447). Barley husk (BH), wheat bran (WB), and oat husk (OH) were selected as substrates and were pretreated using different molar ratios of choline chloride: glycerol (ChCl: Gly) and choline chloride: acetic acid (ChCl: AA). DES pretreatment was followed by dilute sulfuric acid hydrolysis. The fermentable sugar content (FSC), sugar profiles, and inhibitory components in the hydrolysates were analyzed. DES pretreatment improved the FSC in all substrates, with wheat bran showing the highest FSC of 72.54 g/mL. Considering the fermentable sugar contents, ChCl: Gly (1:2) was selected as the most suitable DES. The analysis of biomass composition after pretreatment indicated a reduction in extractives and lignin, along with an increase in cellulose content. Subsequently, inulinase fermentation using Aspergillus niger A42 has demonstrated that DES-pretreated wheat bran was the most effective substrate, yielding 416.05 U/mL inulinase activity (Iase) and 486.22 U/mL invertase-type (Sase) activity. The enzyme solution was partially purified via Spin-X UF membranes (50 kDa cut-off), resulting in Iase activity of 769.93 U/mL and Sase activity of 566.69 U/mL. The purification coefficients were 1.88 and 0.92 for inulinase and invertase-type activity, respectively. In conclusion, DES pretreatment was successfully applied to produce inulinase enzyme from BH, WB, and OH. Among the materials studied, WB emerged as the most suitable biomass for producing inulinase from Aspergillus niger A42.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.139592", "ptypes": [ "Journal Article" ], "first_author": "Yavuz HGH", "dirs": [ "D" ] }, "41732506": { "pmid": "41732506", "title": "Anthocyanins from fruit and vegetable waste: biosynthesis, extraction, and gut health benefits.", "abstract": "Anthocyanins (ANCs), naturally abundant in fruit and vegetable waste, represent a promising class of sustainable functional ingredients with notable gut-related health benefits. This review uniquely focuses on waste-derived anthocyanins and their microbiome-modulating effects, including enhancement of intestinal barrier integrity, prebiotic-like activity, and short-chain fatty acid production. Advances in green extraction and purification techniques such as ultrasound-, microwave-, enzyme-assisted, pulsed electric field, and deep eutectic solvent-based methods have improved recovery efficiency while promoting environmental sustainability. Furthermore, stabilization strategies including acylation, protein/polysaccharide complexation, co-pigmentation, and nano/microencapsulation enhance anthocyanin bioavailability and industrial applicability. By integrating technological innovation with gut health insights, this review highlights emerging opportunities to valorize agro-industrial waste and develop next-generation nutraceutical and functional food systems.", "journal": "Food chemistry: X", "year": "2026", "doi": "10.1016/j.fochx.2026.103614", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ayub H", "dirs": [ "D" ] }, "40222539": { "pmid": "40222539", "title": "Application of magnetic aldehyde-functionalized ionic liquids for immobilization of acetylcholinesterase.", "abstract": "This study presents a simple and efficient approach for enzyme immobilization using magnetic aldehyde-functionalized ionic liquids, with acetylcholinesterase (AChE) as a model enzyme. We synthesized a new magnetic particle, Fe3O4@SiO2@[ImBa][Cl], by modifying silica-coated Fe3O4 with the ionic liquid 4-(imidazol-1-yl)benzaldehyde hydrochloride ([ImBa][Cl]), whose structure was characterized by SEM, TEM, FT-IR, EDS, TGA and VSM. Immobilization of AChE occurred through both physical adsorption and covalent bonding, driven by electrostatic interaction between cations of the ionic liquid and AChE, as well as Schiff base reaction between the aldehyde groups and protein amines. We identified the optimal immobilization conditions including solution pH (7), AChE concentration (0.8 mg/mL) and incubation time (90 min), under which the immobilization yield reached 16.38 μg/mg. Compared to free AChE, the immobilized AChE exhibited superior substrate affinity and catalytic activity based on kinetic study, also outperforming traditional covalent immobilization methods that utilized glutaraldehyde as cross-linker. Furthermore, the immobilized AChE demonstrated excellent reusability, as well as enhanced storage and thermal stability compared to free AChE. Additionally, it was employed for evaluation of drug inhibitory activity, which could be used for relevant drug discovery. This method indicates that magnetic aldehyde-functionalized ionic liquids are simple and efficient carriers for immobilization of enzymes.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.143101", "ptypes": [ "Journal Article" ], "first_author": "Dong M", "dirs": [ "D" ] }, "42536043": { "pmid": "42536043", "title": "A Thermostable Multifunctional GH5 Endoglucanase From Bacillus sp. for Lignocellulosic Biomass Saccharification.", "abstract": "The enzymatic conversion of lignocellulosic biomass to glucose remains a bottleneck for cost‑effective biofuel production, largely due to the complexity and expense of enzymes. Here we describe BsEG2, a multifunctional GH5‑like endoglucanase cloned from Bacillus sp. strain BS and heterologously expressed. BsEG2 displays optimal activity at pH 6.0 and 55°C and efficiently hydrolyzes both amorphous and crystalline cellulose. Kinetic analysis on carboxymethyl cellulose (CMC) indicates high substrate affinity and catalytic efficiency. Unlike typical endoglucanases, BsEG2 combines processive endoglucanase activity with broad substrate specificity. It degrades long cellulose chains and oligosaccharides and also shows β‑glucosidase‑like activity on cellobiose, yielding glucose across a wide range of chain lengths. BsEG2 is highly thermostable (a half-life of ~15 days at 55°C), tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM. It remains active in seawater, suggesting potential to reduce freshwater demand in biomass processing. These properties, namely, multifunctionality, stability, inhibitor tolerance, and halotolerance, make BsEG2 a strong candidate for simplified, cost‑effective enzyme formulations for glucose production from lignocellulosic feedstocks.", "journal": "Biotechnology journal", "year": "2026", "doi": "10.1002/biot.70290", "ptypes": [ "Journal Article" ], "first_author": "Ghosh D", "dirs": [ "D" ] }, "40733425": { "pmid": "40733425", "title": "An Efficient Extraction, Characterization and Antioxidant Study of Polysaccharides from Peucedani Decursivi Radix.", "abstract": "Hot water extraction (HE), enzyme-assisted hot water extraction (EAHE), ultrasonic-assisted extraction with NADES (UAE-NADES) and ultrasonic-assisted extraction with NADES and enzyme pretreatment (UAE-NADES-E) were employed to extract polysaccharides from Peucedani Decursivi Radix (PDR) and their structures were characterized for the first time. UAE-NADES-E was found to be the most effective extraction method, and the extraction process was optimized by Box-Behnken design (BBD)-response surface methodology (RSM) experiments. The optimal extraction process was determined by using a NADES system with a molar ratio of betaine to 1,3-butanediol of 1:3, a water content of 30%, a liquid/solid ratio of 40:1 mL/g, an ultrasound time of 30 min, an ultrasound temperature of 45 °C and an alcohol precipitation time of 6 h; the polysaccharide extraction yield reached 19.93%. Further, the structures of polysaccharides from PDR extracted by the above four methods were characterized by FT-IR, SEM, gel and anion-exchange chromatography. Eight monosaccharides were detected in the PDR polysaccharides extracted by the four methods. The PDR polysaccharides extracted by the UAE-NADES-E method had lower molecular weights compared with those extracted by the other methods. Moreover, the PDR polysaccharides exhibited obvious antioxidant activity, as revealed by DPPH, ABTS+ and hydroxyl radical scavenging experiments, meaning they have the potential to be developed as natural antioxidants.", "journal": "Plants (Basel, Switzerland)", "year": "2025", "doi": "10.3390/plants14142188", "ptypes": [ "Journal Article" ], "first_author": "Li Q", "dirs": [ "D" ] }, "41477550": { "pmid": "41477550", "title": "Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers.", "abstract": "Well-characterized tumor-associated antigens (TAAs) represent targets of interest in many anticancer therapeutic strategies. Although the use of monoclonal antibodies has led this field for years, the development of smaller molecules, with improved properties, holds great potential for specific anticancer applications. Affitins are small (7 kDa), thermostable, and high-affinity nonimmunoglobulin protein scaffolds derived from archaea. Using ribosome display and next-generation sequencing, we isolated the first Affitins specific for human mesothelin (hMSLN), a TAA overexpressed in many solid cancers and currently targeted in clinical trials. The homodimerization of the most promising Affitin (N13) improved affinity by 60-fold (from 35 nM to 0.57 nM). The high specificity of N13 was demonstrated on cell co-cultures under static or dynamic conditions. This Affitin, monomeric or dimeric, was also stable under a wide range of temperatures and upon repeated freeze/thaw cycles. Finally, bispecific natural killer (NK) cell engagers (BiKEs) composed of an anti-cluster of differentiation 16 (CD16) variable heavy domain of heavy chain (VHH) fused to a monomer or a dimer of N13 Affitins were constructed. Using a cytotoxicity assay, we showed the specific lysis of hMSLN-expressing cells in the presence of BiKE and NK cells, supporting the potential therapeutic application of these affinity agents.", "journal": "Molecular therapy. Oncology", "year": "2025", "doi": "10.1016/j.omton.2025.201095", "ptypes": [ "Journal Article" ], "first_author": "Briolay T", "dirs": [ "D" ] }, "40888155": { "pmid": "40888155", "title": "Optimizing colloidal stability and viscosity of multispecific antibodies at the drug discovery-development interface: a systematic predictive case study.", "abstract": "The discovery and development of multispecific antibodies present unique challenges in optimizing their physicochemical properties to enhance developability and manufacturability. Common developability challenges include increased risk of aggregation, high viscosity, poor solubility, low expression yields, complex purification requirements, greater propensity for fragmentation, immunogenicity, or pharmacokinetics. In this study, we systematically investigate the solution behavior of engineered bispecific IgG1-VHH constructs derived from a parental NKp30 ×EGFR natural killer cell engager (NKCE) molecule, focusing on colloidal stability, hydrophobicity, thermal stability, pH sensitivity, and viscosity. By combining in silico predictions and experimental evaluations, we engineered variants with altered isoelectric points (pIs) of the Fab and VHH domains and characterized them across a broad, formulation-relevant pH range (pH 4.5-8.0). Our findings indicate that aligning slightly basic pI profiles (approximately 7.5-9.0) across variable domains within bispecific antibodies can effectively mitigate charge asymmetries in standard acidic formulations that may lead to unfavorable solution behavior. Importantly, rational design and early-stage experimental validation yielded optimized variants exhibiting significantly improved colloidal stability and viscosity compared to the starting molecule. This systematic study, the first of its kind for bispecific antibodies, highlights the value of integrating domain-level in silico assessments early in antibody design, facilitating efficient optimization toward improved solution behavior of multispecific biotherapeutics.", "journal": "mAbs", "year": "2025", "doi": "10.1080/19420862.2025.2553622", "ptypes": [ "Journal Article" ], "first_author": "Siegmund V", "dirs": [ "D" ] }, "41965429": { "pmid": "41965429", "title": "Glycan-based biological degraders targeting the cytokine immune axis.", "abstract": "Targeted degradation of extracellular proteins via endo-lysosomal pathways constitutes a promising therapeutic strategy by enabling irreversible target removal and potentially reducing systemic effects of antigen accumulation. In this study, we present a systematic approach for the design and evaluation of Biological Degraders (BioDegs) that leverage receptor-mediated uptake through triantennary N-acetylgalactosamine (TGN) induced activation of the asialoglycoprotein receptor (ASGPR). While this principle has been established, there is still a lack about molecular determinants that influence uptake efficiency. Using interleukin-6 (IL-6) and its soluble receptor (sIL-6R) as therapeutically relevant model antigens, we systematically compared a range of BioDeg formats, including full-length antibodies (siltuximab, tocilizumab), a VHH against IL-6, and an IL-6 binding decoy-receptor design, with respect to binding affinity, thermal stability, cellular uptake, lysosomal trafficking, and degradation efficiency in HepG2 cells. Our results demonstrate that combined properties of scaffold architecture and receptor engagement critically influence degradation outcomes. This work provides a comparative framework for BioDeg design and highlights key parameters for developing lysosome-targeting degraders for extracellular proteins.", "journal": "Communications biology", "year": "2026", "doi": "10.1038/s42003-026-10001-9", "ptypes": [ "Journal Article" ], "first_author": "Seifert M", "dirs": [ "D" ] }, "41155590": { "pmid": "41155590", "title": "Design and Preclinical Validation of an Anti-B7-H3-Specific Radiotracer: A Non-Invasive Imaging Tool to Guide B7-H3-Targeted Therapies.", "abstract": "Background: B7-H3, an immunoregulatory protein of the B7 family, has been associated with both anti-cancer immunity and tumor promotion, with its expression commonly correlated with poor prognosis. Although it is frequently expressed across cancers, its heterogeneity may limit the effectiveness of B7-H3-targeted therapies. Consequently, a sensitive and non-invasive method is needed to assess B7-H3 expression for patient selection and stratification. Single-domain antibody fragments (sdAbs) offer a promising platform for developing such a diagnostic tool. Methods: To generate B7-H3 sdAbs, two Ilamas were immunized with the recombinant human B7-H3 protein. Positive clones were selected through Phage biopanning and characterized for thermal stability, binding specificity, and affinity to human and murine B7-H3 proteins. Selected sdAbs were radiolabeled with Technetium-99m (99mTc) and evaluated for B7-H3 detection in two xenograft tumor models using micro-SPECT/CT imaging and dissection studies. Results: Sixteen purified sdAbs bound specifically to recombinant B7-H3 proteins and cells expressing native B7-H3 antigens, with nanomolar affinities. The four best-performing sdAbs bound promiscuously to tested mouse and human B7-H3 isoforms. Lead sdAb C51 labeled with 99mTc displayed specific accumulation across two human B7-H3+ tumor models, achieving high contrast with a tumor-to-blood ratio of up to 10 ± 3.16, and a tumor uptake of up to 4.96 ± 1.4%IA/g at 1.5 h post injection. Conclusions: The lead sdAb enabled rapid, specific, and non-invasive imaging of human B7-H3+ tumors. Its isoform promiscuity supports broad applicability across cancers expressing different human B7-H3 isoforms. These results support further development for clinical translation to enable patient selection and improved B7-H3-targeted therapies.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ph18101477", "ptypes": [ "Journal Article" ], "first_author": "Funeh CN", "dirs": [ "D" ] }, "40913862": { "pmid": "40913862", "title": "A synthetic humanized camelid single-domain antibody targeting VEGF exhibits in vitro antiangiogenic effects.", "abstract": "Vascular endothelial growth factor (VEGF) is a key player in the development and progression of several diseases, most notably cancer and retinal disorders. Over the last twenty years, VEGF has emerged as a significant therapeutic target for these conditions. This study reports the isolation and characterization of a fully synthetic, humanized, affinity-matured single-domain antibody fragment (VHH) designed to target VEGF. Our approach involved a two-step strategy to construct a phage-displayed library: first, diversifying the CDR3 region of the NbBCII10FGLA nanobody and then diversifying the CDR1 and CDR2 regions. The selection process involved three rounds of biopanning against VEGF isoform 121. The resulting antibody fragment, NbH1, showed a four-fold increase in affinity for human VEGF compared to the original VHH. Significantly, this enhanced affinity did not compromise the solubility or thermal stability of NbH1. Molecular modeling indicated that NbH1 recognizes a unique epitope on VEGF. Functional assays, including competitive and conventional ELISAs, demonstrated that NbH1 effectively disrupts the interaction between VEGF and its main receptor VEGFR2, and with bevacizumab, making it particularly promising for therapeutic applications. This novel molecule demonstrates cross-species activity and does not interfere with VEGFR1 binding. Furthermore, NbH1 inhibits VEGF-induced phosphorylation of VEGFR2 in human endothelial cells and hampers their sprouting in a 3D spheroid angiogenesis assay, highlighting its potential for treating pathological angiogenesis in vivo.", "journal": "International immunopharmacology", "year": "2025", "doi": "10.1016/j.intimp.2025.115493", "ptypes": [ "Journal Article" ], "first_author": "Gonzalez-Moya I", "dirs": [ "D" ] }, "40347884": { "pmid": "40347884", "title": "Nanobody-based immunotoxins: A precision tool in the treatment of solid tumors.", "abstract": "Solid tumors, the main cause of cancer-related death, represent a significant therapeutic challenge due to the high-density microenvironment and intolerance to conventional treatments. Nanobody-based immunotoxins (NbITs) are an exciting candidate, combining the ultimate specificity of nanobodies (single-domain antibody fragments of camelid antibodies) and detrimental effects of the toxin. These nanobodies are small (one-tenth of conventional antibody size), thermostable with high specificity, high antigen binding affinity which give it the ability to penetrate into solid tumors. Specific delivery to tumor cells is achieved through conjugating nanobodies with cytotoxic agents of bacterial origin or synthetic drugs. This phenomenon is initially attracted to the cells by the antigen-antibody interaction that is further enhanced by receptor-mediated internalization and cytotoxic payload release that subdues essential cellular processes and, as a consequence, damages the cells. This review discusses the mechanisms that underlie the effectiveness of NbITs, such as tumor antigen recognition, toxin release, and cellular signaling pathways elicited by the internalized toxins. We also discuss the application of NbITs in treating cancers such as HER2-positive breast cancer and EGFR-overexpressing lung cancer, and other cancers, highlighting their ability to address limitations of conventional therapies. Key challenges in NbIT development, including stability, immunogenicity, and efficient delivery, are critically evaluated. Current advances such as the creation of bispecific nanobody constructs, optimization of linker strategies, as well as the incorporation of nanoparticle-based delivery systems are maximizing the therapeutic potential of these molecules. This review synthesizes recent progress and addresses current obstacles in NbIT development, showcasing their transformative potential as a targeted therapeutic approach for solid tumors. It also covers future opportunities to develop and advance this emerging treatment strategy.", "journal": "International immunopharmacology", "year": "2025", "doi": "10.1016/j.intimp.2025.114801", "ptypes": [ "Journal Article", "Review" ], "first_author": "Emami A", "dirs": [ "D" ] }, "41496549": { "pmid": "41496549", "title": "Multidimensional maturation of antibody variable domains with machine-learning assistance.", "abstract": "In antibody development, a mutagenesis approach has been widely used to improve affinity, but such mutations often compromise biophysical properties. Here, we combined molecular evolution with machine learning to simultaneously improve affinity and expression level of camelid heavy-chain antibody variable domains (VHHs). Using phage display and deep sequencing, we selected five residues in an anti-SARS-CoV-2 VHH for affinity maturation. We constructed training data using experimentally measured expression levels and target affinities of 117 variants with randomized residues. Machine-learning-predicted top-rank variants showed improved expression level and affinity compared to variants in the training data. Several variants achieved 50-70-fold stronger affinities in the pico-molar range and 4-5-fold higher expression level than wild-type. Furthermore, one variant showed 9.5°C improvement in thermal stability. These results highlight the utility of machine learning-assisted molecular evolution as a strategy for multidimensional optimization of antibody properties.", "journal": "mAbs", "year": "2026", "doi": "10.1080/19420862.2025.2611472", "ptypes": [ "Journal Article" ], "first_author": "Ito T", "dirs": [ "D" ] }, "40600960": { "pmid": "40600960", "title": "Magnetic Nanoworm-Based Screening of Yeast Library for Anti-PEG Nanobodies.", "abstract": "PEGylation is commonly utilized to modify nanoparticles, vaccines, therapeutic proteins, and biomaterials. The increased use of PEG-containing cosmetics and medicines results in anti-PEG immunoglobulins in humans, including IgG, IgM, and IgE types. Recent studies have shown that high-affinity antibodies against PEG can be isolated through immunization. These antibodies have been used in immunoassays and for decorating PEGylated nanoparticles with targeting ligands. However, conventional antibodies have limitations, such as their large size and potential stability issues. Here, we developed a protocol to isolate anti-PEG nanobodies (Nbs) from a yeast library. We prepared ∼60 nm fluorescently labeled PEGylated and non-PEGylated iron oxide nanoworms for negative and positive magnetic selection, followed by multiparameter flow cytometry sorting. The representative Nb clone exhibited highly specific binding to PEGylated liposomes and nanoparticles and was able to compete with commercial antibackbone PEG IgG but not with anti-methoxy PEG IgG for PEG binding. The Nb showed a micromolar dissociation constant, no aggregation, high thermal stability, and reversible structural stability. This work represents a proof-of-concept screening and isolation of anti-PEG Nbs.", "journal": "Molecular pharmaceutics", "year": "2025", "doi": "10.1021/acs.molpharmaceut.5c00540", "ptypes": [ "Journal Article" ], "first_author": "Ray S", "dirs": [ "D" ] }, "39755714": { "pmid": "39755714", "title": "Nanobody-based indirect competitive ELISA for the detection of aflatoxin M1 in dairy products.", "abstract": "Aflatoxin M1 (AFM1) is known to be carcinogenic, mutagenic, and teratogenic and poses a serious threat to food safety and human health, which makes its surveillance critical. In this study, an indirect competitive ELISA (icELISA) based on a nanobody (Nb M4) was developed for the sensitive and rapid detection of AFM1 in dairy products. In our previous work, Nb M4 was screened from a Bactrian-camel-immunized phage-displayed library. It exhibits VH-like features, possesses higher thermal stability than monoclonal antibody (mAb 1E6) and tightly binds to AFM1-BSA with a KD value of 2.5 nM. Under the optimal conditions, its half-maximal inhibitory concentration was 0.338 ng/mL, the limit of detection was 0.051 ng/mL, and linearity was noted in the range of 0.168-0.679 ng/mL. Nb M4 displayed almost no cross-reactivity with other mycotoxins. No matrix effect was observed in milk and milk powder samples, and the matrix effect in yogurt samples could be weakened by 2-fold dilution. Furthermore, validation studies in spiked samples (milk, yogurt, and milk powder) resulted in good recoveries of 95.40-111.33%, with a low coefficient of variation (2.89-6.78%). High-performance liquid chromatography was used to evaluate the accuracy and reliability of the developed icELISA, which indicated a satisfactory consistent correlation (R2 = 0.9722). This study highlights the potential of Nb M4 as a promising component for detecting AFM1 in dairy products.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-024-83869-4", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Yi L", "dirs": [ "D" ] }, "40127552": { "pmid": "40127552", "title": "Development of a nanobody-based immunoassay for the analysis of the disinfectant triclosan in water.", "abstract": "Triclosan (TCS), a broad-spectrum antimicrobial agent in pharmaceuticals and personal care products, necessitates environmental monitoring due to its antimicrobial properties and widespread in the environment. In this study, two anti-TCS single-domain antibodies (i.e., nanobodies, Nbs), T1 and T2, were isolated from a phage-displayed Nb library derived from a camel immunized with a mixture of TCS immunogens. The T1-based enzyme-linked immunosorbent assay (ELISA) exhibited a better sensitivity to TCS than the T2-base ELISA. Motivation at enhancing specificity, sensitivity, and stability of Nb-based immunoassays promoted exploring use of a bivalent strategy to enhance performance. The bivalent Nb T1-T1 was tandemized via a linker-(GGGGS)3- between. The thermal stability of the bivalent Nb was improved in comparison with that of a monovalent Nb. The sensitivity of T1-T1-based ELISA, with an IC50 value of 4.3 ng mL-1 of TCS, was improved approximately 2-3 fold in comparison to those of T1-or T2-based ELISAs (8.5 and 14.6 ng mL-1, respectively). The average recovery of TCS from water samples measured with T1-T1-based ELISA was in the range of 99-125 %, which correlated well with that measured by a high-performance liquid chromatography (HPLC) method (R2 > 0.99). TCS in river water samples was detected by the resultant ELISA and an HPLC method, showing a satisfactory correlation.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2025.127980", "ptypes": [ "Journal Article" ], "first_author": "Xu B", "dirs": [ "D" ] }, "41718525": { "pmid": "41718525", "title": "Preparation, Characterization, and Binding Mechanism of Nanobodies Targeting Multiple Serotypes of Salmonella.", "abstract": "Salmonella Enteritidis and Salmonella Typhimurium both are the most prevalent Salmonella serotypes responsible for human infections. Immunoassays, typically relying on serotype-specific antibodies, often lack the capacity for broad-spectrum Salmonella serotype detection. To address this limitation, in this study, nanobody VHH-II-3 was isolated from a phage display library constructed by immunization with inactivated S. Enteritidis and S. Typhimurium cells, followed by a cross-antigen strategy for biopanning. The VHH-II-3 nanobody could bind against 13 Salmonella serotypes and exhibited excellent thermostability. The antigenic targets of VHH-II-3 were identified as the structural epitopes of flagellar proteins FliC and FlgL through His-tag pull-down assays coupled with mass spectrometry, further verified by Western blotting and VHH ELISA. Biolayer interferometry (BLI) affinity analysis revealed that the binding constant of VHH-II-3 to FliC reached the nanomolar level (10-9 M), with the amino acid residue of Gln105 within the CDR3 loop playing a critical role in antigen recognition. Notably, FlgL likely contributed significantly to the broad-spectrum recognition capability of VHH-II-3, as it was highly conserved across diverse Salmonella serotypes. This study provided a promising nanobody for the broad-serotype detection for Salmonella and offered novel perspectives into their antigen-binding mechanisms for improved diagnostic and monitoring applications.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c07402", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Huang Y", "dirs": [ "D" ] }, "42085164": { "pmid": "42085164", "title": "Decoding the structure of GPR151 via NELiS.", "abstract": "Structure determination of orphan G protein-coupled receptors (GPCRs) is hindered by lack of known ligands and their inherent instability during purification. Conventional thermostability screening requires ligands or purified protein, limiting its utility for orphan GPCRs. Here, we present the Nb6-Enabled Ligand-Free Stabilization Platform (NELiS)-a ligand- and purification-independent method for identifying stabilizing mutations. Applied to GPR151, an orphan GPCR enriched in habenula and implicated in neuropsychiatric disorders, NELiS identified four mutations that significantly improved thermostability and expression, allowing receptor purification. Using the stabilized and purified receptor, we further found a high-affinity, GPR151-specific nanobody that facilitated structural determination. Structural analysis revealed unconventional activation-resistant features across canonical motifs and an autoinhibitory N-terminal region occupying the orthosteric pocket. Functional studies confirmed a unique activation mechanism and the critical role of the N terminus in receptor maturation and trafficking. These results establish NELiS as a generalizable tool for structural and functional investigation of orphan GPCRs.", "journal": "Proceedings of the National Academy of Sciences of the United States of America", "year": "2026", "doi": "10.1073/pnas.2534234123", "ptypes": [ "Journal Article" ], "first_author": "Wang Y", "dirs": [ "D" ] }, "40662802": { "pmid": "40662802", "title": "From high-throughput evaluation to wet-lab studies: advancing mutation effect prediction with a retrieval-enhanced model.", "abstract": "Enzyme engineering is a critical approach for producing enzymes that meet industrial and research demands by modifying wild-type proteins to enhance properties such as catalytic activity and thermostability. Beyond traditional directed evolution and rational design, recent advancements in deep learning offer cost-effective and high-performance alternatives. By encoding implicit coevolutionary patterns, these pretrained models have become powerful tools, with the central challenge being to uncover the intricate relationships among protein sequence, structure, and function. We present VenusREM, a retrieval-enhanced protein language model designed to capture local amino acid interactions in both spatial and temporal scales. VenusREM achieves state-of-the-art performance on 217 assays from the ProteinGym benchmark. Beyond high-throughput open benchmark validations, we conducted a low-throughput post hoc analysis on more than 30 mutants to verify the model's ability to improve the stability and binding affinity of a VHH antibody. We also validated the effectiveness of VenusREM by designing 10 novel mutants of a DNA polymerase and performing wet-lab experiments to evaluate their enhanced activity at elevated temperatures. Both in silico and experimental evaluations not only confirm the reliability of VenusREM as a computational tool for enzyme engineering but also demonstrate a comprehensive evaluation framework for future computational studies in mutation effect prediction. The implementation is available at https://github.com/tyang816/VenusREM.", "journal": "Bioinformatics (Oxford, England)", "year": "2025", "doi": "10.1093/bioinformatics/btaf189", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Tan Y", "dirs": [ "D" ] }, "39968946": { "pmid": "39968946", "title": "AI-enabled alkaline-resistant evolution of protein to apply in mass production.", "abstract": "Artificial intelligence (AI) models have been used to study the compositional regularities of proteins in nature, enabling it to assist in protein design to improve the efficiency of protein engineering and reduce manufacturing cost. However, in industrial settings, proteins are often required to work in extreme environments where they are relatively scarce or even non-existent in nature. Since such proteins are almost absent in the training datasets, it is uncertain whether AI model possesses the capability of evolving the protein to adapt extreme conditions. Antibodies are crucial components of affinity chromatography, and they are hoped to remain active at the extreme environments where most proteins cannot tolerate. In this study, we applied an advanced large language model (LLM), the Pro-PRIME model, to improve the alkali resistance of a representative antibody, a VHH antibody capable of binding to growth hormone. Through two rounds of design, we ensured that the selected mutant has enhanced functionality, including higher thermal stability, extreme pH resistance, and stronger affinity, thereby validating the generalized capability of the LLM in meeting specific demands. To the best of our knowledge, this is the first LLM-designed protein product, which is successfully applied in mass production.", "journal": "eLife", "year": "2025", "doi": "10.7554/eLife.102788", "ptypes": [ "Journal Article" ], "first_author": "Kang L", "dirs": [ "D" ] }, "40099854": { "pmid": "40099854", "title": "Affinity-stability trade-off mechanism of residue 35 in framework region 2 of VHH antibodies with β-hairpin CDR3.", "abstract": "Single-domain VHH antibodies are promising therapeutic and diagnostic tools. The third complementarity-determining region (CDR3) is usually the most critical region for antigen recognition by VHH antibodies. When CDR3 adopts a short and extended β-hairpin conformation, framework region 2 (FR2) often interacts directly with the antigen. However, the importance of these interactions in antigen recognition remains unclear. In this research, we investigated the role of FR2 residues in VHH antibodies with β-hairpin CDR3s. We found that several FR2 residues, particularly at positions 35 and 37, are critical for high-affinity antigen binding. Notably, a trade-off was observed: introducing a charged residue at position 35 enhanced binding affinity but reduced thermal stability. These findings provide insights into optimizing FR2 in single-domain antibodies to improve their functionality for diagnostic and therapeutic applications.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70095", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Yamamoto K", "dirs": [ "D" ] }, "42317504": { "pmid": "42317504", "title": "NbBayesLM: bayesian prediction of nanobody thermostability using protein language model.", "abstract": "Nanobodies, single-domain antibodies derived from camelids, are promising biologics due to their small size and high stability. Accurate prediction of their thermostability is critical for therapeutic and diagnostic applications. Due to their ability to bind conformationally constrained epitopes that are typically inaccessible to conventional antibodies, nanobodies represent a uniquely valuable modality in therapeutic target engagement and drug discovery. Existing methods for predicting nanobody thermostability often rely on limited data, handcrafted features, or black-box machine learning models that lack uncertainty quantification, limiting their generalizability and reliability. To address these gaps, this study, named NbBayesLM, proposes a Bayesian neural network (BNN) approach that integrates protein language model (PLM) embeddings with chemical property features to predict nanobody thermostability. In our formulation, physicochemical properties are incorporated as Bayesian priors, providing biologically meaningful constraints that guide posterior learning and improve model interpretability. Trained on a dataset of 10,630 nanobody sequences with experimentally determined T m values, our model achieves a mean absolute error of 1.89 °C and R 2 score of 0.67, outperforming existing models reported in the literature, while the fusion mechanism enhances performance over unimodal approaches and the BNN architecture provides well-calibrated uncertainty estimates to guide candidate selection and accelerate nanobody engineering.", "journal": "Frontiers in bioinformatics", "year": "2026", "doi": "10.3389/fbinf.2026.1832968", "ptypes": [ "Journal Article" ], "first_author": "Shishir FS", "dirs": [ "D" ] }, "40939862": { "pmid": "40939862", "title": "A universal single-plasmid, single-promoter system for efficient protein display and RNA encapsulation in MS2 virus-like particles.", "abstract": "Bacteriophage MS2 virus-like particles (VLPs) represent a promising platform for biomedical applications. However, challenges remain in integrating protein display, RNA encapsulation, and convenient purification methods. Here, we developed a universal single-plasmid single-promoter system (USS) that enables modular assembly of multifunctional VLPs through designated PstI (protein insertion) and KpnI (RNA encapsulation) sites. The USS system achieved: 1) Efficient display of large protein molecules (e.g., EGFP, Nanobody N16) on each VLP surface while encapsulating target RNA; 2) Rapid one-step purification via His-tag affinity chromatography, bypassing ultracentrifugation; 3) The resulting VLPs exhibit thermal stability (60°C for 15 min) and nuclease resistance. Functional validation demonstrated: 1) Diagnostics: CoviN-encapsulated VLPs served as RT-qPCR controls for COVID-19 detection (linear range: 102-108 copies/μL); 2) Dual functionality for co-delivery: N16-VLPs with shPCSK9 suppressed protein PCSK9 expression by 68 % (p < 0.01) in Hepa1-6 cells and bound PD-L1 with nanomolar affinity (EC50 = 1.6 μg/mL). Crucially, the simplicity of USS eliminated the need for multi-plasmid constructs, providing a streamlined approach to VLPs production that is both efficient and scalable for applications from molecular diagnostics to combination therapies.", "journal": "Journal of biotechnology", "year": "2025", "doi": "10.1016/j.jbiotec.2025.09.004", "ptypes": [ "Journal Article" ], "first_author": "Chen P", "dirs": [ "D" ] }, "42300510": { "pmid": "42300510", "title": "Nanobody-based enzyme immunoassay for naringenin in pummelos and herbal samples.", "abstract": "Nanobodies have emerged as alternatives to conventional antibodies due to their unique advantages. In this study, two naringenin-specific nanobodies, Nb-C9 and Nb-C13, were successfully isolated. The obtained nanobodies demonstrated superior thermal stability compared to the previously reported naringenin monoclonal antibody (mAb). Based on Nb-C9, an indirect competitive enzyme-linked immunosorbent assay (Nb-ELISA) was developed for naringenin detection. The established assay exhibited a half-maximal inhibitory concentration (IC50) of 15.31 ng mL-1, with a linear range spanning from 4.55 to 60.60 ng mL-1 (IC20-IC80). Furthermore, the Nb-ELISA was applied to quantify naringenin levels in peel and pulp samples from six pomelo varieties and four citrus-derived traditional Chinese herbal samples. The detection results showed strong consistency with those obtained by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). All results demonstrated that the developed Nb-ELISA is both accurate and reliable, while the identified naringenin-specific nanobodies provide a valuable foundation for developing novel immunoassays targeting naringenin.", "journal": "Analytical methods : advancing methods and applications", "year": "2026", "doi": "10.1039/d6ay00451b", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Sun F", "dirs": [ "D" ] }, "41874027": { "pmid": "41874027", "title": "Developability Evaluation of Single-Domain Antibody-Chelator Conjugates for Diagnostic Radiotracers.", "abstract": "Background/Objectives: Developability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here, we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. Methods: Assays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency (\"chelatability\"). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze-thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimize experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. Results: A tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. Conclusions: This study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules.", "journal": "Antibodies (Basel, Switzerland)", "year": "2026", "doi": "10.3390/antib15020022", "ptypes": [ "Journal Article" ], "first_author": "Kaiser PD", "dirs": [ "D" ] }, "40920240": { "pmid": "40920240", "title": "Preparation and characterization of a Llama VHH-hFc chimeric antibody recognizing conserved neutralization epitope of H5N1 hemagglutinin with high affinity.", "abstract": "Highly pathogenic avian influenza (HPAI) H5N1 virus poses a continuing global public health threat due to its outbreaks in poultry farms and zoonotic transmission from birds to humans. In the quest of effective therapeutics against H5N1 infection, antibodies with broad neutralizing activity have attracted significant attention. In this study, we employed a phage display technique to select and identify VHH antibodies with specific neutralizing activity against H5N1 hemagglutinin (HA) from an immune llama-derived antibody library. Subsequently, we prepared fusions of VHH10 antibody with human Fc fragment. The chimeric antibody VHH10-hFc was characterized and evaluated for its specificity, binding affinity, serum persistence and antigen recognition epitope. Following purification from 293 F cell cultures, VHH10-hFc chimeric antibody retained its specificity to H5N1 HA. Its antigen-binding affinity was enhanced by up to 130-fold, and its serum persistence was extended by up to 170-fold compared to VHH10. The VHH10-hFc chimeric antibody demonstrated high affinity, excellent thermal stability, and broad reactivity against H5N1 HA in clades 0, 1, 2, and 4. Through epitope mapping, we identified a conformational epitope consisting amino acid residues at positions Q187, K189, L190, Y191, N193, T215, S217 and N220 located on the top region of HA, which was specific and conserved epitopes among H5N1 strains. Consequently, VHH10-hFc, with great specificity, high affinity, prolonged serum persistence and good thermal stability, recognizes a conserved neutralization epitope on the globular head of H5N1 HA, indicating great potential in therapeutic strategies against H5N1 infection.", "journal": "Archives of microbiology", "year": "2025", "doi": "10.1007/s00203-025-04460-7", "ptypes": [ "Journal Article" ], "first_author": "Liu L", "dirs": [ "D" ] }, "41966376": { "pmid": "41966376", "title": "Novel format single-domain antibodies using human VL domain as scaffold.", "abstract": "Single-domain antibodies (sdAbs) represent a unique class of antibody fragments with distinct advantageous characteristics: small monomeric size, excellent solubility and high thermal stability. However, most sdAbs currently available are derived from immunized animals and require humanization for therapeutic use. Notably, sdAbs feature a longer and variable-length third complementarity-determining region (CDR3)-a key structural element for antigen binding but one that increases the technical complexity of library construction. To address these challenges, this study, for the first time, describes the development of synthetic sdAb libraries using a stability-engineered human light-chain variable domain (VL) as the scaffold. We performed limited randomization on three loops located on the opposite side of the CDRs using degenerate codons, which were designed based on the natural length and amino acid properties of these loops. Eukaryotic expression assays confirmed that sdAbs generated via this randomization strategy exhibit excellent secretory expression efficiency. We constructed two ribosome display libraries using two sets of degenerate codon primers and compared the panning efficiency of two strategies against four model antigens (Jo-1, SSB, HAV, and CRP): standalone ribosome display and a combined approach of ribosome display followed by phage pIX display. Both strategies successfully yielded target-specific binders, with the combined panning method demonstrating superior performance to standalone ribosome display. The results demonstrate that synthetic sdAb libraries based on a stability-optimized human VL scaffold provide a facile and reliable source of functional sdAbs. This innovative design is anticipated to serve as a promising platform for the development of next-generation single-domain bispecific antibodies.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.151940", "ptypes": [ "Journal Article" ], "first_author": "Yan X", "dirs": [ "D" ] }, "41149317": { "pmid": "41149317", "title": "Development of Nanobody-Based Sandwich ELISA Resistant to SpA Interference for Sensitive Detection of Staphylococcal Enterotoxin A.", "abstract": "Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples.", "journal": "Biosensors", "year": "2025", "doi": "10.3390/bios15100666", "ptypes": [ "Journal Article" ], "first_author": "Hu C", "dirs": [ "D" ] }, "40260965": { "pmid": "40260965", "title": "Optimization of synthetic human VH affinity and solubility through in vitro affinity maturation and minimal camelization.", "abstract": "An attractive feature of human VHs over camelid VHHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human VHs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to VHHs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human VH phage display library and identified a diverse set of antigen-specific VHs. However, the VHs exhibited low affinity, and many had low solubility; that is, they were prone to aggregation. To explore the feasibility of improving the affinity, we subjected a representative VH to in vitro affinity maturation. We created a yeast surface display library of VH variants employing a site-saturated mutagenesis approach targeting complementarity-determining regions and selected against the target antigen. Next-generation sequencing of the selected variants, combined with structural modeling, identified a set of VHs as potentially improved candidates. Characterization of these candidates revealed several VHs with improved affinities of up to 100-fold (KDs as low as 3 nM) and potent neutralization capabilities; however, they still showed significant aggregation. By introducing as few as two camelid residues into the framework region 2 of a high-affinity VH (a process referred to as camelization), we were able to completely solubilize the VH without compromising its affinity and other important attributes, including thermostability and protein A binding. This study demonstrates the feasibility of generating high-affinity, -solubility, and -stability human VHs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70114", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Bélanger K", "dirs": [ "D" ] }, "42053822": { "pmid": "42053822", "title": "Molecular dynamics simulations along with the synthesis of innovative engineered nanobodies targeting Interleukin-2.", "abstract": "Interleukin-2 (IL-2) is an essential cytokine that plays a significant role in the immune response. The use of anti-IL-2 antibodies to inhibit IL-2 may help mitigate the excessive immune response observed in conditions such as lupus, rheumatoid arthritis, and multiple sclerosis. Single-domain VHH antibodies (nanobodies) offer advantages over traditional antibodies, including their small size, enhanced thermal stability, reduced immunogenicity, superior tissue penetration, and cost-effective production. Consequently, nanobodies have become a promising approach for the diagnosis and treatment of diseases. We employed a structure-guided complementarity-determining region (CDR) grafting approach to design two anti-IL-2 nanobodies, denoted Nb-IL2-01 (derived from 7DR4) and Nb-IL2-02 (derived from 6YE3), by transplanting CDRs from established monoclonal antibodies into a nanobody framework. Molecular dynamics simulations were used to assess the predicted structural stability of the designed constructs and their potential molecular interactions with IL-2. Following expression in E. coli and purification, the capacity of the designed nanobodies to bind immobilized IL-2 was evaluated by enzyme-linked immunosorbent assay (ELISA). Molecular dynamics simulations predicted that the Nb-IL2-02/IL-2 complex would exhibit greater conformational stability than the Nb-IL2-01/IL-2 complex. Experimentally, both nanobodies demonstrated specific, concentration-dependent binding to IL-2 in ELISA, confirming that the designed constructs possess antigen-binding functionality. We successfully generated IL-2-binding nanobody constructs using a CDR grafting-based design approach. The integration of computational design with experimental characterization demonstrates a feasible pipeline for developing functional single-domain binders against therapeutic targets. The specific binding of the designed nanobodies supports their potential as candidates for further development and optimization.", "journal": "Molecular biology reports", "year": "2026", "doi": "10.1007/s11033-026-11824-9", "ptypes": [ "Journal Article" ], "first_author": "Heidari MM", "dirs": [ "D" ] }, "39385626": { "pmid": "39385626", "title": "PLAbDab-nano: a database of camelid and shark nanobodies from patents and literature.", "abstract": "Nanobodies are essential proteins of the adaptive immune systems of camelid and shark species, complementing conventional antibodies. Properties such as their relatively small size, solubility and high thermostability make VHH (variable heavy domain of the heavy chain) and VNAR (variable new antigen receptor) modalities a promising therapeutic format and a valuable resource for a wide range of biological applications. The volume of academic literature and patents related to nanobodies has risen significantly over the past decade. Here, we present PLAbDab-nano, a nanobody complement to the Patent and Literature Antibody Database (PLAbDab). PLAbDab-nano is a self-updating, searchable repository containing ∼5000 annotated VHH and VNAR sequences. We describe the methods used to curate the entries in PLAbDab-nano, and highlight how PLAbDab-nano could be used to design diverse libraries, as well as find sequences similar to known patented or therapeutic entries. PLAbDab-nano is freely available as a searchable web server (https://opig.stats.ox.ac.uk/webapps/plabdab-nano/).", "journal": "Nucleic acids research", "year": "2025", "doi": "10.1093/nar/gkae881", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Gordon GL", "dirs": [ "D" ] }, "41600754": { "pmid": "41600754", "title": "Complete Neutralization of Tetanus Neurotoxin by Alpaca-Derived Trivalent Nanobodies Aimed at Veterinary Medical Applications.", "abstract": "Tetanus is a zoonotic disease posing significant threats to both humans and animals, particularly horses, sheep, and ruminants. Current antitoxin therapies rely on animal-derived immunoglobulins, presenting challenges including animal welfare concerns, pathogen contamination risks, and manufacturing complexity. Alpaca-derived nanobodies (VHH) are promising alternatives owing to their high antigen-binding affinity, thermostability, and potential for microbial production. We developed highly active trivalent VHH antibodies (tVHH) that target multiple epitopes of tetanus neurotoxin (TeNT). Following alpaca immunization with tetanus toxoid, 41 VHH clones were isolated using phage display. Six VHH clones were selected through in vivo neutralization assays, from which three clones of VHH (8, 11, 36) were selected to construct tVHH-8/11/36 and tVHH-8/36/11. Using an improved 21-day mouse neutralization assay, tVHH-8/11/36 demonstrated exceptional neutralizing activity of approximately 1580 IU/mg against 4000 LD50 of toxin, substantially exceeding current human and veterinary anti-tetanus immunoglobulin preparations. Surface plasmon resonance and ELISA confirmed that each VHH recognizes different TeNT domains, producing synergistic neutralizing effects through multimerization. Since antitoxin therapy challenges are common to both animals and humans, this tVHH technology supports One Health by providing a unified therapeutic platform applicable across species through sustainable microbial production.", "journal": "Veterinary sciences", "year": "2026", "doi": "10.3390/vetsci13010098", "ptypes": [ "Journal Article" ], "first_author": "Sakamoto C", "dirs": [ "D" ] }, "39982615": { "pmid": "39982615", "title": "Development and evaluation of Hsp90-targeting nanobodies for visualisation of extracellular Hsp90 in tumours using PET imaging.", "abstract": "The extracellular localisation of the Heat shock protein 90 (Hsp90) is associated with the diseased state and wound healing and presents a promising opportunity for cancer targeting using Positron Emission Tomography (PET) imaging and molecularly targeted radiotherapy. The aim of this work is to develop a radiotracer with low nanomolar binding affinity to target the extracellular and particularly membrane pool of Hsp90, evaluate it in vitro, and conduct preliminary PET studies in vivo in mouse tumour models. Variable Heavy domain of Heavy chain antibodies, often referred to as Nanobodies, are suitable targeting vectors for the extracellular targets due to their favourable pharmacokinetic properties and low nanomolar target affinities. The main objective of the study is to target tumours expressing extracellular and membrane Hsp90 phenotype with minimal tracer accumulation in the non-target organs, which limited the translation of previously studied small molecule cytosolic Hsp90 tracers suffering from high non-Hsp90 specific background in the abdominal area. Six nanobodies were obtained after llama immunization with recombinant Hsp90α and ELISA biopanning, produced in E. coli and screened for stability and affinity. We selected one nanobody, 4DAM26, with good thermal stability, no aggregation at elevated temperatures, and low nanomolar affinity towards Hsp90α and Hsp90β isoforms for translation as a PET radiotracer. The nanobody was bioconjugated to p-NCS-NODAGA and radiolabeled with gallium-68 with 75 ± 11% radiochemical yield and > 99% radiochemical purity and remained stable up to 3 h in phosphate buffered saline and mouse serum. Pilot in vivo evaluation using µPET/CT and ex vivo biodistribution demonstrated a favourable pharmacokinetic profile, but the tumour uptake was non-distinguishable from the background tissue. Compared to the small molecule Hsp90 tracers, the studied Nb-based tracer has improved pharmacokinetics properties including renal clearance and almost no accumulation in the non-target organs. Tumour uptake, on the other hand, was minimal and could not be differentiated from the background in µPET/CT. Our experiments indicate that in the studied models, membrane and extracellular expression of Hsp90 is majorly an artifact of cellular death, as only dead/dying cells had accessible pools of Hsp90 by flow cytometry, a consequence of a leaky membrane. More fundamental research is required to reassess the role of extracellular Hsp90 in cancer, and our future efforts will be focused on improving our inventory of cytosolic Hsp90 tracers with proven Hsp90-specific tumour accumulation.", "journal": "EJNMMI radiopharmacy and chemistry", "year": "2025", "doi": "10.1186/s41181-025-00331-0", "ptypes": [ "Journal Article" ], "first_author": "Narykina V", "dirs": [ "D" ] }, "41562559": { "pmid": "41562559", "title": "Rapid isolation and characterization of high-affinity single-domain antibodies for broad-Spectrum detection of SARS-CoV-2 variants.", "abstract": "The rapid evolution of SARS-CoV-2 presents substantial challenges to maintaining diagnostic accuracy. Single-domain antibodies (variable domains of camelid heavy-chain antibodies, VHHs), commonly referred to as nanobodies, are attractive tools for diagnostic applications due to their high specificity, stability, affinity and expression yield. In this study, three high-affinity VHHs that recognize the receptor-binding domain (RBD) of the SARS-CoV-2 BA.2 variant were isolated from a large naïve alpaca VHH phage display library. These nanobodies exhibited nanomolar affinities and high thermal stability. Notably, reformatting one VHH into a bivalent VHH-Fc fusion construct, K115.2-Fc, enhanced binding affinity 24-fold, achieving an equilibrium dissociation constant of 68.3 picomolar. Functional characterization demonstrated that K115.2-Fc cross-reacted with the Wuhan SARS-CoV-2 RBD and multiple variants, including Alpha, Beta, Gamma, Delta, Kappa, BA.1, BA.2, and BA.4/5. In indirect enzyme-linked immunosorbent assay, K115.2-Fc detected full-length spike protein with a limit of detection of 8.1 pM. Moreover, it enabled sensitive detection in western blotting and flow cytometry, highlighting its applicability across diverse diagnostic applications. Collectively, these findings identify K115.2-Fc as a promising candidate with potential for SARS-CoV-2 diagnostic utility.", "journal": "Protein engineering, design & selection : PEDS", "year": "2025", "doi": "10.1093/protein/gzag001", "ptypes": [ "Journal Article" ], "first_author": "Cho AH", "dirs": [ "D" ] }, "40981273": { "pmid": "40981273", "title": "Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists.", "abstract": "Bispecific antibodies have emerged as a promising class of therapeutics, enabling simultaneous targeting of two distinct antigens. Single-domain antibodies (sdAbs) comprising camelid variable heavy chains (VHHs) provide a compact and adaptable platform for bispecific antibody design due to their small size and ease of linkage. Here we investigate structure-activity relationship of VHH-based cytokine surrogates by combining cell signaling and functional assays with x-ray crystallography and other biophysical techniques. We describe crystal structures of four unique bispecific VHHs that engage and activate the cytokine receptor pairs IL-18Rα/IL-18Rβ and IL-2Rβ/IL-2Rγ. These bispecific VHH molecules, referred to as surrogate cytokine agonists (SCAs), create unique cytokine signals that can be tuned by linker engineering. Our structural analysis reveals multiple dimeric conformations for these bispecific SCAs, where the two VHH domains can interact to form a compact structure. We demonstrate that the dimeric conformation can be enforced via engineering of a non-native disulfide bond between the VHH subunits, thus enhancing molecular thermostability. Our findings have important implications for the design and engineering of bispecific VHHs or sdAbs, offering a novel strategy for tuning their activity and increasing their stability.", "journal": "Antibodies (Basel, Switzerland)", "year": "2025", "doi": "10.3390/antib14030074", "ptypes": [ "Journal Article" ], "first_author": "Trenker R", "dirs": [ "D" ] }, "41513368": { "pmid": "41513368", "title": "Carbon dot-enhanced nanobody-based fluorescence immunoassay for ultrasensitive Listeria monocytogenes detection in dairy products.", "abstract": "Listeria monocytogenes (L. monocytogenes), a psychrophilic Gram-positive bacterium, causes severe foodborne listeriosis outbreaks. However, rapid, accurate diagnostic assays for this pathogen in complex food matrices remain limited. Herein, we established a phage-displayed nanobody (Nb) library by immunizing a bactrian camel with inactivated L. monocytogenes. After biopanning, four specific Nbs were obtained with good specificity and thermal stability. Using soluble Nbs as capture antibodies and phage-displayed nanobodies as detecting antibodies, a fluorescent ELISA for L. monocytogenes was constructed by employing carbon dots as fluorescent probes based on the inner filter effect-mediated quenching. The developed assay exhibited a limit of detection of 3.21 × 103 CFU/mL, demonstrating a 144-fold enhancement over conventional ELISA, enabling highly sensitive and specific pathogen detection. In real sample analysis, the average recoveries ranged from 97.6 to 119.6 % and the relative standard deviation below 6.3 %, showcasing significant potential in food analysis applications.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2025.344954", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Liu X", "dirs": [ "D" ] }, "42178281": { "pmid": "42178281", "title": "Screening and structural characterization of a nanobody targeting a thermostable green fluorescent protein.", "abstract": "Nanobody-fluorescent protein pairs are powerful tools in imaging, protein purification, and structural biology studies. While thermostable green fluorescent protein (TGP) offers improved characteristics over conventional GFP, nanobodies that specifically recognize TGP remain relatively limited. Here, we report the screening and identification of a synthetic nanobody Sb32 that binds TGP with nanomolar affinity using the ribosome technique. The crystal structure of the Sb32-TGP complex, solved at 1.82 Å resolution, reveals an unusual binding mode in which complementarity determining region 2 (CDR2) provides the major contribution, rather than the typically dominant CDR3. Moreover, the interface is stabilized by an extensive hydration network, which compensates for relatively few direct contacts and may explain the fast association and dissociation kinetics observed for this complex. These findings expand the repertoire of TGP-specific nanobodies and highlight an alternative strategy by which nanobodies achieve high affinity. Sb32 provides a new reagent for TGP-based applications, with potential utility in membrane protein purification and structural studies.", "journal": "Acta biochimica et biophysica Sinica", "year": "2026", "doi": "10.3724/abbs.2025256", "ptypes": [ "Journal Article" ], "first_author": "Li Y", "dirs": [ "D" ] }, "39873888": { "pmid": "39873888", "title": "Pharmacoinformatics-based screening and construction of a neutralizing anti-SARS-CoV-2 camelidae nanobody drug conjugate.", "abstract": "Nanobodies or variable antigen-binding domains (VHH) derived from heavy chain-only antibodies (HcAb) occurring in the Camelidae family offer certain superior physicochemical characteristics like enhanced stability, solubility, and low immunogenicity compared to conventional antibodies. Their efficient antigen-binding capabilities make them a preferred choice for next-generation small biologics. In the present work, we design an anti-SARS-CoV-2 bi-paratopic nanobody drug conjugate by screening a nanobody database. SAbDab-nano database was screened based on the physicochemical properties and SARS-CoV-2 binding affinity of the documented nanobodies. Molecular docking, computational modeling, in silico site-directed mutagenesis, and MD simulations were performed to construct an effective nanobody bi-paratope. The construct's physicochemical properties were assessed, and its structural integrity was validated through model energy refinement and quality assessment. The triple-mutant (N78Q K116N T123F) nanobody, based on the bioinformatics analysis, exhibited enhanced binding efficiency against its targets: SARS CoV-2 WT RB (- 353.3), NRP1 (- 376.5) and Omicron RBD (- 380.8), compared to the WT nanobody (SARS CoV-2 WT RBD = - 337.5, NRP1 = - 361.5, Omicron RBD = - 359.5). In silico evaluation also predicted that the construct would demonstrate efficient solubility, high thermostability (Tm 67.4 °C), low molecular weight of 29.36 KDa, and non-toxic, non-allergenic properties. Anti-SARS-CoV-2 neutralizing nanobody-based therapeutics, as demonstrated through this computational work, represents a promising alternative to traditional COVID-19 prophylaxis.", "journal": "Molecular diversity", "year": "2025", "doi": "10.1007/s11030-024-11086-2", "ptypes": [ "Journal Article" ], "first_author": "Kalita E", "dirs": [ "D" ] }, "41593815": { "pmid": "41593815", "title": "VIF-Ig: a novel fc framework for ADCC by incorporation of VHH unit.", "abstract": "Antibody-dependent cellular cytotoxicity is a key mechanism for antibody-based therapeutics, and current engineering strategies to enhance ADCC primarily rely on two approaches: Fc mutations to improve the antibody's intrinsic CD16a affinity, or the fusion of binding-modules targeting NK cell receptors. The former often compromises antibody stability and induces CD16a downregulation; the latter occupies sites critical for target association and limits the assembly of multi-specific therapeutics. Here, we introduce a novel Fc engineering approach wherein the CH2 domain of the Fc region is replaced with an anti-NKp46 VHH named VIF-Ig. It has been reported that NKp46 expression remains unaltered after NK cell activation across various tumor microenvironments, addressing a key limitation of CD16a-dependent strategies. The novel molecules exhibit potent ADCC, high thermal stability, and retain FcRn binding for favorable pharmacokinetic profiles. Furthermore, we demonstrate that VIF-Ig can accommodate VHHs to target various epitopes. Thus, this versatile modular platform is suitable for developing next-generation NK cell or even other cell engagers with enhanced efficacy and tunable specificity, especially for emerging multi-specific immune cell engagers.", "journal": "Protein engineering, design & selection : PEDS", "year": "2025", "doi": "10.1093/protein/gzag003", "ptypes": [ "Journal Article" ], "first_author": "Li Y", "dirs": [ "D" ] }, "42133239": { "pmid": "42133239", "title": "Design and validation of a ribosome display library for synthetic nanobody selection.", "abstract": "Single-domain antibodies (nanobodies) are compact, highly engineerable binding scaffolds widely used in structural biology, biotechnology, and therapeutics. When combined with the Legobody toolkit, they enable high-resolution cryo-electron microscopy (cryo-EM) analysis of small membrane proteins. Ribosome display is a powerful method for generating synthetic nanobodies (sybodies) with exceptionally high library diversity. However, existing sybody libraries are incompatible with the Legobody system, necessitating additional subcloning steps that reduce throughput and limit efficiency. Here, we report the rational design of a new ribosome-display-compatible nanobody library, termed S1.0, engineered for intrinsic compatibility with the Legobody platform through modifications in the C-terminal region. Compared to a benchmark sybody library, S1.0 features increased randomization in the complementarity-determining regions CDR1 and CDR3, while exhibiting reduced variability in CDR2. Selection against calmodulin demonstrated comparable efficiency to the benchmark library. Moreover, selection against thermostable green fluorescent protein (TGP) yielded multiple high-affinity sybodies, with nanomolar dissociation constants validated by size-exclusion chromatography and biolayer interferometry. Importantly, the selected sybodies are directly compatible with the Legobody system without requiring further engineering or subcloning. The S1.0 library represents a valuable resource for nanobody discovery and, in particular, provides an efficient route for generating Legobody-compatible binders suitable for structural studies of challenging small proteins by cryo-EM.", "journal": "Advanced biotechnology", "year": "2026", "doi": "10.1007/s44307-026-00112-z", "ptypes": [ "Journal Article" ], "first_author": "Gu W", "dirs": [ "D" ] }, "42719415": { "pmid": "42719415", "title": "Development and Humanization of a Camelid-Derived Neutralizing Nanobody Targeting Botulinum Neurotoxin Type A.", "abstract": "Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is one of the most potent neurotoxins, causing botulism, a potentially fatal paralytic disease. Among different serotypes, BoNT/A is the most potent and the primary cause of botulism. The receptor-binding domain (Hc) of BoNT is crucial for targeting host cell receptors, making it a key therapeutic target. This study aims to develop neutralizing antibodies against BoNT/A by isolating camelid-derived nanobodies (Nbs) targeting BoNT/A-Hc (AHc). A panel of Nbs specifically targeting AHc was isolated from a camelid immune phage display library. The VHH fragments were fused with human IgG Fc (hFc) to generate VHH-hFc fusion proteins. Neutralizing activity was evaluated using a murine lethality assay following pre-incubation of antibody and toxin. Potent Nbs were humanized through homology modeling, which predicts a theoretically reduced risk of immunogenicity. The affinity of the parent and humanized antibodies to AHc was measured using biolayer interferometry (BLI). Their biophysical properties, including thermal stability and aggregation propensity, were assessed by differential scanning fluorimetry (DSF), static light scattering (SLS), and dynamic light scattering (DLS). Fifty-nine unique VHH clones were identified, four of which provided complete protection against a 20 LD50 BoNT/A challenge in mice. The most potent antibodies, 1A1 and 1A3, exhibited ED50 of 0.47 μg per mouse, corresponding to a neutralizing potency of 4.3 IU/mg. Upon humanization, the variants hA1 and hA3 showed strong binding affinities, with KD ranging from 1 to 2 nM. The humanized variant hA1 demonstrated superior neutralizing activity against BoNT/A, exhibiting an ED50 of 0.12 μg and a potency of 17.1 IU/mg, which represents an approximate four-fold increase compared to the parental antibody (1A1). Additionally, hA1 demonstrated enhanced thermal stability and minimal aggregation propensity. Humanized Nbs could serve as an attractive platform for developing novel botulism therapeutics, with potential advantages over traditional serum antitoxins in safety profile, clinical tolerability and scalable manufacturing. These favorable traits render them viable candidates for next-generation antitoxins against BoNT/A poisoning. Humanized Nb hA1 displays potent neutralizing activity and optimized biophysical properties, implying its value as a preclinical lead for botulism intervention. Nevertheless, systematic follow-up studies covering post-exposure protective efficacy, direct immunogenicity testing and comprehensive safety profiling are required to fully validate its translational prospects.", "journal": "International journal of nanomedicine", "year": "2026", "doi": "10.2147/IJN.S616511", "ptypes": [ "Journal Article" ], "first_author": "Jiang Y", "dirs": [ "D" ] }, "40275961": { "pmid": "40275961", "title": "Evaluation of an anti-CD3 VHH and construction of an anti-CD3/anti-EGFR bispecific tandem VHH as a cancer cell targeting drug construct.", "abstract": "Recently, the development of T-cell engager cancer therapeutics, consisting of anticancer and anti-T-cell antibody parts to engage the T-cell to the cancer site, has gained interest. Anti-CD3 antibodies are predominantly used to achieve specific binding to T-cells for the T-cell engager construction. Various kinds of anti-CD3 IgG clones have been developed and engineered, but the available anti-CD3 VHH, a single variable domain of a dromedary heavy-chain antibody, clones are limited in number. Thus, the assessment of the available anti-CD3 VHHs is important for therapeutic applications. Here, we demonstrated the expression and characterization of an anti-CD3 VHH clone, 117G03, and evaluated this T-cell engager with an anti-EFGR VHH in a bispecific format to assess the developability of this anti-CD3 VHH clone. 117G03 was expressed as a monomer in the soluble fraction and demonstrated decent thermal stability with binding activity against a CD3-positive cell line. The T-cell engager construct was prepared using the refolding method and exhibited enhanced cytotoxic activity against the epidermal growth factor receptor (EGFR)-positive cell line mediated by activated T-cells. These results indicate that 117G03 can be utilized for T-cell engager applications.", "journal": "Biochemistry and biophysics reports", "year": "2025", "doi": "10.1016/j.bbrep.2025.102015", "ptypes": [ "Journal Article" ], "first_author": "Asanuma T", "dirs": [ "D" ] }, "41744901": { "pmid": "41744901", "title": "Analytical Performance of Nanobody-Based Immunoassay and Immunosensing Platforms for Bacteria and Toxin Detection: A Systematic Review.", "abstract": "Background: bacterial pathogens and their toxins present analytical challenges for rapid and specific detection, contributing to over 600 million cases of illness annually and worsening antimicrobial resistance (AMR). Conventional detection methods are useful but limited. Single-domain antibodies (sdAbs) offer alternative recognition elements with unique biochemical and engineering benefits, enabling the development of nanobody-based immunoassays and biosensing platforms that provide fast, highly selective, and reliable detection of bacterial pathogens and toxins in both food and clinical environments. Objectives: this systematic review assesses the analytical and functional performance of nanobody-based immunoassays and sensing formats for detecting bacteria and toxins across food and clinical samples. Methods: following PRISMA guidelines, major scientific databases were used to gather research, resulting in 32 eligible studies published between 2011 and 2025. Results: data collected included assay platforms, target bacteria and toxins, limit of detection, sensitivity, specificity, matrix recovery, and practicality. Risk of bias was evaluated using an adapted QUADAS-2 framework. The review shows that nanobody-based immunoassays have achieved high performance, thermostability, compatibility with genetic engineering, and versatile assay design. When combined with advanced transduction and signal amplification strategies, these systems contribute to the development of highly sensitive and user-friendly bioanalytical platforms for detecting bacteria and toxins. Conclusions: however, most studies relied on spiked samples and lacked large-scale validation, emphasizing the need for standardized benchmarking and real-world testing.", "journal": "Antibodies (Basel, Switzerland)", "year": "2026", "doi": "10.3390/antib15010015", "ptypes": [ "Journal Article", "Review" ], "first_author": "Jalil A", "dirs": [ "D" ] }, "42579767": { "pmid": "42579767", "title": "Isolation of Novel Fully Human Single-Domain Antibodies Targeting GUCY2C from a Heavy-Chain-Only Transgenic Mouse Platform.", "abstract": "IntroductionTraditional natural single-domain antibodies(sdAbs) are primarily derived from camelids and sharks. However, the large size and slow reproduction of these species limit their widespread application in antibody development. In this study, we employed a fully human heavy-chain-only antibody mouse ( huNano mouse ) platform and a rapid, efficient preparation process to successfully develop fully human heavy-chain-only antibodies(HcAbs) targeting the tumor antigen guanylyl cyclase C (GC-C). These sdAbs provide high-quality candidate molecules for subsequent GC-C-based CAR-T therapy and bispecific antibody drug development.MethodsFully huNano mice were immunized with GC-C recombinant protein. SdAb sequences specifically binding to GC-C were selected via phage display technology and subsequently cloned into mammalian expression vectors for expression and purification. The binding activity, affinity, thermal stability, and monomeric dispersion of the antibodies were systematically evaluated using various methods, including ELISA, flow cytometry (FACS), biolayer interferometry (BLI), and size-exclusion chromatography (SEC-HPLC).ResultsMultiple sdAbs with specific recognition of GC-C were successfully obtained and exhibited strong binding affinities. After one-step purification, the monomeric purity of most antibodies exceeded 95%, with aggregate content below 5%. The nanobodies exhibited good thermal stability, with melting temperature (Tm) and aggregation onset temperature (Tagg) both falling within the range of 60-68 °C, demonstrating excellent drug development potential.ConclusionThese results validate the capability of the huNano mouse platform to efficiently produce high-quality, fully human sdAbs.", "journal": "Technology in cancer research & treatment", "year": "2026", "doi": "10.1177/15330338261473054", "ptypes": [ "Journal Article" ], "first_author": "Zhu P", "dirs": [ "D" ] }, "39552758": { "pmid": "39552758", "title": "Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling.", "abstract": "Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.", "journal": "Synthetic and systems biotechnology", "year": "2025", "doi": "10.1016/j.synbio.2024.10.003", "ptypes": [ "Journal Article" ], "first_author": "Wang X", "dirs": [ "D" ] }, "40833585": { "pmid": "40833585", "title": "Side by side comparison of NOTA and DOTA for conjugation efficiency, gallium-68 labeling, and in vivo biodistribution of anti-mesothelin sdAb A1-His.", "abstract": "Mesothelin is a glycoprotein overexpressed in various cancers, with limited expression in healthy tissues. The single-domain antibody (sdAb, or nanobody) A1-His has previously successfully been validated in mice for the SPECT imaging of mesothelin positive tumors following radiolabeling with 99mTc. Our objective was to radiolabel this sdAb with 68Ga for PET imaging, exhibiting superior sensitivity and resolution than SPECT in clinical practice. To this aim, it was conjugated to NOTA chelator that is commonly employed for 68Ga labeling of antibody-derived tracers. In addition, the high affinity and specificity of A1-His sdAb position it as a promising candidate for theranostic applications. In anticipation of future radiolabeling with beta-emitting radionuclides, DOTA-conjugated A1-His was also evaluated. Given the high thermal stability of sdAbs, this DOTA-conjugated sdAb could potentially be implemented in future studies as a theranostic agent with beta-emitting radionuclides. A1-His was successfully conjugated to p-SCN-Bn-DOTA and p-SCN-Bn-NOTA under optimized conditions, achieving chelator-to-sdAb ratios of 1.8 and 1.3, respectively. NOTA-A1-His allowed rapid radiolabeling with 68Ga at room temperature, achieving high radiochemical purity (> 98%) within 5 min. Using DOTA, similar purity was obtained at 60 °C for 15 min. Both radiotracers demonstrated stability over 4 h in the radiolabeling medium and 2 h in human blood. However, some instability was observed in murine blood. Biodistribution and imaging studies in mice bearing mesothelin-expressing tumors showed specific tumor targeting for both tracers. Notably, [68Ga]Ga-DOTA-A1-His exhibited twofold lower kidney uptake compared to [68Ga]Ga-NOTA-A1-His, potentially enhancing imaging contrast and reducing renal radiation exposure. His-tag removal, further improves the biodistribution profile of the 2 tracers. Both p-SCN-Bn-DOTA and p-SCN-Bn-NOTA chelators can be effectively conjugated to the A1 sdAb and radiolabeled with 68Ga, producing stable radiotracers with specific tumor-targeting capabilities. NOTA chelator offers advantages in rapid, room-temperature radiolabeling. However, DOTA would offer the advantage to be employed for theranostic approaches using β- emitters such as 177Lu or 161Tb. The lower kidney retention of DOTA-A1 also suggests that its dosimetry, a key factor in theranostic, would be more favorable.", "journal": "EJNMMI radiopharmacy and chemistry", "year": "2025", "doi": "10.1186/s41181-025-00380-5", "ptypes": [ "Journal Article" ], "first_author": "N'Guessan É", "dirs": [ "D" ] }, "42347766": { "pmid": "42347766", "title": "Reconstructing enzyme evolution by protein engineering.", "abstract": "It is important to understand how the fascinating diversity of modern enzymes has manifested itself since the onset of life. A powerful approach to address this question is the computational and experimental retracing of evolutionary processes by protein engineering techniques such as rational design, directed evolution, and ancestral sequence reconstruction. In this review, we show that these methods were used to elucidate how first enzymes have emerged from ligand-binding protein scaffolds, how the enormous variety of enzymes with similar catalytic mechanisms but different substrate specificities have emerged, and how natural selection has driven the formation of oligomeric enzyme complexes from their monomeric ancestors. A further important aspect of enzyme evolution is the adaptation of catalytic activity and thermostability to changing environmental conditions on geological timescales. We will present work in which the interdependence of these enzymatic properties was analyzed and reproduced. Finally, we will discuss case studies that show how the rapid evolution of new enzymatic functions has allowed for the degradation of anthropogenic substances that have only recently been released into the environment.", "journal": "FEBS letters", "year": "2026", "doi": "10.1002/1873-3468.70400", "ptypes": [ "Journal Article", "Review" ], "first_author": "Drexler L", "dirs": [ "D" ] }, "41052325": { "pmid": "41052325", "title": "ProT-VAE: Protein Transformer Variational AutoEncoder for functional protein design.", "abstract": "Deep generative models have demonstrated success in learning the protein sequence to function relationship and designing synthetic sequences with engineered functionality. We introduce the Protein Transformer Variational AutoEncoder (ProT-VAE) as an accurate, generative, fast, and transferable model for data-driven protein design that blends the merits of variational autoencoders to learn interpretable, low-dimensional latent embeddings for conditional sequence design with the expressive, alignment-free featurization offered by transformer-based protein language models. We implement the model using NVIDIA's BioNeMo framework and validate its performance in retrospective functional prediction and prospective functional design. The model identifies a phenylalanine hydroxylase enzyme with 2.5× catalytic activity over wild-type, and a γ-carbonic anhydrase enzyme with a melting temperature elevation of ΔTm = +61 °C relative to the most thermostable sequence reported to date and activity in 23% v/v methyl diethanolamine at pH 11.25 and 93°C corresponding to industrially relevant conditions for enzymatic carbon capture technologies. The ProT-VAE model presents a powerful and experimentally validated platform for machine learning-guided directed evolution campaigns to discover synthetic proteins with engineered function.", "journal": "Proceedings of the National Academy of Sciences of the United States of America", "year": "2025", "doi": "10.1073/pnas.2408737122", "ptypes": [ "Journal Article" ], "first_author": "Sevgen E", "dirs": [ "D" ] }, "39730046": { "pmid": "39730046", "title": "Engineering thermostability of industrial enzymes for enhanced application performance.", "abstract": "Thermostability is a key factor for the industrial application of enzymes. This review categorizes enzymes by their applications and discusses the importance of engineering thermostability for practical use. It summarizes fundamental theories and recent advancements in enzyme thermostability modification, including directed evolution, semi-rational design, and rational design. Directed evolution uses high-throughput screening to generate random mutations, while semi-rational design combines hotspot identification with screening. Rational design focuses on key residues to enhance stability by improving rigidity, foldability, and reducing aggregation. The review also covers rational strategies like engineering folding energy, surface charge, machine learning methods, and consensus design, along with tools that support these approaches. Practical examples are critically assessed to highlight the benefits and limitations of these strategies. Finally, the challenges and potential contributions of artificial intelligence in enzyme thermostability engineering are discussed.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2024.139067", "ptypes": [ "Journal Article", "Review" ], "first_author": "Xu K", "dirs": [ "D" ] }, "41131942": { "pmid": "41131942", "title": "Functional and structural characterization of F1-ATPase with common ancestral core domains in stator ring.", "abstract": "Extant F1-ATPases exhibit diverse rotational stepping behaviors-3-, 6-, or 9-step cycles-yet the evolutionary origin of these patterns remains unclear. Here, we used ancestral sequence reconstruction to infer the catalytic β and non-catalytic α subunits of a putative ancestral F1-ATPase. We then fused their functionally critical domains into the thermostable F1 from Bacillus PS3, yielding a stable chimeric enzyme. Cryo-EM revealed two distinct conformational states-binding and catalytic dwell states-separated by a ~34° rotation of the γ subunit, suggesting a fundamental six-step mechanism akin to that of extant six-stepping F1-ATPases. Single-molecule rotation assays with ATP and the slowly hydrolyzed ATP analog ATPγS demonstrated that the chimeric motor is intrinsically a six-stepper, pausing at binding and catalytic dwell positions separated by 32.1°, although the binding dwell is significantly prolonged by an unknown mechanism. These findings indicate that F1-ATPase was originally a six-stepper and diversified into 3-, 6- and 9-step forms in evolutionary adaptation. Based on these results, we discuss plausible features of the entire FoF1 complex, along with potential physiological contexts in the last universal common ancestor and related lineages.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70345", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Suzuki AK", "dirs": [ "D" ] }, "39988321": { "pmid": "39988321", "title": "Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis.", "abstract": "Enzymatic DNA synthesis, using stepwise nucleotide addition catalyzed by template-independent polymerases, promises higher efficiency, quality, and sustainability than today's industry-standard phosphoramidite-based processes. We report the directed evolution of a terminal deoxynucleotidyl transferase that uses 3'-phosphate-blocked 2'-deoxynucleoside triphosphates (dNTPs) to control the polymerization reaction. Over 32 iterative rounds of laboratory evolution, 80 amino acid substitutions-constituting ∼20% of the coding protein sequence-were introduced. The engineered polymerase exhibits uniformly high catalytic activity, raising incorporation efficiency by 200-fold to >99% for dNTPs with a 3'-reversible terminator while reducing extension times by >600-fold to 90 s. The same enzyme variant displays improved enzyme robustness, as reflected in the 20°C increase in thermostability. Based on these performance characteristics, the engineered polymerase represents an operational prototype for biocatalytic DNA synthesis at a commercial scale.", "journal": "Nucleic acids research", "year": "2025", "doi": "10.1093/nar/gkaf115", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Forget SM", "dirs": [ "D" ] }, "42178469": { "pmid": "42178469", "title": "Application and Molecular Modification of Enzyme in Textile Degumming.", "abstract": "This review comprehensively examines the application and molecular modification of enzymes in the textile degumming of plant-based natural fibers. Traditional chemical and physical degumming methods are energy-intensive and environmentally detrimental, having negative impact on fibers' mechanical properties and performance. In contrast, enzymatic degumming, utilizing biocatalysts such as pectinases, xylanases, mannanases, and laccases, offers a promising eco-friendly solution due to its specificity, mild conditions, and minimal fiber damage. However, the widespread industrial adoption of enzymatic degumming is hindered by inherent limitations of native enzymes, including poor thermal stability, narrow pH adaptability, and low tolerance to industrial process conditions. To address these challenges, enzyme molecular modification has emerged as a transformative strategy. This review outlines the structure and gum composition of representative bast fibers and details the types, mechanisms, and synergistic actions of key degumming enzymes. It provides an in-depth analysis of advanced enzyme engineering technologies, including directed evolution, rational and semi-rational design, computational-aided methods, and AI-driven approaches, with specific case studies on their application to optimize these enzymes. For instance, engineered pectate lyase V52A showed significantly improved activity and thermostability, while the xylanase Q130K/E195K showed a 9.40‑fold longer pH half‑life and a 2.55‑fold higher specific activity than the wild type. Despite significant progress, current challenges persist, such as insufficient multi-factorial industrial adaptability of engineered enzymes, the complexity of fiber-specific gum structures, high production costs, and underdeveloped industrial application systems. Future prospects lie in integrating multi-technological approaches for precise enzyme engineering, in-depth exploration of gum-enzyme interactions, optimization of fermentation processes, and the development of intelligent, integrated degumming systems. This work aims to provide theoretical support and technical references to advance the industrialization of enzymatic degumming for sustainable textile processing.", "journal": "Applied biochemistry and biotechnology", "year": "2026", "doi": "10.1007/s12010-026-05739-1", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhao W", "dirs": [ "D" ] }, "41342083": { "pmid": "41342083", "title": "Recent Advances in Phospholipase C Applications in Food Industry: From Molecular Mechanisms to Green Processing Technologies.", "abstract": "Phospholipase C has become a pivotal biocatalyst in agricultural and food chemistry, enabling efficient degumming while producing diacylglycerol as a nutraceutical. Over the past two decades, more than 40 microbial, plant, and mammalian PLCs have been characterized, revealing conserved catalytic frameworks but broad diversity in regulatory domains and substrate scope. Advances in protein engineering have yielded thermostable variants active above 70 °C and pH 3.5 and 10.0, while enzymatic routes cut energy use by up to 70% compared with chemical methods. This Perspective presents a comprehensive synthesis that integrates food engineering, sustainability, and enzyme biotechnology. It summarizes PLC's evolutionary origins, structure-function relationships, and production strategies and highlights rational mutagenesis, directed evolution, and AI-guided redesign improving catalytic stability and process compatibility. Industrial applications─including oil refining, dairy optimization, and functional lipid generation, demonstrate measurable efficiency gains and environmental benefits, positioning PLC as a model enzyme for sustainable bioprocessing within a circular bioeconomy.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.5c11096", "ptypes": [ "Journal Article", "Review" ], "first_author": "Pei XD", "dirs": [ "D" ] }, "41870126": { "pmid": "41870126", "title": "Directed Evolution of an Efficient Polycarbonate Depolymerase With Exceptional Operational Stability.", "abstract": "We recently developed a high-throughput directed evolution platform for engineering polymer degrading enzymes, and showcased its utility through the development of an efficient and thermostable variant of IsPETase, termed HotPETase. Here, we show that this platform can be used to re-engineer PET degrading enzymes for the recycling of other aromatic-containing commodity polymers. Promiscuous poly(bisphenol-A carbonate) (PC) depolymerase activity of LCCICCG was enhanced by directed evolution to afford an engineered polycarbonate hydrolase, that also benefits from improved solvent tolerance and operational stability at elevated temperatures. Interestingly, the enzyme-concentration dependent inhibition observed with the parent enzyme is also alleviated through evolution, improving practical utility. PC-2 can achieve rapid and complete depolymerization of a PC film to bisphenol-A (BPA) in just 6 h at 75°C. This study shows how plastic degrading enzymes can be readily adapted through evolution to operate on new and valuable polymer classes.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2026", "doi": "10.1002/anie.202525215", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Jones HA", "dirs": [ "D" ] }, "40505904": { "pmid": "40505904", "title": "Activity and stability improvement: structure-function insights into CotA from Bacillus subtilis.", "abstract": "CotA, a bacterial multicopper oxidase, exhibits exceptional thermal stability, alkali resistance, and substrate versatility, making it valuable for industrial biocatalysis, environmental remediation, and dye degradation. Extensive research has enhanced its activity and stability through directed evolution, semi-rational design, and rational design, yet the underlying structural features remain largely unclear. In this study, we performed site-directed mutagenesis on CotA from Bacillus subtilis and analyzed its effects on stability. Among the CotA single and double mutants we constructed, the T377I/T418G variant exhibited a 6.12-fold increase in activity compared to the wild type and a 50 % improvement in thermal stability at 80 °C. To uncover the relationship between the enzyme activity, stability, and the spatial structure of the CotA mutants, we conducted molecular dynamics (MD) simulations to analyze the catalytic structural features. The results indicated that the increase in ABTS-specific enzyme activity was linked to an expanded binding pocket, while the increased thermal stability was attributed to a higher proportion of random coils in the secondary structure. Our study provides new insights into the catalytic activity and thermostability structural features of CotA, laying the groundwork for its applications in industrial and environmental fields.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.145156", "ptypes": [ "Journal Article" ], "first_author": "He W", "dirs": [ "D" ] }, "41003756": { "pmid": "41003756", "title": "Strategies to improve photosynthesis by modifying the RuBisCO system and its limitations.", "abstract": "RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the primary enzyme responsible for carbon fixation in the Calvin-Benson-Bassham cycle, yet it remains one of the most inefficient enzymes in nature. Its slow catalytic rate, low specificity for CO₂ over O₂, and susceptibility to inhibition and heat stress limit photosynthetic efficiency and crop productivity, particularly under fluctuating environmental conditions. This review highlights current strategies to enhance RuBisCO performance. These include direct mutagenesis, ancestral protein reconstruction, and the incorporation of chimeric or heterologous RuBisCO subunits to improve kinetics and stability. Enhancing the expression of native or foreign RuBisCO subunits, as well as engineering thermostable RuBisCO activase, further supports improved photosynthetic capacity. Additionally, the integration of carbon-concentrating mechanisms such as cyanobacterial carboxysomes and algal pyrenoids offers promising avenues to increase CO₂ availability around RuBisCO. Emerging synthetic biology approaches, including artificial carbon fixation pathways, aim to bypass the limitations of natural photosynthesis altogether. We have also discussed the limitations of each method. Complementing these advances, artificial intelligence and machine learning are being increasingly used to predict beneficial mutations, model enzyme behaviour, and guide protein engineering. Together, these multidisciplinary strategies hold great potential to optimize RuBisCO, improve crop yields, and enhance resilience in a changing climate.", "journal": "Molecular biology reports", "year": "2025", "doi": "10.1007/s11033-025-11075-0", "ptypes": [ "Journal Article", "Review" ], "first_author": "Namachivayam R", "dirs": [ "D" ] }, "39673482": { "pmid": "39673482", "title": "Structural insights into a DNA polymerase reading the xeno nucleic acid HNA.", "abstract": "Xeno nucleic acids (XNAs) are unnatural analogues of the natural nucleic acids in which the canonical ribose or deoxyribose rings are replaced with alternative sugars, congener structures or even open-ring configurations. The expanding repertoire of XNAs holds significant promise for diverse applications in molecular biology as well as diagnostics and therapeutics. Key advantages of XNAs over natural nucleic acids include their enhanced biostability, superior target affinity and (in some cases) catalytic activity. Natural systems generally lack the mechanisms to transcribe, reverse transcribe or replicate XNAs. This limitation has been overcome through the directed evolution of nucleic acid-modifying enzymes, especially polymerases (pols) and reverse transcriptases (RTs). Despite these advances, the mechanisms by which synthetic RT enzymes read these artificial genetic polymers remain largely unexplored, primarily due to a scarcity of structural information. This study unveils first structural insights into an evolved thermostable DNA pol interacting with the XNA 1,5-anhydrohexitol nucleic acid (HNA), revealing unprecedented HNA nucleotide conformations within a ternary complex with the enzyme. These findings not only deepen our understanding of HNA to DNA reverse transcription but also set the stage for future advancements of this and similar enzymes through deliberate design.", "journal": "Nucleic acids research", "year": "2025", "doi": "10.1093/nar/gkae1156", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Gutfreund C", "dirs": [ "D" ] }, "40553201": { "pmid": "40553201", "title": "Engineered β-galactosidase catalyzes lactose to prebiotics in situ in raw milk.", "abstract": "The enzymatic conversion of lactose to galactooligosaccharides (GOS) within raw milk offers a promising avenue for reducing lactose content while enhancing its prebiotic benefits. This process hinges on utilizing enzymes with high transglycosylation activity compatible with current milk processing methods. In the present studies, Bacillus circulans β-galactosidase (BglD) was identified as an effective enzyme, achieving a lactose conversion rate of 73.61% in milk. However, BglD's resistance to pasteurization underscored the need for enzyme modifications. Through molecular directed evolution, a mutant T473L/R484P was engineered. It exhibited improved catalytic performance at lower temperatures, with optimal activity at pH 6.5 and 55 °C. In contrary to the wild-type, mutant T473L/R484P exhibited reduced thermostability, and its activity diminished rapidly above 50 °C. Remarkably, in simulated dairy environments, mutant T473L/R484P converted over 78% to 82% of lactose at low temperatures (5 to 10 °C), reducing lactose to around 10.4 to13.0 g/L while generating approximately 30 to 34 g/L of GOS. These findings highlight the mutant's potential in producing low-lactose, GOS-enriched milk, and pave the way for innovative in situ lactose-to-GOS conversion processes in raw milk.", "journal": "Biotechnology letters", "year": "2025", "doi": "10.1007/s10529-025-03601-8", "ptypes": [ "Journal Article" ], "first_author": "Zhao J", "dirs": [ "D" ] }, "41078880": { "pmid": "41078880", "title": "Discovery and engineering of bifunctional enzymes for lignocellulose degradation: Metagenomic and computational approaches.", "abstract": "Efficient degradation of lignocellulosic biomass is vital for converting plant-based waste into renewable fuels and chemicals. Owing to its complex composition of cellulose, hemicellulose, and lignin, its enzymatic breakdown often requires multiple enzymes to act synergistically. Bifunctional enzymes that combine two catalytic activities in a single protein offer a promising solution. This review highlights recent advances in the identification and engineering of bifunctional enzymes for lignocellulose degradation, particularly through metagenomics, protein fusion and computational design. Functional pairings, such as cellulase/xylanase, were examined with a focus on their synergistic effects, substrate specificity, and stability. Promiscuous and naturally evolved bifunctional enzymes from extreme or uncultured environments are also discussed. Advances in silico modeling and directed evolution have enhanced enzyme properties such as thermostability and substrate range. The review concludes with an outlook on the challenges and opportunities of implementing bifunctional enzymes to improve the economic and technical viability of biomass conversion.", "journal": "Biotechnology reports (Amsterdam, Netherlands)", "year": "2025", "doi": "10.1016/j.btre.2025.e00926", "ptypes": [ "Journal Article", "Review" ], "first_author": "Goudarzi R", "dirs": [ "D" ] }, "40612499": { "pmid": "40612499", "title": "Directed evolution of an ultrastable carbonic anhydrase from Desulfovibrio vulgaris towards enhanced tolerance to flue gas impurities.", "abstract": "Carbonic anhydrases (CAs) have been proved as a highly efficient and selective promoter for conventional Carbon Capture Utilization and Storage (CCUS) industrial processes. The aim of this work was to demonstrate a high-throughput screening system for detecting engineered CAs with resistance to common inhibitors (SO4 2-, SO3 2-, NO3 -, NO2 -) present as major impurities in post-combustion flue gases, maintaining their initial thermostability. We established a screening protocol on solid and liquid assays for selecting mutants generated with error-prone PCR (epCA8.0) maintaining the thermostability of the parent DvCA8.0 but having improved resistance to flue gas inhibitors. A library of around 1000 mutants was created. The mutant E12 (G7D) showed 50 % increased stability for a mix of inhibitors corresponding to total concentration of 300-600 mM and 65 % increased stability to 150 mM, compared to the parent DvCA8.0. To our knowledge, this is the first time that a CA was evolved by protein engineering methods to increase its stability to common flue gas inhibitors. Additionally, we have established a premise for screening and characterization of CA libraries, which has not been clearly addressed previously, as presence of ionic inhibitors significantly change the pH of enzyme assays, while the nature of such screening assays is pH dependent. We envision that this study will open the pathway for the development of highly resistant CAs in the near future, overcoming stability and cost issues that are associated with their limited application in CCUS technologies.", "journal": "Computational and structural biotechnology journal", "year": "2025", "doi": "10.1016/j.csbj.2025.06.007", "ptypes": [ "Journal Article" ], "first_author": "Antonopoulou I", "dirs": [ "D" ] }, "40112285": { "pmid": "40112285", "title": "Efficient C25-Hydroxylation of Vitamin D3 Utilizing an Artificial Self-Sufficient Whole-Cell Cytochrome P450 Biocatalyst.", "abstract": "Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction of self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization to regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic optimization of reaction conditions, including cosolvents, cell concentrations, and plasmid selection, to enhance catalytic performance. Under optimized conditions, the heme domain Vdh-K1 achieved a 91.6% conversion efficiency and was subsequently selected for chimeric enzyme assembly. By employing local energetic frustration analysis to evaluate protein flexibility and allosteric dynamics, we identified chimeric P450 variants with highly frustrated linkers. The optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, catalytic activity, and coupling efficiency, achieving a yield of 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli whole-cell catalysis─the highest reported yield to date. This work underscores the utility of computational frustration analysis in refining linker dynamics for multidomain enzymes and establishes a scalable, cost-effective framework to advance P450s systems for industrial biosynthesis of high-value compounds.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.4c12356", "ptypes": [ "Journal Article" ], "first_author": "Liang Z", "dirs": [ "D" ] }, "40490963": { "pmid": "40490963", "title": "Construction of hyperthermostable d-allulose 3-epimerase from Arthrobacter globiformis M30 using the sequence information from Arthrobacter psychrolactophilus.", "abstract": "d-Allulose is one of the rare monosaccharides and is considered as a safe ingredient in foods. It can be enzymatically produced from d-fructose by the enzyme d-allulose 3-epimerase. More stable enzymes can operate effectively for longer durations, reducing the need for frequent replacements and thereby lowering costs. In addition, the preparation of the recombinant Arthrobacter globiformis M30 (AgDAE) enzyme requires heat treatment at 60-70 °C to remove host cell debris and potential microbial contaminants. Therefore, to address the need for more thermostable enzymes in d-allulose production, we aimed to create thermostable mutants of AgDAE using the protein engineering method. We cloned d-allulose identified from A. globiformis M30 and, using sequence homology, we constructed thermostable mutants by protein engineering. Each effect of the five mutations used was independent and additive. By integrating positive mutations, we succeeded in the construction of a chimeric enzyme exhibiting hyperthermostability without loss of enzymatic activity. The constructed chimera mutant was highly functional above 95 °C and remained stable under 80 °C. Our approach using structural information for the chimeric construction experiments also suggested that incorporating mutations from other homologous enzymes can impart advantages in enzymes in a simple and effective manner.", "journal": "FEBS open bio", "year": "2025", "doi": "10.1002/2211-5463.70060", "ptypes": [ "Journal Article" ], "first_author": "Shimada K", "dirs": [ "D" ] }, "39752562": { "pmid": "39752562", "title": "Thermostability Enhancement of Tagatose 4-Epimerase through Protein Engineering and Whole-Cell Immobilization.", "abstract": "d-Tagatose, a rare sugar endowed with a low-calorie property, superior taste quality, and probiotic functionality, has garnered significant research attention. However, the prevailing biological production methods relying on β-galactosidase and l-arabinose isomerase face challenges including high cost and suboptimal conversion efficiency. Consequently, it is of great research significance to find efficient alternative routes for d-tagatose synthesis. Previously, Thermotoga petrophila tagaturonate 3-epimerase was modified to function as tagatose 4-epimerase (T4E) enabling the direct conversion of d-fructose to d-tagatose. In this study, T4E was further engineered through directed evolution, specifically targeting the enhancement of its thermostability for application. This endeavor yielded promising T4E variants with superiority over those of the original enzyme. T4E I430P exhibits a half-life (t1/2) at 70 °C that is 1.83-fold that of T4E, with an increased melting temperature (Tm) of 5.1 °C compared to T4E. Additionally, T4E G90S/T272A/I430P demonstrated a 21.4% increase in specific activity compared to T4E. At 70 °C, its t1/2 was 1.69-fold that of T4E, and its Tm is 2.9 °C higher than T4E. Furthermore, whole-cell immobilization integrating these engineered T4E variants into a robust biocatalytic system was employed. This innovative approach not only underscores the practical feasibility of modifying enzymes through directed evolution but also establishes a foundation for the cost-effective, large-scale production of d-tagatose.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.4c08630", "ptypes": [ "Journal Article" ], "first_author": "Liu Z", "dirs": [ "D" ] }, "42376006": { "pmid": "42376006", "title": "Evolution of alcohol oxidase for improved methanol bioconversion and formaldehyde tolerance.", "abstract": "Methanol oxidation by alcohol oxidases (AOXs) is a key bottleneck in one-carbon (C1) bioconversion due to limited catalytic efficiency and poor formaldehyde tolerance. Here, we report the directed evolution of an alcohol oxidase from Gloeophyllum trabeum, yielding an optimized variant, GtAOXM3. The engineered enzyme exhibits a sixfold increase in catalytic efficiency (7.7 s-1 mM-1), together with enhanced formaldehyde tolerance, thermostability, and high methanol specificity. Molecular dynamics simulations suggest that increased global rigidity and cooperative residue dynamics contribute to the improved performance. When incorporated in multienzyme cascade systems, GtAOXM3 enables efficient conversion of methanol to the value-added chemicals dihydroxyacetone (34.5 mM) and ethylene glycol (23.3 mM). This work establishes GtAOXM3 as an efficient and cost-effective biocatalyst for methanol-based C1 biotransformation.", "journal": "Synthetic and systems biotechnology", "year": "2027", "doi": "10.1016/j.synbio.2026.05.010", "ptypes": [ "Journal Article" ], "first_author": "Ding XW", "dirs": [ "D" ] }, "41418326": { "pmid": "41418326", "title": "Thermostable amylases from thermophilic microbes: advances in production, engineering, and industrial applications.", "abstract": "Amylases are essential enzymatic macromolecules widely employed in industrial sectors such as starch processing, textiles, detergents, paper manufacturing, pharmaceuticals, and biomedical research. Among α-, β-, and γ-amylases, thermostable α-amylases from thermophilic microbes show high catalytic activity and structural stability under heat, pH variation, and solvent stress. These properties make them valuable for stable, contamination-resistant, and efficient bioprocesses. Thermostable amylases also hold promise in biomedical fields, including diagnostics, enzyme replacement therapy, and nanocarrier-based drug delivery. This review summarizes microbial sources and production approaches for thermostable amylases, highlighting submerged and solid-state fermentation methods. The discussion also outlines optimization of carbon and nitrogen substrates, fermentation duration, and moisture control strategies that directly influence enzyme yield and activity. Factors governing enzyme yield and stability are analyzed, including nutrient balance, pH, temperature, and moisture. Despite their potential, widespread application remains limited by low native production yields, suboptimal heterologous expression, and functional trade-offs between thermostability and enzymatic activity. Recent advances in protein engineering (rational design and directed evolution), omics-driven strain improvement, and nanotechnology integration provide paths to address these limitations. By integrating these strategies, researchers are achieving enzymes with longer operational lifetimes, higher substrate specificity, and improved reusability under industrial and physiological conditions. These advances highlight the growing relevance of thermostable amylases in industrial biotechnology and biomedical research.", "journal": "Nanotechnology", "year": "2025", "doi": "10.1088/1361-6528/ae2f66", "ptypes": [ "Journal Article" ], "first_author": "Vala V", "dirs": [ "D" ] }, "41990964": { "pmid": "41990964", "title": "Directed evolution of a thermostable laccase from Geobacillus stearothermophilus for efficient reduction of oxygen.", "abstract": "The growing demand for laccases with enhanced stability and high redox potential is driven by their potential applications in diverse bioelectrochemical processes. Bacterial laccases are generally more stable than their fungal counterparts but typically exhibit lower redox potentials. To address the need for a laccase with both high redox potential and enhanced stability, we identified a gene encoding a multicopper oxidase in the gram-positive, thermophilic bacterium Geobacillus stearothermophilus. Recombinant heterologous expression and characterization identified the enzyme as a putative laccase (GbsLac). GbsLac showed activity toward phenolic substrates, with a specific activity of 3.3 U/mg. It displayed thermophilic properties, with an optimum temperature of 70 °C at pH 7.5 and a high melting temperature of 91.8 °C, indicating strong thermostability. Electrochemical analysis of oxygen reduction reaction (ORR) revealed a lower onset potential compared to T. versicolor laccase (Tvl). Site-directed mutations-M503L and D438A-caused anodic shifts of ∼120 ± 25 mV and ∼40 ± 10 mV, respectively, indicating enhanced redox potential due to modifications in the T1 copper coordination sphere. These effects are attributed to the removal of weak axial coordination (SMet-T1Cu) via M503 deletion and reduced electron donation by H440 following disruption of its hydrogen bond with D438. Additionally, replacement with non-polar residues likely reduced reorganization energy, contributing to the increased ET1°. These findings highlight GbsLac as a promising biocatalyst for ORR applications.", "journal": "Biochimie", "year": "2026", "doi": "10.1016/j.biochi.2026.04.005", "ptypes": [ "Journal Article" ], "first_author": "Dey B", "dirs": [ "D" ] }, "42884031": { "pmid": "42884031", "title": "Thermostable Carbohydrate-Active Enzymes for High-Temperature Biomass Conversion: From Molecular Design to Process Integration.", "abstract": "High-temperature processing of carbohydrate-rich biomass, including starch liquefaction and lignocellulose saccharification, can improve sugar yields, reduce contamination risks, and facilitate integration with thermochemical pretreatments. However, the operational stability of native carbohydrate-active enzymes (CAZymes) at 60-90 °C remains a major bottleneck for efficient and economically viable biorefineries, and existing reviews rarely integrate multiscale molecular mechanisms with process-relevant engineering strategies. This review systematically examines the multiscale basis of thermostability in CAZymes-covering core hydrophobic packing, electrostatic and metal-mediated networks, disulfide bonds, conformational dynamics, and modular architectures involving catalytic domains, linkers, and carbohydrate-binding modules-and directly links these features to enzyme longevity under high-solids, inhibitor-rich conditions. We then critically compare key engineering strategies, including structure-guided rational design, semi-rational directed evolution, consensus design, ancestral sequence reconstruction, and machine learning-assisted workflows embedded in design-build-test-learn (DBTL) cycles, highlighting their respective strengths, limitations, and complementarity. Representative case studies demonstrate that engineered thermophilic α-amylases and cellulases achieving ΔTm improvements of approximately 10-20 °C can sustain >80-85% residual activity after prolonged exposure at process-relevant temperatures, translating into tangible benefits such as 15-30% reductions in enzyme dosing and measurable decreases in steam consumption, alongside higher sugar titers. By bridging molecular determinants, engineering paradigms, and quantitative process performance, this review provides a pragmatic roadmap for deploying thermostable CAZymes as robust biocatalysts in sustainable high-temperature biomass conversion.", "journal": "International journal of molecular sciences", "year": "2026", "doi": "10.3390/ijms27198788", "ptypes": [ "Journal Article", "Review" ], "first_author": "Hu Y", "dirs": [ "D" ] }, "41863618": { "pmid": "41863618", "title": "Genetically engineered lipases: advances in expression and upscaling for industrial applications.", "abstract": "Lipases are versatile enzymes with widespread industrial applications, including detergents, food processing, pharmaceuticals, biofuels, and environmental cleanup. Their ability to catalyze both hydrolytic and synthetic reactions under diverse conditions underpins their biotechnological significance. Native lipases, however, exhibit limitations such as low stability, narrow substrate spectrum, and low production yields, which limit their large-scale application. Emerging developments in genetic and protein engineering have enabled accurate modulation of enzyme properties and expression systems, offering potential solutions to overcome these challenges. This review presents an integrated view of existing strategies for designing high-performance lipases for industrial applications. It summarizes advancements from metagenomic discovery and gene optimization to expression optimization through codon improvement, promoter adjustment, signal peptide design, and chaperone-mediated folding. Protein engineering strategies, including rational design, directed evolution, and domain recombination; are addressed to enhance catalytic activity, selectivity, and thermostability. Concomitantly, breakthroughs in fermentation optimization, host metabolism engineering, and enzyme immobilization have enhanced the scalability and operational robustness of lipase manufacturing. Novel omics-driven and systems biology platforms now facilitate the rational design of microbial hosts optimized for efficient enzyme biosynthesis. Collectively, these advances outline a coherent blueprint for engineering lipases into strong, industrially applicable biocatalysts.", "journal": "Archives of microbiology", "year": "2026", "doi": "10.1007/s00203-026-04825-6", "ptypes": [ "Journal Article", "Review" ], "first_author": "Majumder D", "dirs": [ "D" ] }, "41873072": { "pmid": "41873072", "title": "[Directed evolution of cellobiose 2-epimerase and its application in the synthesis of lactulose].", "abstract": "We had engineered Caldicellulosiruptor morganii CE (CmCE) to construct the enzyme CmCE/D226G for catalyzing the isomerization of lactose to lactulose. However, the poor thermostability and catalytic efficiency of CmCE/D226G limits its application under high substrate concentrations. To address these issues, we employed directed evolution to improve the catalysis performance of CmCE/D226G. First, we used error-prone PCR to construct a variant library and identified the positive variants through cysteine-tryptophan chromogenic high-throughput screening. Then, aiming the crucial residue positions, we conducted saturation mutagenesis to obtain the variants with improved thermostability and catalysis. Accordingly, we successfully obtained a variant named CmCE/D226G/S180T. Its half-life and catalysis efficiency (kcat/Km) at 70 ℃ were 184.2 min and 36.5 L/(mmol·s), respectively, which were higher than those (117.7 min and 14.96 L/(mmol·s), respectively) of CmCE/D226G. The improvement of isomerization efficiency resulted from the shortening distance between His377 and the O1 of substrate enol intermediate. The improvement of thermostability was attributed to the rigidness enhancement of a flexible loop. With 350 g/L lactose as the substrate, CmCE/D226G/S180T achieved a lactulose yield of 54.8% and an epilactose yield of 9.2%, while CmCE/D226G only reached a lactulose yield of 50% and an epilactose yield of 10.3%. Taken together, this work provides an elite enzyme for catalyzing the transformation of lactose to lactulose, which especially facilitates the innovation of eco-friendly synthesis of lactulose.", "journal": "Sheng wu gong cheng xue bao = Chinese journal of biotechnology", "year": "2026", "doi": "10.13345/j.cjb.250384", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "Jia D", "dirs": [ "D" ] }, "41106182": { "pmid": "41106182", "title": "Development of a portable biosensor for glyphosate detection using bacterial surface-displayed glyphosate oxidase.", "abstract": "Glyphosate persistence in the environment may threaten human health and ecosystems. The development of a cost-effective, portable and sensitive method for glyphosate detection is crucial. In this study, we conducted directed evolution of the highly active glyphosate oxidase B5R18 in our laboratory and obtained a variant with increased enzyme activity and thermostability. Subsequently, via surface display technology for the S-layer protein from the Bacillus thuringiensis (Bt) strain CTC, the glyphosate oxidase variant was actively anchored onto the surface of the Bt strain. Compared with the purified enzyme, the surface-displayed glyphosate oxidase exhibited better storage stability, with more than 95 % activity remaining after 35 days at 28 °C, whereas the free enzyme activity was only 20 %. Under optimal reaction conditions, surface-displayed glyphosate oxidase can catalyze glyphosate to generate H2O2. Using a horseradish peroxidase coupling assay, a visible orange-red substance, which can also be detected spectrophotometrically at 450 nm, was produced. The increase in absorbance was linearly correlated with the concentration of glyphosate (5-400 μM). The detection limit was 0.82 μM, and the recovery rates of real samples reached 97.5-109.1 %. Additional orange-red product is deposited on the surface of the whole-cell catalyst, which can be completely intercepted by a 0.22 μm filter membrane. A portable biosensor and color comparison card for detecting glyphosate were subsequently prepared, which allowed qualitative and semiquantitative analysis of glyphosate levels (0.05-1 mM, LOD of 0.013 mM). This biosensing device provides an easy-to-operate and effective strategy for onsite glyphosate detection.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2025.117974", "ptypes": [ "Journal Article" ], "first_author": "Liu X", "dirs": [ "D" ] }, "39744925": { "pmid": "39744925", "title": "Directed Evolution of an Alginate Lyase from Flammeovirga sp. for Seaweed Fertilizer Production from the Brown Seaweed Laminaria japonica.", "abstract": "An alginate lyase (FsAly7) from Flammeovirga sp. was engineered by directed evolution to improve its optimum temperature and thermostability. The optimum temperature of the positive mutant mFsAly7 (FsAly7-Ser43Pro) was increased by 5 °C, and the thermal inactivation half-lives at 40 and 45 °C were 4.4 and 5.6 times higher than those of FsAly7, respectively. mFsAly7 was expressed in high levels in Pichia pastoris with the highest yield of 3125.5 U·mL-1. On the property-improved enzyme, a bioconversion strategy was developed to prepare the Laminaria japonica hydrolysate, and the highest AOS yield (29.5 mg·mL-1) was 1.5 times higher than that of FsAly7. Moreover, the application potential of the L. japonica hydrolysate as an organic fertilizer was evaluated. The addition of L. japonica hydrolysate improved the sprouting rate, fresh mass, seedling height, and stem diameter of the buckwheat sprouts. Therefore, mFsAly7 may be a good candidate for the production of seaweed fertilizers from L. japonica.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.4c09016", "ptypes": [ "Journal Article" ], "first_author": "Jiang J", "dirs": [ "D" ] }, "40539308": { "pmid": "40539308", "title": "Microfluidic-Assisted Evolution of a Robust NAD+-Dependent Enzyme with Improved Isobutanol Tolerance at Elevated Temperatures.", "abstract": "Prompted by the thermostability issue identified in recent work on enzyme discovery/engineering and its application, the directed evolution of an NAD+-dependent aldehyde dehydrogenase (ALDH) with improved thermostability and isobutanol tolerance at 50 °C, properties required for its successful implementation in cell-free isobutanol biosynthesis, is described herein. ALDH not only plays an important role in multienzyme cascades for the production of platform chemicals but also represents a bottleneck due to its modest stability. Using a custom-built absorbance-activated droplet sorter, ultrahigh-throughput microfluidic screening of a randomized library of 63,000 members is performed, leading to the discovery of a variant with a 250-fold prolonged half-life at 50 °C without significant loss of activity. Subsequently, the most promising mutations are distributed on designer templates in the combinatorial staggered extension process library to create a new generation of variants. One of these variants shows a threefold increase in kcat Km -1. Another shows significantly higher stability in 3% v/v isobutanol, retaining ≈50% of its initial activity after 6 h of incubation at 50 °C. Finally, a cell-free multienzymatic cascade using the ultimate variant demonstrates its superior stability in 4% v/v isobutanol at 50 °C, highlighting the success of engineering to overcome the cofactor-related challenge of establishing cascade balance.", "journal": "ChemSusChem", "year": "2025", "doi": "10.1002/cssc.202501120", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Teshima M", "dirs": [ "D" ] }, "39855695": { "pmid": "39855695", "title": "[Improvement of catalytic activity and thermostability of glucose oxidase from Aspergillus heteromorphus].", "abstract": "Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.", "journal": "Sheng wu gong cheng xue bao = Chinese journal of biotechnology", "year": "2025", "doi": "10.13345/j.cjb.240458", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "Yu S", "dirs": [ "D" ] }, "42121498": { "pmid": "42121498", "title": "α-Amylase: Its Structure, Molecular Modification, and Application in the Food Field.", "abstract": "This review comprehensively examines the structural architecture, catalytic mechanisms, and targeted molecular engineering of α-amylase (primarily the GH13 family), a pivotal biocatalyst in the food industry. We highlight diverse microbial sources of α-amylases and their cost-effective heterologous expression in well-characterized hosts like Bacillus subtilis and Escherichia coli. To overcome extreme operational bottlenecks-such as elevated temperatures and acidic environments-recent advances in protein engineering are critically evaluated. These strategies, including directed evolution, semi-rational design, and advanced immobilization on nanomaterials, synergistically enhance the enzyme's thermostability, catalytic efficiency, and reusability. Furthermore, this paper synthesizes the state-of-the-art applications of engineered α-amylases across key food processing sectors, including baking, sugar refining, and brewing. By integrating structural biology with advanced material science, this review provides a targeted roadmap for developing next-generation, high-performance α-amylases to address current and future challenges in sustainable food processing.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15091555", "ptypes": [ "Journal Article", "Review" ], "first_author": "Liu G", "dirs": [ "D" ] }, "40196513": { "pmid": "40196513", "title": "Conformational Dynamics and Catalytic Backups in a Hyper-Thermostable Engineered Archaeal Protein Tyrosine Phosphatase.", "abstract": "Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid-base catalysis upon loop closure. These enzymes also have a conserved phosphate binding loop that is typically highly rigid and forms a well-defined anion binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP's high sequence similarity to its natural counterparts, ShufPTP presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active site cysteine, mechanistic promiscuity, and most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8°C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme, and showcase the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.03.26.645524", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Yehorova D", "dirs": [ "D" ] }, "41933419": { "pmid": "41933419", "title": "Flavin-dependent monooxygenases as versatile biocatalysts in biomanufacturing: mechanisms, engineering, and applications.", "abstract": "Flavin-dependent monooxygenases (FMOs) are versatile oxidative biocatalysts that catalyze a wide array of oxygenation reactions, such as hydroxylation, epoxidation, Baeyer-Villiger oxidation, and halogenation. These enzymes utilize flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as cofactors to mediate selective incorporation of oxygen into diverse substrates. Owing to their remarkable chemo-, regio-, and stereoselectivity, FMOs have attracted increasing attention as powerful tools for biomanufacturing. Recent advances in enzyme engineering, structural biology, and computational design have expanded the catalytic diversity of FMOs and enabled their integration into biocatalysis frameworks. Moreover, developments in cofactor regeneration, directed evolution, and cell-free biotransformation have improved FMOs' catalytic efficiency and scalability. Despite these advances, challenges such as limited thermostability, oxygen transfer efficiency, and substrate scope remain obstacles for industrial applications of FMOs. This review summarizes the structural characteristics, catalytic mechanisms, and engineering strategies of FMOs, highlights recent progress in their integration into biocatalysis platforms, and discusses current limitations and possible solutions. Insights into improving FMO catalytic performance and expanding their potential as next-generation biocatalysts for biosynthesis will be provided.", "journal": "Biotechnology for biofuels and bioproducts", "year": "2026", "doi": "10.1186/s13068-026-02755-5", "ptypes": [ "Journal Article", "Review" ], "first_author": "Li Y", "dirs": [ "D" ] }, "42065372": { "pmid": "42065372", "title": "Coordinated Engineering of Noncatalytic and Active-Site Regions in a Thermophilic D-Tagatose 4-Epimerase Enhances D-Tagatose Bioconversion from D-Fructose.", "abstract": "D-Tagatose is a low-calorie rare sugar with glycemic control and obesity prevention benefits, making it a promising functional sweetener. One-step epimerization of D-fructose to D-tagatose by D-tagatose 4-epimerase (T4E) is an attractive approach, but current T4Es suffer from low activity and poor thermostability. To address this, a novel T4E from the Thermoproteales archaeon (TaDt4e) was identified and engineered. A truncation variant Δ104-123 increased activity by 76.9%, and directed evolution produced the double mutant S46A/I113M with a 153.8% increase over wild-type enzyme. Using 100 g/L D-fructose, whole-cell biotransformation with S46A/I113M achieved 12.1% conversion within 10 min, and the purified enzyme reached 23.8% within 90 min. These results show that coordinated engineering of noncatalytic and active-site regions can substantially improve the catalytic performance of TaDt4e. This study not only identifies TaDt4e as a promising candidate for D-tagatose production, but also provides insights into the semirational or rational engineering of T4Es and related enzymes.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.6c00985", "ptypes": [ "Journal Article" ], "first_author": "Zhu Z", "dirs": [ "D" ] }, "41863619": { "pmid": "41863619", "title": "Next-generation strategies for PLA degradation: microbial consortia, metagenomics, enzyme engineering and AI-guided approaches.", "abstract": "Polylactic acid (PLA) is one of the most widely used biodegradable bioplastics; however, its slow degradation under natural conditions limits its environmental sustainability. This review summarizes recent advances in microbial and biotechnological strategies that enhance PLA biodegradation across diverse ecosystems. Emerging approaches include screening insect gut microbiota, isolating fungal species with strong adsorption or enzymatic capacities, and exploring soil, compost, and aquatic microbiomes using metagenomics and environmental DNA (eDNA) tools. Microbial consortia, thermophilic degraders, and co-culture systems are highlighted as effective solutions to overcome the intrinsic crystallinity and hydrolysis-dependent breakdown of PLA. Beyond natural systems, this review emphasizes the increasing role of synthetic biology, directed evolution, and artificial intelligence (AI) in engineering high-performance PLA-degrading enzymes. AI-driven structural prediction and machine-learning platforms offer new possibilities for designing robust depolymerases with improved specificity, thermostability, and catalytic efficiency. Collectively, these multidisciplinary strategies provide a roadmap for accelerating PLA degradation in industrial composting, wastewater treatment, and bioremediation. Future integration of ecological screening with computational enzyme engineering is expected to advance scalable and sustainable PLA waste management.", "journal": "Archives of microbiology", "year": "2026", "doi": "10.1007/s00203-026-04726-8", "ptypes": [ "Journal Article", "Review" ], "first_author": "Jonathan AR", "dirs": [ "D" ] }, "41734857": { "pmid": "41734857", "title": "Structural evolution-based ancestral sequence reconstruction of medium-chain alcohol dehydrogenase for efficient synthesis of fused-ring chiral alcohols.", "abstract": "Drug molecules used to treat complex diseases often contain cyclic chiral moieties. Specifically, the synthesis of fused-ring alcohols (e.g., chiral β-tetralol and its derivatives) by chemical methods is challenging. Medium-chain alcohol dehydrogenase (MDR) are widely used in the asymmetric reduction of potential chiral ketones to chiral alcohols, and can be used to synthesize chiral fused-ring alcohols. In this study, we integrated enzyme sequence and structural information in an ancestral sequence reconstruction method. This process identified ancestral medium-chain alcohol dehydrogenase N42, with average conversion and selectivity rates exceeding 98% towards 19 ketone substrates (classified into fused-ring, heterocyclic, and aryl groups), and enhanced thermostability (melting temperature 9.9 °C higher than that of its descendant). Computational analysis explained the origin of the N42 activity and revealed a unique set of functional regulatory sites in the MDR family. This ancestral reconstruction strategy combines sequence and structural information provides novel insights into enzyme discovery and functional regulation, might prove useful for studying enzyme functional evolution in families with deep structural differentiation.", "journal": "Bioresource technology", "year": "2026", "doi": "10.1016/j.biortech.2026.134261", "ptypes": [ "Journal Article" ], "first_author": "Wang J", "dirs": [ "D" ] }, "40145755": { "pmid": "40145755", "title": "Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering.", "abstract": "Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection and screening. To address these challenges, we developed a growth-coupled continuous directed evolution (GCCDE) approach, enabling automated and efficient enzyme engineering. By linking enzyme activity to bacterial growth and utilizing the MutaT7 system, GCCDE combines in vivo mutagenesis and high-throughput selection of superior enzyme variants in a single process. To validate this approach, we evolved the thermostable enzyme CelB from Pyrococcus furiosus to enhance its β-galactosidase activity at lower temperatures while maintaining thermal stability. CelB activity was coupled to the growth of E. coli, allowing variants with improved activity to utilize lactose more efficiently and promote faster growth in a minimal medium. Using a continuous culture system, we achieved automated high-throughput mutagenesis and simultaneous real-time selection of over 10⁹ variants per culture. Integrating in vitro and in vivo mutagenesis further increased genetic diversity, yielding CelB variants with significantly enhanced low-temperature activity compared to the wild type while preserving thermostability. DNA sequencing identified key mutations likely responsible for improved substrate binding and catalytic turnover. This GCCDE approach is broadly applicable for optimizing diverse enzymes, demonstrating the potential of automated continuous evolution for industrial and research applications. Enzyme engineering aims to develop enzymes with improved or novel traits, but traditional methods are slow and require repetitive manual steps. This study presents a faster, automated protein engineering approach. We utilized an in vivo mutagenesis technique, MutaT7 tools, to induce mutations in living bacteria and established a direct link between enzyme activity and bacterial growth. A continuous culture setup enables automated mutagenesis and growth-coupled selection of better-performing variants in real time. Bacteria with improved enzymes grew faster, selecting superior variants without manual intervention. Using this method, we engineered CelB with better performance at lower temperatures while maintaining thermal stability. By combining high-throughput mutagenesis and selection in a single process, this system bypasses iterative cycles of error-prone PCR, transformation, and screening. Our approach is adaptable to various enzymes, providing a faster and more efficient solution for enzyme engineering.", "journal": "Applied and environmental microbiology", "year": "2025", "doi": "10.1128/aem.02491-24", "ptypes": [ "Journal Article" ], "first_author": "Deng Y", "dirs": [ "D" ] }, "41756727": { "pmid": "41756727", "title": "Directed Evolution of T7 RNA Polymerase Minimizes dsRNA By-product and Enables High-Fidelity mRNA Synthesis for Demanding Therapeutic Applications.", "abstract": "T7 RNA polymerase (T7 RNAP) is the most widely used enzyme for synthesizing therapeutic mRNA. However, RNA transcribed by T7 RNAP often contains double-stranded RNA (dsRNA) by-products that trigger innate immune responses and complicate purification. Here, we report an engineered T7 RNAP variant, M30, which exhibits higher catalytic efficiency and reduced dsRNA by-product formation. M30 was developed through 4 rounds of directed evolution using an ultrahigh-throughput aptamer-based fluorescence-activated droplet sorting system. M30 displays a 10-fold increase in catalytic efficiency over wild-type T7 RNAP at 37 °C, along with markedly enhanced thermostability and approximately 10-fold lower production of dsRNA by-products. mRNAs synthesized with M30 achieve efficient protein expression in human cells and in mice, while eliciting reduced immunogenicity compared with mRNAs produced by wild-type T7 RNAP. Biophysical assays and structural analyses suggest that these improvements result from increased DNA template binding affinity and decreased RNA binding affinity. Together, these features make M30 a promising catalyst for high-quality therapeutic mRNA production.", "journal": "Research (Washington, D.C.)", "year": "2026", "doi": "10.34133/research.1172", "ptypes": [ "Journal Article" ], "first_author": "Qin W", "dirs": [ "D" ] }, "40668676": { "pmid": "40668676", "title": "Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay.", "abstract": "Poly(ethylene terephthalate) (PET) hydrolases, which depolymerize PET to its monomers, have gained attention for their potential to facilitate bio-industrial recycling of this waste plastic. Here, we present a protocol for screening large, random mutagenesis enzyme libraries simultaneously for enhanced activity, solubility, and stability. We outline steps for library construction, screening using plate-based split GFP and model substrate assays, and determination of enzyme thermostability. We then detail procedures for validation assays on PET substrates and characterization of final variants. For complete details on the use and execution of this protocol, please refer to Groseclose et al.1 and Groseclose et al.2.", "journal": "STAR protocols", "year": "2025", "doi": "10.1016/j.xpro.2025.103969", "ptypes": [ "Journal Article" ], "first_author": "Groseclose TM", "dirs": [ "D" ] }, "41676228": { "pmid": "41676228", "title": "Advancing NanoLuc Luciferase Stability beyond Directed Evolution and Rational Design through Expert-Guided Deep Learning.", "abstract": "Engineered luciferases have transformed biological imaging and sensing, yet optimizing NanoLuc luciferase (NLuc) remains challenging due to the inherent stability-activity trade-off and its limited sequence homology with characterized proteins. We report a hybrid approach that synergistically integrates deep learning with structure-guided rational design to develop enhanced NLuc variants that improve thermostability and thereby activity at elevated temperatures. By systematically analyzing libraries of engineered variants, we established that modifications to termini and loops distal from the catalytic center, combined with preservation of allosterically coupled networks, effectively increase thermal resilience while maintaining enzymatic function. Our optimized variantsnotably B.07 and B.09exhibit substantial thermostability enhancements (increased melting temperatures of 7.2 and 5.1 °C, respectively), leading to the sustained activity of a high-activity mutant at elevated temperatures. Molecular dynamics simulations and protein folding studies elucidate how these mutations favorably modulate conformational landscapes without perturbing the substrate binding architecture. Beyond providing a thermostabilized tool for bioluminescence applications, our integrated methodology presents a framework for engineering enzymes when traditional homology-based approaches fail and stability-activity constraints present formidable barriers to improvement.", "journal": "ACS catalysis", "year": "2026", "doi": "10.1021/acscatal.5c08789", "ptypes": [ "Journal Article" ], "first_author": "Gardiner S", "dirs": [ "D" ] }, "40862219": { "pmid": "40862219", "title": "Engineering PHL7 for improved poly(ethylene terephthalate) depolymerization via rational design and directed evolution.", "abstract": "Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has emerged as a promising approach for polyester recycling, and, to date, many natural and engineered PET hydrolase enzymes have been reported. For industrial use, PET hydrolases must achieve high depolymerization extent and exhibit excellent thermostability. Here, we engineered a natural PET hydrolase, Polyester Hydrolase Leipzig #7 (PHL7), through rational design and directed evolution using a high-throughput screening platform. Four new enzymes were engineered with enhanced properties compared with the parent enzyme, wild-type PHL7 (PHL7-WT), and other benchmark PET hydrolases, under the tested conditions. In bioreactors, the exemplary engineered enzyme, PHL7-Jemez, exhibited improved ability to depolymerize amorphous PET film compared with PHL7-WT at 2.9% and 20% substrate loadings, with 37% and 270% higher hydrolysis, respectively, after 48 h. This study develops several state-of-the-art PET hydrolases and demonstrates a directed evolution platform to engineer high-performance enzymes, which can accelerate enzyme discovery toward improved biocatalytic recycling.", "journal": "Chem catalysis", "year": "2025", "doi": "10.1016/j.checat.2025.101399", "ptypes": [ "Journal Article" ], "first_author": "Groseclose TM", "dirs": [ "D" ] }, "40611977": { "pmid": "40611977", "title": "Surface-Active Catalysts for Interfacial Gas-Liquid-Solid Reactions.", "abstract": "Multiphase reactions combining gas and liquid phases and a solid catalyst are widespread in the chemical industry. The reactions are typically affected by the low gas solubility in liquids and poor mass transfer from the gas phase to the liquid, especially for fast reactions, leading to much lower activity than the intrinsic catalytic activity. In practice, high pressure, temperature, and cosolvents are required to increase the gas solubility and boost the reaction rate. Gas-liquid-solid (G-L-S) microreactors based on particle-stabilized (Pickering) foams rather than conventional surfactant-stabilized foams can increase the contact between the gas and liquid phases, together with surface-active catalytic particles, and dramatically accelerate G-L-S reactions. Unlike surfactants, surface-active catalytic particles can be recycled and reused and reduce coalescence, Ostwald ripening, and aggregation by adsorbing selectively at the G-L interface, promoting stability. In this Account, we present first a taxonomy of microstructured G-L-(S) interfaces to build G-L-S microreactors (catalytic membrane contactors, microdroplets, micromarbles, microbubbles, and particle-stabilized bubbles/foams). Within this taxonomy, we provide a critical appraisal of surface-active catalytic particles to engineer particle-stabilized aqueous and oil foams. We address the fundamental thermodynamics and dynamics aspects of particle adsorption at the G-L interface and examine the foaming stabilization mechanisms. We further enumerate the possible interactions between particles and G-L interfaces and elucidate how the interfacial self-assembly of surface-active particles can discourage foam destabilization mechanisms. We also discuss strategies for the synthesis of surface-active particles, including surface modification of preformed hydrophilic particles, synthesis of organic-inorganic hybrids, coprecipitation, and bottom-up synthesis, including methods for depositing catalytic centers. Various types of particles capable of stabilizing foams are identified including silica particles modified with hydrophobic and hydrophilic chains, silica particles functionalized with oleophobic and oleophilic chains, biphenyl-bridged organosilica particles, and surface-active polymers. Finally, we highlight recent advances from our group, including catalytic oxidation, hydrogenation, and tandem reactions, facilitated by tailor-designed surface-active particles in aqueous/nonaqueous foam. The relationship between the structure, properties, and foaming performance of surface-active particles, along with their catalytic efficiency within foams, is elucidated. It is our hope that this Account will inspire innovative designs of surface-active particles with tailored properties for the advancement of industrially relevant multiphase reactions. Looking ahead, developing data-driven computational tools would be highly beneficial, allowing the in silico design of particles with tailored foaming, foam stability, and local G-L miscibility for defined G-L systems, thus precluding trial-and-error approaches. Parameters such as the three-phase contact angle of particles, the line tension, and the optimal particle size and shape to ensure gas regeneration could be modeled and implemented.", "journal": "Accounts of materials research", "year": "2025", "doi": "10.1021/accountsmr.5c00026", "ptypes": [ "Journal Article" ], "first_author": "Wang K", "dirs": [ "D" ] }, "41711056": { "pmid": "41711056", "title": "Foam Stability Mediated by Cellulose Nanocrystal-Anionic Surfactant Interactions.", "abstract": "The combination of cellulose nanocrystals (CNC) and anionic alkyl poly(ether) carboxylate surfactants offers a sustainable approach to modulating the interfacial and foaming properties of aqueous systems. This study investigated CNC-surfactant interactions in bulk and at the air-water interface using surface tension, interfacial rheology, foaming tests, particle size, zeta potential, and electron microscopy. CNC slightly increased the critical micelle concentration and reduced surfactant adsorption at the interface, indicating an interaction between the two components. Increasing the surfactant concentration improved CNC dispersion, forming smaller, more uniform aggregates. Although CNC addition slightly reduced foamability due to slower surfactant diffusion and increased surface tension, it significantly enhanced foam stability at low surfactant concentrations. This improvement was attributed to aggregated CNC forming percolated networks and reinforcing interfacial films. Dynamic surface tension and dilatational rheology confirmed an increased interfacial viscoelasticity, evidencing stronger interfacial layers. At high surfactant concentration, CNC was well dispersed, resulting in lower viscoelasticity and reduced foam stability, highlighting the importance of the CNC aggregation state. Overall, CNC-surfactant interactions enable tunable control over interfacial structure and foam stability, offering valuable insights for greener formulations in personal care, food, and household applications.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2026", "doi": "10.1021/acs.langmuir.5c06009", "ptypes": [ "Journal Article" ], "first_author": "Rodrigues PDC", "dirs": [ "D" ] }, "39779035": { "pmid": "39779035", "title": "Interactions between cellulose nanocrystals and conventional/gemini surfactants.", "abstract": "Research on the interaction between surfactants and cellulose nanocrystals (CNC) has mainly focused on the interaction between CNC and conventional surfactants, and there are no reported studies on the interaction between CNC and gemini surfactants. The interactions between CNC and conventional surfactant (tetradecyltrimethylammonium bromide, termed as TTAB), asymmetric gemini surfactant ([C14H29(CH3)2N+(CH2)6N+(CH3)2C6H13]Br (14-6-6)) or symmetric gemini surfactant ([C14H29(CH3)2N+(CH2)6N+(CH3)2C14H29]Br2 (14-6-14)) were examined. With increasing surfactant concentration, interaction of TTAB/CNC was described by three regions, i.e. electrostatic interaction, CNC induced micellization and dilution of free micelles. However, in the case of gemini surfactant/CNC, four binding regimes were observed, i.e. cooperative adsorption, CNC induced micellization, formation and dilution of free micelles. The behavior of 14-6-6/CNC was similar to 14-6-14/CNC where CNC promoted the partition of gemini surfactant to the air-water interface at high surfactant concentration, while it was inhibited at low surfactant concentration. At low CNC concentration, micellization induced by CNC and aggregation of surfactant/CNC complexes were absent. pH had a minimal impact on the binding process at low CNC concentration, but it affected the binding at higher CNC concentration. Additionally, the presence of electrolytes influenced the micellization process induced by CNC by reducing the electrostatic interactions.", "journal": "Carbohydrate polymers", "year": "2025", "doi": "10.1016/j.carbpol.2024.123132", "ptypes": [ "Journal Article" ], "first_author": "Cao G", "dirs": [ "D" ] }, "41466032": { "pmid": "41466032", "title": "Synthesis, characterization and application of new nanodemulsifier in oil industries.", "abstract": "The energy production industry is confronted with a multitude of obstacles, particularly within the field of oil production. One such challenge pertains to the formation and stability of oil emulsions. The water-in-oil (W/O) emulsion is stabilized by native crude oil emulsifiers, which migrate and concentrate at the water/oil interface. The water-in-oil (W/O) emulsion is stabilized by native crude oil emulsifiers, which migrate and concentrate at the water/oil interface and form a barrier preventing droplet coalescence. Asphaltenes, resins, waxes, and fine solid particles are natural emulsifiers that stabilize emulsion. Therefore, it is crucial to address the challenges inherent to oil production by employing efficacious techniques to treat emulsions at the earliest stages of oil extraction. This approach can effectively minimize supplementary production costs associated with the transportation and storage of crude oil. The chemical method of demulsification represents the most prevalent approach to emulsion resolution in oil fields. The unique properties of nanoparticles come from their size, surface area, and many other unique physicochemical properties. A large number of nanoparticles have been investigated in demulsification applications for enhanced oil recovery, either alone or in combination with surfactants. This work presents a discussion of the preparation of a new nanocomposite through the interaction of ZrO₂.B₂O₃ nanoparticles with surfactant cationic cetyltrimethylammonium bromide (CTAB) and evaluates its efficacy as a demulsifier for the separation of water-in-oil emulsions. The structure and morphology of the prepared ZrO₂.B₂O₃-[CTAB] were characterized by XRD, EDS, FESEM, TEM and FT-IR analytical methods. In addition, the effectiveness of the prepared nanocomposite as a demulsifier was tested using the bottle test method. In order to comprehend the mechanism of action of the prepared nanocomposite, the interfacial tension of the crude oil emulsion was measured in the presence and absence of a nanodemulsifier using the Nouy ring method. Additionally, the critical micelle concentration was calculated using the molar conductivity method to determine the minimum concentration required to initiate the aggregation of nanodemulsifier particles. In addition to the semi-experimental determination of the compatibility of the nano-demulsifier for application in the demulsification process, the equilibrium values of the hydrophilic-lipophilic balance were calculated and found to be equal to 9.8, which is deemed suitable for the destabilization of water-in-oil emulsions. Practical measurements demonstrated a gradual decrease in the IFT of the oil/water mixture from 24.22 to 7.56 mN/m in the presence of ZrO₂.B₂O₃-[CTAB]. Moreover, the experimental findings demonstrated that the nanocomposite exhibited optimal demulsification efficiency within a 90-min time frame, 60 °C, and at a concentration of 80 ppm was achieved at 72.4%. The study also investigated the effect of some factors on the demulsification efficiency and identified temperature, settling time, and dosage as influential factors in the process of demulsifying W/O emulsion. A comparative analysis was conducted between the effectiveness of the prepared nanocomposite and the commercial demulsifier (DRCI1286) utilized in Halfaya oil fields.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-31152-5", "ptypes": [ "Journal Article" ], "first_author": "Basheer NA", "dirs": [ "D" ] }, "41157132": { "pmid": "41157132", "title": "Stereoisomeric Effects of Diammoniumcyclohexane Counterions on the Self-Assembly of Amino Acid-Based Surfactants.", "abstract": "The impact of counterion structure, especially variations in constitutional and stereochemical isomers, on the properties and performance of AABSs remains under-explored. This study investigates how structural variations, particularly the stereochemistry of diammonium cyclohexane (DACH) counterions, influence the self-assembly behavior of AABSs. Four AABSs: undecanoyl-glycine, -L-alanine, -L-valine, and -L-leucine, were paired with six DACH counterions representing cis/trans isomers of 1,2-, 1,3-, and 1,4-DACH. Critical micelle concentrations (CMCs) were determined via conductimetry, and micellar sizes were measured using dynamic light scattering. The degree of counterion binding (β) was calculated to probe micelle stability, while geometry-optimized structures of the DACH isomers were obtained using density functional theory. Lastly, pH measurements were taken to probe the protonation of DACH counterions at their natural pH, where both the DACH counterion and AABS headgroups intrinsically behave as buffers. Results indicate that while surfactant hydrophobicity primarily dictates CMC in other AABS/DACH combinations, trans-1,3-DACH leads to consistently higher CMCs. This deviation likely arises from its structural conformation, which positions the amine groups an intermediate distance of ~4.4-4.5 Å apart, allowing a small fraction of divalently charged counterions to form strong electrostatic bridging pockets at the micelle interface. These interactions dominate over headgroup effects, leading to elevated and surfactant-independent CMC values. Regarding size and other unusual trends in the systems, cis- isomers formed slightly larger micelles, and trans-1,4-DACH induces abnormal aggregation in undecanoyl-glycine leading to temperature dependent gel formation. These findings highlight the significant influence of counterion structure on AABS behavior and support counterion design as a strategy for enhancing surfactant performance in sustainable applications.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules30204114", "ptypes": [ "Journal Article" ], "first_author": "Blanco SE", "dirs": [ "D" ] }, "39870255": { "pmid": "39870255", "title": "Effect of formulation composition on trastuzumab stability.", "abstract": "For monoclonal antibody drug products as for other biologics, while the innovator drug products first becomes commercially available, they are often followed by one or more biosimilar products. These biosimilars often differ from the innovator product, as well as from each other, in their formulation composition. However, the impact of the formulation composition on the stability of the active pharmaceutical ingredient subjected to different 'stresses' is still not understood. We have evaluated the effect of different formulations on structural stability and aggregation behavior of a monoclonal antibody, trastuzumab (both the drug substance and the final drug product), against three most common stresses encountered during production, storage, and formulation into a lyophilized product - freeze-thaw, freeze-drying, and agitation. Irrespective of the stabilizer used, the formulations exhibited good conformational stability against all three stresses. However, the freeze-drying process caused a significant increase in the number of soluble aggregates, but only in sucrose containing formulations. On the other hand, agitation in sorbitol containing formulation led to a significant increase in insoluble aggregates. This effect could also be attributed to the absence of surfactant in this formulation composition. The stabilizing effect of trehalose appeared to be independent of its concentration. Therefore, the effect of formulation composition is more pronounced for aggregation of trastuzumab than for its conformational stability. Our findings suggest that formulation design warrants consideration of both conformational stability and aggregation behavior of the active ingredient.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125275", "ptypes": [ "Journal Article" ], "first_author": "Ahmad A", "dirs": [ "D" ] }, "41481806": { "pmid": "41481806", "title": "Membrane-Mimetic Micelles Drive Structural Switching in Uperin 3.5.", "abstract": "Uperin 3.5, an antimicrobial and amyloidogenic peptide from frog skin secretions, may represent an important evolutionary link between host-defense peptides and functional amyloids. Its ability to switch between α-helical and β-sheet conformations in membrane-associated environments makes it a compelling model for understanding how amyloidogenic sequences can acquire physiological antimicrobial roles. Microsecond-scale molecular dynamics simulations were utilized to investigate the adsorption, conformational transitions, and self-assembly behavior of Uperin 3.5 near zwitterionic DPC (dodecylphosphocholine) micelles, which mimic eukaryotic membrane environments. The DPC micelle surface promoted rapid peptide adsorption and α-helix formation, with the helical content reaching ∼40% over the simulation trajectory. Upon saturation of the micelle surface with 6-7 helical peptides, further peptide influx led to β-sheet accumulation through self-assembly at the peptide-micelle interface. This underscores the importance of the peptide-to-surfactant ratio in determining peptide aggregation behavior. The simulations also revealed a mechanism by which Uperin 3.5 peptides adopt an antiparallel cross-α, fibril-like structure near membranes with antiparallel β-sheet dimers serving as intermediate species for further helix formation. Compared with anionic interfaces, peptides near zwitterionic DPC micelles preferentially remained at the surface, offering insights into mechanisms that may mitigate cell cytotoxicity.", "journal": "The journal of physical chemistry. B", "year": "2026", "doi": "10.1021/acs.jpcb.5c05659", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Banerjee S", "dirs": [ "D" ] }, "42870554": { "pmid": "42870554", "title": "Infection-Phase Biomolecular Coronas in Inhaled Nanomedicine.", "abstract": "An inhaled nanomedicine encounters two successive forms of remodeling: device-related changes during aerosol generation and biological conditioning after deposition. Respiratory infection adds a moving interface, as epithelial secretion, surfactant composition, plasma leakage, and neutrophil-derived material vary across airway compartments and over time. Pulmonary and disease-corona studies already establish that host environments influence nanoparticle identity. The next question is how this knowledge should change formulation selection and dosing for infected lungs. We propose an infection-phase framework based on measured, overlapping airway environments. It connects the post-aerosol particle to its surface-associated biomolecules, distinguishes this interface from aggregation and bulk mucus effects, and links analytical measurements to functional delivery. A staged research program begins with one compartment and two contrasting environments, then adds controlled particle-transfer experiments, candidate-component perturbation, and donor-level validation. Lipid nanoparticles, polymeric carriers, and nanozymes require different functional endpoints within this shared framework. The immediate translational output is a biorelevant performance stress test that identifies robust or environment-selective formulations and guides the next experiment.", "journal": "Advanced healthcare materials", "year": "2026", "doi": "10.1002/adhm.71853", "ptypes": [ "Journal Article" ], "first_author": "Wu Z", "dirs": [ "D" ] }, "42028833": { "pmid": "42028833", "title": "Predicting Agitation Stability of Monoclonal Antibodies during Developability Assessment.", "abstract": "Developability assessment facilitates the selection of antibody drug candidates with desirable pharmaceutical properties. However, it remains uncertain whether agitation-induced aggregation can be predicted from standard developability parameters. Here, we investigated whether key biophysical parameters predict agitation-induced aggregation of monoclonal antibodies (mAbs). To this end, we generated a benchmark data set by characterizing the aggregation upon agitation in the presence of an air-liquid interface of ten approved mAbs reformulated in a common surfactant-free buffer. The extent of aggregation varied substantially among mAbs and was primarily dependent on antibody identity. Flow imaging microscopy combined with machine learning revealed micrometre-sized aggregates with distinct morphologies, consistent with aggregation at air-liquid interfaces. Examination of thin liquid films and foams confirmed the presence of aggregates directly at the air-liquid interface and, therefore, the critical role of this interface for antibody aggregation during agitation. We then applied fluorescence-based, light scattering, and chromatographic techniques to determine standard developability parameters for each mAb, including apparent melting temperature (Tm), nonreversibility onset temperature (Tnr), aggregation onset temperature (Tagg), diffusion self-interaction parameter (kD), hydrophobic interaction chromatography retention time, and relative monomer yield after isothermal refolding from chemical denaturants. Notably, none of these parameters correlated with agitation-induced aggregation. Finally, we assessed the surface properties of the mAbs via drop shape analysis and found that the combination of surface pressure and elastic modulus yields a good correlation with the concentration of micrometre-sized aggregates formed due to agitation. Overall, these findings highlight limitations in predicting mAb interfacial stability using standard developability assays and underscore the importance of studying antibody behavior at interfaces.", "journal": "Molecular pharmaceutics", "year": "2026", "doi": "10.1021/acs.molpharmaceut.6c00092", "ptypes": [ "Journal Article" ], "first_author": "Cohrs M", "dirs": [ "D" ] }, "40545071": { "pmid": "40545071", "title": "Mitigation of marine microbial corrosion using natural products based on chitosan polymer.", "abstract": "A major cause of marine corrosion is sulfate-reducing bacteria (SRB). Traditional chemicals may pose an environmental threat to this sector. Therefore, this research aims to develop eco-friendly alternatives to biocides and corrosion inhibitors. This work successfully modified chitosan, a natural and biodegradable biopolymer, into cationic and non-ionic surfactants. H1NMR, FTIR, and elemental analysis characterized the modified compounds. Mixed SRB culture was isolated from rust samples collected from steel immersed in a coastal area in Qingdao, China. Various techniques were used to evaluate the biological activity and corrosion protection provided by the newly modified chitosan surfactants in both abiotic and biotic environments, and their surface parameters in an aqueous solution were studied. SEM was used to display the surface morphology of the carbon steel; corrosion products were analyzed by EDS; surface topography and pitting were visualized by AFM; moreover, TEM was used to investigate the biocidal effect on the bacterial cell wall. Based on surface and thermodynamic calculations, the two surfactants exhibited adsorption behavior at the interface first, followed by aggregation in the bulk of the solution. The findings indicate that the newly modified chitosan-based surfactant displays significant corrosion protection over carbon steel and high antimicrobial activity against the isolated bacteria.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.145429", "ptypes": [ "Journal Article" ], "first_author": "Kobisy AS", "dirs": [ "D" ] }, "40074961": { "pmid": "40074961", "title": "Characterizing the Complex Multi-Step Degradation Kinetics of Amphotericin B in a Microemulsified Drug Delivery System.", "abstract": "Amphotericin B (AmB), a potent amphiphilic drug with antifungal and antileishmanial properties, exhibits reduced nephrotoxicity when delivered via lipid-based systems like microemulsions (ME). However, the complexity of these multi-phasic systems challenges the use of simple schemes and models for describing AmB degradation. The aim of this study was to establish a degradation scheme and model for AmB within a ME, alongside a control micellar formulation. AmB degradation pathways and models in both lipidic and aqueous systems were evaluated based on prior research. Experimental investigations into interface degradation pathways were conducted using a micellar approach. High-Performance Liquid Chromatography (HPLC) was employed for AmB quantification. Oxidation emerges as the principal degradation pathway within micelles, dependent on surfactant-induced aggregation. Considering AmB's behavior in distinct media (lipidic, aqueous, and micellar), an empirical degradation scheme is proposed, translated into a complex multi-pathway mathematical model capable of describing experimental data on AmB degradation in ME under dark conditions. Aggregation and oxidation played significant roles, and kinetic constants were calculated for AmB in ME. The model presented here represents a significant step toward accurately describing the non-linear degradation of AmB in prospective liquid lipid-based dispersions, potentially advancing its market prospects.", "journal": "AAPS PharmSciTech", "year": "2025", "doi": "10.1208/s12249-025-03080-0", "ptypes": [ "Journal Article" ], "first_author": "Santos SRA", "dirs": [ "D" ] }, "41553867": { "pmid": "41553867", "title": "Impact of Molecular Features on the Interfacial Behaviors of Fluorine-Free Firefighting Foam Surfactants.", "abstract": "Finding fluorine-free firefighting formulations that are comparable to their fluorinated counterparts or perform even better against fires atop diverse fuels is a crucial need. To address this need, the fundamental relationships between surfactant molecular features and the interfacial properties that drive foam characteristics must be advanced. Molecular dynamics simulations enable a mechanistic understanding of the molecular phenomena driving surfactant interfacial interactions across a diverse set of fuels. In the present work, it has been shown that there are trade-offs among interfacial activity, surfactant-surfactant interactions, aggregation, and interfacial spreadability. In terms of specifically designing surfactants to retain interfacial stability against polar solvents and alcohols, the following appear to be crucial for enhancing surfactant interfacial performance: increasing the molecular size (up to at least 1200 g/mol, especially sizable hydrophobic groups), maximizing the number of hydrogen bond accepting sites in a small volume, and designing a hydrophilic tail to interact strongly with water up to the point of excluding polar fuel from hydrogen bonding at the interface. These mechanistic insights from simulations, the design of which was informed by previous experimental works, enable the research field to move forward with surfactant screening against a wide range of possible structures, mixtures, and fuels in future efforts to develop fluorine-free foams with increased performance and efficiency.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2026", "doi": "10.1021/acs.langmuir.5c05095", "ptypes": [ "Journal Article" ], "first_author": "Williamson KI", "dirs": [ "D" ] }, "40606552": { "pmid": "40606552", "title": "Chemical incompatibility between formation and injection water: implications for oil recovery in porous media.", "abstract": "Low-salinity water flooding is widely recognized as an effective enhanced oil recovery (EOR) method, primarily by altering wettability and reducing interfacial tension. However, chemical incompatibility between injected water and formation water may induce scale deposition, leading to pore blockage and injectivity impairment, thereby posing significant challenges to EOR efficiency. A better understanding of the interplay between chemical incompatibility and pore-scale oil-water interface dynamics is crucial for optimizing waterflooding performance, particularly in low-permeability reservoirs. This study integrates ion characterization, colloidal analysis, solubility product calculations, and microfluidic visualization to systematically evaluate the compatibility of formation and injected waters, while directly observing pore-scale fluid displacement processes. Results reveal that ionic composition analysis reveals significant incompatibility between the sulfate-rich injection water and calcium/barium-containing formation water, creating conditions favorable for mineral scaling. Subsequent examination of scaling dynamics demonstrates that incompatible fluid mixing initiates nanoparticle formation, which progresses through two distinct growth pathways: coalescence-driven crystal enlargement and aggregation-dominated cluster formation, ultimately leading to pore-throat obstruction. Microfluidic visualization shows residual oil persists primarily as interfacial films and pore-center clusters after initial waterflooding, with their spatial arrangement governed by salinity-dependent wettability alteration and capillary forces. The introduction of incompatible water further exacerbates fluid trapping through capillary valve effects-a capillary-driven resistance occurring when interfacial forces oppose fluid advancement at pore-throat junctions-creating stagnant zones that promote particle accumulation. Pressure monitoring during flooding experiments reveals characteristic response patterns: an initial pressure peak during waterflooding, followed by secondary pressure elevation due to scale deposition, and subsequent partial pressure reduction through surfactant-mediated interfacial tension reduction and wettability modification. A self-reinforcing cycle emerges, coupling ion incompatibility, capillary trapping, and precipitate growth, encapsulated in a colloid-capillary coupling framework. To disrupt this cycle, a synergistic chemical strategy combining surfactants and scale inhibitors is proposed, simultaneously enabling interface modification and nucleation suppression to enhance sweep efficiency. This integrated approach provides a mechanistic foundation for optimizing waterflooding in chemically complex reservoirs, achieving a balanced synergy between interfacial control and scale mitigation.", "journal": "Frontiers in chemistry", "year": "2025", "doi": "10.3389/fchem.2025.1621714", "ptypes": [ "Journal Article" ], "first_author": "Wang J", "dirs": [ "D" ] }, "42054515": { "pmid": "42054515", "title": "The Unusual Surface Behavior of Dendrimer Siloxane Surfactants with Superior Wetting Performance.", "abstract": "A homologous series of dendrimer tetrasiloxane (tris(trimethylsiloxy)silylpropyl) ammonium surfactants are synthesized and investigated for their aqueous surface tension, bulk phase behavior, and wetting behavior. Each surfactant homologue comprises two strongly surface-active components, a tetrasiloxane monoquat and a tetrasiloxane diquat, and the different homologues have different mono-to-diquat ratios. The two components appear to mix nonideally in the bulk solution as well as at the liquid-air interface, as evidenced by the formation at above a critical aggregation concentration of two coexisting association structures in the bulk liquid and two condensed surface states. While the lower surface tension state allows superspreading of water over a solid paraffin surface for all the homologue surfactant in the series, the maximum spreading area differs greatly for the different homologues. Surface tension measurements from this work also confirm a benchmark for the lower bound of aqueous surface tension enabled by the permethylated siloxane surfactant to be near 19.5 mN/m, which is still a significant gap from the lowest possible surface tension exhibited by neat permethylated siloxane oil (15.7 mN/m) and perfluorinated surfactant in aqueous solution.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2026", "doi": "10.1021/acs.langmuir.5c06547", "ptypes": [ "Journal Article" ], "first_author": "Liu Y", "dirs": [ "D" ] }, "41379247": { "pmid": "41379247", "title": "Investigation into the temperature resistance of saturated cardanol surfactants in crude oil-water systems: a molecular dynamics simulation.", "abstract": "Surfactant flooding represents one of the critical methods in chemical enhanced oil recovery (EOR) technology. However, under high-temperature and high-salinity conditions, the interfacial properties of surfactants are prone to alteration, leading to reduced interfacial activity. In this study, three distinct types of surfactants derived from saturated cardanol were selected: 8EO8POH (nonionic surfactant), 8EO8POC2SO3 (sulfonate surfactant), and 8EO8POSO3 (sulfate surfactant). A high-temperature (30 ~ 180 ℃) and high-salinity (NaCl + CaCl2 concentration of 1.0 mol/L) system model was constructed through molecular dynamics simulations to evaluate their thermal resistance. The results indicate that an increase in temperature enhances the aggregation of water molecules and crude oil components around the surfactant molecules, thereby strengthening weak interactions. Under these conditions, the balance between hydrophilic and lipophilic effects becomes the predominant factor determining the superior interfacial performance of the surfactants. Consequently, the order of heat resistance is as follows: 8EO8POC2SO3 > 8EO8POSO3 > 8EO8POH. In this study, Packmol was employed to construct the model, and Gromacs was used to perform molecular dynamics simulations under the GAFF force field. The simulated pressure was set to 1115.0 kPa. The temperatures were set at 303.15 K, 333.15 K, 363.15 K, 393.15 K, 423.15 K, and 453.15 K, respectively. The time step for all simulations was set to 2 fs. In the 1 ns and 15 ns NPT simulations, the Berendsen and Parrinello-Rahman methods were employed to maintain system pressure, and the temperature control was achieved through velocity-rescale. The LINCS algorithm was utilized to constrain molecular bond lengths. Short-range and long-range were used Lennard-Jones potential and Particle-Mesh Ewald (PME) summation method.", "journal": "Journal of molecular modeling", "year": "2025", "doi": "10.1007/s00894-025-06587-2", "ptypes": [ "Journal Article" ], "first_author": "Lu C", "dirs": [ "D" ] }, "39089129": { "pmid": "39089129", "title": "Intrinsic dynamics of emulsions: Experiments in microgravity on the International Space Station.", "abstract": "In order to understand the basic mechanisms affecting emulsion stability, the intrinsic dynamics of the drop population must be investigated. We hypothesize that transient ballistic motion can serve as a marker of interactions between drops. In 1G conditions, buoyancy-induced drop motion obscures these interactions. The microgravity condition onboard the International Space Station enable this investigation. We performed Diffusing Wave Spectroscopy (DWS) experiments in the ESA Soft Matter Dynamics (SMD) facility. We used Monte Carlo simulations of photon trajectory to support data analysis. The analysis framework was validated by ground-based characterizations of the initial drop size distribution (DSD) and the properties of the oil/water interface in the presence of surfactant. We characterized the drop size distribution and found to be bi-disperse. Drop dynamics shows transient ballistic features at early times, reaching a stationary regime of primarily diffusion-dominated motion. This suggests different ageing mechanisms: immediately after emulsification, the main mechanism is coalescence or aggregation between small drops. However at later times, ageing proceeds via coalescence or aggregation of small with large drops in some emulsions. Our results elucidate new processes relevant to emulsion stability with potential impact on industrial processes on Earth, as well as enabling technologies for space exploration.", "journal": "Journal of colloid and interface science", "year": "2025", "doi": "10.1016/j.jcis.2024.07.205", "ptypes": [ "Journal Article" ], "first_author": "Lorusso V", "dirs": [ "D" ] }, "39907695": { "pmid": "39907695", "title": "Enhancing surface/interface activity and wettability via trimeric surfactant-containing mixtures.", "abstract": "The aggregation behaviors of a trimeric surfactant (Citric-3C12) in combination with anionic-nonionic surfactants of varying hydrophobic tail lengths (9-EOS/12-EOS) at different molar fractions were explored by surface tension measurements, dynamic light scattering (DLS), ζ potential analysis, and Cryo-TEM. Surface activity parameters, the interaction parameter (β), and the interfacial and micellar composition were evaluated by both the ideal mixing model and the regular solution model, with the values of βσ and βm found to be negative. The combination of the trimeric cationic surfactant with anionic-nonionic surfactants exhibits a pronounced synergistic effect driven by electrostatic, hydrophobic and hydrogen bonding interactions. The surface tension and critical micelle concentration (CMC) of the mixed system are significantly lower than those of the individual surfactants. Above the CMC, the surfactants form vesicles, with vesicle size tending to decrease as charge neutralization occurred. Furthermore, the adsorption behaviors of the binary mixtures were examined through surface tension, interfacial tension, and contact angle measurements. The synergistic effect is also evident in the interface adsorption behavior, where the mixed system exhibits enhanced oil-water interface activity and superior wettability on the hydrophobic surfaces.", "journal": "Soft matter", "year": "2025", "doi": "10.1039/d4sm01184h", "ptypes": [ "Journal Article" ], "first_author": "Zhang C", "dirs": [ "D" ] }, "41684249": { "pmid": "41684249", "title": "Template-Free Microfluidic Fabrication of Water-in-Water Microcapsules for Controlled Release.", "abstract": "Template-free fabrication of water-in-water microcapsules in completely miscible aqueous one-phase systems (AOPS) remains a long-standing challenge due to the lack of a well-defined interface. Current microcapsule fabrication is typically achieved through immiscible two-phase systems, including oil-water systems and aqueous two-phase systems (ATPS). This study proposes a microfluidic strategy that enables complete template-free microcapsule formation entirely within an aqueous one-phase system. Local coacervation is directly induced at the mixing boundary by controlling streams containing oppositely charged polymers and surfactants, forming a continuous shell that encapsulates the inner volume effectively without the need for an immiscible template or subsequent core material removal. The resulting microcapsules, reaching diameters of several hundred micrometers, exhibited homogeneity and long-term stability in surfactant-rich environments without aggregation and disintegration. Under specific conditions, shell formation was reversible, allowing dissolution in alkaline environments while remaining intact for over 4 months without chemical perturbations. Particle encapsulation experiments demonstrated that this method effectively retains particles larger than 500 nm, while smaller solutes diffuse freely through the shell, indicating size-selective permeability. Unlike traditional aqueous two-phase strategies that rely on pre-existing interfaces, our method operates entirely in a completely miscible aqueous system, with shell formation originating from controlled localized coacervation at the mixing boundary. This simple, materially flexible and environmentally friendly strategy is potentially scalable and broadens possibilities for sustainable next-generation microcapsule design, enabling high-throughput controlled-release applications in aqueous formulations such as food, pharmaceutical, and personal care products.", "journal": "ACS applied materials & interfaces", "year": "2026", "doi": "10.1021/acsami.6c00242", "ptypes": [ "Journal Article" ], "first_author": "Li M", "dirs": [ "D" ] }, "41077160": { "pmid": "41077160", "title": "Effect of temperature on the aggregation of an Fc-fusion protein under agitation.", "abstract": "Mitigating protein aggregation remains a challenge in the development of biopharmaceuticals, and agitation is well known as a stress that can induce protein aggregation. However, the temperature dependence of agitation-induced aggregation is not well understood. In this study, the aggregation of an Fc-fusion protein under agitation stress was investigated at 5, 25, and 40 °C. Soluble and insoluble aggregates were quantified by size-exclusion liquid chromatography and flow imaging microscopy, respectively. Both the aggregation level and the aggregate clusters were temperature dependent. The threshold for the orbital shaking that induced protein aggregation was temperature independent. Although thermal stress at 40 °C increased the number of oligomers, it did not lead to a higher monomer loss in a subsequent agitation at 25 °C. The aggregation induced by agitation stress was suppressed by adding a surfactant or removing the vial headspace, indicating that the aggregation occurred via an interface-mediated pathway. Thus, the observed temperature dependence was attributed to the protein adsorption to the interface and the following interfacial unfolding and aggregation was affected by the temperature. The results emphasized the importance of temperature control during shipping to ensure the quality of drug products. Agitation stability studies at a controlled temperature also provide a deep understanding of the protein aggregation mechanism, which is important for formulation development.", "journal": "European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences", "year": "2025", "doi": "10.1016/j.ejps.2025.107320", "ptypes": [ "Journal Article" ], "first_author": "Wang Z", "dirs": [ "D" ] }, "42648675": { "pmid": "42648675", "title": "The intrinsically disordered C-terminus of AzPhaP phasin is required for emulsion stabilization.", "abstract": "Phasin proteins are attractive candidates for the development of bio-based emulsifiers because they naturally localize at the interface between hydrophobic polyhydroxyalkanoate granules and the bacterial cytoplasm. For most phasins, however, surfactant activity has been described mainly as a macroscopic property, with limited information on which structural elements contribute to emulsion stability after formation. We addressed this question using AzPhaP from Azotobacter sp. FA8 and a C-terminally truncated variant, AzPhaPΔC, lacking the final 27 residues predicted to be intrinsically disordered. The deletion had little effect on the overall secondary structure in solution, with both proteins exhibiting melting temperatures near 54 °C and partial structural recovery after heating to 90 °C. Likewise, surface activity was unaffected, as both proteins reduced surface tension to 43-45 mN m-1 and exhibited an operational critical aggregation concentration of approximately 2.0 mg/mL. The difference, however, emerged after emulsification. Full-length AzPhaP maintained droplet size between 24 and 72 h, whereas droplets stabilized by AzPhaPΔC increased from 2.27 ± 0.26 to 4.30 ± 1.49 μm. In a 21-day refrigerated benchmark assay, AzPhaP produced smaller droplets than bovine serum albumin on days 7 and 14, although Triton X-100 remained superior. These results distinguish initial surface-tension reduction from long-term emulsion stabilization and indicate that the disordered C-terminal region of AzPhaP is required for emulsion persistence after interface formation, uncoupling surface activity from droplet stabilization and providing insight into phasin interfacial function.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.154080", "ptypes": [ "Journal Article" ], "first_author": "Salgado RL", "dirs": [ "D" ] }, "39794155": { "pmid": "39794155", "title": "Boundary Lubrication with Adsorbed Anionic Surfactant Bilayers in Hard Water.", "abstract": "The adsorption behavior of an anionic surfactant, hydroxy alkane sulfonate with an alkyl chain length of 18 (C18HAS), from its hard water solution onto a mica surface and resulting lubrication properties were investigated. Because of the double chain-like chemical structure and aggregation behavior, C18HAS formed vesicles in hard water, which adsorbed onto a negatively charged mica surface via cation (Ca2+) bridging and then transformed into a bilayer film. The number of bilayers formed on the surface was evaluated by force curve measurements using an atomic force microscope (AFM), and the results showed a time-dependent increase of the number of adsorbed bilayers. Friction and lubrication properties were evaluated for the confined film of the C18HAS hard water solution between mica surfaces using the surface forces apparatus (SFA). When the two surfaces were brought into contact under load and sheared against each other, the lubricating film consisted of two adsorbed C18HAS bilayers whose friction coefficient μ was of the order of 10-3 or below. The detailed analysis of the friction features revealed that the slipping in the boundary film does not occur at the interface between the opposed headgroup region of the two adsorbed bilayers, which is the typical mechanism for the low friction of adsorbed phospholipid bilayers extensively studied in the literature. Instead, slipping occurs at the interface between opposed liquid-like alkyl chain tails within the adsorbed bilayers; the low friction coefficient comes from the existence of two slip planes in the boundary film.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.4c04155", "ptypes": [ "Journal Article" ], "first_author": "Toyoda A", "dirs": [ "D" ] }, "41685635": { "pmid": "41685635", "title": "Efficient Photocatalytic H2O2 Production in a Quasi-Homogeneous System Utilizing Conjugated Polymer Nanoparticles.", "abstract": "Conjugated polymers demonstrate potential for photocatalytic hydrogen peroxide (H2O2) generation; however, their inadequate aqueous dispersibility leads to interfacial mismatch and inconsistent hydrophilicity, thereby hindering the migration of charge carriers and ionic species at the water-photocatalyst interface. In this work, three conjugated polymers TAQ, TPPQ, and TMPQ, exhibiting aggregation-enhanced H2O2 production are designed and synthesized. Subsequently, their quasi-homogeneous photocatalytic systems are fabricated in aqueous media via the nanoprecipitation method assisted by an amphiphilic surfactant, facilitating effective H2O2 generation. Under an atmosphere devoid of sacrificial agents, the quasi-homogeneous systems of TAQ, TPPQ, and TMPQ exhibit H2O2 production rates 27-58 times higher than their powder forms. Notably, TAQ achieves a H2O2 production rate of 3915µmol g-1 h-1 and an apparent quantum yield of 24.6% at 400 nm, representing some of the highest values reported for single-junction polymer-based photocatalysts. These findings underscore the efficacy of quasi-homogeneous system design in markedly improving photocatalytic performance.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.202511175", "ptypes": [ "Journal Article" ], "first_author": "Tong Y", "dirs": [ "D" ] }, "41380624": { "pmid": "41380624", "title": "Effect of chain length on the structure of aqueous surfactin solutions: Molecular dynamics studies.", "abstract": "Surfactin, a highly effective biosurfactant synthesized by various strains of Bacillus subtilis, exhibits remarkable biological and physicochemical properties. Molecular dynamics simulations highlight the influence of hydrocarbon chain length on the micellar structure, aggregation behavior, hydrogen bonding patterns, and secondary structure of surfactin molecules. The findings reveal that the aggregates adopt an increasingly spherical shape as the hydrocarbon chain length increases. The aggregation number of surfactin molecules also increases with longer chain lengths, indicating enhanced clustering tendencies. In the peptide ring, the occurrence of γ-turn, identified by the intramolecular hydrogen bond between Leu2 - Val4 residues, decreases with increasing chain length. The β-turn, identified by hydrogen bond between Leu2-Asp5 does not exhibit a clear trend, though the maximum occurrence probability is observed in systems with surfactin having longer alkyl tail. The surface tension of surfactin solutions increases with chain length due to lower coverage of the surface in case of surfactins with longer tails due to the tilted orientation of their hydrophobic tails, which limits efficient packing.", "journal": "Journal of molecular graphics & modelling", "year": "2026", "doi": "10.1016/j.jmgm.2025.109252", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Patro L", "dirs": [ "D" ] }, "42437524": { "pmid": "42437524", "title": "Self-Assembled Wheat Gluten Peptide Nanoparticles as Dual-Functional Stabilizers for the Protection of Omega-3 Polyunsaturated Fatty Acids.", "abstract": "Omega-3 polyunsaturated fatty acids are highly susceptible to oxidation, limiting the shelf life and sensory quality of omega-3-fortified foods. Herein, wheat gluten peptide nanoparticles (WGPNs) with coupled emulsifying and antioxidant functions were produced by sequential pepsin-trypsin hydrolysis. LC-MS/MS showed a peptide pool (1-4 kDa) enriched in Gln/Pro, featuring hydrophobic motifs and C-terminal Lys/Arg residues that confer surfactant-like behavior. WGPNs formed spontaneously in aqueous media, mainly driven by hydrophobic association and hydrogen bonding, with concentration-dependent β-sheet formation and a critical aggregation concentration of ∼0.17 mg/mL. Above this threshold, WGPNs exhibited enhanced radical-scavenging activity and colloidal stability across a wide ionic-strength range. Interfacial measurements demonstrated rapid adsorption of WGPNs at the oil-water interface, reduced interfacial tension, and formation of a stable interfacial layer. Consequently, WGPNs effectively stabilized fish oil emulsions and markedly retarded lipid oxidation. This work establishes WGPNs as a clean-label, dual-function platform for protecting oxidation-sensitive lipids in food systems.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.6c01720", "ptypes": [ "Journal Article" ], "first_author": "Feng G", "dirs": [ "D" ] }, "40834101": { "pmid": "40834101", "title": "Ultrahigh-concentration biologic therapeutics enabled by spray drying with a glassy surfactant excipient.", "abstract": "Biopharmaceuticals such as peptides and antibodies have become critical to health care. Despite their exceptional potency and specificity, biopharmaceuticals are prone to aggregation, which can limit efficacy. These therapies therefore often require low-concentration formulations as well as cold storage to maintain stability; however, high doses are required to treat many diseases. Most approved protein drug products are administered intravenously, imposing excessive burdens on patients. New approaches are needed to formulate proteins at high concentrations to enable less burdensome subcutaneous injection, preferably with an autoinjector that can be used directly by patients. To address this challenge, we report a subcutaneously injectable protein delivery platform composed of spray-dried protein microparticles suspended in a nonsolvent liquid carrier. These microparticles contain only biopharmaceuticals and a high-glass transition temperature polyacrylamide-derived copolymer excipient that affords key benefits over traditional excipients. First, the excipient improved stabilization of biopharmaceuticals through the spray-drying process, and second, it improved morphology and properties of the spray-dried particles, enhancing suspension injectability. We demonstrated with albumin, human immunoglobulin G, and an anti-COVID monoclonal antibody (IDBiologics) that this technology enables ultrahigh-concentration protein formulations (exceeding 500 milligrams per milliliter) that are injectable through standard needles with clinically relevant injection forces. In addition, experiments with two clinically relevant antibody drugs show that these ultrahigh-concentration formulations reduce required injection volumes without altering pharmacokinetics or efficacy in mice. This approach could nearly triple the number of commercial protein drugs amenable to subcutaneous administration, improving access to these critical biopharmaceuticals.", "journal": "Science translational medicine", "year": "2025", "doi": "10.1126/scitranslmed.adv6427", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, U.S. Gov't, Non-P.H.S.", "Research Support, N.I.H., Extramural" ], "first_author": "Jons CK", "dirs": [ "D" ] }, "41507198": { "pmid": "41507198", "title": "Anti-aggregation self-assembled monolayers enable high-performance and scalable perovskite solar cells.", "abstract": "Stable self-assembled monolayers (SAMs), such as (2-(9H-carbazol-9-yl)) ethylphosphonic acid (2PACz), are crucial for reducing interfacial energy loss in high-performance perovskite solar cells (PSCs). However, the inherent aggregation tendency of SAMs at the buried interface hinders the device's performance. Here, we propose a surfactant-assisted strategy to inhibit the aggregation of 2PACz by functionalizing cetyltrimethylammonium bromide (CTAB). Capitalizing on its distinctive electrostatic potential distribution, CTAB engages in non-bonding interactions with 2PACz. Theoretical and experimental characterizations prove that this promotes the uniform dispersion and anchoring of 2PACz on the substrate, leading to the SAM formation with high surface potential and excellent coverage. Moreover, the perovskite films with CTAB-modified SAM exhibit enhanced crystallinity with reduced trap state density and improved hole extraction efficiency. Consequently, the PSCs with a p-i-n architecture achieve a power conversion efficiency (PCE) of 26.20% (0.072 cm²). Scaled-up modules attain a PCE of 22.34% (22.96 cm²), confirming the scalability. Additionally, anti-aggregation-SAMs-integrated devices demonstrate stability, maintaining over 80% and 90% of their initial PCEs after tracking at maximum power point for 800 h and ageing at 65 °C for 1000 h, respectively.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-025-68207-0", "ptypes": [ "Journal Article" ], "first_author": "Du K", "dirs": [ "D" ] }, "41830852": { "pmid": "41830852", "title": "Interfacial modulation of cochleate assemblyviacationic surfactant enables enhanced encapsulation of water-soluble drugs.", "abstract": "Cochleates offer a structurally robust lipid-based platform for drug delivery but remain limited by structural heterogeneity, aggregation, and poor encapsulation of water-soluble therapeutics due to weak bilayer partitioning. Here, we introduce a cationic surfactant-mediated interfacial engineering strategy to modulate cochleate assembly and enhance encapsulation of water-soluble drugs. Using cetyltrimethylammonium bromide (CTAB) as a model cationic surfactant and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as the anionic lipid matrix, we demonstrate controlled modulation of bilayer electrostatics and lamellar organization during Ca²⁺-driven cochleate formation. Multimodal characterization using cryo-TEM, FE-SEM, SAXS, FTIR, and DSC indicates that CTAB incorporation occurs in a manner consistent with interfacial bilayer association, without disrupting Ca²⁺-bridged lamellar architecture, preserving the fundamental lamellar repeat spacing while systematically modulating lamellar coherence and colloidal stability. Using irinotecan as a mechanistically stringent model drug with limited intrinsic bilayer affinity, based on its amphiphilic, weakly basic nature and poor loading in unmodified cochleates, CTAB-engineered cochleates exhibit a marked increase in encapsulation efficiency and drug loading, together with improved structural integrity and sustained release under simulated gastrointestinal conditions. These enhancements arise from interactions that favor drug association at the bilayer-aqueous interface rather than deep hydrophobic partitioning, while maintaining cochleate structural organization. This work establishes interfacial electrostatic modulation as an important design principle for cochleate engineering, enabling efficient encapsulation and controlled release of water-soluble therapeutics without compromising structural integrity.", "journal": "Colloids and surfaces. B, Biointerfaces", "year": "2026", "doi": "10.1016/j.colsurfb.2026.115601", "ptypes": [ "Journal Article" ], "first_author": "Thakur N", "dirs": [ "D" ] }, "41626457": { "pmid": "41626457", "title": "Effect of Hydrocarbon Chain Length on the Performance of Cationic Silicone Surfactants.", "abstract": "Three quaternary ammonium cationic silicone surfactants with different carbon-hydrogen chain lengths are synthesized by a two-step process. The molecular structure of the surfactants has been researched by infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry. Subsequently, the effects of carbon-hydrogen chain length on the surface activity, aggregation behavior, and micellization process of surfactants were investigated through methods such as surface tension, electrical conductivity, and dynamic light scattering, providing theoretical guidance for the application of surfactants. It was found that the three silicone surfactants have excellent surface activity, with their critical micelle concentration (CMC) being 9.83, 6.81, and 4.56 mmol·L-1, respectively, while the γCMC (surface tension at CMC) being as low as 20.95, 20.73, and 20.17 mN·m-1, respectively. As the carbon-hydrogen chain increases, the area occupied by a single silicone surfactant molecule at the gas-liquid interface (A S) increases, and the CMC values decrease significantly. The size distribution of the three silicone surfactants is unimodal, and they can self-assemble into nonuniform sized spheroidal aggregates. Moreover, the values of ΔG m 0 range from -25.85 to -28.45 kJ·mol-1, indicating that the micellization process of SiCHs is spontaneous. Based on the values of ΔH m 0 and -TΔS m 0, we can see that the increase in the carbon-hydrogen chain gradually causes the micellization process to shift from entropy-driven to enthalpy-driven.", "journal": "ACS omega", "year": "2026", "doi": "10.1021/acsomega.5c11169", "ptypes": [ "Journal Article" ], "first_author": "Zhu X", "dirs": [ "D" ] }, "41696523": { "pmid": "41696523", "title": "Synthesis, characterization and surface properties of an organosilicon surfactant.", "abstract": "In order to explore alternative substances for traditional fluorocarbon surfactants while maintaining high surface activity, this study successfully designed and synthesized an organosilicon surfactant (SiCH) containing a hydroxyl quaternary ammonium salt. Its molecular structure was confirmed by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). This surfactant exhibits excellent thermal stability, with an initial decomposition temperature of 167 °C and minimal residue at high temperatures. The critical micelle concentration (CMC) and the surface tension (γ CMC) at the CMC of SiCH are 9.10 mmol/L and 22.81 mN/m, respectively. The organosilicon surfactant-sodium hexanesulfonate (SiCH-SHS) exhibits significant synergistic effect, reducing the CMC and γ CMC to 6.88 mmol/L and 21.03 mN/m respectively. The area occupied by a single surfactant molecule at the air/water interface (A S) of the SiCH-SHS is 1.24 nm2, and the pC20 is 2.91. The size distribution of SiCH is unimodal, and they can self-assemble into non-uniform sized spheroidal aggregates (122-295 nm). Moreover, the thermodynamic process of micelle formation of the surfactant was studied through conductivity tests conducted at different temperatures (298.15 - 303.15 K). Thermodynamic analysis indicates that micellization is a spontaneous ( Δ G m 0 <0), entropy-driven process.", "journal": "Frontiers in chemistry", "year": "2026", "doi": "10.3389/fchem.2026.1744604", "ptypes": [ "Journal Article" ], "first_author": "Zhu X", "dirs": [ "D" ] }, "42626992": { "pmid": "42626992", "title": "Bridging the Gap: Heterogeneous Micro-Confined Palladium Catalysts Surpassing Homogeneous Systems for C-N Cross-Coupling in Water.", "abstract": "Homogeneous systems, while highly active, are inherently fragile and prone to catalyst deactivation through metal aggregation, ligand dissociation, and irreversible formation of catalytically inactive palladium black, thereby limiting recyclability during cross-coupling. In contrast, supported heterogeneous catalysts often sacrifice reactivity due to restricted site accessibility, perturbed electronic environments, and uncontrolled metal leaching. Overcoming this dichotomy requires control over palladium speciation under turnover conditions, an objective that remains unmet for C─N cross-coupling. Herein, we report a heterogeneous palladium catalyst embedded in micro-confined spaces to resolve this long-standing limitation and establish a new paradigm for C─N cross-coupling. A hydrophobic confined environment provided by a hyper-crosslinked polymer integrated with t-BuXPhos, and the system selectively stabilises catalytically competent low-valent Pd while suppressing aggregation, ligand dissociation, and metal leaching. Interestingly, this confinement preserves substrate accessibility via dynamic exchange at the material-water interface with the synergy of a non-ionic surfactant CNSL-1000-M, enabling C─N coupling at parts-per-million (ppm) palladium loadings. Our catalyst operating at ≤500 ppm exhibits initial rates up to 31-fold higher than its homogeneous analogue. We have demonstrated the recyclability over five successive cycles, affording a cumulative turnover number (TON) of 2.2 × 104, the highest value reported to date for heterogeneous C─N cross-coupling.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.74969", "ptypes": [ "Journal Article" ], "first_author": "Yadav R", "dirs": [ "D" ] }, "41905308": { "pmid": "41905308", "title": "Disruption of pulmonary surfactant function under oxidative stress: structural and biophysical insights.", "abstract": "Pulmonary surfactant forms surface-active films at the air-liquid interface which are essential for alveolar stability but are highly susceptible to oxidative damage under inflammatory conditions. The reactive species hypochlorous acid (HClO/ClO-), generated by activated neutrophils, is a potent oxidant that may compromise surfactant integrity; however, its molecular and functional impact remains incompletely understood. In this study, the effects of oxidative stress induced by HClO/ClO- on the biophysical and structural properties of a fully native porcine surfactant were investigated using Langmuir balance, captive bubble surfactometry, differential scanning calorimetry, FTIR-ATR spectroscopy, and epifluorescence microscopy. Oxidation induced concentration- and time-dependent inhibition of surfactant adsorption and interfacial film stability. Langmuir balance experiments revealed selective disruption of films containing unsaturated phospholipids or native mixtures, while saturated monolayers remained largely unaffected. FTIR-ATR analysis showed perturbations in lipid headgroup regions and protein secondary structure rearrangements towards β-sheet-rich aggregates, consistent with oxidative unfolding and aggregation. These molecular changes compromise protein-lipid interactions, delaying adsorption and respreading at the air-liquid interface. Epifluorescence microscopy further revealed disruption of lateral lipid organization and altered formation of three-dimensional surfactant reservoirs enriched in disordered components. Functional assays confirmed impaired reduction of surface tension during quasi-static cycling, partially alleviated under dynamic cycling, suggesting exclusion or depuration of oxidized components from the interface. Together, these findings demonstrate that neutrophil-derived oxidants progressively compromise pulmonary surfactant by targeting lipid-protein cooperativity, leading to early interfacial dysfunction followed by structural reorganization. This mechanistic insight advances understanding of oxidative surfactant inactivation and supports the development of antioxidant strategies or oxidant-resistant surfactant formulations as potential therapeutic interventions for inflammatory lung injury.", "journal": "Journal of colloid and interface science", "year": "2026", "doi": "10.1016/j.jcis.2026.140353", "ptypes": [ "Journal Article" ], "first_author": "Cejas JDP", "dirs": [ "D" ] }, "39800502": { "pmid": "39800502", "title": "Digital recombinase polymerase amplification chip based on asymmetric contact angle composite interface.", "abstract": "Digital recombinase polymerase amplification (dRPA) is an effective tool for the absolute quantification of nucleic acids and the detection of rare mutations. Due to the high viscosity or other physical properties of the reagent, this can compromise the accuracy and reproducibility of detection results, which limits the broader adoption and practical application of this technology. In this study, we developed an asymmetric contact angle digital isothermal detection (ACA-DID) chip and optimized the ACA-DID chip structure to achieve rapid digital recombinase polymerase amplification. We designed with a pressure-driven and highly asymmetric contact angle composite interface to enable robust digital RPA. The addition of surfactants to the PDMS creates an asymmetric contact angle between the upper and lower surfaces of the fluid channel, improving reagent flow and facilitating entry into microwells. This design addresses the challenges posed by high-viscosity reagents, which typically complicate effective digital discretization and lead to fluorescence signal aggregation. By diluting specific components of the RPA reagent, we improved the uniformity of amplification and effectively reduced signal aggregation. The hydrophobic surface of results strong adsorption to biological macromolecules, such as nucleic acids and proteins, which will decrease the efficiency of dRPA amplification. To achieve efficient amplification of reagents in the microchamber, this work uses a surface modification strategy of PDMS doped surfactant, which eliminates the issue of PDMS materials hindering dRPA amplification efficiency. The ACA-DID chip demonstrated excellent analytical accuracy in the quantification of African swine fever DNA samples, highlighting its potential to enhance the accessibility and effectiveness of dRPA technology. This innovation promises to overcome key limitations in current digital RPA amplification platforms, driving broader adoption and application, especially in cost-sensitive or resource-limited settings.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2024.343543", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Zhang Z", "dirs": [ "D" ] }, "40572555": { "pmid": "40572555", "title": "Counterion-Mediated Assembly of Fluorocarbon-Hydrocarbon Surfactant Mixtures at the Air-Liquid Interface: A Molecular Dynamics Study.", "abstract": "This study employs molecular dynamics simulations to investigate counterion effects (Li+, Na+, K+) on the interfacial aggregation of mixed short-chain fluorocarbon, Perfluorohexanoic acid (PFHXA), and Sodium dodecyl sulfate (SDS) surfactants. Motivated by the need for greener surfactant alternatives and a fundamental understanding of molecular interactions governing their behavior, we demonstrate that counterion hydration radius critically modulates system organization. K+ ions induce superior monolayer condensation and interfacial performance compared to Li+ and Na+ counterparts, as evidenced by threefold analysis: (1) RMSD/MSD-confirmed equilibrium attainment ensures data reliability; (2) 1D/2D density profiles and surface tension measurements reveal K+-enhanced packing density (lower solvent-accessible surface area versus Na+ and Li+ systems); (3) Electrostatic potential analysis identifies synergistic complementarity between SDS's hydrophobic stabilization via dodecyl chain interactions and PFHXA's charge uniformity, optimizing molecular-level charge screening. Radial distribution function analysis demonstrates K+'s stronger affinity for SDS head groups, with preferential sulfate coordination reducing surfactant-water hydration interactions. This behavior correlates with hydrogen-bond population reduction, attributed to SDS groups functioning as multidentate ligands-their tetrahedral oxygen arrangement facilitates cooperative hydrogen-bond networks, while counterion-specific charge screening competitively modulates bond formation. The resultant interfacial restructuring enables ordered molecular arrangements with lower system curvature than those observed in Li+ and Na+-containing systems. These findings elucidate counterion-mediated interfacial modulation mechanisms and establish K+ as an optimal candidate for enhancing PFHXA/SDS mixture performance through hydration-radius screening. The work provides molecular-level guidelines for designing eco-friendly surfactant systems with tailored interfacial properties.", "journal": "Molecules (Basel, Switzerland)", "year": "2025", "doi": "10.3390/molecules30122592", "ptypes": [ "Journal Article" ], "first_author": "Quan X", "dirs": [ "D" ] }, "39927480": { "pmid": "39927480", "title": "Surfactant-Free Emulsion Polymerization of Styrene in Ethanol-Water Mixtures: The Role of Mesostructures in the Formation of Polystyrene.", "abstract": "Although the synthesis of monodisperse polystyrene nanoparticles (MPSPs) in surfactant-free systems has been widely investigated, the role of mesostructures in forming MPSPs is still unclear. Herein, the styrene/ethanol-water (St/EtOH-H2O) ternary system with certain mesostructures was employed as a model to investigate the correlation between the mesostructure of systems and the properties of polystyrene products. The mesostructures of the ternary system, including styrene droplets, a sponge-like structure, and water droplets, were investigated by transmission electron microscope (TEM) with negative staining, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Classical molecular dynamics (MD) simulations showed the spontaneous aggregation of ethanol at the styrene-water interface, which may be one of the factors stabilizing the mesostructures. We demonstrated that the formation of MPSPs can only occur in the systems containing styrene droplets. Cooling the reactants of styrene droplet systems in the early stages of polymerization resulted in incomplete polystyrene nanospheres (PSNSs), indicating that the formation of MPSPs originated from the interface of styrene droplets. Subsequently, the MPSPs were gradually formed through self-templating polymerization. This study offers valuable insights into the preparation and understanding of the formation process of polymer nanoparticles in other similar surfactant-free systems.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.5c00080", "ptypes": [ "Journal Article" ], "first_author": "Zhang A", "dirs": [ "D" ] }, "40799195": { "pmid": "40799195", "title": "Characterisation of potential interactions between urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and whey protein isolate.", "abstract": "Urea fertilizer augmented with the urease inhibitor (UI) N-(n-butyl)-thiophosphoric triamide (NBPT), is used to stabilize nitrogen and reduce greenhouse gas emissions. Although studies have shown that NBPT does not enter milk via cattle grazing on NBPT-treated fields, questions remain about its potential to impact milk components were it inadvertently to enter the dairy supply chain. The aim of this study was to examine possible effects of NBPT on the chemistry and functionality of whey protein isolate (WPI). NBPT raised the peak denaturation temperature of WPI (p < 0.05) and resulted in increased hydrogen bonding and β-sheet formation. NBPT reduced the viscosity of WPI and caused faster gelation. NBPT had a competitive adsorption advantage at the air-water interface (p < 0.05) compared to WPI. Microscopy imaging of WPI-NBPT gels revealed increased protein aggregation. Overall, NBPT modified the structure and functionality of WPI, primarily through non-covalent interactions.", "journal": "Food chemistry: X", "year": "2025", "doi": "10.1016/j.fochx.2025.102800", "ptypes": [ "Journal Article" ], "first_author": "Byrne MP", "dirs": [ "D" ] }, "40160740": { "pmid": "40160740", "title": "Molecular Dynamics Simulation of the Effects of Anionic-Nonionic Surfactants on Interfacial Properties of the Oil-Water Interface.", "abstract": "Surfactant oil drive is a crucially enhanced oil recovery method that improves oil recovery rates. The aggregation behavior of surfactant molecules at the oil-water interface significantly influences oil repulsion. In this study, a molecular dynamics simulation is used to investigate this repellent behavior of single and binary surfactants of alkanolamides (6501) and fatty alcohol polyoxyethylene ether sodium sulfate (AES). The oil-water interface is characterized by density distribution, interfacial thickness, radial distribution function, interfacial tension, and interfacial generation energy. The results reveal that the dodecanolamide surfactant (126501) and AES effectively reduce interfacial tension. In the binary 126501/AES system, the interfacial film thickness increases to 18.08 Å, and the diffusion coefficient increases to 0.186 Å2/ps. The radial distribution function shows that oil molecules are located 4.2 Å from the anionic head of AES, which weakens the intermolecular forces within the oil layer. In the 126501/AES system, the interfacial energy of -96.12 kJ/mol indicates a stable interface. Moreover, both the 126501/AES and tetradecanolamide surfactant (146501)/AES systems exhibit excellent resistance to metal ions. The molecular-level mechanism provides useful guidance for designing the surfactant systems for enhanced oil recovery.", "journal": "ACS omega", "year": "2025", "doi": "10.1021/acsomega.4c11148", "ptypes": [ "Journal Article" ], "first_author": "Chen Z", "dirs": [ "D" ] }, "41179157": { "pmid": "41179157", "title": "Enhanced Viscosity Reduction of Heavy Oil by a Synthesized Extended Anionic Surfactant: Mechanistic Exploration and Performance Evaluation.", "abstract": "The exploitation and transportation of heavy oil are severely hindered by its high viscosity. In this study, an extended anionic surfactant PE-54 was synthesized via sulfuric acid esterification to improve the flowability of heavy oil. The molecular structure of the surfactant (PE-54) was confirmed by Fourier transform infrared, 1H nuclear magnetic resonance (NMR) and 13C NMR, elemental analysis, and gel permeation chromatography. The critical micelle concentration of PE-54 was determined to be 1.02 mmol/L. The effects of surfactant dosage, oil-water ratio, and salinity on viscosity reduction were systematically investigated. Under optimal conditions (salinity of 10,023 mg/L, temperature of 50 °C, oil/water ratio of 6:4, and dosage of 1500 mg/L, the viscosity reduction rate reached 94%). The viscosity reduction mechanism was further elucidated through particle size analysis, Zeta potential measurements, interfacial tension tests, rheological characterization, and molecular dynamics simulations (MD). PE-54 molecules rapidly adsorbed at the oil-water interface, competitively interacting with asphaltenes and resins. The surfactant formed a stable interfacial configuration, hindering the aggregation of heavy components and enhancing the interfacial stability. These synergistic effects effectively improved emulsion stability and reduced viscosity, providing theoretical and practical guidance for the development of heavy oil viscosity reduction technologies.", "journal": "ACS omega", "year": "2025", "doi": "10.1021/acsomega.5c03136", "ptypes": [ "Journal Article" ], "first_author": "Wu H", "dirs": [ "D" ] }, "40006191": { "pmid": "40006191", "title": "Impact of Surfactants on Silk Fibroin Self-Assembly at the Air-Water Interface.", "abstract": "Silk fibroin (SF)-based materials attract significant interest because of their biocompability and great diversity of possible morphologies. One of the approaches to obtain SF materials is the use of an air-water or oil-water interface as a template for protein self-assembly. Surfactants can change the surface properties of adsorbed SF layers by promoting or preventing the formation of SF fiber networks. This study focuses on the influence of two typical ionic surfactants, cationic cetyltrimethylammonium bromide (CTAB) and anionic sodium dodecyl sulfate (SDS), on the dynamic properties of SF layers adsorbed at the air-water interface. The dynamic surface elasticity, surface tension, ellipsometric angle Δ, and the film thickness were measured as a function of the surface age and surfactant concentration. The morphology of the layers was evaluated by atomic force microscopy (AFM). For the adsorption layers of globular proteins, the main effect of the surfactants consists in the protein unfolding at high concentrations and in a decrease in the electrostatic adsorption barrier. In the case of SF layers, CTAB and SDS strongly influence the protein aggregation at the air-water interface. Regardless of the sign of the surfactant charge, its addition to SF solutions results in a decrease in the surface elasticity and the destruction of the ordered structure of protein fibers at concentrations higher than 1 × 10-4 M. With the further increase in the surfactant concentration, the thread-like aggregates disappear, the packing of thin fibers becomes less tight, a uniform layer disintegrates into separate islands, and finally, the protein is displaced from the interface.", "journal": "Polymers", "year": "2025", "doi": "10.3390/polym17040529", "ptypes": [ "Journal Article" ], "first_author": "Milyaeva OY", "dirs": [ "D" ] }, "40263889": { "pmid": "40263889", "title": "Natural Polyphenol-Assisted Dispersing Liquid Metal/Carbon Composite Electrode for the Superior Interface in Carbon-Based Perovskite Solar Cells.", "abstract": "Carbon-based perovskite solar cells (PSCs) present lower power conversion efficiency (PCE) compared with that of metal-based PSCs. One of the main reasons is the limited conductivity of carbon materials. Liquid metals (LMs) with good fluidity and excellent conductivity can be applied as superior electrode materials for a broad range of applications. Herein, LMs are combined with a carbon slurry to improve their conductivity and enhance interfacial contact for high photovoltaic performance of carbon-based PSCs. In order to well disperse LMs and avoid their aggregation, three natural polyphenols, namely, gallic acid (GA), tannic acid (TA), and tea polyphenols (TP), with different molecular structures are adopted to disperse Gallium nanoparticles (Ga NPs) and form an encapsulation layer. As a novel surfactant, the TP molecule with moderate numbers of hydroxyl groups and steric hindrance presents the best property to disperse Ga NPs among these natural polyphenols. It can tightly bind with Ga3+ to form a core-shell structure on the surface of Ga NPs. Its durable outer shell, around 11 nm, enables TP@Ga NPs to have superior structural stability, effectively inhibiting the invasion of water and oxygen, even after 10 days. With an optimal mass ratio of TP@Ga NPs to the carbon slurry (1:40), C-PSCs based on the TP@Ga40/C electrode achieve a champion PCE of 16.58%, significantly higher than that of the control devices (13.27%). In addition, the long-term stability of C-PSCs has also been increased, and after being stored at room temperature and a relative humidity of 35% for 35 days, the PCE can remain above 85% of the initial PCE. This work proposes a new method to well disperse LMs by TP surfactants and explores its promising application on improving the efficiency and stability of carbon-based PSCs.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.5c00860", "ptypes": [ "Journal Article" ], "first_author": "Lu D", "dirs": [ "D" ] }, "39749771": { "pmid": "39749771", "title": "Adsorption kinetics and solubilisation of ciprofloxacin in quaternary ammonium-based surface-active compounds: experimental and computational study.", "abstract": "The adsorption and aggregation of amphiphiles at different solvent interfaces are of great scientific and technological importance. In this study, interfacial tension measurements of surface-active compounds-ionic liquid 2-dodecyl-2,2dimethylethanolammonium bromide (12Cho.Br) and cationic surfactant cetyltrimethylammonium bromide (CTAB)-were conducted both in the absence and presence of ciprofloxacin (CIP). Equilibrium interfacial tension (EIFT) measurements and conductivity data demonstrate the effect of CIP on the critical micellar concentration and surface excess concentration of 12Cho.Br and CTAB. Additionally, dynamic interfacial tension (DIFT) measurements were performed to compare the interfacial tension of pure 12Cho.Br and CTAB solutions, as well as those in the presence of the drug (with and without 0.3% acetic acid), as a function of time. The DIFT analysis revealed that the adsorption of 12Cho.Br and CTAB at the air-water interface followed a mixed diffusion-adsorption controlled mechanism. The adsorption processes of 12Cho.Br and CTAB molecules were studied over short time intervals (t → 0) and longer time intervals (t → ∞). The adsorption behaviour was correlated with concentration and the presence of energy barriers. In the presence of CIP, the diffusion coefficient was compared to that of the pure 12Cho.Br and CTAB systems to assess its effect on adsorption and to validate the participation of CIP in DIFT relaxations. Additionally, DIFT measurements were employed to investigate CIP solubility in different SAC systems. Data from the relaxation profiles across a range of concentrations were used to determine the solubility limit of the drug molecules. The solubility data obtained from DIFT correlates strongly with the UV spectroscopy results. Furthermore, DFT calculations provide insights into the frontier orbital structures and physicochemical parameters of complex formations.", "journal": "Soft matter", "year": "2025", "doi": "10.1039/d4sm00936c", "ptypes": [ "Journal Article" ], "first_author": "Bhat AR", "dirs": [ "D" ] }, "40499765": { "pmid": "40499765", "title": "Lyophilized SARS-CoV-2 self-amplifying RNA vaccines for microneedle array patch delivery.", "abstract": "mRNA vaccines have emerged as a pivotal tool to respond to global pandemics like SARS-CoV-2. However, RNA vaccines face challenges with limited duration of immunogenicity and reliance on a special cold-chain for long term storage. Self-amplifying (saRNA) vaccines have shown sustained antigen expression and durable immune responses. Herein we developed lyophilization formulations for a SARS-CoV-2 saRNA ionizable lipid nanoparticle (LNP) to help reduce dependence on the cold chain. Our results show the induction of robust immune responses in mice by saRNA-LNPs when delivered either intramuscularly or intradermally following lyophilization and storage for up to 15 weeks at above freezing temperatures. Additionally, lyophilized saRNA-LNPs were efficiently delivered into the skin via microfluidic microarray patches (M-MAPs), inducing strong humoral and cellular immunity. M-MAPs offer a painless, self-administered alternative to traditional injections for transdermal drug delivery. Our work highlights the potential for a thermostable, self-administered RNA-LNP vaccine to improve vaccine coverage.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2025", "doi": "10.1016/j.jconrel.2025.113944", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Driskill MM", "dirs": [ "D" ] }, "42198221": { "pmid": "42198221", "title": "Emerging Non-Conventional Approaches in mRNA-LNP Formulation for Therapeutic Applications.", "abstract": "Lipid nanoparticles (LNPs) have become the cornerstone of nucleic acid delivery platforms, particularly in RNA-based vaccines and therapeutics. However, the conventional methods of LNP production, which are primarily reliant on microfluidic mixing of aqueous and organic solvent phases, pose limitations in terms of mRNA stability, residual organic contamination, scalability, cost, and environmental impact. These limitations prompted a renewed search for non-conventional strategies with the promise of improving mRNA-LNP encapsulation approaches. These emerging approaches aim to address key bottlenecks, including mRNA hydrolysis-driven degradation, high production losses, and complex downstream purification. Moreover, the ability to decouple LNP synthesis from mRNA encapsulation could enable streamlined, modular manufacturing workflows and customizable payload delivery, including single- or multiple-mRNA payloads, thereby expanding the therapeutic scope of LNPs. This review offers an early insight into the design principles and scalability potential of emerging non-conventional LNP encapsulation approaches, including solvent-free and microfluidics-free methodologies, and pre-built LNP workflows. We also examine trends in emerging LNP encapsulation tools, including high-shear mixing, sonication, membrane contraction, and other approaches. Finally, we extrapolate the suitability of the methods for scale-up approaches and their economic implications based on the process information.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18050527", "ptypes": [ "Journal Article", "Review" ], "first_author": "Zhang Y", "dirs": [ "D" ] }, "40786068": { "pmid": "40786068", "title": "Fabrication and optimization of freeze-dried isoniazid-loaded poly-ε-caprolactone nanoparticles.", "abstract": "Microfluidic nanoprecipitation followed by freeze-drying would yield uniformly sized, stable nanoparticles by preserving their physicochemical property without compromising therapeutic performance. The isoniazid (INH)-loaded poly-ε-caprolactone (PCL) nanoparticles could be developed using a microfluidic technique for the management of tuberculosis. The INH-loaded nanoparticles were fabricated via a microreactor-assisted nanoprecipitation method and optimization using a design of experiments factorial design approach. The resulting INH-PCL nanoformulation was characterized for particle size, polydispersity index (PDI), zeta potential (surface charge), Fourier-transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction analysis and field emission scanning electron microscope. The optimized nanoparticles exhibited an average particle size (248.4 ± 5.372 nm) and high encapsulation efficiency (82.26 ± 4.36 %). Thermal and spectroscopic analyses confirmed the absence of drug-polymer interactions, ensuring formulation integrity; stability studies under accelerated conditions demonstrated negligible changes in particle size, PDI, and zeta potential over the period of 6 months, indicating robust colloidal stability. A scanning electron microscopy study revealed rod-shaped nanoparticles with smooth surfaces. Lyophilization (freeze-drying) enhanced long-term stability, yielding a readily re-dispersible powder (reconstitution index ~1.066). Following diffusion-controlled kinetics, in vitro drug release studies in phosphate buffer saline (pH 7.4) showed sustained drug release (92.45 % cumulative release over 48 h). Our results confirm that the INH-loaded PCL nanoformulation combines excellent stability, high drug-loading capacity, and sustained release, key attributes of effective tuberculosis therapy.", "journal": "ADMET & DMPK", "year": "2025", "doi": "10.5599/admet.2774", "ptypes": [ "Journal Article" ], "first_author": "Kole E", "dirs": [ "D" ] }, "40414439": { "pmid": "40414439", "title": "Lipid-Polyhydroxyalkanoate Hybrid Nanoparticles as Sustainable Platform for mRNA delivery.", "abstract": "The success of mRNA vaccines for the treatment of COVID-19 has generated enormous interest in mRNA therapeutics for various diseases, highlighting the need for robust delivery platforms. Combining the excellent transfection properties of lipids with the high stability of polymeric nanoparticles in a single hybrid system has become an attractive approach to generate next generation mRNA delivery systems. We introduce a modular lipid-polymer hybrid nanoparticle (LPHNP) design based on medium-chain-length polyhydroxyalkanoates (mcl-PHAs) as sustainable alternative to poly(lactic-co-glycolic acid) (PLGA), and a polymer-lipid (DMG-PEG, or poly(2-ethyl-2-oxazoline-myristic acid (PEtOx-MA)). A small library of LPHNPs containing the cationic lipid DOTAP were synthesized using a scalable microfluidic process. Physico-chemical and biological properties of mRNA-LPHNPs were studied. Results showed that the mcl-PHAs (PHAF10 and PHAGlu) derived from Pseudomonas putida can form distinct LPHNPs depending on polymer-to-lipid ratios, and mcl-PHA composition. Cell toxicity, transfection efficiency, serum stability, and the effect of protein interactions was investigated in HeLa cells and human brain endothelial (hCMEC/D3) cells. Importantly, the particles' biophysical properties and transfection efficacies were not affected after lyophilization and storage at various temperatures for two months. Reporter mRNA was identified across all major organs collected after intravenous injection into mice with limited protein expression in the blood, highlighting the excellent stability of hybrid nanoparticles against blood cell interactions. This approach holds promise for accelerating development of a novel sustainable polymer for mRNA delivery that differs from conventional lipid nanoparticle systems.", "journal": "European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V", "year": "2025", "doi": "10.1016/j.ejpb.2025.114755", "ptypes": [ "Journal Article" ], "first_author": "Mazrad ZAI", "dirs": [ "D" ] }, "38872047": { "pmid": "38872047", "title": "Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy.", "abstract": "Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by conventional single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60-70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology.", "journal": "Drug delivery and translational research", "year": "2025", "doi": "10.1007/s13346-024-01644-4", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "González-García D", "dirs": [ "D" ] }, "42345446": { "pmid": "42345446", "title": "Multimodal Detection of α-Amanitin Using a Cell-Free Biosensing Platform.", "abstract": "This study developed a transcription-based, multimodal cell-free biosensing platform for the sensitive detection of α-amanitin, a deadly mushroom toxin. The core design of the platform converts α-amanitin-dependent RNA polymerase inhibition into tunable, genetically encoded outputs. The system uses programmable DNA templates to produce specific RNA sequences, which are then processed in parallel by three independent signal transduction modules: fluorescence using the Malachite Green (MG) light-up RNA aptamer; colorimetry through RNA-directed gold nanoparticle aggregation; and enzymatic catalyzed by an in vitro transcribed G-quadruplex/hemin (G4/hemin) RNAzyme. This multimodal output strategy provides built-in self-validation for a single detection event, significantly enhancing result reliability. Evaluations demonstrated that the biosensor exhibits high sensitivity, with limits of detection (LODs) of 1.30 μg/mL for fluorescence detection, 0.69 μg/mL for AuNP-based colorimetric detection, and 6.29 μg/mL for RNAzyme-based colorimetric detection, and showed good selectivity and robust performance in complex matrices. Lyophilization tests confirmed operational stability for potential on-site applications. This work establishes a versatile, modular framework for transcription-based biosensing, with potential extension to other RNA polymerase inhibitors.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.6c01287", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Huang J", "dirs": [ "D" ] }, "42645059": { "pmid": "42645059", "title": "A Ready-to-Use Recombinant Yeast Two-Hybrid Assay for Thyroxine Detection.", "abstract": "We report a freeze-dried ready-to-use yeast thyroid screen (YTS), preserving the general dose-response characteristics of the freshly prepared counterpart. This field-deployable method reduces the assay time of the overall procedure from several days to 5 h with no requirement for sterile conditions, thus fulfilling key requirements for on-site implementation in a biosensor array. The effects of cell density and concentration of the cryoprotectant trehalose on median effective concentrations (EC50), limit of detection (LOD) and biosensor induction (IF) were determined and monitored over a storage period of 5 months. In addition, the impact of these parameters was monitored on the biosensor survival rate during freeze-drying and the subsequent storage process. Throughout the 5-month study, the freeze-dried recombinant yeast assay retained comparable dose-response characteristics to those of the freshly prepared counterpart, displaying median values of EC50 in the range of 350 nM to 550 nM and LODs in the range of 20 nM to 45 nM of the reference compound thyroxine (T4). Long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4-equivalent activities were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively. In summary, we have successfully demonstrated a proof of principle of a field-deployable yeast thyroid screen (YTS) by using freeze-dried cells and trehalose as a cryoprotectant to achieve storability for up to 5 months at 4 °C.", "journal": "Biosensors", "year": "2026", "doi": "10.3390/bios16080441", "ptypes": [ "Journal Article" ], "first_author": "Danhausen M", "dirs": [ "D" ] }, "41011863": { "pmid": "41011863", "title": "Optimization of PLGA Nanoparticle Formulation via Microfluidic and Batch Nanoprecipitation Techniques.", "abstract": "Polymeric nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) are widely used in drug delivery, yet scalable and reproducible production methods remain a major challenge. In this study, we combine experimental nanoprecipitation and computational fluid dynamics (CFD) modeling to optimize PLGA nanoparticle formulation using both traditional batch and microfluidic methods. While Design of Experiments (DoE) was used to optimize the batch process, microfluidic mixing was systematically explored by varying flow parameters such as the flow rate ratio (FRR) and total flow rate (TFR). We compared two microfluidic mixer designs with Y-junction and three-inlet junction geometries to evaluate their impact on the mixing efficiency and nanoparticle formation. Experimental results revealed that the three-inlet design produced smaller, more uniform nanoparticles with superior post-lyophilization stability. CFD simulations confirmed these findings by displaying velocity fields and PLGA concentration gradients, demonstrating significantly more homogeneous mixing and efficient interfacial contact in the three-inlet configuration. Furthermore, simulated outlet concentrations were used to predict the nanoparticle size via theoretical modeling, which closely agreed with the experimental data. This integrated approach highlights the importance of microfluidic geometry in controlling nanoparticle nucleation dynamics and provides a framework for rational design of scalable nanomedicine production systems.", "journal": "Micromachines", "year": "2025", "doi": "10.3390/mi16090972", "ptypes": [ "Journal Article" ], "first_author": "Kozalak G", "dirs": [ "D" ] }, "40179701": { "pmid": "40179701", "title": "Collaborative light-off and light-on bacterial luciferase biosensors: Innovative approach for rapid contaminant detection.", "abstract": "Bioluminescence-based light-off and light-on biosensors are widely used in environmental monitoring due to their rapid, cost-effective, real-time, and easy operation. However, stability and sensitivity issues in detecting real samples remain challenging. This study introduces a novel approach utilizing combined light-off and light-on biosensors for rapid and sensitive contaminants detection within 45 min in real samples. First, a recombinase-based state machine (RSM) was used to construct light-off RSM biosensors (light-off RSMs) for continuously strong light emission and overexpressing of outer membrane porins OmpC and OmpF enhanced their sensitivity to toxic contaminants. Additionally, a new experimental protocol containing the cell culture, collection, preparation, and the contaminant measurement was established for bioluminescent light-on whole-cell biosensors (light-on WCBs) in contaminant detection, initially developed using cadmium (Cd) and later applied to lead (Pb) and mercury (Hg). For Cd light-on WCBs, overexpressing OmpC and knocking out the contaminant exporter ZntA enhanced the accumulation of intracellular Cd in WCB cells, resulting in increased sensitivity to low concentrations of contaminants. Further, metabolic modifications in light-on WCBs significantly boosted luminescence. These genetic modified bacterial strains, whether freshly harvested or as freeze-dried powders, showed rapid luminescent responses to contaminants in the picomolar (pM) to nanomolar (nM) range within 45 min. Finally, the combined use of light-off RSMs and light-on WCBs successfully assessed toxicity and detected specific contaminants in real environmental and food samples. These strategies could be applied to developing other bacterial luciferase-based biosensors and even other types such as colorimetric biosensors.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2025.117369", "ptypes": [ "Journal Article" ], "first_author": "Yang J", "dirs": [ "D" ] }, "42104082": { "pmid": "42104082", "title": "Double-coated PLGA nanoparticles with hierarchical surface architecture for CD44-targeted siRNA delivery.", "abstract": "Efficient delivery of small interfering RNA (siRNA) remains a materials challenge because it requires nanocarriers that stabilize polyanionic cargo, support cellular interactions, and enable cytosolic delivery. Although poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are used due to biocompatibility, biodegradability, and regulatory acceptance, siRNA delivery with PLGA requires interfacial engineering to meet these constraints. Here, a modular double-coated PLGA NP platform (dcNPs2.0) is developed and optimized for siRNA complexation, surface functionalization, and scalable manufacturing. The system comprises a PLGA core coated with a polyethyleneimine (PEI) interlayer to mediate siRNA binding, followed by a hyaluronic acid (HA) outer layer, which improves colloidal stability and promotes CD44-mediated uptake. Process optimization, including transition from batch nanoprecipitation to microfluidic fabrication, provides high yield, excellent reproducibility, narrow size distributions, and increased siRNA loading. X-ray photoelectron spectroscopy confirms hierarchical multilayer assembly. The optimized dcNPs2.0 formulation exhibited robust physicochemical stability during storage, in serum-containing media, and following lyophilization with appropriate cryoprotection. Functional evaluation of dcNPs2.0 demonstrated efficient HA-mediated cellular uptake and effective silencing following siRNA delivery in both two-dimensional monolayers and three-dimensional spheroids of MDA-MB-231 cells. Overall, this work establishes a scalable, rationally engineered PLGA nanoplatform that integrates extracellular targeting with intracellular delivery requirements for siRNA therapeutic applications.", "journal": "Drug delivery and translational research", "year": "2026", "doi": "10.1007/s13346-026-02115-8", "ptypes": [ "Journal Article" ], "first_author": "Longobardi G", "dirs": [ "D" ] }, "41155946": { "pmid": "41155946", "title": "Physiological Barriers to Nucleic Acid Therapeutics and Engineering Strategies for Lipid Nanoparticle Design, Optimization, and Clinical Translation.", "abstract": "Lipid nanoparticles are a clinically validated platform for delivering nucleic acids, but performance is constrained by multiscale physiological barriers spanning circulation, vascular interfaces, extracellular matrices, cellular uptake, and intracellular trafficking. This review links composition-structure-function relationships for ionizable lipids, helper phospholipids, cholesterol, and PEG-lipids to systemic fate, endothelial access, endosomal escape, cytoplasmic stability, and nuclear transport. We outline strategies for tissue and cell targeting, including hepatocyte ligands, immune and tumor selectivity, and selective organ targeting through compositional tuning, together with approaches that modulate escape using pH-responsive chemistries or fusion-active peptides and polymers. We further examine immunomodulatory co-formulation, route and schedule effects on biodistribution and immune programming, and manufacturing and stability levers from microfluidic mixing to lyophilization. Across these themes, we weigh trade-offs between stealth and engagement, potency and tolerability, and potency and manufacturability, noting that only a small fraction of endosomes supports productive release and that protein corona variability and repeat dosing can reshape tropism and clearance. Convergence of standardized assays for true cytosolic delivery, biomarker-guided patient selection, and robust process controls will be required to extend LNP therapeutics beyond the liver while sustaining safety, access, and scale.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17101309", "ptypes": [ "Journal Article", "Review" ], "first_author": "Kim Y", "dirs": [ "D" ] }, "41401494": { "pmid": "41401494", "title": "Enhanced amperometric tyramine sensing via tyrosinase entrapment in polyethylene glycol-modified methacrylate microgels.", "abstract": "A highly sensitive and selective tyrosinase-based electrochemical biosensor for tyramine detection in complex food matrices was developed using polyethylene glycol-poly(methacrylic acid) microgels (PEG-pMAA) as enzyme immobilization matrix. The morphology and particle size of the obtained PPO-PEG-pMAA microgel were studied by SEM and particle analyzer system, obtained polydisperse microgels in the range of 2-18 μm with a 6.2 μm of average particle size. Incorporation of PEG during microgel polymerization provided a favorable microenvironment for enzyme activity, resulting in a 22 % higher amperometric response toward tyramine compared to microgels without PEG (PPO-pMAA), without compromising reproducibility (RSD ≤6.1 %). The PPO-PEG-pMAA biosensor's performance was optimized in terms of cross-linking degree, PEG %, pH, potential, temperature, enzyme loading and microgel deposition. A complete enzyme entrapment at pH 6 in microgel synthesis with a cross-linking ratio of 2 %, which balanced enzyme retention and substrate accessibility is obtained. Under optimal conditions, the device exhibited a wide linear range (2.0 × 10-7 - 4.7 × 10-5 M), low detection limit (45 nM) and rapid amperometric responses (50 s). The freeze-dried microgels were stable 9 months and the microgel-based biosensor retained 95-100 % of its initial response during 35 days of storage, confirming excellent stability. Interference studies showed negligible effects from the most abundant acids and amino acids present in cheese, highlighting the device's selectivity. Application to cheese samples, including Brie and Gouda, yielded recoveries between 97.6 and 105.6 %, and allowed monitoring of tyramine accumulation during storage, demonstrating the biosensor's reliability and suitability as a rapid and practical tool for tyramine screening in complex food matrices.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2025.129253", "ptypes": [ "Journal Article" ], "first_author": "Hervás-Pérez JP", "dirs": [ "D" ] }, "41707426": { "pmid": "41707426", "title": "Programmable AND-gate strategy to reduce false positives in rolling circle amplification.", "abstract": "Improving the specificity of biosensors by reducing their false positive rate is a long-standing challenge, particularly for point-of-care (POC) diagnostics. An emerging and potentially impactful class of POC biosensor is those that can detect specific nucleic acid sequences. These sensors often use isothermal amplification of nucleic acid sequences, which is considered suitable for POC use due to its relative simplicity and its lack of requirement for specialized equipment or highly trained personnel. However, isothermal amplification methods are still quite susceptible to false positives, especially those like Rolling Circle Amplification (RCA) that amplify nucleic acids under mild temperature conditions that are more prone to near-target amplification. To address this limitation, we introduced an AND-gate Boolean logic to create Multi-Key RCA, which uses detection of multiple nucleic acid sequences as a prerequisite to amplification. Multi-Key RCA requiring two DNA sequences not only markedly reduced false positives compared to conventional RCA but also achieved high specificity in single-nucleotide polymorphism (SNP) detection. We showed that Multi-Key RCA also can detect RNA and can be expanded to use AND gates with more than two inputs. We integrated Multi-Key RCA with a lateral flow assay to create a user-friendly synthetic biology-based diagnostic platform requiring no specialized equipment, and we showed that this diagnostic can be implemented as a one-pot reaction that integrates all RCA steps and can be lyophilized for eventual long-term storage stability. Overall, this Boolean logic-based synthetic biology approach shows significant promise to reduce false positives in field-deployable nucleic acid biosensors.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.118515", "ptypes": [ "Journal Article" ], "first_author": "Seok J", "dirs": [ "D" ] }, "42653700": { "pmid": "42653700", "title": "Exploration of Development Parameters for IgG Microspheres Using an Automated Platform for High-Throughput Formulation Screening.", "abstract": "Background/Objectives: PLGA/PLA microspheres are attractive candidates for depot delivery of protein therapeutics, but formulation development for antibodies remains challenging because protein encapsulation, burst release, stability, and polymer composition must be balanced across a large design space. This study evaluated an automated high-throughput formulation workflow for preparing and screening IgG-loaded pegylated PLGA/PLA microspheres, with emphasis on loading and early release behavior. Methods: Microspheres were prepared using an automated W/O/W emulsion workflow consisting of primary emulsion formation, microfluidic secondary emulsification, solvent removal, washing, and lyophilization. A formulation library was generated by varying polymer chemistry, polymer concentration, polymer blends, IgG concentration, and additives. Formulations were evaluated for IgG loading and in vitro release over the first 7 days using automated sampling and protein quantification. Statistical analyses were used to identify formulation variables associated with loading, burst release, day-7 release, and Weibull-derived kinetic descriptors. Results: Across 129 formulations, IgG loading was generally in the low-to-mid single-digit percentage range. Additive class and IgG concentration in the dispersed phase were the formulation variables most strongly associated with early release behavior. Additive-free systems were associated with lower burst release, whereas non-polymeric additives were associated with higher burst and faster early release. Day-7 release differences were less robust after correction for variance heterogeneity and resampling. Conclusions: The automated workflow enabled rapid preparation and comparative screening of IgG-loaded PLGA/PLA microspheres. The findings support the use of high-throughput automated screening to identify formulation variables associated with burst modulation and early release behavior. Because release was evaluated over 7 days and only one model IgG was used, the results should be interpreted as an early-stage formulation screening study rather than definitive evidence of long-term antibody depot performance.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2026", "doi": "10.3390/ph19081203", "ptypes": [ "Journal Article" ], "first_author": "Tyagi P", "dirs": [ "D" ] }, "41981865": { "pmid": "41981865", "title": "A Cell-Resolved Ultrastable Biosensor Enables One-Step Detection of Gene-Fusion Transcripts in Unprocessed Whole Blood.", "abstract": "Cell-specific detection of aberrant mRNA in blood is essential for diagnosing and treating hematological malignancies. However, current sensors are unable to function in unprocessed whole blood due to limitations in chemical stability and cell-targeting capability. Here, we engineer a cell-resolved ultrastable sensor for hematology (CRUSH) via spherical-nucleic-acid (SNA) technology, which enables live-cell detection of leukemia fusion transcripts in unprocessed whole blood. CRUSH employs stoichiometrically controlled thiol protector to achieve a defect-free thiol monolayer on AuNPs. This design feature endows CRUSH with a record-breaking stability, withstanding 0.1 M dithiothreitol, a 10,000-fold improvement over conventional SNAs. We also demonstrate that the phagocytic bias of myeloid cells over lymphoid cells drives selective internalization of CRUSH in myeloid lineages in whole blood. Leveraging cellular selectivity and engineered stability, CRUSH offers a mixed-and-read diagnostic test, where lyophilized sensors are directly mixed with whole blood samples, followed by standard flow cytometry analysis. This one-step test detects BCR-ABL1 fusions in living myeloid cells with high specificity and robustness, enabling accurate discrimination of multilineage acute lymphoblastic leukemia within 1 h. Our study bridges biosensing innovation with urgent diagnostic needs, offering a rapid, specific, and robust tool for accurate diagnosis and treatment of hematologic malignancies.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2026", "doi": "10.1002/anie.1339483", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Kong C", "dirs": [ "D" ] }, "41439322": { "pmid": "41439322", "title": "An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.", "abstract": "Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.", "journal": "Lab on a chip", "year": "2026", "doi": "10.1039/d5lc01096a", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Wang Z", "dirs": [ "D" ] }, "40603400": { "pmid": "40603400", "title": "Double emulsion templated monodisperse antibubbles via combined high-shear homogenization and T-junction microfluidics.", "abstract": "Antibubbles are unique bubbles (within a bulk liquid) that comprise liquid core(s). This study focused on creating and evaluating antibubbles with multiple cores using double emulsion (DE) templating. The primary emulsion (PE) was made using high-shear homogenization and then passed through a T-junction to form the DE. These DEs were then freeze-dried and rehydrated to form antibubbles. The study examined the effect of PE parameters (homogenization speed (rpm), internal phase ([Formula: see text], %), and interfacial particle concentration ([Formula: see text], %)), second emulsification parameters (flow rate ratio, interfacial particle concentration ([Formula: see text], %), microchannel size, and additional cryoprotectants in continuous phase), and post-emulsification freezing temperature on DE and the antibubble. The best conditions for yielding small-sized DE and the antibubble with maximum reconstitution coefficient (RC) were selected to evaluate the encapsulation efficiency (EE) of calcein in [Formula: see text]. The study found that antibubbles could provide better EE than DEs for storage longer than 7 days. Future studies should focus on scaling up production, improving EE during the DE-to-antibubble transition, and evaluating stability and release dynamics under in-vitro gastrointestinal simulations using human digestive fluids and tissues for more accurate in vivo predictions.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-04009-0", "ptypes": [ "Journal Article" ], "first_author": "Niroula A", "dirs": [ "D" ] }, "42594724": { "pmid": "42594724", "title": "Development of a microfluidic isothermal amplification nucleic acid detection (mFIND) platform for simultaneous detection of respiratory pathogens.", "abstract": "We developed the mFIND platform, a centrifugal microfluidic isothermal amplification system for simultaneous detection of influenza A/B (FLUAV/FLUBV), SARS-CoV-2, and RSV - key agents of acute respiratory infections. The chip integrates four independent reaction chambers preloaded with lyophilized reagents, enabling room-temperature storage and a simplified two-step workflow. A portable analyzer automates sample metering, isothermal amplification (41 ± 0.4 °C), and real-time fluorescence monitoring, completing detection within 15 min with an ultra-low reaction volume of 2.5 μL per chamber. The platform achieved limits of detection of 2 copies/test for FLUAV/FLUBV and 5 copies/test for SARS-CoV-2/RSV, with no cross-reactivity against 23 non-target pathogens. Clinical validation using 236 positive samples showed 100% positive and negative agreement with qPCR. The mFIND platform combines rapid turnaround, simplified workflow, low reagent consumption, and parallel processing of four samples (16 targets), positioning it as a practical point-of-care nucleic acid testing solution for decentralized settings.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.119096", "ptypes": [ "Journal Article" ], "first_author": "He P", "dirs": [ "D" ] }, "39849850": { "pmid": "39849850", "title": "2-Propanol Suspension Method to Increase Acetylcholinesterase and Flow Stability on μPADs.", "abstract": "Ensuring detection performance and shelf life is crucial for analytical devices. Advances in materials and reaction mechanisms have improved detection performance, yet extending the operational lifetime of microfluidic paper-based analytical devices (μPADs)─especially those reliant on sensitive enzymes─remains a challenge. Here, we present an alternative to air-drying and lyophilization: loading enzymes suspended in 2-propanol (iPrOH). By suspending the enzyme in iPrOH, we circumvent the enzyme activity losses commonly associated with freeze-thawing and freeze-drying. Accelerated aging tests, supported by statistical analyses of long-term activity retention (including comparisons over multiple time points), indicate that while conventional methods do not sustain consistent superiority, the iPrOH suspension method maintains higher enzymatic activity over extended periods. By avoiding stabilizers and circumventing the limitations of other techniques, our method enables μPADs to achieve both longevity and stable fluid flow. Thus, we provide a more robust, on-site analytical platform capable of reliable on-site detection.", "journal": "ACS applied bio materials", "year": "2025", "doi": "10.1021/acsabm.4c01879", "ptypes": [ "Journal Article" ], "first_author": "Yamaguchi A", "dirs": [ "D" ] }, "41693084": { "pmid": "41693084", "title": "An Easy-To-Use and Field-Deployable Cell-Free Bioreporter for Environmental Detection of Tetracyclines and Macrolides.", "abstract": "The widespread use of antibiotics in agriculture, livestock production, and clinical medicine has resulted in their continuous release into the environment, posing substantial ecological and public health risks. Although chromatographic approaches remain the gold standard for antibiotic detection due to their high accuracy and sensitivity, they require expensive instrumentation, labor-intensive workflows, and trained personnel, limiting their suitability for rapid or on-site monitoring. Here, we report a synthetic biology-based cell-free bioreporter that integrates antibiotic-responsive transcription factors, RNA polymerase, and engineered DNA transcription templates to generate a visible fluorescent readout upon exposure to tetracycline or macrolide antibiotics through an in vitro transcription reaction. This system eliminates biocontainment concerns inherent to whole-cell biosensors and demonstrates high sensitivity, specificity, and accuracy when applied to real water samples. In addition, the biosensor can be freeze-dried without substantial loss of activity, enabling long-term storage and convenient deployment outside laboratory settings. Together, these features establish a portable and robust assay for antibiotic monitoring in environmental applications.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c04800", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Cui R", "dirs": [ "D" ] }, "40416977": { "pmid": "40416977", "title": "Simultaneously ultrasensitive and differential detection of SARS-CoV-2, adenovirus and influenza a virus using multiplex fluorescence lateral flow immunoassay.", "abstract": "The rapid and precise differential diagnosis of respiratory diseases is crucial to impede the spread of the viruses, considering the substantial demand resulting from frequent co-infections. A quantum dot nanobeads (QBs)-based multiplex fluorescence lateral flow immunoassay (QBs-based MF-LFA) biosensor was developed. The MF-LFA biosensor enabled simultaneous and sensitive quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Adenovirus (ADV), and Influenza A Virus (IAV), boasting low limit of detection (LOD) of 56, 120, and 41 copies/mL, respectively. Compared to colloidal gold LFA, the LOD was improved by 200, 417, and 1220 times, respectively, while maintaining sensitivity comparable to PCR techniques. The biosensor provided results within 20 minutes, exhibited good reproducibility, and boasted high accuracy with recoveries ranging from 96% to 105%. Additionally, the biosensor had a shelf life of up to 8 months, attributed to the use of freeze-dried probes with minimal water content, ensuring enhanced stability. Clinical samples of SARS-CoV-2, ADV and IAV infections were tested, the results were consistent with both PCR testing and clinical diagnostic tests. This highlights the considerable potential of our biosensor for early and rapid differential detection of respiratory viruses.", "journal": "Frontiers in immunology", "year": "2025", "doi": "10.3389/fimmu.2025.1540676", "ptypes": [ "Journal Article" ], "first_author": "Li X", "dirs": [ "D" ] }, "40967322": { "pmid": "40967322", "title": "Fabrication of freezed-dried docetaxel liposomal powders through a microfluidic technology with enhanced blood circulation and antitumor efficacy.", "abstract": "The industrial scalability and clinical translation of liposomal formulations are significantly hindered by challenges in stability and batch-to-batch variability. To address these limitations, docetaxel (DTX)-loaded long-circulating liposomal freeze-dried powders were engineered via a synergistic integration of microfluidic technology and lyophilization, optimized through an orthogonal experimental design focusing on particle size, polydispersity index (PDI), and entrapment efficiency (EE). The optimized formulation, incorporating hydroxypropyl-β-cyclodextrin (HP-β-CD) as a lyoprotectant, demonstrated nanoscale particle diameter (∼120 nm), monodisperse distribution (PDI < 0.2), and elevated EE (>90 %), with physicochemical integrity preserved post-lyophilization. Reconstituted DTX liposomes (DTX-Lip) exhibited pH-dependent sustained release kinetics, with enhanced drug elution at acidic pH (5.5) versus physiological pH (7.4). Solid-state characterization (XRD, FT-IR, DSC) confirmed molecular interactions between DTX and lipid-excipient matrices. In vitro cytotoxicity assays revealed superior apoptotic induction in A549 human lung carcinoma cells by DTX-Lip compared to free DTX solution (DTX-Sol). Pharmacokinetic profiling in vivo demonstrated prolonged systemic circulation of DTX-Lip, marked by significantly elevated area under the curve (AUC0-∞), maximum drug concentration (Cmax), mean retention time (MRT0-∞), longer half-life (T1/2), lower clearance (CL) and steady-state volume of distribution (Vss) relative to DTX-Sol (p < 0.05). In a C57BL/6 murine lung cancer model, DTX-Lip elicited significant tumor suppression (p < 0.05) without mortality, underscoring enhanced therapeutic efficacy and mitigated systemic toxicity. Collectively, the engineered DTX-Lip system combines optimal nano-pharmaceutical attributes (size, homogeneity, EE), controlled release, and favorable in vitro-in vivo performance, offering a robust platform for industrial-scale production of stable, efficacious liposomal DTX formulations. These findings advance translational strategies for liposome-based chemotherapeutics with improved reproducibility and clinical safety.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.126190", "ptypes": [ "Journal Article" ], "first_author": "Lan LJ", "dirs": [ "D" ] }, "42135423": { "pmid": "42135423", "title": "Improving the compatibility of INFOGEST digesta with intestinal epithelial models through post-digestion storage conditions.", "abstract": "The INFOGEST protocol is widely used for standardized in vitro digestion studies, but its application to cell models remains challenging due to residual enzyme activity, bile salts, and matrix effects that impair cell metabolism. This study investigated a post-digestion processing strategy to enable direct testing of INFOGEST digesta on 2D Caco-2 monolayers and intestine-on-chip systems without chemical inhibitors. Five post-digestion storage methodologies were compared: A, no storage; B, liquid nitrogen at - 80 °C; C, liquid nitrogen/freeze-drying; D, - 20 °C/freeze-drying; and E, - 20 °C. Residual enzymatic activities and effects on epithelial cell morphology and viability were evaluated, and the optimized workflow was validated in a microfluidic intestine-on-chip, including plant matrices. The condition D significantly reduced residual enzymatic activity, especially trypsin, identified as a key mediator of epithelial cell disruption. This storage method preserved Caco-2 monolayer integrity at a 25% dilution, with no cytotoxicity or cell detachment. When applied to digested Brassica oleracea and Malus domestica matrices, condition D did not compromise cell viability as evidenced by the results obtained with INFOGEST digesta in the absence of plant food matrices. In intestine-on-chip experiments, treated digesta produced stable TEER values and continuous ZO-1 localization, confirming barrier integrity. This simple freeze-then-lyophilize workflow enables a scalable and biocompatible integration of INFOGEST digestion with advanced cellular models for studying food bioaccessibility and intestinal absorption.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-51799-y", "ptypes": [ "Journal Article" ], "first_author": "Troisi F", "dirs": [ "D" ] }, "42789142": { "pmid": "42789142", "title": "Portable bioactive capsule-integrated electrochemical-colorimetric dual-mode biosensor based on Mn/Ce MOF nanozyme for rapid detection of malathion in water.", "abstract": "To enable rapid and reliable on-site monitoring of organophosphorus pesticides a novel portable dual-mode biosensor has been developed which integrates electrochemical and colorimetric detection into a single freeze-dried bioactive capsule. This innovative design supports instrument-free operation and provides dual-signal cross-validation. The sensor is based on a newly synthesized Mn/Ce bimetallic metal-organic framework (MOF) nanozyme with high oxidase-like activity, co-immobilized with acid phosphatase (ACP) within a mannitol- and sucrose-stabilized lyophilized capsule to ensure long-term stability and field readiness. In the presence of malathion, the pesticide selectively inhibits ACP, thereby restoring the MOF's catalytic activity and enabling the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). The resulting signal is detected both visually and through chronoamperometry after conversion to a yellow diimine product. This approach achieves a wide linear detection range of 1-2000 nM and an exceptionally low detection limit of 0.06 nM for malathion, along with high accuracy evidenced by recoveries of 93.31%-104.03% in real water samples. This work establishes a field-deployable and user-friendly sensing platform that combines innovative material design with integrated dual-mode detection, offering a robust and practical strategy for on-site environmental monitoring.", "journal": "Mikrochimica acta", "year": "2026", "doi": "10.1007/s00604-026-08398-5", "ptypes": [ "Journal Article" ], "first_author": "Li S", "dirs": [ "D" ] }, "40262634": { "pmid": "40262634", "title": "Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.", "abstract": "Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR-Cas enzymes1-4 to enable diverse genome-editing applications. However, such enzymes have caveats such as an increased risk of off-target editing3,5,6. Here, to enable scalable reprogramming of Cas9 enzymes, we combined high-throughput protein engineering with machine learning to derive bespoke editors that are more uniquely suited to specific targets. Through structure-function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif (PAM)7 requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM machine learning algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico-directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHOP23H allele in human cells and mice. Together, PAMmla integrates machine learning and protein engineering to curate a catalogue of SpCas9 enzymes with distinct PAM requirements, motivating a shift away from generalist enzymes towards safe and efficient bespoke Cas9 variants.", "journal": "Nature", "year": "2025", "doi": "10.1038/s41586-025-09021-y", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "Silverstein RA", "dirs": [ "D" ] }, "41415723": { "pmid": "41415723", "title": "Recent advances on protein engineering for improved stability.", "abstract": "Stability is a crucial factor influencing applicability of industrial biocatalysts, and enzymatic operations in partial organic solvents and higher temperatures are preferable since organic solvents may improve the solubility of the substrates, and the reaction rates are often higher at elevated temperatures. This review aims to summarize recent advances for engineering enzymes to meet the industrial needs for solvent and thermostability and offer insights in the advances in methodologies utilizing B-factors, ancestral reconstructions, or machine learning approaches.", "journal": "Biodesign research", "year": "2025", "doi": "10.1016/j.bidere.2025.100005", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shi J", "dirs": [ "D" ] }, "40935922": { "pmid": "40935922", "title": "Biophysics-based protein language models for protein engineering.", "abstract": "Protein language models trained on evolutionary data have emerged as powerful tools for predictive problems involving protein sequence, structure and function. However, these models overlook decades of research into biophysical factors governing protein function. We propose mutational effect transfer learning (METL), a protein language model framework that unites advanced machine learning and biophysical modeling. Using the METL framework, we pretrain transformer-based neural networks on biophysical simulation data to capture fundamental relationships between protein sequence, structure and energetics. We fine-tune METL on experimental sequence-function data to harness these biophysical signals and apply them when predicting protein properties like thermostability, catalytic activity and fluorescence. METL excels in challenging protein engineering tasks like generalizing from small training sets and position extrapolation, although existing methods that train on evolutionary signals remain powerful for many types of experimental assays. We demonstrate METL's ability to design functional green fluorescent protein variants when trained on only 64 examples, showcasing the potential of biophysics-based protein language models for protein engineering.", "journal": "Nature methods", "year": "2025", "doi": "10.1038/s41592-025-02776-2", "ptypes": [ "Journal Article" ], "first_author": "Gelman S", "dirs": [ "D" ] }, "41074875": { "pmid": "41074875", "title": "Enriching stabilizing mutations through automated analysis of molecular dynamics simulations using BoostMut.", "abstract": "Thermostability is a critical goal in protein engineering for applications of biocatalysts and biomedicines. Despite striking advances in biomolecular predictive modeling, reliably identifying stabilizing mutations remains challenging. Previously, molecular dynamics (MD) simulations and visual inspection have been used as a secondary filter to improve the success rate of mutations pre-selected by thermostability algorithms. However, this approach suffers from low throughput and subjectivity. Here, we introduce Biophysical Overview of Optimal Stabilizing Mutations (BoostMut), a computational tool that standardizes and automates mutation filtering by analyzing dynamic structural features from MD. BoostMut formalizes the principles guiding manual verification, providing a consistent and reproducible stability assessment. Rigorous benchmarking across multiple datasets showed that integrating BoostMut's biophysical analysis improves the prediction rate regardless of the initial thermostability predictor. Given a modest amount of existing mutant stability data, BoostMut's performance can be further enhanced with a lightweight machine learning model. Upon experimentally validating BoostMut predictions on the enzyme limonene epoxide hydrolase, we identified stabilizing mutations previously overlooked by visual inspection and achieved a higher overall success rate. We foresee BoostMut being used for mutation filtering, as an integrated step in thermostability prediction workflows, and for labeling data to train future predictors.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70334", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Korbeld KT", "dirs": [ "D" ] }, "38559182": { "pmid": "38559182", "title": "Biophysics-based protein language models for protein engineering.", "abstract": "Protein language models trained on evolutionary data have emerged as powerful tools for predictive problems involving protein sequence, structure, and function. However, these models overlook decades of research into biophysical factors governing protein function. We propose Mutational Effect Transfer Learning (METL), a protein language model framework that unites advanced machine learning and biophysical modeling. Using the METL framework, we pretrain transformer-based neural networks on biophysical simulation data to capture fundamental relationships between protein sequence, structure, and energetics. We finetune METL on experimental sequence-function data to harness these biophysical signals and apply them when predicting protein properties like thermostability, catalytic activity, and fluorescence. METL excels in challenging protein engineering tasks like generalizing from small training sets and position extrapolation, although existing methods that train on evolutionary signals remain powerful for many types of experimental assays. We demonstrate METL's ability to design functional green fluorescent protein variants when trained on only 64 examples, showcasing the potential of biophysics-based protein language models for protein engineering.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2024.03.15.585128", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Gelman S", "dirs": [ "D" ] }, "41422228": { "pmid": "41422228", "title": "Generalizable and scalable protein stability prediction with rewired protein generative models.", "abstract": "Predicting changes in protein thermostability caused by amino acid substitutions is essential for understanding human diseases and engineering proteins for practical applications. While recent protein generative models demonstrate impressive zero-shot performance in predicting various protein properties without task-specific training, their strong unsupervised prediction ability remains underexploited to improve protein stability prediction. We present SPURS, a deep learning framework that rewires and integrates two complementary protein generative models-a protein language model and an inverse folding model-and reprograms this unified framework for stability prediction through supervised fine-tuning on mega-scale thermostability data. SPURS delivers accurate, efficient, and scalable stability predictions and generalizes to unseen proteins and mutations. Beyond stability prediction, SPURS enables broad applications in protein informatics, including zero-shot identification of functional residues, improved low-N protein fitness prediction, and systematic dissection of stability-pathogenicity for human diseases. Together, these capabilities establish SPURS as a versatile tool for advancing protein stability prediction and protein engineering at scale.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-67609-4", "ptypes": [ "Journal Article" ], "first_author": "Li Z", "dirs": [ "D" ] }, "40027759": { "pmid": "40027759", "title": "Rewiring protein sequence and structure generative models to enhance protein stability prediction.", "abstract": "Predicting changes in protein thermostability due to amino acid substitutions is essential for understanding human diseases and engineering useful proteins for clinical and industrial applications. While recent advances in protein generative models, which learn probability distributions over amino acids conditioned on structural or evolutionary sequence contexts, have shown impressive performance in predicting various protein properties without task-specific training, their strong unsupervised prediction ability does not extend to all protein functions. In particular, their potential to improve protein stability prediction remains underexplored. In this work, we present SPURS, a novel deep learning framework that adapts and integrates two general-purpose protein generative models-a protein language model (ESM) and an inverse folding model (ProteinMPNN)-into an effective stability predictor. SPURS employs a lightweight neural network module to rewire per-residue structure representations learned by ProteinMPNN into the attention layers of ESM, thereby informing and enhancing ESM's sequence representation learning. This rewiring strategy enables SPURS to harness evolutionary patterns from both sequence and structure data, where the sequence likelihood distribution learned by ESM is conditioned on structure priors encoded by ProteinMPNN to predict mutation effects. We steer this integrated framework to a stability prediction model through supervised training on a recently released mega-scale thermostability dataset. Evaluations across 12 benchmark datasets showed that SPURS delivers accurate, rapid, scalable, and generalizable stability predictions, consistently outperforming current state-of-the-art methods. Notably, SPURS demonstrates remarkable versatility in protein stability and function analyses: when combined with a protein language model, it accurately identifies protein functional sites in an unsupervised manner. Additionally, it enhances current low-N protein fitness prediction models by serving as a stability prior model to improve accuracy. These results highlight SPURS as a powerful tool to advance current protein stability prediction and machine learning-guided protein engineering workflows. The source code of SPURS is available at https://github.com/luo-group/SPURS.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.02.13.638154", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Li Z", "dirs": [ "D" ] }, "40662839": { "pmid": "40662839", "title": "OrgNet: orientation-gnostic protein stability assessment using convolutional neural networks.", "abstract": "Accurately predicting the impact of single-point mutations on protein stability is crucial for elucidating molecular mechanisms underlying diseases in life sciences and advancing protein engineering in biotechnology. With recent advances in deep learning and protein structure prediction, deep learning approaches are expected to surpass existing methods for predicting protein thermostability. However, structure-based deep learning models, specifically convolutional neural networks, are affected by orientation biases, leading to inconsistent predictions with respect to the input protein orientation. In this study, we present OrgNet, a novel orientation-gnostic deep learning model using 3D convolutional neural networks to predict protein thermostability change upon point mutation. OrgNet encodes protein structures as voxel grids, enabling the model to capture fine-grained, spatially localized atomic features. OrgNet implements spatial transforms to standardize input protein orientations, thus eliminating orientation bias problem. When evaluated on established benchmarks, including Ssym and S669, OrgNet achieves state-of-the-art performance, demonstrating superior accuracy and robust performance compared to existing methods. OrgNet is available at https://github.com/i-Molecule/OrgNet.", "journal": "Bioinformatics (Oxford, England)", "year": "2025", "doi": "10.1093/bioinformatics/btaf252", "ptypes": [ "Journal Article" ], "first_author": "Buyanov I", "dirs": [ "D" ] }, "40388995": { "pmid": "40388995", "title": "Multidimensional computational strategies enhance the thermostability of alpha-galactosidase.", "abstract": "Alpha-Galactosidase has significant industrial application value in food processing, animal nutrition and medical applications. Microbial-derived α-galactosidases predominate industrial implementation due to high productivity, yet their inherent thermal instability necessitates systematic protein engineering. In this study, we established a dual-strategy protein engineering framework to enhance the thermostability of Aspergillus tubingensis α-galactosidase (AtWU_04653). Strategy I employed integrative computational design tools (ABACUS2/PROSS/DBD2) for mutational library construction, which yielded the dominant mutant A169P exhibiting remarkable performance: 78.52 % enhancement in thermal half-life at 55 °C (pH 4.0) and 52.04 % increase in catalytic efficiency (kcat /Km). Strategy II implemented a physics-based computational methodology combining GROMACS molecular dynamics simulations with Rosetta unfolding free energy calculations and SPIRED machine learning predictions, successfully deriving three stabilized variants (E429I, N380L, T64P) displaying 57.33 %, 67.17 %, and 41.34 % extended half-lives respectively. Notably, E429I and T64P demonstrated concurrent 85.25 % and 65.90 % catalytic activity augmentation (kcat /Km). Both strategies achieved substantial reduction in experimental screening workload while enabling synergistic thermostability-activity optimization. This study uses sequence conservation analysis, unfolding free energy calculation, molecular dynamics simulation, and innovative protein prediction models to establish multidimensional computational strategies for designing mutants, providing new and important technical references for computational design and functional optimization of enzymes.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.144316", "ptypes": [ "Journal Article" ], "first_author": "Zou Y", "dirs": [ "D" ] }, "39665261": { "pmid": "39665261", "title": "Amino acid sequence encodes protein abundance shaped by protein stability at reduced synthesis cost.", "abstract": "Understanding what drives protein abundance is essential to biology, medicine, and biotechnology. Driven by evolutionary selection, an amino acid sequence is tailored to meet the required abundance of a proteome, underscoring the intricate relationship between sequence and functional demand. Yet, the specific role of amino acid sequences in determining proteome abundance remains elusive. Here we show that the amino acid sequence alone encodes over half of protein abundance variation across all domains of life, ranging from bacteria to mouse and human. With an attempt to go beyond predictions, we trained a manageable-size Transformer model to interpret latent factors predictive of protein abundances. Intuitively, the model's attention focused on the protein's structural features linked to stability and metabolic costs related to protein synthesis. To probe these relationships, we introduce MGEM (Mutation Guided by an Embedded Manifold), a methodology for guiding protein abundance through sequence modifications. We find that mutations which increase predicted abundance have significantly altered protein polarity and hydrophobicity, underscoring a connection between protein structural features and abundance. Through molecular dynamics simulations we revealed that abundance-enhancing mutations possibly contribute to protein thermostability by increasing rigidity, which occurs at a lower synthesis cost.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.5239", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Buric F", "dirs": [ "D" ] }, "41455072": { "pmid": "41455072", "title": "Evaluation of ADP Glucose Pyrophosphorylase Subunit Interaction in Wheat by Binding Free Energy Calculations.", "abstract": "ADP-glucose pyrophosphorylase (AGPase; E.C. 2.7.7.27) is the rate-limiting enzyme catalyzing the first committed step of starch biosynthesis in higher plants. The enzyme functions as a heterotetramer comprising two large (LS) and two small (SS) subunits that share 47.02% sequence identity in wheat. To elucidate the structural mechanism underlying heterotetramer assembly, we generated six possible dimeric conformations based on two-fold symmetry, three side-by-side (D1, D2, D3), and three upside-down (D4, D5, D6) orientations and evaluated their stability through all-atom molecular dynamics (MD) simulations combined with MM-GBSA and MM-PBSA free energy analyses. Among all configurations, the D2 heterodimer emerged as the most stable, exhibiting the lowest binding free energy (-15.2 kcal·mol⁻1), largest interface area (1757.2 Å2), and strongest predicted affinity (Kd = 2.1 × 10⁻11 M). Interaction energy analysis revealed that D2 stability is primarily governed by an extensive network of 25 hydrogen bonds and seven salt bridges at the LS-SS interface. Together, these results provide the first comprehensive molecular insight into the assembly and stabilization of wheat AGPase, a central determinant of starch biosynthetic efficiency. These results provide the first deep-learning-based molecular model of wheat AGPase, offering detailed structural insight into its subunit assembly and stability mechanisms. By identifying key interfacial residues that govern complex formation, this study establishes a foundation for rational protein engineering aimed at enhancing AGPase thermostability and catalytic efficiency, traits directly linked to improved starch accumulation and grain yield in cereal crops.", "journal": "Molecular biotechnology", "year": "2026", "doi": "10.1007/s12033-025-01545-y", "ptypes": [ "Journal Article" ], "first_author": "Pandey B", "dirs": [ "D" ] }, "40314227": { "pmid": "40314227", "title": "Semantical and geometrical protein encoding toward enhanced bioactivity and thermostability.", "abstract": "Protein engineering is a pivotal aspect of synthetic biology, involving the modification of amino acids within existing protein sequences to achieve novel or enhanced functionalities and physical properties. Accurate prediction of protein variant effects requires a thorough understanding of protein sequence, structure, and function. Deep learning methods have demonstrated remarkable performance in guiding protein modification for improved functionality. However, existing approaches predominantly rely on protein sequences, which face challenges in efficiently encoding the geometric aspects of amino acids' local environment and often fall short in capturing crucial details related to protein folding stability, internal molecular interactions, and bio-functions. Furthermore, there lacks a fundamental evaluation for developed methods in predicting protein thermostability, although it is a key physical property that is frequently investigated in practice. To address these challenges, this article introduces a novel pre-training framework that integrates sequential and geometric encoders for protein primary and tertiary structures. This framework guides mutation directions toward desired traits by simulating natural selection on wild-type proteins and evaluates variant effects based on their fitness to perform specific functions. We assess the proposed approach using three benchmarks comprising over 300 deep mutational scanning assays. The prediction results showcase exceptional performance across extensive experiments compared to other zero-shot learning methods, all while maintaining a minimal cost in terms of trainable parameters. This study not only proposes an effective framework for more accurate and comprehensive predictions to facilitate efficient protein engineering, but also enhances the in silico assessment system for future deep learning models to better align with empirical requirements. The PyTorch implementation is available at https://github.com/ai4protein/ProtSSN.", "journal": "eLife", "year": "2025", "doi": "10.7554/eLife.98033", "ptypes": [ "Journal Article" ], "first_author": "Tan Y", "dirs": [ "D" ] }, "41793568": { "pmid": "41793568", "title": "Thermostability engineering of microbial transglutaminase using artificial intelligence and investigation of its underlying mechanisms.", "abstract": "The application of artificial intelligence in enzyme molecular evolution has emerged as a research hotspot. However, applying machine learning to enzyme molecular modification still presents many challenges. In particular, accelerating the integration of machine learning and rational design is one of the important development trends in the field of protein engineering. In this study, we experimentally validated key amino acid mutations (E164L, E164P, S199E, and S199Q) predicted by a lab-developed sparse convolutional neural network to enhance the thermostability of microbial transglutaminase. We further investigated the molecular basis of this enhanced stability using molecular dynamics simulations. Compared with the wild type MTG, the thermal stability of the four mutants was significantly improved, and S199E showed the most remarkable improvement. At 60 °C and 50 °C, the half-lives of S199E were 2.3 times and 5.8 times those of the wild type, respectively, and the enzyme activity was increased by 1.4-fold. Molecular dynamics simulations showed that the binding free energy of S199E was - 28.68 kcal/mol, slightly lower than that of the wild type (- 27.96 kcal/mol). The root mean square deviation and root mean square fluctuation of the S199E mutant were 0.25 nm and 0.0566 nm, respectively, showing no significant changes compared with the wild type. LigPlot analysis indicated that E199 formed one hydrogen bonds with A309 and three salt bridges with H201, which might enhance local stability. These findings indicate that the improved thermal stability of the S199E mutant arises from enhanced local structural stability, not from major changes in overall protein structure, and accounts for its slightly lower binding free energy compared to the wild type.", "journal": "Bioresources and bioprocessing", "year": "2026", "doi": "10.1186/s40643-026-01024-5", "ptypes": [ "Journal Article" ], "first_author": "Song X", "dirs": [ "D" ] }, "42136527": { "pmid": "42136527", "title": "PythiaStudio: a one-stop protein engineering platform powered by Pythia model suite.", "abstract": "Predicting how mutations affect protein stability and protein-protein binding affinity is crucial for protein engineering and drug development. Although several computational tools have been developed for these tasks, they often require specialized expertise and are difficult to integrate into unified workflows. Here, we present PythiaStudio (https://pythiastudio.wulab.xyz), a comprehensive web platform that integrates our recently developed Pythia, Pythia-PPI, and Pythia-Pocket models with complementary protein analysis tools. The platform enables users to predict mutational effects on protein stability and protein-protein binding affinity, ligand binding pocket, through an intuitive interface. Additional features include fitness and structure prediction. PythiaStudio provides interactive visualization tools, including mutation heatmaps, sortable result tables, and structure viewers. Importantly, the platform offers an integrated engineering workflow that combines stability and fitness predictions to guide rational protein design. We demonstrate the utility of this workflow through multiple cases, including different glycoside hydrolases and amidases. In these cases, the two-step computational redesign strategy successfully improved both thermostability and catalytic activity. PythiaStudio democratizes access to state-of-the-art deep learning-based protein engineering methods, enabling researchers without computational expertise to perform sophisticated protein engineering.", "journal": "Nucleic acids research", "year": "2026", "doi": "10.1093/nar/gkag408", "ptypes": [ "Journal Article" ], "first_author": "Sun J", "dirs": [ "D" ] }, "39571721": { "pmid": "39571721", "title": "Deep learning for NAD/NADP cofactor prediction and engineering using transformer attention analysis in enzymes.", "abstract": "Understanding and manipulating the cofactor preferences of NAD(P)-dependent oxidoreductases, the most widely distributed enzyme group in nature, is increasingly crucial in bioengineering. However, large-scale identification of the cofactor preferences and the design of mutants to switch cofactor specificity remain as complex tasks. Here, we introduce DISCODE (Deep learning-based Iterative pipeline to analyze Specificity of COfactors and to Design Enzyme), a novel transformer-based deep learning model to predict NAD(P) cofactor preferences. For model training, a total of 7,132 NAD(P)-dependent enzyme sequences were collected. Leveraging whole-length sequence information, DISCODE classifies the cofactor preferences of NAD(P)-dependent oxidoreductase protein sequences without structural or taxonomic limitation. The model showed 97.4% and 97.3% of accuracy and F1 score, respectively. A notable feature of DISCODE is the interpretability of its transformer layers. Analysis of attention layers in the model enables identification of several residues that showed significantly higher attention weights. They were well aligned with structurally important residues that closely interact with NAD(P), facilitating the identification of key residues for determining cofactor specificities. These key residues showed high consistency with verified cofactor switching mutants. Integrated into an enzyme design pipeline, DISCODE coupled with attention analysis, enables a fully automated approach to redesign cofactor specificity.", "journal": "Metabolic engineering", "year": "2025", "doi": "10.1016/j.ymben.2024.11.007", "ptypes": [ "Journal Article" ], "first_author": "Kim J", "dirs": [ "D" ] }, "40706456": { "pmid": "40706456", "title": "Microbial and enzymatic strategies for aflatoxin control: Integrating intelligent detection and computational design.", "abstract": "Aflatoxins (AFs), potent carcinogenic mycotoxins, pose a major global threat to human health. This review offers an in-depth summary of microorganisms capable of degrading AFs, including bacteria, probiotics, and fungi, and highlights the key enzymes responsible for detoxification. We propose an integrated system combining smartphone-based detection, machine learning-driven enzyme discovery, and computationally optimized biocatalyst design for effective AFs mitigation. Microbial degraders facilitate aflatoxin B1 (AFB1) detoxification through extracellular enzymatic activity or cell surface adsorption mechanisms. Enzymes such as laccase, peroxidase, reductase, and lactonase effectively convert AFB1 into less toxic metabolites. However, industrial application of AFs-degrading enzymes remains constrained by their instability and insufficient efficiency. Emerging technologies, including machine learning-driven enzyme discovery and computer-aided protein engineering demonstrate significant potential for enhancing enzyme performance. This review highlights that integrating intelligent detection systems with computer-aided enzyme design offers a transformative framework for proactive AF control throughout food and feed supply chains.", "journal": "Food chemistry", "year": "2025", "doi": "10.1016/j.foodchem.2025.145584", "ptypes": [ "Journal Article", "Review" ], "first_author": "Ouyang B", "dirs": [ "D" ] }, "42038007": { "pmid": "42038007", "title": "From machine learning to multimodal models: The AI revolution in enzyme engineering.", "abstract": "Protein engineering is a powerful tool for applications spanning synthetic biology, biocatalysis, and drug discovery. Recent advances in artificial intelligence (AI), from conventional machine learning (ML) algorithms to large-scale pre-trained protein models, have greatly accelerated enzyme engineering field entering a data-driven era. This review provides a guidance map of current enzyme engineering tasks and builds an integrative perspective on AI methods, model types, landmark tasks, and data resources. We begin by delineating the core modeling tasks in enzyme engineering, which include encompassing function annotation, structural modeling, and property prediction and by reviewing recent advances alongside dominant algorithmic frameworks. Next, we outlined the evolution of AI into enzyme engineering, tracing its progression through four stages: classical machine learning approaches, deep neural networks, protein language models (pLMs), and emerging multimodal architectures. Finally, we highlight four trends that are redefining the landscape of AI-driven enzyme design: (i) the replacement of handcrafted features with unified, token-level embeddings; (ii) a shift from single-modal models toward multimodal, multitask systems; (iii) the emergence of intelligent agents capable of reasoning; and (iv) a movement beyond static structure prediction toward dynamic simulation of enzyme function. Together, these developments are paving the way for intelligent, generalizable, and mechanistically interpretable AI platforms poised to synthetic biology.", "journal": "Biodesign research", "year": "2026", "doi": "10.1016/j.bidere.2025.100044", "ptypes": [ "Journal Article", "Review" ], "first_author": "Shi Z", "dirs": [ "D" ] }, "42439171": { "pmid": "42439171", "title": "Molecular Adaptations of Thermophilic Proteins-From Mechanistic Understanding to Machine Learning Approaches.", "abstract": "Thermophilic proteins are known for their stability and activity at elevated temperatures and hence serve as useful models for engineering enzymes with enhanced stability. The stability of these proteins is governed by both thermodynamic and kinetic factors that affect the folding-unfolding equilibrium and the rate of irreversible denaturation. Structural studies have shown that enhanced thermostability often results from multiple cooperative interactions, including improved hydrophobic packing, strengthened electrostatic interactions, and optimized hydrogen-bonding networks. Comparative studies between thermophilic and mesophilic proteins have helped to identify general mechanisms that support thermal adaptations. Mutational and protein-engineering approaches have further demonstrated the influence of specific amino acid residues or motifs on the stability through altered or modified local structural interactions. In recent years, computational approaches and machine learning methods have emerged as inevitable tools to predict protein stability. Although these approaches have improved the predictions, there still exist several limitations like limited training datasets, bias toward certain protein families, difficulties in model interpretation, etc. Therefore, combining experimental thermodynamic data, structural studies, and modern AI-based computational tools, it is possible to develop more reliable strategies to design thermostable proteins, in particular, for various industrial and biotechnological applications.", "journal": "ChemistryOpen", "year": "2026", "doi": "10.1002/open.70234", "ptypes": [ "Journal Article", "Review" ], "first_author": "Arunachalam S", "dirs": [ "D" ] }, "42422031": { "pmid": "42422031", "title": "Growth factor thermostability dataset derived from experimental and computational approaches to accelerate cultured meat development.", "abstract": "This project compiled experimentally measured and computationally predicted thermostability data for growth factors (GFs) commonly used in cultured meat and seafood (CM) production. Data were generated on recombinant GFs using thermal shift assays and combined with publicly available data using a structured literature curation workflow developed using a Large Language Model (LLM) with manual validation. In addition, in silico protein stability calculations were generated using FoldX. Data such as GF identity, species origin, recombinant host, variant information, method of measurement, and experimental conditions were collected and standardized into a tabular format. The combined dataset contains 513 melting temperature (Tm) values across diverse GFs and variants, along with additional thermodynamic parameters on a subset of 15 GFs. The data were harmonized, standardized, and released as an open-source dataset suitable for computational modeling, machine learning workflows, culture media design, and safety assessment frameworks. This resource supports research evaluating GF stability under production- and processing- relevant conditions and provides a foundation for future data expansion, including additional GF variants and other growth factors. By consolidating dispersed thermostability measurements into a unified dataset, supported by experimental data, the resource supports accessibility and reuse across the CM, protein engineering, and regulatory science communities.", "journal": "Data in brief", "year": "2026", "doi": "10.1016/j.dib.2026.113042", "ptypes": [ "Journal Article" ], "first_author": "Ng W", "dirs": [ "D" ] }, "42620015": { "pmid": "42620015", "title": "HaloMPNN: retraining ProteinMPNN on halophilic proteomes for salt-tolerant enzyme design.", "abstract": "Machine learning-guided protein sequence redesign is now routinely used to optimize multiple properties relevant for protein engineering, most prominently thermostability and recombinant expression levels. Salt tolerance is a valuable property for \"blue-biotechnology\"- enabled biomanufacturing, yet no generative computational method exists to redesign proteins for increased salt tolerance. We hypothesized that a training dataset heavily biased toward salt-adapted proteomes would yield a model capable of designing proteins with halophilic properties. To test this, we retrained the sequence redesign model ProteinMPNN on proteins from \"salt-in\" extreme halophiles such as Haloarcula marismortui , a Dead Sea archaeon that grows optimally near 3-4 M NaCl, roughly six times the salinity of seawater, and accumulates molar concentrations of salts in its cytoplasm. Our model, HaloMPNN, redesigns non-halophilic proteins so that their properties shift towards those of natural halophilic proteins: lower predicted isoelectric point, greater surface acidity, and reduced surface and core hydrophobicity. Redesigning a broad range of non-halophilic proteins with SolubleMPNN, ProteinMPNN, and HyperMPNN shows that this shift is specific to HaloMPNN rather than a generic consequence of sequence redesign. HaloMPNN therefore offers both a route to designing candidate salt-tolerant enzymes and a means of identifying the characteristics that underlie halophilic adaptation.", "journal": "bioRxiv : the preprint server for biology", "year": "2026", "doi": "10.64898/2026.08.02.742362", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "Lee AL", "dirs": [ "D" ] }, "40414395": { "pmid": "40414395", "title": "ActiMut-XGB: Predicting thermodynamic stability of point mutations for CALB with protein language model.", "abstract": "Predicting the functional impact of single-point mutations on protein residual activity, especially after high-temperature incubation, is critical in protein engineering. We present an innovative machine learning model based on eXtreme Gradient Boosting that leverages protein sequence data to predict thermostability, circumventing the need for three-dimensional structural information. Our model integrates features from the ESM2 language model, physicochemical properties, evolutionary features, and positional features. A key advancement is the use of transfer learning with thermal stability data from various proteins, which enhances prediction accuracy and generalizability. To fine-tune and validate the model, we used experimental data from Candida antarctica lipase B single-point mutants, a widely studied enzyme in biocatalysis and industrial applications. Despite potential limitations of Gibbs free energy values in capturing all factors influencing thermostability, our model represents a significant improvement over traditional approaches, providing valuable insights for protein engineering, enzyme optimization, and therapeutic protein development.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.144609", "ptypes": [ "Journal Article" ], "first_author": "Jiang Y", "dirs": [ "D" ] }, "41873756": { "pmid": "41873756", "title": "Deep learning-guided dual-fitness evolution of T7 RNA polymerase for enhanced stability and activity.", "abstract": "In protein engineering, simultaneously improving multiple fitness attributes is a critical yet challenging goal, largely due to the vastness of sequence space, the multifaceted interplay among different traits, and the complexity of non-linear mutational effects (epistasis). To address this, we developed a data-driven evolutionary strategy that couples in silico deep learning with a wet-lab multi-objective selection workflow. By employing independent model fine-tuning for distinct traits, our approach facilitates navigating the fitness landscape to identify beneficial mutation combinations. We applied this strategy to T7 RNA polymerase (T7 RNAP), performing dual-fitness evolution to simultaneously enhance thermostability and activity at elevated temperatures. After five rounds of iterative evolution, we obtained T7 RNAP mutants exhibiting a melting temperature (Tm) increase of >10°C, a 60-fold enhancement in high-temperature activity, and a 70% reduction in by-product content. Validation in cell transfection demonstrated their potential for producing high-quality mRNA for industrial applications.", "journal": "Nucleic acids research", "year": "2026", "doi": "10.1093/nar/gkag259", "ptypes": [ "Journal Article" ], "first_author": "Jiang F", "dirs": [ "D" ] }, "40769241": { "pmid": "40769241", "title": "Computational-assisted protein engineering to develop thermostable and highly active catalase for industrial and biocatalytic applications.", "abstract": "Catalases are ideal biocatalysts for hydrogen peroxide removal in industrial wastewater and enzymatic processes, yet the practical application is hindered by poor thermostability. In this study, a highly active catalase was identified from Fictibacillus enclensis using a computer-assisted screening strategy. To overcome thermal instability, a computational design framework integrating ProteinMPNN-based sequence optimization with physics-based energy calculations was developed. The engineered variant, D78P/K201R/E384Y/T435A, exhibited a 1.9-fold increase in catalytic efficiency and a 4.9-fold extension of half-life at 40°C. Molecular dynamics simulations and structural analyses revealed that the mutations conferred enhanced global rigidity through stabilized hydrogen-bond networks. Moreover, the variant was employed for the treatment of industrial effluents containing hydrogen peroxide residues andthe biocatalytic upgrading of glycerol. This study not only paved the way for industrial applications of catalase but also established a strategic framework for enhancing both stability and catalytic activity in enzyme engineering.", "journal": "Bioresource technology", "year": "2025", "doi": "10.1016/j.biortech.2025.133081", "ptypes": [ "Journal Article" ], "first_author": "Xu S", "dirs": [ "D" ] }, "40938899": { "pmid": "40938899", "title": "Resolution of physics and deep learning-based protein engineering filters: A case study with a lipase for industrial substrate hydrolysis.", "abstract": "Computational enzyme design remains a powerful yet imperfect tool for optimizing biocatalysts, especially when targeting non-natural substrates. Using design tools we investigated Pseudomonas aeruginosa LipA, a lipase with a flexible lid domain crucial for substrate binding and turnover, aiming to enhance its hydrolysis of the industrially relevant substrate Roche ester. We generated an initial set of single-point mutations based on structural proximity to the active site and evaluated their effects using a computational pipeline integrating molecular dynamics (MD) simulations, density functional theory (DFT) calculations, and ensemble-based energy scoring. While we identified several active variants, attempts to rank them by activity using structural features, such as hydrogen bond formation or residue flexibility, failed. Deep learning models, applied post hoc for structural analysis via AlphaFold3, produced nearly identical active site geometries across variants, irrespective of activity. Reaction pathway analysis revealed energy barriers varying by 5-15 kcal/mol depending on substrate conformation, with the nucleophile addition step consistently rate-limiting. However, these small energetic shifts, likely critical for incremental activity changes, were indistinguishable by current computational or deep learning methods. Our results highlight the limitations of existing approaches in resolving subtle functional differences and underscore the need for improved benchmarks, reactive force fields, and more sensitive ranking metrics. Advancing these areas will be essential for designing enzymes with gradual, evolution-like activity improvements and for bridging the gap between structural prediction and catalytic function.", "journal": "PloS one", "year": "2025", "doi": "10.1371/journal.pone.0332409", "ptypes": [ "Journal Article" ], "first_author": "Gardiner S", "dirs": [ "D" ] }, "40643334": { "pmid": "40643334", "title": "Identifying pivotal sites affecting thermostability of GH11 xylanase via conventional and deep learning-based energy calculation.", "abstract": "The GH11 xylanase XynCDBFV, derived from Neocallimastix patriciarum, is widely used in various industries. However, its relatively low thermostability limits its potential. In this study, two computational approaches-Rosetta Cartesian_ddG and the deep learning-based tool Pythia-were employed to identify key residues affecting XynCDBFV thermostability. Both methods highlighted residues D57 and G201 as promising targets. Site-saturation mutagenesis at these positions yielded 18 variants with improved thermostability. Notably, three D57 variants (D57N/S/T) exhibited a 10°C increase in optimal temperature and retained 3.4%-21.7% higher residual activity than the wild type after 1-h incubation at 80°C. Five G201 variants (G201A/C/F/I/V) showed 5°C/10°C enhancements in optimal temperatures, with 10.1%-22.6% improved residual activity. These findings validate D57 and G201 as pivotal sites influencing thermostability. However, combining beneficial mutations from both sites led to reduced thermostability due to negative epistatic interactions. Comparative analysis revealed that while Rosetta Cartesian_ddG offers broader screening, it suffers from a high false discovery rate. In contrast, Pythia provides a balanced trade-off between precision and speed. This study offers a robust framework for enzyme thermostability enhancement and underscores the value of integrating computational predictions with experimental validation in protein engineering.", "journal": "FEMS microbiology letters", "year": "2025", "doi": "10.1093/femsle/fnaf072", "ptypes": [ "Journal Article" ], "first_author": "Zhang S", "dirs": [ "D" ] }, "41071611": { "pmid": "41071611", "title": "Data-efficient protein mutational effect prediction with weak supervision by molecular simulation and protein language models.", "abstract": "Machine learning-based protein mutational effect prediction is widely used in protein engineering and pathogenicity prediction, but training data scarcity remains a major challenge due to high costs of experimental measurements. A previous study proposed data augmentation using computational estimates by molecular simulation. However, this approach has been limited to predicting mutational effects on thermostability. Here, we present a new data augmentation method that combines molecular simulation with zero-shot prediction computed by protein language models. These computational estimates serve as 'weak' training data to supplement experimental training data. Our method dynamically adjusts the weight and inclusion of weak training data based on available experimental training data. This reduces potential negative impacts of weak training data while extending applicability to diverse protein properties such as binding affinity and enzymatic activity. Benchmark tests demonstrate that our method improves prediction accuracy particularly when experimental training data are scarce. These results indicate the capability of our approach to advance protein engineering and pathogenicity prediction in small data regimes.", "journal": "Briefings in bioinformatics", "year": "2025", "doi": "10.1093/bib/bbaf536", "ptypes": [ "Journal Article" ], "first_author": "Deguchi T", "dirs": [ "D" ] }, "40247745": { "pmid": "40247745", "title": "Deep mutational scanning reveals a de novo disulfide bond and combinatorial mutations for engineering thermostable myoglobin.", "abstract": "Engineering protein stability is a critical challenge in biotechnology. Here, we used massively parallel deep mutational scanning (DMS) to comprehensively explore the mutational stability landscape of human myoglobin (hMb) and identify key mutations that enhance stability. Our DMS approach involved screening over 10,000 hMb variants by yeast surface display, single-cell sorting, and high-throughput DNA sequencing. We show how surface display levels serve as a proxy for thermostability of soluble hMb variants and report strong correlations between DMS-derived display levels and top-performing machine learning stability prediction algorithms. This approach led to the discovery of a variant with a de novo disulfide bond between residues R32C and C111, which increased thermostability by >12°C compared with wild-type hMb. By combining single stabilizing mutations with R32C, we engineered combinatorial variants that exhibited predominantly additive effects on stability with minimal epistasis. The most stable combinatorial variant exhibited a denaturation temperature exceeding 89°C, representing a >17°C improvement over wild-type hMb. Our findings demonstrate the capabilities in DMS-assisted combinatorial protein engineering to guide the discovery of thermostable variants and highlight the potential of massively parallel mutational analysis for the development of proteins for industrial and biomedical applications.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70112", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "Küng C", "dirs": [ "D" ] }, "42239726": { "pmid": "42239726", "title": "Dynamics-informed multigraph neural networks for protein thermostability prediction and residue-level interpretation.", "abstract": "Thermostability is crucial for protein engineering, but experimental determination of melting temperatures is costly and time-consuming. Most existing machine learning approaches for melting temperature prediction rely on sequential and structural features while largely overlooking protein dynamics, a key determinant of conformational stability. Here, we introduce three dynamics-informed graphs and evaluate their utility relative to sequential or structural representations. Specifically, we propose three dynamical graphs derived from normal mode analysis-co-directionality, coordination, and deformation graphs-and assess whether they can serve as alternatives to contact graphs in predicting melting temperatures. We then integrate sequential, structural, and dynamical information within a unified multigraph learning framework. Our results show that dynamical graphs achieve comparable predictive performance to conventional contact graphs, and that combining structural and dynamical graphs yields consistent, albeit modest, improvements over contact-only models. Furthermore, Laplacian centrality analysis on coordination graphs reveals enrichment tendencies and mechanical signals, providing interpretability. Overall, this work demonstrates the value of protein dynamics-informed multigraph representations for learning protein properties.", "journal": "iScience", "year": "2026", "doi": "10.1016/j.isci.2026.116089", "ptypes": [ "Journal Article" ], "first_author": "Chen YL", "dirs": [ "D" ] }, "41422407": { "pmid": "41422407", "title": "EvoZymePro-Cat: A Protein-Ligand-Aware Deep Learning Framework for Predicting Mutation Effects in Enzyme Function.", "abstract": "Enzymes are biological catalysts that speed up chemical reactions in an eco-friendly way. Precise enzyme design is hindered by vast sequence space and intricate sequence-structure-function interdependencies. To address these challenges, we developed EvoZymePro-Cat (EZPro-Cat), a deep learning platform for enzyme mutant screening. Conventional methods for predicting absolute mutant activities suffer from systematic errors and limited generalizability. Our pairwise comparison framework directly models relative activity superiority between variants, eliminating dependence on absolute value predictions. The framework integrates full sequence and local structure semantics of protein and ligand information using bilinear attention mechanisms. Protein sequences are encoded using the ESM1b transformer model. Ligands are represented through MolT5 embeddings and MACCS molecular fingerprints. The adaptability of protein residues to their microenvironments is captured by integrating structural features and site-specific evolutionary characteristics. Bilinear attention mechanisms capture long-range intermolecular interactions during catalysis by bidirectional projection and weighted fusion of protein-ligand features. Compared to existing methods, our model exhibits superior performance in identifying improved enzyme mutants through comparative prediction of mutation effects on activity, such as Km and kcat. For deep mutation scanning data sets, a few-shot learning strategy combined with the EZPro-Cat framework boosts prediction precision (AUC 0.908). By using integrated multimodal representations, EZPro-Cat offers a mechanistic and practical solution for functional profiling of intraprotein variants, driving paradigm shifts in highly efficient enzyme discovery and directed evolution.", "journal": "ACS synthetic biology", "year": "2026", "doi": "10.1021/acssynbio.5c00755", "ptypes": [ "Journal Article" ], "first_author": "Xu R", "dirs": [ "D" ] }, "42173379": { "pmid": "42173379", "title": "Computational and deep-learning-guided design of raspberry ketone synthase towards concurrent gains in activity and thermostability.", "abstract": "Raspberry ketone synthase is the key enzyme for raspberry ketone biosynthesis, yet its activity and thermostability remain suboptimal for industrial use. By combining computational and deep-learning-guided design, we obtained a Rubus idaeus raspberry ketone synthase (RiRZS1) mutant M3 with 12.06-fold higher relative activity and 38.71-fold improved thermostability compared with the wild type. Kinetic analysis showed that M3 exhibited 14.89-fold higher kcat and 5.91-fold higher catalytic efficiency(kcat/Km), indicating markedly enhanced catalytic competence. Structural inspection and molecular dynamics simulations revealed that M3 induced only subtle conformational rearrangements but reduced global flexibility, accounting for its improved thermostability. Meanwhile, tunnel and substrate-pocket reshaping in M3 was associated with increased prereaction-state populations, which may contribute to enhanced catalytic performance. In whole-cell biotransformation, the raspberry ketone titer of M3 was 2.38-fold that of wild-type, and the corresponding space-time yield was 3.57-fold higher. This computational and artificial intelligence-assisted framework enabled a synergistic optimization of RiRZS1 activity and stability, and can serve as a transferable design framework for protein engineering in sustainable bio-manufacturing.", "journal": "Bioresource technology", "year": "2026", "doi": "10.1016/j.biortech.2026.134952", "ptypes": [ "Journal Article" ], "first_author": "Yang Y", "dirs": [ "D" ] }, "42649115": { "pmid": "42649115", "title": "FireProtASR 2.0: evolution-guided Design of Protein Ancestors and Successors with phylogenetics and machine learning.", "abstract": "Evolution-guided protein design remains one of the most effective strategies for engineering proteins with enhanced stability, activity, or specificity. To make these approaches more accessible, we previously developed FireProtASR-a fully automated pipeline for ancestral sequence reconstruction (ASR). Here, we present FireProtASR 2.0, a significantly enhanced version that extends the design space beyond ancestral inference by integrating a successor sequence predictor (SSP) and a generative model based on variational autoencoders (VAEs). These new modules enable both 'prospective' and 'retrospective' evolutionary design strategies. The SSP module predicts likely future mutations based on site-wise evolutionary trends, and the method was previously validated through in silico benchmarks, demonstrating improvements in thermostability and activity. The VAE module captures global evolutionary constraints in a low-dimensional latent space, from which novel functional ancestral-like variants can be sampled. The VAE-based design strategy was previously validated experimentally on the haloalkane dehalogenase family, yielding variants with enhanced thermostability while maintaining catalytic activity. Both these modules are newly available in FireProtASR in a fully automated pipeline, guiding the users via an interactive graphical user interface. With expanded functionality, modernized user interface, and a more robust backend, FireProtASR 2.0 provides a comprehensive, accessible, and fully automated platform for evolutionary-based protein engineering (https://loschmidt.chemi.muni.cz/fireprotasr/).", "journal": "Briefings in bioinformatics", "year": "2026", "doi": "10.1093/bib/bbag436", "ptypes": [ "Journal Article" ], "first_author": "Kohout P", "dirs": [ "D" ] }, "42850317": { "pmid": "42850317", "title": "Integrative Approaches in Protein Structure/Function Analysis, Design, and Engineering.", "abstract": "Over the past decade, protein design has drawn considerable attention and emerged as a promising tool for sustainable and efficient biocatalytic processes, driven by breakthroughs in DNA sequencing, gene synthesis, bioinformatics, and artificial intelligence. Enabled by strategies such as directed evolution, structure/function analysis, and (semi-)rational design, engineered enzymes can now accommodate nonnatural substrates, catalyze novel reactions, and operate under harsh conditions including elevated temperatures and organic solvents.This chapter provides a comprehensive overview of major strategies in protein design including directed evolution, rational design, and semi-rational design. Emphasis is placed on recent advances in high-throughput screening technologies, identification of functionally critical residues, and optimization of site-specific mutations. Furthermore, the integration of artificial intelligence and multiscale modeling to enzyme design is discussed, highlighting data-driven decision-making and deep learning-assisted prediction of enzymatic performance. Looking forward, biocatalysis is poised to further expand its applications in valorization of renewable resources, biofuels, and synthetic chemistry. Powered by ongoing advances in AI and computational protein design, the development of enzymes with unprecedented catalytic functions is anticipated to accelerate rapidly.", "journal": "Advances in biochemical engineering/biotechnology", "year": "2026", "doi": "10.1007/10_2026_369", "ptypes": [ "Journal Article" ], "first_author": "Sun Z", "dirs": [ "D" ] }, "42063854": { "pmid": "42063854", "title": "Engineering Plant-Derived P450 Enables the Efficient Production of Berberine through a Concise and Modular Enzymatic Cascade in E. coli.", "abstract": "Sustainable and scalable production of plant natural products remains a major challenge at the interface of synthetic biology, metabolic engineering, and drug discovery, often constrained by plant-membrane-bound cytochrome P450s. To overcome the bottleneck in P450-catalyzed methylenedioxy-bridge (MDB) formation, we designed a concise, modular Escherichia coli pathway to the alkaloid berberine that bypasses multiple native steps and enables high-level accumulation of the P450-dependent intermediate. We then implemented integrated engineering of CjCYP719A1, including expression optimization, structure-guided design, consensus mutagenesis, and machine learning, to obtain a variant with 8.9-fold higher activity and improved thermostability. This enabled a berberine titer of 353 mg/L in scaled-up preparations, the highest reported to date, demonstrating industrial potential. Furthermore, mechanistic studies reveal their improved catalytic activity and thermostability. Together, this work provides a practical framework for functional expression and systematic optimization of plant-derived P450s to enable efficient production of plant natural products in E. coli.", "journal": "JACS Au", "year": "2026", "doi": "10.1021/jacsau.6c00169", "ptypes": [ "Journal Article" ], "first_author": "Qin Y", "dirs": [ "D" ] }, "41817278": { "pmid": "41817278", "title": "Computational Redesign of an Ancestral Xylose Isomerase: Tuning the Substrate Preference and Thermostability for Biomass Valorization.", "abstract": "Xylose isomerase is a promising biocatalyst for lignocellulose valorization, but natural enzymes are limited by a preference for either d-xylose or d-glucose. Here, we integrated ancestral sequence reconstruction with deep learning methods to identify ASR285, an enzyme active toward both d-glucose and d-xylose. To enhance its potential for practical applications, we designed a flexible lid by truncating the α3-helix and reducing steric hindrance through the W140F mutation. Guided by computational analysis, the ASR285-M2 mutant (ASR285-Δhelix9/W140F/S147A/W189Y) was engineered, resulting in a 7.85-fold increase in the catalytic activity toward d-glucose while preserving the native d-xylose isomerization capacity. It also exhibited an approximately 2-fold longer half-life, indicating improved thermostability. In real lignocellulosic hydrolysates, ASR285-M2 achieved a 9-fold higher d-fructose yield than ASR285, enabling simultaneous production of d-fructose and d-xylulose. This semirational strategy successfully optimized both substrate preference and stability, providing a practical biocatalyst for biomass valorization.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.5c17228", "ptypes": [ "Journal Article" ], "first_author": "Yue S", "dirs": [ "D" ] }, "42861354": { "pmid": "42861354", "title": "Integrated computation-guided thermostabilization of the fungal sweet protein Honey Truffle Sweetener.", "abstract": "Thermostability limits the use of sweet proteins as sugar substitutes in thermally processed foods. Here, we report an integrated computation-guided workflow for thermostabilizing Honey Truffle Sweetener (HTS), a recently identified fungal sweet protein with low intrinsic stability. We determined the HTS crystal structure and used it to guide subsequent computational analyses. Rosetta and ThermoMPNN evaluated candidate substitutions from complementary physics-based and deep-learning-based perspectives, and the selected single-mutant library was experimentally characterized. The resulting melting-temperature data were then used to develop SMRC-Net, which combines mutation-centered features from the ESM2 protein language model with molecular dynamics-derived structural descriptors to support mutation prioritization. Repeated nested cross-validation supported quantitative Tm prediction within the HTS scaffold, while prospective testing showed 9/10 directional agreement in a small, computationally selected variant set. Validated substitutions were then assembled stepwise in small modules, followed by the introduction of an engineered disulfide bond. The final variant, FM, had a melting temperature of 82.1 °C, 27.5 °C higher than that of wild-type HTS. Untreated FM retained sweetness, while separate heat- and pH-treatment assays showed retained solubility and largely preserved secondary-structure profiles. Sweetness after these treatments was not evaluated. Molecular dynamics analyses indicated that FM was not uniformly rigidified but instead exhibited localized flexibility redistribution and a more consolidated conformational ensemble. This work provides a practical computation-guided framework for protein thermostabilization, with potential applicability to other compact proteins.", "journal": "Current research in food science", "year": "2026", "doi": "10.1016/j.crfs.2026.101584", "ptypes": [ "Journal Article" ], "first_author": "Wang Z", "dirs": [ "D" ] }, "40235597": { "pmid": "40235597", "title": "ProstaNet: A Novel Geometric Vector Perceptrons-Graph Neural Network Algorithm for Protein Stability Prediction in Single- and Multiple-Point Mutations with Experimental Validation.", "abstract": "Proteins play a critical role in biology and biopharma due to their specificity and minimal side effects. Predicting the effects of mutations on protein stability is vital but experimentally challenging. Deep learning offers an efficient solution to this problem. In the present work, we introduced ProstaNet, a deep learning framework that predicts stability changes resulting from single- and multiple-point mutations using geometric vector perceptrons-graph neural network for 3-dimensional feature processing. For training ProstaNet, we meticulously crafted ProstaDB, a comprehensive and pristine thermodynamics repository, including 3,784 single-point mutations and 1,642 multiple-point mutations. We also created thermodynamic looping for enlarging the limited data size of multiple-point mutation and applied an innovative clustering method to generate a standard testing set of multiple-point mutation. Besides, we identified residue scoring as the most important encoding method in protein properties prediction. With these innovations, ProstaNet accurately predicts thermostability changes for both single-point and multiple-point mutations without showing any bias. ProstaNet achieves an accuracy of 0.75, outperforming existing methods for single-point mutation prediction, including ThermoMPNN (0.63), PoPMuSiCsym (0.66), MUPRO (0.52), and FoldX (0.71). ProstaNet also achieves a 1.3-fold increase in accuracy compared to FoldX for multiple-point mutation predictions. Validated by experiment, 4 out of 5 single-point mutation predictions (80%) and all multiple-point mutation predictions (100%) for HuJ3 mutants were accurate, demonstrating the potential benefits of ProstaNet for protein engineering and drug development.", "journal": "Research (Washington, D.C.)", "year": "2025", "doi": "10.34133/research.0674", "ptypes": [ "Journal Article" ], "first_author": "Liang T", "dirs": [ "D" ] }, "40955661": { "pmid": "40955661", "title": "Blocking extracellular glycine uptake mediated by GlyT1 mitigates protoporphyria.", "abstract": "Accumulation of the light-reactive heme precursor protoporphyrin IX (PPIX) in blood causes protoporphyria, a disease characterized by severe pain resulting from sunlight exposure, as well as by the occurrence of liver failure in some patients. Thus, decreasing PPIX biosynthesis is a promising strategy to treat protoporphyria. In this issue of the JCI, Ducamp et al. report that inhibition of the glycine plasma membrane transporter GLYT1 using bitopertin decreased PPIX accumulation and ameliorated liver disease using human in vitro and mouse in vivo models. Their findings support the ongoing development of bitopertin to treat protoporphyria, while concurrently pointing to underexplored roles of glycine in erythroid cells.", "journal": "The Journal of clinical investigation", "year": "2025", "doi": "10.1172/JCI197344", "ptypes": [ "Journal Article", "Comment", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "42705751": { "pmid": "42705751", "title": "Enhancing the functionality and pH responsiveness of anthocyanin-based intelligent packaging for food quality monitoring.", "abstract": "Anthocyanins, naturally occurring flavonoid pigments, have attracted substantial interest for intelligent food packaging due to their pronounced halochromic behavior, antioxidant activity, and biocompatibility. This review critically evaluates recent advances in green extraction technologies, including ultrasound-, microwave-, enzyme-, pulsed electric field-, and supercritical fluid-assisted extraction, with emphasis on their effects on anthocyanin yield, structural integrity, and pH-responsive performance. Furthermore, the influence of polymer matrices and fabrication techniques (e.g., solvent casting, layer-by-layer assembly, and electrospinning) on film microstructure, physicochemical properties, and sensing behavior is systematically discussed. Material engineering strategies, including copigmentation, inclusion complexation, nanoencapsulation, and metal-organic framework immobilization, are systematically evaluated for their roles in enhancing color stability, pH and volatile amine sensitivity, controlled release, and multifunctionality. Environmental and safety aspects, including migration behavior, biocompatibility, biodegradability, and regulatory considerations, are also critically assessed to facilitate industrial implementation. Overall, this review establishes a comprehensive structure-property-performance framework linking anthocyanin chemistry, extraction processes, material engineering, and sensing functionality, thereby providing strategic insights for the rational design and commercialization of next-generation intelligent packaging systems for real-time food freshness monitoring.", "journal": "Food research international (Ottawa, Ont.)", "year": "2026", "doi": "10.1016/j.foodres.2026.120396", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "40590173": { "pmid": "40590173", "title": "Cerium Oxide Nanozymes: Exploring Synthesis, Catalytic Activity, and Biomedical Potential.", "abstract": "Cerium oxide (CeO2) nanozymes have emerged as a novel class of nanozyme materials distinguished by their tunable valence states (Ce3+/Ce4+) and abundant surface oxygen vacancies (OVs), demonstrating exceptional chemical stability, cost-effectiveness, and scalable manufacturability. Precise control of reaction temperature or implementation of carrier-mediated crystal growth strategies effectively enhances nanoparticle dispersion and mitigates aggregation. This review systematically summarizes recent advancements in CeO2 nanozymes: First, it elaborates on cutting-edge synthetic methodologies, encompassing self-supported strategies (calcination, hydrothermal, and sol-gel) and carrier-mediated approaches (small-molecule compounds, macromolecular compounds, and nanomaterials), with comparative analysis of their respective merits and limitations. Second, the catalytic mechanism is comprehensively elucidated, particularly focusing on the modulation of electron transfer kinetics and surface adsorption energies in metal/nonmetal hybrid systems. By optimizing oxygen vacancy concentration to regulate Ce3+/Ce4+ redox cycling dynamics, significant enhancement in redox efficiency and catalytic stability can be achieved, enabling precise enzyme-like activity regulation. Furthermore, the review comprehensively summarizes breakthrough applications in multidimensional biomedical fields, including precision tumor therapy, intelligent antibacterial systems, inflammatory microenvironment regulation, and ultrasensitive biosensing. This work establishes a theoretical framework for structure-activity relationship-guided rational design of high-performance CeO2 nanozymes and delineates their potential translational applications in precision medicine.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2025", "doi": "10.1002/smll.202504436", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40990394": { "pmid": "40990394", "title": "An Automated Multi-Sample Digital LAMP Platform for Quantitative Detection of SDC2 Methylation in Colorectal Cancer.", "abstract": "DNA methylation of the SDC2 gene has emerged as a critical biomarker for early detection of colorectal cancer (CRC). However, effective clinical application demands analytical platforms that offer enhanced accuracy, speed, and sample high-throughput capacity. In this study, we present an automated multisample digital loop-mediated isothermal amplification (dLAMP) platform designed to meet these requirements. Our platform integrates a dual-volume microfluidic chip capable of analyzing 4 or 8 samples in parallel, significantly enhancing sample throughput and efficiency. Integrated with automatic sample injection and advanced image processing, the platform achieves streamlined, multistep analysis of SDC2 methylation levels. We designed methylation-specific (MS) and unmethylation-specific (UMS) LAMP primers with high specificity. Validation using CRC cell lines (HCT116 and Caco-2) and clinical tissue samples demonstrated robust performance with high sensitivity, specificity and reproducibility. The results showed elevated SDC2 methylation was detected in tumor tissues across all CRC stages. This integrated automated dLAMP platform provides a robust, multisample high-throughput tool for methylation-based diagnostics and holds strong potential for noninvasive early cancer screening and clinical biomarker applications.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c03244", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "39914541": { "pmid": "39914541", "title": "Insight to critical role of surface tension for cellulose-based film: A review.", "abstract": "Cellulose-based films are increasingly popular in material science due to their abundance, biodegradability, and strong mechanical properties, making them ideal for food packaging, pharmaceuticals, and biomedical engineering applications. However, these films often experience excessive shrinkage during formation, which can reduce their effectiveness. This review focuses on the importance of surface tension in the formation and properties of cellulose films, showing how it affects film thickness, uniformity, and mechanical strength. The review explores how chemical modifications, environmental factors, and solvent choices affect surface tension and film properties. Then, it discusses various strategies to control surface tension and reduce shrinkage, such as the controlled drying conditions, plasticizers and use of surfactants. Managing the drying environment, including temperature, humidity, and method, is crucial for controlling surface tension and shrinkage. Plasticizers enhance flexibility, allowing cellulose chains to rearrange during drying, while cationic surfactants reduce shrinkage by modifying cellulose surfaces and stabilizing film structures. Future developments could include new surface modification techniques and the use of nanocellulose to further refine film characteristics. Overall, the ability to control surface tension is key to improving the performance of cellulose-based films, meeting the rising demand for sustainable materials in various industries.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.140680", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "42334550": { "pmid": "42334550", "title": "Development of a digital loop-mediated isothermal amplification using a digital polymerase chain reaction device.", "abstract": "This study established a robust digital loop-mediated isothermal amplification (dLAMP) workflow using the QIAcuity nanoplate-based dPCR system to overcome the limitations of traditional isothermal reactions and low partition counts. By optimizing reactions targeting the mitochondrial CO1 region of Limnoperna fortunei with diluted EvaGreen Master Mix, the method achieved high-throughput absolute quantification across a wide dynamic range without requiring standard curves. Results demonstrated that while conventional turbidimetric LAMP showed moderate variability accompanied by occasional delayed amplification at low concentrations, dLAMP maintained stable and clear detection at 1.52 × 101 copies. Despite minor saturation effects at high concentrations and stochasticity at extremely low levels, the QIAcuity-based dLAMP demonstrated enhanced quantitative stability compared to the conventional bulk assay. This user-friendly platform potentially reduces the risk of molecular dropout through high partition density (up to 26,000 partitions), offering a practical and precise tool for diagnostics, food safety, and environmental DNA (eDNA) monitoring, especially for low-copy target detection.", "journal": "Analytical sciences : the international journal of the Japan Society for Analytical Chemistry", "year": "2026", "doi": "10.1007/s44211-026-00943-9", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41497180": { "pmid": "41497180", "title": "Characterization of the ketoreductase domain of pikromycin module 2.", "abstract": "Polyketides represent a diverse class of natural products that serve as major sources of medicinal compounds. Their biosynthesis is generally catalyzed by multimodular polyketide synthase comprising functional domains, such as a β-ketosynthase, an acyltransferase, and an acyl carrier protein. These domains mediate the elongation of polyketide chains via decarboxylative Claisen-like condensation. A reductive loop comprising β-ketoreductase (KR), dehydratase (DH), and enoyl reductase domains converts the β-keto group into a hydroxy group, alkene, and alkane, respectively. Particularly, the KR domains are pivotal in determining the stereochemical configurations of the hydroxy and methyl groups on the macrolide backbone and are classified into A1, A2, B1, B2, and C types. In this study, we performed a reductive loop exchange using pikromycin PKS, PikAIII module 5 (PikAIII-M5) as a template. The PikAIII-M5 was derived from the pikromycin biosynthetic gene cluster of the pikromycin-producing Streptomyces sp. AM4900. Next, we constructed a chimeric enzyme by replacing the KR domain of PikAIII-M5 with a DH-KR di-domain derived from PikAI module 2 (PikAI-M2), followed by the artificial addition of a thioesterase domain derived from PikAIV module 6. Thereafter, we evaluated the enzymatic activity of the construct using various chemically synthesized N-acetylcysteamine substrate analogs. This demonstrated that the chimeric module enzyme catalyzed the formation of (2R,3R,4S)-3-hydroxy-2,4-dimethylheptanoic acid, indicating that the KR domain of PikAI-M2 is a B1-type. These findings offer insights into the unresolved classification of KR domains that do not strictly conform to the Caffrey motif.", "journal": "Chemical science", "year": "2026", "doi": "10.1039/d5sc07470c", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41915416": { "pmid": "41915416", "title": "Integrating experimental and theoretical approaches to unveil structure-bioactivity relationships in silver(I) N-substituted glycine complexes.", "abstract": "N-methyl and N-glycyl glycine derivatives - namely (trimethylammonium)acetate (N,N,N-trimethylglycine, betaine, Bet), N,N-dimethylglycine (Dmg), N-methylglycine (sarcosine, Sar), and glycylglycylglycine (GlyGlyGly) - as naturally occurring glycine metabolites, were employed as stabilizing ligands for silver(I) ions, leading to the formation of water-soluble polymeric coordination compounds: {[Ag(HSar)(NO3)]}n (AgSar), {[Ag(HDmg)(NO3)]}n (AgDmg), {[Ag3(HBet)2(NO3)3]}n (AgBet), and [Ag(HGlyGlyGly)(NO3)] (AgGlyGlyGly). The composition and structures of the resulting complexes were unambiguously confirmed using attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), elemental analysis, thermogravimetric analysis (TGA), and single-crystal X-ray diffraction (SC-XRD). Speciation and stability studies in relevant test and growth media were conducted using a novel multi-technique approach combining potentiometric titrations, electrochemical measurements, 1H NMR and UV-Vis spectroscopy and were additionally supported by theoretical calculations. From a biological evaluation standpoint, all complexes demonstrated significantly enhanced antistaphylococcal activity (including S. aureus L12) compared to silver(I) nitrate (AgNO3), with AgBet and AgGlyGlyGly exhibiting approximately 10-fold, and AgSar and AgDmg approximately 5-fold, increased efficacy. Moreover, AgBet and AgGlyGlyGly were nearly twice as active as silver(I) sulfadiazine (AgSD), a clinically used but poorly water-soluble antimicrobial agent. Remarkably, AgBet and AgGlyGlyGly also showed unusual potent antifungal activity, being 10- to 100-fold more effective than both AgNO3 and AgSD against Rhizopus oryzae, the causative agent of mucormycosis. In cytotoxicity assays, AgSar exhibited the greatest selectivity and sensitivity, being over three times higher than cisplatin (cisPt), against the human breast adenocarcinoma cell line MDA-MB-231. New approaches from the perspectives of structure-activity relationships and bioavailability of the novel silver(I) complexes were explored through human serum albumin (HSA) binding studies and molecular docking calculations, as well as experimental and computational evaluations of lipophilicity and additional Lipinski parameters. Furthermore, the silver(I) complexes' action mechanism was assessed via β-galactosidase inhibition and PCR amplification inhibition in E. coli, intracellular reactive oxygen species (ROS) production, their effects on cell cycle progression and binding to ctDNA.", "journal": "Dalton transactions (Cambridge, England : 2003)", "year": "2026", "doi": "10.1039/d5dt03032c", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41184176": { "pmid": "41184176", "title": "Harnessing Multiplexed Proteolysis-Targeting Chimera for Comprehensive Influenza A Virus Targeting.", "abstract": "Influenza A virus (IAV) remains a global health threat due to its high mutation rate and immune evasion. Current therapies, limited by single-target mechanisms and drug resistance, are insufficient. To address this, we developed a multiplexed PROTAC strategy that simultaneously targets and degrades multiple components of the viral ribonucleoprotein (vRNP) complex, the core machinery of IAV replication. Our PROTAC molecule leverages the highly conserved 5' untranslated region of viral RNA, integrating an oligonucleotide targeting ligand with an E3 ligase-recruiting unit to precisely bind and degrade vRNP components, including NP and polymerase subunits (PB1, PB2, PA). This multitarget approach disrupts viral replication at multiple levels, establishes a high genetic barrier to resistance, and demonstrates broad-spectrum activity against IAV strains. In antiviral studies, PROTAC2 exhibits exceptional potency at low concentrations (0.8 μM), sustaining inhibition for over 48 h─four times longer than ribavirin─ and shows an extended therapeutic window during late-stage infection. By simultaneously degrading eight vRNP complexes, this strategy achieves superior efficacy and virtually eliminates the risk of resistance. This innovative approach provides a durable, resistance-resistant, and highly effective therapeutic candidate, addressing the limitations of current treatments and advancing influenza therapy.", "journal": "Journal of the American Chemical Society", "year": "2025", "doi": "10.1021/jacs.5c09384", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "42357361": { "pmid": "42357361", "title": "Development and Characterization of a Stable Oil-in-Water Nanoemulsion Using Impingement Jet Mixing and Lyophilization Techniques.", "abstract": "Nanoemulsion (NEM) is an effective adjuvant and delivery system for vaccines and nucleic acids, capable of inducing immune responses against diverse pathogens. Background/Objectives: Conventional NEM manufacture uses multi-step operations, typically high-shear homogenization and then microfluidization (HSHM), thereby increasing process complexity and contamination risk. As water-rich colloidal dispersions, NEM is prone to microbial proliferation and droplet coalescence; freezing further disrupts microstructure, causing phase fusion and separation, so NEM adjuvants are often stored separately from antigens in multi-vial formats. Lyophilization could reduce cold-chain dependence and enable single-vial products, but there is no systematic study on lyoprotectants comparation and process optimization of lyophilized NEM. Methods: An impingement jet mixing (IJM) process was evaluated as a simplified, scalable route for NEM production. Key IJM parameters, including flow ratio, total flow rate, preparation temperature, microchannel type, and shear mode-were examined to match attributes of conventional HSHM. Lyophilized and reconstituted NEM were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, differential scanning calorimetry and/or in vitro potency to inform lyoprotectant selection, and Taguchi Design of Experiment (DOE) methodology guided lyophilization processes. Results: IJM yielded NEM with droplet size, polydispersity index (PDI) and morphology comparable to HSHM, with higher throughput and fewer unit operations. Optimized lyophilization technique with designed lyoprotectant and process formed closed structures to prevent the easy-to-flow monolayer of the emulsion from fusing, producing robust and stable NEM. Conclusions: Coupling IJM with targeted lyophilization establishes a scalable, lower-risk manufacturing paradigm for NEM that preserves critical quality attributes, reduces cold-chain reliance and enables single-vial adjuvanted vaccine formats with tangible industrial and clinical benefits.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18060745", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40800615": { "pmid": "40800615", "title": "Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples.", "abstract": "Digital polymerase chain reaction (dPCR) is a DNA quantification technology that offers absolute quantification of DNA templates. In this study, we optimized and validated a chip-based dPCR EvaGreen assay with commonly used 16S rRNA gene primer pairs and compared its performance to quantitative real-time PCR (qPCR). We compared measurements of low amounts of template DNA using a newly designed synthetic DNA standard to assess precision, accuracy, and sensitivity. Optimization approaches were tested to minimize partitions with intermediate fluorescence levels between true positive and true negative partitions (so-called \"rain\") for dPCR. Both dPCR and qPCR demonstrated similar quantification performance, with variability in accuracy increasing for samples containing fewer than 30 copies μl-1 template concentrations. Both tested 16S rRNA gene primer sets amplified non-target template contaminants within both qPCR and dPCR mixtures, which could not be eliminated by ultraviolet light or DNAse treatment and negatively affected the apparent sensitivity of both PCR assays. Digital PCR was less susceptible to common PCR inhibitors, such as ethanol and humic acids, but was more susceptible to tannic acid inhibition than qPCR. These findings demonstrate the suitability of dPCR for 16S rRNA gene quantification of low biomass environmental samples.", "journal": "ISME communications", "year": "2025", "doi": "10.1093/ismeco/ycaf115", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40968728": { "pmid": "40968728", "title": "A Set of Rapid Diagnostic Tool for Babesia microti Infection.", "abstract": "Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis, with a global pooled prevalence of 2.23% and regional peaks in Europe (4.17%) and North America (1.54%). Traditional diagnostics like microscopy and polymerase chain reaction (PCR) suffer from low sensitivity in low-parasitemia cases or high costs ($230/test), necessitating accessible, rapid assays for resource-limited regions. A cross-priming amplification combined with vertical flow visualization (CPA-VF) assay, a straightforward molecular method targeting the 18S rRNA gene of B. microti, requires minimal equipment and facilitates rapid detection. Sensitivity/Specificity: The CPA-VF assay detected 2.56 fg/reaction (equivalent to 0.000004% parasitic red blood cells), with a sensitivity of 95.5% matching that of RT-PCR but at a 60-fold lower cost ($3.8/test). It showed no cross-reactivity with B. duncani, B. divergens, or Plasmodium. Clinical Validation: Testing 49 positive samples (19 experimentally infected mice +30 artificially spiked) and 492 field samples, CPA-VF demonstrated 95.5% sensitivity (95% CI: 88.2-98.7) and 95.5% specificity compared to nested PCR (nPCR). Intra-assay coefficients of variation (CV) was 2.1%-7.2% and inter-assay kappa coefficient was 0.94, confirming reliability. CPA-VF is a rapid, low-cost ($3.8/test), and instrument-free diagnostic tool for B. microti, particularly suitable for endemic regions where timely diagnosis reduces mortality risks from misdiagnosis as malaria. Its portability and visual readout address critical gaps in resource-constrained settings.", "journal": "Journal of clinical laboratory analysis", "year": "2025", "doi": "10.1002/jcla.70102", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42765333": { "pmid": "42765333", "title": "Mechanistic interpretability of fine-tuned protein language models for nanobody thermostability prediction.", "abstract": "While Protein Language Models (PLMs) fine-tuned on biophysical data achieve high predictive accuracy, the physical principles underlying their predictions remain obscure. Deciphering these representations offers a unique opportunity to not only interpret model decisions but also to discover novel biophysical insights governing protein properties. Here, we present a framework using Sparse Autoencoders (SAEs) to extract mechanistic knowledge from PLMs fine-tuned for nanobody thermostability. We fine-tuned the ESM-2 model on the nanobody thermostability dataset, achieving superior performance compared to significantly larger state-of-the-art models. SAE analysis successfully decomposed the model's dense embeddings into sparse, interpretable features without loss of predictive accuracy. We characterized these features through both global and local analyses. Global analysis provided an aggregate map of position-dependent feature contributions, whereas local analysis identified specific residue-level patterns, including known determinants such as the VHH-tetrad and critical disulfide bonds, as well as candidate stabilizing residues. Free Energy Perturbation calculations supported the structural plausibility of selected residue-level hypotheses. These results show that SAE-based interpretation can generate testable, structurally grounded hypotheses for rational protein engineering. The data and source code of the proposed method are available at GitHub (https://github.com/matsunagalab/paper_nanobody-thermostability-sae) and Zenodo (DOI: 10.5281/zenodo.18012027).", "journal": "Bioinformatics (Oxford, England)", "year": "2026", "doi": "10.1093/bioinformatics/btag685", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42654144": { "pmid": "42654144", "title": "Comprehensive Evaluation of Storage Stability and Cytotoxicity of Co-Spray-Dried Theophylline Dry Powders for Inhalation: Follow-Up Study.", "abstract": "Background/Objectives: The stability and biological safety of newly developed formulations must be established to support their therapeutic efficacy and clinical translation in pulmonary drug delivery. Therefore, this follow-up study comprehensively evaluated the short- and long-term stability and the in vitro cytotoxicity of optimized, co-spray-dried theophylline (THN) dry powders for inhalation against A549 lung epithelial cells. Methods: Two established formulations were selected: THN-RAF (raffinose-leucine-glycine based) and THN-TRE (trehalose-leucine based). Stability was assessed under accelerated conditions (40 °C/75% RH, 3 months) and long-term desiccator storage (25 °C, 1 year) using laser diffraction, SEM, XRPD, FTIR, DSC, TGA, and Andersen Cascade Impaction. As THN-TRE had been previously confirmed to be cytocompatible, only THN-RAF and its components were evaluated against A549 human alveolar epithelial cells using the MTT assay. Results: Under accelerated conditions, both formulations exhibited pronounced recrystallization (Xc up to 89.9%), agglomeration (D [0.9] up to 217.08 µm for THN-TRE), and deterioration in aerodynamic performance (FPF as low as 11.55%, MMAD up to 6.68 µm). By contrast, long-term desiccator storage induced substantial recrystallization (Xc up to 80.7%) while preserving thermal, chemical, and aerodynamic performance (FPF ≈ 40%; MMAD 4.99-5.21 µm). THN-RAF was more resistant to stress-induced agglomeration than THN-TRE. Cytotoxicity assessment confirmed cytocompatibility of THN-RAF, with cell viability exceeding 70.99% at all tested concentrations (up to 500 µg/mL). Conclusions: These findings reveal a marked discrepancy between the outcomes of ICH accelerated testing and long-term desiccator storage. They underscore the importance of considering moisture-protective packaging configurations when designing stability protocols for amorphous inhalable formulations.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18081027", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41992105": { "pmid": "41992105", "title": "High-temperature ssBP-LAMP breaks the barrier of nonspecific amplification and unlocks the full potential of LAMP diagnostics.", "abstract": "BACKGROUND: Loop-mediated isothermal amplification (LAMP) is a highly attractive nucleic acid detection method for decentralized molecular diagnostics, but its broader adoption has been limited by unpredictable nonspecific amplification (NSA). Here, we dissect the fundamental determinants of LAMP specificity and present a new high-temperature ssBP-LAMP technique to suppress NSA without compromising assay sensitivity or speed. METHODS: Ubiquitous bioinformatic tools for prediction and design of primer sets and their characteristics were employed to generate a panel of 21 primer sets targeting diverse bacterial, viral, fungal, and human targets. Fluorometric ssBP-LAMP was implemented with a series of coordinated modifications, from increased temperature (only possible owing to a novel, engineered thermostable strand displacement Bst polymerase enzyme) to fine-tuning of additives and auxiliary enzymes. Finally, proof-of-concept validation using both fluorometric and colorimetric approaches for ssBP-LAMP was performed, and their resistance to common inhibitors of molecular diagnostics was evaluated. RESULTS: We show for the first time that primer design alone is insufficient to ensure specificity, and bioinformatic predictions are insufficient predictors of efficiency and, more importantly, of specificity, even when using elevated reaction temperatures. This suggests that NSA frequently originates during the thermal ramp-up phase, before the assay reaches its operational temperature. To address this, we employed a thermolabile single-stranded DNA-binding protein (ssBP) from bacteriophage T7, which transiently sequesters primers during temperature ramp-up. When combined with conventional LAMP primers and elevated assay temperatures (≥ 68 °C), ssBP consistently suppressed NSA across all tested targets. This approach translated directly to both liquid and lyophilized master mixes, enabling highly specific ssBP-LAMP detection by real-time fluorescence and colorimetry. These formulations retained robust sensitivity (down to 10–100 copies/reaction for real-time detection), eliminated false positives, and exhibited tolerance to clinically relevant inhibitors such as plasma, hemoglobin, saliva, and urine, especially in real-time fluorescence detection. CONCLUSIONS: Together, the results establish high-temperature ssBP-LAMP as a rapid (< 15 min), specific, inhibitor-tolerant, and highly adaptable molecular diagnostic technology that overcomes key limitations of conventional LAMP while achieving analytical performance comparable to qPCR, with broad relevance for both clinical and point-of-care testing.", "journal": "Cellular & molecular biology letters", "year": "2026", "doi": "10.1186/s11658-026-00926-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41435648": { "pmid": "41435648", "title": "Hydration-mediated stabilization and anti-aggregation of soy and whey protein isolates, insulin, and collagen in hydrophilic deep eutectic solvents.", "abstract": "In deep eutectic solvent (DES)-H2O systems, protein stability is not determined solely by chemical composition but by a general colloid-interface mechanism: there exists a moisture-dependent hydrogen-bonding network reorganization point at which interfacial hydration forces and bulk DES structure are co-optimized. At this \"critical point\", protein-solvent interfacial properties-wettability, hydration layer structure, and interfacial tension-are tuned to form stable colloid systems. We investigated three hydrophilic DESs (choline chloride:glycerol, betaine:glycerol, L-carnitine:glycerol) across a moisture gradient. Bulk and interfacial properties were probed via viscosity, density, refractive index, conductivity, contact angle, interfacial tension, and AFM force spectroscopy. Aggregation and secondary structure of protein colloid systems were evaluated for four proteins using TEM, PSD, UV-Vis, FTIR, and CD. MD simulation was used to demonstrate the supramolecular interaction between proteins and DESs. Hydrophilic DES-H₂O systems undergo hydrogen-bonding network reorganization at ∼16 wt% moisture content, where bulk and interfacial properties of all DESs display marked changes. These interfacial transitions correlate directly with maximal anti-aggregation effects and preservation of native protein structures. Our results suggest a mechanism whereby water actively reorganizes the internal hydrogen-bonding network of hydrophilic DESs, leading to protein colloids that are both structurally and colloidally stable. This study established a general colloid-interface framework for protein stabilization in DES-H₂O systems, with potential applications in biomedical, pharmaceutical, cosmetic, and food products.", "journal": "Journal of colloid and interface science", "year": "2026", "doi": "10.1016/j.jcis.2025.139683", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39521398": { "pmid": "39521398", "title": "Vadadustat Three Times Weekly in Patients With Anemia Due to Dialysis-Dependent CKD.", "abstract": "Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) may offer an alternative to erythropoiesis-stimulating agents (ESAs) for the treatment of anemia in the setting of chronic kidney disease (CKD). The objective of this study was to investigate the efficacy and safety of conversion from the long-acting ESA methoxy polyethylene glycol-epoetin-ß (MPG-EPO) to the oral HIF-PHI vadadustat 3 times weekly versus maintenance therapy with MPG-EPO. Phase 3b, open-label, noninferiority trial. Multicenter study in the United States in 456 adult participants with anemia and dialysis-dependent CKD. Participants were randomized 1:1:1 to vadadustat at a starting dose of 600mg thrice weekly, vadadustat at a starting dose of 900mg thrice weekly, or MPG-EPO for up to 52 treatment weeks and 4 safety follow-up weeks after the end of treatment or early termination. Primary and secondary efficacy end points were the mean change in hemoglobin concentration from baseline during primary (weeks 20-26) and secondary (weeks 46-52) evaluation periods, respectively. Noninferiority was specified as a lower bound of the 95% CI above -0.75g/dL for the difference in mean change in hemoglobin concentration from baseline. Other efficacy end points were the proportion of participants with hemoglobin levels within the target range and the proportions of participants requiring ESA or red blood cell transfusion rescue for anemia during the evaluation periods. Primary safety end points were any treatment-emergent and serious adverse events (AEs). After combining the vadadustat groups (600mg and 900mg thrice weekly; n=304), vadadustat was noninferior to MPG-EPO (n=152) for primary (least-squares mean treatment difference, -0.33; 95% CI, -0.53 to-0.13) and secondary (-0.33; -0.56 to-0.09) efficacy end points. Mean hemoglobin concentrations were stable for all groups except for an initial slight decrease in the vadadustat 600mg group, which stabilized by week 12. ESA rescue for anemia was more frequent in the MPG-EPO group (primary evaluation period, 27.7%; secondary evaluation period, 16.2%) than in the combined vadadustat (14.2%; 7.3%) groups. Transfusion rates were low and occurred at similar rates across treatment groups (2.7% and 4.0% in the combined vadadustat and MPG-EPO groups, respectively). The incidences of any treatment-emergent and serious treatment-emergent AEs were similar across treatment groups. Potential errors in attribution of AEs as drug-related. Three-times-weekly vadadustat was noninferior to MPG-EPO in its effect on hemoglobin levels without detectable differences in AEs. Funding from a private entity (Akebia Therapeutics, Inc). Registered at ClinicalTrials.gov with study identifier NCT04707768. Vadadustat, taken 3 times a week, was noninferior to methoxy polyethylene glycol-epoetin-ß (MPG-EPO) in treating anemia. Vadadustat is an oral drug used to treat anemia in people with chronic kidney disease. This study enrolled participants undergoing dialysis for kidney failure and compared the efficacy and safety of continuing to receive MPG-EPO, a long-acting injectable anemia treatment, or switching to vadadustat, taken by mouth 3 times per week for 1 year. Vadadustat was noninferior to MPG-EPO in maintaining hemoglobin levels. Hemoglobin levels remained stable across all groups, except for a slight decrease in the lower-dose vadadustat group, which stabilized with dose titration by week 12. The occurrence of new or serious side effects was similar across treatment groups.", "journal": "American journal of kidney diseases : the official journal of the National Kidney Foundation", "year": "2025", "doi": "10.1053/j.ajkd.2024.09.006", "ptypes": [ "Journal Article", "Multicenter Study", "Clinical Trial, Phase III", "Randomized Controlled Trial", "Equivalence Trial" ], "first_author": "", "dirs": [ "B" ] }, "41076639": { "pmid": "41076639", "title": "Flow-LAMP: Label-Free Digital LAMP Using Scatter-Based Flow Cytometry on Vortex-Generated Polydisperse Gel Beads.", "abstract": "Accurate nucleic acid quantification is vital for clinical diagnostics, yet the widespread adoption of digital PCR remains limited due to its reliance on fluorescence detection and specialized microfluidics. We present Flow-LAMP, a label-free digital assay integrating loop-mediated isothermal amplification with scatter-based flow cytometric analysis of agarose gel beads. Polydisperse gel beads are formed by vortex emulsification and retain magnesium pyrophosphate precipitate in positive reactions. Flow cytometry enables volume and amplification readouts via forward (FSC) and side scattering (SSC) signals, respectively. We confirmed that SSC was strongly correlated with amplification products, while FSC-Height accurately reflected the bead volume. Using Epstein-Barr virus plasmid, Flow-LAMP achieved accurate quantification with a limit of detection of 38.15 copies/μL. Results from testing clinical plasma samples correlated well with qPCR and digital PCR. By eliminating fluorescent labeling and microfluidics, Flow-LAMP offers a cost-effective and accessible platform for digital nucleic acid detection using standard lab equipment.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c04768", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "39905842": { "pmid": "39905842", "title": "Glutamate dehydrogenase 1-dependent α-ketoglutarate promotes hepatitis B virus transcription by modulating histone methylations on the covalently closed circular DNA minichromosome.", "abstract": "Hepatitis B virus (HBV) hijacks host cell metabolism, especially host glutamine metabolism, to support its replication. Glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme crucial for glutamine metabolism, can interact with histone demethylases to regulate gene expression through histone methylation. However, the mechanisms underlying GDH1-mediated glutamine metabolism reprogramming and the roles of key metabolites during HBV infection remain unclear. Transcriptomic and metabolomic analyses of HBV-infected cell were performed. Both HBV-infected cells and humanized liver chimeric mice were used to elucidate the effect of glutamine metabolism on HBV. HBV infection leads to the abnormal activation of glutamine metabolism, including upregulation of key enzymes and metabolites involved in glutamine metabolism. The viral core protein (HBc) mediates the translocation of GDH1 into the nucleus, where GDH1 activates covalently closed circular DNA (cccDNA) transcription by converting glutamate to α-ketoglutarate (αKG). Mechanistically, the promoting effect of GDH1-derived αKG on cccDNA transcription is independent of its conventional role. Rather, αKG directly interacts with the lysine-specific demethylase KDM4A and enhances KDM4A demethylase activity to regulate αKG-dependent histone demethylation, controlling cccDNA transcription. Our findings highlight the importance of glutamine metabolism in HBV transcription and suggest that glutamine deprivation is a potential strategy for silencing cccDNA transcription.", "journal": "Clinical and molecular hepatology", "year": "2025", "doi": "10.3350/cmh.2024.0694", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40750194": { "pmid": "40750194", "title": "CRISPR-based sensing platform for the Group B streptococcus screening in pregnant women.", "abstract": "Group B Streptococcus (GBS) is a major cause of perinatal infections. Prenatal screening is critical to prevent maternal and neonatal GBS infections, reduce adverse outcomes, and guide clinical interventions. While bacterial culture is the gold standard, it is time-consuming and delays decision-making. Rapid molecular tests like PCR are sensitive and specific but require expensive equipment and skilled personnel. Point-of-care tests (e.g., Xpert GBS LB) offer speed and sensitivity but remain costly and underutilized. This study aims to develop a portable CRISPR/Cas13a-based device for rapid, on-site GBS detection. A palm-sized CRISPR detection platform, PalmCS, was developed. PalmCS includes two key components: an integrated tube for nucleic acid extraction, gene amplification, and CRISPR-based reaction, and a multifunctional device for thermal regulation, fluorescence detection, and automatic result interpretation. The sealed plug needle valve controls fluid flow for sample input and result output. By optimizing crRNA selection, Cas13a/crRNA ribonucleoprotein (RNP) complex concentrations, and isothermal recombinase polymerase amplification primers, we established a one-pot CRISPR/Cas13a-RPA method for GBS detection. Results showed that 3 M guanidinium salt combined with 5 % Tween 20 achieved the highest extraction efficiency. The system extracted nucleic acids from samples in 5 min at room temperature, demonstrating its potential for rapid diagnostics. PalmCS was validated using 40 clinical samples, achieving a limit of detection (LOD) of 20 copies/reaction, 97.5 % sensitivity, and 100 % specificity. This study used a one-step nucleic acid extraction combined with a closed one-pot RPA-CRISPR reaction for rapid, sensitive, and specific GBS detection. This provides a new platform for prenatal screening in pregnant women. In resource-limited settings with emergency deliveries, PalmCS enables on-site GBS testing, allowing antibiotic administration during delivery for positive cases to significantly reduce neonatal infection risk.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344390", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "40140580": { "pmid": "40140580", "title": "Gene-modified pig-to-human liver xenotransplantation.", "abstract": "The shortage of donors is a major challenge for transplantation; however, organs from genetically modified pigs can serve as ideal supplements1,2. Until now, porcine hearts and kidneys have been successively transplanted into humans3-7. In this study, heterotopic auxiliary transplantation was used to donate a six-gene-edited pig liver to a brain-dead recipient. The graft function, haemodynamics, and immune and inflammatory responses of the recipient were monitored over the subsequent 10 days. Two hours after portal vein reperfusion of the xenograft, goldish bile was produced, increasing to 66.5 ml by postoperative day 10. Porcine liver-derived albumin also increased after surgery. Alanine aminotransferase levels remained in the normal range, while aspartate aminotransferase levels increased on postoperative day 1 and then rapidly declined. Blood flow velocity in the porcine hepatic artery and portal and hepatic veins remained at an acceptable level. Although platelet numbers decreased early after surgery, they ultimately returned to normal levels. Histological analyses showed that the porcine liver regenerated capably with no signs of rejection. T cell activity was inhibited by anti-thymocyte globulin administration, and B cell activation increased 3 days after surgery and was then inhibited by rituximab. There were no significant peri-operative changes in immunoglobulin G or immunoglobulin M levels. C-reactive protein and procalcitonin levels were initially elevated and then quickly declined. The xenograft remained functional until study completion.", "journal": "Nature", "year": "2025", "doi": "10.1038/s41586-025-08799-1", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40147392": { "pmid": "40147392", "title": "Green synthesis of carbon dots and their application as fluorescent probes for rutin detection.", "abstract": "Rutin has antioxidant, antitumor, antibacterial, antiviral, anti-aging, and analgesic effects, and it is a drug used to treat various circulatory disorders. However, rutin overdosage can seriously harm the health. The rutin detection method has drawbacks, including expensive costs, low sensitivity, and complicated steps. Therefore, given that carbon dots have superior fluorescence characteristics, the development of nanofluorescent probes for rutin detection is the research motivation of this article. N-CDs and N, B-CDs were produced by a hydrothermal approach using o-phenylenediamine, glucose, urea, and boron oxide, commonly available in the laboratory. The morphology and composition of CDs have been analyzed. According to the findings, the surfaces of the N-CDs and N, B-CDs were abundant in functional groups that contained oxygen, and they were spherical, uniform in size, and well distributed, which is beneficial for enhancing its fluorescence response to the detection object. In addition, two kinds of CDs emit bright yellow and blue-green fluorescence under the ultraviolet lamp at 365 nm. Rutin was detected in solution using synthesized N-CDs and N, B-CDs as fluorescent probes. Rutin was discovered to have a fluorescence quenching effect on N-CDs and N, B-CDs. Within a specific concentration range, the N-CDs and N, B-CDs fluorescence intensities demonstrated an excellent linear relationship with the rutin concentration, and the corresponding correlation coefficients R2 were 0.9897 and 0.9409, respectively. To verify the detection accuracy of the experiment, we used the random forest algorithm to predict the detection model. The results showed that the experimental and predicted values were coincident, suggesting the possibility of using N-CDs and N, B-CDs as fluorescent probes to detect rutin. This work offers experimental procedures and data references to create doping CDs with superior fluorescence qualities. It broadens the use of doped CDs in the detection of rutin.", "journal": "Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy", "year": "2025", "doi": "10.1016/j.saa.2025.126084", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39615902": { "pmid": "39615902", "title": "Hydrogel loop-mediated isothermal amplification for ultra-fast diagnosis of Helicobacter pylori in stool samples without nucleic acid extraction.", "abstract": "The gastrointestinal diseases caused by Helicobacter pylori (H. pylori) infection made the accurate detection of H. pylori infection more important. Non-invasive methods, such as molecular diagnostic methods, had become a promising method for detection of H. pylori. Stool samples combined with loop-mediated isothermal amplification (LAMP), showed potential practicability for real-time detection. However, complex nucleic acid extraction steps were required to remove the large numbers of amplification inhibitors in stool samples before LAMP reaction. And the limited number of H. pylori made the detection with long reaction time and low sensitivity. The problems mentioned above were urgently to be solved. In this study, we proposed a strategy for ultra-rapid sensitive detection of H. pylori in stool samples by hydrogel LAMP (hLAMP) without extraction. The hydrogel was combined with stool samples after simple thermal cracking, and amplification spaces were formed in its nanopore structures by nano-localization. The LAMP reaction was accelerated by nano space-localization. Besides, this method based on hLAMP could specifically and sensitively detect as low as 100 CFU/mL H. pylori within 40 min from sampling to result due to good anti-inhibition effect on complex samples of hydrogel. The whole process involved sample simple disposal for 10 min and LAMP reaction for 30 min. Furthermore, the excellent anti-inhibition mechanism of hydrogel was discussed, and the mechanism of hydrogel accelerating LAMP was explored. This is the first application of that hydrogel and LAMP systematically combined to detect H. pylori in stool samples. The developed method had been verified in actual clinical applications that the accuracy rate reached 88.9 % compared with routine histopathology. And it also provided a potential idea for the diagnosis and prevention of H. pylori.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2024.343384", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40586435": { "pmid": "40586435", "title": "Tandem-Activatable PROTAC Prodrug for Tumor Biomarker-Driven Near-Infrared Opto-Proteolysis.", "abstract": "Tumor-specific protein degradation is crucial for successful cancer treatment by proteolysis-targeting chimera (PROTAC), which however still remains challenging. Here, we report a tandem-activatable PROTAC prodrug (TAP) strategy for precise on-tumor proteolysis in vivo. TAP is constructed by caging PROTAC with a tumor-homing cyclopeptide through a tandem-locking linker, which comprises a singlet-oxygen (1O2) cleavable moiety, a near-infrared (NIR) photosensitizer (PS) and a Cathepsin B (CatB)-cleavable dipeptide substrate. The proteolytic activity of TAP is initially turned off but can be sequentially switched on by tumor biomarker enzyme CatB and NIR light. Such a tandem-lock design ensures that PROTAC exclusively degrade oncoprotein target in tumor. The results revealed that TAP carried out a tumor-selective degradation of bromodomain-containing protein 4 (BRD4) under the cooperative action by CatB and NIR light, which further synergized with photodynamic therapy (PDT) by the PS to suppress tumor growth. This work thus presents the first tandem-activatable approach for spatiotemporally controlled proteolysis to minimize the off-tumor toxicity of PROTAC.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2025", "doi": "10.1002/anie.202510641", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "42492273": { "pmid": "42492273", "title": "Effects of carriers and addition modes on the physical and chemical properties of green pepper (Piper nigrum L.) powders treated by freeze-far infrared drying during storage.", "abstract": "Green pepper powder (GPP) via freeze-far infrared drying (FD-FIRD) features unique pungency, but easily suffers flavor loss and quality decline in storage. Thus, this study investigated the effects of three carriers-maltodextrin, trehalose, and inulin-and two application methods, mixed pulping (MP) and osmotic soaking (OS), on the 70-day storage stability of FD-FIRD GPPs. Trehalose best maintained color and bioactive components due to its high glass transition temperature and matrix-stabilizing effect, while maltodextrin mainly improved particle-size uniformity by narrowing the D10-D90 range by 30-60%. Among all treatments, OS-trehalose showed the highest initial lightness (L⁎ = 64.26), the lowest color change (ΔE = 5.87), and the most stable flowability. XRD and FTIR analyses further showed that the carriers and application methods mainly affected crystalline domain formation without altering the amorphous structure. Overall, OS-trehalose is a promising strategy for producing shelf-stable, high-quality GPPs.", "journal": "Food chemistry", "year": "2026", "doi": "10.1016/j.foodchem.2026.150442", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "A" ] }, "40215221": { "pmid": "40215221", "title": "Bacterial viability retention in probiotic foods: a review.", "abstract": "Probiotics offer substantial health benefits, leading to their increased consumption in various food products. The viability of probiotics is a critical factor that influences the nutritional and therapeutic efficacy of these foods. However, as probiotics often lose viability during production and oral administration, effective preservation and encapsulation technologies are needed to overcome this challenge. This review elucidates the diverse sources and incorporation strategies of probiotics, while systematically analyzing the effects of water transformation (ice front velocity, glass transition temperature, and collapse temperature), processing conditions (food matrix, temperature, and dissolved oxygen), and gastrointestinal challenges (gastric fluid, digestive enzymes, and bile salts) on probiotic viability. Effective strategies to strengthen probiotic viability encompass three primary domains: fermentation processes, production techniques, and encapsulation methods. Specifically, these include meticulous fermentation control (nitrogen sources, lipids, and carbon sources), pre-stress treatments (pre-cooling, heat shock, NaCl stress, and acid stress), optimized lyoprotectant selection (carbohydrates, proteins, and polyols), synergistic freeze-drying technologies (infrared technology, spray drying, and microwave), bulk encapsulation approaches (polysaccharide or protein-based microencapsulation), and single-cell encapsulation methods (self-assembly and surface functionalization). Despite these advancements, targeting specific probiotics and food matrices remains challenging, necessitating further research to enhance probiotic viability.", "journal": "Critical reviews in food science and nutrition", "year": "2025", "doi": "10.1080/10408398.2025.2488228", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40101367": { "pmid": "40101367", "title": "Osteogenic function of BMP2-modified PEEK scaffolds for orbital fracture repair.", "abstract": "This study aimed to investigate the osteogenic function of polyetheretherketone (PEEK) scaffolds modified with bone morphogenetic protein 2 (BMP2) and its possibility for orbital fracture repair. The 3D-printed PEEK sheets were combined with BMP2-loaded hyaluronic acid hydrogel (HAH) to fabricate PEEK-BMP2-HAH composite scaffolds. Bone marrow mesenchymal stem cells (BMSCs) were seeded onto PEEK or PEEK-BMP2-HAH scaffolds. Cell adhesion and cell proliferation were measured by transmission electron microscopy and CCK-8 assay. Alkaline phosphatase (ALP) chromogenic, alizarine red S staining, and PCR analysis of Runt-related transcription factor 2 (Runx2), collagen-I (Col-I), Osterix, and osteopontin (OPN) were performed to assess osteogenic activity. The rat orbital fracture defect model is proposed for evaluating the biocompatibility, osteogenic integration, and functional recovery of PEEK orbital implants. Compared with PEEK, cell adhesion and cell proliferation were increased in PEEK-BMP2-HAH scaffolds. ALP activity and mineralized nodule formation were increased in PEEK-BMP2-HAH scaffolds than that in PEEK the mRNA expression of Runx2, Osterix, Col-I and OPN was increased on PEEK-BMP2-HAH scaffolds than that on PEEK at 14 d of osteogenic induction. Besides, a bone defect animal model revealed that BMP2-HAH-modified PEEK scaffolds could effectively facilitate the repair of the orbital bone defect, with increased expression of OPN and Runx2. BMP2-loaded HAH effectively increased adhesion, proliferation, and osteogenic differentiation of BMSCs on PEEK. PEEK-BMP2-HAH scaffolds are expected to become new materials for orbital fracture repair.", "journal": "Biomedical materials (Bristol, England)", "year": "2025", "doi": "10.1088/1748-605X/adc220", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41005298": { "pmid": "41005298", "title": "Vorapaxar enhanced mitochondria-associated ferroptosis primes cancer immunotherapy via targeting FOXO1/HMOX1 axis.", "abstract": "Immunotherapy has revolutionized cancer treatment, yet challenges persist, such as resistance and lethal thromboembolism, necessitating dual-purpose strategies. Targeting ferroptosis emerges as a promising strategy to enhance immunotherapy efficacy, prompting our investigation of antiplatelet agents that simultaneously promote ferroptosis and mitigate thromboembolic risks. Through systematic screening of 20 Food and Drug Administration (FDA)-approved antiplatelet agents, we identify vorapaxar as a potent pro-ferroptotic drug. Mechanistically, vorapaxar binds forkhead box O1 (FOXO1), inhibits its phosphorylation at Ser256, and facilitates nuclear translocation to upregulate heme oxygenase 1 (HMOX1), promoting mitochondrial iron overload and mitochondria-associated ferroptosis. Vorapaxar enhances immunotherapy-induced tumor ferroptosis and antitumor immunity across diverse melanoma models, including B16F10 tumor-bearing mice, Braf/Pten-driven spontaneous melanoma mice, and peripheral blood mononuclear cell (PBMC)-humanized mice. Clinically, high FOXO1/HMOX1 co-expression correlates with improved immunotherapy response and progression-free survival. These findings position vorapaxar as a promising adjunct to immunotherapy, offering a dual benefit for cancer patients requiring both antithrombotic therapy and immunotherapy.", "journal": "Cell reports. Medicine", "year": "2025", "doi": "10.1016/j.xcrm.2025.102371", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39607469": { "pmid": "39607469", "title": "A sustainable and Integrated Microbial Biocatalysis of Resveratrol from Polygonum cuspidatum Siebold & Zucc Using Cellulose-Based Immobilised Aspergillus niger with Deep Eutectic Solvent-Assisted Microreactors.", "abstract": "An efficient and green method was developed using deep eutectic solvent assistance to enhance the biotransformation method of producing resveratrol from Polygonum cuspidatum Siebold & Zucc, using cellulose-based immobilised Aspergillus niger in the process. Various deep eutectic solvents (DES) were screened to obtain a superior biocatalytic effect. The increase in DES concentration aggravated the degree of cell membrane damage. Natural deep eutectic solvents (NADES) exhibited a more favourable catalytic effect than DES due to their excellent biocompatibility. This enhancement is associated with the hydrogen bonding donor components present in NADES, with catalytic ability ranking as alcohol-based > sugar-based > organic acid. CHCL/EG exhibited the maximum catalytic effect at 1.0 wt%. Under optimal conditions (pH 6.5; temperature, 29.5 °C; ratio of liquid to solid 20:1 (mL/g), and time 47 h), the resveratrol yield reached 32.79 mg/g, which was 13.06-fold to that of the untreated sample (2.51 mg/g). The residual activity of the cellulose-based microreactor was 81.46% after ten trials. The proposed method was successfully employed, demonstrating higher biocatalysis efficiencies and superior environmental protection compared to conventional solvents for resveratrol biocatalysis.", "journal": "Applied biochemistry and biotechnology", "year": "2025", "doi": "10.1007/s12010-024-05118-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42141568": { "pmid": "42141568", "title": "Comprehensive interscale energy transfer in homogeneous isotropic turbulence.", "abstract": "This study examines interscale energy transfer, conventionally denoted as the \"energy cascade,\" in forced homogeneous isotropic turbulence. By employing spatial filtering techniques, the turbulent kinetic energy is decomposed into distinct large- and small-scale components, as well as local- and subfilter-scale constituents-specifically encompassing large-scale, small-scale, subfilter-large-scale, and subfilter-small-scale terms. Transport equations for each component are derived to establish an analytical framework elucidating the mechanisms governing cross-scale energy transfer. Within this framework, energy is primarily transferred scale-by-scale from the large-scale and subfilter-small-scale components to the small-scale and subfilter-large-scale portions of the turbulent kinetic energy. Building upon this framework, the following results and discussions are presented. First, the scaling laws governing interscale energy transfer and viscous dissipation rates are elucidated across both the dissipative and inertial subranges. Second, via Leonard decomposition, the subfilter-large-scale and subfilter-small-scale stress tensors are partitioned into Leonard, cross, and Reynolds components. In the dissipative range, interscale energy transfer is predominantly governed by the Leonard and cross-stress terms, whereas all three components contribute significantly in the inertial subrange, with their respective contributions quantitatively assessed. Third, the accuracy of several classical models in predicting interscale energy transfer rates-both from large to subfilter-large scales and from subfilter-small to small scales-is validated. Finally, investigation of extreme dissipation events reveals substantial amplification at small scales and marked alterations in scaling behavior near the inertial subrange, which are closely associated with turbulent intermittency, as evidenced by probability density distributions.", "journal": "Physical review. E", "year": "2026", "doi": "10.1103/n6sj-w2hf", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40613834": { "pmid": "40613834", "title": "An Integrative Strategy Enhancing Nanobody Thermostability via CDR Grafting, In Silico Mutagenesis Screening, and Multiplex Evaluation.", "abstract": "Nanobodies are transformative tools in biomedical research and therapy due to their structural advantages and exceptional stability. However, their intrinsic stability varies significantly, while existing stabilization strategies often face various limitations. Here, we report a computational-experimental integrative approach that combines complementarity-determining region (CDR) grafting with virtual mutagenesis for stabilization. Using A4.2m as the framework region donor and Nb20, a SARS-CoV-2 spike protein-targeting nanobody, as the CDR source, Nb20-4.2m was engineered and demonstrated a 10 °C enhancement in melting temperature (Tm) and 55% improvement of refolding efficiency. Subsequently, through a computational pipeline, experimental validations, and a combination of mutations, the final construct was yielded with 68 °C Tm and >82% refolding efficiency. A molecular dynamics simulation indicated that the stability enhancement originates from optimized intramolecular hydrogen bonding networks. With a higher efficiency than conventional methods, this approach offered a paradigm shift in engineering and established a versatile platform for nanobody optimization to fit broad applications in clinics and industry.", "journal": "ACS synthetic biology", "year": "2025", "doi": "10.1021/acssynbio.5c00112", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40450980": { "pmid": "40450980", "title": "Mechanism of puerarin alleviating myocardial remodeling through NSUN2-mediated m5C methylation modification.", "abstract": "Myocardial remodeling is a primary contributor to the development and progression of heart failure. Reversing this process is crucial for the prevention and treatment of heart failure. Puerarin (Pue), a traditional Chinese medicine, has been widely utilized in the management of cardiovascular diseases; however, its potential mechanisms for reversing myocardial remodeling remain unclear. To investigate the effect of Pue in myocardial remodeling and the potential mechanism with m5C methylation. A transverse aortic coarctation (TAC)-induced myocardial hypertrophy model was established in vivo, while angiotensin II (AngII)-induced cardiomyocyte hypertrophy was examined in vitro using H9c2 and NRCM cells. Various doses of Pue were administered to assess their effects. Myocardial remodeling was evaluated through echocardiography, H&E staining, WGA staining, Masson staining, PSR staining, and Western Blot (WB) analysis in mice, alongside mean cell area measurement and WB analysis in cultured cells. The m5C level was quantified using Dot Blot and immunohistochemistry (IHC). RNA sequencing, molecular docking, microscale thermophoresis (MST), transfection, immunofluorescence (IF), and WB were employed to confirm that Pue reverses myocardial remodeling via NSUN2-mediated m5C methylation modification in both in vitro and in vivo settings. The methylation modification was further validated using methylation immunoprecipitation (MeRIP-qPCR). In both in vitro and in vivo experiments, we found that long-term administration of Pue can alleviate myocyte hypertrophy and fibrosis while improving both diastolic and systolic function in mice. Additionally, Pue was shown to upregulate the m5C modification level in cardiomyocytes. RNA sequencing, along with docking and MST assays, revealed that Pue upregulates and targets NSUN2. Furthermore, the protective effect of Pue was diminished by the knockdown of NSUN2 expression. Pue was found to inhibit the activation of NF-κB, and the knockdown of NSUN2 prolonged the half-life of NF-κB mRNA. The enrichment of m5C mRNA was confirmed through MeRIP-qPCR. This study suggests that Pue enhances the m5C methylation of NF-κB mRNA by targeting NSUN2. The m5C methylation facilitates the degradation of NF-κB mRNA and suppresses NF-κB activation, thereby modulating the expression of factors related to myocardial remodeling. Consequently, this process alleviates myocardial remodeling and prevents the progression of heart failure, providing a novel perspective for the clinical treatment of heart failure.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2025.156849", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "39903905": { "pmid": "39903905", "title": "Tracking Water Transport with Short-Wave Infrared: Kinetic Phase Diagrams, Dissolution, and Drying.", "abstract": "Short-wave infrared (SWIR) imaging has been extensively used in defense applications but remains underutilized in the study of soft materials and the broader consumer product industry. Water molecules absorb around ∼1450 nm, making moisture-rich objects appear black, whereas surfactants and other common molecules in consumer products do not absorb and provide a good contrast. This experimental study showcases the varied capabilities of SWIR imaging in tracking water transport in soft material systems by analyzing dissolution dynamics, tracking phase transitions (when combined with cross-polarized optical imaging), and monitoring drying kinetics in the surfactant and polymer solutions. The dynamic phase evolution to equilibria of a binary aqueous solution of a nonionic surfactant hexaethylene glycol monododecyl ether (C12E6) is presented. The influence of confined hydration in dynamic-diffusive interfacial transport capillaries was investigated by tracking the micellar to hexagonal phase transition concentration (C*). The effects of varying concentrations of an industrially relevant additive─monovalent common salt (NaCl) on the radial (2D) dissolution of lamellar-structured concentrated sodium lauryl ether sulfate (70 wt % SLE1S) pastes was studied. An equation was developed to estimate the radial dissolution coefficients based on total dissolution time and surfactant concentrations in the sample and solvent. Water loss was investigated by tracking the drying of aqueous poly(vinyl) alcohol films. In situ monitoring of drying kinetics is used to draw correlations between the solution viscosity and drying time. SWIR imaging has already revealed previously inaccessible insights into surfactant hydration and holds the potential to become a turnkey method in tracking water transport, enabling better quality control and product stability analysis.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.4c05057", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41436541": { "pmid": "41436541", "title": "Reduced graphene oxide promoted by SnO2 for photodegradation of tetracycline in water.", "abstract": "Reduced graphene oxide, with a high specific surface area and conductivity, also exhibits photocatalytic activity, and its photocatalytic performance improves with the addition of semiconductors. In this study, SnO2/reduced graphene oxide (RGO) composite was applied for tetracycline removal from water under visible light irradiation. The photocatalytic performance of RGO was enhanced through SnO2 deposition. RGO was synthesized using Hummer’s method and subsequently impregnated with SnCl2 solution to deposit SnO2. The effect of SnO2 loading was investigated, and the calcination temperature was set at 400 °C. FTIR, XRD, Raman spectroscopy, SEM, TEM, BET, PL, and DRS analyses characterized the SnO2/RGO photocatalyst. SEM and TEM confirmed RGO layered morphology. PL analysis revealed an electron-hole recombination peak at 394 nm. DRS results showed a reduction in the band gap of SnO2/RGO composite compared to pristine SnO2 and RGO. The effects of photocatalyst dosage, initial tetracycline concentration, and pH were studied and optimized using Design-Expert software (version 11) to maximize degradation efficiency. Under optimized conditions, with a tetracycline concentration of 10 mg/L, catalyst dosage of 4 g/L, pH of 7, and 10 min exposure to a 125 W LED lamp, 92% degradation was achieved. Kinetic analysis followed a second-order rate model. The reusability and regenerability of the SnO2/RGO composite were evaluated over five cycles, yielding tetracycline removal efficiencies of 91%, 73.25%, 65.7%, 60.3%, and 57%, respectively. Tetracycline removal was further assessed in the presence of Na+, Cl−, Mg2+, and NO3−, as well as using tap water. Compared with previous studies, this system demonstrated comparable removal efficiency within a substantially shorter reaction time under visible light irradiation, underscoring its economic potential for water treatment applications.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-32230-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40405695": { "pmid": "40405695", "title": "Universal Metal-Exchange Strategy for Room Temperature Synthesis of Single-Atom Nanozymes.", "abstract": "The high-temperature limitations of single-atom catalysts (SACs) synthesis, primarily thermal aggregation and low metal loadings, are overcome by a novel room-temperature metal-exchange strategy. We leverage the weak Zn─N coordination in high-loading Zn SACs (12.10 wt%, synthesized via controlled ZIF-8 pyrolysis) as a platform for the facile exchange with a broad range of metals (Mn, Fe, Co, Ni, Cu, Ru, Pt, up to 12.67 wt%) and the creation of multi-metallic species. This ambient-temperature approach significantly reduces aggregation, enhancing the exposure of catalytic active sites and delivering superior multi-enzyme-like activities (catalase, peroxidase, oxidase). Our work not only provides a scalable, low-temperature route to high-performance SACs but also reveals crucial insights into the stability of SACs under metal-ion conditions.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2025", "doi": "10.1002/anie.202505123", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41495819": { "pmid": "41495819", "title": "Zhimu-Huangbai codecoction for the treatment of type II diabetes mellitus through its self-assembling nanoparticles.", "abstract": "Traditional Chinese medicine (TCM) decoctions represent a complex system comprising multiple phases, and their therapeutic effects cannot be fully elucidated solely at the molecular level. The nanophase within TCM decoctions plays a significant role in mediating their pharmacological activities. Zhimu and Huangbai herbal decoction (ZBD), traditionally used to treat type 2 diabetes mellitus (T2DM), is a formulation combining Rhizoma Anemarrhenae and Cortex phellodendri chinensis. Our prior research identified natural nanoparticles (N-ZBD) within ZBD. However, the role of these nanoparticles in ZBD's anti-T2DM effects and their potential impact on the oral bioavailability of its active components remain unclear. Therefore, this study aimed to examine the anti-T2DM effects of N-ZBD and to assess whether N-ZBD could enhance the bioavailability of its primary active compounds. Initially, we employed a dialysis centrifugation method to isolate the N-ZBD (nanoparticles derived from Zhimu-Baihu Decoction) from ZBD. The separated N-ZBD exhibited an average particle size of 234.4 ± 1.04 nm, a polydispersity index (PDI) of 0.56 ± 0.06, and a zeta potential of - 12 .97 ± 1.46 mV. Then, a type 2 diabetes mellitus (T2DM) rat model was created by administering a low dose of streptozotocin (35 mg/kg) intraperitoneally following 4 weeks on a high-fat diet. After a 7-week treatment period, compared with the model group, the levels of FBG, TC, TG and LDL in MET group, N-ZBD group and ZBD group were significantly reduced, and the morphology of islets was significantly improved. In all phases state of ZBD, N-ZBD had a significant therapeutic effect on T2DM rats, which was comparable to that of ZBD. In order to explore whether N-ZBD has the effect of promoting absorption, we assessed the intestinal absorption properties of N-ZBD, ZBD, and free drugs (mangiferin, timosaponin BII, berberine, phellodendrine, neomangiferin and jatrorrhizine) utilizing a single-pass intestinal perfusion (SPIP) model. Compared to free drugs, N-ZBD enhanced the absorption of active ingredients in the jejunum and ileum. In order to explore whether N-ZBD can improve the bioavailability of active ingredients, a method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was also established to compare the pharmacokinetic profiles of six primary active compounds in plasma after the oral administration of ZBD, N-ZBD and free drugs. The pharmacokinetic profiles of N-ZBD and ZBD in rats were comparable. Notably, N-ZBD exhibited higher Cmax, AUC0-t, and T1/2 values for the index compounds compared to free drugs and a reduced plasma clearance rate. In conclusion, N-ZBD is primarily responsible for ZBD's anti-T2DM effects and significantly enhances the bioavailability of active ingredients, highlighting the essential role of natural nanoparticles in the therapeutic effectiveness of decoctions.", "journal": "Chinese medicine", "year": "2026", "doi": "10.1186/s13020-025-01290-z", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41209703": { "pmid": "41209703", "title": "Tooth-on-a-chip to engineer early dental epithelial-mesenchymal interaction.", "abstract": "Tooth enamel-the highly mineralized outer layer shielding teeth from mechanical and chemical wear-is produced during development by specialized dental epithelial (DE) cells called ameloblasts. Functional enamel is challenging to regenerate because the enamel-forming cells-DE-derived ameloblasts-disappear even before tooth eruption. To achieve regeneration, DE and dental mesenchymal (DM) need to interact. Current in-vitro models encompass only limited DE-DM interaction as they offer little control over the interface geometry and usually force both lineages to share a single, sub-optimal medium, leading to variable outcomes. Hence, herein we developed a micro-engineered \"tooth-on-a-chip\" that shapes the DE-DM interface while independently perfusing lineage-specific media. A three-channel polydimethylsiloxane (PDMS) device, fabricated by rapid 3-D printing and soft lithography, traps DM cells in a fibrin hydrogel between two fluid channels. Within 3 days the DM cells condensed into a papilla-like core and secreted collagen, creating a signaling niche for sequential seeding of DE cells. Quantitative PCR (qPCR) of DM cells showed progressive up-regulation of odontogenic markers, while immunofluorescence confirmed ameloblast differentiation of the DE layer through AMELX expression. Alizarin Red staining, calcium assays, SEM confirmed progressive, spatially confined mineralization at the engineered interface. Although this platform now is in its proof-of-concept stage, it provides the first controllable, dual-medium microenvironment that recapitulates early odontogenesis, amelogenesis, and mineral deposition in a single device, offering a versatile tool for dissecting tooth morphogenesis and accelerating true enamel-regeneration strategies.", "journal": "Materials today. Bio", "year": "2025", "doi": "10.1016/j.mtbio.2025.102451", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41176836": { "pmid": "41176836", "title": "Nano/micro-encapsulated enzymes as engineered biocatalysts for the food industry.", "abstract": "Enzymes are indispensable tools in modern food processing, offering precise catalytic functions that improve product quality, texture, and preservation. Nevertheless, their industrial utility is often constrained by poor stability under thermal, oxidative, or acidic/alkaline conditions. Nano/microencapsulation technologies have emerged as robust strategies to overcome these limitations, enhancing enzyme resilience, prolonging activity, and enabling targeted delivery. This review presents a thorough examination of both established and novel encapsulation approaches for enzymes, i.e. coacervation, spray drying, liposomal systems, supercritical CO₂ processing, sol-gel techniques, and metal-organic frameworks. The role of various biomolecules e.g. polysaccharides, proteins, lipids, and nucleic acids in encapsulation systems is discussed in the context of improving enzyme protection and functional integration. Mechanisms for environmental shielding and stimulus-responsive release are also highlighted. Applications of encapsulated enzymes in diverse food sectors, including dairy, baking, beverages, meat processing, and functional food development, are explored, showcasing their potential in optimizing food formulation and processing efficiency. Although these technologies offer significant promise, hurdles like production scalability, material safety, economic feasibility, and regulatory compliance remain. Future research aimed at refining encapsulation systems and designing intelligent, cost-effective carriers will be pivotal in advancing the use of engineered enzymes in food technology.", "journal": "Advances in colloid and interface science", "year": "2026", "doi": "10.1016/j.cis.2025.103697", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "41254318": { "pmid": "41254318", "title": "A Tale of Two Stressors in Biologic Drug Product Development: Shaking Mode and Primary Packaging.", "abstract": "Mechanical, interfacial, and shear stresses encountered during development, manufacturing and transportation of biologics can compromise monoclonal antibody (mAb) stability. However, most scale-down shaking models often depend solely on orbital agitation and overlook the effect of the solid-liquid interface. To study this gap, stress conditions were applied to simulate early-stage product development and real-world transportation in this work. Accordingly, the aggregation profiles of Cetuximab and Tocilizumab formulations, with and without polysorbate 80 (PS80), were systematically compared after applying horizontal and orbital shaking. Protein aggregation was assessed using orthogonal techniques such as size-exclusion chromatography, dynamic light scattering, flow imaging microscopy, ultraviolet-visible spectroscopy, and visual inspection. Horizontal shaking more effectively revealed Cetuximab's susceptibility to aggregation under mechanical and interfacial stress whereas orbital shaking conditions were not as discriminative. Furthermore, to explore the effect of vial surface chemistry on subsequent protein aggregation, Cetuximab was subjected to horizontal shaking stress using both untreated and silanized glass vials. Interestingly, hydrophobic silanized vials without surfactant resulted in increased Cetuximab aggregation compared to untreated vials. In contrast, Cetuximab with PS80 showed fewer aggregates in silanized vials than in glass vials. These results underscore the value of selecting right-for-purpose agitation models and highlight the need to explore the triple interface for improving stress screening in drug product development.", "journal": "Pharmaceutical research", "year": "2026", "doi": "10.1007/s11095-025-03959-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42081956": { "pmid": "42081956", "title": "Qinggan Jiangzhi Cha ameliorates NAFLD by modulating the AGE-RAGE/PRKCA/MAPK3/AP-1 signaling axis.", "abstract": "Qinggan Jiangzhi Cha (QGJZC), a compound formulation rooted in Traditional Chinese Medicine, is traditionally employed to clear heat, soothe the liver, and reduce lipid accumulation to alleviate hepatic stagnation and indigestion, aligning with modern NAFLD therapeutic strategies targeting lipid metabolism and inflammation. This study aimed to evaluate the efficacy of QGJZC against NAFLD and to elucidate the underlying mechanisms. Rats with high-fat diet-induced NAFLD were treated with QGJZC. Therapeutic efficacy was assessed by serum biochemical markers, histopathological staining, and inflammatory cytokines. An integrative approach combining serum chemical analysis, network pharmacology, transcriptomics, and molecular validation was employed to elucidate the mechanism. QGJZC intervention markedly lowered the elevated serum ALT and AST levels in NAFLD model rats, ameliorated lipid metabolism disorders (decreased TG, TC, and LDL-C; increased HDL-C), and dose-dependently alleviated pathological damage such as hepatic steatosis and inflammatory cell infiltration. Serum pharmacochemical analysis identified 109 absorbed components, with flavonoids being predominant. Integrated network pharmacology and transcriptomic analyses linked the therapeutic mechanism to the AGE-RAGE signaling pathway, among others. Experimental validation demonstrated that QGJZC significantly inhibited the expression of key mediators of the hepatic AGE-RAGE signaling axis (AGEs, RAGE, PRKCA), reduced MAPK3 phosphorylation levels, and attenuated downstream AP-1 nuclear translocation and activation. This study demonstrates that QGJZC ameliorates metabolic disturbances and hepatic pathological injury in NAFLD rats. Its therapeutic effects are associated with the synergistic actions of multiple absorbed bioactive constituents and may involve regulation of the AGE-RAGE/PRKCA/MAPK3/AP-1 signaling pathway, thereby attenuating hepatic inflammatory responses. These findings provide pharmacological support for the potential clinical use of QGJZC in NAFLD treatment.", "journal": "Journal of ethnopharmacology", "year": "2026", "doi": "10.1016/j.jep.2026.121783", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42609682": { "pmid": "42609682", "title": "Experimental and computational investigation to elucidate the structure and dynamics of amino acid-based deep eutectic system.", "abstract": "Deep Eutectic Solvents derived from amino acids are considered environmentally friendly because they are biodegradable, recyclable, and exhibit acceptable levels of toxicity. These solvents have various applications in various fields, such as biocatalysis, electrochemistry, extraction, and pharmaceuticals. Glutamine is an amino acid commonly found in human blood, and glycerol is a conventional solvent with rapid biodegradability. In this study, biodegradable amino acids based deep eutectic solvent (DES) was prepared from the mixture of glutamine and glycerol at a 1:3 (Glu: Gly) molar ratio, and differential scanning calorimetry (DSC) was used to confirm the phase transition. Exothermic and endothermic phase transitions detected at -5.12 °C and 2.83 °C respectively. Moreover, peak broadening due to spectral shifts of functional groups in the FTIR data confirmed the formation of DES, and PCA on the IR data concluded that the DES formation was glutamine-dominated. 1H NMR experiment was conducted to understand the chemical structure of the DES system. Molecular dynamics (MD) simulation and density functional theory were applied to study the structure and interaction of DES. The radial distribution function from the MD simulation confirmed the existence of three hydrogen bonds that stabilized the complex. Independent gradient model based on Hirshfeld partition (IGMH) analysis visualized the strongest interaction between the carboxylic acid of the glutamine and the alcoholic group of glycerol. Charge transfer analysis identified glycerol as a hydrogen bond donor and glutamine as a hydrogen bond acceptor. The application of our DES system was evaluated by measuring Density, pH and Conductivity. All these extensive insights will contribute to a deeper understanding of other biodegradable amino acid-based DESs and provide a broader approach to DES research.", "journal": "Journal of molecular liquids", "year": "2026", "doi": "10.1016/j.molliq.2026.129832", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42713007": { "pmid": "42713007", "title": "Generation and evaluation of multimers of anti C-reactive protein Nb and alkaline phosphatase based on three self-assembly peptides.", "abstract": "Nanobodies (Nbs) have been extensively utilized in medical diagnosis and therapies due to their advantages, such as ease of genetic manipulation and efficient soluble expression in prokaryotic systems. However, enhancing their binding affinity to antigens to match or even exceed that of polyclonal antibodies for high-sensitivity bioassays remains a challenge. Therefore, in the present study, a novel strategy to enhance the binding affinity of antibodies to antigens was developed by fusing an anti-C-reactive protein Nb (CRPNb) to the N-terminus of self-assembling peptides such as right-handed coiled coil [RHCC; derived from a right-handed coiled-coil peptide of an archaebacterium (Staphylothermus marinus)], verotoxin (VTB; the B-subunit of Escherichia coli verotoxin) and C4-binding protein (C4bp; derived from human plasma C4-binding protein α-chain). For functional detection, alkaline phosphatase (AP) was further fused to the C-terminus of RHCC, VTB and C4bp, respectively. This approach enabled the formation of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. These were then expressed as soluble cytoplasmic proteins in the E. coli strain BL21 (DE3) and purified by imidazole elution. The protein expression was assessed by western blotting. Additionally, the stability of the multimeric constructs was evaluated using a direct ELISA, whilst their sensitivity was assessed using a competitive ELISA. The CRPNb-VTB-AP pentamers and CRPNb-RHCC-AP tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer based on a direct ELISA. Furthermore, they exhibited good thermal stability based on a direct ELISA. At 80˚C, CRPNb-VTB-AP, CRPNb-AP and CRPNb-RHCC-AP retained ~85, 75 and 60% activity, while CRPNb-H only retained ~40%. After incubation at 80˚C for 55 min, the two multimers still maintained ~40% activity. The results suggested them to be suitable for storage and transportation at ambient temperatures. In conclusion, a novel nanobody-fusion protein platform was established in the present study. The fusion proteins functioned as high-affinity binders for target antigens and heat-stable signal tracers for immunoassay detection and quantitative analysis, providing a novel type of thermostable immunoreagent.", "journal": "Experimental and therapeutic medicine", "year": "2026", "doi": "10.3892/etm.2026.13279", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42874971": { "pmid": "42874971", "title": "Amorphous trehalose as a superior lyoprotectant for high-temperature long-term storage of freeze-dried mRNA-lipid nanoparticles.", "abstract": "The large-scale global application of messenger ribonucleic acid-lipid nanoparticles (mRNA-LNPs) is severely restricted by their mandatory ultra-low-temperature storage requirement, which poses critical logistics and transportation bottlenecks, especially in resource-limited regions. Lyophilization serves as a mild and well-established strategy to improve the thermal stability of biotherapeutics; nevertheless, complex physicochemical stresses during the lyophilization process may disrupt the structural integrity of mRNA and destroy the colloidal stability of LNP systems. Sucrose has been widely recognized as an efficient lyoprotectant, whereas the underlying mechanisms responsible for the stabilizing/destabilizing effects of crystalline and amorphous excipients remain incompletely understood. In this work, differential scanning calorimetry (DSC) and X-ray diffraction (XRD) were utilized to characterize lyophilized samples with diverse formulations. Combined with stability evaluation at 4 °C, 25 °C and 40 °C, the correlations between excipient crystallization behavior, thermodynamic properties and storage stability were systematically clarified. The results revealed that trehalose, with an intrinsically higher glass transition temperature in the amorphous state, exhibits superior lyoprotective capacity compared with sucrose under extreme storage conditions (40 °C), making it a more promising candidate for mRNA-LNP lyophilization. As a typical crystalline excipient, mannitol is inappropriate for single-component lyoprotectant application, as it severely impairs the long-term storage stability of mRNA-LNPs by damaging nanoparticle morphology and reducing nucleic acid encapsulation efficiency.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127546", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39885054": { "pmid": "39885054", "title": "Efficient secretory expression, purification, and characterization of lipase in Pseudomonas aeruginosa M18, with multifunctional applications in diagnostics.", "abstract": "Lipase (EC 3.1.1.3) is a crucial hydrolase with broad industrial and clinical applications. In this study, the lipA and lipH genes from Pseudomonas aeruginosa were cloned into the pBBR1MCS-2 vector and expressed under the regulation of the highly efficient BSFP_0720 promoter from Burkholderia stabilis FERMP-21014. This system allowed for efficient secretory expression in Pseudomonas aeruginosa without requiring an inducer. The recombinant lipase exhibited both lipase and cholesteryl esterase activities, making it suitable for triglyceride and cholesterol assay kits. Additionally, gene editing was used to knock out the endogenous cholesterol oxidase gene in Pseudomonas aeruginosa, eliminating cross-interference in different assay kits. High-density fermentation using glucose as the carbon source resulted in lipase activity reaching 68 kU/L and cholesteryl esterase activity reaching 214 kU/L after 30 h of fermentation, representing a 356-fold increase compared to natural production. By combining ammonium sulfate precipitation, hydrophobic interaction chromatography, and anion exchange chromatography, a purity of 94.32% was achieved (as determined by CE-SDS). Accelerated stability tests showed that the lyophilized lipase retained over 96% residual activity after storage at 37 °C for 21 days and at 45 °C for 7 days, suggesting its suitability for long-term storage. The enzymatic properties of the lipase demonstrated resistance to common chemicals, high activity in buffers with pH values between 7 and 9, and short-term tolerance to high temperatures (60 °C). These characteristics make the lipase highly adaptable for use in complex clinical samples and various industrial applications. The successful high-efficiency expression and multifunctional utility of this lipase highlight its significant commercial potential in diagnostics and other fields.", "journal": "World journal of microbiology & biotechnology", "year": "2025", "doi": "10.1007/s11274-025-04279-w", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40874181": { "pmid": "40874181", "title": "Usefulness and Limitations of Polymerase Chain Reaction (PCR) for the Diagnosis and Management of Toxoplasmosis Following Allogeneic Hematopoietic Cell Transplant: Single-center Experience With 31 Patients Over 16 Years.", "abstract": "Toxoplasmosis is an early post-transplant complication in recipients of allogeneic hematopoietic cell transplant (HCT), typically arising from reactivation of latent infection. Toxoplasma gondii polymerase chain reaction (PCR) has improved detection. Single-center, retrospective review of allogeneic HCT recipients who developed toxoplasmosis from August 2008 to November 2024. We identified 31 cases of toxoplasmosis among 1235 HCT recipients. Ten had infection and 21 had end-organ disease. Fever was the most common clinical manifestation (74.2%). Patients with pulmonary or central nervous system disease often lacked organ-specific symptoms. Toxoplasmosis primarily occurred in patients not on prophylaxis (90.3%), at a median of 28 days post-HCT (interquartile range 20-69 days). Whole blood Toxoplasma PCR diagnosed 80.6% cases and showed a cumulative sensitivity of 93.3%. However, PCR was not always positive at symptom onset, and some asymptomatic patients already had end-organ disease at the time of first PCR positivity. Trimethoprim-sulfamethoxazole (TMP-SMX) was the most used treatment (48.4%). Mortality directly attributable to toxoplasmosis was 12.9%, but all-cause mortality was 61.3%. Toxoplasmosis is an early post-HCT complication with high morbidity and mortality. Prophylaxis is essential. TMP-SMX is effective, but sometimes it is withheld early post-HCT due to potential myelotoxicity. Given the short window between infection and progression to disease, we recommend twice-weekly monitoring with whole blood PCR while off TMP-SMX and early initiation of TMP-SMX post-HCT for Toxoplasma seropositive patients. Atovaquone may be considered as a bridging prophylaxis until TMP-SMX is started, but its absorption may be compromised early post-HCT and breakthrough cases have been reported.", "journal": "Open forum infectious diseases", "year": "2025", "doi": "10.1093/ofid/ofaf462", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41790919": { "pmid": "41790919", "title": "Rapid detection of carbapenem resistance genes using multiplex LAMP and melt curve analysis in clinical specimens.", "abstract": "Carbapenem-resistant Enterobacteriaceae have become a major clinical and public health challenge due to the need for rapid administration of effective antimicrobials and implementation of supplemental infection control practices. Identifying genes associated with carbapenem resistance is crucial for managing these cases. The timely initiation of effective antimicrobial therapy and infection control interventions to prevent spreading are critical. Therefore, rapid diagnostic tests for detecting carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are necessary. This study aimed to evaluate the use of LAMP and melting curve analysis in real-time polymerase chain reaction (RT-PCR) to identify carbapenem resistance genes New Delhi metallo-β-lactamase (NDM) and oxacillinase (OXA) in clinical specimens. This study emphasized the benefits of the LAMP strategy in comparison to traditional methods, demonstrating its effectiveness and practicality. LAMP was proven to be more sensitive than RT-PCR and conventional multiplex PCR, with a detection rate of 96.7% and 91.6% respectively. While LAMP had a slightly lower specificity rate of 88.7%, it remains a promising method for rapid and accurate identification. Furthermore, the study found that LAMP could detect bacterial DNA even in low quantities, with a limit of detection of 102 CFU/mL for both K. pneumoniae and E. coli. This capability is crucial for early diagnosis and treatment in settings where multidrug-resistant (MDR) bacteria are prevalent. RT-PCR systems that combine melting curve analysis with LAMP offer promise for quick and precise identification of carbapenem resistance genes in clinical specimens. This approach can enhance the diagnosis and management of multidrug-resistant bacterial infections.", "journal": "Journal of infection in developing countries", "year": "2026", "doi": "10.3855/jidc.21220", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "C" ] }, "39946350": { "pmid": "39946350", "title": "Rational Design and Optimization of a Potent IDO1 Proteolysis Targeting Chimera (PROTAC).", "abstract": "Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive protein that inhibits antitumor immunity through both tryptophan metabolism and nonenzymatic functions. Drugs targeting IDO1 enzyme activity have failed to improve the overall survival of patients with cancer. Developing new therapeutics that neutralize both enzyme- and nonenzyme-derived immunosuppressive IDO1 effects is therefore of high interest. We previously described a novel proteolysis targeting chimera (PROTAC), NU223612, that degrades IDO1 in cultured human glioblastoma (GBM) cells, as well as in well-established brain tumors, in vivo. In this study, we rationally optimized the structure of our lead series to create NU227326, which degrades IDO1 with a DC50 of 5 nM in human GBM cells. Mechanistic studies showed that IDO1 degradation occurred through the ubiquitin-proteasome system and was sustained for at least 2 days, supporting NU227326 as a highly potent IDO1 PROTAC suitable for further studies in GBM and other human cancers.", "journal": "Journal of medicinal chemistry", "year": "2025", "doi": "10.1021/acs.jmedchem.5c00026", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40225561": { "pmid": "40225561", "title": "ROS-responsive MnO2 mesoporous hydrogel to modulate liver-muscle crosstalk and mitigate NAFLD-associated sarcopenia via exosomal miR-582-5p delivery.", "abstract": "Background: The interrelation between non-alcoholic fatty liver disease (NAFLD) and sarcopenia has emerged as a significant concern due to its systemic impact on metabolic health. However, therapeutic approaches targeting the liver-muscle axis remain underdeveloped. Oxidative stress and inflammatory pathways are key mediators of this crosstalk, exacerbating disease progression. This study aims to develop a reactive oxygen species (ROS)-responsive MnO2 mesoporous PD (HA) hydrogel to modulate this axis and investigate its therapeutic efficacy in NAFLD-associated sarcopenia. Methods: MnO2 mesoporous PD (HA) hydrogels were synthesized with ROS-sensitive properties and characterized for rheological, fluorescence, and conductivity responses. A HepG2-C2C12 co-culture model mimicked the NAFLD-muscle wasting interplay, while high-fat diet (HFD)-induced NAFLD mouse models were used for in vivo evaluations. Cellular stress markers, exosomal miR-582-5p signaling, and atrogenic pathways were assessed using immunofluorescence, qRT-PCR, and histological analyses. Results: Pathway analysis of HFD-induced NAFLD showed upregulation of lipid metabolism and inflammatory signaling, promoting muscle atrophy via exosomal miR-582-5p. The MnO2 mesoporous hydrogel significantly reduced oxidative stress and inflammation in the HepG2-C2C12 co-culture. In vivo, hydrogel implantation in HFD mice mitigated hepatic fibrosis, reduced ROS accumulation, preserved muscle fiber integrity, and downregulated atrogenic markers. Conclusions: The MnO2 mesoporous PD (HA) hydrogel presents a dual-targeting therapeutic strategy for NAFLD and sarcopenia by attenuating oxidative stress and modulating liver-muscle axis signaling. These findings provide a foundation for innovative interventions targeting metabolic comorbidities.", "journal": "Theranostics", "year": "2025", "doi": "10.7150/thno.108280", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42083836": { "pmid": "42083836", "title": "Modulating Photons and Phonons in Graphene-Based Phase Change Materials for Solar-Thermal-Electrical Generation.", "abstract": "Solar-thermal-electrical generators offer a promising route for the conversion of solar energy into electricity. Their reliability can be enhanced by integrating phase change materials (PCMs) to buffer thermal fluctuations and ensure stable operation, yet conventional PCMs often suffer from low thermal conductivity and poor solar capture. Herein, photosensitive Co/C-anchored reduced graphene oxide (rGO) conductive framework was fabricated via metal-organic framework (MOF) pyrolysis-assisted zinc volatilization strategy. This design enables the in situ formation of MOF-derived uniformly dispersed cobalt nanoparticles within rGO framework. After the encapsulation of paraffin wax (PW), the resultant rGO@Co/C-PW composite PCMs demonstrate a remarkable solar-thermal conversion efficiency of 92.5% under 100 mW·cm-2 irradiation, attributed to the synergistic interplay between the broadband absorption and non-radiative relaxation of graphitic carbon and the localized surface plasmon resonance of Co nanoparticles. rGO@Co/C-PW also exhibits enhanced thermal conductivity, high phase change enthalpy, and excellent long-term cycling stability, supported by regulated non-isothermal phase transition kinetics via heterogeneous nucleation and spatial confinement. When integrated into a thermoelectric module, a sustained and stable power output of 8.82 mW under 100 mW·cm-2 is generated, capable of powering small electronic devices. This study provides valuable insights into developing next-generation PCMs integrating solar-thermal conversion, thermal energy storage, and thermoelectric output.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.73606", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40045363": { "pmid": "40045363", "title": "IDO1 inhibition enhances CLDN18.2-CAR-T cell therapy in gastrointestinal cancers by overcoming kynurenine-mediated metabolic suppression in the tumor microenvironment.", "abstract": "Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in hematologic malignancies but faces significant limitations in gastrointestinal tumors due to the immunosuppressive tumor microenvironment (TME). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme in the TME, suppresses T cell efficacy by catalyzing tryptophan degradation to kynurenine (Kyn), leading to T cell exhaustion and reduced cytotoxicity. This study investigates the role of IDO1 inhibition in overcoming metabolic suppression by kynurenine and enhancing Claudin18.2 (CLDN18.2) CAR-T cell therapy in gastric and pancreatic adenocarcinoma models. We evaluated the impact of genetic knockdown and pharmacological inhibition of IDO1 (using epacadostat) on CAR-T cell functionality, including cytokine production and exhaustion marker expression. The effects of fludarabine and cyclophosphamide preconditioning on IDO1 expression, CAR-T cell infiltration, and antitumor activity was also examined. In vivo tumor models of gastric and pancreatic adenocarcinomas were used to assess the efficacy of combining IDO1 inhibition with CLDN18.2-CAR-T therapy. IDO1 inhibition significantly enhanced CAR-T cell function by increasing cytokine production, reducing exhaustion markers by decreasing TOX expression and improving tumor cell lysis. Preconditioning with fludarabine and cyclophosphamide further suppressed IDO1 expression in the TME, facilitating enhanced CAR-T cell infiltration. In vivo studies demonstrated that combining IDO1 inhibition with CAR-T therapy led to robust tumor growth suppression and prolonged survival in gastric and pancreatic tumor models. Targeting IDO1 represents a promising strategy to overcome immunosuppressive barriers in gastrointestinal cancers, improving the efficacy of CLDN18.2-CAR-T therapy. These findings highlight the potential for integrating IDO1 inhibition into CAR-T treatment regimens to address resistance in treatment-refractory cancers.", "journal": "Journal of translational medicine", "year": "2025", "doi": "10.1186/s12967-025-06276-x", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40317860": { "pmid": "40317860", "title": "Control of Buckling of Colloidal Supraparticles.", "abstract": "The properties of clusters of colloidal particles, often termed supraparticles, are determined by the arrangement of the primary particles. Therefore, controlling the structure formation process is of key importance. While buckled morphologies can result from fast drying kinetics as found in spray drying, controlling the morphology under slow drying conditions remains a challenge. The final morphology of a supraparticle formed from an emulsion droplet can be controlled by manipulating particle-surfactant interactions. Water/oil emulsions are used to template supraparticle formation. The interactions of negatively charged colloidal particles with the surfactants stabilizing the water/oil-interface are tailored via the local pH within the aqueous droplet. At low pH, protonation of the anionic headgroup of the surfactant decreases electrostatic repulsion of the particles, facilitates interfacial adsorption, and subsequently causes buckling. The local pH of the aqueous droplet phase continuously changes during the assembly process. The supraparticle formation pathway can therefore be controlled by determining the point in time at which interfacial adsorption is enabled by adjusting the initial pH. Consequently, the final supraparticle morphology can be tailored at will, from fully buckled structures, via undulated surface morphologies to spherically rough and spherically smooth supraparticles and crystalline colloidal clusters.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2025", "doi": "10.1002/smll.202411772", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42354098": { "pmid": "42354098", "title": "Screening of Superior Lactic Acid Bacteria from Sourdough and Optimization of Freeze-Dried Bacterial Powder Preparation Technology for Steamed Buns.", "abstract": "To address the industrialization limitations of traditional sourdough and develop high-performance direct-vat-set (DVS) starters for steamed buns, this study screened superior lactic acid bacteria (LAB) and optimized the preparation technology of freeze-dried bacterial powder. The results demonstrated that the compound system of Lactiplantibacillus plantarum and Streptococcus thermophilus at a 1:1 ratio achieved favorable synergistic fermentation with sourdough, which effectively regulated dough acidification and improved the comprehensive sensory quality of steamed buns. The critical processing parameters, including centrifugation conditions and compound lyoprotectant composition containing trehalose, lactose and skim milk powder, were systematically optimized to significantly enhance the freeze-drying survival ability of compound LAB, and the high fitting degree of the established regression model confirmed the stability and reliability of the optimized formulation. The optimal dosage of freeze-dried DVS powder was verified to rationally modulate dough acidity and comprehensively promote the specific volume, structural characteristics and sensory performance of steamed buns. This systematic study provides robust theoretical and technical support for the industrial application of DVS LAB starters in high-quality steamed bun production, facilitating the modernization of traditional fermented wheat products.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15122130", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40788622": { "pmid": "40788622", "title": "PFKFB3-Inhibitor 3PO-Mediated Glycolytic Reprogramming Promotes Inflammatory Dental Pulp Repair: An In Vitro and In Vivo Study.", "abstract": "This study explores the role of PFKFB3 in pulpitis and its potential as a therapeutic target by modulating glycolytic reprogramming in dental pulp stem cells (DPSCs). Pulpitis, a common inflammatory disease, causes long-term damage to dental structures. Recent evidence suggests that metabolic reprogramming can modulate inflammatory responses and promote tissue repair. This study aims to investigate the anti-inflammatory and reparative effects of glycolysis inhibitors on inflamed dental pulp through in vivo and in vitro experiments. In vitro, hDPSCs were stimulated with lipopolysaccharide (LPS) (1 μg/mL, 3 h) to mimic pulpitis. The effects of the PFKFB3 inhibitor 3PO (10 μM) and siRNA targeting PFKFB3 (50 nM) on glycolysis were assessed using Seahorse analysis, while their impacts on inflammation were evaluated via ELISA and qRT-PCR. A co-culture of DPSCs and macrophages was used to study 3PO's effects on inflammation interactions and glycolytic reprogramming of the inflammatory microenvironment. The influences of 3PO on odontogenic differentiation were examined through qRT-PCR, Western blotting, ALP staining and ARS staining. The related signalling pathways were validated through Western blot (WB) experiments. An SD rat model was employed to validate the in vivo efficacy of 3PO@GelMA pulp capping, and HE staining and immunohistochemistry were used to evaluate the degree of pulp inflammation and reparative dentine formation. In vitro, LPS elevated glycolytic activity and inflammatory factors (IL-6, IL-1β, TNF-α) in DPSCs. 3PO and si-PFKFB3 mitigated these factors and promoted odontogenic differentiation, evidenced by increased DSPP and ALP expression. Metabolic reprogramming mediated by 3PO and si-PFKFB3, as assessed by Seahorse XF analysis, indicated a shift from glycolysis to oxidative phosphorylation. Additionally, 3PO induced metabolic reprogramming of the co-culture system, reduced the levels of pro-inflammatory cytokines, and promoted the polarisation of macrophages towards the M2 phenotype. PFKFB3 inhibition activated the AMPK/SIRT1/PGC-1α/NF-κB and AMPK/mTOR/NF-κB signalling pathways. In vivo, in the rat pulpitis model, 3PO@GelMA hydrogel application resulted in significantly decreased IL-6 and increased DSPP expression compared to the LPS group (p < 0.001), accompanied by reduced inflammation and enhanced reparative dentine formation. The PFKFB3 inhibitor 3PO reduces inflammation and promotes reparative dentine formation in pulpitis via metabolic reprogramming and specific signalling pathways, offering a new therapeutic strategy.", "journal": "International endodontic journal", "year": "2025", "doi": "10.1111/iej.70016", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41152542": { "pmid": "41152542", "title": "Simultaneous Detection of Four Madurella Species Using Loop-Mediated Isothermal Amplification (LAMP) for Eumycetoma Diagnosis.", "abstract": "Eumycetoma is a neglected tropical disease caused primarily by a Madurella mycetomatis infection, besides other related species. In this study, we designed a novel loop-mediated isothermal amplification (LAMP) primer set capable of simultaneously detecting four Madurella species (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii). Genomic sequencing of M. pseudomycetomatis strain and comparative genome analysis revealed the candidate genes that were common among and specific to Madurella species. The 3 LAMP primer sets targeting these genes detected up to 1 pg of the genomic DNA of all 4 Madurella species, exhibiting no cross-reactivity toward other pathogenic fungi. Among these, one primer set showing better reactivity was selected as a candidate used for diagnosis. Therefore, we developed novel primer sets which enabled the simultaneous detection of four Madurella species. Our present findings will lead to a faster and simpler diagnostic tool for eumycetoma detection, especially in rural clinical settings.", "journal": "Mycopathologia", "year": "2025", "doi": "10.1007/s11046-025-01019-4", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "C" ] }, "42400699": { "pmid": "42400699", "title": "A novel DREB transcription factor enhances salt tolerance in peanut (Arachis hypogaea L.) through upregulation of AhP5CR and AhBADH, leading to increased osmolyte accumulation.", "abstract": "Soil salinity is a major abiotic stress that severely restricts peanut (Arachis hypogaea L.) growth, yield, and quality. As peanut is highly sensitive to saline-alkaline conditions, identifying transcription factors involved in salt stress tolerance is of considerable importance. Dehydration-responsive element-binding (DREB) transcription factors are known regulators of abiotic stress responses; however, their functional roles in peanut remain poorly characterized. In this study, the AhDREB gene, encoding a DREB transcription factor, was overexpressed in peanut to investigate its regulatory function under salt stress. Transgenic T1 lines (TA1-1, TA1-6, and TA1-10) were generated via Agrobacterium-mediated transformation and verified by PCR, RT-PCR, and Western blot analyses. Under salt stress (250 mM NaCl), AhDREB transcript levels increased by 3.39-, 3.27-, and 4.06-fold in TA1-1, TA1-6, and TA1-10, respectively, compared with non-stressed controls. The downstream osmolyte biosynthetic genes AhP5CR and AhBADH were significantly upregulated, with expression increases of 2.66-3.07-fold and 2.01-4.04-fold, respectively. Consistently, proline and glycine betaine contents were markedly elevated, reaching up to 9.71- and 9.37-fold higher levels in TA1-10 plants than in controls. Among the transgenic lines, TA1-10 exhibited the highest transcriptional activity and osmolyte accumulation. These results demonstrate that AhDREB enhances salt tolerance in peanut by transcriptionally activating AhP5CR and AhBADH, thereby promoting osmolyte biosynthesis and improving osmotic adjustment under saline conditions. This study provides the first functional characterization of AhDREB as a novel transcription factor regulating salt stress responses in peanut.", "journal": "Molecular biology reports", "year": "2026", "doi": "10.1007/s11033-026-12295-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40884606": { "pmid": "40884606", "title": "Bioactivity of mycosynthesized nanoparticles assists photothermal therapy of breast cancer cells.", "abstract": "Chaga mushroom (Inonotus obliquus) exhibits cytotoxic effects against breast cancer cells. Mycosynthesized nanoparticles, owing to their biodegradability, biocompatibility, and low toxicity, present a promising therapeutic approach. This study explored the cytotoxic potential of gold nanoparticles synthesized using Chaga mushroom extract (AuCh-NPs) combined with Light Emitting Diode (LED) irradiation (530 nm) on human breast cancer (MCF-7) cells, aiming to develop a safe and effective sensitizer for photothermal therapy. The AuCh-NPs were characterized using UV-visible spectroscopy, FTIR, TEM, particle size analysis, and zeta potential measurements. Cytotoxicity was evaluated via MTT assay under LED irradiation with total light exposure 325.8 and 488.7 J cm-2, alongside mechanistic studies involving wound healing, autophagy, cell cycle arrest, annexin V analysis, real-time PCR, and comet assays.TEM revealed spherical AuCh-NPs with sizes ranging from 15.4 to 28.9 nm. The MTT assay demonstrated enhanced cytotoxicity under LED irradiation, with AuCh-NPs exhibiting a lower IC50 (5.56 µM) than citrate-capped gold nanoparticles (AuCit-NPs, 7 µM). Cell cycle analysis revealed significant arrest in G0/G1 (91.68%) and S (7.55%) phases, while annexin V analysis confirmed apoptosis induction. Real-time PCR showed upregulation of the pro-apoptotic genes BAX, and the comet assay indicated increased double-strand DNA damage in MCF-7 cells treated with AuCh-NPs compared to AuCit-NPs. These findings highlight the superior selective cytotoxicity of AuCh-NPs against MCF7 cells, positioning them as a promising targeted agent for photothermal therapy in breast cancer treatment.", "journal": "Apoptosis : an international journal on programmed cell death", "year": "2025", "doi": "10.1007/s10495-025-02145-6", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42737657": { "pmid": "42737657", "title": "Development of Loop-Mediated Isothermal Amplification with a Lateral Flow Dipstick for Detection of the BlaOXA-23-like Gene from Carbapenem-Resistant Acinetobacter baumannii.", "abstract": "Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major cause of hospital-acquired infections. Conventional phenotyping for carbapenem resistance requires more than 24 h, which delays clinical decisions and increases the risk of outbreaks. This study aims to develop a lateral flow dipstick (LFD) assay that detects loop-mediated isothermal amplification (LAMP) products targeting the blaOXA-23-like gene, providing results within 1-1.5 h. Primer specificity was evaluated using seven Gram-positive and nine Gram-negative species commonly present in clinical samples, as well as carbapenem-susceptible A. baumannii and CRAB isolates harboring other resistance genes. The assay detected the blaOXA-23-like gene at concentrations as low as 1.57 pg/µL, demonstrating ~10-fold greater sensitivity than conventional PCR. The gene was present in 224/268 CRAB samples (83.6%) and absent in 100 carbapenem-susceptible A. baumannii samples. Results were fully concordant with PCR, yielding 100% sensitivity and specificity. The method requires no expensive or complex instrumentation. Future studies should validate the assay across multiple hospitals and clinical specimens to confirm its generalizability and clinical utility. Multiplex LAMP with dual LFD readouts could also enable simultaneous detection of additional carbapenem resistance genes, supporting rapid CRAB surveillance and outbreak control.", "journal": "International journal of molecular sciences", "year": "2026", "doi": "10.3390/ijms27177761", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41202380": { "pmid": "41202380", "title": "Chinese medicine formulation Mailuoning attenuates high-fat diet-induced obesity by regulating thermogenesis through activating the peroxisome proliferator-activated receptor α/fibroblast growth factor 21 axis.", "abstract": "Mailuoning is a Chinese medicine formulation known for its efficacy in clearing heat, nourishing yin, activating blood circulation, and resolving blood stasis. In traditional Chinese medicine, Mailuoning is considered to have potential benefits in improving obesity. Our previous research has demonstrated that Mailuoning could effectively alleviate non-alcoholic fatty liver disease (NAFLD), a metabolic disorder with pathophysiological similarities to obesity. However, the alleviating effects of Mailuoning on obesity have yet to be fully elucidated, and further investigation is required to confirm its therapeutic effect. To assess the impact and underlying mechanisms of Mailuoning on obesity, and to investigate its potential active components. Mice were fed with a high-fat diet (HFD) to induce obesity. The anti-obesity effects of Mailuoning were assessed by monitoring body weight, food intake, and hepatic lipid accumulation, as well as performing insulin tolerance test (ITT) and glucose tolerance test (GTT). Real-time PCR and western-blot analyses were utilized to detect gene and protein expression, respectively. The metabolic rate of mice was assessed using metabolism cages. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed to identify compounds in inguinal white adipose tissue (iWAT). Additionally, Oil Red O staining assay was conducted on 3T3-L1 cells to detect cellular lipid accumulation and screen for active compounds. Mailuoning significantly reduced the weight of mice, iWAT, and epididymis white adipose tissue (eWAT), lowered the levels of hepatic triglyceride (TG) and non-esterified fatty acid (NEFA), and improved glucose regulation and homeostasis, thereby exerting its efficacy in alleviating HFD-induced obesity. Mechanistically, Mailuoning upregulated hepatic expression of peroxisome proliferator-activated receptor α (PPARα), facilitated its nuclear translocation, and enhanced the expression of its downstream fatty acids oxidation-related genes. Moreover, Mailuoning increased the expression of hepatic fibroblast growth factor (FGF) 21 and promoted its release into the blood. In addition, Mailuoning increased the protein expression of FGF21, FGF receptor (FGFR) 1, and Klotho/β-Klotho (KLB), as well as enhanced the phosphorylation of intracellular extracellular signal-regulated kinase 1/2 (ERK1/2), consequently elevating the expression of thermogenic genes in both brown adipose tissue (BAT) and iWAT. Energy metabolism results revealed that Mailuoning increased energy expenditure in mice. Subsequent research suggested that Lonicerae Japonicae Flos and Lonicerae Flos might be the primary bioactive herbal medicines in Mailuoning for treating obesity. A total of 52 compounds were detected in iWAT, among which 18 were identified in these two herbal medicines. Among these, loganic acid, luteolin, loganin, chlorogenic acid, caffeic acid, and macranthoidin B effectively lowered intracellular lipid levels in 3T3-L1 cells. Mailuoning improved obesity in HFD-fed mice by establishing crosstalk between the liver and adipose tissue by activating PPARα/FGF21 axis, thereby enhancing energy expenditure. Among the constituent herbs, Lonicerae Japonicae Flos and Lonicerae Flos were identified as the primary herbal medicines responsible for the anti-obesity efficacy of Mailuoning.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2025.157474", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40001484": { "pmid": "40001484", "title": "\"One Pot\" Enzymatic Synthesis of Caffeic Acid Phenethyl Ester in Deep Eutectic Solvent.", "abstract": "Caffeic acid phenethyl ester (CAPE) represents a valuable ester of caffeic acid, which, over time, has demonstrated remarkable pharmacological properties. In general, the ester is obtained in organic solvents, especially by the esterification reaction of caffeic acid (CA) and 2-phenylethanol (PE). In this context, the purpose of this study was the use of the \"one pot\" system to synthesize CAPE through biocatalysis with various lipases in a choline-chloride-based DES system, employing the \"2-in-1\" concept, where one of the substrates functions as both reactant and solvent. The synthesis process of CAPE is contingent on the molar ratio between CA and PE; thus, this factor was the primary subject of investigation, with different molar ratios of CA and PE being studied. Furthermore, the impact of temperature, time, the nature of the biocatalyst, and the water loading of the DES system was also examined. This 'green' synthesis method, which has demonstrated encouraging reaction yields (%), could secure and maintain the therapeutic potential of CAPE, mainly due to the non-toxic character of the reaction medium.", "journal": "Biomolecules", "year": "2025", "doi": "10.3390/biom15020181", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "42671244": { "pmid": "42671244", "title": "Uracil-DNA Glycosylase-Enhanced Loop-Mediated Isothermal Amplification Assays for Detection of Zoonotic Malaria Plasmodium Parasites.", "abstract": "Zoonotic malaria caused by simian Plasmodium species presents increasing diagnostic challenges in endemic regions. This study developed and evaluated malachite green-based loop-mediated isothermal amplification (MG-LAMP) assays for detecting five zoonotic malaria parasites (P. knowlesi, P. cynomolgi, P. inui, P. coatneyi, and P. fieldi). The assays enable rapid visual detection through a colorimetric change without requiring specialized equipment. To reduce carryover contamination, deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) were incorporated into the reaction. All five UDG MG-LAMP assays demonstrated high analytical sensitivity, with limits of detection of 1 copy/µL for P. cynomolgi, P. coatneyi, and P. fieldi, and 10 copies/µL for P. knowlesi and P. inui. The assays showed 100% specificity, with no cross-reactivity among target species. Malachite green enabled clear visual discrimination between positive and negative reactions. A simplified direct blood lysis method requiring less than 20 min was also developed to enable rapid DNA preparation without column-based purification. Overall, the MG-LAMP assays combined with UDG and rapid lysis provide a sensitive, specific, and practical approach for diagnosing zoonotic malaria, particularly in resource-limited endemic settings.", "journal": "Vector borne and zoonotic diseases (Larchmont, N.Y.)", "year": "2026", "doi": "10.1177/15303667261484456", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39523151": { "pmid": "39523151", "title": "Efficacy of different disinfecting methods for contact lenses against Acanthamoeba castellanii.", "abstract": "To analyze the efficacy of different disinfecting methods for contact lenses (CL) against Acanthamoeba castellanii (AC) using quantitative PCR (qPCR) based on RNA detection. Three CL materials: rigid gas permeable (RGP), hydrogel (Hy), and silicone hydrogel (SiHy), were contaminated with 1x105 amoebae/ml and incubated for 24 h at 30 °C. After contamination, pre-cleaning steps were performed before using four maintenance solutions based on hydrogen peroxide (HP), sodium hypochlorite (SH), povidone-iodine (PI), and a multipurpose solution (MS). The pre-cleaning steps involved using a cleaner (20 % isopropyl alcohol) and rinsing the CL. Disinfection systems 1 and 2 involved no cleaner and rinsed the CL with tap water or saline solution, respectively. Systems 3 and 4 included a cleaner and rinsed with tap water or saline, respectively. After cleaning, A. castellanii was extracted and stored for qPCR analysis, using Hsp70 and TPBF genes to detect RNA A. castellanii. Results were presented as the percentage of positives or negatives (presence or absence of amoebae), with a p-value < 0.05 considered statistically significant. Disinfection system 1 with MS resulted in 56 % positives for RGP lenses and 100 % positives for both hydrogel materials. When MS was combined with a cleaner, 12.5 % positives were found for SiHy and 100 % negatives for Hy and RGP lenses (p < 0.05). PI solution alone yielded 38 % and 12 % positives for hydrogel and silicone hydrogel lenses, respectively, but was 100 % effective when combined with a cleaner. HP and SH, whether combined with a cleaner or not, were effective against AC for all CL materials, except HP without a cleaner for Hy lenses. All disinfection methods showed some efficacy against Acanthamoeba on any CL material. The most effective solutions were those based on hydrogen peroxide and sodium hypochlorite. Using a cleaner enhanced the final disinfecting efficacy, especially with the multipurpose solution.", "journal": "Contact lens & anterior eye : the journal of the British Contact Lens Association", "year": "2025", "doi": "10.1016/j.clae.2024.102326", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41266742": { "pmid": "41266742", "title": "Probiotic modulation of central and peripheral sensitization in a migraine model via the gut-brain-trigeminal axis.", "abstract": "The gut-brain axis has emerged as a pivotal modulator of migraine pathophysiology. This study examined the neurophysiological effects of targeted probiotic supplementation-Lactobacillus plantarum, Bifidobacterium longum, their combination, and a multi-strain formulation (Pro-14)-in a rat model of cortical spreading depression (CSD), a hallmark trigger of migraine. B.longum significantly reduced CSD frequency (p = 0.0045), while L.plantarum and its combination with B.longum markedly attenuated CSD amplitude (p = 0.0003, p = 0.01, respectively), reflecting suppression of cortical hyperexcitability. In small-to-medium-sized trigeminal ganglion (TG) neurons, probiotics significantly lowered total spike counts (p < 0.0001), and induced hyperpolarization of resting membrane potential (p < 0.0001), consistent with peripheral desensitization. Additionally, the threshold-Resting Membrane Potential (RMP) gap increased significantly across all treated groups (p < 0.0001), indicating reduced neuronal excitability. Only B.longum prolonged action potential duration and depolarization time (p = 0.0454 and p = 0.034, respectively), suggesting enhanced modulation of nociceptive signaling. In large-sized TG neurons, similar hyperpolarizing trends in RMP (p < 0.0001) and increased excitability thresholds (p < 0.0001) were observed without changes in spike frequency. These findings reveal strain-specific modulation of central and peripheral sensitization via the gut-brain-trigeminal axis and support the development of precision-targeted, microbiota-based therapies as non-pharmacologic strategies for migraine prophylaxis.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-24928-2", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39803216": { "pmid": "39803216", "title": "From Soil to Salad: Strategies for Reducing Foodborne Illness Outbreaks.", "abstract": "This study addresses the global issue of foodborne illness, specifically focusing on those resulting from the consumption of leafy green vegetables. It explores the rising trend of consuming minimally processed or raw foods and the imperative of maintaining safety standards starting at the preharvest stage to prevent pathogenic bacterial contamination. The study identifies soil and irrigation water as key sources of pathogens and emphasizes the need for strict preventive measures during production and preharvest. It discusses the challenges of postharvest decontamination and highlights the importance of early-stage prevention strategies. The paper also examines advanced pathogen detection methods and food safety practices recommended by USDA and FSMA's PSR, including HACCP and LGMA strategies. Aimed at providing insights for consumers and producers, the study underscores the necessity of effective manufacturing strategies to ensure the safety of leafy greens.", "journal": "Food science & nutrition", "year": "2025", "doi": "10.1002/fsn3.4521", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "42326795": { "pmid": "42326795", "title": "Vascular Phenotyping in Parkinson's Disease: Diabetes Mellitus Operationalizes a Microvascular Metabolic Syndrome Cluster Across PPMI Diagnostic Cohorts.", "abstract": "Diabetes mellitus elevates Parkinson's disease (PD) risk, via hypothesized cerebrovascular mediation. Whether the diabetes/prediabetes vascular-risk phenotype concentrates in cardiometabolic risk or macrovascular events across prodromal and clinically diagnosed PD remains unresolved. To quantify the vascular-risk burden associated with diabetes/prediabetes across the PPMI diagnostic cohorts to test whether this association differs by cohort. Cross-sectional analysis of 413 PPMI participants (76 healthy controls, 145 prodromal PD, 192 clinically diagnosed PD) examined diabetes/prediabetes (n = 73) and seven vascular risk factors. The Vascular Burden Score (0 to 7) was a priori partitioned into microvascular and macrovascular sub-scores. Modified Poisson regression estimated adjusted prevalence ratios (aPR), adjusted for age, sex, and body mass index. A cohort-by-diabetes interaction tested cross-cohort consistency. Sensitivity analyses incorporated nigral diffusion tensor imaging (PD-risk biomarker) and FreeSurfer white matter hypointensity volume (cerebrovascular marker). Diabetes/prediabetes elevated Vascular Burden Score (β = 0.53, 95% CI 0.29 to 0.77, p < 0.001) versus non-diabetic participants, with a non-significant cohort-by-diabetes interaction (F = 0.29, p = 0.747). Three microvascular factors survived false discovery rate correction: obesity (aPR 2.28), hypertension (aPR 1.60), and hyperlipidemia (aPR 1.45). Macrovascular events showed no diabetic amplification (β = -0.06, p = 0.25). In the imaging-phenotyped subset, Vascular Burden Score components contributed classifier variance distinct from nigral microstructure. Diabetes/prediabetes operationalize a microvascular cluster stable across prodromal and idiopathic PD. Cardiometabolic phenotyping may complement established PD-risk biomarkers (dopamine transporter SPECT, nigral diffusion), pending longitudinal validation linking vascular phenotype to dopaminergic markers.", "journal": "medRxiv : the preprint server for health sciences", "year": "2026", "doi": "10.64898/2026.06.09.26355285", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "C" ] }, "40884671": { "pmid": "40884671", "title": "The Evolutionary Potential of Chromoanagenesis.", "abstract": "Many genomics specialists recognize that the massive genome rearrangements grouped under the term \"chromoanagenesis\" are a path to rapid evolutionary change by restructuring chromosomes, creating chimeric sequence combinations, and altering regulatory interactions leading to novel phenotypes. Less attention has been paid to the role of ubiquitous eukaryotic double-strand DNA break repair functions known as \"alternative end-joining\" (AltEJ) in generating additional DNA sequence innovation. A close look at some examples of chromoanagenesis rearrangements in the human germline and tumor cells illustrates how diverse these novel sequences can be. AltEJ creates sequence duplications and DNA insertions into junctions between chromosome breakpoints; these insertions range in size from a few base pairs up to many kilobases in length. The AltEJ insertions can come from nearby or distal sequences on one of the rearranged chromosomes, from another chromosome, or from de novo replication without template carried out by the versatile DNA polymerase theta, an enzyme known for its activity as an untemplated DNA terminal transferase. In a significant number of cases, the AltEJ insertions contain novel combinations of several segments copied from more than one region of the genome by a phenomenon known as \"template switching.\" By virtue of the combinatorial and creative actions of AltEJ in chromosome restructuring, massive genome change by chromoanagenesis contributes an apparently limitless range of DNA sequence innovation to eukaryotic evolutionary novelty.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2025", "doi": "10.1007/978-1-0716-4750-9_37", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39951758": { "pmid": "39951758", "title": "Differentiable pixel-super-resolution lensless imaging.", "abstract": "Conventional lensless imaging systems require complex phase diversity measurements and sequential processing steps, limiting their practical application despite their compact design. We present a differentiable end-to-end pixel-super-resolution (dPSR) technique that unifies PSR hologram synthesis, autofocusing, and complex-field reconstruction within a single optimization framework. By jointly optimizing these traditionally separate processes, our method eliminates both phase diversity requirements and error accumulation from sequential processing. Our method achieves superior position estimation accuracy (mean error 0.0282 pixels versus 0.1172 pixels with conventional methods), delivering precise autofocusing with accuracy better than 0.3 µm, and enabling a twofold resolution enhancement beyond the sensor's native pixel size. This robust performance is validated through both simulated and experimental results, including challenging phase objects and label-free cell imaging, establishing dPSR as a practical solution for high-resolution microscopy applications.", "journal": "Optics letters", "year": "2025", "doi": "10.1364/OL.552086", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41692188": { "pmid": "41692188", "title": "Stress-dependent interfacial competition with direct binding enhances IgG4 stabilization.", "abstract": "This study elucidates the stress-dependent stabilization mechanisms of nonionic surfactants, polysorbates (Tween 20/80), and poloxamer 188 (P188) for IgG4 monoclonal antibodies (mAbs), a clinically important subclass with distinct structural vulnerabilities. To investigate these mechanisms, we employed a multi-method approach that integrated isothermal titration calorimetry (ITC), critical micelle concentration (CMC) measurements, and a multi-stress model encompassing orbital shaking, vortex stress, and freeze-thaw cycles. Our results demonstrate that polysorbates bind to IgG4 via entropy-driven hydrophobic interactions, whereas no such interaction was observed for P188. Under mild thermodynamically controlled stresses, both surfactants stabilized the protein through saturable interfacial adsorption once their concentrations reached the respective CMC, despite a pronounced difference in CMC values (Tween 20/80 ≪ P188). In contrast, under intense, kinetically controlled vortex stress, their mechanisms diverge: polysorbates require supra-CMC concentrations (4 × CMC) to act as dynamic monomer reservoirs and provide full protection, whereas P188 provides only limited stabilization because of its inherently slower adsorption kinetics. These findings establish that IgG4 stabilization is governed by distinct, stress-dependent pathways. The resulting framework directly connects fundamental protein-surfactant interactions with rational formulation design, facilitating a paradigm shift from empirical screening to mechanism-guided selection of stabilizers.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.150932", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41319638": { "pmid": "41319638", "title": "Superior stability and pollutant degradation via chitosan-immobilized laccase: A comparative study with NH2-MIL-125 MOF.", "abstract": "The accumulation of emerging pollutants (EPs) in aquatic environments poses significant threats to both human health and ecosystem integrity, highlighting the urgent need for effective and sustainable remediation strategies. Among the available approaches, biological systems such as laccase-based bioremediation have emerged as a promising greener solution, particularly when the enzyme is immobilized to enhance its stability and reusability. Although laccase immobilization on various carriers have been widely studied, published comparisons of their performance remain limited and insufficiently studied. In this study, we evaluated and compared the degradation efficiency of 15 EPs by laccase immobilized on two supports: chitosan and the metal-organic framework NH2-MIL-125. Our results showed that laccase immobilized on chitosan exhibited superior thermal and pH stability, along with enhanced recyclability, outperforming both free and MOF-immobilized enzymes. In the presence of a model redox mediator hydroxybenzotriazole (HOBT), chitosan-immobilized laccase achieved complete degradation of paracetamol and 48 % degradation of sulfamethoxazole in 1 h, both of which were not degraded by either free laccase or the MOF-immobilized form. While NH2-MIL-125 allowed for higher enzyme loading and was easier to handle, it significantly compromised the thermal stability of the enzyme. This study demonstrates, for the first time, the superior performance of laccase immobilized on chitosan, as directly compared to NH2-MIL-125, for the degradation of a diverse class of EPs. By combining enhanced stability, reusability, and broad pollutant coverage, the system highlights strong potential for large-scale and sustainable wastewater treatment, advancing enzyme-based bioremediation as a practical and environmentally friendly technology.", "journal": "Journal of environmental management", "year": "2025", "doi": "10.1016/j.jenvman.2025.128129", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40861759": { "pmid": "40861759", "title": "Optimizing SARS-CoV-2 Detection: A Rapid, Cost-Effective Reverse Transcription Polymerase Chain Reaction (RT-PCR) Method Without RNA Extraction.", "abstract": "Introduction In December 2019, the emergence of a novel coronavirus, SARS-CoV-2, led to the global COVID-19 pandemic. Early diagnostic efforts included molecular tests such as reverse transcription polymerase chain reaction (RT-PCR). However, testing faced challenges including RNA reagent shortages and prolonged processing times. Antigen card tests, though reliable for positive results, had limitations. Tata MD CHECK RT-PCR XF emerged as a versatile diagnostic assay, capable of detecting SARS-CoV-2 with or without RNA extraction. This facilitated efficient testing amid ongoing resource constraints. This study was planned to compare the diagnostic accuracy of the Tata MD CHECK RT-PCR XF kit, a direct RT-PCR assay that eliminates the RNA extraction step, with conventional RT-PCR incorporating RNA extraction for detecting SARS-CoV-2 RNA. The direct RT-PCR approach demonstrated a sensitivity of 93.9%, closely aligning with conventional RT-PCR results, and showed comparable performance in lower Ct ranges (0-30), though reduced detection was observed at higher Ct values. Methods A cross-sectional study was conducted in the Department of Microbiology, Santosh Medical College, Ghaziabad. Nasopharyngeal and oropharyngeal samples were collected in viral transport media (VTM). Samples were processed in duplicate: one underwent RNA extraction, while the other was directly tested, without RNA extraction, using the Tata MD CHECK RT-PCR XF kit. Results A total of 110 samples were evaluated, with 62.7% from males and 37.3% from females, primarily in the 21-40 age group. The Tata MD Direct PCR demonstrated high diagnostic accuracy, with a sensitivity of 93.9%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 84.4%. Discussion This cost-effective and rapid method significantly reduced turnaround times compared to conventional RT-PCR.", "journal": "Cureus", "year": "2025", "doi": "10.7759/cureus.88654", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41089093": { "pmid": "41089093", "title": "Bridging the Diagnostic Gap: A Rapid, Cost-Effective, and Equitable Hepatitis C Virus RNA Detection Method for Resource-Limited Settings.", "abstract": "Hepatitis C virus (HCV) remains a major global health challenge, particularly in resource-limited settings where access to molecular diagnostics is restricted. The loop-mediated isothermal amplification (LAMP) assay offers a rapid, cost-effective alternative to polymerase chain reaction (PCR) for HCV RNA detection. Unlike PCR, LAMP uses isothermal amplification (60-65°C), eliminating the need for expensive thermal cyclers and yielding results in under 60 minutes. We evaluated two LAMP detection methods - hydroxynaphthol blue (HNB, colorimetric) and calcein (fluorescent) - against an in-house TaqMan qPCR assay. Both LAMP methods demonstrated high sensitivity (99.6%) and specificity (95.6% for HNB, 99.2% for calcein), with a broad dynamic range (10-10⁶ copies/mL). Clinical validation against real-time PCR showed strong agreement, with a positive predictive value of 98.6% and a negative predictive value of 94.4%. The HNB-LAMP assay, in particular, provides a simple visual readout (blue to sky blue), requiring no specialized equipment, while calcein-LAMP offers fluorescence-based detection under UV light. Both methods showed no significant correlation (p > 0.5), confirming their reliability. LAMP's advantages - minimal infrastructure, ambient temperature stability, and low cost (<$5 per test) - make it ideal for decentralized testing in low-resource settings. This approach could revolutionize HCV diagnosis by enabling same-day test-and-treat strategies, improving linkage to care, and supporting global HCV elimination efforts. Future steps include field validation in remote clinics, manufacturing scale-up, and integration into point-of-care platforms to maximize accessibility. By bridging the diagnostic gap, LAMP can potentially transform HCV management in underserved regions worldwide.", "journal": "Cureus", "year": "2025", "doi": "10.7759/cureus.92101", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41347631": { "pmid": "41347631", "title": "Discovery of a Novel DNMT1 Inhibitor with Improved Efficacy in Treating β-Thalassemia.", "abstract": "β-thalassemia is a recessively inherited blood disorder affecting millions worldwide. Pharmacological induction of fetal hemoglobin (HbF) is an effective therapeutic strategy, yet existing DNA methyltransferase (DNMT) inhibitors, although effective HbF inducers, currently are not approved for β-thalassemia treatment. Here, we report that DMT207, a novel non-nucleoside DNMT1 inhibitor, robustly reactivates HbF in HUDEP-2 cells and adult primary erythroblasts with minimal toxicity. In a mouse model of β-thalassemia, DMT207 effectively elevates the levels of mouse fetal- and embryonic-type hemoglobin, promotes the maturation of erythroid cells, and alleviates the splenomegaly. Further multi-omics analyses expose γ-globin as one of the most sensitive genes with promoter demethylation and transcriptional activation following DMT207 treatment. Mechanistically, DMT207 traps DNMT1 into a catalytically inactive conformation and concurrently enhances its interaction with UHRF1, which partially contributes to DNMT1 degradation. These findings highlight the therapeutic potential of DMT207 for β-thalassemia and support its further preclinical development.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2026", "doi": "10.1002/advs.202513469", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41139261": { "pmid": "41139261", "title": "Validated isocratic stability-indicating RP-HPLC method for concurrent remdesivir estimation in drug products used in bioavailability studies.", "abstract": "This study presents a stability-indicating, isocratic RP-HPLC method for the quantitative analysis of remdesivir (RMD) in pharmaceutical formulations and bioavailability studies. The method was developed to ensure robustness, precision and accuracy across diverse matrices, including freeze-dried powders, microspheres, novasomes and biological samples like blood. Chromatographic separation was achieved using a Zorbax Eclipse Plus C-18 column (4.6 × 250 mm, 5 μm) with a mobile phase of 1% trifluoroacetic acid (TFA) and acetonitrile (55:45) at a 1 mL/min flow rate. Detection was carried out at 254 nm using a variable wavelength detector (VWD). Validation followed current good manufacturing practices (cGMP), covering parameters such as system suitability, linearity, specificity, precision, accuracy, robustness and forced degradation. The method showed a retention time consistent with RMD standards, a limit of detection (LOD) of 0.73 µg/mL and a limit of quantification (LOQ) of 2.22 µg/mL. Excellent linearity (R² = 0.999) was observed in the 12-120 µg/mL range. Long-term and accelerated stability studies confirmed the method's capability to detect degradation products without interference. The validated method supports reliable routine quality control testing and pharmacokinetic studies, making it suitable for in-vivo bioavailability assessments of RMD formulations.", "journal": "Pakistan journal of pharmaceutical sciences", "year": "2025", "doi": "10.36721/PJPS.2025.38.6.REG.14360.1", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "", "dirs": [ "A" ] }, "41353895": { "pmid": "41353895", "title": "Calpain-1 Potentiates Periodontal Regeneration via PHLPP1-ERK-Driven Osteogenesis in Periodontal Ligament Stem Cells.", "abstract": "In recent years, human periodontal ligament stem cells (hPDLSCs) are considered a promising seed cell source for bone tissue engineering, offering a prospective treatment for periodontal bone defects. The osteogenic differentiation of hPDLSCs is a critical process in bone tissue engineering. Calcium signalling regulates a myriad of cellular processes, including osteogenic differentiation; however, the function of calpain-1 (CAPN1), a calcium-dependent protein, remains incompletely elucidated. Intracellular Ca2+ levels were evaluated using Fluo-4 AM assay. CAPN1 was overexpressed by transfection of lentivirus. Knockdown of CAPN1 was performed using siRNA transfection. mRNA and protein expression were detected through Real-time PCR and Western blot, respectively. Immunofluorescence assay was performed using anti-CAPN1 and anti-phosphatase PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) antibodies. Hydroxyapatite/beta-tricalcium phosphate loaded with CAPN1-overexpressing hPDLSCs were implanted into the dorsal region of nude mice. Periodontal bone defects were prepared using a trephine drill, and GelMA loaded with CAPN1-overexpressing hPDLSCs were implanted into the defect area. Fluo-4 AM assay showed a continuous upregulation of intracellular Ca2+ levels, CAPN1 expression, and calpain activity during osteogenic induction of hPDLSCs. Overexpression of CAPN1 enhanced osteogenic differentiation of hPDLSCs and involved activation of the ERK1/2 signalling pathway. Knockdown CAPN1 or inhibiting CAPN1 by chemical inhibitor decreased osteogenic differentiation of hPDLSCs through ERK1/2 signalling pathway. Immunofluorescence staining and molecular docking analysis suggested that CAPN1 was colocalized and bound with PHLPP1. Furthermore, the combination of CAPN1-overexpressing hPDLSCs with hydroxyapatite/beta-tricalcium phosphate or GelMA hydrogel significantly enhanced bone regeneration in both subcutaneous implantation and periodontal bone defect models. Our study reveals the role and mechanism of CAPN1 in the osteogenic differentiation of hPDLSCs, which provides a theoretical and technical foundation for targeting CAPN1 in bone regeneration.", "journal": "International dental journal", "year": "2026", "doi": "10.1016/j.identj.2025.109309", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40921034": { "pmid": "40921034", "title": "Bimorph Soft Actuators Based on Isostructural Heterogeneous Janus Films.", "abstract": "Bimorph soft actuators, traditionally composed of two materials with distinct responses to external stimuli, often face durability challenges due to structural incompatibility. Here, we propose an alternative design employing free-standing, isostructural heterogeneous Janus (IHJ) films that harmonize stability with high actuation efficiency. These IHJ films were fabricated through a vacuum self-assembly process, consisting of Ti3C2Tx MXene nanosheets and hybrid graphene oxide (GO)-biomass bacterial cellulose (BC), with a well-matched two-dimensional lattice structure. The special structure of the film, featuring continuous laminar layers and one-dimensional (1D) BC nanofibers intertwining with two-dimensional (2D) GO and MXene nanosheets, promotes interlayer slip during deformation, enhancing stability and deformability. The IHJ film responds swiftly to electrical stimuli, humidity, and light, causing layer expansion or contraction through the rapid absorption and release of water molecules and enabling controllable actuation. The IHJ film can be utilized as actuators in various soft robotic applications, such as a \"flower robot\" under electrical stimulus, a \"passive switch\" under humidity, and a \"worm robot\" under infrared lamp exposure. The design of the IHJ film achieves large-scale deformation, low power consumption, high responsiveness, and enhanced real-time sensing capabilities, representing an important development in soft actuator design.", "journal": "ACS nano", "year": "2025", "doi": "10.1021/acsnano.4c16626", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40709741": { "pmid": "40709741", "title": "Molecular insights into particular mutations impact on Taq polymerase dynamics and structure: a molecular dynamics simulation study.", "abstract": "Taq DNA polymerase (Taq pol), a useful enzyme in biotechnology, has been the subject of an extensive investigation to create enzymes with altered characteristics, notably the enhancement of Taq pol's thermostability, But the demand for such enzymes remains unmet. In this study, we aimed to introduce several mutations to the Taq pol structure to improve its structural stability. We combined the sequence-based and structure-based rational design mutagenesis techniques to incorporate five mutations into the Taq pol structure. The impact of the mutations on the enzyme was examined by the utilization of molecular dynamics simulations. All mutant enzymes exhibited a more compact structure and a greater abundance of internal hydrogen bonds and secondary structure contents. While the mutants TaqG389E and TaqS290K|A353K|L365E|N384E|G389E demonstrated the least structural dynamics, the other mutant enzymes displayed higher or comparable flexibility compared to the wild-type. Furthermore, a reduction in the distance between mutation positions and neighboring charged amino acids was observed in most of the mutant structures. The enhancement of non-bonded interactions resulted in the evaluation of structure stability. Based on this fact, all of the suggested mutations had positive effects on the stability of the Taq pol structure. Additionally, a decrease in structural flexibility led to a more stable structure; hence, TaqG389E and TaqS290K|A353K|L365E|N384E|G389E mutants were considered more stable compared to wild-type Taq pol. The mutants TaqG389E and TaqS290K|A353K|L365E|N384E|G389E exhibited excellent performance in terms of reduced structural flexibility, evaluated internal hydrogen bonds, and the capacity to establish multiple stabilizable interactions when compared to the wild-type.", "journal": "Journal of biomolecular structure & dynamics", "year": "2026", "doi": "10.1080/07391102.2025.2524436", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41077252": { "pmid": "41077252", "title": "Loop-mediated isothermal amplification (LAMP): A promising tool for rapid, point-of-care diagnosis of parasitic diseases in low-income countries.", "abstract": "Parasitic diseases pose a major burden throughout the globe, especially in low-income countries, which are disproportionately affected due to factors such as overpopulation, vector-control challenges, financial difficulties, etc. Because access to advanced diagnostic technologies is limited in low-income regions, early and accurate diagnosis, which is critical in preventing diseases from progressing to their fatal stages, is not possible. Methods such as polymerase chain reaction (PCR) are often impractical in such regions due to their complexity and high running costs. Loop-mediated isothermal amplification (LAMP) is a promising alternative diagnostic technology that amplifies and detects a specific DNA sequence from a sample. Its features, such as high sensitivity and specificity, affordability, simplicity and versatility, make it an attractive alternative for poorer countries. Moreover, LAMP operates under isothermal conditions, thereby reducing the need for expensive equipment. This review evaluates the applicability of LAMP to be used as an alternative POC diagnostic method for parasitic infections by comparing it with routinely used diagnostic methods. Furthermore, several emerging trends in LAMP technology, such as multiplex LAMP, reverse transcription LAMP, microfluidic chip-incorporated LAMP, digital LAMP, and single nucleotide polymorphism LAMP, are discussed, further illustrating the potential of improving the diagnostic accuracy and affordability of LAMP. The need for cost-effective and accurate diagnostic tools is critical, particularly in regions where parasitic diseases are most prevalent. LAMP represents a viable solution to reduce the burden of parasitic diseases through improved diagnostic capabilities.", "journal": "Molecular and biochemical parasitology", "year": "2025", "doi": "10.1016/j.molbiopara.2025.111704", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "39667227": { "pmid": "39667227", "title": "Artificial intelligence-driven quantification of antibiotic-resistant Bacteria in food by color-encoded multiplex hydrogel digital LAMP.", "abstract": "Antibiotic-resistant bacteria pose considerable risks to global health, particularly through transmission in the food chain. Herein, we developed the artificial intelligence-driven quantification of antibiotic-resistant bacteria in food using a color-encoded multiplex hydrogel digital loop-mediated isothermal amplification (LAMP) system. The quenching of unincorporated amplification signal reporters (QUASR) was first introduced in multiplex digital LAMP. During amplification, primers labeled with different fluorophores were integrated into amplicons, generating color-specific fluorescent spots. While excess primers were quenched by complementary quenching probes. After amplification, fluorescent spots in red, green, and blue emerged in hydrogels, which were automatically identified and quantified using a deep learning model. Methicillin-resistant Staphylococcus aureus and carbapenem-resistant Escherichia coli in real fruit and vegetable samples were also successfully detected. This artificial intelligence-driven color-encoded multiplex hydrogel LAMP offers promising potential for the digital quantification of antibiotic-resistant bacteria in the food industry.", "journal": "Food chemistry", "year": "2025", "doi": "10.1016/j.foodchem.2024.142304", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "C" ] }, "42224782": { "pmid": "42224782", "title": "Rapid multiplex detection of Echinococcus granulosus and Echinococcus multilocularis using a one-pot RPA-assisted CRISPR-Cas12a/Cas13a assay in a portable multi-tube device.", "abstract": "Echinococcosis, caused by Echinococcus granulosus and Echinococcus multilocularis, remains a significant zoonotic threat, particularly in pastoral regions where rapid environmental surveillance is essential yet technically constrained. Here, we report a rapid and integrated one-pot recombinase polymerase amplification -assisted, orthogonal CRISPR-Cas12a/Cas13a platform for rapid and specific discrimination of these two species in environmental samples. Coupled with a simplified NaOH-based DNA extraction method, the assay enables a streamlined workflow completed within 60 min, achieving a detection limit of as low as 1 copy/μL without observable cross-reactivity. To facilitate point-of-care deployment, we further developed a low-cost, miniaturized handheld device capable of parallel analysis of up to eight samples with dual-target readout. The platform was validated using field samples, including canine feces, pasture grass, and vegetables, demonstrating complete agreement with quantitative PCR results, with 100% sensitivity and specificity. This integrated CRISPR-based biosensing system provides a robust and field-deployable solution for on-site echinococcosis surveillance and offers a scalable framework for multiplex environmental pathogen detection.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.118857", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41960158": { "pmid": "41960158", "title": "Stage-matched conductive hydrogel with pH/ROS/MMP9 triple-responsive salidroside release for post-infarction myocardial repair.", "abstract": "Myocardial infarction (MI) triggers massive cardiomyocyte loss, inflammatory and fibrotic remodeling, and disruption of electrical conduction, which are difficult to reverse with a single therapeutic modality. Pathologically, the infarcted myocardium undergoes a sequential transition from local acidosis to oxidative/inflammatory injury and finally matrix metalloproteinase-driven scar formation. Guided by these stage-specific cues and the need to restore electrical continuity, we designed an injectable conductive hydrogel (PGO/CAM@Sal) that integrates a polypyrrole-based conductive network with pH/reactive oxygen species (ROS)/matrix metalloproteinase-9 (MMP9) triple-responsive salidroside delivery. The hydrogel matches myocardial stiffness, exhibits favorable injectability, self-healing and tissue adhesion, shows electrical conductivity comparable to native myocardium, and enables on-demand salidroside release under MI-mimicking conditions. In vitro, PGO/CAM@Sal effectively scavenges ROS, suppresses pro-inflammatory cytokine production, promotes M2 macrophage polarization and angiogenesis, and enhances connexin-43 expression and cardiomyocyte electrical coupling. In a rat MI model, local injection of PGO/CAM@Sal bridges the non-conductive infarct, stabilizes ventricular electrophysiology, improves conduction velocity and homogeneity, reduces arrhythmia vulnerability, and concurrently attenuates fibrosis, hypertrophy, and adverse left ventricular remodeling, leading to marked recovery of systolic function. Transcriptomic analysis suggests a potential involvement of the Mmp12/Cybb axis and related pathways involved in inflammation, oxidative stress, fibrosis, calcium handling, and ion transport. These molecular alterations were further validated by qRT-PCR and immunohistochemical analyses, collectively supporting the integrated therapeutic benefits observed. Accordingly, this stage-matched conductive smart hydrogel provides a comprehensive, spatiotemporally controlled strategy for precise post-MI repair.", "journal": "Materials today. Bio", "year": "2026", "doi": "10.1016/j.mtbio.2026.103016", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40268096": { "pmid": "40268096", "title": "Whole yeast cell synthesis of 5-hydroxymethylfurfural-derivatives from apple waste processed by green technologies.", "abstract": "The transition to renewable resources and bioprocesses is essential to address climate change and meet energy demands. Biorefineries offer a sustainable solution by valorising agro-industrial wastes, such as apple pomace, a by-product of the juice and cider industries. This study presents an efficient process to produce 5-hydroxymethylfurfural (HMF) derivatives using deep eutectic solvent (DES) and microwave-assisted heating. Under optimal conditions (160 °C, 30 min, and 50 % Choline Chloride: Lactic acid (ChCl:LA) DES) HMF yield reached 34.6 %. Engineered Saccharomyces cerevisiae strains expressing HMF oxidase enzyme facilitated the bioconversion of HMF from apple pomace hydrolysate into high-value derivatives, particularly furandicarboxylic acid (FDCA), a precursor for bio-based polymers. The best-performing strain achieved 10.8 mM FDCA and 15.5 mM 5-formyl-2-furancarboxylic acid (FFCA) under optimised conditions. This integrated approach-combining catalytic HMF production via DES microwave-assisted technology with microbial biocatalysis-offers a sustainable pathway for agro-waste valorisation and the production of renewable chemicals.", "journal": "Bioresource technology", "year": "2025", "doi": "10.1016/j.biortech.2025.132570", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40421621": { "pmid": "40421621", "title": "Covalent Immobilization of Lipase on NH2-MIL-125(Ti) through Ugi Reaction for Biodiesel Production.", "abstract": "In this study, heterogeneous biocatalysts were produced by successfully synthesizing the metal-organic framework (MOF) NH2-MIL-125(Ti) as a support, followed by the chemical stabilization of the lipase enzyme using the Ugi four-component reaction (Lipase-NH2-MIL-125), resulting in a stabilization efficiency of 87%. The amine group in MOF plays one of the reactants in the Ugi reaction, and a firm covalent bond is created between the enzyme and the support, which avoids enzyme leaching and leads to a stable biocatalyst. Enzyme efficiency, reusability, pH, and temperature stability of Lipase-NH2-MIL-125 have been investigated, and their high performance has been proven for the biocatalyst. The biodiesel production process using oleic acid has been utilized to evaluate the catalytic activity of the designed biocatalyst, and different parameters have been optimized. The results confirmed the good activity of Lipase-NH2-MIL-125 in biodiesel production, and even after 6 cycles, the activity slightly decreased, which confirmed the stability of the biocatalyst during the reaction.", "journal": "ACS applied bio materials", "year": "2025", "doi": "10.1021/acsabm.5c00321", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41388303": { "pmid": "41388303", "title": "SIRT2 Inhibition promotes microglia LC3-associated phagocytosis via NRF2/CD36 after the experimental subarachnoid hemorrhage.", "abstract": "BACKGROUND: Subarachnoid hemorrhage (SAH) results in the accumulation of blood in the subarachnoid space, which can trigger inflammatory responses and oxidative damage, thereby exacerbating secondary brain injury. Microglia play a critical role in clearing hematomas and cellular debris during pathological recovery. This study aimed to investigate the regulatory role of SIRT2 in phagocytic function and its potential mechanisms. METHODS: The SAH model of endovascular perforation in Sprague-Dawley (SD) rats and in vitro model of primary microglia culture with Oxygenated hemoglobin (OxyHb) stimulation were used. Flow cytometry, Western blot, immunofluorescence, ELISA were used to detect the microglial phagocytic ability, expressions of SRIT2 and LC3-associated phagocytosis (LAP)-related proteins. Molecular docking, and immunoprecipitation were used to explore the underlying mechanism of the SIRT2/NRF2/CD36 signaling pathway. Behavioral experiments were conducted to evaluate changes in neurological function. RESULTS: Within 24 h after SAH, the expression of SIRT2 in brain and microglia was significantly increased, while the phagocytic ability and the expression of LAP-related proteins Rubicon, NOX2 were decreased. After inhibiting SIRT2, microglial LAP function was enhanced and the phagocytic index was increased. Mechanistically, SIRT2 inhibited NRF2 nuclear translocation by deacetylation, thereby downregulating CD36 expression and suppressing microglia LAP. ML385 reversed the enhancement of LAP and the improvement in neurological function induced by SIRT2 inhibition. CONCLUSION: SIRT2 suppresses microglial LAP by deacetylating NRF2, thereby impairing its nuclear translocation and reducing transcriptional expression of CD36 subsequently. Inhibiting SIRT2 may effectively enhance microglial LAP function via activating NRF2/CD36 pathway, promoting blood clearance and improving neurological prognosis after SAH.", "journal": "Journal of neuroinflammation", "year": "2025", "doi": "10.1186/s12974-025-03623-z", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41210715": { "pmid": "41210715", "title": "Isolation and characterization of Stenotrophomonas rhizophila T3E: a multifunctional rhizobacterium enhancing tomato growth and soil health.", "abstract": "Plant growth-promoting rhizobacteria (PGPR) serve as sustainable alternatives to chemical fertilizers and pesticides for improving crop productivity. Among them, Stenotrophomonas rhizophila has demonstrated considerable potential to enhance plant growth, yet the mechanisms underlying its effects on plant development and soil health remain insufficiently elucidated. In this study, S. rhizophila strain T3E was isolated and identified from the tomato (Solanum lycopersicum) rhizosphere based on morphological and phylogenetic analyses. The growth-promoting traits of T3E were comprehensively characterized through germination and pot experiments, integrated with phytohormone profiling, gene expression and biochemical analyses, soil assessment, and whole-genome sequencing. Inoculation with T3E significantly enhanced radicle length, primary root length, plant height, and seedling fresh weight, while quantitative assays revealed increased levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in the roots. qRT-PCR analysis showed that T3E upregulated multiple root growth-related genes (e.g., EAT2, LAX2, GTS1, GRFs) and T3E colonization-related genes (pyrB, flmH, pilR, bopD), supporting its strong root colonization ability. Physiologically, T3E treatment enhanced SOD activity, increased glycine betaine and soluble sugar levels, and reduced MDA content, suggesting improved root health and stress resistance. Whole-genome sequencing revealed a 4.31 Mb circular chromosome with 3,744 coding sequences, diverse secondary metabolite biosynthetic gene clusters, and abundant carbohydrate-active enzyme (CAZyme) genes. Soil assays demonstrated that T3E inoculation improved key physicochemical properties (TN, TP, TK, AK) and significantly enhanced the activities of soil enzymes, such as phosphatase, CAT, urease, and sucrase. These findings identify S. rhizophila T3E as a promising multifunctional PGPR that promotes plant growth, strengthens rhizosphere colonization, and enhances soil fertility. This study provides a solid theoretical basis for developing microbial biofertilizers and advancing sustainable agricultural practices.", "journal": "Frontiers in plant science", "year": "2025", "doi": "10.3389/fpls.2025.1692957", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39966067": { "pmid": "39966067", "title": "Leveraging non-enzymatic functions of LSD1 for novel therapeutics.", "abstract": "Lysine-specific demethylase 1 (LSD1) is a key enzyme that removes the methylation marks from lysines in the histone tails of nucleosomes. Emerging evidence suggests that LSD1 exhibits both enzyme-dependent and independent functions across various diseases. However, most LSD1-targeted therapies in clinical trials focus on its classic demethylase activity. Only one allosteric inhibitor (SP-2577) and two nonproteolysis-targeting chimera (PROTAC) LSD1 degraders (BEA-17 and UM171), which target its enzyme-independent functions, have entered clinical assessment. Given the limited exploration of therapeutic strategies targeting the non-enzymatic functions of LSD1, in this opinion, we summarize current insights into its biological roles and structural characteristics. We also highlight potential therapeutic interventions targeting the non-enzymatic functions of LSD1, including allosteric inhibitors, protein-protein interaction (PPI) inhibitors, and small-molecule degraders, and discuss challenges and future directions in drug discovery targeting these functions.", "journal": "Trends in pharmacological sciences", "year": "2025", "doi": "10.1016/j.tips.2025.01.006", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41378743": { "pmid": "41378743", "title": "Simultaneous Recovery of Mannooligosaccharides and Chlorogenic Acids from Spent Coffee Grounds: Integration of Enzymatic Hydrolysis with Natural Deep Eutectic Solvent Extraction.", "abstract": "Although mannooligosaccharides (MOSs) and chlorogenic acids (CGAs) from spent coffee grounds (SCGs) possess complementary bioactivities, their simultaneous recovery is hindered by entrapment within fibrous matrices and distinct polarities. This study developed a two-step strategy combining enzymatic hydrolysis and extraction using natural deep eutectic solvents (NADESs). Optimal enzymatic conditions were 6.51 U/g of Viscozyme L and 15.84 U/g of mannanase at 45 °C for 14 h. The conductor-like screening model for real solvents (COSMO-RS) predicted that choline chloride-organic acid NADESs had the highest solubilities for five MOSs and nine CGAs among 27 candidates. Extraction with choline chloride-citric acid NADES achieved the highest MOS and CGA yields at 114.81 and 2.11 mg/g of SCG, respectively, with an antioxidant capacity of 0.14 mmol of Trolox equivalent/g of SCG. This work demonstrates that integrating biocatalysis with solvent informatics can overcome matrix and polarity limitations, offering a sustainable strategy for upcycling food waste into functional ingredients without generating harmful residues.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.5c11408", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "D" ] }, "40647828": { "pmid": "40647828", "title": "Development of a Continuous Extrusion Process for Alginate Biopolymer Films for Sustainable Applications.", "abstract": "This study presents a novel method for producing extrudable alginate-based films using continuous thermo-mechanical mixing, providing a scalable alternative to conventional solvent-casting techniques. The effects of glycerol concentration (30-50 wt%) and processing temperature (110-120 °C) on the films' thermal, mechanical, and structural properties were systematically investigated. Structural characterization was performed using 1H NMR and FT-IR, and thermal transitions were analyzed via DSC (Differential Scanning Calorimetry) and DMA (Dynamic Mechanical Analysis). The glass transition temperature (Tg) of the alginate/glycerol/water system was modeled using the Gordon-Taylor equation. Glycerol incorporation significantly reduced Tg-by up to 76 °C with 40 wt% glycerol-and enhanced ductility and toughness, reaching 3.26 MJ/m3 at the optimal level. The influence of processing temperature was found to depend on plasticizer content: at lower glycerol levels, elevated temperatures decreased Tg and elongation at break, likely due to thermal degradation. However, films with higher glycerol content retained stable mechanical and thermal behavior across both temperature profiles. This work is among the first to explore how processing temperature affects extruded, plasticized pure alginate films. The findings provide key insights into the formulation and scalable production of bio-based packaging materials, highlighting the importance of optimizing both plasticizer concentration and processing parameters.", "journal": "Polymers", "year": "2025", "doi": "10.3390/polym17131818", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41467489": { "pmid": "41467489", "title": "Protein Engineering of Taq DNA Polymerase for Ultrafast PCR and Its Application in Molecular Diagnostics of Genetically Modified Crops.", "abstract": "To shorten the turnaround time of molecular diagnostics, an ultrafast polymerase chain reaction (PCR) technique has been developed in recent years, which requires thermostable DNA polymerases with high kinetic properties. In the present study, Thermus aquaticus (Taq) DNA polymerase was engineered through two strategies: fusion with CL7, a thermostable nonspecific DNA-binding tag, and rational design in its N-terminal exonuclease domain. The results indicated that the CL7 tag promoted the thermostability of this polymerase dramatically, while triple substitutions, L208I/E209 K/E210R, increased its polymerase activity by approximately 5-fold. Moreover, this engineered enzyme demonstrated a strong resistance to crude plant tissue lysate up to 30% (v/v) in the PCR system. Therefore, an ultrafast qPCR system was established to detect exogenous genes in genetically modified (GM) crops with this variant. The whole detection procedure was shortened to <30 min. Moreover, the limit of detection reached approximately 0.1 copies/μL. This study established a highly simple and accurate system suitable for rapid molecular diagnosis. More importantly, this is the first report about improving the kinetic properties of Taq polymerase through rational design of its exonuclease domain.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.5c12875", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "D" ] }, "42114182": { "pmid": "42114182", "title": "Deep eutectic solvent tailoring the electrode-enzyme interface for phenylalanine dehydrogenase immobilization and efficient bioelectrosynthesis.", "abstract": "Deep eutectic solvents (DESs) are a group of emerging solvents for biocatalysis with tailored performance. Herein, we report the usage of hierarchical zeolitic imidazole framework-8 (HZIF-8) and halogenated DES for the immobilization of phenylalanine dehydrogenase. Effects of three different halogenated DESs (I, Cl and Br) on the activity of immobilized enzymes for production of homophenylalanine were studied. The optimal activity towards with the model reaction of catalyzing ethyl 2-oxo-4-phenylbutyrate (EOPB) to L-homophenylalanine was 6.11 U/mg at 70 °C, 5.4-fold of that of the free enzyme, with 78.4% of its original activity retained after six recycles. The immobilized enzymes were further loaded on a poly(methylene blue) electrode with electrochemical NAD+ regeneration, leading to bioelectrosynthesis of phenylpyruvate. Using phenylalanine-ethanolamine (Phe-MEA) as the substrate, 13.85 mM phenylpyruvate was obtained after 8 h, with a total turnover number (TTN) value of 138.5 for NADH regeneration, while a TTN of 102.2 using L-Phe as the substrate.", "journal": "Bioelectrochemistry (Amsterdam, Netherlands)", "year": "2026", "doi": "10.1016/j.bioelechem.2026.109315", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41601090": { "pmid": "41601090", "title": "Metal-organic framework-hybridized immobilized laccase for the efficient degradation of industrial wastewater pollutants.", "abstract": "Natural laccases from Lentinula edodes exhibit remarkable catalytic versatility in degrading various organic pollutants in industrial wastewater. However, their practical applications are limited by their inherent structural instability and poor reusability. To address these limitations, we designed a robust hybrid biocatalyst, Lac@UiO-66-NH₂, through optimized immobilization strategies. Optimal conditions for achieving an immobilization efficiency of 90.16 % were as follows: enzyme loading of 7 mg/mL, incubation temperature of 25 °C, and incubation time of 1 h. The immobilized laccase exhibited significantly enhanced stability and reusability, along with remarkable biocatalytic activity, achieving degradation efficiencies of 84.87 % for malachite green and 93.54 % for methylene blue in simulated wastewater. Comprehensive characterization techniques-including scanning electron microscopy, X-ray diffraction, and Brunauer-Emmett-Teller analysis-validated successful laccase immobilization and elucidated the microstructure-performance relationship, highlighting the structural advantages of the composite. This study demonstrates the potential of Lac@UiO-66-NH₂ as a novel, highly stable, and reusable hybrid biocatalyst for efficient removal of phenolic contaminants, offering a sustainable solution for wastewater treatment. The findings underscore its practical applicability in environmental remediation and its alignment with green chemistry principles.", "journal": "Ecotoxicology and environmental safety", "year": "2026", "doi": "10.1016/j.ecoenv.2025.119668", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40312414": { "pmid": "40312414", "title": "N 6-methyladenosine modifications stabilize phosphate starvation response-related mRNAs in plant adaptation to nutrient-deficient stress.", "abstract": "N6-methyladenosine (m6A), an abundant internal mRNA modification, is induced by various stress conditions and post-transcriptionally regulates gene expression. However, how m6A modifications help plants respond to nutrient-deficiency stress remains unclear. Here, we profile high-confidence m6A modifications in Arabidopsis transcriptome-wide under normal and inorganic orthophosphate (Pi)-deficient conditions (-P). High-confidence m6A modifications are identified using synthetic modification-free RNA libraries for systematic calibration. Pi starvation induces widespread m6A modifications, mediated by the Pi starvation response (PSR) master regulator PHOSPHATE STARVATION RESPONSE1 (PHR1) and its family members. Many Pi starvation-induced (PSI) m6A modifications occur on PSR-related mRNAs, including PHR1. In addition, PHR1 proteins interact with the m6A writers MRNA ADENOSINE METHYLASE (MTA) and METHYLTRANSFERASE B (MTB) in nuclei under -P conditions. m6A modifications facilitate systemic PSR signaling, as reflected by the reduced Pi content and PSR signaling in a knockdown artificial miRNA line targeting MTA, which shows a global decrease in m6A. Transcriptome-wide mRNA decay analysis reveals that PSI-m6A increases the stability of PSR-related mRNAs, but not through alternative polyadenylation site shifts. Analysis of transgenic plants with mutations in m6A loci demonstrates that m6A stabilizes PHR1 transcripts via a positive feedback loop. Our findings indicate that PSI-m6A modifications facilitate PSR signaling by enhancing the stability of certain mRNAs, shedding light on the role of m6A modifications in nutrient stress responses in plants.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-59331-y", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42187059": { "pmid": "42187059", "title": "CDs-PEI/siIhh Delivery System for the Treatment of Osteoarthritis.", "abstract": "Osteoarthritis (OA) is a prevalent degenerative cartilage disorder with few effective therapeutic options. Overexpression of Indian hedgehog protein (Ihh) is linked to OA progression, and inhibiting Ihh expression has been shown to alleviate OA symptoms. However, the dense cartilage structure made it difficult to efficiently deliver nucleic acids to deeper cartilage regions. In this study, we developed a nucleic acid delivery system, CDs-PEI/siIhh. The regulatory effect of the CDs-PEI/siIhh on gene expression was assessed using PCR and western blotting. The results demonstrated that CDs-PEI/siIhh effectively modulated both genes and protein expression in chondrocytes. Cartilage explant penetration experiments confirmed that CDs-PEI/siIhh successfully delivered siIhh to chondrocytes in the deeper regions of cartilage. In vivo experiments indicated that CDs-PEI/siIhh inhibited cartilage degeneration and matrix degradation. In conclusion, CDs-PEI/siIhh represents a promising strategy for the alleviation of OA.", "journal": "ACS applied bio materials", "year": "2026", "doi": "10.1021/acsabm.5c02517", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42587992": { "pmid": "42587992", "title": "Effect of Galacto-oligosaccharides with Different Degrees of Polymerization on the Storage Stability of Freeze-Dried Lecithin Liposomes.", "abstract": "Galacto-oligosaccharides (GOS) are widely recognized as prebiotic ingredients, whereas their technological role in stabilizing lipid-based systems remains poorly understood. In this study, GOS were synthesized by enzymatic transgalactosylation of lactose and enriched by ethanol precipitation. Carbohydrate composition, glass transition temperature (Tg), and their influence on the formation of primary lipid oxidation were evaluated using freeze-dried lecithin liposomes as a model membrane system. Ethanol precipitation increased the GOS content from 22.8% to 50.7% (w/w) while reducing the proportion of monosaccharides from 40.0% to 15.5% (w/w). Tg was strongly influenced by carbohydrate composition and relative humidity (RH), with GOS-enriched preparations exhibiting higher Tg values than the crude reaction mixture. The formation of primary lipid oxidation products, assessed by UV spectroscopy through conjugated diene formation at 234 nm, was significantly lower in liposomes freeze-dried with GOS-rich preparations or sucrose than in phosphate-buffered saline (PBS) controls, particularly under storage conditions in which the carbohydrate matrices remained in the glassy state. At higher RH, transition to the rubbery state was associated with increased conjugated diene formation. These findings suggest that the protective effect of GOS-rich formulations is associated with the physicochemical properties of the carbohydrate matrix and is consistent with the proposed role of the glassy state in limiting molecular mobility, rather than direct antioxidant activity. Beyond their established prebiotic properties, GOS-rich preparations may also contribute to the stability of dehydrated lipid-containing systems, supporting their use as multifunctional food ingredients.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15152734", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40964000": { "pmid": "40964000", "title": "Ti3C2Tx MXene Potentiates PSAT1-Mediated Osteogenesis Through miR-665/GSK-3β/β-Catenin Axis to Counteract Inflammation-Induced Bone Loss.", "abstract": "Inflammatory signaling-induced stem cell dysfunction severely impairs bone regeneration. This study aimed to develop a combinatorial strategy using Ti3C2Tx MXene scaffolds and PSAT1-engineered dental pulp stem cells (oe-PSAT1 DPSCs) to counteract inflammation-mediated osteogenic suppression. Dental pulp stem cells (DPSCs) were treated with TNF-α to simulate an inflammatory microenvironment. miR-665 expression and its targeting relationship with PSAT1 were analyzed via qRT-PCR, dual-luciferase reporter assay, and Western blot. The role of the miR-665/PSAT1/GSK-3β/β-catenin axis in osteogenic differentiation was evaluated using ALP activity, alizarin red staining, and immunofluorescence. Ti3C2Tx MXene was synthesized and characterized, and its effects on ROS scavenging and osteogenesis were assessed in vitro. In vivo efficacy was validated using a rat calvarial defect model with micro-CT, histological staining, and immunohistochemistry. TNF-α stimulation upregulated miR-665, which directly targeted PSAT1 and inhibited the GSK-3β/β-catenin pathway, suppressing DPSCs osteogenic differentiation. PSAT1 overexpression rescued this suppression. Ti3C2Tx MXene scavenged ROS, enhanced calcium-dependent mineralization, and synergized with oe-PSAT1 DPSCs to amplify β-catenin activation. In rat models, the Ti3C2Tx MXene /oe-PSAT1 DPSCs combination achieved superior bone defect closure (higher BV/TV, Tb. Th, and mature collagen deposition) compared to Ti3C2Tx MXene alone. This study identifies the miR-665/PSAT1/GSK-3β/β-catenin axis as a key regulator of inflammatory osteogenesis. The Ti3C2Tx MXene/oe-PSAT1 DPSCs strategy concurrently neutralizes oxidative stress and activates osteogenic signaling, providing a translatable platform for inflammatory bone regeneration.", "journal": "International journal of nanomedicine", "year": "2025", "doi": "10.2147/IJN.S521957", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41802812": { "pmid": "41802812", "title": "Targeting endosomal trafficking-mediated antigen escape to resensitize myeloma to CAR-T therapy.", "abstract": "Antigen escape is one of the leading causes of relapse following chimeric antigen receptor (CAR)-T therapy, particularly in multiple myeloma. A critical gap persists in understanding the tumor-intrinsic pathways that trigger antigen loss, insight essential for devising strategies to resensitize tumors to immune attack. We identify a previously uncharacterized post-translational mechanism centered on the metabolic enzyme ribonucleotide reductase subunit M2 (RRM2), termed trafficking-mediated antigen escape, to enhance cellular therapy efficacy. We combined single-cell RNA sequencing analysis with multiplex immunofluorescence to identify a clinically relevant RRM2+ myeloma subpopulation exhibiting low MICA/B abundance. Functional validation included induced pluripotent stem cell-derived myeloma organoids monitored by real-time imaging and disseminated xenograft models to assess the effect of subtoxic osalmid treatment on NKG2D CAR-T cell activity. Co-immunoprecipitation, guanosine 5'-triphosphate pulldown, and confocal microscopy were used to investigate the underlying trafficking mechanism. Single-cell analysis uncovered a clinically prevalent RRM2+ myeloma subpopulation with profoundly reduced MICA/B surface abundance, which established tumor-intrinsic heterogeneity as one of fundamental causes of NKG2D CAR-T resistance. We further demonstrated RRM2's non-canonical role as a trafficking regulator that actively shuttles MICA/B toward lysosomal degradation via RAB7A activation while simultaneously blocking RAB11-mediated recycling. Therapeutic intervention using subtoxic osalmid, a clinically approved drug and previously characterized as an RRM2 inhibitor, successfully reversed this trafficking defect, restored MICA/B membrane presentation and synergized with NKG2D CAR-T cells to enhance their expansion, polyfunctional cytokine secretion, and stem-like properties. This combination strategy achieved durable tumor remission in vivo by sustaining T-cell fitness while reducing exhaustion, offering an immediately actionable solution to clinical antigen escape. Our study establishes RRM2-driven trafficking as a novel and targetable mechanism of antigen escape in CAR-T therapy. By repurposing osalmid to restore MICA/B surface presentation, we provide a clinically translatable strategy that specifically potentiates NKG2D CAR-T cell efficacy in multiple myeloma and could potentially enhance the efficacy of CAR-T across diverse antigens. This work highlights the therapeutic potential of modulating intracellular trafficking to overcome resistance in cellular immunotherapy.", "journal": "Journal for immunotherapy of cancer", "year": "2026", "doi": "10.1136/jitc-2025-014040", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41037727": { "pmid": "41037727", "title": "[Evaluation of the accelerated stability of an allergen extract in combination with an inactivated bacterial suspension using the Advanced Kinetic Model (AKM)].", "abstract": "The coadministration of allergen extracts with bacterial preparations has gained clinical relevance in subcutaneous immunotherapy for respiratory allergic diseases. However, there are no systematic studies evaluating the physicochemical stability or preservation of immunological activity of these mixtures. This study aimed to characterize the stability of a combined formulation using the Advanced Kinetic Model (AKM), a mathematical approach to shelf-life prediction based on nonlinear kinetics. A lyophilized mite extract (D. pteronyssinus/D. farinae) and an inactivated bacterial suspension (IPI-Asac) were used. Incubations were performed at 4, 15, 30, 37, and 45°C for up to 90 days, with chromatographic (SEC-HPLC), electrophoretic (SDS-densitometric PAGE), and functional (ELISA-IgE) analyses. The samples were studied separately and in a 4:1 ratio (extract:bacteria). The AKM model was applied to the IgE-binding loss data to extrapolate long-term stability. No significant differences were observed between the extract alone and the mixture in terms of aggregation, protein degradation, or loss of IgE-binding capacity. At 15°C, >90% activity was retained for up to 90 days. Conclusion: The AKM model predicted a retention of 75% functional activity at 4°C for up to 1.5 years. The bacterial suspension did not alter the degradation kinetics or biophysical profiles. The data obtained suggest that the inclusion of an inactivated bacterial suspension does not compromise the conformational or functional stability of the allergens. The maintenance of IgE-specific activity under simulated storage conditions supports the technical feasibility of a coformulation. The application of the AKM model provided robust predictions of biological longevity without requiring prolonged stability studies under real-world conditions. The blending of allergenic extracts with inactivated bacterial suspensions preserves their immunological and biophysical properties under accelerated thermal conditions. These findings support the possibility of formulating combination products without negatively impacting immunotherapeutic efficacy, justifying additional clinical studies and multi-batch validation.", "journal": "Revista alergia Mexico (Tecamachalco, Puebla, Mexico : 1993)", "year": "2025", "doi": "10.29262/ram.v72i3.1543", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41418753": { "pmid": "41418753", "title": "Stress to exhaustion: Proteotoxicity in T cells.", "abstract": "In a recent Nature paper, Yi et al.1 uncover that a noncanonical proteotoxic stress response (PSR) in exhausted T cells (Tex), termed \"Tex-PSR,\" drives T cell exhaustion. This response is characterized by sustained global protein synthesis, accumulation of protein aggregate, and selective upregulation of chaperone proteins.", "journal": "Molecular cell", "year": "2025", "doi": "10.1016/j.molcel.2025.11.016", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42269198": { "pmid": "42269198", "title": "Topology-guided hard example mining for cell detection.", "abstract": "Automatic cell detection is a key task in digital pathology, where manual counting remains impractical due to its time-consuming nature and susceptibility to variability and error. Current deep learning approaches still have difficulty achieving accurate detection, particularly in images with crowded cell distributions. In such settings, capturing the global organization of cells within tissue becomes critical; however, the topological structure underlying cell arrangements is often ignored by existing models. To address these limitations, we propose topology-guided hard example mining (TG-HEM), a novel training strategy that incorporates topological constraints into the training of cell detection networks through loss reweighting. In contrast to pixel-centric HEM techniques, TG-HEM identifies challenging regions by quantifying topological discrepancies between ground truth and predicted cell distributions using persistent homology, rather than relying solely on local pixel-wise errors. By assigning higher importance to regions with larger topological inconsistencies and further emphasizing hard-to-learn pixels within these regions, the proposed approach guides backpropagation toward regions that reflect structural differences in cell distributions. This formulation enables HEM at both the region and pixel levels, allowing the network to better capture higher-order organization in crowded cell distributions. We evaluate TG-HEM across multiple network architectures and on two datasets: the publicly available BRCA-M2C dataset and our in-house KUCell dataset, which we release as part of this work. The experimental results show that the proposed TG-HEM approach consistently improves both cell counting and localization accuracy compared to existing HEM strategies. These improvements are achieved without introducing additional model complexity or inference-time overhead, and with only negligible impact on training time. The KUCell dataset is available at https://mysite.ku.edu.tr/cgunduz/downloads/KUCell, and the codes are available at https://github.com/caki35/TGHEM.", "journal": "Medical image analysis", "year": "2026", "doi": "10.1016/j.media.2026.104155", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39670656": { "pmid": "39670656", "title": "Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics.", "abstract": "Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.", "journal": "ACS synthetic biology", "year": "2025", "doi": "10.1021/acssynbio.4c00469", "ptypes": [ "Journal Article", "Research Support, U.S. Gov't, Non-P.H.S.", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "40617264": { "pmid": "40617264", "title": "Monoacyl phospholipids to replace polysorbates as interfacial stabilizers in parenteral monoclonal antibody formulations.", "abstract": "Protein-based drugs are prone to both physical and chemical instability in aqueous solutions. Surfactants, such as polysorbates (PS), are commonly employed to mitigate interfacial stress, thus preventing protein aggregation and particle formation. However, polysorbates can undergo enzymatic hydrolysis by residual host cell proteins and oxidation during long-term storage in parenteral formulations. This can lead to the generation of free fatty acid particles, inadequate protein stabilization, and protein oxidation. In this study, we investigated several monoacyl phospholipids (MAPLs) with varying fatty acid chains as potential alternative surfactants for monoclonal antibody (mAb) formulations and compared their efficacy to the industry standard, polysorbate 80. The hemolytic activity of MAPLs was tested using erythrocytes in 95 % plasma. All MAPLs prevented mAb particle formation during shaking and freezing-thawing at surfactant concentrations several orders of magnitude below the threshold for hemolysis, suggesting that the risk of erythrocyte damage from MAPLs is non-critical. Stabilization of mAbs occurred around the critical micelle concentration, which were comparable to that of PS80, but MAPLs achieved lower interfacial tension values. MAPLs were found to be more resistant to enzymatic hydrolysis by porcine liver esterase and forced oxidation than PS80. After long-term liquid storage, lyso-myristoyl-phosphatidylcholine (LPC 14:0) at low concentrations provided superior mAb stabilization to PS80, which exhibited substantial chemical degradation. At higher concentrations, both PS80 and LPC 14:0 showed a decrease in surfactant concentration. Lyophilization enhanced mAb stabilization relative to liquid formulations, with MAPLs performing as well as PS80 at high concentrations and outperforming PS80 at low concentrations. MAPLs also better preserve the siliconization in pre-filled syringe (PFS) barrels compared to PS80. In short, MAPLs demonstrate mAb stabilization and chemical stability comparable to, and in some cases superior to, PS80, making them a promising alternative as interfacial stabilizers in parenteral protein formulations and warranting further exploration.", "journal": "European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences", "year": "2025", "doi": "10.1016/j.ejps.2025.107191", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40598551": { "pmid": "40598551", "title": "A Self-Generated Electricity-Driven Sclera reinforcement bionic piezoelectric patch for Management of High Myopia.", "abstract": "High myopia (HM) is a progressive ocular condition characterized by excessive axial elongation and severe refractive errors, often leading to sight-threatening complications. The underlying pathological driver of HM is the weakening of scleral biomechanics, making the sclera a key therapeutic target. While posterior scleral reinforcement (PSR) has been established as an effective intervention to strengthen the sclera, currently available PSR materials often fail to fully meet clinical demands. Inspired by the electric eel, which generates surface electrolytes to facilitate electric discharge and influence interactions with its surroundings, we developed a biomimetic piezoelectric patch (BPP@PVDF) for HM treatment. This patch integrates a bovine pericardium (BPP) scaffold with a piezoelectric polyvinylidene fluoride (PVDF) film, endowing the BPP with electrical properties and improved cell adhesion. Through electrical activation, the BPP enhances scleral mechanical strength and promotes collagen synthesis, effectively mitigating axial elongation in myopia. Both in vitro and in vivo experiments demonstrate that our precisely designed patch provided a stable and effective solution for reducing progressive axial elongation in HM. By leveraging nanotechnology, electrical stimulation, and scleral reinforcement surgery, this study offers a groundbreaking approach with significant implications for both scientific research and clinical practice. Our strategy paves the way for enhanced surgical outcomes in HM treatment, offering a promising avenue for future therapeutic advancements.", "journal": "Journal of nanobiotechnology", "year": "2025", "doi": "10.1186/s12951-025-03493-w", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41882404": { "pmid": "41882404", "title": "In vivo generation of anti-BCMA CAR-T cells in relapsed or refractory multiple myeloma: a phase 1 study.", "abstract": "In vivo chimeric antigen receptor (CAR)-T cell generation can bypass ex vivo manufacturing and lymphodepletion, potentially simplifying and accelerating access to cellular therapy; preliminary clinical experience supports feasibility and suggests preliminary efficacy. This phase 1, single-arm, open-label trial evaluated the safety and tolerability of ESO-T01, a nanobody-directed, immune-shielded lentiviral vector encoding a humanized anti-B cell maturation antigen (BCMA) CAR, in adults with relapsed or refractory multiple myeloma. ESO-T01 was administered as a single intravenous infusion of 0.2 × 109 transduction units without leukapheresis, ex vivo manufacturing or lymphodepleting chemotherapy. Five heavily pretreated male patients (median three prior lines) were consecutively enrolled and followed for a median of 6.0 months. The trial was stopped early in 2025, and no further enrollment was performed. The primary endpoint was safety and tolerability, and secondary endpoints included efficacy, pharmacokinetics and pharmacodynamics of ESO-T01. No dose-limiting toxicities occurred. All patients developed grade 3 or higher adverse events. Cytokine release syndrome occurred in four patients (three grade 3 and one grade 2) and was managed with corticosteroids, tocilizumab, or supportive care. The most frequent toxicities were transient cytopenias and reversible hepatic enzyme elevations, and three patients experienced grade 2 infections. One patient developed grade 1 immune effector cell-associated neurotoxicity and died from extramedullary lesion-related spinal cord compression. Preliminary antimyeloma activity was observed: four of five patients achieved objective responses, including three stringent complete remissions, with minimal residual disease negativity (10-5) in all evaluable responders (4/4) by day 60. These findings provide preliminary evidence on the feasibility and safety of in vivo CAR-T generation using an immune-shielded vector. ClinicalTrials.gov registration: NCT06791681 .", "journal": "Nature medicine", "year": "2026", "doi": "10.1038/s41591-026-04244-6", "ptypes": [ "Journal Article", "Clinical Trial, Phase I" ], "first_author": "", "dirs": [ "D" ] }, "42070720": { "pmid": "42070720", "title": "Investigating adeno-associated virus stabilization at the air-water interface through competitive adsorption.", "abstract": "This study investigated the stabilization of adeno-associated viruses (AAVs) at the air-water interface (AWI), focusing on the interplay between surfactant and capsid adsorption during agitation stress. AAV-9 and its engineered variant, AAV-PHP.eB-known for enhanced central nervous system tropism-were subjected to orbital shaking to simulate interfacial stress encountered during manufacturing and transportation. The impact of surfactant type and concentration was systematically evaluated using polysorbates 20 and 80, Poloxamer 188 (P 188), and Kolliphor® HS15. Size-exclusion chromatography-multi-angle light scattering (SEC-MALS) and total holographic characterization (THC) provided insights into capsid titer recovery and subvisible particle (SVP) formation. Hydrophobic interaction chromatography (HIC) and pendant drop shape analysis revealed striking differences in surface hydrophobicity and activity between the AAV variants. Our results suggested that AAV-PHP.eB, with higher surface hydrophobicity and activity, suffered greater adsorption and SVP formation than AAV-9, requiring higher P 188 concentrations for effective stabilization. At 0.1 % (w/v) P 188, a shift in SVP populations was detected by THC and Dynamic Light Scattering with the emergence of rod-like and irregularly shaped particles, likely representing P 188 aggregates. The findings suggest that greater P 188 concentration not only offers protection by saturating the AWI and direct binding to the capsid, but may also form aggregate structures in presence of AAVs that further impede capsid adsorption and aggregation.", "journal": "Journal of pharmaceutical sciences", "year": "2026", "doi": "10.1016/j.xphs.2026.104304", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40358119": { "pmid": "40358119", "title": "M2 Macrophage-Derived Extracellular Vehicles-Loaded Hyaluronic Acid-Alginate Hydrogel for Treatment of Osteoarthritis.", "abstract": "Osteoarthritis (OA), a high-prevalence degenerative cartilage disease, urgently requires novel therapeutic strategies. M2 macrophage-derived exosomes (M2-Exo) demonstrate therapeutic potential for OA, though their regulatory mechanisms in chondrocyte-macrophage (Mφ) interactions remain to be elucidated. To investigate the regulatory effects of M2-Exo on chondrocytes and Mφ in vitro, and to evaluate the therapeutic effect of the M2-Exo-loaded hydrogel system (ALG-M2Exo) on cartilage damage in a rat OA model. In the cell experiment, M2-Exo were extracted and characterized using ultracentrifugation. Different concentrations of M2-Exo were co-cultured with inflammatory chondrocytes or M1Mφ to evaluate their direct anti-inflammatory effects and the ability to promote M1Mφ repolarization to the M2 phenotype, using methods such as EdU, TUNEL, qRT-PCR, and Western blot. Then, the repolarized RM2Mφ were co-cultured with inflammatory chondrocytes to verify their anti-inflammatory efficacy, employing similar detection methods. In the in vivo experiment, sodium iodoacetate was injected to establish a rat knee OA model, followed by interventions including ALG-M2Exo. After 4 and 8 weeks, samples were collected for gross observation and histological staining to assess cartilage damage repair. In the cell experiment, M2-Exo exhibited typical exosomal characteristics, directly promoting the proliferation of inflammatory chondrocytes, inhibiting their apoptosis, reducing the expression of TNF-α, iNOS, and MMP-13, and increasing the expression of IL-10 and COL II. RM2Mφ showed similar therapeutic effects on inflammatory chondrocytes as M2-Exo. In the in vivo experiment, the ALG-M2Exo group demonstrated superior repair effects on cartilage damage compared to other groups, with the treatment effect at 8 weeks being better than at 4 weeks. ALG-M2Exo effectively promotes the repair of cartilage damage in OA through both a direct pathway by releasing M2-Exo that act on chondrocytes and an indirect pathway that facilitates the repolarization of M1Mφ to M2Mφ.", "journal": "Orthopaedic surgery", "year": "2025", "doi": "10.1111/os.70059", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39775496": { "pmid": "39775496", "title": "Sample-to-answer microfluidic device towards the point-of-need detection of Staphylococcus aureus enterotoxin genes in ruminant milk.", "abstract": "Milk is commonly screened both for indicators of animal disease and health, but also for foodborne hazards. Included in these analyses is the detection of Staphylococcus aureus, that can produce an enterotoxin, causing staphylococcal food poisoning (SFP), which often leads to sudden onset of significant gastrointestinal symptoms in humans. Epidemiological data on SFP are limited, particularly in low- and middle-income countries. Many conventional assays for the detection of staphylococcal enterotoxins rely on the detection of the genes coding for them, either directly in food samples or after bacterial culture. Currently, many of the nucleic acid-based methods used require specific expertise and equipment, whilst bacterial culture takes 24-48 hours; both are contributory factors that limit efforts either during food safety emergencies or routine screening. Here we present the development of a \"sample-to-answer\" isothermal nucleic acid loop-mediated amplification (LAMP) assay in a microfluidic device for the detection of Staphylococcus aureus enterotoxin genes in ruminant milk. A multiplex LAMP assay targeting two of the most prevalent S. aureus enterotoxin-encoding genes (A and B) was integrated into a microfluidic device combining simple 1 : 10 dilution for sample preparation and a lateral flow assay for easy readout. We achieved a limit of detection of 104 colony forming units per ml in spiked cow and goat milk samples, an order of magnitude more sensitive than the European recommendation for the maximum allowable presence of coagulase-positive staphylococci in raw milk. The assay showed no cross-reactivity in detecting other tested non-enterotoxigenic S. aureus strains or associated foodborne pathogens. The test integrated the simplicity of use of microfluidic devices with the sensitivity, specificity and rapidity of a nucleic acid-based assay, and a simple lateral flow readout to provide an appropriate device to ensure the safety of milk for human consumption. To illustrate its potential for point-of-need practical applications, the test was performed in agricultural settings in rural Turkey in a limited feasibility exercise.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d4lc00907j", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42852765": { "pmid": "42852765", "title": "Comparative Evaluation of Surfactants and Stabilizers in Preventing Protein Aggregation in Insulin Formulations.", "abstract": "Protein aggregation is an issue that affects the stability and effectiveness of biopharmaceutical products. Proteins easily undergoes denaturation, due to mechanical agitation and prolonged storage times. Although polysorbates have been extensively used to stabilize proteins, there is still room to identify other highly effective additives. This research aimed to compare the efficacy of hydroxypropyl-β-cyclodextrin (HP-β-CD) in preventing protein aggregation in recombinant human insulin with polysorbate 20 (PS-20), polysorbate 80 (PS-80), and Poloxamer 188 (P188). Recombinant human insulin formulations with the addition of PS-20, PS-80, P188, or HP-β-CD, or the absence of any additive (control group), were prepared. The prepared samples were subjected to mechanical stress through shaking at 100 rpm for 24 hours. Thereafter, a sixmonth accelerated stability test was performed at 25±2°C/65±5% RH. Protein aggregation and particle formation were measured using size-exclusion high-performance liquid chromatography (SEHPLC) to detect HMWP, dynamic light scattering (DLS) for submicron aggregate size, and light obscuration (LO) of subvisible particles. Mechanical agitation caused protein aggregation in all formulations; however, the degree of aggregation depended on the stabilizer used. HP-β-CD showed the highest stabilization activity, with only a 1.2% increase in HMWP level, whereas the non-stabilized control increased by 12.3%. LO analysis showed that formulations containing HP-β-CD had the least subvisible particles. In addition, DLS analysis showed no changes in aggregate size. Accelerated stability study results showed even better performance of HP-β-CD, with HMWP levels ranging from 2.60% to 2.64% over six months of storage, exceeding the performance of PS-20, PS-80, and P188. The stabilization effect exerted by HP-β-CD could be assumed due to its capacity to decrease protein unfolding and prevent aggregation by the interfacial stress. Compared with the other surfactants, HP-β-CD provided better protection against mechanical and storage-related degradation of the protein while limiting particle formation. Under the conditions of agitation and storage stress, the use of HP-β-CD provides more efficient stabilization of recombinant human insulin as compared with polysorbate surfactants and Poloxamer 188.", "journal": "Current protein & peptide science", "year": "2026", "doi": "10.2174/0113892037484972260921041034", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40699866": { "pmid": "40699866", "title": "Design of New Primer Sets for the Development of a Loop-Mediated Isothermal Amplification for Rapid Detection of Neisseria meningitidis.", "abstract": "Neisseria meningitidis is a pathogenic bacterial agent that causes meningococcal meningitis in humans. Developing a rapid and low-cost N. meningitidis detection method is crucial, especially for developing countries. This study focuses on the development of an efficient loop-mediated isothermal amplification (LAMP) method for accurate N. meningitidis identification. A new LAMP primer set was designed, and a LAMP reaction was optimized. The colorimetric detection method was also applied, and the assay characteristics were evaluated using clinical samples. The results demonstrated a specific LAMP reaction for N. meningitidis detection of genotypes A, B, and C, with a limit of detection of 102 cfu/mL, 100% specificity and sensitivity, and a rapid detection time of only 40 min by colorimetric visual inspection. No cross-reactivity with reference strains of Streptococcus pneumoniae, Staphylococcus aureus, Neisseria lactamica, Mycobacterium tuberculosis, and Haemophilus influenzae type b was observed in the LAMP reaction with the new primer set. This result suggests that the LAMP reaction could be a promising tool for developing a rapid N. meningitidis detection method suitable for use in Vietnam and other developing countries.", "journal": "Current issues in molecular biology", "year": "2025", "doi": "10.3390/cimb47060467", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42551106": { "pmid": "42551106", "title": "Antiphasic Arctangent-Arccotangent (AAA) probes: A kinetically orthogonal design for single-tube, real-time, and low-crosstalk multiplex LAMP.", "abstract": "The rapid and discriminative detection of multiple pathogens is crucial for effective disease control, yet remains a significant challenge for point-of-care testing (POCT). Herein, we report a novel probe design strategy-Antiphasic Arctangent-Arccotangent (AAA) probes-for kinetically orthogonal, low-crosstalk multiplex detection in a single closed tube via loop-mediated isothermal amplification (LAMP). The AAA probe system comprises two kinetically complementary probes: the Arctangent Probe (ATP), a stem-loop probe that yields a rising fluorescence signal, and the Arccotangent Probe (ACP), a guanine-quenched linear probe that produces a falling fluorescence signal. Their inherently antiphasic kinetic trajectories effectively reduce false positives arising from nonspecific amplification. This system requires no additional enzymes or complex operations, preserving the simplicity of conventional LAMP. As a proof of concept, we demonstrated its utility by simultaneously detecting Japanese encephalitis virus (JEV) and Plasmodium vivax (P. vivax), two representative mosquito-borne pathogens, with high specificity and a sensitivity of 5 copies/μL. The assay performed robustly with clinical samples and in complex matrices (e.g., serum, tissue homogenates). Beyond the specific duplex detection, the AAA probe principle establishes a general framework for designing orthogonal probe sets, offering a promising and versatile strategy for multiplex nucleic acid detection in resource-limited settings.", "journal": "Biosensors & bioelectronics", "year": "2026", "doi": "10.1016/j.bios.2026.119092", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41697503": { "pmid": "41697503", "title": "Deep Eutectic Solvent Pretreatment to Enhance the Enzymatic Saccharification Efficiency of Cotton Fibers.", "abstract": "Cotton fibers constitute one of the most abundant cellulose-rich resources, offering potential as feedstock for bioethanol production through enzyme-catalyzed hydrolysis into fermentable sugars. However, the inherent structural limitations of fibers considerably restrict cellulase accessibility, leading to low saccharification efficiency. To address this limitation, a pretreatment strategy employing a deep eutectic solvent (DES), comprising choline chloride (ChCl) and p-toluenesulfonic acid (TsOH) at a molar ratio of 1:1 and 75% (v/v) concentration, was investigated. Suitable pretreatment conditions, 90 ℃ for 1 h with a solid-liquid ratio of 1:30, effectively enhanced the yield of enzyme-catalyzed hydrolysis by approximately 1.8 times. Additionally, the recyclability assessment indicated sustained pretreatment efficacy over at least three cycles of DES. Moreover, a notable synergistic effect was observed when cellulase and cellobiase were compounded at a volume ratio of 4:1, which further enhanced the yield by around 1.4 times. The integration of DES pretreatment with compound enzyme system resulted in a remarkable 2.4 times increase in saccharification efficiency, achieving within 2 h a hydrolysis yield equivalent to that obtained after 12 h using cellulase alone. This study provides critical insights into efficient and sustainable strategies for enzyme-catalyzed hydrolysis of cotton fibers and demonstrates the promising applicability of DES in cotton biomass valorization.", "journal": "Applied biochemistry and biotechnology", "year": "2026", "doi": "10.1007/s12010-026-05613-0", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41514392": { "pmid": "41514392", "title": "Efficacy of phospholipid-bound omega-3 versus standard omega-3 in patients with hypertriglyceridemia: a randomized clinical trial.", "abstract": "BACKGROUND: Hypertriglyceridemia is a modifiable risk factor for cardiovascular disease, and while omega-3 fatty acids (omega-3 FAs) are known to lower triglyceride (TG) concentrations, their effectiveness is influenced by formulation and bioavailability. Phospholipid-bound (PL) omega-3 FAs—such as those found in krill oil—demonstrate superior intestinal absorption and membrane integration compared with TG- and ethyl ester omega-3 FAs based forms. Preclinical studies have shown that PL omega-3 FAs exhibit anti-inflammatory, antithrombotic, and TG-lowering properties; however, clinical data remain limited. This pilot randomized clinical trial compared the efficacy of PL omega-3 FAs with standard omega-3 FAs in patients with mild to moderate hypertriglyceridemia. METHODS: We performed a randomized, controlled, double-blind, parallel-group pilot trial involving patients aged 18–65 years with fasting TG between 150 and 499 mg/dL. Patients were randomized to receive PL omega-3 FAs (eicosapentaenoic acid [EPA) + docosahexaenoic acid [DHA), 825 mg/day) or standard omega-3 FAs (EPA + DHA, 903 mg/day). Primary outcomes were changes in fasting TG and omega-3 index (O3I) after 12 weeks. Secondary outcomes included changes in lipid profile, glycemic markers, inflammatory markers, anthropometric measures, hemodynamic variables, and safety parameters. RESULTS: Of the 47 randomized participants, 44 (22 per group) completed the study and were included in the analysis. Mean TG levels decreased slightly in the PL group (-9.1 mg/dL) and increased in the standard group (+ 15.2 mg/dL), with no statistically significant difference between-groups (p = 0.416). Although the O3I increased significantly in both groups, the between-group difference was not statistically significant, despite a greater rise in the PL group (+ 1.78% vs. +1.50%, p = 0.446). At week 12, 36.4% of participants in the PL group achieved TG ≤ 150 mg/dL, compared with 13.6% in the standard group (p = 0.041). No serious adverse events were reported, and treatment adherence exceeded 95%. CONCLUSIONS: In summary, this pilot randomized trial did not demonstrate statistically significant differences between the PL and TG formulations in triglyceride or Omega-3 Index reduction. However, the numerically higher responder rate and favorable biochemical trends observed with the PL formulation warrant further investigation in larger, adequately powered studies. TRIAL REGISTRATION: NCT06749028 (retrospectively registered on December 19, 2024, after enrollment of the first participant). Available at: https://clinicaltrials.gov/study/NCT06749028 . EUROPEAN FOOD SAFETY AUTHORITY (EFSA): EFSA-2024-00030369.", "journal": "BMC complementary medicine and therapies", "year": "2026", "doi": "10.1186/s12906-026-05245-1", "ptypes": [ "Journal Article", "Randomized Controlled Trial" ], "first_author": "", "dirs": [ "A" ] }, "41931046": { "pmid": "41931046", "title": "Base Editing of HBG1 and HBG2 Promoters for Sickle Cell Disease.", "abstract": "Sickle cell disease is characterized by chronic hemolytic anemia and recurrent severe vaso-occlusive crises. Ristoglogene autogetemcel (risto-cel) includes autologous CD34+ hematopoietic stem and progenitor cells that have been base-edited to target the HBG1 and HBG2 promoters and inhibit BCL11A binding without altering BCL11A expression, yielding a switch in hemoglobin production from sickle hemoglobin (HbS) to antisickling fetal hemoglobin (HbF). In this phase 1-2 study, we enrolled patients 12 to 35 years of age with sickle cell disease who had had at least four severe vaso-occlusive crises in the 2 years before enrollment. After myeloablative conditioning with pharmacokinetically guided administration of busulfan, patients received a single infusion of risto-cel (at a dose of ≥3.0×106 viable CD34+ cells per kilogram of body weight). The primary efficacy end point was freedom from severe vaso-occlusive crises for 12 consecutive months, starting later than 60 days after the last red-cell transfusion. This interim analysis was unplanned; here, we describe safety, editing, engraftment, and hemoglobin production and the number of severe vaso-occlusive crises starting later than 60 days after the last red-cell transfusion. A total of 31 patients received risto-cel and were followed for a mean of 6.6 months (range, 0.3 to 20.4). A median of one cycle (range, one to five) was required for stem-cell collection. Neutrophil engraftment occurred at a median of 17.5 days, and platelet engraftment at a median of 19 days. One patient died from idiopathic pneumonia syndrome. All 31 patients had at least one adverse event, 27 (87%) had an adverse event of grade 3 or higher, and 12 (39%) had a serious adverse event. At 6 months, the mean fraction of on-target edited alleles in peripheral blood was 67.4%, the mean HbF as a fraction of total hemoglobin was more than 60%, and the HbS as a fraction of total hemoglobin was less than 40% (among 13 patients); these levels were maintained throughout follow-up. No investigator-reported severe vaso-occlusive crises occurred later than 60 days after the last red-cell transfusion. Treatment with risto-cel was followed by rapid engraftment and durable expression of HbF and reduction in HbS. These data support further investigation of risto-cel to treat sickle cell disease. (Funded by Beam Therapeutics; BEACON ClinicalTrials.gov number, NCT05456880.).", "journal": "The New England journal of medicine", "year": "2026", "doi": "10.1056/NEJMoa2504835", "ptypes": [ "Journal Article", "Clinical Trial, Phase I", "Clinical Trial, Phase II", "Multicenter Study" ], "first_author": "", "dirs": [ "B" ] }, "40583153": { "pmid": "40583153", "title": "Single-Droplet Dual-Target Quantification of circRNA Biomarkers for Colorectal Cancer Screening.", "abstract": "Circular RNAs (circRNAs) play a crucial role in the oncogenesis, progression, and chemoresistance of tumors. Here, two plasma circRNA biomarkers, circCK1γ3 and circWDR37 is identified and validated. Simultaneous analysis of these biomarkers has the potential to significantly improve the sensitivity of colorectal cancer (CRC) screening. Leveraging the distinct base-cutting preferences of these two Cas proteins, it is successfully expressed and purified LwaCas13a and PsmCas13b, and developed a one-pot Cas13a/13b-RPA assay for the simultaneous detection of circCK1γ3 and circWDR37. To achieve precise detection of circCK1γ3 and circWDR37, the Casµchip, which integrates droplet generation and digital quantification functionalities is designed and fabricated. The Casµchip enables the simultaneous detection of circCK1γ3 and circWDR37 within 70 min. This method exhibits excellent repeatability and high specificity, with a detection limit of 100 copies/reaction. The correlation coefficients for circWDR37 and circCK1γ3 detected by Casµchip and qPCR are 0.9113 and 0.8933, respectively. Casµchip analysis is conducted on a cohort of 60 clinical samples, and the findings indicate that this methodology holds promise for the early detection of colorectal cancer. Casµchip represents the profound integration of CRISPR diagnostics and microfluidic technology, offering a novel paradigm characterized by rapid, precise, and user-friendly features for molecular diagnosis.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2025", "doi": "10.1002/advs.202506159", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42584135": { "pmid": "42584135", "title": "Efficient Biosynthesis of Biobased Organic Amines from Lignocellulosic Biomass-Derived Aldehydes via Recombinant Escherichia coli-Mediated Bio-Amination in a Ternary Deep Eutectic Solvent System.", "abstract": "Lignocellulosic biomass, which consists primarily of cellulose, hemicellulose, and lignin, is a promising renewable feedstock for producing high-value biobased amines. In this study, an integrated chemobiocatalytic strategy was developed to synthesize furfurylamine from corncob using a newly formulated deep eutectic solvent, [Bet][FA][LA], that provides both catalytic and solubilizing functions. Under optimized conditions, corncob was efficiently converted into furfural, which was subsequently aminated by recombinant Escherichia coli BM-AlaDH coexpressing an ω-transaminase and alanine dehydrogenase. The whole-cell catalyst enabled in situ regeneration of L-alanine, thereby reducing amine-donor requirements and improving biocatalytic efficiency. Integration of [Bet][FA][LA]-mediated chemocatalysis with whole-cell biocatalysis converted corncob into furfurylamine in [Bet][FA][LA]. The biocatalytic amination system could convert biomass-derived 5-hydroxymethylfurfural (320 mM) and vanillin (20 mM) into the corresponding amines in yields of 95.4% and 91.0%, respectively. This work establishes a sustainable route for converting lignocellulose-derived aldehydes into value-added biobased amines and advances the comprehensive valorization of lignocellulosic biomass.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.6c05396", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41355325": { "pmid": "41355325", "title": "Distinct 5' UTR Requirements for Translation of the Bicistronic X/P mRNA Among Avian Orthobornaviruses.", "abstract": "Orthobornaviruses express X and the phosphoprotein (P) from a bicistronic X/P mRNA, and these proteins regulate polymerase activity. In mammalian orthobornaviruses, the 5' untranslated region (5' UTR) of the X/P mRNA controls the translational balance between X and P and thereby promotes efficient replication. Avian bornaviruses (ABVs) belong to two clades, clade-2 and -3, that differ in the structure and length of the 5' UTR of the X/P mRNA. However, the functional consequences of these differences remain unclear. Using reverse genetics, we generated chimeric viruses by reciprocally exchanging the 5' UTR of the X/P mRNA among clade 2 parrot bornavirus 5 (PaBV-5) and aquatic bird bornavirus 1 (ABBV-1) and clade 3 PaBV-4. In PaBV-5, a long 5' UTR with a stem-loop and an upstream ORF was required to maintain the X-to-P translational balance. On the other hand, replacing the 5' UTR of the X/P mRNA from PaBV-5 with that from PaBV-4 reduced X expression and markedly impaired viral growth. However, PaBV-4 tolerated the 5' UTR of the X/P mRNA from PaBV-5 without detectable effects on translation or replication, which suggests that translation of PaBV-4 X/P mRNA does not depend on the origin of the 5' UTR. Furthermore, ABBV-1 replicated efficiently with the 5' UTR of the X/P mRNA from PaBV-5 but was strongly attenuated with that from PaBV-4. Taken together, these results demonstrate a clade-dependent requirement for the 5' UTR for translation of the X/P mRNA and provide novel insights into the evolution of translational control in orthobornaviruses.", "journal": "Microbiology and immunology", "year": "2026", "doi": "10.1111/1348-0421.70031", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41182678": { "pmid": "41182678", "title": "Multi-Strain Probiotics Alleviate Alcoholic Liver Disease Via Microbiota-Metabolism Axis: A Randomized Controlled Study.", "abstract": "Alcoholic liver disease (ALD) remains a major global health burden, with over 283 million individuals affected by alcohol use disorder (AUD). Early-stage ALD is characterized by gut microbiota dysbiosis, intestinal barrier dysfunction, and endotoxemia. Traditional therapies focusing solely hepatoprotection or lipid reduction often show limited efficacy due to their inability to restore the gut-liver axis balance. This study aimed to evaluate the adjuvant efficacy of a multi-strain probiotic formulation (including Weizmannia coagulans BC99, Lacticaseibacillus rhamnosus LRa05, Bifidobacterium animalis subsp. lactis BLa80, and Weizmannia coagulans BC179) combined with polyene phosphatidylcholine (Essentale®) in improving liver function and metabolic profiles in ALD patients.A randomized, single-blind, placebo-controlled clinical trial was conducted in 42 ALD patients. Participants received either Essentale® plus probiotics or Essentale® plus placebo for 30 days. Liver function tests, serum lipids, fecal microbiota (16 S rRNA sequencing), and fecal metabolites (GC-MS) were assessed at baseline, day 15, and day 30.Compared to placebo, the probiotic group showed significant reductions in ALT, AST, GGT, and TG, along with increased HDL-C levels. Probiotics promoted the enrichment of Bifidobacterium, Faecalibacterium, and Akkermansia, and improved microbial diversity. Metabolomic profiling revealed upregulation of anti-inflammatory and antioxidant metabolites (e.g., EGCG, S-methylglutathione) and downregulation of pro-inflammatory lipotoxic intermediates. Spearman analysis confirmed correlations between key bacterial genera and liver/metabolic biomarkers.Multi-strain probiotics effectively modulate the gut-liver axis by reshaping gut microbiota and metabolic networks, thereby enhancing the therapeutic efficacy of conventional hepatoprotective drugs in ALD. These findings support their clinical potential as a safe and complementary strategy for managing ALD.", "journal": "Probiotics and antimicrobial proteins", "year": "2026", "doi": "10.1007/s12602-025-10818-6", "ptypes": [ "Journal Article", "Randomized Controlled Trial" ], "first_author": "", "dirs": [ "A" ] }, "40480571": { "pmid": "40480571", "title": "Rapid field visual detection of avian metapneumovirus by integrating MIRA pre-amplification with CRISPR-Cas13a to enhance sensitivity and specificity: Innovative technologies well-suited for real-time large-scale epidemiological surveillance.", "abstract": "Avian metapnemovirus (aMPV) is associated with swollen head syndrome in poultry and causes significant economic losses due to respiratory disease and reduced egg production, hence the urgent need for rapid and reliable detection methods. To address this challenge, we developed two novel CRISPR-Cas13a combined multiplex isothermal recombinase amplification (MIRA) assays: a fluorescence-based method (Fluorescent-MIRA-Cas13a) and a lateral flow strip-based platform (LF-MIRA-Cas13a), representing the first application of CRISPR-Cas13a for aMPV detection. Through systematic screening of 21 primer pairs and 4 crRNAs, optimal combinations were identified, achieving excellent specificity with no cross-reactivity against 12 common poultry pathogens. The assays demonstrated high sensitivities of 2 copies/reaction (Fluorescent-MIRA-Cas13a) and 5 copies/reaction (LF-MIRA-Cas13a), outperforming qPCR (20 copies/reaction), while maintaining reproducibility across inter- and intra-assay tests. Clinical evaluation using 200 suspected samples revealed 21 % (Fluorescent-MIRA-Cas13a) and 17.5 % (LF-MIRA-Cas13a) positivity rates, aligning closely with qPCR (19 %) and showing strong diagnostic concordance (κ > 0.93). Notably, both methods enabled detection within 1 h, without requiring specialized plug-in instruments (only a handheld UV emitter or lateral flow strips), significantly reducing operational complexity compared to conventional techniques. These field-deployable assays provide a cost-effective solution for rapid on-site diagnosis and large-scale epidemiological surveillance, particularly in resource-limited settings.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.144966", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41121340": { "pmid": "41121340", "title": "Exosomes derived from Schwann cells (SCDE) promote tendon repair through the PTEN/PI3K/Akt signaling pathway.", "abstract": "Tendon injury is one of the most frequently encountered soft tissue injuries with slow repair and poor functional recovery. Over the past few years, there's been a buzz about how exosomes might boost tissue repair and regeneration. This research dive delves into the question of whether Schwann cell-derived exosomes (SCDEs) can jumpstart tendon healing by tweaking the PTEN/PI3K/Akt signaling chain.been concentrated on the effects of exosomes in promoting tissue regeneration and repair. In the present study, we would like to explore whether exosomes derived from Schwann cells (SCDE) could promote tendon repair via modulation of PTEN/PI3K/Akt signaling cascade. This study integrates experiments performed both in vitro and in vivo. Tendon cells were categorized into the NC and SCDE groups, with a scratch assay employed.vitro and in vivo conditions tendo cells were divided into the NC group and SCDE group, and scratch assay, Transwell migration assay, flow cytometry for cell cycle detection, and Western blot and qRT-PCR analyses were performed to measure SCX, DCN, COL1A1, p-AKT, PTEN, and other markers.In the in vivo experiments, a rat model of 1/3 patellar tendon defect was established, and hydrogel or hydrogel + SCDE was injected. Tissues were gathered at 2, 4, and 8 weeks following the surgical procedure, and HE, Masson, and Sirius Red stains were applied to gauge the healing process. Important protein levels were assessed at the 2 week mark after the operation.Masson, and Sirius Red staining were performed to evaluate tissue repair. Key protein expression was measured at 2 weeks post-surgery.", "journal": "Journal of orthopaedic surgery and research", "year": "2025", "doi": "10.1186/s13018-025-06352-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41034580": { "pmid": "41034580", "title": "Proteotoxic stress response drives T cell exhaustion and immune evasion.", "abstract": "Chronic infections and cancer cause T cell dysfunction known as exhaustion. This cell state is caused by persistent antigen exposure, suboptimal co-stimulation and a plethora of hostile factors that dampen protective immunity and limit the efficacy of immunotherapies1-4. The mechanisms that underlie T cell exhaustion remain poorly understood. Here we analyse the proteome of CD8+ exhausted T (Tex) cells across multiple states of exhaustion in the context of both chronic viral infections and cancer. We show that there is a non-stochastic pathway-specific discordance between mRNA and protein dynamics between T effector (Teff) and Tex cells. We identify a distinct proteotoxic stress response (PSR) in Tex cells, which we term Tex-PSR. Contrary to canonical stress responses that induce a reduction in protein synthesis5,6, Tex-PSR involves an increase in global translation activity and an upregulation of specialized chaperone proteins. Tex-PSR is further characterized by the accumulation of protein aggregates and stress granules and an increase in autophagy-dominant protein catabolism. We establish that disruption of proteostasis alone can convert Teff cells to Tex cells, and we link Tex-PSR mechanistically to persistent AKT signalling. Finally, disruption of Tex-PSR-associated chaperones in CD8+ T cells improves cancer immunotherapy in preclinical models. Moreover, a high Tex-PSR in T cells from patients with cancer confers poor responses to clinical immunotherapy. Collectively, our findings indicate that Tex-PSR is a hallmark and a mechanistic driver of T cell exhaustion, which raises the possibility of targeting proteostasis pathways as an approach for cancer immunotherapy.", "journal": "Nature", "year": "2025", "doi": "10.1038/s41586-025-09539-1", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "C" ] }, "41609094": { "pmid": "41609094", "title": "Neuroprotective Effects of the SIRT3/AMPK Axis in Experimental Glaucoma.", "abstract": "Glaucoma is a major cause of irreversible blindness, characterized by the progressive degeneration of retinal ganglion cells (RGCs), with oxidative stress and apoptosis playing central roles in its pathogenesis. Sirtuin 3 (SIRT3) has demonstrated antioxidant and anti-apoptotic effects in various neurodegenerative diseases; however, its precise role in glaucoma remains unclear. This study aimed to elucidate the neuroprotective function and mechanistic basis of the SIRT3/AMP-activated protein kinase (AMPK) axis in glaucoma. A rat model of chronic ocular hypertension (COH) was generated using cross-linked hydrogel injection, while an N-methyl-D-aspartate (NMDA)-induced RGC injury model was developed in vitro. SIRT3 overexpression was achieved using adeno-associated virus (AAV) transfection, either alone or combined with the AMPK inhibitor Compound C. Functional and molecular analyses were performed, including intraocular pressure (IOP) measurement, hematoxylin-eosin (H&E) staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunofluorescence, Cell Counting Kit-8 (CCK-8) cell viability assay, flow cytometry, Quantitative real-time PCR (qRT-PCR), and western blotting. In the COH model, both SIRT3 expression and the p-AMPK/AMPK ratio were significantly reduced at weeks 2, 4, and 6 (p < 0.05). Overexpression of SIRT3 lowered IOP, preserved retinal thickness, and decreased the number of TUNEL-positive cells (p < 0.001), while Compound C partially reversed these effects (p < 0.05). In addition, SIRT3 overexpression markedly educed reactive oxygen species (ROS) accumulation (p < 0.001) and restored the p-AMPK/AMPK ratio (p < 0.001), both of which were partially inhibited by Compound C. In NMDA-induced RGCs, SIRT3 overexpression significantly increased SIRT3 mRNA levels (p < 0.01), enhanced cell viability (p < 0.001), and suppressed apoptosis (p < 0.001), with these effects attenuated by Compound C (p < 0.01). The reduction of ROS and activation of AMPK by SIRT3 in this model were also partly reversed by AMPK inhibition (p < 0.01). This study provides the first comprehensive in vivo and in vitro evidence in glaucoma models that SIRT3 confers neuroprotection in experimental glaucoma, primarily through activation of the AMPK signaling pathway. These findings identify the SIRT3/AMPK axis as a novel mechanistic target and suggest a promising therapeutic strategy for IOP-independent neuroprotection in glaucoma.", "journal": "Frontiers in bioscience (Landmark edition)", "year": "2026", "doi": "10.31083/FBL46525", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41581989": { "pmid": "41581989", "title": "Cell free systems for biodesign.", "abstract": "Cell-free systems (CFS) have emerged as transformative tools in synthetic biology, enabling the execution of complex biological reactions such as transcription and translation outside the confines of living cells. By eliminating the cellular membrane, CFS allows unprecedented control over biochemical conditions, facilitating rapid prototyping (up to 10x faster than traditional in vivo systems), streamlined design-build-test cycles, and the direct production of proteins, including those that are toxic or difficult to express in vivo. Rooted in pivotal discoveries from the 1960s, CFS technologies have evolved to include refined systems like the PURE system, freeze-dried diagnostics, and programmable biosynthesis platforms, integrating seamlessly with automation, artificial intelligence, and microfluidics. Modern CFS platforms support a broad range of applications, from on-demand vaccine and therapeutic production to environmental monitoring, protein engineering, and sustainable biomanufacturing. Their modular nature makes them ideal for developing genetic circuits, metabolic pathways, and biosensors, while also accelerating high-throughput screening and educational access through platforms like BioBits. Despite challenges such as reagent costs, batch variability, and scalability, recent advances in energy regeneration, lyophilization, and predictive modelling are progressively addressing these hurdles. Ultimately, CFS is not just a powerful research tool; it represents a paradigm shift toward decentralized, programmable biotechnology. From field-deployable diagnostics to space-based biomolecule synthesis, cell-free systems are paving the way for a more responsive, accessible, and innovative future in biological engineering.", "journal": "Progress in molecular biology and translational science", "year": "2026", "doi": "10.1016/bs.pmbts.2025.08.010", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40488088": { "pmid": "40488088", "title": "Development and Characterization of Zein/Eudragit Composite Nanoparticles for Insulin Intranasal Delivery.", "abstract": "Diabetes mellitus (DM) is a chronic metabolic disorder characterized by elevated blood glucose levels, primarily due to impaired insulin secretion or action. The standard treatment for Type 1 Diabetes Mellitus still involves daily parenteral insulin administration, which presents several challenges including patient discomfort, reduced adherence, and the potential for peripheral hyperinsulinemia. Intranasal administration has emerged as a promising alternative due to the nasal cavity's high vascularization, ease of access, and significant absorption capacity, though certain physiological barriers remain. This study aimed to develop and characterize Zein-Eudragit nanoparticles (NPS) as carriers for insulin (ZEU/INS NPS) intended for intranasal administration. The NPS were prepared using a liquid-liquid dispersion method, and the production process was optimized through a 24 factorial design. The resulting NPS were evaluated in terms of physicochemical properties, including particle size, polydispersity index (PDI), zeta potential, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TG), and morphology. Additionally, the physical stability of the NPS during storage, in vitro insulin release, and in vitro mucoadhesion were assessed. The optimized nanoparticle formulation exhibited a mean particle size below 200 nm, a PDI of less than 0.3, a zeta potential of approximately +30 mV, and an encapsulation efficiency of 42%. FTIR analysis confirmed the interaction between nanoparticle components following insulin encapsulation, and DSC/TG analysis demonstrated the thermal stability of the system. NPS stored under refrigerated conditions maintained their stability for up to 60 days. In vitro release studies revealed that about 60% of the encapsulated insulin was released over a 24 h period. The in vitro mucoadhesion assay further supported the potential of these NPS to enhance the residence time in the nasal cavity. Overall, ZEU/INS NPS successfully demonstrated favorable physicochemical characteristics for the intranasal delivery of insulin. These findings suggest that these NPS offer significant promise as an effective and noninvasive delivery system for insulin.", "journal": "ACS omega", "year": "2025", "doi": "10.1021/acsomega.4c10474", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42198267": { "pmid": "42198267", "title": "Optimization of Sugar-Derivatives Mixtures for Stabilizing Polyclonal Immunoglobulin G in Spray-Dried Inhalable Powders During Processing and Long-Term Storage.", "abstract": "Background/Objectives: The development of dry powder formulations for pulmonary delivery of therapeutic antibodies requires careful stabilization strategies to preserve protein integrity during spray-drying and long-term storage. This study investigates the impact of various sugar-derivatives, a polyol (D-mannitol), a disaccharide (D-sucrose) and a polysaccharide (dextran 10 kDa), used individually or in combination, on the physical stability of bovine polyclonal immunoglobulin G (pAb) in dry powders for inhalation (DPIs). Methods: A design of experiments (DoE) approach was employed to evaluate the effects of these excipients on residual moisture (RM), low-order aggregates (LOA) and high-order aggregates (HOA), immediately after spray-drying (T0) and after 10 months of storage at room temperature in a desiccator (T10). Results: All DPIs exhibited a high amorphous content and a favorable glass transition temperature, with RM decreasing over time. The combination of D-mannitol and dextran 10 kDA (DPI-MD) demonstrated the most effective stabilization, minimizing LOA and HOA formation at T0 and T10. Although the ternary mixture, including D-sucrose (DPI-MSD) exhibited higher process stability, it was less stable over time in comparison to the binary mixture. The aerodynamic performance of these carrier-free DPIs, assessed via laser diffraction (% ˂ 5 µm), were between 51 ± 3 (DPI-MD) and 67 ± 4 (DPI MSD) and a Next Generation Impactor, confirmed that formulation produced aerosol with suitable size distribution and fine particle fractions (FPFn upt to 71 ± 5% for DPI-MSD), for deep pulmonary deposition. Conclusions: These findings highlight the importance of combining excipients with complementary physical properties to achieve robust protein stabilization. The DPI-MD emerged as the most promising candidate for pAb lung delivery, balancing protein integrity, powder stability, and aerodynamic efficiency.", "journal": "Pharmaceutics", "year": "2026", "doi": "10.3390/pharmaceutics18050573", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39965366": { "pmid": "39965366", "title": "An RPA-CRISPR/Cas12a-assisted method for nucleic acid detection of Haemonchus contortus in sheep.", "abstract": "Haemonchus contortus (H. contortus), a highly pathogenic and blood-feeding nematode, could cause haemonchosis，resulting in tens of billions of dollars in production losses and significantly impacting the development of sheep husbandry. Rapid and accurate detection methods were particularly important for the prevention and control of haemonchosis. In this study, we developed a one-pot effective detection method that integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology based on the conserved region of ITS2 of H. contortus, with readout through fluorescence signals visualized by lateral flow strips (LFS) and observable under UV or blue light. The detection procedure was successfully finished in within 1 h and demonstrated high specificity and sensitivity, with no cross-reactivity detected with nine other common ovine pathogens and a detection limit as low as 0.1 copies/μL for fluorescence and 100 copies/μL for LFS. Validation with 89 sheep fecal samples revealed a 46.07 % positivity rate, fully consistent with quantitative PCR results. In summary, the RPA-CRISPR/Cas12a method for H. contortus detection exhibited the advantages of high specificity, high sensitivity, and low device dependence, portable and visible results. The technique presented significant potential for large-scale clinical application and provided novel point-of-care testing for clinical use in remote rural and resource-constrained areas.", "journal": "Veterinary parasitology", "year": "2025", "doi": "10.1016/j.vetpar.2025.110421", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40666886": { "pmid": "40666886", "title": "Comprehensive Cross-Domain Taxonomic Classification of Microbiotas using Partitioned Amplification Multiplexed Amplicon Sequencing (PAMA-seq).", "abstract": "Microbial communities encompass diverse bacteria, archaea, and eukaryotes that play vital roles in ecosystems and host health. Comprehensive analysis of these communities requires accurate, quantitative, and cross-domain profiling, yet current sequencing methods-metagenome shotgun sequencing (MGS) and ribosomal RNA (rRNA) amplicon sequencing-struggle to achieve these goals in a single assay. MGS provides broad functional insights but suffers from high cost, computational burden, and reliance on incomplete reference databases. rRNA amplicon sequencing, while more taxonomically targeted, typically profiles either prokaryotes or eukaryotes separately, depending on the chosen primer sets, and thus lacks simultaneous cross-domain resolution. To address these limitations, we developed PAMA-seq, a droplet-digital multiplex PCR technique. PAMA-seq partitions DNA sample from microbial communities into nanoliter droplets, each containing a single template molecule, and independently amplifies both the 16S and 18S rRNA genes, resulting in uniform amplification efficiency and accurate quantification. We validated PAMA-seq on a synthetic microbial community, a stool sample from a patient with colorectal cancer, and a coastal seawater sample. The method provided stable, cross-domain taxonomic profiles at sequencing depths as low as 104 reads-one to two orders of magnitude fewer than comparable shotgun metagenomics approaches-while significantly reducing variability between replicates. By combining comprehensive domain coverage with low sequencing depth requirements, PAMA-seq offers an efficient, cost-effective, and scalable method for monitoring microbial communities across diverse ecosystems, from clinical samples to global marine environments.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.06.18.660470", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "C" ] }, "39838702": { "pmid": "39838702", "title": "In Vitro Assessment of Chitosan-PEG Hydrogels Enriched with MSCs-Exosomes for Enhancing Wound Healing.", "abstract": "Regenerating skin tissue remains a major challenge in medical science, especially due to the risk of scarring and prolonged healing, which becomes even more complicated in people with diabetes. Recent advancements have led to the creation of therapeutic dressings incorporating drug-delivery systems to tackle these issues. Exosomes (Exos) derived from mesenchymal stem cells (MSCs) have gained significant attention for mediating therapy without directly using cells, thanks to their natural anti-inflammatory and tissue repair properties mirroring those of MSCs. In this study, an advanced wound dressing combines chitosan (CS) and polyethylene glycol (PEG) hydrogel with adipose MSCs-derived Exos (ADMSCs-Exos). This composite, formed using a straightforward blending technique, is engineered to improve the healing process of severe skin injuries by steadily releasing Exos as the hydrogel degrades. The in vitro studies demonstrate that this hydrogel-exosome dressing greatly enhances endothelial cell migration, reduces oxidative stress, and promotes angiogenesis, crucial for effective wound healing. Additionally, real time-polymerase chain reaction (RT-PCR) analysis revealed significant upregulation of key genes involved in these processes, supporting the therapeutic potential of the hydrogel-Exo combination. These findings emphasize the potential of this hydrogel-Exos combination as an innovative and promising solution for advanced wound care.", "journal": "Macromolecular bioscience", "year": "2025", "doi": "10.1002/mabi.202400609", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40580289": { "pmid": "40580289", "title": "Probe-Based qPCR Workflow Using Fast Nucleic Acid Sampling and Molecular Genotyping in a Dioecious Plant.", "abstract": "This chapter presents a protocol for probe-based quantitative polymerase chain reaction (qPCR) genotyping, focusing on male-specific genotype identification in plant samples. DNA is isolated from raw tissue or FTA cards and subjected to a preamplification enrichment reaction. The diluted product is then used as input for qPCR detection, employing specific probes to identify the presence or absence of the target genotype. This robust and high-throughput method offers increased specificity for genotyping various sample types, including crude nucleic acid extracts. An advantage of qPCR is that genotyping can be performed on various sample types, including from crudely prepared nucleic acid extracts used as input with inhibitor-tolerant polymerases. In this workflow, we describe an extraction and probe-based qPCR method used to detect male-specific genotype identification in plant sample DNA extracted from raw tissue or FTA imprinted cards.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2025", "doi": "10.1007/978-1-0716-4642-7_9", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42273395": { "pmid": "42273395", "title": "Hydrogel-based multiplexed high-risk human papillomavirus DNA/RNA detection for cervical cancer screening.", "abstract": "Rapid triaging of high-risk human papillomavirus (HPV) infections, especially in resource-limited regions, can reduce cervical cancer deaths by identifying high-risk patients and minimizing pathology bottlenecks and overtreatment. Here, we developed a simple, rapid method for parallel detection of HPV DNA and RNA subtypes associated with a high risk of developing cervical cancer. Specifically, we applied loop-mediated isothermal nucleic acid amplification (LAMP) in a hydrogel-based platform (LAMPGel) for HPV DNA or RNA detection using distinct hydrogel chambers within a single chip. The LAMPGel platform locally restricts single HPV gDNA or mRNA strands with a LAMP cocktail, which is then distributed within a thin hydrogel to locally form single fluorescent spots after a short isothermal incubation of <30 min. Detecting and counting these spots identifies HPV infection, viral load, and, more importantly, RNA expression as a predictive marker for cervical cancer development. LAMPGel is a sensitive, specific, easy-to-use, and rapid method for a point-of-care (POC) platform for potential applications in low- and middle-income countries once the technology is further validated with clinical samples in those settings. Furthermore, it could find broader applications in POC nucleic acid assays.", "journal": "Biosensors & bioelectronics: X", "year": "2025", "doi": "10.1016/j.biosx.2025.100664", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41905654": { "pmid": "41905654", "title": "Aggregation of monoclonal and bispecific antibodies during pneumatic tube transport of IV bags in a hospital: Mitigation by polysorbate 80, poloxamer 188, and headspace removal.", "abstract": "Pneumatic tube systems (PTS) are being increasingly used in hospitals to rapidly transport critical packages, but their mechanical stresses raise concerns for the safe transport of protein therapeutics. This study examined the impact of the PTS at the University of Iowa Hospital on protein admixtures of a monoclonal antibody (mAb) and a bispecific antibody (BsAb) prepared in IV-bags and benchmarked it against manual transport and shaking. The PTS ride imposed sharp shocks nearly tenfold higher than either shaking or manual transport. Flow imaging microscopy revealed increased microparticle formation for both proteins, though with distinct profiles. The mAb generated more small aggregates, whereas the BsAb produced fewer yet larger particles. Surfactants reduced particle formation, with Polysorbate 80 (PS80) more effective than poloxamer 188. For BsAb, PS80 lowered microparticles by ∼70% but failed to prevent large particle formation, in contrast to its near-complete protection for mAb. Headspace removal further reduced particle formation, fully preventing the formation of large aggregates when combined with PS80. Size exclusion chromatography revealed no detectable increase in soluble aggregates or monomer loss. These findings show that PTS presents a unique stress profile not replicated by shaking and highlight the need for case-by-case evaluation of PTS compatibility based on protein, container, and excipients.", "journal": "Journal of pharmaceutical sciences", "year": "2026", "doi": "10.1016/j.xphs.2026.104264", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41792088": { "pmid": "41792088", "title": "Effect and safety evaluation of MIL-101 loaded with prochloraz against rice sheath blight.", "abstract": "Rice sheath blight (ShB), a fungal disease caused by Rhizoctonia solani, seriously threatens rice yield and quality. Prochloraz (PRO), a broad-spectrum imidazole fungicide, may cause some harm to the environment and organisms when overused. To improve its efficiency and reduce environmental impact, MIL-101, a metal-organic framework, was chosen as carrier to load prochloraz to make PRO@MIL-101. Characterizations including high-performance liquid chromatography (HPLC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) confirmed successful drug loading, with MIL-101 maintaining a regular octahedral structure and good stability. In vitro release studies showed that PRO@MIL-101 exhibited a rapid release behavior, with a faster release under acidic conditions, and fitted the Ritger-Peppas model. PRO@MIL-101 also showed better wetting properties in contact angle and adhesion function assays. Antifungal tests indicated that PRO@MIL-101 achieved strong inhibitory effects at relatively low concentrations. Safety evaluations demonstrated that MIL-101 promoted rice growth and chlorophyll synthesis at low-to-medium concentrations, which may enhance rice disease resistance by increasing antioxidant enzyme activities. The study successfully constructs a prochloraz delivery system based on MIL-101. The system has a drug-loading rate of ≈10%, and exhibits excellent drug release performance in an acidic environment and strong adhesion to leaf surfaces. MIL-101 is safe for rice, as it can promote rice growth and enhance stress resistance. Overall, these results suggest that the drug-loading system shows an excellent control effect on ShB, providing a new idea for reducing pesticide usage while improving efficacy of its control effect. © 2026 Society of Chemical Industry.", "journal": "Pest management science", "year": "2026", "doi": "10.1002/ps.70659", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40074752": { "pmid": "40074752", "title": "Development of a rapid malaria LAMP-MS assay for diagnosis of malaria infections.", "abstract": "Malaria remains a critical global health concern, especially in tropical and subtropical regions, where it causes substantial morbidity and mortality. Current diagnostic methods, such as microscopy and PCR-based assays, are reliable but often impractical in resource-limited settings due to their dependency on complex equipment and skilled personnel. This study developed a novel malaria diagnostic platform by combining the Chelex-100/boiling DNA extraction method with a Loop-mediated Isothermal Amplification-MicroScanner (LAMP-MS) assay. The Chelex-100/boiling method is simpler and more cost-effective than conventional DNA extraction processes, making it suitable for use in resource-limited settings. The LAMP-MS assay enables multiplex detection through a microchip design with four chambers. Each chamber of the microchip is preloaded with specific primers targeting Pan, Plasmodium falciparum (Pf), Plasmodium vivax (Pv), and an internal control, minimizing non-specific amplification in multiplex LAMP reactions. In a clinical evaluation of 260 samples, the assay demonstrated a sensitivity of 97.5% for the Pan target and 100% for the Pf-specific target in the 80 Plasmodium falciparum (Pf) clinical samples. Similarly, for the 80 Plasmodium vivax (Pv) clinical samples, the assay achieved a sensitivity of 95% for the Pan target and 94% for the Pv-specific target. Notably, in the 100 non-infected clinical samples, the assay exhibited 100% specificity, with no false positives observed. These findings suggest that LAMP-MS is a rapid and reliable alternative to PCR-based methods, especially in resource-limited environments.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-92935-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41678212": { "pmid": "41678212", "title": "Plant-derived betaine enhances barrier integrity and immune responses in canine intestinal epithelial cells and macrophages under endotoxin challenge.", "abstract": "Interest in the utilization of novel plant-based extracts for supporting gut health and nutrition in companion animals is growing. The effect of betaine on canine intestinal barrier integrity and immune-related gene and protein expression by epithelial and macrophage-like cells was evaluated under conditions of inflammatory challenge from lipopolysaccharide (LPS). We evaluated: (1) the effect of betaine pre-treatment (0 to 1,000 µg/mL for 24 h) on the viability of canine intestinal epithelial MCA-B1 and macrophage-like DH82 cells; (2) epithelial barrier function of LPS-challenged MCA-B1 cells pre-treated with betaine, by measurement of 4-kDa fluorescein isothiocyanate (FITC-dextran [FD4] flux); (3) gene and protein expression of tight junction proteins and adhesion molecules using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and proteomics; (4) anti- and pro-inflammatory and regulatory gene expression in LPS-challenged, betaine pre-treated, MCA-B1, DH82 and peripheral blood mononuclear cells and; (5) the phagocytic activity of DH82 cells pre-treated with betaine. No cytotoxicity of betaine against MCA-B1 or DH82 cells were observed. Betaine pre-treatment of MCA-B1 reduced FD4 permeability compared with untreated cells (P < 0.05), accompanied by increased (mRNA) expression of claudin-1 (P < 0.001) and E-cadherin (P < 0.05) and increased protein expression of junctional adhesion molecules (P < 0.05), vinculin (P < 0.05) and aquaporin-3 (AQP3; P < 0.01). Betaine pre-treatment of LPS-challenged cells also increased the expression of regulatory cytokines TGF-β1 (P < 0.01), TGF-β2 (P < 0.01), and IL-33 (P < 0.05) in MCA-B1 cells and reduced the expression of IL-6 (P < 0.05) and MyD88 (P < 0.01) in DH82 cells compared with untreated cells. In addition, betaine enhanced the phagocytic activity of DH82 cells (P < 0.01) compared with untreated cells. Confirmatory studies in vivo are needed but these results suggest that plant-derived betaine has potential as a nutritional supplement for companion animals to support gut health and immunity.", "journal": "Journal of animal science", "year": "2026", "doi": "10.1093/jas/skag031", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40659571": { "pmid": "40659571", "title": "Pharmacological prolyl hydroxylase inhibition enhances high-intensity exercise capacity in mice.", "abstract": "Increased blood hemoglobin concentration theoretically promotes oxygen delivery to the periphery. Clinically, in addition to recombinant human erythropoietin analogs, prolyl hydroxylase (PH) inhibitors to stabilize the expression of hypoxia-inducible factor (HIF) protein were recently approved for the treatment of anemia in chronic kidney disease. However, whether the new agent helps enhance physical exercise capacity remains a matter of controversy. Treatment of C57BL/6J mice via oral gavage with roxadustat of 30 mg/kg three times per week for 5 weeks elevated the blood hemoglobin concentration, upregulated the HIF-downstream gene expression of the muscle, and enhanced a high-intensity exercise performance measured with a treadmill running exhaustion test, compared to those treated with vehicle, while total body weight or skeletal muscle mass was comparable. This physical effect depended on the increase in blood hemoglobin concentration, as confirmed by mice hemodiluted to control hemoglobin concentrations through phlebotomy followed by infusion of the approximate volume of phosphate-buffered saline. In conclusion, oral administration of the HIF-PH inhibitor to mice increased high-intensity exercise capacity as well as elevated blood hemoglobin concentration. The finding implies the versatile effects on humans taking HIF-PH inhibitors, as well as the risk of them being abused for blood doping.", "journal": "Physiological reports", "year": "2025", "doi": "10.14814/phy2.70464", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "41077875": { "pmid": "41077875", "title": "Evaluation of various formulations of Contagious Bovine pleuropneumonia vaccine (T1/44) for thermotolerance and shelf life.", "abstract": "Contagious Bovine Pleuropneumonia (CBPP) remains a significant livestock disease in sub-Saharan Africa, with the T1/44 vaccine's heat-lability and reliance on cold-chain infrastructure limiting its efficacy in tropical regions. This study evaluated the thermotolerance and shelf-life of seven CBPP vaccine formulations containing different stabilizing agents. Seven different vaccine formulations, incorporating stabilizing agents such as lactalbumin, casein, gelatin, skimmed milk, maltose, and sucrose, were tested. The formulations were subjected to accelerated stability testing at temperatures of 4°C, 25°C, and 37°C over a 14-day period. The viability of Mycoplasma mycoides was assessed by measuring colony-forming units (CFU) at each time point. Statistical analysis was performed using two-way ANOVA with a significance level of p < 0.05. All formulations showed a decrease in mycoplasma viability over time, with the most significant loss occurring at 37°C. Formulation 2 (Casein + Gelatin) showed the most promising results, with a stable mycoplasma count of 1.09×107 CFU/mL post-lyophilization and only minor reductions in CFU at various temperatures. After 14 days, Formulation 2 maintained the highest titres, with counts of 4.4×107 CFU/mL at 4°C, 1.2×106 CFU/mL at 25°C, and 8.0×106 CFU/mL at 37°C. Other formulations, such as Formulation 1 (Lactalbumin + Gelatin), Formulation 3 (Skimmed Milk), and Formulation 4 (Skimmed Milk + Maltose), showed moderate stability but experienced greater decreases in viability, particularly at elevated temperatures. For example, Formulation 3 (Skimmed Milk) had a significant decline in titre at 37°C, with counts falling to 1.05×105 CFU/mL by Day 14. Formulations 5, 6, and 7 (which included combinations of skimmed milk, sugars, and gelatin) showed poor stability at 37°C, with some formulations losing all viability at higher temperatures. This study highlights the effectiveness of casein and gelatin as stabilizers for enhancing the thermotolerance and shelf life of the T1/44 CBPP vaccine. These findings provide a valuable foundation for the development of more resilient CBPP vaccines that can be more widely used in resource-limited settings, especially in regions with unreliable cold-chain systems.", "journal": "Veterinaria italiana", "year": "2025", "doi": "10.12834/VetIt.3773.35869.2", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40220418": { "pmid": "40220418", "title": "Multi-approach analysis reveals the mechanism by which Shugan Xiaozhi decoction protects against metabolic dysfunction-associated steatohepatitis.", "abstract": "Metabolic dysfunction-associated steatohepatitis (MASH) is a human health-threatening hepatic disease with limited treatment strategies. As a clinical Traditional Chinese Medicine compound for MASH, Shugan Xiaozhi (SGXZ) decoction has a definite effect, but its mechanism in treating MASH is still not very clear. Exploring the potential mechanism of SGXZ decoction in treating MASH through multiomics and animal experimental validation. UPLC-ESI-MS method was used to identify the main components of SGXZ decoction. Periodic acid-schiff (PAS), picrosirius red (PSR), and oil red o staining were used to assess the effect of SGXZ decoction on MCD-induced MASH mouse model. The mechanism of SGXZ decoction on MASH was analyzed using multiomics techniques. TUNEL staining, western blot (WB), immunohistochemistry (IHC), kits, transmission electron microscopy (TEM), and immunofluorescence (IF) were used to validate the mechanism of SGXZ decoction on MASH. Finally, molecular docking and molecular dynamics simulation were used to verify the targeting between key components of SGXZ decoction and important targets for intervention. Through UPLC-ESI-MS analysis, 30 main active ingredients were obtained from SGXZ decoction. SGXZ decoction improved MASH, as evidenced by the improvement in histopathology, hepatic function indexes, lipid and fibrosis indicators. Both proteomic and transcriptomic results suggested an important role for ferroptosis in SGXZ decoction intervention in MASH, ferroptosis-related pathways were the main significant pathways obtained from these analyses. In addition, SGXZ decoction treatment reduced cell death, inflammation, and oxidative stress levels and restored impaired mitochondrial morphology in MCD-induced MASH mice. Furthermore, Mechanism experiments proved that SGXZ decoction treatment improved iron metabolism and lipid peroxidation imbalance and activated the Xc- system in MASH mice. SGXZ decoction does have a therapeutic effect on MASH, and its mechanism may be related to its regulation of p53/ SLC7A11/GPX4 pathway to reduce ferroptosis.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2025.156712", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41810001": { "pmid": "41810001", "title": "Graphene Oxide Potentiates Induction Efficiency and Functional Maturation of Induced Mammary Epithelial Cells.", "abstract": "This study aims to explore the effects of graphene oxide (GO) particles on the RepSox-mediated transdifferentiation of fibroblasts into mammary epithelial cells. GO was synthesized using the Hummers method, and its structure was characterized by Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. Its biocompatibility was verified through CCK - 8 and EdU assays. The effects of the GO/RepSox composite system on the transdifferentiation process of fibroblasts and the potential regulatory mechanisms were comprehensively evaluated using morphological observation, immunofluorescent staining, Western blot analysis, real - time quantitative PCR (qRT - PCR), and RNA sequencing techniques. The synthesized GO not only had good biocompatibility but also promoted cell proliferation. GO significantly improved the efficiency of RepSox-mediated transdifferentiation of fibroblasts into mammary epithelial cells and enhanced the lactation function of mammary epithelial cells. Mechanistically, GO may create favorable conditions for transdifferentiation by coordinately regulating mitochondrial energy metabolism (the ATP level was significantly increased in the R + GO group) and cell cycle progression (the proportion of cells in the G1 phase was significantly increased). This study first elucidates the regulatory role of GO in cell fate determination and provides innovative research ideas and experimental evidence for the application of nanomaterials in cell reprogramming and transdifferentiation.", "journal": "International journal of nanomedicine", "year": "2026", "doi": "10.2147/IJN.S560553", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40446539": { "pmid": "40446539", "title": "3D printed GelMA/HAMA based mechanical microenvironment boosted PDAC chemoresistance via NRF2-repressed ferroptosis.", "abstract": "As a highly aggressive solid tumor, pancreatic ductal adenocarcinoma (PDAC) is featured by a conspicuous tumor microenvironment (TME) with abundant extracellular matrix (ECM) deposition, such as collagen and hyaluronic acid (HA). Accompanying with the pathological process, the dense matrix caused stiffen microenvironment, which leads to poor chemotherapy. Therefore, understanding the potential mechanism of mechanical microenvironment on PDAC chemoresistance is of great significance. To this end, 3D printed methacrylated gelatin (GelMA) and hyaluronic acid methacryloyly (HAMA) hydrogel model was fabricated to resemble the mechanical microenvironment of PDAC. The results presented that the PDAC cell lines (MIA-PaCa2 and PANC-1) embedded in the hydrogel were distinctively less sensitive to gemcitabine compared to tissue culture plate (TCP) group. Interestingly, immunofluorescence staining and qRT-PCR assay demonstrated that the NRF2 level was elevated, which further upregulated the downstream effectors, such as SLC7A11 and GPX4. Meanwhile, the GSH concentration and GPX4 activity of hydrogel group were increased, while the lipid peroxidation level was reduced, which indicated that the matrix stiffness microenvironment mediated ferroptosis phenotype. Furthermore, RNA interference experiment elaborated that shNRF2 group showcased significantly enhanced sensitivity to gemcitabine, which demonstrated that the biomimetic microenvironment modulated chemoresistance through ferroptosis. All in all, the 3D printed model may serve as a new platform for illustrating the underlying mechanism of the TME induced chemoresistance, and also proposed a novel therapeutic strategy for PDAC through NRF2 inhibition, which effectively promotes ferroptosis to overcome chemoresistance.", "journal": "Colloids and surfaces. B, Biointerfaces", "year": "2025", "doi": "10.1016/j.colsurfb.2025.114816", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41208485": { "pmid": "41208485", "title": "Quantitative Analysis and Multicomponent Formulation of Ultra-Adsorptive Pore-Space-Partitioned Metal-Organic Frameworks.", "abstract": "One tenet of isoreticular chemistry is the use of longer framework-building units to increase unit-cell and pore volume. While this belief has led to tremendous success as shown by isoreticular series such as IRMOF-1 to IRMOF-16 and MOF-74-I to MOF-74-XI, it can nevertheless be flawed as we move to multimodule systems, for which the quantitative analysis on the correlation between cell volume and ligand dimension can offer guidance on the rational formulation of MOF components. Here, through the synthesis of 60 new pacs materials, we report a broad expansion of pacs-type MOFs by extensively varying metal-trimer compositions, framework-forming ligands (L1), and pore-partitioning ligands (L2). This expanded structural library provides the experimental basis to highlight a nonmonotonic trend between unit-cell volumes and ligand lengths as an intrinsic property of the pacs platform. We further reveal an amplification of the stability-enhancing effect by the NiV trimer over other trimers as we transition to a greater pore-size regime, showing that the nickel-vanadium combination provides a crucial stabilizing factor for large pacs materials. The new structural family includes members with record-setting properties, including ultrahigh BET surface areas (>4400 m2/g) which is likely the highest among high-connected (>6) frameworks based on low-valent (≤3) metals. Multiple members of this family are believed to have the all-time-high propane uptake capacity (e.g., 15.6 mmol/g or 350.1 cm3/g at 298 K and 1 bar; 6.72 mmol/g or 150.7 cm3/g at 298 K and 0.1 bar), especially among C3H8-selective MOFs. The exceptional uptake capacity, coupled with inverse propane/propylene selectivity, enables efficient separation.", "journal": "Journal of the American Chemical Society", "year": "2025", "doi": "10.1021/jacs.5c14861", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "39630034": { "pmid": "39630034", "title": "Curcumin-carbon dots suppress periodontitis via regulating METTL3/IRE1α signaling.", "abstract": "This study aimed to investigate the effects of curcumin-carbon dot conjugates (CUR-CD) on periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS) was used to establish periodontitis mode in vivo and in vitro. Histological analysis was conducted using hematoxylin and eosin (HE) staining. Bone morphogenetic protein 2 (BMP2) and secreted phosphoprotein 1 (OPN) expression was determined using immunohistochemistry. mRNA levels were detected using reverse transcription quantitative real-time PCR (RT-qPCR). Protein expression was determined using Western blot. N6-methyladenosine (m6A) enrichment was determined using methylated RNA immunoprecipitation (MeRIP) assay. Cytokine release was determined using enzyme-linked immunosorbent assay (ELISA) assay. Osteogenic differentiation was detected using alkaline phosphatase (ALP) staining. The results showed that CUR-CD inhibited the inflammatory response, as well as promoted bone healing in vivo and osteogenic differentiation in vitro. Moreover, CUR-CD downregulated methyltransferase 3 (METTL3), which inhibited m6A modification of endoplasmic reticulum to nucleus signaling 1 (IRElα) and downregulated IRElα expression. However, overexpression of IRElα reversed the effects of CUR-CD, stimulating inflammatory response and inhibiting bone healing and osteogenic differentiation. Collectively, CUR-CD inhibits the progression of periodontitis via downregulating IRElα. Therefore, CUR-CD may be an alternative strategy for periodontitis.", "journal": "Critical reviews in eukaryotic gene expression", "year": "2025", "doi": "10.1615/CritRevEukaryotGeneExpr.2024054463", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39714110": { "pmid": "39714110", "title": "Development of Thermosensitive Mucoadhesive Gel Based Encapsulated Lipid Microspheres as Nose-to-Brain Rivastigmine Delivery System.", "abstract": "Alzheimer's disease (ALZ) is a neurodegenerative disease that damages neuronal cells and causes decline in cognitive abilities. Administration of cholinesterase inhibitor compounds is the primary choice in the treatment of ALZ, one of which is rivastigmine (RVT). Several routes of administration of RVT are available, such as oral and transdermal. However, in the oral route, RVT has low bioavailability, undergoes first-pass metabolism, and the presence of the blood-brain barrier (BBB) reduces the therapeutic concentration of RVT. The transdermal route is nonselective target in the brain. This study aims to combine thermosensitive mucoadhesive gel (TG) and lipid microspheres (LM) as a drug delivery system to improve the efficacy of RVT. Combining these will prevent systemic side effects of RVT and increase drug concentration in the brain. LM was formulated with varying concentrations of Compritol polymer. The results of LM evaluation showed the values of particle size, PDI, and %EE and %DL were 8.519 μm, 0.018 ± 0.004, 72.54%, and 76.43%, respectively. The TG formulation can provide a liquid form at room temperature (25 °C) and a gel at nasal temperature (35 °C). Hemolytic and HET-CAM tests confirmed TG RVT LM's safety for use. Ex vivo studies showed controlled and sustained release of TG RVT LM, and in vivo studies showed TG RVT LM a higher pharmacokinetic profile in the brain than oral formulations and injections. The Cmax was found to be 7.05 ± 0.55 μg/cm3, Tmax was 24 h, and AUC0-24, which is related to the effectiveness of brain targeting, was 225.73 μg/cm3. In conclusion, this study shows the successful development of TG RVT LM, as evidenced by improved drug delivery to the brain, which is characterized by higher concentrations of RVT in the brain compared with oral and injectable RVT, this delivery system shows potential as a future treatment for Alzheimer's disease.", "journal": "Langmuir : the ACS journal of surfaces and colloids", "year": "2025", "doi": "10.1021/acs.langmuir.4c03530", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "39441765": { "pmid": "39441765", "title": "RGD peptide promotes follicle growth through integrins αvβ3/αvβ5 in three-dimensional culture.", "abstract": "Three-dimensional ovarian tissue culture is a unique model to define the effects of molecules on folliculogenesis. Using this model, we determined that RGD-integrin interaction plays a role in antrum formation and theca cell differentiation. We recently developed a three-dimensional (3D) ovarian tissue culture system supported by bacterial-derived dextran hydrogel. Arg-Gly-Asp (RGD) is an extracellular matrix-derived triple peptide. Immature ovarian tissues cultured in RGD-modified dextran hydrogel significantly promoted antral follicle growth and oocyte quality compared with those cultured in dextran hydrogel alone. In this study, we examined the mechanism of follicle growth stimulated by RGD treatment in the 3D system. First, we detected that direct contact between RGD-modified dextran hydrogel and ovarian interstitial cells is necessary to promote antral follicle growth. Therefore, we hypothesized that RGD stimulates antral follicle growth through RGD-binding integrin receptors expressed in the interstitial cell mass. Using quantitative PCR (qPCR) and immunochemical staining, we identified that integrins ⍺vβ3 and ⍺v5 are predominantly expressed in the ovarian interstitial compartment. To assess the effect of RGD-integrin interaction on follicle growth, ovarian tissues were cultured with cilengitide (Ci), an inhibitor specific for ⍺vβ3 and ⍺vβ5. Ci treatment suppressed RGD-induced follicle growth and oocyte quality in a dose-dependent manner. When the interstitial cell aggregates were cultured with RGD, cell migration and theca-related gene expression were significantly upregulated. Ci treatment dramatically suppressed these RGD-induced activities. In coculturing the interstitial aggregate and secondary follicles with RGD, migrating cells formed the outermost cell layers around the follicles, like theca layers, which were totally blocked by Ci treatment. In conclusion, our results suggest that RGD stimulates theca cell differentiation in the ovarian interstitial cells through integrins ⍺vβ3 and ⍺v5 to promote antral follicle growth in our 3D system.", "journal": "Reproduction (Cambridge, England)", "year": "2025", "doi": "10.1530/REP-24-0151", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "D" ] }, "40090014": { "pmid": "40090014", "title": "Rapid Detection of Frogeye Leaf Spot Pathogen in Seeds by LAMP Assays to Protect Soybean Production.", "abstract": "Frogeye leaf spot (FLS), caused by Cercospora sojina, is a significant disease affecting soybean crops economically. Seed inspection plays a vital role in reducing the incidence of this disease. However, limited research has been conducted on the fast and accurate identification of C. sojina in soybean seeds. In this study, we developed a rapid diagnostic method to detect C. sojina in soybean seeds using loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was utilized to identify specific sequences belonging to C. sojina. The LAMP primer set designed based on the selected specific sequence proved to be highly specific, distinguishing C. sojina from other common soybean pathogens and two maize pathogens of the Cercospora genus. The primer set exhibited remarkable sensitivity, detecting 16 copies per reaction, which is approximately 1,000 times more sensitive than conventional PCR. The LAMP assays were applied for direct inspection of soybean seeds, revealing that 20 of the 30 tested soybean varieties were contaminated with C. sojina, with contamination rates ranging from 10 to 60%. The seeds of TongNong 943 and JiNong 38 had the highest contamination rate at 60%. Seeds coated with fungicide showed a reduced disease incidence, as demonstrated on PDA medium and in soil pots. In summary, our results indicate that rapid seed diagnosis using LAMP assays can effectively screen out seeds carrying C. sojina, providing a preventive strategy in managing FLS disease.", "journal": "Plant disease", "year": "2025", "doi": "10.1094/PDIS-10-24-2156-SR", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40490041": { "pmid": "40490041", "title": "Protein stability and critical stabilizers in frozen solutions.", "abstract": "Freezing is a common unit operation during the processing and storage of therapeutic proteins. Upon freezing, protein molecules and other solutes are excluded from the ice crystals, resulting in the formation of a freeze concentrated solution (FCS). Protein destabilization can be prevented due to the low temperature and the formation of a viscous FCS. However, the changes in temperature, pH, salt concentration, and the formation of ice-solution interfaces can induce protein destabilization. The current paper reviews the stresses associated with freezing and thawing and discusses the stabilization strategies. At subambient conditions, protein unfolding is thermodynamically favored when the solution is cooled below the cold denaturation temperature. The formation of a viscous FCS, specifically at a temperature below the glass transition temperature (Tg'), immobilizes and stabilizes the proteins. Protein can adsorb at the ice/solution interface, followed by unfolding and aggregation. Therefore, freezing and thawing rates need to be carefully controlled to minimize the ice surface area (due to the formation of small ice crystals) and to avoid ice recrystallization. Besides, stabilizers, including sugars, surfactants, and amino acids, are added as stabilizers (cryoprotectants) to prevent protein destabilization upon freezing and frozen storage. In situ spectroscopic (infrared, Raman, and solid-state NMR) and scattering (X-ray and neutron) techniques provide useful insights into protein stability and phase transitions of excipients in frozen solutions. Finally, we discuss the importance of the freezing process and excipient selection in the successful lyophilization of protein formulations. The review paper comprehensively discusses the challenges during the cryopreservation of therapeutic proteins and provides valuable insights into the critical stabilizers in frozen protein solutions.", "journal": "European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V", "year": "2025", "doi": "10.1016/j.ejpb.2025.114764", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "42114400": { "pmid": "42114400", "title": "Optimization of lyophilization medium for Vibrio harveyi phage and application in oral delivery to groupers.", "abstract": "Vibrio harveyi is a major pathogen in grouper aquaculture, and the rise of antibiotic resistance necessitates the development of new control strategies. Phage therapy is considered a promising alternative, but formulation stability limits its practical application. This study aimed to optimize the lyoprotectant formulation for the V. harveyi phage V-YDF132 and to evaluate its immunomodulatory effects as a feed additive in grouper, as well as its prophylactic efficacy against V. harveyi infection. Among nine candidate composite protectants screened, formula P9 (consisting of inulin, skim milk, gelatin, and sodium L-glutamate) was identified as the optimal formulation. The resulting preparation exhibited good stability: the phage titer decreased by 0.51 log immediately after lyophilization; after 21 d at 37 °C in an accelerated stability test, the titer decreased by only 0.90 log; and after 9 months at 4 °C, the titer decreased by 0.51 log. During a 14-d feeding trial, there were no differences in growth performance between the phage-fed and control groups. Feeding phage-supplemented diets significantly increased the activities of serum alkaline phosphatase (AKP), nitric oxide synthase (NOS), and lysozyme (LYZ), and significantly upregulated the expression of immune-related genes (CD4, CD8, IgM, and TCRβ) and inflammatory cytokines (IL-6 and IL-8) in the spleen. Challenge tests showed that the phage-fed group achieved a relative percent survival (RPS) of 28.6%. In summary, the lyophilized phage V-YDF132 formulation can serve as a potential immunomodulator and, when administered orally, enhances grouper resistance to V. harveyi.", "journal": "Fish & shellfish immunology", "year": "2026", "doi": "10.1016/j.fsi.2026.111415", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39895263": { "pmid": "39895263", "title": "Advanced multiplexed electrochemical nanobiosensors for simultaneous detection of BK polyomavirus miRNAs in renal transplants.", "abstract": "BK Polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) is a leading cause of kidney transplant graft loss. Although polymerase chain reaction (PCR) is a common technique for quantifying BK Polyomavirus (BKPyV) viral loads, it lacks optimal sensitivity due to variations in sample type and source, DNA extraction techniques, primer and probe sequences, and the BKPyV strain DNA used for standard-curve genotype variance, all of which can affect the results. Studies have shown that bkv-miR-B1-5p and bkv-miR-B1-3p can be suitable candidates for the diagnosis of nephropathy in kidney transplant recipients, especially in the early stages of the disease. This research introduces an innovative electrochemical nanobiosensor that can detect BKPyV-related miRNAs, including bkv-miR-B1-5p and bkv-miR-B1-3p, with high sensitivity in renal transplant patients. By using a biotin-modified molecular beacon (biotin-MB) and gold nanoparticles (AuNP)/silver nanoparticles (AgNP), this sensor is able to detect viral miRNA with unparalleled sensitivity. Using square-wave voltammetry (SWV), the system can detect miRNA targets simultaneously, surpassing the constraints of traditional techniques like PCR. The sensor displays high linearity with detection limits of 0.3 fM for bkv-miR-B1-5p and 3.6 fM for bkv-miR-B1-3p. This technology offers the potential for diagnosing BKPyVAN earlier and more accurately, offering important information to enhance the lifespan of kidney transplants.", "journal": "Analytical methods : advancing methods and applications", "year": "2025", "doi": "10.1039/d4ay02000f", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "40155088": { "pmid": "40155088", "title": "Recent advances in carbohydrate phase behavior and rheology.", "abstract": "The past decades have seen major advances in the understanding of the role of phase and state transitions of food carbohydrates on the behavior during processing and on product characteristics. Specifically, the awareness of the importance of the glass transition temperature and the plasticization by water and its study for a variety of food system is having major impact on the formulation and processing of foods, and in defining shelf-life conditions. This has led to the use of phase and state diagrams in the analysis and prediction of the behavior of food systems during processing and storage. This review first summarizes the current understanding of the food carbohydrate phase behavior and rheology, with emphasis on the concentrated states close to the glass transition and in the glassy state. Several pertinent topics, including the modeling of the rheological properties close to the glass transition, the strongly non-linear diffusion of water in the rubbery and glassy states, the aging and antiplasticization of glassy carbohydrate matrices, and consequences of amorphous-amorphous phase separation for the behavior of carbohydrate blends in concentrated states are discussed. Applications in food processing and product development are discussed, including the spray drying and freeze drying, powder agglomeration of food powders, powder caking, encapsulation, baked goods, crystallization and extrusion.", "journal": "Advances in food and nutrition research", "year": "2025", "doi": "10.1016/bs.afnr.2024.11.002", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "42867920": { "pmid": "42867920", "title": "Simplified bacterial identification and antimicrobial resistance detection in bacteraemia by on-chip multiplexed LAMP from blood cultures.", "abstract": "Bacteraemia is a severe infection associated with high risks of complications and mortality. Diagnosis relies mainly on blood cultures, followed by culture-based methods, which remain the gold-standard but are time-consuming. Isothermal bacterial DNA amplification provides a rapid, reliable and cost-effective alternative for identifying and characterising bacteria directly from positive blood cultures, enabling a timelier diagnosis, particularly in resources-limited settings. However, routine implementation has been hindered by sample processing requirements and its limited multiplexing capacity. Here, we developed an on-chip loop-mediated isothermal amplification (LAMP) workflow, ALADIM (accessible and integrated LAMP analysis for multiplex detection of infectious agents in microfluidics), that integrates bacterial extraction and multiplex LAMP, followed by instrument-free amplicons detection using lateral flow immunoassay (LFIA), all within two hours (vs. 24-48 hours for the gold-standard). To demonstrate the capabilities of our device, we targeted the most common bacteria found in bacteraemia, E. coli, the co-amplification of two highly prevalent antimicrobial resistance genes, blaCTX-M-15 and blaOXA-48, and the detection of a positive control. Multiplex LAMP followed by LFIA detection was first evaluated using 14 strains cultured in LB. On-chip results were comparable to those obtained with our reference method (tube-based LAMP in a thermocycler followed by LFIA detection), showing 100% sensitivity, >84% specificity for the detection of malF and blaOXA-48, and 68% sensitivity, 83% specificity for blaCTX-M-15. As a proof of concept for the applicability of the complete ALADIM workflow, we analysed positive overnight blood cultures spiked with 15 strains resulting in 61 independent experiments (DNA extraction/amplification/LFA readout). In this complex matrix, ALADIM specificity remained above 77% for all targets, while sensitivity exceeded 94% for malF and blaOXA-48 but decreased to 56% for blaCTX-M-15. A similar decrease in overall sensitivity was observed for the in-tube method (>68%), highlighting the challenges associated with direct whole-blood analysis. This feasibility study shows the integration of multiplex DNA amplification, microfluidic processing, and LFIA detection in a single platform. Future work will focus on improving sample preparation, assay performance in complex matrices, and expanding multiplexing capabilities of ALADIM towards additional targets, to deliver a clinically reliable point-of-care testing device for low- and middle-income countries.", "journal": "The Analyst", "year": "2026", "doi": "10.1039/d5an01310k", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40668152": { "pmid": "40668152", "title": "Alkali-Soluble Resins as pH-Responsive Protective Colloids.", "abstract": "Emulsion polymers are widely present in numerous applications such as adhesives, coatings, paints, and cementitious materials. In applications such as construction, they are favored in dry form that can be restabilized in the final formulation in the presence of water. This offers advantages in terms of storage, transportation, and final application. This work investigates a pH-switchable stabilizer. Turning off the stabilizer leads to latex particle coagulation and dry polymer isolation. Then, turning it back on recovers the restabilized particles in the form of an emulsion. The success of this process relies on the switchability of the colloidal stabilization system and the surface protection of low glass transition temperature (Tg < 20 °C) particles through a hard shell, ensuring particle integrity during coagulation and polymer isolation. Carboxyl-containing alkali-soluble resins are identified as promising candidates capable of providing both requirements simultaneously. Low Tg (14 °C) polymer particles of vinyl acetate and vinyl neodecanoate (VeoVa10) stabilized by high Tg alkali-soluble resins were synthesized in seeded semicontinuous emulsion polymerization. The polymer isolation was performed through a sequence of steps, including latex coagulation at pH ≤ 3, water filtration (resulting in approximately 70% water removal), drying, grinding, and reinforcement with kaolin as an anticaking agent. The isolated polymer product was then successfully restabilized in the presence of water by applying energy and increasing the pH.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c12131", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40593724": { "pmid": "40593724", "title": "Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.", "abstract": "The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&PS DNA and restored through light-triggered chemical bond breakage of PC&PS DNA. The universality of DNACas is demonstrated by adopting the PC&PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-61094-5", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41073868": { "pmid": "41073868", "title": "Digital Droplet PCR (ddPCR) for Absolute Quantification of 16S rRNA Copy Number in Metagenomic Data.", "abstract": "Digital Droplet PCR (ddPCR) is a quantitative PCR method that offers high sensitivity and accuracy in measuring the amount of nucleic acid in a sample, without the need of a standard curve. In ddPCR, a single sample is partitioned into up to 20,000 droplets, using the water-oil emulsion technology, and the amplification reaction occurs within each droplet using a fluorescent hydrolysis probe (Taqman) or a DNA-binding fluorescent dye. Following PCR, the emitted signals are individually measured in each droplet. Here, we describe a ddPCR optimized protocol for accurately quantifying the total copy number of the 16S rRNA gene in a metagenomic DNA sample. The protocol utilizes a primer pair, targeting the 16S V5-V6 hypervariable regions, in combination with a double-strand DNA-binding fluorescent dye.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2026", "doi": "10.1007/978-1-0716-4767-7_14", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41346060": { "pmid": "41346060", "title": "Digital-to-Droplet Microfluidic Platform Powered Digital Isothermal Nucleic Acid Amplification for On-Demand Detection of Post-Transplantation Infection-Causing Viruses.", "abstract": "The etiological diagnosis of severe infectious diseases is complicated by variability in the timing of sample submissions, heterogeneity in detection targets, and stringent requirements for rapid turnaround times, thereby necessitating the development of on-demand detection methodologies. To address these challenges, we introduce an automated digital isothermal nucleic acid amplification (dINA) assay facilitated by a digital-to-droplet (D2D) microfluidic platform. This D2D system permits the continuous introduction and extraction of water-in-oil droplets, enabling the digital microfluidics (DMF) module to alternately interface with multiple droplet microfluidic units. Within this framework, samples or reagents encapsulated in droplets are sequentially delivered to the DMF module, where nucleic acid extraction and subsequent mixing with loop-mediated isothermal amplification (LAMP) reagents are accomplished through a series of precise droplet manipulations. The resultant microliter-scale LAMP reaction droplets are then dispensed into the sample inlets of individual droplet microfluidic modules, where they are partitioned into picoliter-scale droplets for dINA analysis. The principal advantages of this system include (1) the ability to process samples immediately upon receipt, (2) substantial scalability in throughput, and (3) absolute quantification independent of calibration standards. As a proof of concept, this platform was employed to detect viruses responsible for post-transplantation infections, demonstrating a markedly reduced turnaround time for specimens submitted at variable intervals. These findings underscore the potential of this system as an efficient and adaptable diagnostic tool for the on-demand detection of pathogens implicated in severe infectious diseases.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c05853", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42554489": { "pmid": "42554489", "title": "Rapid, low-cost, and portable molecular detection of HIV-1 subtype B and HCV 1a directly from blood.", "abstract": "Accessible molecular diagnostics for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) remain constrained by the cost and infrastructure requirements of existing molecular point-of-care platforms. In this study, we describe a portable, proof-of-concept molecular workflow with a total runtime of 32 min for the simultaneous qualitative detection of HIV-1 subtype B and HCV genotype 1a directly from contrived whole blood. The workflow employs a battery-powered heating device, ambient-stable reagents, and lateral-flow dipstick readout. Contrived whole blood samples were diluted twofold with nuclease-free water and added directly to rehydrated reverse-transcription loop-mediated amplification (RT-LAMP) reactions without nucleic acid extraction, and reaction parameters were systematically varied to determine lower limits of detection. Both viral targets were consistently detected at clinically relevant input levels following a 30 min isothermal amplification step, with visual lateral flow results available within 2 min. In addition, CRISPR-Cas12a detection was used as an orthogonal method to verify RT-LAMP target amplicons during assay development. Together, this work establishes the analytical feasibility of a rapid, low-cost, portable molecular workflow and provides a foundation for future development of decentralized HIV-1 and HCV molecular diagnostics.IMPORTANCEOngoing changes in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) epidemiology highlight the need for more adaptable and accessible diagnostic tools. In this study, we developed a fast, low-cost test that detects HIV-1 and HCV from a small blood sample. The test is designed to work outside traditional laboratories, using minimal equipment. It does not require complex sample preparation, refrigeration, or expensive machines, and produces results in about 30 min. The workflow combines a rapid genetic amplification method with an easy-to-read test strip and a small, battery-powered heater, making it portable and suitable for use in low-resource or remote settings. An additional molecular method was used during development to confirm that the test was accurately detecting the correct viral targets. By focusing on simplicity, speed, and affordability, this work aims to expand access to reliable HIV and HCV testing in settings where these infections often occur together and laboratory access is limited.", "journal": "Microbiology spectrum", "year": "2026", "doi": "10.1128/spectrum.00416-26", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "41968146": { "pmid": "41968146", "title": "Efficient genome editing with chimeric oligonucleotide-directed editing.", "abstract": "Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report several nCas9-DNA polymerase fusion proteins and their engineered versions to create a simple and efficient two-component chimeric oligonucleotide-directed editing (CODE) system. CODE contains a derivative of Bst DNA polymerase engineered for increased thermostability and processivity as well as a chimeric pegRNA (cpegRNA) for programmable search and replace genome editing. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate that CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci in HEK293T cells with plasmid- and RNP-based delivery. We also show that CODEMax can successfully modify mouse and bovine embryos with up to 9.3% precise editing. Further optimization of CODEMax systems may enhance editing outcomes in embryos and other challenging contexts. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-71624-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40680952": { "pmid": "40680952", "title": "A one-pot CRISPR-Cas12a-based assay for rapid, on-site detection of African swine fever virus.", "abstract": "African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and devastating disease threatening global swine production. The disease has caused substantial economic losses, driving the need for efficient diagnostic tools to enhance surveillance and control. Despite various available assays for ASF, field-deployable tools enabling rapid, accurate, and user-friendly detection remain urgently needed. Here, we developed and validated a novel one-pot recombinase polymerase amplification (RPA)-CRISPR-Cas12a assay for rapid and sensitive detection of ASFV by integrating all components into a single sealed tube, which requires only isothermal heating and ultraviolet visualization. The assay demonstrated a detection limit of 56 TCID50/mL and could be completed within 35 min, and without cross-reactivity with non-ASFV porcine viruses. In comparative testing of 150 clinical samples, the one-pot RPA-CRISPR-Cas12a assay exhibited 100 % agreement with gold standard quantitative PCR (qPCR). Notably, the assay identified ASFV genomic DNA in whole blood as early as 3 days post-infection with sensitivity comparable to the qPCR. This early detection capability, combined with a field-deployable format, provides a robust tool for implementing timely containment measures against ASF, especially in resource-limited setting.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.146109", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42198399": { "pmid": "42198399", "title": "A Three-Arm, Tiered Comparability Strategy Bridging Post-Approval Process Changes for an Omalizumab Biosimilar (CMAB007).", "abstract": "Background: Post-approval manufacturing changes for biologics require rigorous comparability assessments to ensure uninterrupted quality and clinical performance. CMAB007 (Aomaishu®), a China-approved (2023) omalizumab biosimilar, underwent process enhancements-including media optimization and anion-exchange chromatography substitution-yielding a 5-fold increase in production without altering the host cell line. Methods: A novel three-arm tiered strategy was adopted to compare post-change CMAB007, pre-change CMAB007, and reference (Xolair®) products. Critical quality attributes (CQAs) were classified into tiers based on risk impact, with tier-specific acceptance criteria. Comprehensive analytics assessed structure, post-translational modifications, purity/impurities, activity, and Fc-mediated functions. Forced degradation (lyophilized/reconstituted states) and accelerated stability studies were evaluated. Based on the high degree of CMC similarity and to prevent \"biological drift\", the pharmacokinetic (PK) and safety comparability of the post-change CMAB007 versus the reference product (Xolair®) was confirmed in a randomized, double-blind, two-arm study in healthy males (N = 114; single 150 mg subcutaneous administration). The pre-change product was not included in this clinical PK study. Results: Post-change CMAB007 exhibited analytical similarity within tiered acceptance criteria for all CQAs. Stability studies confirmed enhanced robustness under stress conditions. PK equivalence was demonstrated for AUC0-inf (GMR: 99.82%; 90% CI: 91.46~108.94%), AUC0-t (99.54%; 91.40~108.41%), and Cmax (101.88%; 95.21~109.01%). Immunogenicity (ADA incidence: 10.5% vs. 12.5%, p = 0.742) and safety profiles were comparable. Conclusions: This study pioneers a tiered three-arm comparability strategy for post-approval changes, integrating advanced analytics, risk-based quality assessment, and clinical validation. The approach mitigates \"biological drift\" risks, ensuring biosimilar quality, efficacy, and safety while enabling sustainable production scalability.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2026", "doi": "10.3390/ph19050724", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41133096": { "pmid": "41133096", "title": "Synbiotic modulation of adult gut microbiome by 2'-fucosyllactose and Bifidobacterium longum subsp. infantis EFEL8008.", "abstract": "Aim: This study aimed to evaluate the combination of 2'-fucosyllactose (2'-FL) and Bifidobacterium longum subsp. infantis (B. infantis) EFEL8008 as a synbiotic pair for adult gut health, using an in vitro digestion and fecal fermentation model. Methods: The resistance of 2'-FL to digestion was evaluated through simulated digestion encompassing oral, gastric, intestinal, and brush border membrane phases. Fecal fermentation was conducted using adult microbiota to investigate taxonomic and metabolic alterations following treatment with 2'-FL, EFEL8008, or their combination. Microbial composition was profiled using 16S rRNA gene sequencing and quantitative PCR targeting B. infantis. Short-chain fatty acids (SCFAs) and trimethylamine (TMA) levels were quantified by 1H-NMR. Results: A total of 86.67% of 2'-FL remained intact after digestion, demonstrating its resistance to digestion throughout the upper gastrointestinal tract. The synbiotic combination significantly increased Bifidobacterium abundance and improved alpha diversity compared to single treatments. Heat tree and correlation analyses indicated selective enrichment of commensal taxa including Bifidobacterium and Lactobacillus, accompanied by a reduction in the abundance of potentially pathogenic genera such as Escherichia-Shigella. In addition, co-treatment markedly elevated the concentrations of acetate, propionate, lactate, and butyrate, and suppressed the microbial conversion of betaine to TMA, suggesting a favorable metabolic outcome. Conclusion: These results demonstrate that the synbiotic combination of 2'-FL and EFEL8008 promotes beneficial microbial modulation, enhances metabolite production, and supports gut health, highlighting its potential as a next-generation synbiotic strategy.", "journal": "Microbiome research reports", "year": "2025", "doi": "10.20517/mrr.2025.35", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41564239": { "pmid": "41564239", "title": "Thermostability Prediction Powered by Synergistic Deep Learning at Experimental and Theoretical Levels for Nanobodies.", "abstract": "Nanobodies have emerged as powerful biorecognition elements in various fields, while thermostability is a key factor in practical application. Experimental screening remains costly and low throughput, while the scarcity of thermostability data poses a significant challenge for machine learning application. In this work, we innovatively propose a dual-scale synergistic deep learning strategy to improve prediction reliability, including a NBsTem_Tm model trained on 514 experimental melting temperature (Tm) data and a NBsTem_Q model with a theoretical indicator inferred from conformation changes of extensive MD simulations on 704 nanobody structures. Their synergy can alleviate the scarcity of experimental data and the risk of low generalization inherent in small-data NBsTem_Tm models to unseen samples. The two models are constructed by integrating the antibody language model into a joint deep learning architecture to sufficiently learn the feature embedding at different levels. Consequently, NBsTem_Tm achieves a Pearson value of 0.83 on the external test, significantly outperforming three reported competitive models. NBsTem_Q obtains accuracy of 0.84, also exhibiting applicable potential. In addition, the two models can be applied for nanobodies with missing residues, thus being robust to wide application. Benefiting from the two synergistic models, a more reliable screening criterion (Tm > 65 °C and Qclass IV) is proposed for determining highly thermostable nanobodies. The dual-scale framework coupled with a proposed screening strategy is further applied to explore the INDI database with tens of millions of unexplored nanobodies to fill the absence of its thermostability property, discovering approximately 12% thermostable nanobodies. Finally, a user-friendly web server NBsTem is developed to serve as a high-throughput screening platform for nanobody design and development.", "journal": "ACS applied materials & interfaces", "year": "2026", "doi": "10.1021/acsami.5c19073", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42734768": { "pmid": "42734768", "title": "Decoding Nano-Bio Interactions: Gene Regulatory Networks in Advanced Drug Loading.", "abstract": "Conventional treatments often face challenges such as the limited ability to penetrate the blood-brain barrier (BBB). The Doxorubicin-loaded graphene oxide/magnetite (DOX/GO/Fe3O4) nanocomplex offers a promising platform due to GO's high surface area and pH-sensitive release, and Fe3O4's magnetic properties. This protocol describes the methodology for evaluating the cytotoxicity of free DOX versus the DOX/GO/Fe3O4 nanocomplex in the A-172 glioblastoma cell line, followed by advanced bioinformatics analysis to identify gene networks and indirect pathways that enhance the nanomaterial's biocompatibility. The methodology integrates the MTT assay, real-time PCR for apoptosis genes (Casp3, Bax, and Bcl-2), and advanced analysis, including protein-protein interaction (PPI) networking, clustering, and promoter motif analysis. The analysis indicated that miR-92a-2-5p is a potential therapeutic target for preventing myocardial damage and enhancing biocompatibility. The findings highlight key regulatory pathways that indirectly boost nanodrug biocompatibility through the modulation of secondary components like miRNAs and cellular stress mechanisms.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2027", "doi": "10.1007/978-1-0716-5539-9_28", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41265503": { "pmid": "41265503", "title": "Transcriptomic, artificial neural network and Chou-Talalay analysis based investigation of potential mechanism and active components of Qi-Lin pill in treating diminished ovarian reserve.", "abstract": "Qi-Lin pill (QLP), which is derived from the renowned Wu-Zi-Yan-Zong formulation of the Tang Dynasty, has been used for tonifying the kidneys, replenishing vital essence, and treating infertility and menstrual disorders. However, its mechanisms and active components for treating diminished ovarian reserve (DOR) remain unclear. Herein, we integrated transcriptomics, artificial neural network (ANN), and Chou-Talalay analysis to elucidate the potential mechanisms and active components responsible for the effects of QLP. First, QLP was orally administered to a cisplatin (CDDP)-induced mouse model of DOR; ovarian index, estrous cycle, ovarian tissue morphology, and biochemical parameters were observed to assess efficacy. Second, transcriptomics analysis was performed to reveal the possible mechanisms of QLP, and qRT-PCR and western blotting were used to validate the expressions of key signaling molecules in vivo. Third, the aforementioned mechanisms of QLP were further investigated in CDDP-induced functional impairment of estradiol (E2) synthesis in ovarian granulosa-like KGN cells via using siRNA-mediated depletion and specific inhibition. Subsequently, UPLC-QQQ-MS was used to quantify 33 compounds in 13 batches of QLP. Then, the ANN with feature importance analysis was used to correlate the chemical constituents in the content assay with pharmacological indicators to screen for potential active ingredients. Finally, the synergistic effect of the selected components was investigated using the Chou-Talalay index method in the CDDP-induced functional damage model of human ovarian granulosa-like tumor cells (KGN). QLP restored estrous cyclicity, ovarian index, serum hormones (follicle-stimulating hormone, anti-Müllerian hormone, E2), and follicular development (antral follicles and corpora lutea). Mechanistically, QLP ameliorated CDDP-induced functional damage of the ovaries in vivo and in vitro by upregulating follicle-stimulating hormone receptor (FSHR) expression and activating the ADCY1/cAMP/PKA/CREB signaling pathway, which resulted in upregulated aromatase expression and increased E2 synthesis. Subsequently, ten potentially active compounds, including paeoniflorin, albiflorin, danshensu, gallic acid, betaine, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, salvianolic acid A, hesperidin, nobiletin, and epimedin C, were selected by ANN combined with feature importance analysis. Finally, these ten components were divided into two mixture forms, one of which comprised monoterpenoids (paeoniflorin and albiflorin) in a mixture. Two optimized mixtures synergistically amplified FSHR expression and cAMP/PKA/CREB signaling via Chou-Talalay analysis, thus boosting E2 and aromatase in KGN cells. These findings reveal that QLP could exert its effects through a multicomponent synergistic interaction involving cAMP-mediated steroidogenesis, thus providing new insights into the mechanism by which this traditional Chinese medicine prescription treats ovarian dysfunction.", "journal": "Journal of ethnopharmacology", "year": "2026", "doi": "10.1016/j.jep.2025.120935", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40580290": { "pmid": "40580290", "title": "Probe-Based RT-qPCR Workflow for Dual Interrogation of Virus/Viroid Genotype in Molecular Diagnostics Multiplex.", "abstract": "This chapter presents a protocol for dual interrogation reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) genotyping, a rapid, high-throughput, and economical molecular diagnostic approach for the simultaneous detection of viroid and virus genotypes in plant samples. The method utilizes crude nucleic acid extracts from raw tissue or FTA cards, streamlining sample preparation and enabling field-deployable testing. The incorporation of dual interrogation molecular assays and inhibitor-tolerant polymerases ensures high accuracy and robustness, even with challenging sample types. The protocol details the extraction process and subsequent RT-qPCR detection, facilitating efficient and reliable pathogen screening for plant diagnostics and disease management. In this workflow, we describe an extraction and probe-based RT-qPCR method used to detect viroid and virus genotypes in plant samples extracted from raw tissue or FTA imprinted cards.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2025", "doi": "10.1007/978-1-0716-4642-7_10", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41021005": { "pmid": "41021005", "title": "Nattokinase-driven remodeling of tumor microenvironment enhances the efficacy of MSLN-targeted CAR-T cell therapy in solid tumors.", "abstract": "Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the treatment of hematological malignancies but faces significant challenges in solid tumor therapy. The tumor microenvironment (TME) of solid tumors, including the dense extracellular matrix (ECM) and immunosuppressive factors, creates physical and biochemical barriers that hinder CAR-T cell infiltration, function, and persistence. Despite advances in CAR-T cell engineering, overcoming these TME barriers remains a major obstacle. The ECM in solid tumors restricts CAR-T cell penetration and limits their antitumor efficacy. Recent studies have explored ECM modulation as a strategy to enhance CAR-T therapy. Nattokinase (NKase), a natural fibrinolytic enzyme, has shown potential in degrading ECM components, promoting TME remodeling, and improving therapeutic responses. In this study, we evaluate the therapeutic efficacy of NKase in combination with MSLN-targeted CAR-T cells for solid tumors. We hypothesize that NKase will reduce tumor fibrosis by degrading ECM components, facilitating CAR-T cell infiltration and enhancing their cytotoxic activity. In vitro and in vivo experiments will assess the effects of NKase on CAR-T cell infiltration, activation, memory phenotype maintenance, and antitumor activity. This study aims to optimize CAR-T therapy for solid tumors and supports the clinical potential of NKase as an adjunctive therapeutic agent.", "journal": "Cancer immunology, immunotherapy : CII", "year": "2025", "doi": "10.1007/s00262-025-04167-0", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39848611": { "pmid": "39848611", "title": "Single Nucleotide Polymorphism Highlighted via Heterogeneous Light-Induced Dissipative Structure.", "abstract": "The unique characteristics of biological structures depend on the behavior of DNA sequences confined in a microscale cell under environmental fluctuations and dissipation. Here, we report a prominent difference in fluorescence from dye-modified single-stranded DNA in a light-induced assembly of DNA-functionalized heterogeneous probe particles in a microwell of several microliters in volume. Strong optical forces from the Mie scattering of microparticles accelerated hybridization, and the photothermal effect from the localized surface plasmons in gold nanoparticles enhanced specificity to reduce the fluorescence intensity of dye-modified DNA to a few %, even in a one-base mismatched sequence, enabling us to clearly highlight the single nucleotide polymorphisms in DNA. Fluorescence intensity was positively correlated with complementary DNA concentrations ranging in several tens fg/μL after only 5 min of laser irradiation. Remarkably, a total amount of DNA in an optically assembled structure of heterogeneous probe particles was estimated between 2.36 ymol (2.36 × 10-24 mol) and 2.36 amol (2.36 × 10-18 mol) in the observed concentration range. These findings can promote an innovative production method of nanocomposite structures via biological molecules and biological sensing with simple strategies avoiding genetic amplification in a PCR-free manner.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.4c02119", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "40374565": { "pmid": "40374565", "title": "Development of Zn-Based Tower-like Photoelectrochemical Sensor Array for Sandwich-Type Immunosensing Analysis of Tau.", "abstract": "A novel tower-like photoelectrochemical (PEC) sensor array, incorporating a self-calibration strategy, has been developed. In this study, Zn foil was folded into a quadrangular prism, upon which ZnO nanorods were grown in situ via a hydrothermal process. Subsequently, these ZnO nanorods were sensitized with narrow bandgap CdSe nanospheres. The resultant Zn/ZnO/CdSe have a stepped bandgap structure, effectively minimizing electron-hole pair recombination, enhancing electron transfer, and boosting PEC efficiency. The quadrangular structure is partitioned into four layers by light shields, creating a tower-like electrode equipped with 16 detection points. Furthermore, ZnFe2O4 is used as a detection antibody label for signal amplification. By rotating and moving the tower-like electrode, continuous multiple points detections are achieved, which reduces the operation time and helps to minimize the batch-to-batch variability. Additionally, the integration of a self-calibration strategy allows for selecting a point on the working electrode as a reference for calibration, thereby enhancing detection accuracy and sensitivity. In addition, the PEC immunosensor demonstrates a good linear correlation with microtubule-associated protein tau (Tau) within a measurement range of 0.01-50 ng·mL-1, with a detection limit of 1.1 pg·mL-1. This immunosensor is portable, highly stable, repeatable, and has strong specificity, with good storage stability.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c05455", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40839293": { "pmid": "40839293", "title": "Autonomous microfluidic influenza A/B subtyping system using on-chip multiplex isothermal amplification for field-deployable surveillance.", "abstract": "Influenza viruses present significant challenges to global health. A rapid recombinase polymerase amplification (RPA)-based detection system for multiple influenza strains (A, H1N1/H2N2/H3N2/H5N1/H7N9 and B) has been developed to address the limitations of 2-h Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) tests. By using optimized primers targeting key viral proteins (M, NA, HA, PA), the method achieves detection in 10 min with 0.99 log linearity (1-106 copies/μL). Clinical validation demonstrated 100% sensitivity and specificity, effectively distinguishing infections from healthy controls. This portable platform shows strong potential for point-of-care (POC) applications in resource-limited settings, offering timely diagnosis and epidemic control through its rapid and accurate detection capabilities.", "journal": "AMB Express", "year": "2025", "doi": "10.1186/s13568-025-01933-2", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40927825": { "pmid": "40927825", "title": "Parasagittal dural space and arachnoid granulations morphology in pre-clinical and early clinical multiple sclerosis.", "abstract": "There is limited knowledge on the post-glymphatic structures such as the parasagittal dural (PSD) space and the arachnoid granulations (AGs) in multiple sclerosis (MS). To evaluate differences in volume and macromolecular content of PSD and AG between people with newly diagnosed MS (pwMS), clinically isolated syndrome (pwCIS), or radiologically isolated syndrome (pwRIS) and healthy controls (HCs) and their associations with clinical and radiological disease measures. A total of 69 pwMS, pwCIS, pwRIS, and HCs underwent a 3.0 Tesla magnetic resonance imaging (MRI) including selective inversion recovery quantitative magnetization transfer imaging to compute the macromolecular-to-free water pool-size-ratio (PSR) and the free water relaxation rate (R1f), T2-weighted (T2-w) volumetric isotropic turbo-spin-echo to segment the PSD and AG, T2-w fluid-attenuated inversion recovery images to compute T2-lesions burden, and a T1-w sequence to quantify chronic black holes (cBHs) burden and brain parenchymal fraction. After adjusting for age and sex, larger frontal (p = 0.006) and parietal (p = 0.047) PSD volumes along with larger parietal AG volumes (p = 0.033) were measured in patients compared to HCs. Parietal PSD volume correlated with cBH PSR (r = -0.430, p = 0.032). Upon diagnosis, enlarged structures in the post-glymphatic system may be observed, indicating a need for further investigation of their role.", "journal": "Multiple sclerosis (Houndmills, Basingstoke, England)", "year": "2025", "doi": "10.1177/13524585251364720", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40538744": { "pmid": "40538744", "title": "Graphene oxide-carbon nanotube-magnetite nanocomposites for efficient arsenic removal from aqueous solutions.", "abstract": "In this study, graphene oxide-carbon nanotube-magnetite (GO/CNT/Fe3O4) nanocomposites were synthesized via a co-precipitation method for wastewater treatment. The obtained results indicated that Fe3O4 nanoparticles exhibited a spherical-shaped morphology, with an average diameter of around 20 nm, and decorated the surface of GO and CNTs. The adsorption data fitted well with the Langmuir isothermal model, exhibiting a coefficient of R 2 > 0.998, and the adsorption kinetics followed the pseudo-second-order model. This indicated that the adsorption mechanism involved surface complexation between adsorbents and As(iii) ions rather than electrostatic interactions. The As(iii) removal results also indicated that the GO/CNT/Fe3O4 nanocomposites exhibited a significantly enhanced adsorption capacity (q max) of 128.5 mg g-1 compared with those of CNT/Fe3O4 (106.3 mg g-1) and GO/Fe3O4 (113.9 mg g-1) composites. In addition, GO/CNT/Fe3O4 nanocomposites exhibited the highest adsorption efficiency of up to 99.18%. The coexisting ions, such as phosphate and sulfate, showed a negligible influence on the adsorption of As(iii) in solutions. The obtained results demonstrated that the GO/CNT/Fe3O4 nanocomposites could be promising candidates for the removal of arsenic and other contaminants from aqueous solutions.", "journal": "RSC advances", "year": "2025", "doi": "10.1039/d5ra02925b", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41738053": { "pmid": "41738053", "title": "Integrated physiological, biochemical, and molecular analysis of drought tolerance in soybean cultivars.", "abstract": "Drought stress is an important constraint to soybean (Glycine max L. Merr.) production, specifically in arid and semi-arid regions. This study integrated physiological, biochemical, and molecular approaches to elucidate the adaptive mechanisms of soybean genotypes against drought stress. Seven genotypes, four tolerant, 'Ajmeri', 'NARC-21', 'DMX4561' and 'Rawal', and three susceptible 'Anjasmoro', 'Grobogan', 'Dering-1' were evaluated under control and drought conditions in a pot experiment conducted under controlled conditions. Physiological traits, including relative water content (RWC), chlorophyll content, photosynthetic rate (Pn), and cell membrane stability (CMS), were quantified alongside biochemical indicators such as proline, glycine betaine (GB), and antioxidant enzyme activities. The relative expression of drought-responsive genes, GmDREB2, GmLEA-D11, GmP5CS, GmBADH2, GmSOD1, GmCAT1, GmPOD1, GmPIP2;9, GmCHLG was conducted via qRT-PCR. Results indicated that tolerant genotypes kept higher RWC (>80%), chlorophyll (≥1.73 g g-1 FW), Pn (≥30 μmol m-2 s-1), and CMS (>70%) under drought. They also accumulated more proline (up to 47.5 µg g-1 FW) and GB (up to 157 µg g-1 FW). The activities of antioxidant enzymes, in tolerant genotypes were markedly higher (CAT up to 15.3 U mg-1 protein; SOD >50 U mg-1 protein) than in susceptible genotypes. Besides, the multivariate analyses (correlation, PCA, hierarchical clustering) grouped tolerant genotypes to osmolytes (proline, GB), antioxidant enzymes, and physiological traits, proving strong drought trait association. The expression analysis showed high upregulation of stress-related genes (e.g., GmP5CS ~3.8-fold; GmBADH2 ~3.4-fold; GmSOD1 ~3.5-fold) in tolerant genotypes, rectified the physiochemical findings. Overall, these results proved that drought tolerance in soybean is regulated by the co-ordination of osmolytic adjustment, enhanced antioxidant activities, maintenance of photosynthetic traits, and transcriptional activation of stress-responsive genes. The identified tolerant genotypes will serve as promising breeding resources for the development of stress-tolerant soybean cultivars.", "journal": "Frontiers in plant science", "year": "2026", "doi": "10.3389/fpls.2026.1730570", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41345818": { "pmid": "41345818", "title": "Redox Modulation of Erythropoiesis by Selenoproteins.", "abstract": "Erythroblasts are constantly challenged by oxidative stress arising from the accumulation of free heme and reactive radicals, particularly during stress erythropoiesis, which is triggered by inflammatory signals. In response to infection or tissue damage, stress erythropoiesis serves as a compensatory mechanism to overcome inhibition of steady-state erythropoiesis by pro-inflammatory cytokines to sustain abundant red blood cell (RBC) production. Maturation of erythroblasts during stress erythropoiesis critically depends on the erythroblastic island (EBI) microenvironment, composed of a central macrophage and surrounding immature progenitors. Within this niche, the dynamic balance between inflammatory and resolution pathways is essential for proper erythroblast development and adaptation under stress. Selenium (Se), an essential trace element, incorporated into selenoproteins via the 21st amino acid selenocysteine, imparts antioxidant and redox-regulatory activity, which govern erythroblast-intrinsic signaling as well as microenvironmental regulation. Depletion of the selenoproteome or selenium in the diet results in mild anemia and impaired stress erythropoiesis. However, the contributions of individual selenoproteins to erythroblast development is not well understood. Given the importance of selenoproteins in influencing macrophage polarization, its contribution to the regulation of the erythropoietic microenvironment deserves further study. In this review, we highlight the role of selenium and selenoproteins in stress erythropoiesis, emphasizing their functions in supporting erythroblast development and modulating the erythropoietic niche.", "journal": "Biological trace element research", "year": "2026", "doi": "10.1007/s12011-025-04923-4", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41933161": { "pmid": "41933161", "title": "Bacterial species differentiation via real-time detection of microbial volatile organic compounds using a wavelength multiplexed photoionization detector and AI image-based analysis.", "abstract": "Healthcare-associated infections (HCAIs) contribute significantly to global mortality, driven by the increasing antimicrobial resistance. Rapid, high-throughput bacterial detection is crucial for infection control and patient care. We report a real-time, multiplex lamp-based Photoionization Detector (PID) assisted by AI-image-based analysis for bacterial identification. Using four lamps with varying ionization energies, the sensor selectively ionizes VOCs emitted by bacteria, producing four distinct current curves for each target species (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). These curves were transformed into image representations, capturing their spectral patterns for bacterial differentiation. A pre-trained ResNet-18 Convolutional Neural Network (CNN) within a Few-Shot Learning (FSL) framework extracted key features, enabling accurate (> 88%) bacterial differentiation even with limited labeled data. This sensor detected bacterial concentrations as low as 10² CFU and distinguished contamination levels. The synergistic integration of PID sensing with AI-driven analysis offers a powerful approach to rapid bacterial diagnostics, demonstrating strong potential for clinical implementation and improved patient care. This study marks an early step toward AI-based VOC sensing, where FSL acts as a proof-of-concept under data scarcity.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-46818-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42282845": { "pmid": "42282845", "title": "Heme orchestrates a tissue stress response to proteolytic damage.", "abstract": "Whereas cellular stress responses are well defined, tissue-level stress remains poorly understood. Proteases are among the most widespread enzymes, and excessive proteolytic activity drives diseases such as arthritis and chronic obstructive pulmonary disease, yet unifying features of this stress are unclear. Here, using the lung and diverse proteases, we identify a conserved injury signature of proteolytic stress marked by vascular disruption, red blood cell extravasation, and heme release that triggers oxidative stress. We show that alveolar macrophages act as primary sensors of this stress response, activating NRF2-dependent heme detoxification program and fibroblasts produce protease inhibitors to limit damage. Repeated exposure to proteolytic stress induces tissue adaptation and protects against subsequent injury and infection. These findings define a unifying framework for tissue-level proteolytic stress sensing and adaptation.", "journal": "bioRxiv : the preprint server for biology", "year": "2026", "doi": "10.64898/2026.05.31.729126", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "B" ] }, "39506098": { "pmid": "39506098", "title": "Human immune organoids to decode B cell response in healthy donors and patients with lymphoma.", "abstract": "Antibodies are produced when naive B cells differentiate into plasma cells within germinal centres (GCs) of lymphoid tissues. Patients with B cell lymphoma on effective immunotherapies exhibit diminished antibody production, leading to higher infection rates and reduced vaccine efficacy, even after B cell recovery. Current ex vivo models fail to sustain long-term GC reactions and effectively test B cell responses. Here we developed synthetic hydrogels mimicking the lymphoid tissue microenvironment, enabling human GCs from tonsils and peripheral blood mononuclear cell-derived B cells. Immune organoids derived from peripheral blood mononuclear cells maintain GC B cells and plasma cells longer than tonsil-derived ones and exhibit unique B cell programming, including GC compartments, somatic hypermutation, immunoglobulin class switching and B cell clones. Chemical inhibition of transcriptional and epigenetic processes enhances plasma cell formation. While integrating polarized CXCL12 protein in a lymphoid organ-on-chip modulates GC responses in healthy donor B cells, it fails with B cells derived from patients with lymphoma. Our system allows rapid, controlled modelling of immune responses and B cell disorders.", "journal": "Nature materials", "year": "2025", "doi": "10.1038/s41563-024-02037-1", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "40460301": { "pmid": "40460301", "title": "Phytic Acid-Polypeptide Network-Promoted Deposition of Photoactive Agents for the Construction of Synergistic Bactericidal Coatings.", "abstract": "Constructing antimicrobial surface coatings is essential to protect biomedical implants and devices from biological contamination or microbial infections. In this study, a simple technique was developed to prepare phytic acid (PA) and ε-polylysine (Ply)/hyperbranched polylysine (HPL) network coatings on implant surfaces due to the intrinsic surface affinity of PA and electrostatic attraction between Ply/HPL and PA. The PA-Ply (or PA-HPL) coating facilitates the encapsulation of diverse photoactive agents, including gold nanoparticles (Au NPs), multiwalled carbon nanotubes (MWNTs), graphene oxide (GO), indocyanine green (ICG), and chlorophyll copper sodium salt (SCC) onto substrate surfaces in a matrix-independent manner. The physicochemical characterization confirms that PA-Ply network coatings accommodate high quantities of photoactive components. Although multiple substrates and photoactive agents are initially explored to compare coating formation and loading capacity, the primary focus of this study is the in vitro and in vivo antibacterial performance of SCC- and ICG-functionalized coatings. In vitro studies demonstrate that PA-Ply exerts more detrimental effects on bacterial pathogens compared to PA-HPL network coatings. Additionally, PA-Ply (or HPL)-SCC generates singlet oxygen (1O2) under xenon lamp irradiation, while the PA-Ply-SSC-modified titanium (Ti) surface achieves enhanced bacterial inactivation through a synergistic mechanism of contact killing and photodynamic action. Similarly, the PA-Ply (or HPL)-ICG-modified implants exhibit favorable biocompatibility and synergistic antibacterial effects in vivo under near-infrared (NIR) irradiation. The findings present a feasible strategy for surface modification, endowing medical implants with enhanced antibacterial protection.", "journal": "ACS applied materials & interfaces", "year": "2025", "doi": "10.1021/acsami.5c09482", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41906410": { "pmid": "41906410", "title": "UGT1A1 Fragment Analysis: Genotyping the (TA)n Variable Repeat Polymorphism for Clinical Applications.", "abstract": "UGT1A1 is involved in glucuronidation of drugs and plays a large role in metabolism. Genotyping variants of differing dinucleotide TA repeat length within the TATA box of the UGT1A1 region (dbSNP ID rs3064744) are utilized in the dosing of irinotecan and atazanavir for pharmacogenomics purposes. Here we describe a CLIA-validated PCR and fragment analysis assay that distinguishes between UGT1A1*36 (TA)5, UGT1A1*1 (TA)6, UGT1A1*28 (TA)7, and UGT1A1*37 (TA)8, as well as potential heterozygous combinations. This development offers a more determinant genotype than assays like PharmacoScan can accurately establish alone. PCR was performed using 2X Platinum SuperFi PCR Master Mix, 5X SuperFi GC Enhancer, a FAM-labeled forward primer, reverse primer, and 0.5 ng/μL-20 ng/μL DNA. MiniGene plasmids for each variant served as controls. QC was performed using the Agilent Bioanalyzer 2100 or TapeStation 4200; fragment analysis was performed with HiDi Formamide and LIZ 500 size standard and processed via capillary electrophoresis. Data were analyzed using the GeneMapper version 5 software and peaks with the largest area under the curve (AUC) were identified and compared with known sizes of controls. Per CLIA regulations, the assay passed validation with 100% concordance for all parameters: accuracy, precision, reportable range, reference range, analytical specificity, and analytical sensitivity. Following validation, clinical assay implementation for 940 samples run on PharmacoScan showed disagreement for 74 samples at UGT1A1 rs3064744. Clinical implementation as a supplemental assay to other pharmacogenomics offerings resulted in a correction rate of the loci within patient samples of 7.8%, with 2.77% caused by miscalling UGT1A1*37 variant.", "journal": "Journal of clinical laboratory analysis", "year": "2026", "doi": "10.1002/jcla.70214", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41431195": { "pmid": "41431195", "title": "MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.", "abstract": "Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2026", "doi": "10.1002/advs.202517799", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "39907539": { "pmid": "39907539", "title": "Selective endothelin A receptor antagonism in chronic kidney disease: improving clinical application.", "abstract": "Endothelin-1 (ET-1) is a peptide that is involved in various chronic diseases including cardiovascular and kidney disease. ET-1 can bind to two receptors, endothelin A (ETA) and endothelin B (ETB), which are found in different organs and tissues. When ET-1 binds to the ETA receptor, it causes blood vessels to narrow, while binding of ET-1 to the ETB receptor causes blood vessels to widen. These receptors help regulate fluid and electrolyte balance in the kidneys, as well as the kidney's ability to filter various substances out of the body. Overactivation of ET-1 can occur in people with diabetes or obesity, which can damage the structure and function of the kidney.Studies in mice and humans with kidney disease have shown that blocking the ETA receptor improves kidney health. As a result, medicines that specifically block the ETA receptor, known as endothelin receptor antagonists (ERAs), are a promising option for treating these kidney diseases. The first ERA (sparsentan) is now available for use in patients with immunoglobulin A (IgA) nephropathy, a specific type of kidney disease. It should be noted that ERAs can cause side effects. Fluid retention, which can increase the risk of heart failure, is a side effect that is particularly observed in patients with type 2 diabetes and severe kidney disease. This side effect is less often observed in patients with IgA nephropathy or patients without diabetes.Treatment strategies to optimize safe and effective use of ETA blockers are being developed. Overall, these insights offer hope for better care of patients with kidney disease. Endothelin-1 (ET-1) is a 21-amino acid peptide involved in numerous cardiovascular and renal processes. ET-1 can bind to endothelin receptor A (ETA) and endothelin receptor B (ETB), which are found in various organs and tissues. In general, binding of ET-1 to the ETA receptor causes vasoconstriction, whereas activation of the ETB receptor leads to vasodilation. In the kidney, endothelin receptors regulate fluid and electrolyte balance, regional blood flow and glomerular filtration rate. In pathological conditions, ET-1 promotes kidney injury through adverse effects on the endothelial glycocalyx, podocytes and mesangial cells, and stimulating inflammation and fibrosis in the tubules. In experimental and clinical studies, inhibition of the ETA receptor has been shown beneficial in a variety of kidney diseases. These include diabetic kidney disease, immunoglobulin A nephropathy, focal segmental glomerulosclerosis and Alport syndrome. Accordingly, selective ETA endothelin receptor antagonists (ERA) may prove a viable therapeutic option in these diseases. However, clinical application is challenged by the occurrence of fluid retention which can lead to heart failure, in particular in patients with severe CKD. Concomitant use of sodium-glucose cotransporter 2 inhibitors (SGLT2i) may mitigate these adverse effects through their diuretic actions. The development of highly selective ETA antagonists, such as atrasentan and zibotentan, and the opportunities of combining these with SGLT2i, holds promise to optimize efficacy and safety of ERAs in clinical practice.", "journal": "Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association", "year": "2025", "doi": "10.1093/ndt/gfae214", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "42448178": { "pmid": "42448178", "title": "Enhancing lyophilized cake morphology in biopharmaceutical products through stepped annealing at freezing phase.", "abstract": "Lyophilization is widely used to stabilise biopharmaceuticals, yet split‑cake formation remains a challenge. Cake elegance, a critical quality attribute (CQA), is influenced by both formulation and process parameters. As biosimilar manufacturers are constrained from altering formulation composition, optimisation of process parameters becomes the primary strategy. We hypothesised that precooling combined with two‑step annealing during freezing would facilitate freeze‑concentrate migration into microchannels, yielding homogeneous ice crystallisation and elegant cakes. rt‑PA protein was produced using CHO cells and the protein formulation contained L-Arginine and Polysorbate 20. Critical temperatures were determined using Freeze‑Drying Microscopy and Integrated Impedance‑DTA. The formulation was filled into 20 mL Lyo glass vials and lyophilization parameters were optimized to obtain an elegant cake of the rt-PA protein. Protein aggregation and potency were assessed by size‑exclusion chromatography and biological assays respectively. Replacing traditional one‑step annealing at -10 °C with precooling at -8 °C combined with two‑step annealing at -15 °C and -10 °C promoted uniform ice crystallisation, and decreased split‑cake formation. As expected, the residual moisture decreased, with a slight increase in reconstitution time for elegant cake. Robustness of the optimized method was confirmed by varying annealing conditions (±2 °C). Optimisation of freezing‑phase parameters significantly influenced cake morphology, producing elegant lyophilized matrices with desirable quality attributes. Such physical appearance is considered a CQA and is highly valued by regulatory agencies, as it reflects appropriate process control and product quality in biopharmaceutical products. This study thus establishes a process development framework that can be extended to other molecules to confirm broader applicability.", "journal": "Journal of pharmaceutical sciences", "year": "2026", "doi": "10.1016/j.xphs.2026.104416", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41477126": { "pmid": "41477126", "title": "Protective Effects of Berberine-Loaded Black Seed (Nigella sativa L.) Oil Nanoemulsion in the Atherosclerotic Mouse Model.", "abstract": "This study investigated atherosclerosis (AS), a pivotal contributor to the onset of coronary artery disease and other cardiovascular ailments.‎. The presents study examined a novel therapeutic strategy combining black seed oil [Nigella sativa L. (NS)] and berberine (BBR) in a C57BL/6J mouse model of AS. After preparing the NS nanoemulsion, gas chromatography-mass spectrometry (GC-MS) was used for analysis. A BBR-loaded black seed oil-based nanoemulsion (BBS) was developed and evaluated for its thermodynamic stability, viscosity, particle size, and dynamic light scattering (DLS) properties. Forty-eight male C57BL/6J mice (8 weeks old, 18 - 20 g) were divided into six groups and fed a modified AIN-76 diet with 25% dietary fat for 16 weeks to induce AS. Treatment began after 8 weeks through oral gavage for the remaining 8 weeks. Levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), and total cholesterol were measured, along with the atherogenic coefficient and cardiac risk ratio. We also quantified malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities, and assessed pathological changes in cardiac and aortic tissues using hematoxylin and eosin (H&E) staining. The GC-MS analysis confirmed that NS oil met quality benchmarks, displaying stability with a zeta potential of -18.4 mV. The study showed a Polydispersity Index (PDI) of 0.3019 and a Z-average of 250.4 nm. The BBS improved lipid profiles in mice, increasing HDL and decreasing LDL, TG, and total cholesterol, thereby reducing cardiac risk. The formulation exhibited strong antioxidant effects, with reduced MDA levels and enhanced SOD, GPX, and CAT activities. Pathological observations supported these biochemical results. Our findings suggest that BBS offers valuable insights into the mechanisms underlying atherosclerotic disease. This understanding could pave the way for novel approaches to cardiovascular health and the development of effective preventive strategies.", "journal": "Iranian journal of pharmaceutical research : IJPR", "year": "2025", "doi": "10.5812/ijpr-162867", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42834897": { "pmid": "42834897", "title": "Recent advances in AI-driven deep eutectic solvent design for biocatalysis: Co-design of extremozyme and reaction medium.", "abstract": "Deep eutectic solvents (DES) have emerged as sustainable, highly tunable alternatives to conventional organic media for non-aqueous biocatalysis. DES dynamically interact with enzymes to shift reaction equilibria, enhance thermostability and tailor stereoselectivity. However, the vast combinatorial space and complex physicochemical properties of DES make traditional trial-and-error solvent engineering highly inefficient. This review systematically examines recent advancements in DES-driven biocatalysis and highlights the role of artificial intelligence (AI) for rational DES design. Specifically, it synthesizes evidence that DES composition and water content jointly shape enzyme-specific microenvironments and thereby affect catalytic performance. Extremozymes are also evaluated as candidate catalysts for DES-containing media and as sources of potentially transferable adaptation features for enzyme engineering. It then discusses how AI can support DES formation screening, prediction of physicochemical properties using quantitative structure-property relationship (QSPR) models, and candidate formulation generation at the solvent level. At the system level, the focus shifts to predicting how a specific enzyme responds to a defined formulation in terms of activity and stability. Finally, we outline a mechanistically informed, multi-objective framework that integrates DES composition, enzyme sequence and structure, and operating conditions with physicochemical calculations, AI-based prediction, and experimental validation, providing a practical route toward genuine DES-enzyme co-design.", "journal": "Biodesign research", "year": "2026", "doi": "10.1016/j.bidere.2026.100109", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "42556675": { "pmid": "42556675", "title": "Gradient-channel quaternized chitosan/corncob-based aerogel membranes for efficient oil-water separation and dye removal.", "abstract": "Oil-containing wastewater, particularly surfactant-stabilized oil-in-water emulsions, remains extremely challenging to treat due to its high stability and complex composition. In this study, a gradient porous aerogel membrane was prepared using quaternized chitosan and corn cobs via directed freezing and freeze-drying techniques. Quaternized chitosan endows the membrane with inherent hydrophilicity and a positively charged surface, while the incorporation of corn cobs enhances mechanical strength and improves dye removal capacity. The gradient pore structure further generates a capillary-assisted transport effect, thereby enhancing separation performance. This combination of structure and function confers the membrane with underwater superoleophobicity and electrostatic interaction capabilities, enabling efficient oil-water separation and dye adsorption. The membrane exhibited the most outstanding separation performance in both immiscible oil-in-water mixtures (11,190 L m-2 h-1, 99%) and oil-in-water emulsions prepared with anionic surfactants (1200 L m-2 h-1, 97%), while effectively removing various dyes and demonstrating good cycling stability. This study proposes a bio-based strategy that integrates structural regulation with interfacial functionality, providing a highly promising platform for multifunctional wastewater treatment.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.153854", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42283702": { "pmid": "42283702", "title": "A Nanoconfined, Hydrogel-Partitioned LAMP Platform for Rapid, Extraction-Free Quantification of Mycoplasma pneumoniae.", "abstract": "Accurate quantification of Mycoplasma pneumoniae (M. pneumoniae) is important because pathogen load is associated with disease severity and treatment outcomes. However, quantitative polymerase chain reaction (qPCR) requires DNA extraction and standard-curve calibration, whereas digital polymerase chain reaction (dPCR) remains costly and less accessible. Here, we developed gLAMP-X, an extraction-free hydrogel-limited amplification (gLAMP) platform for rapid quantification of M. pneumoniae from clinical throat swabs. The \"X\" component denotes extraction-free lysis that disrupts the lipid bilayer of M. pneumoniae while preserving nucleic acid integrity. In gLAMP-X, a polyethylene glycol (PEG)-based nanoporous hydrogel partitions loop-mediated isothermal amplification (LAMP) into isolated nanoscopic microreactors, converting homogeneous amplification into a digital-like system in which each event forms a discrete fluorescent cluster for end point counting. Under optimized conditions, gLAMP-X achieved a limit of detection of 1.7 copies/μL within 20 min. In 90 pediatric throat swabs, gLAMP-X showed 100% sensitivity, 98.1% specificity, and strong quantitative agreement with qPCR.", "journal": "ACS infectious diseases", "year": "2026", "doi": "10.1021/acsinfecdis.6c00203", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41299842": { "pmid": "41299842", "title": "One-Pot CRISPR-Based Isothermal Amplification for Nucleic Acid Detection: A Comparative Review of Different Strategies.", "abstract": "Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.", "journal": "ACS sensors", "year": "2025", "doi": "10.1021/acssensors.5c00806", "ptypes": [ "Journal Article", "Review", "Comparative Study" ], "first_author": "", "dirs": [ "C" ] }, "40512366": { "pmid": "40512366", "title": "Mechanism of Sangqi Qingxuan Liquid in Alleviating Vascular Endothelial Injury in Hypertension Focuses on β-Catenin.", "abstract": "To explore the main components and potential mechanisms of Sangqi Qingxuan Liquid in the treatment of arterial vascular endothelial cells (AVECs) injury in hypertension through network pharmacology. Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID) were used to screen the active components of Sangqi Qingxuan Liquid (SQQX), which met the oral utilization rate and drug similarity criteria. An active component-target network was constructed using Cytoscape 3.6 software. A protein-protein interaction (PPI) network of targets associated with SQQX treatment for hypertension was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The Metascape database was used to perform enrichment analysis of gene ontology biological functions and MSigDB pathway enrichment analysis of proteins in the PPI network. Further analysis of the main components of SQQX was performed using UPLC-MS. Based on the results of network pharmacology, the mechanism of SQQX to improve the injury of AVECs in hypertension was verified through lentiviral transfection by Wnt/ β -catenin signaling pathway. AVECs induced by angiotensin II (Ang II ) was used to establish a model of endothelial function injury in hypertension. Cell viability, intracellular nitric oxide content, malonaldehyde content, and superoxide dismutase activity were measured to determine the optimal induction conditions. The optimal intervention conditions for SQQX were determined based on cell viability, cellular DNA activity, and the gradient method. The cells were further divided into blank, model, overexpression lentivirus negative control, overexpression lentivirus, overexpression lentivirus + SQQX intervention (2.47 mg/mL, 12 h), inhibition lentivirus negative control, inhibition lentivirus, and inhibition lentivirus + SQQX intervention (2.47 mg/mL, 12 h) groups. Finally, quantitative real-time PCR and Western blotting were performed to analyze the molecular mechanisms of SQQX in the Wnt/ β -catenin signaling pathway. The main SQQX components were betaine, buddleoside, and chlorogenic acid, in descending order. Network pharmacology analysis screened 12 pathways associated with the hypertensive vascular endothelium. The results showed that 1 µ mol/L for 12 h was the optimal condition for Ang II to induce AVECs injury, and 2.47 mg/mL SQQX intervention for 12 h was the optimal condition for treating AVECs injury. In the experimental validation based on the interaction network of the Wnt/ β -catenin signaling pathway, SQQX significantly decreased the expressions of β -catenin, Smad2, peroxisome proliferator-activated receptors (PPARs), endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) caused by the β -catenin overexpression lentivirus (P<0.05 or P<0.01). The function of vascular endothelial cells can be improved by the β -catenin inhibition lentivirus, and no obvious changes were observed after further intervention with SQQX. SQQX may protect against AVECs injury by regulating the Wnt/β -catenin signaling pathway.", "journal": "Chinese journal of integrative medicine", "year": "2025", "doi": "10.1007/s11655-025-4013-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39561905": { "pmid": "39561905", "title": "Exploring the impact of material selection on the efficacy of hot-melt extrusion.", "abstract": "Hot-melt extrusion (HME) has emerged as a versatile and efficient technique in pharmaceutical formulation development, particularly for enhancing the solubility and bioavailability of poorly water-soluble drugs. This review delves into the fundamental principles of HME, exploring its application in drug delivery systems. A comprehensive analysis of polymers utilized in HME, such as hydroxypropyl methylcellulose, ethyl cellulose, hydroxypropyl cellulose, and polyvinylpyrrolidone, is presented, highlighting their roles in achieving controlled drug release and improved stability. The incorporation of plasticizers, such as triacetin, poly(propylene glycol), glycerol, and sorbitol, is critical in reducing the glass transition temperature (Tg) of polymer blends, thereby enhancing the processability of HME formulations. A comparison of Tg values for various polymer-plasticizer combinations is discussed using different predictive models. For researchers and industry professionals looking to optimize drug formulation strategies, this article offers valuable insights into the mechanisms through which HME enhances drug solubility and bioavailability two critical factors in oral drug delivery. Furthermore, by reviewing recent patents and marketed formulations, the article serves as a comprehensive resource for understanding both the technical advancements and commercial applications of HME. Readers will gain a deep understanding of the role of polymers and additives in HME, alongside future perspectives on how emerging materials and techniques could further revolutionize pharmaceutical development. This review is essential for those aiming to stay at the forefront of pharmaceutical extrusion technologies and their potential to improve therapeutic outcomes. The review concludes that meticulous material selection is vital for advancing pharmaceutical manufacturing processes and ensuring optimal outcomes in HME applications, thereby enhancing the overall efficacy of drug delivery systems.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2024.124966", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40178804": { "pmid": "40178804", "title": "GalNAc-functionalized metal-organic frameworks for targeted siRNA delivery: enhancing survivin silencing in hepatocellular carcinoma.", "abstract": "Small interfering RNA (siRNA) is a potent method for silencing survivin mRNA within cells, offering a promising option for treating hepatocellular carcinoma (HCC) since survivin is specifically overexpressed in HCC cells. However, the clinical use of gene therapy with siRNA is limited by factors such as rapid enzyme degradation, low cell uptake, and non-specific distribution in the body. In this study, we investigate the use of a specially selected metal-organic framework (MOF) to encapsulate siRNA, with the aim of silencing survivin mRNA in HCC cells and reducing the survivin protein level. The MOF was functionalized with triantennary N-acetylgalactosamine (GalNAc), which has high affinity for asialoglycoprotein receptors that are overexpressed in HCC cells. Both in vitro and in vivo experiments showed that the GalNAc-decorated MOF specifically accumulated in HCC tumor tissue and was effectively endocytosed by HCC cells. The protective properties of the MOF increased the stability of siRNA and allowed for significant downregulation of survivin expression in HCC tumors, contributing to tumor inhibition through the suppression of cell proliferation and the induction of apoptosis. These findings highlight the potential of MOF-based siRNA delivery systems for targeted cancer therapy.", "journal": "Biomaterials science", "year": "2025", "doi": "10.1039/d5bm00363f", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39936600": { "pmid": "39936600", "title": "Buddleoside alleviates nonalcoholic steatohepatitis by targeting the AMPK-TFEB signaling pathway.", "abstract": "Nonalcoholic steatohepatitis (NASH) is a combination of hepatic steatosis, inflammation, and fibrosis, and it often follows simple hepatic steatosis in nonalcoholic fatty liver disease (NAFLD). However, no pharmacological treatment is currently available for NASH. Given the important role of TFEB (transcription factor EB) in regulating the macroautophagy/autophagy-lysosomal pathway, TFEB is potentially a novel therapeutic target for treatment of NASH, which function can be regulated by AMP-activated protein kinase (AMPK) and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1). Buddleoside (Bud), a natural flavonoid compound, has recently emerged as a promising drug candidate for liver diseases. Here, we shown that Bud treatment alleviated hepatic steatosis, insulin resistance, inflammation, and fibrosis in mice fed a high-fat and high-cholesterol (HFHC) diet. Notably, Bud activated AMPK, inhibited MTORC1, and enhanced TFEB transcriptional activity as well as autophagic flux in vivo and in vitro. Inhibition of AMPK or knockout of hepatic Tfeb abrogated the alleviation effects of Bud on hepatic steatosis, insulin resistance, inflammation, and fibrosis. Mechanistic investigation revealed that Bud bound to the PRKAB1 subunit via Val81, Arg83, and Ser108 residues and activated AMPK, thereby eliciting phosphorylation of RPTOR (regulatory associated protein of MTOR complex 1) and inhibiting the kinase MTORC1, which activated the TFEB-mediated autophagy-lysosomal pathway and further ameliorated HFHC-induced NASH in mice. Altogether, our results indicate that Bud ameliorates NASH by activating hepatic the AMPK-TFEB axis, suggesting that Bud is a potential therapeutic strategy for NASH.Abbreviations: ACAC, acetyl-CoA carboxylase; ADaM, allosteric drug and metabolite; AICAR, 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside; AKT, AKT serine/threonine kinase; ALP, autophagy-lysosomal pathway; AMPK, AMP-activated protein kinase; Bud, buddleoside; CAMKK2, calcium/calmodulin dependent protein kinase kinase 2; CC, compound C; CETSA, cellular thermal shift assay; Cmax, maximum concentration; CQ, chloroquine; DARTS, drug affinity responsive target stability assay; EIF4EBP1, eukaryotic translation factor 4E binding protein 1; GOT1, glutamic-oxaloacetic transaminase 1; GPT, glutamic-pyruvic transaminase; GSK3B, glycogen synthase kinase 3 beta; GTT, glucose-tolerance test; HFD, high fat diet; HFHC, high-fat and high-cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; IKBKB, inhibitor of nuclear factor kappa B kinase subunit beta; INSR, insulin receptor; ITT, insulin-tolerance test; LDH, lactate dehydrogenase; STK11, serine/threonine kinase 11; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MTORC1, MTOR complex 1; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; ND, normal diet; NFKB, nuclear factor kappa B; PA, palmitic acid; PSR, picrosirius red; RRAG, Ras related GTP binding; RPTOR, regulatory associated protein of MTOR complex 1; RPS6, ribosomal protein S6; RPS6KB, ribosomal protein S6 kinase B; SMAD2, SMAD family member 2; SMAD3, SMAD family member 3; SQSTM1, sequestosome 1; TFEB, transcription factor EB; tfeb-HKO, hepatocyte-specific tfeb knockout; TSC2, TSC complex subunit 2.", "journal": "Autophagy", "year": "2025", "doi": "10.1080/15548627.2025.2466145", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "40917009": { "pmid": "40917009", "title": "Rolling circle amplification for next-generation molecular diagnostics, genome analysis, and spatial transcriptome profiling.", "abstract": "Rolling circle amplification (RCA) has emerged as a highly versatile and robust isothermal amplification technology, offering exceptional sensitivity, specificity, and scalability for next-generation molecular diagnostics and multi-omics research. Its ability to generate long, repetitive DNA sequences with high fidelity has made it a pivotal tool in disease diagnostics, genomic analysis, and spatial transcriptome profiling. Recent advancements have expanded RCA into various formats, including solution-phase, solid-phase, hydrogel-based, and digital RCA, enhancing its analytical performance and adaptability across diverse biological applications. The integration of RCA with CRISPR-based detection, biosensors, and nanoparticle-assisted signal amplification has significantly improved its diagnostic potential, enabling ultrasensitive detection of nucleic acids, proteins, extracellular vesicles, and single cells. In addition to diagnostics, RCA has profoundly impacted genomic research, particularly in extrachromosomal circular DNA sequencing, offering insights into genome instability and cancer evolution. In spatial transcriptomics, RCA-based in situ RNA sequencing and hybridization have enabled high-resolution gene expression mapping in tissue architecture, providing invaluable insights into cellular heterogeneity and disease microenvironments. This review comprehensively explores the fundamental principles, design modifications, and advanced signal amplification strategies of RCA, with a focus on its applications in molecular diagnostics, genome analysis, and spatial transcriptome profiling. Additionally, it addresses key challenges, including reaction specificity, automation, and cost-effectiveness, and discusses future directions for integrating RCA into high-throughput molecular diagnostics, biomarker discovery, and single-cell and spatial multi-omics platforms. Through continuous innovation, RCA is poised to play a transformative role in precision medicine, next-generation sequencing, and biomedical research.", "journal": "Nanoscale", "year": "2025", "doi": "10.1039/d5nr03032c", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "41286502": { "pmid": "41286502", "title": "Noncanonical amino acid-aided synthesis of anti-PD-L1 bispecific nanobody for colon cancer immunotherapy.", "abstract": "Colorectal cancer (CRC) is the second leading cause of cancer-related mortality, and the incidence of early-onset colon cancer has been increasing globally in recent years. The development of immunotherapies for colon cancer is critical for providing new treatment strategies to combat drug resistance. Here, a bispecific nanobody against PD-L1 (BsNb-PD-L1) constructed using genetically encoded noncanonical amino acids (ncAAs) is reported. A computational protocol was developed to identify appropriate sites in the nanobody for incorporating p-acetylphenylalanine (pAcF). Variants of nanobodies PV2 and PV3 with pAcF incorporated were conjugated with linkers containing an aminooxy functionality to enable oxime ligation. The resulting PV2-S71 + PV3-N77 bispecific nanobody (BsNb-ncAA) exhibited higher thermostability and binding affinity compared to the nanobody monomers and the BsNb constructed by simply fusing two proteins. Moreover, in an in vitro phagocytosis model, the BsNb-ncAA exhibited improved capability to inhibit immune evasion and showed stronger biological activity compared to the fusion protein PV2-PV3. Furthermore, the BsNb-ncAA resulted in a marked increase in the number of CD8+ T cells within tumor tissues and demonstrated efficient inhibitory effects against colon tumor growth in vivo. Our study provides a general strategy for constructing BsNbs, which has potential applications in other cancer immunotherapy.", "journal": "Communications biology", "year": "2025", "doi": "10.1038/s42003-025-09222-1", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40328992": { "pmid": "40328992", "title": "Design, Development, and Characterization of High Drug-Loaded Drug-Drug-Polymer Ternary Amorphous Solid Dispersions.", "abstract": "Ternary amorphous solid dispersions (TASD) are a three-component system that incorporates at least one drug in an amorphous form, offering potential advantages over conventional binary amorphous solid dispersions (ASD). This study aimed to design, characterize, and evaluate a stable and soluble high drug-loaded TASD combining two poorly water-soluble drugs, curcumin (CUR) and resveratrol (RES), with a hydrophilic polymer. Polymer screening studies, including miscibility, crystallization tendency, Flory-Huggins interaction parameter, and solubility parameter, were complemented by advanced techniques such as crystallization kinetics and molecular interaction analysis to assess drug-polymer interactions and amorphous stability. After selecting the optimal polymer, TASDs were prepared by rotary evaporation. Pure drugs, physical mixtures, binary, and ternary ASDs were characterized using X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier-transform infrared (FTIR) spectroscopy, and Raman spectroscopy. In-vitro dissolution under non-sink conditions was performed using a USP-II apparatus, and centrifuged samples were analyzed by UV-Vis spectroscopy. Long-term physical stability was assessed over 12 months at room temperature. Eudragit EPO was identified as the optimal polymer among EPO, HPMCAS, and S100. The resulting high drug-loaded (50% w/w) TASD was amorphous, exhibiting a single glass transition temperature (Tg) with strong drug-polymer interactions. In dissolution studies, the 50% drug-loaded TASD showed a ~ 197-fold and ~ fourfold increase in dissolved CUR and RES, respectively, compared to the crystalline drugs in the physical mixture after 1 h in acidic condition. Despite a lower RES release, it was still twice the release from binary ASDs. The TASD formulation remained physically amorphous for 12 months at room temperature storage.", "journal": "AAPS PharmSciTech", "year": "2025", "doi": "10.1208/s12249-025-03123-6", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41815860": { "pmid": "41815860", "title": "Screening and Identification of Triglyceride-Lowering Lactic Acid Bacteria and Preparation of Probiotic Agents.", "abstract": "To isolate lactic acid bacteria with significant triglyceride-degrading ability from traditional fermented foods in Guizhou and develop probiotic tablets, providing a basis for the development of functional probiotics. Strains with triglyceride-degrading ability were screened from 26 isolated strains. Morphological analysis, 16S rRNA gene sequencing, and stress resistance tests were conducted. The lyoprotectant composition for tablet preparation was optimized, and the stability and survival rate of the probiotic tablets were evaluated. Six LAB strains were selected, all exhibiting a triglyceride degradation rate exceeding 60%. Among them, PYC02 (Limosilactobacillus fermentum), SZ02 (Weissella cibaria), and PLHB02 (Weissella cibaria) showed degradation rates greater than 80%. PYC02 and PLHB02 maintained survival rates above 80% under conditions of pH 3 and 0.3% bile salts. The optimal lyoprotectant composition for maximum probiotic survival was 8% defatted milk powder, 5% sodium alginate, 2% maltodextrin, and 8% trehalose. After 30 days of storage at 4°C and 25°C, the survival rates of the probiotic tablets exceeded 80% and 60%, respectively, with triglyceride degradation rates greater than 60%. The triglyceride-degrading effects of PYC02 and PLHB02 were significant. These strains exhibited strong stress resistance and high safety, showing potential for the development of functional probiotics, offering new approaches for the prevention and treatment of hypertriglyceridemia.", "journal": "Food science & nutrition", "year": "2026", "doi": "10.1002/fsn3.71609", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41250864": { "pmid": "41250864", "title": "Growing Pains: GH-induced Fibrosis Across Multiple Organs in bGH Mice.", "abstract": "Fibrosis, excessive extracellular matrix deposition, disrupts normal tissue function. It has been observed in select tissues of individuals with acromegaly and in transgenic mouse models of acromegaly, suggesting a role of GH and/or IGF-1. However, analysis across multiple tissues and ages has not been reported. This study evaluated fibrosis in 6 tissues -lung, kidney, liver, spleen, quadriceps, and heart-from young (3 months) and aged (12-15 months) bovine GH transgenic and wild-type mice of both sexes. Fibrosis was assessed using hydroxyproline content, picrosirius red (PSR) staining, and serum biomarkers of collagen turnover (PINP, ICTP, and FAP). Hydroxyproline assays showed collagen content significantly increased with age across all tissues and both sexes. Compared to wild-type, aged male bGH mice had elevated hydroxyproline in the lung, kidney, liver, and quadriceps; aged female bGH mice showed increases in kidney, liver, and quadriceps. PSR staining showed minimal differences in young mice. In aged bGH mice, males exhibited increased PSR staining in all tissues except lung; females showed increases in all tissues except lung and heart. Serum biomarkers showed sex- and age-specific patterns: PINP decreased with age in both sexes; ICTP increased with age in both sexes; FAP was lower in bGH mice and decreased with age in females. In conclusion, excess GH promotes fibrosis in most tissues studied and becomes more pronounced with advancing age, suggesting fibrosis is a common outcome of excess GH. Whether fibrosis is directly caused by GH/IGF-1 or secondary to poor health of bGH mice requires further investigation.", "journal": "Endocrinology", "year": "2025", "doi": "10.1210/endocr/bqaf170", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41775128": { "pmid": "41775128", "title": "Testing the waters: A comparison of DNA extraction methods for the metabarcoding of microbial communities.", "abstract": "DNA metabarcoding is an emerging technique for enhancing microbial community monitoring programs that assess water quality and environmental health. However, comparability between studies is limited by a lack of standardized laboratory protocols. To contribute to protocol optimization and decision making, we compare seven commercially available DNA extraction kits, and evaluate their impact on DNA yield, PCR inhibitor removal, and taxonomic resolution of microplankton, microalgae, and particle-associated bacterial communities using 18S V9 and 16S V3-V4 amplicon sequencing of samples from two contrasting estuarine environments varying in salinity and productivity. We observed greater DNA yields and detected PCR inhibition at the higher productivity, lower salinity site. The Qiagen DNeasy PowerSoil Pro and Omega E.Z.N.A. Tissue DNA kits both yielded greater amounts of DNA, but only the soil- and plant-specific kits effectively removed PCR inhibitors. Notably, differences in taxonomic richness between kits were statistically significant but small (often <10 taxa). Additionally, beta diversity was more strongly influenced by abundance than presence/absence of taxa. All extraction methods shared 84-90% of resolved taxa and detected all major eukaryotic divisions/phyla identified by microscopy, as well as cyanobacteria. Furthermore, all kits detected several potentially harmful taxa including Akashiwo sanguinea and Vibrio sp. These results suggest that DNA extraction methods should be chosen based on environmental conditions, and we recommend the Qiagen PowerSoil Kit for obtaining high yields of uninhibited DNA from productive estuarine water samples. While alpha- and beta-diversity are largely similar between extraction methods, pilot studies should be performed to confirm resolution of taxa of interest.", "journal": "Marine environmental research", "year": "2026", "doi": "10.1016/j.marenvres.2026.107926", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "", "dirs": [ "B" ] }, "39492149": { "pmid": "39492149", "title": "Hydrogel interfaced glass nanopore for high-resolution sizing of short DNA fragments.", "abstract": "Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2024.116895", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41430003": { "pmid": "41430003", "title": "Long-term progression-free survivors (\"super responders\") to olaparib maintenance in recurrent ovarian cancer: a multicenter real-world study (KCOG-G2101s).", "abstract": "PARP inhibitors have been shown to improve progression-free survival in patients with recurrent ovarian cancer. However, their potential for long-term response and cure remains unclear in real-world practice. We conducted a multicenter, retrospective study of patients with recurrent ovarian, fallopian tube, or peritoneal cancer who were treated with olaparib maintenance therapy in the Kansai Clinical Oncology Group. We analyzed clinical outcomes according to histological tissue type, platinum sensitivity, BRCA mutation status, and SLFN11 expression. Epithelial ovarian cancers were classified into type I (low-grade serous carcinoma, clear cell carcinoma, low-grade endometrioid carcinoma) and type II (high-grade serous carcinoma, high-grade endometrioid carcinoma and undifferentiated carcinoma). A total of 72 patients were registered. The median progression-free survival and overall survival were 12.0 and 42.0 months, respectively. Type II tumors exhibited significantly longer progression-free survival than type I tumors (p = 0.027). Among type II tumors, those with platinum-sensitive recurrence and a response to chemotherapy (PSR-R, n = 51) had significantly better progression-free survival than non PSR-R (p < 0.0001). Notably, eight PSR-R patients (15.7%) achieved greater than five years of progression-free survival (\"super responders\"), and all had no evidence of disease at the last follow-up. BRCA mutations and SLFN11 expression were not associated with progression-free survival or super responders. In this real-world cohort, a subset of patients with recurrent ovarian cancer achieved durable, potentially curative responses with olaparib maintenance, regardless of their BRCA mutation status. When evaluating PARP inhibitor therapy, long-term progression-free survival should be considered a key endpoint.", "journal": "International journal of clinical oncology", "year": "2026", "doi": "10.1007/s10147-025-02942-8", "ptypes": [ "Journal Article", "Multicenter Study", "Observational Study" ], "first_author": "", "dirs": [ "C" ] }, "39279552": { "pmid": "39279552", "title": "An Enzyme-Encapsulated MOF@MOF Nanocomposite for Detecting H2O2 Derived From Superoxide Anion Released by Mitochondria of HeLa Cells.", "abstract": "In this work, a horseradish peroxidase (HRP)-encapsulated metal organic framework (MOF)@MOF nanocomposite is developed for detecting H2O2 converted by dismutation of superoxide anions released from live HeLa mitochondria. Initially, an HRP-polyacrylic acid cluster is incorporated on a mesoporous, peroxidase-like Cu/Co-1,4-benzenedicarboxylate (BDC) MOF platform to avoid structural change and deactivation of HRP through its interactions with MOF metal ions. Additionally, a Cu/Co-BDC(HRP)@1,3,5-benzenetricarboxyate (BTC) core-shell MOF/MOF structure, also with peroxide-like properties, serves as a protective matrix for HRP. Then, ultrathin porous carbon shells (UPCS) are adopted to improve the electrical conductivity of the MOF@MOF. The Cu/Co-BDC(HRP)@BTC|UPCS sensing platform exhibits two linear ranges of 0.05-1 µM and 1-1000 µM with a sensitivity of 172 mA mM-1 cm-2 and 1.63 mA mM-1 cm-2, respectively. A limit of detection of 0.057 µM, good selectivity and stability over 35 days for H2O2 detection are also achieved. After treating the mitochondrial complex with specific inhibitors, amperometric results at the sensing platform confirmed complex I and III within mitochondria as the main electron leakage sites in the electron transfer chains. Therefore, this sensing platform provides a tool that may aid in predicting and even developing treatments for some oxidative stress diseases caused by mitochondrial abnormalities.", "journal": "Small methods", "year": "2025", "doi": "10.1002/smtd.202401070", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40884263": { "pmid": "40884263", "title": "One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and Perspectives.", "abstract": "The cutting-edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR-based assays typically require the combination with an additional pre-amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one-pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost-effective, and high-sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one-pot CRISPR-based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one-pot CRISPR/Cas detection as the next generation of diagnostic tools.", "journal": "Advanced science (Weinheim, Baden-Wurttemberg, Germany)", "year": "2025", "doi": "10.1002/advs.202506716", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "39955325": { "pmid": "39955325", "title": "Numerical tests on dynamic response of embankment with pile-supported foundation for high-speed railway in soft ground using soil-water coupling elastoplastic FEM.", "abstract": "Embankment with pile-supported foundation (PSF) is widely used for high-speed railway (HSR) built in soft ground. Although extensive studies have been conducted on the effectiveness of PSF in reducing ground vibration via numerical analyses, the soil-water coupling in soft ground and response discrepancies among different types of PSF are seldom considered. In this study, 2D elastoplastic FEM was conducted to verify the necessity of soil-water coupling analysis and clarify the response discrepancies among different types of PSF, such as pile-net foundation, pile-raft foundation and pile-plate foundation. Numerical results indicate that liquid phase in soft ground plays an important influence on the dynamic response of HSR, the vertical acceleration and displacement will be overestimated while EPWP will be underestimated if soil-water coupling is not considered. Besides, single-phase analysis exaggerates the acceleration attenuation and underestimates the vibration amplification in soft ground. PSF induces significant stress fluctuation in the embankment and stronger vibration beneath the pile end compared to the unreinforced ground, moreover, the distributions of vertical acceleration and EPWP from PSF are partitioned sharply by the piles while vertical dynamic displacement becomes more uniform within the pile reinforced area. The peak acceleration and EPWP in soft ground are significantly different among different types of PSF, overall, the larger stiffness of PSF is, the smaller the peak acceleration and EPWP may be. However, the peak displacements are similar in different cases for the dynamic displacement is basically controlled by the piles rather than the slab or pile cap. The present research gives an insight to analyze high-speed train induced EPWP and settlement accumulation in soft ground.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-89619-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42466327": { "pmid": "42466327", "title": "Light-Activated Nucleic Acid Amplification Systems Using Photo-Caged DNA Polymerase or Primers.", "abstract": "Precise control over nucleic acid amplification is essential for achieving reliable, quantitative, high-throughput, and multiplexed molecular diagnostics, particularly in point-of-care and field settings. However, conventional isothermal amplification methods, especially rapid assays at ambient temperature such as recombinase-aided amplification (RAA), suffer from spontaneous initiation and lack of synchronization across parallel reactions. Here, light-activatable RAA systems employing two photocaging strategies to achieve spatiotemporal control are reported. In the first, a photocaged DNA polymerase is engineered via site-specific incorporation of p-azido-L-phenylalanine and conjugation with a 2-nitrobenzyl-modified ssDNA blocker through click chemistry. This approach offers high stability, making it advantageous for long-term storage and lyophilized formats. In the second, 6-nitropiperonyloxymethyl-modified primers are synthesized to block hybridization or extension until UV activation, offering operational simplicity and rapid integration into existing workflows. Both strategies independently suppressed background activity in the dark and restored full amplification efficiency upon exposure at 365 nm. The systems exhibited high sensitivity, low background, and compatibility with portable diagnostics. A custom-built device integrating UV activation and real-time fluorescence detection enabled seamless, on-demand operation. This modular platform provides flexible light-gated amplification solutions, allowing the selection of polymerase- or primer-based caging according to storage stability or workflow requirements, thereby advancing precise and field-ready molecular diagnostics.", "journal": "Advanced genetics (Hoboken, N.J.)", "year": "2026", "doi": "10.1002/ggn2.202500055", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42030301": { "pmid": "42030301", "title": "Development of a Loop-Mediated Isothermal Amplification (LAMP) for the screening of Candida auris.", "abstract": "Candida auris is an emerging multidrug-resistant yeast associated with invasive infections, healthcare-associated outbreaks, and high mortality, and is often misidentified by conventional diagnostic methods. Rapid, accurate, and scalable screening tools are essential for effective infection control, particularly in high-risk settings. We developed a multiplex loop-mediated isothermal amplification (LAMP) assay that combines a broad-range Candida Pan target with a C. auris-specific target in a single isothermal reaction. Assay conditions were optimized for primer ratio and temperature, and analytical sensitivity was evaluated using serial dilutions of culture-derived C. albicans and C. auris DNA, as well as contrived specimens consisting of urine, swab, and whole-blood matrices. Clinical performance was assessed using 35 Candida-positive clinical specimens (blood, urine, ear swabs) and 94 non-infectious controls. Results were compared with Candida Pan qPCR and C. auris qPCR. Cross-reactivity was tested against common bacterial isolates. Under optimized conditions (1:1 primer ratio, 64 °C), the assay allowed species-level discrimination, with C. auris positive for both Pan and auris channels and C. albicans positive only for the Pan channel. The C. auris-specific LAMP probe detected approximately 10²-10³ cells/mL in culture-derived and contrived specimens, showing a 1-2 log improvement over C. auris qPCR (10⁴-10⁵ cells/mL), while the Pan LAMP channel detected C. auris at around 10⁵ cells/mL. In clinical specimens, Pan LAMP detected Candida spp. in 34/35 cases (97.14%) versus 32/35 (91.14%) for Pan qPCR. All C. auris-positive specimens (9/9) were detected by the multiplex LAMP assay, compared with 6/9 (66.7%) by Pan qPCR. All 94 non-infectious controls and all bacterial isolates tested negative, indicating 100% clinical specificity and absence of cross-reactivity. The multiplex Candida Pan/auris LAMP assay provides a rapid, highly sensitive, and specific alternative to qPCR for C. auris screening, while preserving broad Candida detection in a single isothermal reaction. Its improved analytical and clinical sensitivity suggests strong potential for use in active surveillance and infection-control programs, particularly in settings where timely identification and containment of C. auris are critical.", "journal": "PloS one", "year": "2026", "doi": "10.1371/journal.pone.0348003", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42356829": { "pmid": "42356829", "title": "Quantum Dot-Based Dual-Fluorescence Aptasensing Platform Using Interface-Engineered MXene for Multiplex Protein Detection.", "abstract": "Antigen detection provides rapid and convenient diagnosis of respiratory infections. This study develops an innovative dual-fluorescence aptasensing method based on polydopamine-functionalized MXene (PDA-MXene) for the simultaneous detection of spike protein and hemagglutinin protein. The method employs green- and red-emitting quantum dot (QD) probes as fluorescence reporters, and the PDA-MXene as an effective adsorption and separation substrate. Coupled with a centrifugation-assisted separation strategy, this design method reduces background interference and enhances detection reliability. The method demonstrates good analytical performance, with detection limits of 0.82 ng/mL for spike protein and 2.11 ng/mL for hemagglutinin protein in single-channel mode. The dual-channel mode enables reliable and simultaneous quantification of both target proteins with minimal spectral cross-talk. Furthermore, this method exhibits high specificity against interferents including ions, proteins, and toxins. Artificial saliva, chosen as real sample, is spiked with target proteins to investigate the practical applicability of the method, showing recovery rates for both target proteins between 100 and 114 sensing strategy is simple to operate and allows the detection of new targets by simply replacing the azide-modified aptamer lyophilized powder. It therefore holds promising application for the simultaneous detection of multiple proteins in point-of-care testing and health monitoring fields.", "journal": "Sensors (Basel, Switzerland)", "year": "2026", "doi": "10.3390/s26123856", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40618012": { "pmid": "40618012", "title": "Validation of stool and blood analysis compared to Inv A and ttr based direct blood qPCR assay as diagnostic tools for typhoid fever.", "abstract": "The development of molecular techniques, such as polymerase chain reaction (qPCR), provides a promising approach for detecting S. enterica due to its rapidity, sensitivity, specificity, and resistance to interference from antibiotics, surpassing the routinely used stool and blood culture methods. This study aims to compare different stool and blood culturing methods with the direct blood qPCR-based assay of Salmonella Invasion A (Inv A) and Salmonella Tetrathionate Respiration (ttr) virulence genes as diagnostic tools for typhoid fever. One hundred clinically diagnosed typhoid fever (CDTF) cases were subjected to conventional stool and blood culture, followed by standard biochemical and serotyping identification of S. enterica, compared to direct blood and enriched blood-based ttr and Inv A qPCR-based techniques. Using the enriched blood-based ttr and Inv A qPCR technique, 90% and 85% of cases, respectively, were positive and confirmed as typhoid fever. While the blood-based ttr and Inv A qPCR technique showed 82% and 80% positivity, respectively, with a Ct threshold value of 37 cycles for both techniques. The stool culture method revealed S. enterica infection in only 32% of cases, while blood culture methods identified S. enterica in 48% of cases. The enriched blood-based ttr and Inv A qPCR method is a promising rapid tool for diagnosing typhoid fever, offering high sensitivity, specificity, accuracy and unaffected by antibiotic intake.", "journal": "BMC microbiology", "year": "2025", "doi": "10.1186/s12866-025-04127-9", "ptypes": [ "Journal Article", "Comparative Study", "Validation Study" ], "first_author": "", "dirs": [ "B" ] }, "41666411": { "pmid": "41666411", "title": "Ultrasensitive toltrazuril sulfone detection in eggs via a AuNPs/NH2-Ce-MOF-enhanced electrochemical immunosensor.", "abstract": "The widespread use of toltrazuril (TOL) as a low-toxicity anticoccidial agent in livestock production necessitates sensitive monitoring of its persistent metabolite, toltrazuril sulfone (TOS), which exhibits hepatotoxic and reproductive risks at subchronic exposure levels. Here, we proposed a novel electrochemical immunosensor for the ultrasensitive detection of TOS in eggs, utilizing a decorated amino-functionalized cerium metal-organic framework-gold nanoparticle (NH2-Ce-MOF/AuNP) nanocomposite. The NH2-Ce-MOF was synthesized via hydrothermal crystallization of (NH4)2Ce(NO3)6 and 2-aminoterephthalic acid, exhibiting exceptional stability and an expansive surface area. Compared to conventional electrodes, NH2-Ce-MOF-modified electrodes promoted AuNP nucleation and provided more active sites for HAuCl4 deposition, while also enhancing antibody binding, thereby improving the detection sensitivity of electrochemical immunosensors. Subsequently, electrodeposited HAuCl4 enhanced both electron transfer kinetics (charge transfer resistance decreased by 68% compared to a bare electrode) and bioreceptor loading capacity. Electrochemical evaluation demonstrated superior sensor performance, achieving a wide linear range of 0.05-100 ng mL-1 (R2 = 0.998) with an ultralow limit of detection of 33.2 pg mL-1 (S/N = 3), representing a 3-fold improvement over conventional enzyme-linked immunosorbent assays. The electrochemical immunosensor exhibited excellent selectivity and reproducibility (relative standard deviation <10%). This platform establishes a robust foundation for monitoring TOS residues and other hazardous contaminants in complex food matrices.", "journal": "Analytical methods : advancing methods and applications", "year": "2026", "doi": "10.1039/d5ay01781e", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40310665": { "pmid": "40310665", "title": "Discrepant Effects of Hydrated and Neat Reline on the Conformational Stability of a Knotted Protein.", "abstract": "Although knotted proteins are rare in number, their peculiar topology has long intrigued the scientific community. In this study, we have explored the conformational stability of a trefoil-knotted protein, YbeA, in reline (choline chloride:urea in a 1:2 ratio), a well-characterized deep eutectic solvent, using classical molecular dynamics simulation. Deep eutectic solvents (DESs) are explored as a reliable alternative to conventional solvents, effectively altering a protein's structural stability and activity, either stabilizing its native state or disordering its conformation depending on the relevant interactions involved. Here, using pure and hydrated concentrations of reline, we observe the conflicting effect of the DES on the knotted protein's stability. Our studies at room temperature and elevated temperatures show that in pure reline, the protein is conformationally stable and rigid. In contrast, the protein tends to lose its structural integrity in hydrated reline. The stable knotted topology also gets untied as the protein, solvated in hydrated reline, is exposed to an elevated temperature. Using Minimum Distance Distribution Functions and Kirkwood-Buff Integrals, we analyzed the solvation pattern of the DES constituents around the protein. We expect that this study will lead to more effective strategies in developing tailored solvent systems for comprehending the conformational behavior of knotted proteins.", "journal": "The journal of physical chemistry. B", "year": "2025", "doi": "10.1021/acs.jpcb.5c00148", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41501182": { "pmid": "41501182", "title": "Rapid evolution of a highly efficient RNA polymerase by homologous recombination.", "abstract": "Engineering DNA polymerases to efficiently synthesize artificial or noncognate nucleic acids remains an essential challenge in synthetic biology. Here we describe an evolutionary campaign designed to convert a family of highly selective DNA polymerases into an unnatural homolog with strong RNA synthesis activity. Starting from a homologous recombination library, a short evolutionary path was achieved using a single-cell droplet-based microfluidic selection strategy to produce C28, a newly engineered polymerase that can synthesize RNA with a rate of ~3 nt s-1 and of >99% fidelity. C28 is capable of long-range RNA synthesis, reverse transcription and chimeric DNA-RNA amplification using the PCR. Despite strong discrimination against other genetic systems, C28 readily accepts several 2'F and base-modified RNA analogs. Together, these findings highlight the power of directed evolution as an approach for reprogramming DNA polymerases with activities that could help drive future applications in biotechnology and medicine.", "journal": "Nature chemical biology", "year": "2026", "doi": "10.1038/s41589-025-02124-7", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42865592": { "pmid": "42865592", "title": "Effects of lyophilized Lactiplantibacillus plantarum DC2 probiotic powder on gastrointestinal morphology and microbiota in mice.", "abstract": "Probiotics have potential benefits in modulating gastrointestinal function. However, their efficacy and application depend largely on specific bacterial strains and environmental contexts. In our previous study, a strain of Lactiplantibacillus plantarum (L. plantarum) DC2 exhibited superior functionality in pork fermentation processes and was granted a Chinese patent (ZL202110743486.8). However, its storage and application characteristics remain unclear. To evaluate its direct application potential for gastrointestinal regulation, in this study, the formula and process of lyophilized L. plantarum DC2 powder were optimized via single-factor design (SFD) and response surface methodology (RSM). The optimized protectant consisted of fat-free milk powder, trehalose, glycerine, and L-sodium glutamate. The survival rate and storage stability of lyophilized bacterial powder were maintained at optimal level under optimized conditions. Intragastric administration experiments revealed that the abundance of Lactobacillus in both the stomach and jejunum significantly increased, accompanied by shifts in intestinal morphological features and modulation of the intestinal microbial composition and abundance. In conclusion, a reliable lyophilization protocol was established for optimal survival rate and storage stability of lyophilized L. plantarum DC2 powder, which was capable of modulating intestinal morphology and gut microbial profiles in mice. This study lays a foundation for its further development as a probiotic candidate.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1858099", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40768597": { "pmid": "40768597", "title": "Regulatory KIR+CD8+ T cells are elevated during human pregnancy.", "abstract": "During pregnancy, immune responses must balance protection from infections with tolerance of the semiallogeneic fetus. However, the mechanisms regulating maternal-fetal tolerance remain poorly understood. Recently, we identified CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) as a regulatory subset important for suppressing self-reactivity in human autoimmune and infectious diseases. To better understand what other roles these cells might play, we asked whether they are active during pregnancy. We first observed an increased frequency of KIR+CD8+ T cells in the peripheral blood of pregnant people in the second trimester, especially in those carrying a male fetus. In vitro, KIR+CD8+ T cells inhibited the alloreactive responses of maternal T cells against irradiated cord blood cells and selectively suppressed CD8+ T cells targeting male-specific proteins in mothers with male pregnancies. Therefore, the higher induction of KIR+CD8+ T cells in mothers carrying a male fetus may help suppress additional allogeneic responses triggered by male-specific alloantigens. Longitudinal analysis showed that KIR+CD8+ T cells undergo expansion and differentiate into functional cytotoxic cells during pregnancy. Single-cell RNA sequencing of decidual CD8+ T cells from early pregnancy revealed elevated numbers and increased expression of activation markers in KIR+CD8+ T cells at the maternal-fetal interface. In addition, a higher frequency of KIR+CD8+ T cells was associated with spontaneous abortion and preeclampsia. Together, our findings suggest that KIR+CD8+ T cells may contribute to maternal tolerance by modulating fetal-specific alloreactive T cell responses. They may also be useful as candidate biomarkers or therapeutic targets for human pregnancy disorders.", "journal": "Science translational medicine", "year": "2025", "doi": "10.1126/scitranslmed.adm7697", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "B" ] }, "42599007": { "pmid": "42599007", "title": "Field-Deployable Paper-Based Detection of Vibrio vulnificus via Mechanically Automated Sequential RPA/CRISPR-Cas12a.", "abstract": "Vibrio vulnificus (V. vulnificus) is a highly virulent marine pathogen that causes severe infections, including sepsis and necrotizing fasciitis, with high fatality rates in susceptible individuals. Rapid and sensitive on-site detection is therefore essential, yet existing methods often depend on laboratory-based equipment and trained personnel, limiting field applicability. Here, we present an automated, paper-based RPA/CRISPR-Cas12a diagnostic platform that integrates a microfluidic paper pad with a spiral-spring-driven mechanical actuator. The device enables sequential execution of recombinase polymerase amplification (RPA), CRISPR-Cas12a-mediated detection, and lateral flow assay (LFA) readout through a single user-initiated winding step, without the need for external power or instrumentation. The multistep process was achieved through automatic reconfiguration of the paper pads, enabling pipette-free transfer of RPA amplicons from the paper-based RPA pad to the paper-based CRISPR pad, followed by transfer of the reporter probes to the LFA strip. The paper-based system is preloaded with lyophilized reagents and designed to autonomously control fluid flow and reaction timing. Targeting the vvhA gene specific to V. vulnificus, the platform achieved a limit of detection of 1 colony-forming unit per reaction (CFU/reaction) in pure cultures. This sensitivity was consistently reproduced in gill swab samples from oysters exposed to artificially contaminated seawater at 20 CFU/mL, confirming that reliable detection was achievable in oyster gill swabs under these conditions. Overall, this work establishes a fully mechanical, instrument-free RPA/CRISPR-based diagnostic system for V. vulnificus, offering a rapid, portable, and highly sensitive approach for point-of-care testing and food safety applications.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.6c01273", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41095356": { "pmid": "41095356", "title": "Mechanical and Anti-Icing Properties of Polyurethane/Carbon Fiber-Reinforced Polymer Composites with Carbonized Coffee Grounds.", "abstract": "Spent coffee grounds represent an abundant waste resource with potential for sustainable material applications. This study investigates the use of carbonized spent coffee grounds (CSCG) as fillers in polyurethane (PU) coatings for carbon fiber-reinforced polymer (CFRP) substrates to enhance mechanical durability and anti-icing performance. SCGs were dried, sieved (<100 µm), and oxidatively carbonized in air at 100-300 °C for 60-120 min, then incorporated into PU at 1 or 5 wt.% and applied by spray-coating. A full-factorial design was employed to evaluate the effects of carbonization temperature, particle size, and filler loading. The optimized formulation (300 °C, 100 µm, 5 wt.%) showed the highest water contact angle (103.5°), lowest work of adhesion (55.8 mJ/m2), and improved thermal stability with 60% char yield. Mechanical testing revealed increased tensile modulus with reduced strain, and differential scanning calorimetry indicated an upward shift in glass-transition temperature, suggesting restricted chain mobility. Ice formation at 0 °C was sparse and discontinuous, attributed to lowered polar surface energy, rough surface texture, and porous carbon morphology. These results demonstrate that CSCGs are effective sustainable fillers for PU coatings, offering combined improvements in mechanical, thermal, and anti-icing properties suitable for aerospace, wind power, and other icing-prone applications.", "journal": "Materials (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ma18194533", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41262327": { "pmid": "41262327", "title": "Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.", "abstract": "The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.", "journal": "Molecular systems design & engineering", "year": "2026", "doi": "10.1039/d5me00140d", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41400985": { "pmid": "41400985", "title": "Multifunctional DNA Nanonets for a Novel Self-Powered Biosensor.", "abstract": "Multifunctional DNA nanonets were used to design an ultrasensitive self-powered biosensor for tobramycin (TOB) detection based on a bimetallic metal-organic framework, i.e., Fe/Co-MIL-88(NH2). In the bioanode, Fe/Co-MIL-88(NH2) shows peroxidase-like activity toward H2O2 catalysis and cascades with glucose oxidase to catalyze glucose oxidation. In the biocathode, carbon nanotubes decorated with gold nanoparticles (CNTs@AuNPs) are used to improve the conductivity. When TOB is present, DNA nanonets form double-stranded structures to adsorb [Ru(NH3)6]3+, amplifying the signal and generating a high open-circuit voltage (EOCV). Moreover, a capacitor is used to amplify the instantaneous current by storing charges from enzyme biofuel cells. A smartphone allows real-time readout of the test's instantaneous current via Bluetooth technology. The self-powered biosensor exhibits ultrasensitive TOB detection with a limit of detection of 0.41 fM in the range of 1.0-107 fM and possesses excellent selectivity, good stability, high reproducibility, and outstanding applicability to monitor TOB in actual samples.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c03455", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41104384": { "pmid": "41104384", "title": "CAAT box-derived polymorphism (CBDP) marker system exhibited a clear classification pattern in a global mini core collection of Aegilops tauschii Coss. populations.", "abstract": "In this study, the population structure and genetic diversity of Aegilops tauschii individuals were investigated using CAAT box-derived polymorphism markers. Fifteen primers produced one hundred forty-nine amplification fragments, out of which one hundred forty were polymorphic, yielding a polymorphism rate of 94%. Total amplified fragments (TAF) per primer varied from 8 to 13, with polymorphic fragments ranging from 8 to 12. The mean resolving power (RP) was 12.07, while the average polymorphism information content (PIC) and marker index (MI) were 0.37 and 3.44, respectively. Analysis of molecular variance revealed that 85% of the total genetic variation occurred within three geographical regions (Central and Inner Asia (CI), Caucasus and Caspian Region (CC), and the Middle East (MED)), whereas 58% of the variance was partitioned among countries. The CI region displayed the highest genetic diversity, evidenced by the greatest number of effective alleles, gene diversity, percentage of polymorphic loci, and Shannon's diversity index. At the country level, China and Afghanistan exhibited the highest diversity parameters. Principal coordinates analysis (PCoA) explained 30.25 and 57.60% % of the total variance and delineated three major geographic clusters corresponding to CI, CC, and MED. Cluster analysis corroborated these distinct regional patterns with minimal overlap among groups. Bayesian population structure analysis further supported these findings by assigning accessions to three primary subpopulations (K = 3) and revealing finer substructure at K = 9. Overall, our results demonstrate the effectiveness of CBDP markers for evaluating genetic diversity and geographic structuring in Ae. tauschii, providing valuable insights for its conservation and breeding.", "journal": "3 Biotech", "year": "2025", "doi": "10.1007/s13205-025-04557-2", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40667092": { "pmid": "40667092", "title": "A Clarifying Perspective on Bacterial Pseudo-Receiver Domains.", "abstract": "Two-component regulatory systems typically consist of a sensor kinase and a response regulator. All response regulators contain a receiver domain; most also contain an output domain. Response regulator activity is controlled by the phosphorylation state of the receiver. Receivers contain five conserved active site residues that catalyze phosphorylation and dephosphorylation reactions. Some protein domains identified computationally as receivers (PF00072) lack one or more of the five key conserved residues and are termed pseudoreceivers (PsRs). Because receivers are among the most abundant protein domains in nature, PsRs are also common. PsRs are especially common in plants, where they control circadian rhythms. Little is known about PsRs in fungi and archaea. Here, we focused on bacterial PsRs. We created representative datasets of 9,153 PsR and 143,116 true receiver domain sequences from bacteria. Comparison of amino acid composition of PsR and true receiver domains at each position showed (i) many differences at positions known to be important for phosphorylation-mediated signaling in true receiver domains, consistent with diminished importance in PsRs; and (ii) greatest differences between PsR and true receiver domains in the β3α3 and β4α4 loops, potentially highlighting functionally important regions of PsRs. We also performed covariation analyses of PsR and true receiver domains, which suggested six networks of linked residues that may be important for PsR function. Our analyses lay the foundation for rational experimental approaches to investigate molecular mechanisms of signaling by bacterial PsRs.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.06.23.661182", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "C" ] }, "41957466": { "pmid": "41957466", "title": "Taurine stimulates EPO production in feline renal cells through the HIF pathway.", "abstract": "Chronic kidney disease (CKD) affects approximately 30% of elderly felines, impairing erythropoietin (EPO) secretion and leading to anemia, which significantly compromises feline health. Betaine, docosahexaenoic acid (DHA), and taurine are bioactive compounds known to modulate physiological processes, exhibiting anti-inflammatory and immunomodulatory properties. This study aimed to investigate their potential effects on EPO secretion in renal cells and their therapeutic potential for hematopoietic system improvement and anemia treatment. Two feline renal cell lines, PETCC191 and PETCC3002, were cultured and treated with betaine, DHA, and taurine. EPO expression was analyzed using western blot (WB) and quantitative reverse transcription PCR (qRT-PCR). EPO secretion was quantified by enzyme-linked immunosorbent assay (ELISA). The involvement of the HIF1α pathway was examined using the HIF1α inhibitor YC-1. Among the three compounds, taurine significantly upregulated both mRNA and protein levels of EPO and enhanced its secretion in both PETCC191 and PETCC3002 cells, even under hypoxic conditions. Mechanistic studies revealed that taurine-mediated EPO production in feline renal cells occurs through the HIF1α pathway. Our findings demonstrate that taurine significantly enhances the synthesis and secretion of EPO in feline renal cells, suggesting its potential as a renal protective agent and a promising bioactive compound for the treatment of renal anemia in felines.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-46877-0", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42634078": { "pmid": "42634078", "title": "GC-rich regions affect amplification efficiency of neighboring amplicons in qPCR and digital PCR.", "abstract": "DNA polymerase chain reaction (PCR) has been widely used for the quantitation of DNA and RNA samples. Multiple factors can affect amplification efficiency, including GC-rich regions. The proximity effects of GC-rich regions on the amplification efficiency of neighboring amplicons that are not themselves GC-rich are not fully understood. Using a GFP-6.0Kb recombinant adeno-associated virus (rAAV) vector plasmid containing a 300 bp region with 79% GC content, we evaluated the effect of this GC-rich region on six amplicons (38-61% GC) at varying distances from it in nanoplate digital PCR (ndPCR), real-time quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In ndPCR, amplification efficiency decreased progressively with proximity to the GC-rich region, with the nearest amplicon yielding only 9% of the reference value. Restriction enzyme digestion that physically separated amplicons from the GC-rich region restored efficiency to approximately 100%. In qPCR, the effect on quantitation depended on whether the standard curve material and the sample shared similar neighboring GC-rich sequence context. The effect was not observed in ddPCR under the conditions tested. Additives including DMSO and betaine, and physical separation by restriction enzyme digestion or sonication, mitigated the effect in ndPCR and qPCR. These findings may have broad practical implications for PCR-based quantitation, as GC-rich regions are prevalent in many mammalian promoters, enhancers, and other gene regulatory elements.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-55452-6", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41784267": { "pmid": "41784267", "title": "crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.", "abstract": "CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.", "journal": "Nucleic acids research", "year": "2026", "doi": "10.1093/nar/gkag167", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42839898": { "pmid": "42839898", "title": "Pesticide droplet interfacial behavior and drying deposition characteristics on apple leaf surfaces at different growth stages: role of nonionic surfactant self-assembly.", "abstract": "The physicochemical properties of pesticide solutions can be effectively altered via surfactant addition, which significantly influences the wetting, bouncing behavior and deposition efficiency of pesticide droplets. We combined high-speed imaging, contact angle measurements, and infrared thermography to track droplet impact, spreading, and drying on apple leaves at three growth stages-flower bud, young fruit, and fruit enlargement. Deep-focus microscopy observations revealed that leaf surfaces at different growth and developmental stages possess trichomes of varying densities, which exert a non-negligible influence on droplet bouncing, wetting, and drying deposition behaviors. Cryo-TEM showed that AEO-4, AEO-5, and AEO-7 assembled into spherical micelles, while AEO-6 formed vesicles in pesticide solutions. The small micellar structures facilitated droplet penetration into trichome structures on leaf surfaces, whereas the vesicle elasticity reduced droplet bouncing. AEO-6 enhances Marangoni convection within the droplet, accelerates the drying rate, and promotes uniform deposition, whereas the micelles formed by AEO-4 and AEO-5 are susceptible to capillary flow within the droplet and the micelles formed by AEO-7 are more susceptible to gravitational effects, leading to non-uniform deposition upon drying and a \"coffee ring\" pattern. Through a comprehensive analysis of factors such as the self-assembly morphology of surfactants, the heterogeneity characteristics of leaf surfaces at different growth stages, and the Marangoni flow patterns within droplets, the wetting behavior on adaxial versus abaxial leaf surfaces varied markedly with trichome density. Notably, AEO-6 vesicles demonstrate superior performance, via an energy storage-release mechanism, reducing the coffee-ring effect and bouncing behavior, and enabling more uniform pesticide deposition. This offers a theoretical foundation for customizing surfactant selection based on leaf growth stage and surface location.", "journal": "Soft matter", "year": "2026", "doi": "10.1039/d6sm00743k", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39531721": { "pmid": "39531721", "title": "Epstein-Barr Virus in the RIVERA Case-Control Study of Acute Febrile Illness: Acute Mononucleosis Nearly Absent as an Etiology in the Peruvian Amazon.", "abstract": "Large diagnostic panels allow for pathogens with high or low likelihood of causing attributable illness to be tested simultaneously. Infectious mononucleosis (IM) due to primary infection with Epstein-Barr virus (EBV) is a common cause of acute febrile illness (AFI) in case series from high-income countries, though its contribution to AFI in tropical low-income settings is unclear. As part of a case-control study using multiplex quantitative polymerase chain reaction (qPCR) diagnostics, we set out to determine if primary EBV infection was an underrecognized cause of AFI in the Peruvian Amazon. Presence of EBV DNA in whole-blood samples was equally prevalent among febrile cases and afebrile controls (34.6% [247/714] versus 35.7% [248/695]) and was not correlated with classic IM symptoms. Given the clear lack of clinical significance of the whole-blood PCR results, additional testing was pursued to ascertain the true prevalence of IM among cases of AFI in this population. The presence of EBV DNA in plasma, a marker of active EBV-related processes, was detected in 7% (5/68). Anti-EBNA-1 IgG, a late marker of prior infection, was tested via ELISA and detected in 4/5 of the plasma-positive patients, thereby excluding an acute primary EBV infection in all but one patient. Infectious mononucleosis due to primary infection with EBV was not an important etiology of AFI in the Peruvian Amazon, despite high rates of initial test positivity.", "journal": "The American journal of tropical medicine and hygiene", "year": "2025", "doi": "10.4269/ajtmh.24-0051", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39552097": { "pmid": "39552097", "title": "Alpha-Ketoglutarate Alleviates Systemic Lupus Erythematosus-Associated Periodontitis in a Novel Murine Model.", "abstract": "To establish a reproducible experimental animal model for systemic lupus erythematosus (SLE)-associated periodontitis (PD), investigate the effects of SLE on PD and assess the therapeutic potential of alpha-ketoglutarate (αKG) for SLE-PD treatment. An SLE-PD murine model was established via ligature-induced PD in MRL-lpr strain, with MRL/MpJ strain as a non-SLE control. The periodontal state was assessed using micro-CT, real-time PCR, histology, immunofluorescence and flow cytometry assays. αKG levels were analysed, and a thermoresponsive gel was designed as a periodontal dimethyl (DM)-αKG delivery system. αKG levels were analysed in gingival crevicular fluid (GCF) of PD patients with or without SLE. SLE significantly increased the periodontal inflammation and bone resorption in the SLE-PD model. αKG levels in GCF were lower in PD patients with SLE than in PD patients without SLE. Decreased αKG levels in the gingiva and macrophage M1/M2 imbalance were observed in SLE-PD mice. However, DM-αKG thermoresponsive gel effectively alleviated the periodontal inflammation, bone resorption and macrophage M1/M2 imbalance in SLE-PD mice. Our study established, for the first time, a novel SLE-PD murine model and revealed that SLE increases the severity of PD in vivo. Our findings highlight the therapeutic potential of αKG for SLE-associated PD.", "journal": "Journal of clinical periodontology", "year": "2025", "doi": "10.1111/jcpe.14080", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41944788": { "pmid": "41944788", "title": "Fusogenic Liposome-Mediated Extracellular Vesicles MicroRNA Profiling for Lung Cancer Diagnosis.", "abstract": "Profiling circulating extracellular vesicle-associated microRNAs (EVs-miRNA) is essential for improving lung cancer (LC) diagnosis and prognosis in clinical tests. However, the complexity of RNA extraction procedures and the lack of an LC-specific EVs-miRNA signature limit the clinical applicability of the current liquid biopsy tools. Herein, we develop a fusogenic liposome nanoreactor for the direct profiling of plasma EVs-miRNAs. This platform integrates reagent-encapsulating liposomes, isothermal rolling circle amplification, and CRISPR-Cas12a endonuclease cleavage in a one-pot manner, allowing for ultrasensitive measurements down to fM levels. A LC-specific EVs-miRNA signature was identified through a designed four-phase screening procedure, which was validated by using both public databases and clinical samples. In a proof-of-concept cross-sectional and longitudinal study involving a clinical cohort (n = 141), the profiled signature enabled early diagnosis, therapy monitoring, and prognosis evaluation with an accuracy of up to 93.1%. The platform streamlines the diagnostic workflow into a single fusogenic step, providing an RNA extraction-free and proof-of-concept liquid biopsy framework for potential LC management.", "journal": "ACS nano", "year": "2026", "doi": "10.1021/acsnano.6c00879", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41526713": { "pmid": "41526713", "title": "Enhancing stability and safety of chimeric peptidoglycan hydrolases by linker engineering.", "abstract": "Spread of antimicrobial resistance and lack of new antibiotics have brought attention to alternative strategies of combating pathogenic bacteria. One of these strategies takes advantage of the bacteriolytic activity of peptidoglycan hydrolases. The enzymes allow efficient elimination of pathogenic bacteria while preserving the natural microflora. Such enzymes must meet specific criteria of activity, stability, and safety to become efficient enzybiotics. In our previous work (10.1128/spectrum.03546-23), we have created three chimeric enzymes and demonstrated their high efficacy in the elimination of Enterococcus faecalis and Staphylococcus aureus. In this work, we investigated and addressed issues related to the stability and safety of these enzymes. To improve the stability, we engineered the linkers and optimized storage conditions. Moreover, we demonstrated that such enzymes do not have any cytotoxic effects on eukaryotic cells, Danio rerio or Galleria mellonella. We also investigated the prevalence of resistance development, a particularly important feature for new antimicrobials. In conclusion, we here propose efficient, safe, and stable chimeric enzybiotics to eliminate E. faecalis and S. aureus. KEY POINTS: • Optimized linker design enhances enzyme stability. • Generated chimeric lysins do not display cytotoxicity. • Chimeras with minimal risk of resistance development were selected.", "journal": "Applied microbiology and biotechnology", "year": "2026", "doi": "10.1007/s00253-025-13651-7", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40846335": { "pmid": "40846335", "title": "Flow cytometric lyophilised reagent tube assay for peripheral blood neutrophil myeloperoxidase expression to rule out myelodysplastic neoplasms at a university hospital: a diagnostic accuracy study.", "abstract": "Although flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression can accurately rule out myelodysplastic neoplasms (MDS), it lacks reliability and efficiency due to the practical limitations of laboratory-developed liquid reagent-based assays. This study aimed to quantify the agreement and comparative discriminatory accuracy between a single-use flow cytometric lyophilised reagent tube (BD Lyotube Stain 468) and its laboratory-developed liquid reagent counterpart. Cross-sectional diagnostic accuracy study of two index tests against a reference diagnosis. A university hospital in France. Consecutive adult patients with an indication for bone marrow aspiration due to suspected MDS and unexplained peripheral blood cytopenia. MDS confirmed by cytomorphological evaluation of the bone marrow aspirate performed in duplicate by experienced haematopathologists blinded to the index test. Of 103 participants enrolled between July 2020 and August 2021, 37 had MDS (prevalence, 36%). The median intra-individual robust coefficient of variation (RCV) for myeloperoxidase expression was 30.9% using the BD Lyotube Stain 468 and 31.2% using the laboratory-developed liquid reagent assay, with an intraclass correlation coefficient of 0.94 (95% CI 0.91 to 0.96). The areas under the receiver operating characteristic curves were 0.83 (95% CI 0.74 to 0.90) and 0.82 (95% CI 0.73 to 0.89), respectively. Using a prespecified threshold of 30.0%, the corresponding sensitivity estimates were 89% (95% CI 75% to 97%) and 95% (95% CI 82% to 99%). BD Lyotube Stain 468 performs as well as its laboratory-developed liquid reagent counterpart for the quantification of myeloperoxidase expression by peripheral blood neutrophils. It may obviate the need for invasive bone marrow aspiration in up to 40% of patients with suspected MDS. NCT04399018.", "journal": "BMJ open", "year": "2025", "doi": "10.1136/bmjopen-2024-095640", "ptypes": [ "Journal Article", "Observational Study" ], "first_author": "", "dirs": [ "A" ] }, "41546941": { "pmid": "41546941", "title": "Elucidating the mechanism of fulvic acid-mediated PCR inhibition and its mitigation through nanoparticle-assisted amplification.", "abstract": "Fulvic acid is a common humic contaminant found in soil-matrix forensic samples and is a highly effective PCR inhibitor. This work uses computational analysis to investigate its molecular interaction with Taq DNA polymerase and evaluate nanoparticle-based facilitation strategies. Molecular docking and molecular dynamics simulations proposed selective binding of fulvic acid with catalytic residues (ARG587, ASP610, PHE667, TYR671, ASP785, GLU786), affecting DNA binding, active-site geometry, and Mg²⁺ coordination. Tryptophan quenching assays confirmed a low-affinity (∼251.6 ± 2.2 µM), reversible interaction. Functionally, fulvic acid reduced the peak height by > 80 % within the DNA profile and affected critical loci-D18S51, Penta D, D22S1045, FGA, CSF1PO, and D21S11. Nano-based facilitators-bare and BSA-coated AuNPs-were compared with the commonly employed BSA; the latter showed a statistically significant (p < 0.0001) 25 % TPH improvement compared to the inhibited level but did not affect allelic balance. These findings establish the inhibitory mode for fulvic acid and confirm the effectiveness of BSA-coated AuNPs as a scalable solution to restore PCR efficiency to environmentally compromised forensic DNA samples over highly concentrated application of BSA as a facilitator.", "journal": "Forensic science international", "year": "2026", "doi": "10.1016/j.forsciint.2026.112825", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41927655": { "pmid": "41927655", "title": "Proof of concept for aqueous two-phase system-based extraction of cell-free DNA from plasma for liquid biopsy applications.", "abstract": "Efficient extraction of circulating cell-free DNA (cfDNA) from plasma is critical for liquid biopsy applications, but remains challenging due to cfDNA's short fragment size, low concentration, and the presence of interfering proteins and genomic DNA. This study presents a simple extraction process based on aqueous two-phase system (ATPS) capture and reverse elution purification as a potentially lysis-free alternative to adsorption-based methods. Short DNA fragments selectively partitioned into the salt-rich bottom phase, achieving up to 65% recovery after purification while removing 99.7% of plasma proteins. Reverse elution enabled efficient desalting and up to fourfold DNA concentration under mild, aqueous conditions. The extracts showed no qPCR inhibition and displayed a cfDNA-like size profile with a cutoff around 750 bp, indicating selective enrichment of short fragments. Although the total yield was slightly lower than that of the QIAamp Circulating Nucleic Acid Kit (QIAGEN), the ATPS workflow substantially reduced processing time, equipment needs, and cost. Next-generation sequencing of cfDNA reference material extracted from DNA-free plasma confirmed compatibility with library preparation and sequencing workflows, providing proof of concept for a simplified cfDNA extraction strategy for amplification-based liquid biopsy applications.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-45585-z", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42716166": { "pmid": "42716166", "title": "Balanced regulation of UvsX activity and thermostability based on active learning.", "abstract": "Recombinase polymerase amplification (RPA) enables isothermal nucleic acid detection. However, the key enzyme UvsX of RPA exhibits suboptimal efficiency at low-temperature conditions and low template concentration. Here, we employed a directed engineering strategy combining energy-based screening and protein language models (PLMs). Through semi rational design and iterative optimization with EVOLVEpro, we obtained a triple mutant, TIG (S233T/A311I/A341G), which achieved 1.89-fold RPA relative activity of the wild-type (WT) at a template concentration of 1 × 105 copies reaction-1, and showed higher amplification activity to WT across 37.5-44.3 °C at a template concentration of 1 × 104 copies reaction-1. Molecular dynamics simulations revealed that TIG enhances residue flexibility and remodels the ATP-binding pocket, thereby improving DNA-binding pathway connectivity and strengthening the coupling between ATP hydrolysis and DNA strand exchange. Per-residue energy decomposition and protein structure network analyses further elucidate how key mutations modulate DNA-binding pathways. Finally, under optimized expression conditions (20 °C, 12 h, 0.1 mM IPTG), the yield reached 206.87 ± 3.56 mg L-1 of TIG. Collectively, this study provides a strategy for UvsX engineering and establishes a foundation for the development of more robust RPA assays.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.154387", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40714403": { "pmid": "40714403", "title": "Metal-organic framework-mediated antioxidant enzyme delivery in disease treatment.", "abstract": "Excessive reactive oxygen species (ROS) induce oxidative stress (OS), resulting in biomolecular damage. ROS are closely linked to diseases such as inflammatory bowel disease (IBD), diabetes, and cancer. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glucose oxidase (GOX) play crucial roles in alleviating OS by effectively reducing ROS levels through synergistic action. However, these antioxidant enzymes exhibit poor stability in complex biological environments and lack adequate targeting capability, thereby limiting their applications in disease treatment. Metal-organic frameworks (MOFs) serve as carriers that significantly enhance enzyme stability and targeting ability, thereby boosting catalytic efficiency and therapeutic outcomes. This review discusses the classification of antioxidant enzymes and their role in ROS regulation, the types and physicochemical properties of MOFs, and the assembly methods of enzyme@MOF systems. Furthermore, it examines the potential of integrating MOFs with antioxidant enzymes for the treatment of diseases associated with OS. Although the strategy of enzyme immobilization with MOFs shows great application prospects, further optimization is required in terms of biocompatibility, long-term stability, and delivery efficiency to promote the clinical translation of these results.", "journal": "Redox biology", "year": "2025", "doi": "10.1016/j.redox.2025.103778", "ptypes": [ "Journal Article", "Review", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "42046310": { "pmid": "42046310", "title": "Metal-Organic Frameworks (MOF)-Based Nanozymes: Types, Engineering, and Recent Advances in Biosensing.", "abstract": "Nanozymes, an emerging class of nanomaterials with natural enzyme-mimicking activity, have received immense recognition due to their promising physicochemical properties, easy synthesis, modification, cost-effectiveness, and stability. Metal-organic frameworks (MOFs) with unique structural properties, porosity, customizability, and composition can efficiently mimic the active center of natural enzymes. Rational design of MOF structures with natural enzyme-like activity has opened up a new route in the field of biosensors. In this review, we first classified the MOF nanozymes based on their catalytic activity. Then, we systematically summarized the strategies to augment the enzyme-mimicking performance of MOF nanozymes. Moreover, their applications in the biosensing field have been reviewed. Finally, we discussed the challenges and prospects for the development of MOF nanozymes toward superior application performance.", "journal": "Chemistry (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/chem.71075", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "39817409": { "pmid": "39817409", "title": "Efficacy and safety of daprodustat in patients on peritoneal dialysis in the ASCEND-D trial.", "abstract": "Daprodustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, is approved for treatment of anemia in dialysis patients with CKD in some parts of the world. This subgroup analysis examined the efficacy and safety of daprodustat versus darbepoetin alfa in patients with anemia of CKD undergoing peritoneal dialysis (PD). ASCEND-D (NCT02879305) was an open-label, Phase 3 trial; patients with CKD were randomized to daprodustat daily and epoetin alfa (HD patients) or darbepoetin alfa (PD patients). In PD patients, prespecified analyses of the co-primary endpoints of mean change in hemoglobin from baseline to Weeks 28-52 using an ANOVA model and first occurrence of a major cardiovascular event (MACE) using a Cox proportional hazards model were conducted. The secondary endpoints were average monthly intravenous iron dose to Week 52 and treatment-emergent adverse events. Additional post hoc analyses were conducted. Overall, 340 PD patients (daprodustat n = 171, darbepoetin alfa n = 169) were randomized. Mean age was 53.6 years (±14 SD), 55% male, 56% White. For daprodustat and darbepoetin alfa groups respectively, mean change in hemoglobin was 0.38 and 0.23 g/dL [adjusted mean difference 0.15, 95% confidence interval (CI), -0.04, 0.34], and first occurrence of adjudicated MACE occurred in 40 (23.4%) and 46 (27.2%) patients (HR 0.84; 95% CI, 0.55-1.28). No heterogeneity was observed between PD and HD patients for these endpoints in ASCEND-D. Serum hepcidin was lower with daprodustat; there was no difference in other iron parameters, intravenous iron usage, transfusion requirement, blood pressure, or quality of life. There were no differences in adverse events or incidence of peritonitis between the groups. This subgroup analysis of the ASCEND-D trial demonstrated comparable efficacy and safety of daprodustat versus darbepoetin alfa in PD patients, supporting its use in the treatment of anemia in these patients.", "journal": "Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association", "year": "2025", "doi": "10.1093/ndt/gfae273", "ptypes": [ "Journal Article", "Randomized Controlled Trial", "Clinical Trial, Phase III", "Multicenter Study" ], "first_author": "", "dirs": [ "B" ] }, "40065170": { "pmid": "40065170", "title": "LILRB3 genetic variation is associated with kidney transplant failure in African American recipients.", "abstract": "African American (AA) kidney transplant recipients exhibit a higher rate of graft loss compared with other racial and ethnic populations, highlighting the need to identify causative factors. Here, in the Genomics of Chronic Allograft Rejection cohort, pretransplant blood RNA sequencing revealed a cluster of four consecutive missense single-nucelotide polymorphisms (SNPs), within the leukocyte immunoglobulin-like receptor B3 (LILRB3) gene, strongly associated with death-censored graft loss. This SNP cluster (named LILRB3-4SNPs) encodes missense mutations at amino acids 617-618 proximal to a SHP1/2 phosphatase-binding immunoreceptor tyrosine-based inhibitory motif. The LILRB3-4SNPs cluster is specifically enriched within AA individuals and exhibited a strong association with death-censored graft loss and estimated glomerular filtration rate decline in the AA participants from multiple transplant cohorts. In two large Biobanks (BioMe and All-of-Us), the LILRB3-4SNPs cluster was associated with the early onset of end-stage renal disease and acted synergistically with the apolipoprotein L1 (APOL1) G1/G2 allele to accelerate disease progression. The SNPs were also linked to multiple immune-related diseases in AA individuals. Last, on multiomics analysis of blood and biopsies, recipients with LILRB3-4SNPs showed enhanced inflammation and monocyte ferroptosis. While larger and prospective studies are needed, our data provide insights on the genetic variation underlying kidney transplant outcomes.", "journal": "Nature medicine", "year": "2025", "doi": "10.1038/s41591-025-03568-z", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39820854": { "pmid": "39820854", "title": "A Rapid PCR-LAMP Assay for the Early Detection of Lasiodiplodia theobromae from Basal Stem Rot-Infected Passion Fruit Plants.", "abstract": "Lasiodiplodia theobromae is an emerging threat and the main pathogenic fungi associated with basal stem rot of passion fruit in Guangxi Zhuang Autonomous Region, China. Current pathogen identification protocols are labor-intensive and time-consuming, emphasizing the need for more efficient methods to enable precise surveillance of L. theobromae for early detection and warning. The present study sought to develop a rapid colorimetric LAMP assay for early detection and surveillance of L. theobromae in passion fruit plants. For that, amplifications of ITS locus were performed on fungal genomic DNA using conventional PCR, with the specific primer pair ITS1 and ITS4. The hydroxy naphthol blue (HNB)-dependent colorimetric LAMP assay was then optimized by varying primer sets, inner primers concentration, reaction temperatures and incubation time. A microbial lysis buffer was employed to extract genomic DNA from stems infected with L. theobromae. The prime LAMP primer set targeting the ITS region of L. theobromae was designed and an HNB based colorimetric LAMP assay was optimized. Various optimization parameters were evaluated, with the optimal conditions determined as 1.6 μM of each FIB and BIP, 0.2 μM of each F3 and B3, and incubation at 65 °C for 40 min. This ITS-based LAMP assay could effectively distinguish L. theobromae from less dominant pathogens in passion fruits with a detection limit of 3 pg for ITS locus amplicons. Our proposed method utilizing a microbial lysis buffer enables rapid and cost-effective detection of L. theobromae DNA in early-infected passion fruit plants, eliminating the need for microbial cultivation and DNA purification.", "journal": "Molecular biotechnology", "year": "2026", "doi": "10.1007/s12033-024-01363-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40423947": { "pmid": "40423947", "title": "An efficient and green pretreatment of Astragalus membranaceus fermentation with magnetic cellulose-immobilized Bacillus natto using deep eutectic solvent assisted for improving thrombolytic activity and evaluation of its antioxidant activity.", "abstract": "This study aimed to improve the thrombolytic activity of Astragalus membranaceus using magnetic cellulose-immobilized Bacillus natto fermentation. The fermentation parameters: time, temperature, pH, inoculum amount and solid-liquid ratio were screened by one-way experiments, and Plackett-Burman experiments were performed to determine the fermentation time, temperature, and inoculum amount as the key influencing factors, and the steepest-climbing experiments were performed to optimize the parameters, and then Box-Behnken design (BBD) experiments were conducted to determine the optimal conditions, which significantly increased the thrombolytic efficiency of Astragalus membranaceus immobilized natto fermentation to 311.156 IU/mg and exhibited superior antioxidant activity at 52.23 h of fermentation, an inoculum volume of 1.54 g/g, and a liquid-solid ratio of 30.61 mL/g. In addition, introducing deep eutectic solvent (DES) further enhanced the damage effect. The optimal type and concentration of DES were determined by screening. The magnetic cellulose system exhibited excellent thrombolytic activity and reusability compared to the calcium alginate immobilized system. This study provides a new strategy for immobilizing Bacillus natto provides a scientific basis for developing novel and efficient thrombolytic agents and highlights the potential of magnetic cellulose systems for biocatalysis and biomedical applications.", "journal": "Preparative biochemistry & biotechnology", "year": "2026", "doi": "10.1080/10826068.2025.2509897", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42785846": { "pmid": "42785846", "title": "Enhanced enzymatic hydrolysis of chitosan via MOF-mediated activation of chitosanase.", "abstract": "Chitosanases are attractive biocatalysts for producing bioactive chito-oligosaccharides (COS), but their applications are limited by insufficient catalytic efficiency, poor stability, and challenging recovery. Although many chitosanases can be activated by metal ions, the direct addition of free ions into reaction systems suffers from low efficiency and separation challenges. This study presented a MOF-based strategy to create a Cu-enriched coordination microenvironment, enabling simultaneous immobilization and activation of marine polysaccharide hydrolases. Taking chitosanase OUC-CsnCA as a model, the immobilization on Cu-trimesic acid (Cu-BTC) doubled its activity (413.5 ± 34.7 U/mg) and increased kcat from 361.8 ± 8.0 to 704.4 ± 16.1 min-1, along with improved thermostability and pH tolerance. MALDI-TOF-MS and HPLC confirmed deeper substrate depolymerization upon immobilization. Kinetic and spectroscopic analyses revealed that Cu2+-binding modulated enzyme conformation and promoted enzyme-substrate interactions. Molecular dynamics simulations further indicated that Cu2+ coordination was associated with adjustments in catalytic cleft geometry (D102-E84) and reinforcement of hydrogen-bond interactions within the substrate-binding pocket. Enhanced activities were further observed for immobilized ι-carrageenase and α-agarase, demonstrating the potential applicability of this strategy to other metal-responsive marine polysaccharide hydrolases.", "journal": "Carbohydrate polymers", "year": "2026", "doi": "10.1016/j.carbpol.2026.125796", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41254639": { "pmid": "41254639", "title": "A point-of-care testing device for monkeypox virus integrating recombinase polymerase amplification and lateral flow assay.", "abstract": "The global spread of monkeypox virus (MPXV) underscores the need for rapid, accessible diagnostic tools to control outbreaks. Current diagnostics, relying on centralized laboratory testing, require specialized personnel and equipment. We aimed to develop a point-of-care testing (POCT) kit that simplifies nucleic acid extraction, minimizes contamination, and enables immediate, accurate MPXV detection. We introduced a novel MPXV POCT kit, featuring a separate MPXV DNA rapid release step and a closed integrated device combining recombinase polymerase amplification (RPA) and lateral flow assay (LFA). A fluorescent RPA assay identified F3L-targeting primers and probes with superior amplification efficiency. The RPA system was coupled with LFA to assess the visual diagnostic performance, and then integrated into a POCT device, complemented by a nucleic acid release agent to form the kit. The kit's specificity, sensitivity, and diagnostic performance were validated through clinical body fluid samples. Our nucleic acid release agent streamlines sample processing without compromising detection accuracy, eliminating the need for traditional nucleic acid extraction. The RPA reaction system was formulated into freeze-dried microspheres and integrated directly into the reaction chamber of the POCT device, ensuring the stability and shelf-life of the reagents and simplifying the preparation and configuration process. The POCT device's enclosed design eradicates transfer steps, significantly reducing RPA-induced aerosol contamination. Results can be visually interpreted within 25 min, a substantial improvement over existing methods. In clinical sample validation, the POCT kit showed high specificity and sensitivity, distinguishing other pathogens, with 100% consistency with RT-qPCR results in detecting mpox patient samples. Compared to RT-qPCR and ddPCR, our POCT kit demonstrates stronger diagnostic performance, with the lowest positive concentration we detected being 41 copies/mL (2.1 copies/reaction) in clinical samples and 2.5 copies/μL (2.5 copies/reaction) of DNA plasmid standards, significantly increasing the detection rate of mpox. This compact, portable POCT kit, requiring no professional operation or precision instruments, offers a promising solution for on-site and home self-testing. Its high sensitivity and specificity, coupled with the capacity to swiftly adapt to new MPXV strains, positions it as an effective tool for early detection, vital for controlling mpox outbreaks.", "journal": "BMC medicine", "year": "2025", "doi": "10.1186/s12916-025-04412-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40605160": { "pmid": "40605160", "title": "Sustained Delivery of Dolutegravir Sodium for Better Management of HIV/AIDS via Solid Lipid Nanoparticles.", "abstract": "Human immunodeficiency virus (HIV) is a primary health concern that leads to Acquired immunodeficiency syndrome (AIDS), with more than 39.9 million people living with HIV globally. Dolutegravir sodium is a lipophilic compound with a log P value of 2.2. The current research aimed at augmenting the solubility, dissolution, and therapeutic benefits of Dolutegravir sodium through Solid lipid nanoparticles. The solid lipid nanoparticles (SLN) of Dolutegravir sodium were developed using highspeed homogenization and probe sonication methods. The solid lipid and surfactant were scrutinized for the development of SLN. The optimization of SLN was established using the Box- Behnken design model. The effects of lipid, surfactant, and homogenization speed on particle size and entrapment efficiency were evaluated. The colloidal dispersion was lyophilized, and accelerated stability was assessed. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the interactions between the drug excipients. The thermal behavior and crystalline nature were checked with Differential Scanning Calorimetry (DSC). Among the several tested solid lipids, the highest solubility was observed in glyceryl monostearate (GMS). The colloidal dispersion was stabilized by the Tween 20. Accordingly, the Box-Behnken design model and the analysis of variance (ANOVA) model were applied. The p-values for the particle size and entrapment efficiency were 0.0050 and 0.0010, respectively. The optimized batch D5 showed a particle size of 189 nm, zeta potential (ZP) of -24.6 mV, entrapment efficiency of 85.94%, and drug release of 87.02%. The optimized batch D5 was further lyophilized and analyzed with scanning electron microscopy (SEM), which confirmed the nanoscale range for SLN of Dolutegravir sodium. A significant enhancement in solubility and dissolution was achieved with the solid lipid nanoparticles. The sustained delivery of 24 hours reduces the dosage frequency and minimizes the viral load for the effective therapy of HIV, thereby improving patients' comfort and compliance.", "journal": "Current HIV research", "year": "2025", "doi": "10.2174/011570162X369356250613053801", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41748695": { "pmid": "41748695", "title": "Mutant SRSF2-associated impaired erythropoiesis is defined by increased mTORC1 signaling due to FYN missplicing.", "abstract": "Somatic mutations in RNA splicing regulators, including the serine/arginine-rich protein SRSF2, are frequently observed in myeloid malignancies. Using mouse models and primary human samples, we investigated the impact of SRSF2 mutations on erythropoiesis. We found reduced erythropoiesis in Srsf2P95H versus wild-type mice upon stress-induced erythropoiesis and identified that SRSF2 mutations correlate with reduced hemoglobin in JAK2-mutant patients with myeloproliferative neoplasms (MPN). Consistent with this, Jak2V617F-Srsf2P95H versus Jak2V617F mice displayed reduced red blood cell counts and erythroid precursor frequencies. RNA-sequencing on erythroid precursors showed reduced expression of heme metabolism and mitotic spindle-related genes, and increased expression of mTORC1 signaling in Srsf2P95H versus wild-type cells. RNA splicing analyses on the same cells and on human patient samples identified aberrant FYN splicing in SRSF2mut cells, with increased aberrant FYNB over normal FYNT transcripts. FYNB, but not FYNT, expression resulted in reduced erythroid differentiation and increased phosphorylation of mTORC1 downstream target S6. Additionally, increased S6 phosphorylation was confirmed in primary Srsf2P95H erythroid cells. mTORC1 pathway inhibition using rapamycin normalized FYNB- and Srsf2P95H-induced impaired erythropoiesis and significantly increased erythroid colony formation of SRSF2-mutant myelodysplastic neoplasm (MDS) bone marrow cells. Our data reveal targetable molecular mechanisms of impaired erythropoiesis in SRSF2-mutant cells.", "journal": "Leukemia", "year": "2026", "doi": "10.1038/s41375-026-02892-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41286218": { "pmid": "41286218", "title": "Gold nanoflowers (AuNFs) as enhancers for improved LAMP performance.", "abstract": "AuNFs (gold nanoflowers) are widely applied in the field of biosensing; their unique morphological characteristics and high specific surface area enable them to provide a large number of active sites, thereby enhancing the efficiency of catalytic reactions. These features make AuNFs a promising material for improving the performance of loop-mediated isothermal amplification (LAMP). Firstly, the reduction of 4-NP catalyzed by nano-gold (NG) with different morphologies showed that AuNFs made the yellow solution almost transparent in 4 min, similar to AuNPs (gold nanoparticles), with higher catalytic efficiency compared to AuNRs (gold nanorods) (> 8 min). The incorporation of AuNFs increased the activity of Bst DNA polymerase, and the kinetic parameters showed a Vmax/Km of 0.021, which was higher than that of AuNPs (0.015), AuNRs (0.013), and the LAMP system without NGs (0.013). Moreover, at ambient temperature, the UV-vis intensity of the characteristic peak indicates that the loading capacity of ssDNA is approximately 1.6 × 105 per AuNF, and when the temperature rises to 65 °C, ssDNA is almost completely released from the AuNFs. This mechanism may be beneficial for suppressing the generation of non-specific amplification. Dynamic light scattering (DLS) and circular dichronism (CD) results indicate that AuNFs alter the α-helix structure upon binding with Bst, potentially thereby enhancing enzyme activity. LAMP amplification was performed at different temperatures, and the results illustrate that the Tt values of AuNFs@LAMP were lower than those of AuNRs@LAMP (P < 0.0001), AuNPs@LAMP (P = 0.0082), and classical LAMP (P < 0.0001) at 40, 45, 50, 55, and 60 ℃. Thus, we proposed the AuNFs@LAMP (AuNF-mediated LAMP) method and applied it to the detection of AHSV (African horse sickness virus) standard plasmid sample and spiked dust sample with a sensitivity as low as 10 copies/μL. Intra-day, inter-day, and intra-batch precision showed RSD < 5%, with RSD ranging from 2.51 to 8.75% across low, medium, and high plasmid template concentrations, respectively. Moreover, the specific AuNFs@LAMP system inhibits non-specific amplification in negative samples and in samples containing other equine virus plasmids (WNV, JEV, VSV, NIV, EEEV, WEEV).", "journal": "Mikrochimica acta", "year": "2025", "doi": "10.1007/s00604-025-07618-8", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "42684932": { "pmid": "42684932", "title": "PMAxx dye-assisted colorimetric dual DNAzyme-LAMP (PD-cDDLAMP) for rapid phenotypic antimicrobial susceptibility testing of Escherichia coli.", "abstract": "Rapid antimicrobial susceptibility testing (AST) is crucial for combating antimicrobial resistance and guiding effective therapy. Conventional phenotypic AST methods are often time-consuming, requiring 18-72 h for results, while genotypic approaches often depend on pre-existing knowledge of resistance markers. Although nucleic acid-based phenotypic AST methods have reduced turnaround time, attempts to shorten antibiotic exposure often produce only small differences between susceptible and resistant bacteria. Consequently, most loop-mediated isothermal amplification (LAMP)-based phenotypic AST assays still require prolonged antibiotic exposure (up to 4 h) or rely on sophisticated high-resolution nucleic acid detection, such as digital LAMP, to accurately detect these subtle changes, limiting their simplicity. Therefore, there is a critical need to develop a rapid assay with both short antibiotic exposure times and a simple readout. To address this unmet need, we developed PMAxx dye-assisted colorimetric Dual DNAzyme-LAMP (PD-cDDLAMP), a rapid nucleic acid-based phenotypic AST method that enhances suppression of nucleic acid amplification from antibiotic-susceptible bacteria through the selective binding of photoreactive DNA-crosslinker dye (PMAxx) to the DNA of membrane-compromised cells. This strategy enables reliable discrimination between antibiotic-resistant bacteria (\"signal-on\") and antibiotic-susceptible bacteria (\"signal-off\") after only 30 min of antibiotic exposure using a user-friendly colorimetric DNAzyme-LAMP readout system. PD-cDDLAMP achieved 91.7% sensitivity, 83.3% specificity, and 87.5% accuracy in detecting Escherichia coli exposed to ampicillin and tetracycline, outperforming current methods by reducing antibiotic exposure time to just 30 min. The method was further validated in spiked milk samples, demonstrating its potential for on-site AST in dairy farming. PD-cDDLAMP's simplicity, speed, and reduced workflow complexity make it a promising tool for rapid AST. Rapid, reliable antimicrobial susceptibility testing (AST) is essential for guiding targeted therapy and combating the escalating threat of antimicrobial resistance (AMR). However, current phenotypic ASTs are too slow for urgent decision-making, and genotypic assays cannot fully capture emerging or unknown resistance mechanisms. The PMAxx dye-assisted colorimetric dual DNAzyme-loop-mediated isothermal amplification (PD-cDDLAMP) assay introduced in this work provides a rapid nucleic acid-based phenotypic AST capable of distinguishing antibiotic-resistant from susceptible bacteria within 30 min of antibiotic exposure. By combining PMAxx-mediated suppression of nucleic acid amplification in susceptible cells with equipment-free colorimetric DNAzyme-LAMP readout, PD-cDDLAMP bridges the gap between speed and phenotypic accuracy. Its high diagnostic performance, compatibility with complex matrices such as milk, and minimal instrumentation requirements position this method as a promising point-of-care tool for clinical microbiology laboratories and on-site testing environments. PD-cDDLAMP has the potential to significantly shorten time-to-result while maintaining reliability, thereby supporting antimicrobial stewardship.", "journal": "Microbiology spectrum", "year": "2026", "doi": "10.1128/spectrum.01212-26", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40769552": { "pmid": "40769552", "title": "[Effects of vacuum freeze-drying based on different lyoprotectants on the stability of foot-and-mouth disease virus-like particles].", "abstract": "Vaccination is a crucial strategy for the prevention and control of infectious diseases. Virus-like particles (VLPs), composed of structural proteins, have garnered significant attention as a novel type of vaccine due to their excellent safety and immunogenicity. However, similar to most vaccine antigens, VLPs exhibit insufficient thermal stability, which not only restricts the widespread application of vaccines but also increases the risk of vaccine inactivation. This study aims to enhance the stability and shelf life of VLPs derived from type A foot-and-mouth disease virus (FMDV) by employing vacuum freeze-drying technology. The optimal lyoprotectant formulation was determined through single-factor and combinatorial screening. Subsequently, the correlation between the immunogenicity of the freeze-dried vaccine and the content of FMDV VLPs was evaluated via a mouse model. The stability of FMDV VLPs before and after freeze-drying was further assessed by storing them at 4, 25, and 37 ℃ for varying time periods. Results indicated that the lyoprotectant formulation No.1, composed of 7.5% trehalose, 0.1% Tween 80, 50 mmol/L glycine, 1% sodium glutamate, and 3% polyvinylpyrrolidone (PVP), effectively preserved the content of FMDV VLPs during the vacuum freeze-drying process. The immunization trial in mice revealed that the levels of specific antibodies, immunoglobulin G1 (IgG1), interleukin-4 (IL-4), and neutralizing antibodies induced by freeze-dried FMDV VLPs were comparable to those induced by non-freeze-dried FMDV VLPs. The heat treatment results showed that the storage periods of freeze-dried FMDV VLPs at 4, 25, and 37 ℃ were significantly longer than those of non-freeze-dried FMDV VLPs. In conclusion, the selected lyoprotectant formulation effectively improved the stability of FMDV VLPs vaccines. This study provides valuable insights for enhancing the stability of novel subunit vaccines.", "journal": "Sheng wu gong cheng xue bao = Chinese journal of biotechnology", "year": "2025", "doi": "10.13345/j.cjb.250156", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40804923": { "pmid": "40804923", "title": "LAMPOX: A Portable and Rapid Molecular Diagnostic Assay for the Epidemic Clade IIb Mpox Virus Detection.", "abstract": "The global spread of Mpox virus (MPXV) underscores the urgent need for rapid, field-deployable diagnostic tools, especially in low-resource settings. We evaluated a loop-mediated isothermal amplification (LAMP) assay, termed LAMPOX, developed by Coris BioConcept. The assay was tested in three formats-two liquid versions and a dried, ready-to-use version-targeting only the ORF F3L (Liquid V1) or both the ORF F3L and N4R (Liquid V2 and dried) genomic regions. Analytical sensitivity and specificity were assessed using 60 clinical samples from confirmed MPXV-positive patients. Sensitivity on clinical samples was 81.7% for Liquid V1 and 88.3% for Liquid V2. The dried LAMPOX assay demonstrated a sensitivity of 88.3% and a specificity of 100% in a panel of 112 negative controls, with most positive samples detected in under 7 min. Additionally, a simplified sample lysis protocol was developed to facilitate point-of-care use. While this method showed slightly reduced sensitivity compared to standard DNA extraction, it proved effective for samples with higher viral loads. The dried format offers key advantages, including ambient-temperature stability and minimal equipment needs, making it suitable for point-of-care testing. These findings support LAMPOX as a promising tool for rapid MPXV detection during outbreaks, especially in resource-limited settings where traditional PCR is impractical.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2025", "doi": "10.3390/diagnostics15151959", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42508408": { "pmid": "42508408", "title": "Tumor-infiltrating plasma cell profiling after PD-1 blockade reveals tumor-specific antibodies.", "abstract": "The role of tumor-infiltrating B cells (TIL-Bs) in shaping anti-tumor responses in the context of immune checkpoint blockade remains incompletely understood. Here, we interrogate the humoral response in resected lung tumors from patients with non-small cell lung cancer (NSCLC) treated with neoadjuvant PD-1 blockade. We find that tumors orchestrate tertiary lymphoid structures with CD138+ plasma cells, from which we clone recombinant monoclonal antibodies (mAbs) using B cell receptors (BCRs) exhibiting somatic hypermutation and class switching. Several mAbs bind cell-surface citrullinated proteins, characteristic of cancer cells. Chimeric antigen receptor (CAR) T redirected with the soluble chain fragment variable (scFv) of our lead candidate antibody (PC-1) specifically target tumor cells and tumor-promoting myeloid cells in vivo without off-target activity. Moreover, ablation of the citrullination enzyme PADI2 in tumor-bearing mice eliminates reactivity to PC-1 and cytotoxic killing by the CAR. Our results implicate a therapeutic potential for tumor-infiltrating plasma cells that may be harnessed for cancer treatment.", "journal": "Cancer cell", "year": "2026", "doi": "10.1016/j.ccell.2026.07.001", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41237871": { "pmid": "41237871", "title": "Ling-Gui-Zhu-Gan decoction improves hepatic steatosis through ALKBH1-mediated DNA N6-methyladenosine modification.", "abstract": "Ling-gui-zhu-gan (LGZG) decoction, a traditional Chinese medicinal formulation with a long-established history, is frequently utilized in the treatment of fluid retention disorders, with a particular focus on metabolic dysfunction-associated steatotic liver disease (MASLD). Although its clinical efficacy has been substantiated, the precise mechanisms underlying its therapeutic effects on MASLD remain inadequately understood. This study sought to elucidate the functional role and regulatory mechanisms of LGZG decoction in the treatment of MASLD. A murine model of MASLD was induced through the administration of a high-fat diet (HFD), followed by intervention with LGZG decoction. The therapeutic efficacy of LGZG decoction in mice with MASLD was assessed through a comprehensive methodology, including triglyceride quantification, biochemical assays, hematoxylin-eosin staining, and oil red O staining. Additionally, dot blotting, methylated DNA immunoprecipitation sequencing, real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence were employed to investigate the potential mechanisms of action of LGZG decoction in the context of MASLD. ALKBH1 knockdown or overexpression in AML12 cells was performed to verify the function of ALKBH1 on steatosis, and regulation of downstream targets. The administration of LGZG decoction markedly ameliorated hepatic steatosis in mice with MASLD and enhanced the expression of AlkB homolog 1 (ALKBH1). ALKBH1 serves as a demethylase for DNA N6-methyladenosine (6 mA), facilitating the demethylation of DNA 6 mA and playing a pivotal role in the regulation of 6 mA modifications. The study demonstrated that LGZG decoction significantly diminished DNA 6 mA modification by upregulating ALKBH1 expression. Furthermore, LGZG treatment led to a reduction in 6 mA methylation on the promoters of pleckstrin and Sec7 domain containing 4 (PSD4) and ribosomal protein S23 (RPS23), which subsequently resulted in increased expression of PSD4 and RPS23 at both mRNA and protein levels. In addition, protein levels of ALKBH1 and PSD4 were significantly reduced in AML12 cells with free fatty acid treatment. Overexpression of ALKBH1 led to a significant decrease in TG levels, whereas ALKBH1 knockdown resulted in a significant increase in TG levels in AML12 cells. Overexpression of ALKBH1 also enhanced PSD4 expression, while ALKBH1 knockdown significantly suppressed PSD4 level. The findings suggest that LGZG decoction represents an effective therapeutic strategy for MASLD, alleviating hepatic steatosis through the enhancement of ALKBH1-mediated 6 mA modification, reduction of 6 mA methylation on the PSD4 promoter, and upregulation of PSD4 expression.", "journal": "Journal of ethnopharmacology", "year": "2026", "doi": "10.1016/j.jep.2025.120893", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41897819": { "pmid": "41897819", "title": "Anti-Inflammatory Effects of Lactiplantibacillus plantarum Strain FS4722 Through MAPK and NF-κB Signaling Pathways and Its Lyophilization Optimization.", "abstract": "Probiotics hold considerable promise for treating and preventing inflammatory disease; however, their application is often limited by unclear anti-inflammatory mechanisms and reduced viability following lyophilization. In this study, I thoroughly evaluated the anti-inflammatory potential of Lactiplantibacillus plantarum FS4722 (L. plantarum FS4722) and substantially enhanced strain viability through optimization of the lyoprotectant formulation. Functional assays demonstrated that the fermented supernatant, heat-inactivated bacterial suspension, and cell lysate derived from L. plantarum FS4722 effectively suppressed transcription and expression of inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages. The fermented supernatant exhibited the strongest inhibitory effects, surpassing the reference probiotic Lacticaseibacillus rhamnosus GG (LGG). Mechanistic investigations revealed that anti-inflammatory activity is primarily mediated via inhibition of the MAPK and NF-κB signaling pathways. Furthermore, using component screening combined with response surface methodology, the lyoprotectant formulation (10.00% trehalose, 1.00% sodium carboxymethyl cellulose, and 5.00% skim milk) was optimized, resulting in a lyophilization survival rate of 82.32% while maintaining cellular integrity; in this accelerated stability assessment, the strain retained 78.89% of its activity after 28 days of storage at 4 °C. Collectively, this study provides a robust and efficient approach for probiotic formulation while systematically elucidating the underlying anti-inflammatory mechanisms, thereby offering practical guidance for the development and clinical application of high-performance probiotic products.", "journal": "Foods (Basel, Switzerland)", "year": "2026", "doi": "10.3390/foods15061096", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41287386": { "pmid": "41287386", "title": "Isothermal recombinase polymerase amplification based diagnostics for female genital schistosomiasis and human papillomavirus: a review of combined molecular diagnostic opportunities.", "abstract": "Women in sub-Saharan Africa face complex, multifaceted challenges to their health, including a high burden of infectious diseases aggravated by socioeconomic factors. Parasitic and sexually transmitted infections both cause significant morbidity and mortality. Co-infections compound these effects, leading to high rates of chronic illness and making diagnosis and treatment challenging. There are no integrated approaches for the detection of female genital schistosomiasis (FGS), a gynaecological condition caused by Schistosoma haematobium, and high-risk human papillomavirus (HR-HPV), responsible for over 90% of all cervical cancer cases worldwide. FGS is a chronic condition with health outcomes such as infertility and abortion and remains severely under-reported. HR-HPV infection is the main aetiological agent of cervical cancer, the leading cause of cancer death in women in sub-Saharan Africa. Both can be disabling and stigmatizing to the sufferer. A key to disease management at patient and community levels is accurate and available diagnostics. Due to both FGS and HPV diagnostics utilising cervicovaginal samples, they are ideal candidates for a multiplex molecular diagnostic. The standard molecular diagnostics (namely PCR), through the detection of pathogen DNA, are constrained in low resource settings by requirement of a highly reliable source of energy, reliance on a cold-chain, and prohibitive costs. Isothermal molecular diagnostics are an alternative method to PCR that are more suited to basic settings. This review explores current isothermal diagnostics, with a focus on RPA/RAA, a very simple isothermal technology, for FGS and HPV and proposes the development of a multiplex isothermal diagnostic test to enable integrated screening.", "journal": "Parasitology", "year": "2025", "doi": "10.1017/S0031182025101248", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "41800537": { "pmid": "41800537", "title": "High-Throughput Centrifugal Microfluidic Nucleic Acid Diagnostic System Based on an Integrated Spatial Encoding Strategy for Simultaneous Detection of Gut Bacteria and Gut Virome.", "abstract": "Infectious intestinal disease (IID) poses a significant threat to human health and development due to its high morbidity and mortality rates. However, current diagnostics are challenged by diverse human enteropathogens and difficulties in simultaneously detecting gut bacteria and the virome. Herein, we propose a high-throughput centrifugal microfluidic nucleic acid diagnostic system that couples an integrated spatial encoding strategy with loop-mediated isothermal amplification (LAMP) for the simultaneous detection of 12 human enteropathogens (termed MiLASD) via a novel \"nucleic acid extraction-targeted amplification-spatial coding\" strategy. The MiLASD system integrates automated Chelex-100 resin-based nucleic acid purification, programmable LAMP reactions, and real-time fluorescence signal analysis into a single CD-shaped microfluidic chip. Positioned in the chip's inner circle, Chelex-100 resin efficiently removes proteins and salts, demonstrating superior sensitivity and faster kinetics than the thermolysis method. Using a single fluorescent dye, the system converts nucleic acid amplification into fluorescence signals while simultaneously detecting up to 48 targets via microfluidic spatial encoding, overcoming the multiplexing limitations inherent to conventional LAMP assays. Additionally, screened multiplex LAMP primer sets enable comprehensive human enteropathogen profiling. Post-optimization, MiLASD achieves detection limits of 102 CFU/mL for bacterial targets and 10 copies/μL for viral targets and completes multiplex identification within 45 min. Qualitative analysis of 125 clinical stool samples showed 100% positive predictive agreement and 100% negative predictive agreement. Requiring neither complex instruments nor specialized operators, MiLASD promises significant potential for enhanced diagnosis and epidemiologic surveillance of human enteropathogens.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c03991", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42723409": { "pmid": "42723409", "title": "Synergizing Nanozyme-Enzymes Catalytic Cascade by Spatial Segregation in a Hollow MOF Microreactor.", "abstract": "The integration of enzyme-nanozyme cascades presents a powerful route for diversifying catalytic conversions, yet its potential is often limited by mutual inactivation and mass transfer constraints. Here, we report a spatial compartmentalized core-shell microreactor to overcome these challenges using a metal-organic framework (MOF) hollowing and heterometallation strategy. The resulting MOF cascade microreactor features a \"dynamic\" core (enzyme)-\"static\" shell (nanozyme) nanoarchitecture. This hierarchical nano-architectonics not only mitigates diffusion limitations and keeps enzyme in a high freedom state but, more critically, shields the enzyme from deactivation by reactive oxygen species generated at the nanozyme shell. The resulting microreactor exhibits a 5.25-fold enhancement in cascade efficiency over its solid co-localized counterpart and a 1.95-fold increase over a co-localized hollow system. We demonstrate the general adaptability of this approach with multiple oxidases and leverage the catalytic cascade principle to construct a portable hydrogel-based glucose sensor with smartphone readout, achieving high sensitivity and stability for point-of-care testing. This work establishes rational spatial compartmentalization as a critical principle for developing efficient and durable enzyme-abiotic catalyst hybrids.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.75745", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41080608": { "pmid": "41080608", "title": "GSTP1 improves CAR-T cell proliferation and cytotoxicity to combat lymphoma.", "abstract": "The exhaustion of chimeric antigen receptor T cells (CAR-T) hampers the efficacy of CAR-T cell therapy. Persistent antigen stimulation in T cells results in a surge of intracellular reactive oxygen species (ROS). ROS, as mitochondrial metabolites, alter the integrity of the mitochondrial membrane and promote T-cell exhaustion. Glutathione S-transferase Pi-1 (GSTP1), a member of the glutathione S-transferase family, is an important enzyme in the intracellular clearance of ROS. Overexpression of GSTP1 may enhance the antitumor capability of CAR-T cells. The correlations between GSTP1 and genes related to T-cell exhaustion were analyzed using the TIMER database. Peripheral blood mononuclear cells (PBMCs) were collected from patients with hematologic malignancies (n = 61) and healthy donors (n = 45) to measure GSTP1, B-lymphocyte maturation protein 1 (BLIMP1), and programmed cell death protein 1 (PD-1) expression by qRT-PCR. A T-cell exhaustion model was established to assess GSTP1 expression by Western blotting. The dual-luciferase assay and ChIP-qPCR were used to determine whether the transcription factor BLIMP1 negatively regulated the activity of the GSTP1 promoter. CD19 CAR-T, GSTP1 overexpressing CAR-T (GSTP1 CAR-T), and GSTP1-knockdown CAR-T (shGSTP1 CAR-T) cells were generated to evaluate their antitumor capacity. GSTP1 expression was downregulated when BLIMP1 and PD-1 were upregulated in PBMCs of cancer patients and in the in vitro T-cell exhaustion model. Meanwhile, ROS levels in the T-cell exhaustion model increased. Mechanistically, the BLIMP1 transcription factor negatively regulated the activity of the GSTP1 promoter. Based on these findings, we engineered GSTP1 CAR-T cells, which exhibited improved functionality. GSTP1 CAR-T cells increased the TEMRA population, enhanced proliferation and cytotoxicity, elevated antioxidant capacity, increased IL-2 and IFN-γ secretion, reduced the expression of immune checkpoints, and decreased apoptosis. In vivo, the residual levels of GSTP1 CAR-T cells were higher than those of Cluster of Differentiation 19 (CD19) CAR-T cells and shGSTP1 CAR-T cells, indicating that GSTP1 CAR-T cells exhibited a strong antitumor capacity. BLIMP1 directly suppressed GSTP1 transcription, whereas GSTP1 overexpression enhanced the antitumor capacity of CAR-T cells and maintained redox homeostasis, providing a novel therapeutic strategy to improve CAR-T cell immunotherapy.", "journal": "Frontiers in immunology", "year": "2025", "doi": "10.3389/fimmu.2025.1665407", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41045143": { "pmid": "41045143", "title": "NIR-Driven Nanomotors Integrating With Platelet-Thylakoid Hybrid Membranes for Synchronized Thrombolysis and Vascular Remodeling.", "abstract": "The pathological interplay of oxidative stress, inflammation, and thrombosis driven by endothelial injury creates a self-perpetuating cycle that undermines conventional thrombolytic therapies. Herein, near-infrared (NIR)-responsive nanomotors are constructed by integrating thylakoid and platelet membranes on strontium-doped mesoporous polydopamine nanoparticles (PSr@PT NPs), which enables synchronized thrombus dissolution and vascular microenvironment restoration. Following intravenous administration, PSr@PT nanomotors exhibited preferential accumulation within thrombi and were internalized by injured endothelial cells. Meanwhile, thylakoid-embedded catalase catalyzed endogenous hydrogen peroxide to oxygen, which can not only suppress oxidative damage and neutralize to disrupt platelet-endothelium interactions, but also generate self-propulsive forces via gas propulsion, facilitating deep intrathrombus penetration of PSr@PT NPs. Proteomic analysis revealed that PSr@PT NPs inhibited thrombosis progression by downregulating platelet activation and modulating JAK-STAT/PI3K-Akt signaling pathways, thereby reducing inflammation and fostering angiogenesis. Local NIR irradiation induced mild photothermal conversion of PSr@PT NPs, which softened fibrin networks, enhanced intrathrombus infiltration, and accelerated localized thrombolysis. In both FeCl3-induced murine carotid thrombosis and arachidonic acid-induced zebrafish thrombosis models, the PSr@PT NPs effectively resolved occlusions and restored endothelial function. By coupling ROS-powered propulsion with adaptive microenvironment remodeling, this nanomotor transcends conventional 1D clot lysis, offering a dynamic strategy to prevent thrombosis recurrence and accelerate functional vascular recovery.", "journal": "Advanced materials (Deerfield Beach, Fla.)", "year": "2026", "doi": "10.1002/adma.202511733", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42226203": { "pmid": "42226203", "title": "Comparison of two menopausal hormone therapy regimens in managing menopausal symptoms: a retrospective study.", "abstract": "Femoston (estradiol/dydrogesterone) and Climen (estradiol valerate/cyproterone acetate) are two widely prescribed sequential menopausal hormone therapy (MHT) regimens that differ in estrogen formulation, progestogen component, and sequential pattern. Head-to-head comparative data on their clinical efficacy and safety remain scarce. This single-center retrospective study enrolled 80 perimenopausal and early postmenopausal women who received Femoston (n = 42) or Climen (n = 38) for six consecutive treatment cycles between January 2022 and January 2025. The primary outcome was post-treatment Kupperman Menopausal Index (KMI) score assessed by multivariable linear regression. Secondary outcomes included individual symptom response rates, serum sex hormone levels, lipid profile, hepatic enzymes, fasting plasma glucose, endometrial thickness, and adverse events. Both regimens produced substantial reductions in KMI scores from baseline (Femoston: 26.90 ± 5.72 to 10.48 ± 3.72; Climen: 27.53 ± 6.08 to 11.74 ± 4.12; both P < 0.001). After multivariable adjustment, treatment group was not independently associated with post-treatment KMI score (β = 0.58, 95% CI: -0.82 to 1.98, P = 0.412). Response rates for hot flashes/sweating, insomnia, and mood symptoms were comparably high between groups (all P > 0.05). Both regimens significantly suppressed FSH and LH and elevated E₂. Regarding lipid profiles, Femoston significantly increased HDL-C (P = 0.001) whereas Climen did not (P = 0.402); Climen significantly increased TG (P = 0.048) whereas Femoston did not (P = 0.583). After Benjamini-Hochberg correction, the between-group difference in HDL-C remained significant (adjusted P = 0.024). No clinically significant changes in hepatic enzymes, fasting glucose, or endometrial thickness were observed in either group, and no serious adverse events occurred. Femoston and Climen provide comparable short-term menopausal symptom relief. The more favorable lipid profile with Femoston, particularly its HDL-C benefit, may inform individualized regimen selection, especially in women without indications for antiandrogenic therapy. Prospective studies with longer follow-up are warranted to evaluate hard cardiovascular endpoints.", "journal": "BMC women's health", "year": "2026", "doi": "10.1186/s12905-026-04576-y", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "", "dirs": [ "A" ] }, "41603430": { "pmid": "41603430", "title": "An Extraction-free One-Pot Assay for Rapid Field Discrimination of African Swine Fever Virus Variants by a Single-Step RPA-CRISPR/Cas12a Strategy.", "abstract": "African swine fever virus (ASFV) causes devastating outbreaks in swine populations worldwide. The co-existence of wild-type and emerging gene-deleted variants (e.g., ASFV-ΔI177L) necessitates rapid on-site diagnostic tools for strain identification, which is critical for timely disease control and tailored farm management. Here, we developed a field-deployable, extraction-free one-pot assay (CORDSv2) that combines multiplex RPA and CRISPR/Cas12a to target conserved ASFV sequences and specific fluorescent markers (eGFP/mCherry) of gene-deleted variants. The assay achieved ultrasensitive detection (LOD: 6 copies/μL) and demonstrated 97.9% accuracy with 96 field samples. To streamline field operations, we integrated an extraction-free protocol (for serum/saliva) with freeze-dried reagent microspheres, enabling stable storage and direct use with minimal manual handling. When paired with a portable fluorometer (hippoCORDS), the system completes the entire sample-to-answer workflow within 1 h with a single step: addition of lysate to the microspheres. This robust, portable system addresses the urgent need for simple, on-site ASFV variant surveillance and is adaptable for other high-threat pathogens.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c03287", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39772905": { "pmid": "39772905", "title": "Prediction of protein biophysical traits from limited data: a case study on nanobody thermostability through NanoMelt.", "abstract": "In-silico prediction of protein biophysical traits is often hindered by the limited availability of experimental data and their heterogeneity. Training on limited data can lead to overfitting and poor generalizability to sequences distant from those in the training set. Additionally, inadequate use of scarce and disparate data can introduce biases during evaluation, leading to unreliable model performances being reported. Here, we present a comprehensive study exploring various approaches for protein fitness prediction from limited data, leveraging pre-trained embeddings, repeated stratified nested cross-validation, and ensemble learning to ensure an unbiased assessment of the performances. We applied our framework to introduce NanoMelt, a predictor of nanobody thermostability trained with a dataset of 640 measurements of apparent melting temperature, obtained by integrating data from the literature with 129 new measurements from this study. We find that an ensemble model stacking multiple regression using diverse sequence embeddings achieves state-of-the-art accuracy in predicting nanobody thermostability. We further demonstrate NanoMelt's potential to streamline nanobody development by guiding the selection of highly stable nanobodies. We make the curated dataset of nanobody thermostability freely available and NanoMelt accessible as a downloadable software and webserver.", "journal": "mAbs", "year": "2025", "doi": "10.1080/19420862.2024.2442750", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41789075": { "pmid": "41789075", "title": "Glycoengineering CAR-T cells to overcome galectin-3-mediated immunosuppression.", "abstract": "Chimeric antigen receptor (CAR)-T cell therapy has transformed treatment for relapsed or refractory B-cell malignancies; however, limited in vivo persistence and treatment-limiting toxicities continue to constrain durable efficacy. Because T cell glycan signatures and related galectin-binding properties impact their effector function, we postulated that CAR-T cells similarly displayed signature glycan features that govern their vulnerability to immunosuppressive galectins. In this report, public data mining, galectin-binding and glycosyltransferase expression assessments and glycomics showed that galectin (Gal)-3 was elevated in lymphoma-associated microenvironments and that anti-CD19 CAR-T cells displayed abundant Gal-3-binding glycans, reduced expression of the Gal-3-inhibitory enzyme α2,6-sialyltransferase 1 (ST6GAL1), and heightened susceptibility to Gal-3-mediated immunoregulation. To further explore this association, we enforced ST6GAL1 expression in anti-CD19 CAR-T cells and found that Gal-3-binding was obstructed and Gal-3-mediated cell death and IL-5-induction were reversed. Enforcing ST6GAL1 in CAR-T cells did not weaken tumoricidal activity and significantly improved anti-tumor responses and in vivo persistence. Collectively, this study identifies Gal-3 as a key extrinsic suppressor of CAR-T cell function and establish targeted cell surface α2,6 sialylation as a strategy to enhance CAR-T cell resistance to galectin-rich immunosuppressive microenvironments.", "journal": "Frontiers in immunology", "year": "2026", "doi": "10.3389/fimmu.2026.1766555", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40378394": { "pmid": "40378394", "title": "Re-Engineered β-Agarase for Efficient Agarose Oligosaccharide Preparation with Enhanced Thermostability.", "abstract": "Agarases are enzymes that serve as valuable catalysts in producing bioactive agar oligosaccharides. The thermostability of agarase is crucial for its industrial application since the sol-gel transition of the agar substrate is temperature-dependent. An exo-β-agarase, Aga50D, from Saccharophagus degradans 2-40 was re-engineered based on a single mutation near the interface. The A86D mutant exhibited a melting temperature increase of 23.3 °C with more than a 7-fold improvement in catalytic performance for agarose hydrolysis at 50 °C for 1 h. Small-angle X-ray scattering (SAXS), differential fluorimetry scanning (DFS), and size exclusion chromatography (SEC) analyses provided convincing evidence that the mutation significantly promoted the dimerization. Molecular dynamics simulations of the enzyme further supported our findings, demonstrating that the single mutation near the interface induced the rearrangement of the intrachain salt bridges between the loops and strengthened the interchain salt-bridge interaction at the interface, which were identified as critical mechanisms for improving its thermostability. Meanwhile, the stable interface interaction may play an important role in the dimerization of A86D. These results highlight the design of intersubunit interactions to obtain high-stability enzymes via targeted mutations near the subunit interface. The robust A86D mutant shows great potential in industrial agar oligosaccharide production.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.4c12797", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41135509": { "pmid": "41135509", "title": "Bacterial effector OspB hijacks apoptosis through peptide-bond recombination of BH3 domain proteins.", "abstract": "Apoptosis is a defense response involving key players, including BH3-only proteins that engage BCL-2 family proteins BAX and BAK, initiating mitochondrial outer membrane permeabilization and caspase activation. However, Shigella flexneri subverts these death pathways to promote infection. Here, we identify the Shigella type III secretion system effector OspB as an enzyme that suppresses apoptosis by targeting BAX and BAK. OspB recognizes BAX/BAK in complex with BH3-only activators, notably tBID, and catalyzes a peptide-bond recombination between their BH3 domains. This reaction generates chimeric proteins comprising the N-terminal BH3-only segment fused to the C-terminal region of BAX or BAK, irreversibly inhibiting protein function and thus mitochondrial outer membrane permeabilization and apoptosis. OspB-mediated apoptosis inhibition enhances S. flexneri virulence in vivo. Homologous effectors with similar catalytic activity are conserved across various bacterial species. These findings reveal a bacterial strategy for apoptosis inhibition via remodeling of BCL-2 family proteins, offering avenues for therapeutic intervention.", "journal": "Cell host & microbe", "year": "2025", "doi": "10.1016/j.chom.2025.09.018", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40318934": { "pmid": "40318934", "title": "Fungi deploy host phosphate signaling disrupter.", "abstract": "Plants have evolved sophisticated phosphate starvation response (PSR) mechanisms to maintain intracellular phosphate (Pi) homeostasis for proper growth and survival. Recently, McCombe et al. demonstrated that phytopathogens employ a novel strategy to promote disease progression by secreting conserved Nudix hydrolase effector proteins, which disrupt host Pi signaling pathways.", "journal": "Trends in plant science", "year": "2025", "doi": "10.1016/j.tplants.2025.04.010", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "41213386": { "pmid": "41213386", "title": "Lyophilization enables thermostable formulation of dengue virus-derived defective interfering particles.", "abstract": "Defective interfering particles (DIPs), containing truncated defective viral genomes (DVGs), are natural byproducts of RNA virus replication with potent antiviral activity. We recently developed a virus-free, dengue virus (DENV)-based platform producing antiviral DIPs with DI290 DVG, termed DIP-DI290. In this study, we established a scalable purification and lyophilization workflow for creating thermostable DIP-DI290 formulations for long-term storage. DIP-DI290 particles were purified via tangential flow filtration and ceramic hydroxyapatite chromatography, then lyophilized and reconstituted for biological assessment. After three months of storage at -80 °C, 4 °C, or room temperature, reconstituted DIP-DI290 retained biological activity, significantly upregulating interferon-stimulated genes (ISGs) and suppressing DENV-2 replication. These findings demonstrate that lyophilization preserves DIP-DI290's antiviral efficacy across diverse storage conditions, supporting its development as a thermostable, field-deployable therapeutic platform for virus infections.", "journal": "Antiviral research", "year": "2025", "doi": "10.1016/j.antiviral.2025.106308", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41072281": { "pmid": "41072281", "title": "Tangeretin formulated in a self-nano-emulsifying drug-delivery system (SNEDDS) alleviates symptoms of rheumatoid arthritis by antagonizing M1 macrophages polarization.", "abstract": "As targeting glycolytic reprogramming for antagonizing the polarization and functions of M1 macrophages represents a novel therapeutic strategy, finding new targets or drug candidate on glycolytic modulation is appealing. Tangeretin (TG), a natural polymethoxylated flavone with broad anti-inflammatory bioactivities, however, its low solubility limits its therapeutic applications in rheumatoid arthritis (RA). This study aimed to develop a self-nano-emulsifying drug-delivery system (SNEDDS) for enhancing the bioavailability and the anti-arthritic effects of TG, and to reveal the mechanisms underlying the inhibition of M1 macrophage polarization by targeting glycolytic reprogramming. In vivo pharmacokinetic and the anti-arthritic effect in adjuvant-induced arthritis (AIA) rats were evaluated for the optimized TG-SNEDDS formulation. Moreover, the inhibiting effects of TG on M1 macrophages polarization and function via modulating glycolytic reprogramming were evaluated In vivo and in vitro with Raw264.7 macrophages and bone marrow-derived macrophages (BMDMs). SNEDDS improved the bioavailability of TG compared to 0.5 % CMCNa or a surfactant solvent system, which leads to a significant enhancement of the therapeutic outcomes for RA symptoms and reduced the joint infiltration of M1 macrophage in AIA rats. Moreover, TG prevented M1 polarization and inhibited M1 markers including iNOS, TNF-α, and IL-6. Mechanistically, TG modulated the energy metabolism including the decreased basal and maximal respiration as well as the secretion of lactate during M1 polarization or after M1 polarization by down-regulating the glycolysis level via inhibiting LDHA, G6PD, PDH and HIF-1α. Furthermore, we revealed that TG suppressed M1 macrophage polarization by targeting LDHA, which was confirmed by DARTS and BLI experiments as well as siRNA interference. In sum, by using the formulation of SNEDDS which enhanced the solubility and bioavailability of TG, we demonstrated that TG suppressed the polarization and function of M1 macrophages via modulating metabolic reprogramming as the inhibitor of LDHA, providing a promising approach to effectively inhibit M1 macrophages polarization and alleviate symptoms of rheumatoid arthritis.", "journal": "Phytomedicine : international journal of phytotherapy and phytopharmacology", "year": "2025", "doi": "10.1016/j.phymed.2025.157313", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40618105": { "pmid": "40618105", "title": "LUPP-LAMP: one-pot multiplatform universal LAMP assay for simultaneous detection and genotyping of DENV and ZIKV.", "abstract": "Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause severe health problems upon infection, necessitating timely and accurate diagnostic testing for effective prevention and control of their transmission. In this study, a novel multiplex nucleic acids detection method was introduced based on ligation-based universal primer and probe loop-mediated isothermal amplification (LUPP-LAMP) for the simultaneous detection and genotyping of DENV and ZIKV. By employing ligation of universal primers and probes using splint DNA ligase, LUPP-LAMP simplifies primer design, eliminates primer dimer formation, and achieves high sensitivity and specificity at low temperatures, with a detection limit of 10 copies/reaction. The robustness of this strategy allows for precise differentiation even at low concentrations and in cases of co-infection, which is crucial for addressing early diagnostic challenges due to similar symptoms. LUPP-LAMP is further adapted for point-of-care testing (POCT) by integrating lateral flow test strips and fluorescence imaging, offering rapid and user-friendly results in resource-limited settings. This method is also applied to DENV genotyping (serotypes 1-4) using melting curve analysis (MCA) in cases of co-infection, aiding in epidemiological surveillance and intervention strategies. In summary, LUPP-LAMP represents a significant advancement in the detection and genotyping of DENV and ZIKV, offering a versatile tool for laboratory and POCT applications, thereby enhancing public health outcomes in the control of mosquito-borne diseases.", "journal": "Journal of nanobiotechnology", "year": "2025", "doi": "10.1186/s12951-025-03581-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40988335": { "pmid": "40988335", "title": "Chimeric enzymes enhance treatment potential for globoid cell leukodystrophy through hematopoietic stem cell gene therapy.", "abstract": "Globoid cell leukodystrophy (GLD) is a fatal lysosomal storage disorder caused by a deficiency in the β-galactosylceramidase (GALC) enzyme, leading to severe demyelination and neurodegeneration, and often death before the age of 2 years. Hematopoietic stem/progenitor cell transplantation (HSPC-T) has limited efficacy due to inadequate GALC delivery to the central (CNS) and peripheral nervous systems (PNS) and associated risks. In vivo gene therapy (GT) using adeno-associated viral vectors shows promise, but safety concerns persist. This research presents a strategy using lentiviral (LV) vector-mediated ex vivo HSPC-GT with a chimeric GALC enzyme that incorporates peptides from α-l-iduronidase (IDUA) and apolipoprotein E II (APO) to enhance expression and blood-brain barrier penetration. The chimeric IDUAsp.GALC.APO enzyme exhibited superior production and secretion compared to native GALC and previous chimeric variants in LV-transduced HSPCs, resulting in improved cross-correction and normalization of GALC activity in GLD neural cells. Proof-of-concept studies demonstrated effective enzyme production, secretion, and cross-correction capability of macrophages from GLD patients. In vivo results showed stable gene marking, sustained enzyme production, and efficient delivery of the chimeric GALC in affected organs, including the CNS and PNS. These findings highlight the potential of HSPC-GT using chimeric GALC enzymes as an innovative therapeutic approach for treating GLD.", "journal": "Molecular therapy : the journal of the American Society of Gene Therapy", "year": "2025", "doi": "10.1016/j.ymthe.2025.09.030", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41571297": { "pmid": "41571297", "title": "RIG-I agonists promote antigen-spreading and facilitate durable CAR-T responses in pancreatic ductal adenocarcinoma.", "abstract": "Pancreatic ductal adenocarcinoma (PDAC) remains largely refractory to chimeric antigen receptor (CAR)-T cell therapy. Insufficient T cell infiltration, a highly immunosuppressive microenvironment, and antigen loss pose major challenges for CAR-T cell therapy. We investigated therapeutic synergies of synthetic 5'-triphosphate RNA (3p-RNA), an agonist of the cytoplasmic double-stranded RNA sensor Retinoic Acid Inducible Gene I (RIG-I), and CAR-T cell therapy using syngeneic and human xenograft PDAC models. Tumor growth, chemokine secretion, immune-cell composition, CAR-T persistence, and endogenous T cell responses were assessed by flow cytometry, multiplex cytokine arrays, Enzyme-linked Immunospot (ELISpot), and vaccination-challenge. 3p-RNA provoked rapid type I interferon accompanied with chemokine ligand CCL5 and CXCL9/10/11 secretion, creating chemokine gradients that recruited chemokine receptor CCR5+/CXCR3+ CAR-T cells into tumors. RIG-I activation enhanced CAR-T cell proliferation, activity, and CAR-T persistence. Combination therapy eradicated established tumors in 60%-70% of mice, whereas either monotherapy was largely ineffective. Cured animals rejected CAR antigen-negative tumor cell rechallenge, demonstrating antigen-spreading and endogenous T cell responses. Intratumoral RIG-I priming reprograms the PDAC microenvironment, transforming a non-responsive cancer into a CAR-T-permissive one, supporting durable, poly-antigenic immunity. These findings position 3p-RNA as a rapid, clinically tractable co-therapy to extend CAR-T efficacy to solid tumors.", "journal": "Journal for immunotherapy of cancer", "year": "2026", "doi": "10.1136/jitc-2025-013282", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41127119": { "pmid": "41127119", "title": "Enzyme-mimicking metal-organic framework-enabled microsensor for specific electrochemical monitoring in vivo.", "abstract": "The pursuit of specific and stable electrochemical sensing techniques is vital for probing physiological and pathological processes in the living brain. Although the widespread adoption of natural enzymes is the cornerstone of current neurochemical recognition strategies, their sensing capabilities are susceptible to failure in complex biological environments. Herein, we have synthesized an ascorbate (AA) oxidase mimic, specifically a copper metal-organic framework functionalized with histidine and tryptophan (CuMOF-HT), using a chelation-assisted selective etching method. The simulant exhibits ultrafast AA sensing properties, with a response time of 0.1 seconds, along with exceptional selectivity and reliability. This is attributed to its robust Cu catalytic centers, specific amino acid recognition sites, and water-stable MOF architectures. Furthermore, the enzyme-like CuMOF-HT is integrated onto the tip of a minimally invasive acupuncture needle, forming a microelectrode that demonstrates superior electron transfer rates, enzymatic reactivity, and monitoring stability, making it ideal for subsequent in vivo AA sensing. As a demonstration, the microsensor can keenly track fluctuations in AA concentrations in mouse brain models such as epileptic seizures and cytotoxic edema. More importantly, the specific recognition strategy, which mimics the function of natural enzymes, holds broad applicability for analyzing a wide range of analytes in the field of brain science.", "journal": "Chemical science", "year": "2025", "doi": "10.1039/d5sc03207e", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40546239": { "pmid": "40546239", "title": "Buyang Huanwu Decoction Modulates the Gut Microbiota-C/EBPβ/AEP Axis to Ameliorate Cognitive Impairment in Alzheimer's Disease Mice.", "abstract": "Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and behavioral disturbances. Buyang Huanwu Decoction (BYHWD), a traditional Chinese herbal formulation, has demonstrated potential neuroprotective effects. This study aims to evaluate the therapeutic impact of BYHWD on cognitive impairments in 3×Tg mice and to investigate its underlying mechanism through modulation of the gut microbiota-C/EBPβ/AEP signaling pathway. In two independent experiments, we assessed the effects of BYHWD and its derived fecal microbiota transplantation (FMT-BYHWD) on behavioral performance, neuropathological alterations, and signaling pathways in 3×Tg mice. Treatment with BYHWD significantly improved cognitive function in 3×Tg mice and mitigated AD-like pathological changes. By suppressing the C/EBPβ/AEP signaling pathway, BYHWD reduced pathological Aβ plaque deposition, diminished tau hyperphosphorylation, and inhibited the release of pro-inflammatory cytokines. Further analysis revealed that BYHWD restored gut microbiota balance and suppressed the activation of the C/EBPβ/AEP pathway in the hippocampus. Moreover, transplanting FMT-BYHWD from BYHWD-treated mice to germ-free 3×Tg mice also ameliorated their cognitive deficits and AD-like pathology, suggesting that the anti-AD effects of BYHWD are mediated through the gut-brain axis by regulating the interplay between gut microbiota and the C/EBPβ/AEP signaling pathway. This study uncovers the mechanism by which BYHWD improves cognitive deficits and neuropathological changes in 3×Tg mice via the gut-brain axis, mediated by the modulation of the gut microbiota-C/EBPβ/AEP signaling pathway, providing a novel therapeutic strategy for AD.", "journal": "CNS neuroscience & therapeutics", "year": "2025", "doi": "10.1111/cns.70480", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "40518918": { "pmid": "40518918", "title": "Temporal- and concentration-dependent effects of transforming growth factor-beta 1 (TGF-β1) in primary human knee fibroblasts.", "abstract": "To characterize the effects of varying experimental parameters of transforming growth factor-beta 1 (TGF-β1) in primary knee fibroblasts as an in vitro model of arthrofibrosis. Primary knee fibroblasts were isolated from one patient undergoing primary total knee arthroplasty (TKA) and one patient undergoing revision TKA for arthrofibrosis, respectively. Fibroblasts were stimulated with varying concentrations and durations of the pro-fibrotic cytokine TGF-β1. Picrosirius red staining (PSR) was conducted to assess collagen deposition, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine cellular gene expression levels. A collagen gel contraction assay was used to assess TGF-β1-induced fibroblast contraction. In our experiments, TGF-β1-mediated collagen deposition was consistent across a vast concentration range (0.5 to 40 ng/ml). While α-smooth muscle actin (ACTA2) protein levels and SMAD2 phosphorylation were induced by low concentrations (1 ng/ml), robust ACTA2 transcription required higher concentrations (5 ng/ml) of TGF-β1. Evaluation of TGF-β1-mediated cell and cytoskeletal contractility shows that in the measured fibroblasts, this process occurs within three hours following TGF-β1 administration. Of note, TGF-β1-differentiated myofibroblasts exhibited greater contractile properties than naïve fibroblasts in the presence of TGF-β1. Taken together, this study elucidates key TGF-β1 experimental parameters that will inform the development and interpretation of in vitro models of arthrofibrosis.", "journal": "Bone & joint research", "year": "2025", "doi": "10.1302/2046-3758.146.BJR-2024-0387.R3", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40223663": { "pmid": "40223663", "title": "Survey of pathogens and human fecal markers in stormwater across a highly populated urban region.", "abstract": "Microbial contamination of urban stormwater, rivers, and creeks during rainstorms is a persistent and widespread problem. Remediation of these waters has proven to be challenging since there are many potential sources for the fecal indicator bacteria on which water quality is regulated. Microbial source tracking markers have allowed for improved identification and quantification of the sources of contamination, but the majority of the source-attributed microbial targets are not responsible for causing the illnesses associated with waterborne human fecal contamination. Thus there is a need to better understand the relationships of human pathogens and human fecal markers in stormwater. In this study, we used a spatially-intensive sampling approach (31 sites) across southern California for the analysis of stormwater. During three storms from 2021-2023, we used droplet digital PCR to quantify the human fecal markers HF183 and Lachno3 along with human adenovirus, human norovirus, Campylobacter spp., and Salmonella spp. This spatially intensive sampling design captures information from a 5900 km2 area with ∼22 million people. We detected human markers HF183 and Lachno3 genes at 90% and 97% of the sites; concentrations ranged from below detection to 104 and 105 gene copies per 100 mL, respectively. We found variable concentrations of human bacterial and viral pathogen genes. HF183 was significantly correlated to human adenovirus and Lachno3. Lachno3 was also significantly correlated with Salmonella. We reported PCR inhibition in 83-90% of the samples but found that separating sediment and adding proteinase K during lysis improved DNA/RNA extraction efficiency and reduced inhibition.", "journal": "Environmental science. Processes & impacts", "year": "2025", "doi": "10.1039/d4em00578c", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40073838": { "pmid": "40073838", "title": "Heme metabolism mediates RANKL-induced osteoclastogenesis via mitochondrial oxidative phosphorylation.", "abstract": "Bone undergoes life-long remodeling, in which disorders of bone remodeling could occur in many pathological conditions including osteoporosis. Understanding the cellular metabolism of osteoclasts (OCs) is key to developing new treatments for osteoporosis, a disease that affects over 200 million women worldwide per annum. We found that human OC differentiation from peripheral blood mononuclear cells derived from 8 female patients is featured with a distinct gene expression profile of mitochondrial biogenesis. Elevated mitochondrial membrane potential (MMP, Δψm) was also observed in receptor activator of NF-κB ligand (RANKL)-induced OCs. Interestingly, the gene pathways of heme synthesis and metabolism were activated upon RANKL stimulation, featured by transcriptomic profiling in murine cells at a single-cell resolution, which revealed a stepwise expression pattern of heme-related genes. The real-world human data also divulges potential links between heme-related genes and bone mineral density. Heme is known to have a role in the formation of functional mitochondrial complexes that regulate MMP. Disruption of heme biosynthesis via genetically silencing Ferrochelatase or a selective inhibitor, N-methyl Protoporphyrin IX (NMPP), demonstrated potent inhibition of OC differentiation, with a dose-dependent effect observed in NMPP treatment and a substantial efficacy even at a single dose. In vivo study further showed the protective effect of NMPP on ovariectomy-induced bone loss in female mice. Collectively, we found that RANKL-mediated signaling regulated mitochondrial formation and heme metabolism to synergistically support osteoclastogenesis. Inhibition of heme synthesis impaired OC formation and reversed excessive bone loss, representing a new therapeutic target for metabolic skeletal disorders.", "journal": "Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research", "year": "2025", "doi": "10.1093/jbmr/zjaf040", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40089980": { "pmid": "40089980", "title": "Sunlight-sensitive carbon dots for plant immunity priming and pathogen defence.", "abstract": "Global food production faces persistent threats from environmental challenges and pathogenic attacks, leading to significant yield losses. Conventional strategies to combat pathogens, such as fungicides and disease-resistant breeding, are limited by environmental contamination and emergence of pathogen resistance. Herein, we engineered sunlight-sensitive and biodegradable carbon dots (CDs) capable of generating reactive oxygen species (ROS), offering a novel and sustainable approach for plant protection. Our study demonstrates that CDs function as dual-purpose materials: priming plant immune responses and serving as broad-spectrum antifungal agents. Foliar application of CDs generated ROS under light, and the ROS could damage the plant cell wall and trigger cell wall-mediated immunity. Immune activation enhanced plant resistance against pathogens without compromising photosynthetic efficiency or yield. Specifically, spray treatment with CDs at 240 mg/L (2 mL per plant) reduced the incidence of grey mould in N. benthamiana and tomato leaves by 44% and 12%, respectively, and late blight in tomato leaves by 31%. Moreover, CDs (480 mg/L, 1 mL) combined with continuous sunlight irradiation (simulated by xenon lamp, 9.4 × 105 lux) showed a broad-spectrum antifungal activity. The inhibition ratios for mycelium growth were 66.5% for P. capsici, 8% for S. sclerotiorum and 100% for B. cinerea, respectively. Mechanistic studies revealed that CDs effectively inhibited mycelium growth by damaging hyphae and spore structures, thereby disrupting the propagation and vitality of pathogens. These findings suggest that CDs offer a promising, eco-friendly strategy for sustainable crop protection, with potential for practical agricultural applications that maintain crop yields and minimize environmental impact.", "journal": "Plant biotechnology journal", "year": "2025", "doi": "10.1111/pbi.70050", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42341328": { "pmid": "42341328", "title": "Decoding fibrosis: Transcriptomic and clinical insights via AI-derived collagen deposition phenotypes in MASLD.", "abstract": "Histological assessment is foundational to multi-omics studies of liver disease, yet conventional fibrosis staging lacks resolution, and quantitative metrics like collagen proportionate area (CPA) fail to capture tissue architecture. While recent artificial intelligence (AI)-driven approaches offer improved precision, they are proprietary and not accessible to academic research. Here, we present a novel, interpretable AI-based framework for characterizing liver fibrosis from picrosirius red (PSR)-stained slides. By identifying distinct data-driven collagen deposition phenotypes (CDPs) that capture distinct morphologies, our method substantially improves the sensitivity and biological specificity of downstream transcriptomic and proteomic analyses compared with CPA and traditional fibrosis scores. Pathway analysis reveals that CDPs 4 and 5 are associated with active extracellular matrix remodeling, while phenotype correlates highlight links to liver functional status. Importantly, selected CDPs demonstrated prognostic associations in the discovery cohort, with attenuation of discrimination in the external validation cohort. We've developed a novel digital pathology framework for liver fibrosis quantification. All models and tools are made freely available to support transparent and reproducible multi-omics pathology research.", "journal": "Hepatology (Baltimore, Md.)", "year": "2026", "doi": "10.1097/HEP.0000000000001811", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41758981": { "pmid": "41758981", "title": "Phase 2 study of the lysine-specific demethylase 1 inhibitor bomedemstat for essential thrombocythemia.", "abstract": "Novel treatments that can improve outcomes of essential thrombocythemia (ET) are needed. In a phase 2 trial, participants with ET who required cytoreduction and had inadequate response to, or were intolerant of, ≥1 standard therapy received bomedemstat at a starting dose of 0.6 mg/kg per day, titrated to achieve a target platelet count (200 × 109/L to 400 × 109/L). Primary end points were safety and response, defined as a platelet count of ≤400 × 109/L without new thromboembolic events. Of 73 who received bomedemstat, at 24 weeks, 49 of 64 evaluable participants (77%) achieved a response. Durable reductions in platelet count (≤400 × 109/L for ≥12 weeks) were observed in 52 of 72 participants (72%). Durable reduction in white blood cell count (<10 × 109/L for ≥12 weeks) was observed in 61 of 72 participants (85%); of 10 participants with elevated white blood cell count at baseline, 9 had normal white blood cell count (<10 × 109/L) at week 24. Hemoglobin levels remained stable. After 24 weeks of treatment, a decrease in variant allele frequency of CALR, JAK2, or MPL was observed in 39 of 46 (85%) evaluable participants. By week 24, 2 of 73 participants (3%) had experienced ≥1 thrombotic event, and 15 of 73 (21%) experienced ≥1 hemorrhagic event. During overall treatment period, grade 3 or 4 adverse events (AEs) occurred in 34 of 73 participants (47%). AEs led to temporary treatment interruption in 29 participants (40%) and permanent discontinuation in 11 (15%). No participants died due to AEs. Bomedemstat had clinically relevant activity and manageable safety in participants with ET. This trial was registered at www.clinicaltrials.gov as #NCT04254978.", "journal": "Blood advances", "year": "2026", "doi": "10.1182/bloodadvances.2025017575", "ptypes": [ "Journal Article", "Clinical Trial, Phase II", "Multicenter Study" ], "first_author": "", "dirs": [ "B" ] }, "40977164": { "pmid": "40977164", "title": "Perspectives on deciphering thermotolerance mechanisms in Heliotropium thermophilum: integrating biochemical responses and gene expression patterns.", "abstract": "High temperature stress significantly impacts plant viability and productivity. Understanding thermotolerance mechanisms is essential for developing resilient crops. Heliotropium thermophilum , endemic to geothermal areas with extreme soil temperatures, serves as a model for studying plant high temperature stress responses. We aim to elucidate the biochemical and molecular mechanisms underlying thermotolerance in H. thermophilum . Biochemical assays quantified osmoprotectants (proline, soluble sugars, glycine-betaine, and total phenolics) and lipid peroxidation in H. thermophilum under different soil temperatures. Transcriptome analysis and quantitative Real-Time PCR were performed to validate the expression of genes involved in osmoprotectant biosynthesis, antioxidant defense, and cell wall modification. Glycine-betaine and proline levels increased by up to 189% and 104%, respectively, during peak stress. Elevated total phenolics correlated with reduced lipid peroxidation, indicating effective oxidative stress mitigation. Transcriptome analysis revealed significant upregulation of genes related to osmoprotectant biosynthesis, antioxidant defense, and cell wall modification, with notable expression of heat shock proteins and sugar transport genes. H. thermophilum employs an integrative biochemical and molecular strategy to withstand high soil temperatures, involving osmoprotectant accumulation, enhanced antioxidant defenses, and dynamic cell wall remodeling. These findings provide insights into thermotolerance mechanisms, offering potential targets for enhancing high temperature stress resilience in other crops. This study contributes to understanding plant-soil interactions and developing strategies to ensure agricultural productivity amid global climate change.", "journal": "Functional plant biology : FPB", "year": "2025", "doi": "10.1071/FP24288", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41395373": { "pmid": "41395373", "title": "RNA detection on a microfluidic platform using Thyclotides.", "abstract": "Nucleic acid detection is commonly used to diagnose pathogenic and genetic diseases. Diagnostics based on polymerase chain reaction (PCR) are routinely used to amplify and signal the presence of a target nucleic acid, but PCR-based methods are difficult to deploy at the patient's point-of-care and in resource-limited environments. This study reports a novel nucleic acid detection assay that utilizes chemically modified PNAs to directly detect a target RNA without enzymatic amplification. By incorporating trans-3,4-diaminotetrahydrofuran units into a PNA backbone, THF-PNAs (also called thyclotides) were designed with both enhanced binding affinity to target nucleic acids and bio-orthogonal properties that promote thyclotide-to-thyclotide binding over nucleic acid binding. Specifically, incorporation of R,R-THF monomers within the thyclotide backbone promotes a right-handed helix favoring binding to target nucleic acids. Bio-orthogonal thyclotide containing S,S-THF monomers promotes a left-handed helix, preventing their binding to natural nucleic acids. Complementary bio-orthogonal thyclotides can bind to each other in the presence of competing RNA sequences, and these unique properties were used in combination with gold nanoparticles to develop a detection signal. By employing both THF stereochemistries in different thyclotide sequences, a prototype microfluidic assay was developed to detect synthetic HIV-1 RNA and signal its presence using silver-based enhancement of surface-bound gold nanoparticles. The limit of detection for this assay was 0.5 pM of synthetic HIV-1 RNA, which is a significant (~100-fold) improvement over earlier PNA-based detection system. Concentration-dependent variation in detection signal intensity allows for semi-quantitative determination of different RNA concentrations. A scrambled RNA control sequence does not interfere with detection. All results were obtained without using enzymatic amplification. The thyclotides in this study may be used in more advanced diagnostic technologies.", "journal": "Sensors and actuators reports", "year": "2026", "doi": "10.1016/j.snr.2025.100410", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41060066": { "pmid": "41060066", "title": "A clarifying perspective on bacterial pseudo-receiver domains.", "abstract": "Two-component regulatory systems typically consist of a sensor kinase and a response regulator. Phosphorylation of the receiver domain controls response regulator activity. Pseudo-receivers (PsRs) are identified computationally as receivers but lack key residues to catalyze phosphotransfer reactions. Although PsRs are common, molecular mechanisms that activate and inactivate bacterial PsRs remain a mystery. We untangled four potentially related but distinct concepts: bacterial PsRs, receivers with regulatory mechanisms in addition to phosphorylation, receivers that are active without phosphorylation, and orphan receivers without an obvious partner sensor kinase. We also analyzed bacterial PsR sequences and structures to identify regions of likely functional significance.", "journal": "Journal of bacteriology", "year": "2025", "doi": "10.1128/jb.00261-25", "ptypes": [ "Journal Article", "Review", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42218183": { "pmid": "42218183", "title": "Development of loop-mediated isothermal amplification (LAMP)-based assay for rapid detection of fosfomycin resistance gene fosA.", "abstract": "Fosfomycin is an old treatment agent that has been reintroduced as a first-line antibiotic for the treatment of acute uncomplicated urinary tract infections caused by multidrug-resistant bacteria. However, resistance to fosfomycin is rising, and limited options for its detection are available. In this study, we developed a molecular technique based on loop-mediated isothermal amplification (LAMP) as a rapid and easy-to-perform method for the detection of the fosA resistance gene. LAMP primers were designed using the Primer Explorer V5 software. A total of 63 bacterial strains (34 Klebsiella pneumoniae and 29 Escherichia coli) previously characterized by whole genome sequencing, with different MLST and resistant to fosfomycin were used to evaluate the performance of the new LAMP assay. The fosA LAMP-based assay was standardized for optimum primer concentration and time of reaction. Its sensitivity and specificity were also characterized. LAMP products were detected by visual detection (positive reactions turned yellow while negative controls remained pink) and by electrophoresis. Positive LAMP reaction occurred with incubation at 65 °C for 40 min. Nevertheless, amplification products were already observed at 20 min. The overall sensitivity and specificity of the LAMP assay were 90.5% and 90.9%, respectively. For Klebsiella pneumoniae, sensitivity reached 100%, while specificity could not be determined due to the absence of negative samples. In Escherichia coli, sensitivity was 43% and specificity was 90.9%. The limit of detection of LAMP reaction was 8 pg/µL. To the best of our knowledge, this is the first study to develop a LAMP methodology to detect the fosA gene. This LAMP-based assay is a promising rapid molecular method for fosfomycin detection, which could be implemented for on-site analysis in resource-limited settings, with the potential to improve antibiotic therapy interventions for urinary tract infections.", "journal": "Scientific reports", "year": "2026", "doi": "10.1038/s41598-026-52613-5", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42779094": { "pmid": "42779094", "title": "Amorphization and microencapsulation of β-carotene with improved physical stability and bioaccessibility for emulsion-based 3D printing.", "abstract": "β-Carotene is widely used as a natural colorant and nutritional ingredient, but its poor water dispersibility, high crystallinity and low stability limit its application in functional foods and three-dimensional (3D)-printed food systems. In this study, a solvent-free hot-melt/quenching strategy was combined with wet milling and spray drying to fabricate amorphous β-carotene microcapsules using octenyl succinic anhydride-modified starch (OSA-starch) and trehalose as wall materials. Hot-melt/quenching treatment effectively transformed crystalline β-carotene into an amorphous state, and the optimal condition was determined as 220 °C for 22 s followed by liquid nitrogen quenching. Compared with crystalline β-carotene microcapsules, amorphous β-carotene microcapsules showed reduced crystallinity and improved encapsulation behavior. The incorporation of trehalose into the OSA-starch wall matrix further decreased particle aggregation, improved redispersibility and enhanced storage stability. After 40 days of storage, amorphous β-carotene microcapsules prepared with the OSA-starch/trehalose composite wall materials (AB-Tre) retained 82.2% and 58.9% of β-carotene at 25 °C and 55 °C, respectively, which were higher than those of the other formulations. During in vitro digestion, AB-Tre showed better colloidal stability and higher β-carotene bioaccessibility than crystalline β-carotene microcapsules prepared with the OSA-starch/trehalose composite wall materials (CB-Tre). Furthermore, AB-Tre-loaded high internal phase emulsions showed good printability and could be used to fabricate customized 3D structures. The combination of hot-melt-induced amorphization and OSA-starch/trehalose microencapsulation provides an effective strategy to improve the stability, bioaccessibility and 3D printing applicability of β-carotene. © 2026 Society of Chemical Industry.", "journal": "Journal of the science of food and agriculture", "year": "2026", "doi": "10.1002/jsfa.71114", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41508269": { "pmid": "41508269", "title": "[Mechanistic study on combined application of Puerariae Lobatae Radix polysaccharide and betaine in ameliorating hepatic and intestinal injury via regulating TLR4 signaling pathway in alcoholic liver disease mice].", "abstract": "This study aims to investigate the synergistic protective effects and underlying mechanisms of the combined application of Puerariae Lobatae Radix polysaccharide 1(PLP1) and betaine in alcohol-induced liver and intestinal injuries in mice and AML-12 hepatocytes. An alcoholic liver disease(ALD) mouse model was established according to the NIAAA protocol using chronic ethanol feeding combined with acute ethanol gavage. Mice were randomly divided into six groups: control, model, PLP1 monotherapy, betaine monotherapy, low-dose combination(LD-Comb), and high-dose combination(HD-Comb). Complementary in vitro studies involved ethanol-injured AML-12 mouse hepatocytes, divided into eight groups: control, model, PLP1, betaine, LD-Comb, medium-dose(MD-Comb), HD-Comb, and LD-Comb + lipopolysaccharide(LPS, 1 μg·mL~(-1)). Hepatic and intestinal histopathological changes were evaluated using hematoxylin-eosin(HE) staining, Sirius red staining, and immunohistochemistry. Alanine aminotransferase(ALT), aspartate aminotransferase(AST), alcohol dehydrogenase(ADH), and aldehyde dehydrogenase(ALDH) were quantified by biochemical assays. Enzyme-linked immunosorbent assays(ELISA) measured tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-1β(IL-1β), and LPS levels. Protein expressions of cytochrome P450 2E1(CYP2E1), zonula occludens-1(ZO-1), Occludin, mucin-2(MUC-2), Toll-like receptor 4(TLR4), nuclear factor-κB(NF-κB), and myeloid differentiation primary response 88(MyD88) were analyzed using Western blot, immunohistochemistry, and immunofluorescence. The mRNA expression of TLR4, NF-κB, and MyD88 was analyzed by quantitative PCR(qPCR). The results showed that, compared with the model group, PLP1 or betaine monotherapy significantly alleviated hepatic edema and inflammatory infiltration, reduced collagen deposition, increased intestinal mucus thickness, restored goblet cell count, and decreased serum levels of IL-6, TNF-α, IL-1β, LPS, ALT, and AST, while enhancing ADH and ALDH activity. Concurrently, CYP2E1, TLR4, NF-κB, and MyD88 expression levels were downregulated, and the expression of ZO-1, occludin, and MUC-2 in colon tissues was upregulated. The combined treatment of PLP1 and betaine further enhanced these improvements compared with monotherapies, although the difference between the LD-Comb and HD-Comb groups was not statistically significant. In AML-12 cells, PLP1 or betaine monotherapy significantly reduced ALT, AST, IL-6, and TNF-α levels in the culture supernatant and downregulated the expression of TLR4, NF-κB, and MyD88. The combined treatment showed stronger effects than monotherapy, while co-treatment with the TLR4 agonist LPS partially reversed these benefits. In conclusion, the combination of PLP1 and betaine exerts synergistic protective effects by enhancing ethanol metabolism, promoting tight junction protein synthesis, repairing intestinal barrier function, reducing LPS translocation, and suppressing the activation of the TLR4 signaling pathway, thereby lowering inflammatory cytokine levels. This multi-targeted synergy effectively alleviates liver and intestinal injury in ALD mice and ethanol-injured AML-12 hepatocytes.", "journal": "Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica", "year": "2025", "doi": "10.19540/j.cnki.cjcmm.20250725.703", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40203083": { "pmid": "40203083", "title": "Rapid tuberculosis diagnosis from respiratory or blood samples by a low cost, portable lab-in-tube assay.", "abstract": "Rapid portable assays are needed to improve diagnosis, treatment, and reduce transmission of tuberculosis (TB), but current tests are not suitable for patients in resource-limited settings with high TB burden. Here we report a low complexity, lab-in-tube system that is read by an integrated handheld device that detects Mycobacterium tuberculosis (Mtb) DNA in blood and respiratory samples from a variety of clinical settings. This microprocessor-controlled device uses an LCD user interface to control assay performance, automate assay analysis, and provide results in a simple readout. This point-of-care single-tube assay uses a DNA enrichment membrane and a low-cost cellulose disc containing lyophilized recombinase polymerase amplification and CRISPR-Cas12a reagents to attain single-nucleotide specificity and high sensitivity within 1 hour of sample application, without a conventional DNA isolation procedure. Assay results obtained with serum cell-free DNA isolated from a cohort of children aged 1 to 16 years detected pulmonary and extrapulmonary TB with high sensitivity versus culture and GeneXpert MTB/RIF results (81% versus 55% and 68%) and good specificity (94%), meeting the World Health Organization target product profile criteria for new nonsputum TB diagnostics. Changes in assay results for serum isolated during treatment were also highly predictive of clinical response. Results obtained with noninvasive sputum and saliva specimens from adults with bacteriologically confirmed pulmonary TB were also comparable to those reported for reference methods. This rapid and inexpensive lab-in-tube assay approach thus represents one means to address the need for point-of-care TB diagnostics useable in low-resource settings.", "journal": "Science translational medicine", "year": "2025", "doi": "10.1126/scitranslmed.adp6411", "ptypes": [ "Journal Article", "Research Support, U.S. Gov't, Non-P.H.S.", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "A" ] }, "41656649": { "pmid": "41656649", "title": "Cosmeceutical and Dermatological Potential of Shiitake Mushroom (Lentinula edodes) Extract.", "abstract": "Shiitake mushroom (Lentinula edodes) contains polysaccharides, phenolics, and β-glucans known for their antioxidant and nutraceutical properties; however, its dermatological applications remain limited. This study aimed to evaluate the antioxidant potential, enzyme inhibition activities, and cytotoxicity of freeze-dried Shiitake extract and to develop and characterize a topical cream formulation containing the extract for potential cosmetical and dermatological use. Shiitake mushrooms were extracted with 75% ethanol and freeze-dried. Antioxidant capacity was assessed using a total antioxidant assay. Elastase, collagenase, and tyrosinase inhibition activities were evaluated using fluorometric and spectrophotometric assays. Cytotoxicity was tested on HaCaT human keratinocytes using the AlamarBlue assay. A topical cream formulation containing the shiitake extract was prepared, and its pH, viscosity, density, and stability were assessed through accelerated stability tests. The extract showed moderate antioxidant capacity (107 μM Trolox equivalent). It inhibited elastase activity by 51.99% and induced a concentration-dependent suppression of collagenase activity, reducing measurable enzyme activity to levels below the detection limit of the assay at higher concentrations. No significant tyrosinase inhibition was observed. Cytotoxicity assays confirmed that the extract was non-toxic, maintaining > 90% HaCaT viability across all tested concentrations. The formulated cream exhibited skin-compatible pH (≈5.5), suitable viscosity, homogeneous texture, and remained stable without phase separation or microbial growth under accelerated aging conditions. Shiitake extract appears to be a safe and naturally derived bioactive ingredient, exhibiting notable elastase and collagenase inhibitory activities and demonstrating promising potential for incorporation into anti-aging and nutraceutical skin-care formulations.", "journal": "Journal of cosmetic dermatology", "year": "2026", "doi": "10.1111/jocd.70724", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39990222": { "pmid": "39990222", "title": "The sentinel against brain injury post-subarachnoid hemorrhage: efferocytosis of erythrocytes by leptomeningeal lymphatic endothelial cells.", "abstract": "Rationale: The clearance of extravasated erythrocytes represents the most reasonable strategy against brain injury post-subarachnoid hemorrhage (SAH). There is little knowledge about the autologous clearance of extravasated erythrocytes post-SAH. The leptomeningeal lymphatic endothelial cells (LLECs) have been less studied functionally, which were firstly harvested and cultured in vitro by our group previously and are probably related to the clearance of extravasated erythrocytes post-SAH for they closely surround subarachnoid space. Methods: We established a SAH animal model, employed primary LLECs in vitro, mimicked the conditions of the SAH in vitro, performed RNA sequencing, and transfected LLECs with adenovirus and adeno-associated virus both in vivo and in vitro to reveal the molecular mechanisms of efferocytosis of erythrocytes by LLECs and its neuroprotection post-SAH. Results: Firstly, we demonstrated the eryptosis-initiated degradation of extravasated erythrocytes in vitro. Furthermore, we found LLECs preferentially adhered and engulfed apoptotic erythrocytes in vivo and in vitro while sparing from intact erythrocytes, suggesting their novel capacity in the efferocytosis of erythrocytes. Additionally, the efferocytosis of erythrocytes by LLECs plays a role on neuroprotection via improving neurological functions, maintaining neurostructural integrity, and alleviating neuropathological consequences post-SAH. During efferocytosis, phosphatidylserine (PS) and phosphatidylserine receptor (PSR) mediated the recognition of apoptotic erythrocytes by LLECs. We also confirmed that NHL repeat-containing 2 (NHLRC2) positively regulated the efferocytosis of erythrocytes by LLECs to serve as a central regulator in it mediated neuroprotection post-SAH. Conclusions: This study elucidated the efferocytosis of erythrocytes by LLECs and subsequently neuroprotection post-SAH. These findings highlight a prompt, efficient, and regulable pathway for the autologous clearance of extravasated erythrocytes that performs as a sentinel against brain injury post-SAH.", "journal": "Theranostics", "year": "2025", "doi": "10.7150/thno.103701", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41599470": { "pmid": "41599470", "title": "Bio-Based Viscoelastic Polyurethane Foams: Functional Behavior Across Application Temperatures.", "abstract": "Viscoelastic polyurethane foams were prepared using four different bio-based polyols derived from coconut oil (CO), palm oil (PO), duck fat (DF), and pork fat (PF), employing up to 20 wt.% of the polyol component in a conventional formulation. The introduction of bio-polyols into the polyurethane formulation gave rise to an early minor decomposition of modified foams at low temperatures; however, the overall thermal stability improved slightly by the elimination of some intermediate decomposition stages. The glass transition temperature of foams was only moderately influenced and remained in the typical temperature range (around 10 °C). The effect of biopolyol type and content (5-20 wt.%) on the mechanical properties of the foams was investigated over the temperature range -20 to 40 °C. At 20 and 40 °C, all foams exhibited comfortable viscoelastic properties suitable for furniture applications. Hysteresis and the damping behavior of foams were also influenced by biopolyol type and concentration, with CO and DF providing enhanced energy absorption. Overall, these bio-based foams demonstrate potential for eco-friendly, high-performance applications, although their use at temperatures below 10 °C may be limited by increased stiffness.", "journal": "Polymers", "year": "2026", "doi": "10.3390/polym18020174", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41538097": { "pmid": "41538097", "title": "Optimization of Expression and Thermostability of Terminal Deoxynucleotidyl Transferase Through Iterative Mutagenesis and Computational Design.", "abstract": "Terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase that catalyzes the addition of deoxynucleoside triphosphates to the 3'-terminus of a DNA strand. While TdT is a key enzyme for developing enzymatic DNA synthesis technologies, its inherent low thermal stability presents a significant limitation. This study aims to improve the thermostability of TdT from Mus musculus through a combination of site-saturation mutagenesis and rational design. Residues for saturation mutagenesis were identified using B-factor analysis and the B-FITTER program, while promising substitutions for rational design were selected using Foldit, ProteinMPNN and CARBonAra. Through several iterations of mutagenesis, we obtained two highly promising variants. The first one, dubbed A4, obtained solely through saturation mutagenesis, showed a 26-fold higher expression level than the WT protein. The second one, dubbed mutant 275, demonstrated exceptional stability, showing no significant loss of activity after 180 min of incubation at 45 °C - conditions under which the wild-type enzyme's half-life was less than 2 min. This corresponds to a > 120-fold increase in stability. Additionally, its melting temperature (Tm) was increased by 6.5 °C. Moreover, mutant 275 demonstrated a 4- to 6-fold increase in catalytic activity at 37 °C. This significant enhancement in thermostability was achieved after four rounds of iterative mutagenesis and is attributed to the formation of a stabilizing salt bridge network on the protein surface, distant from the active site. The obtained thermostable TdT variants serve as robust scaffolds for further engineering to improve activity towards the 3'-reversibly blocked nucleotides required for next-generation enzymatic DNA synthesis.", "journal": "Applied biochemistry and biotechnology", "year": "2026", "doi": "10.1007/s12010-025-05569-7", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41581244": { "pmid": "41581244", "title": "IL-6 blockade at the fracture site accelerates bone healing via inflammatory modulation of sensory nerve CGRP signaling.", "abstract": "Delayed fracture healing and nonunion are commonly associated with impaired callus remodeling and dysregulated interleukin-6 (IL-6) signaling; however, the precise role of IL-6 in fracture repair remains controversial. Calcitonin gene-related peptide (CGRP), a sensory neuropeptide expressed at fracture sites, has been shown to suppress IL-6 expression and promote bone regeneration. Nevertheless, whether IL-6 reciprocally regulates CGRP expression or function, thereby influencing fracture healing, remains unclear. This study aimed to determine whether local inhibition of IL-6 enhances CGRP expression and subsequently promotes femoral fracture healing. Adult male C57BL/6 J mice were randomly assigned to four groups to receive different post-fracture treatments. On day 7 after femoral fracture, MR16-1 (a monoclonal anti-mouse IL-6 receptor antibody) was locally administered using a poloxamer 407 hydrogel delivery system, with or without the CGRP receptor antagonist BIBN4096. The poloxamer 407 hydrogel was characterized In vitro for encapsulation efficiency, drug-loading capacity, gelation time, swelling behavior, degradation at 37 °C, and drug-release profile. Fracture healing was assessed at 4 weeks post-fracture by X-ray imaging, micro-computed tomography, and histological analysis. Immunofluorescence staining was performed to examine the expression of bone-related proteins, CGRP, and IL-6. Protein expression levels of IL-6, CGRP, Alkaline Phosphatase (ALP), Runt-related transcription factor 2 (Runx2), Osteocalcin (OCN), Nuclear Factor of Activated T cells Cytoplasmic 1 (NFATC1), Cathepsin K (CTSK), Sclerostin, Cluster of Differentiation 68 (CD68), Cluster of Differentiation 86 (CD86), Cluster of Differentiation 206 (CD206), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1 beta (IL-1β), Inducible Nitric Oxide Synthase (iNOS), Interleukin-10 (IL-10), Transforming Growth Factor-beta (TGF-β), and Arginase-1 (Arg1) were analyzed by Western blotting. Quantitative real-time PCR (qRT-PCR) was used to quantify mRNA expression of inflammatory cytokines, including the pro-inflammatory markers TNF-α, IL-1β, and iNOS, as well as the anti-inflammatory markers IL-10, TGF-β, and Arg1. X-ray assessment and healing scores showed that MR16-1 markedly accelerated fracture repair and the effects were largely reversed when combined with BIBN4096. μCT analysis further revealed that MR16-1 significantly increased callus volume and mineralization, while bone mass at distant sites, including the L5 vertebra and contralateral femur, remained unchanged. In contrast, its combination with BIBN4096 resulted in a reduction of callus volume and mineralization. Histological analysis showed that MR16-1 promoted collagen deposition, reduced cartilage content, and enhanced new bone formation, whereas combined treatment delayed bone maturation. At the molecular level, IL-6 expression was decreased, while CGRP and OCN levels were elevated and osteoclast activity reduced. Pro-inflammatory cytokines (TNF-α, IL-1β) and M1 macrophage markers (iNOS, CD86) were downregulated, whereas anti-inflammatory cytokines (IL-10, TGF-β) and M2 macrophage markers (Arg1, CD206) were upregulated; these effects were partially reversed by co-administration of the CGRP receptor antagonist BIBN4096.Western blot analysis showed that ALP, Runx2, and OCN expression were upregulated, while RANKL, CTSK, and sclerostin expression were downregulated, which may be beneficial for bone formation. However, simultaneous exposure to BIBN4096 weakened these effects and may impair bone regeneration to some extent. These results suggest that local administration of MR16-1 may enhance CGRP expression, promote callus maturation, and accelerate fracture healing, indicating a potential role in bone repair.", "journal": "International immunopharmacology", "year": "2026", "doi": "10.1016/j.intimp.2026.116258", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41916461": { "pmid": "41916461", "title": "High efficiency biosynthesis of 1α,25-Dihydroxyvitamin D3 via engineered dihydroxylase and deep eutectic solvent based resting cell bioprocess.", "abstract": "1α,25-dihydroxyvitamin D3 (1α,25(OH)2VD3), the biologically active form of Vitamin D3 (VD3), plays a crucial role in a wide range of physiological processes. However, the efficient biosynthesis of 1α,25(OH)2VD3 is hindered by the low activity of natural enzymes, poor solubility of substrate VD3, and its intracellular metabolic loss. Herein, we developed an integrated bioprocess by synergizing engineered dihydroxylase (CYP109A2-H) with a deep eutectic solvent (DES) based resting cell system to overcome these limitations. A semi-rational design strategy was employed to enhance the performance of the enzyme, which yielded several beneficial mutants including E81G that increased the catalytic titer by 43.6%. Concurrently, a DES resting cell system was constructed, wherein DES dramatically increased VD3 solubility and resting cells eliminated metabolic loss. This synergistic strategy achieved a record-high 1α,25(OH)2VD3 space-time yield of 33.15 mg·L-1·d-1, establishing the most efficient single-batch bioconversion reported to date. This work provides a sustainable and efficient platform for 1α,25(OH)2VD3 production and a general strategy for the biohydroxylation of hydrophobic compounds.", "journal": "Bioresource technology", "year": "2026", "doi": "10.1016/j.biortech.2026.134526", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41040984": { "pmid": "41040984", "title": "Rapid identification of major Mycobacterium species by loop-mediated isothermal amplification assay using novel species-specific genomic targets.", "abstract": "Rapid and precise identification of Mycobacterium species is critical for appropriate clinical management and epidemiological surveillance. However, conventional methods often fail to differentiate closely related nontuberculous mycobacteria (NTM) species, limiting their clinical utility. We developed a multiplex loop-mediated isothermal amplification (LAMP) assay targeting newly identified species-specific genomic markers for simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and six clinically important NTM species. Analytical performance was assessed using serial dilutions of bacterial cultures and 36 reference strains. Clinical validation was performed on 52 cultured isolates and 349 sputum samples, compared to GeneXpert MTB/RIF and a commercial PCR-reverse dot blot assay. The assay showed high analytical sensitivity, with limits of detection ranging from 76.013 CFU/mL (95% CI: 60.329-113.924 CFU/mL) for MTBC to 166.602-690.629 CFU/mL for NTM species. All reference strains were correctly identified with no cross-reactivity. Among the clinical isolates, all targeted species were accurately detected. One isolate misidentified as M. abscessus by an ITS-based assay was confirmed by sequencing to be M. massiliense, demonstrating the assay's superior discriminatory capacity. For sputum samples, the assay achieved 90.32% sensitivity and 97.55% specificity for MTBC, with an overall agreement of 93.70% (κ = 0.8740). This multiplex LAMP assay offers a rapid, accurate, and field-deployable tool for species-level identification of MTBC and major NTM pathogens. Its simplicity, stability, and compatibility with low-resource settings support its application in routine diagnostics and decentralized tuberculosis programs.", "journal": "Frontiers in cellular and infection microbiology", "year": "2025", "doi": "10.3389/fcimb.2025.1653602", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41205369": { "pmid": "41205369", "title": "Matrix Stiffness-Induced Mechanical Memory of Periodontal Ligament Cell Promotes Osteogenesis in Periodontal Bone Repair.", "abstract": "Human periodontal ligament cells (hPDLCs) can sense and respond to local mechanical stimuli, which further influence the cellular behaviors, including cell proliferation, apoptosis, differentiation. However, when the microenvironment changes, instead of responding to the change instantly, cells harbor a memory of their past microenvironment, namely mechanical memory, which could regulate the cellular fates and outcomes. This study aimed to explore how mechanical memory, induced through matrix stiffness, affects the osteogenic potential of hPDLCs and their role in periodontal bone defect repair. We built different substrate stiffness models by adjusting the concentration of GelMA hydrogel (soft matrix: 12 kPa; stiff matrix: 159 kPa), thereby endowing hPDLCs with different mechanical memories. Live/dead staining and CCK8 assays were performed to investigate the biocompatibility of GelMA hydrogels and cell proliferation. ALP staining and real-time PCR were performed to determine the osteogenic differentiation ability. Furthermore, we transplanted hPDLC sheets preconditioned on different substrates into a rat alveolar bone defect model, in which standardized defects (5 × 2 × 1 mm³) were surgically created on the buccal side of the mandibular incisor. Micro-CT, HE, and Masson staining confirmed that stiff-matrix groups promoted superior alveolar bone regeneration compared to soft-matrix and control groups. Both types of GelMA hydrogels exhibited favorable biocompatibility, and the stiff matrix significantly enhanced the osteogenic differentiation of hPDLCs compared to the soft matrix, as evidenced by increased ALP activity, upregulation of osteogenic markers (ALP, COL-1, OPN, RUNX2), and improved mineral deposition. In vivo, stiff matrix-induced mechanical memory drove persistent osteogenesis even after environmental withdrawal via cell sheet transplantation. Stiff matrix-induced mechanical memory in hPDLCs promotes osteogenic differentiation in vitro and enhances alveolar bone regeneration in vivo. These findings suggest that mechanical memory can regulate the osteogenic behavior of hPDLCs, offering a promising strategy for periodontal bone regeneration.", "journal": "International dental journal", "year": "2026", "doi": "10.1016/j.identj.2025.103992", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42248829": { "pmid": "42248829", "title": "Identification of a mechanism-based binding mode for a histone deacetylase 6 inhibitor.", "abstract": "Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that deacetylates non-histone substrates such as α-tubulin and cortactin. HDAC6 contains two catalytic domains, each containing a catalytic zinc ion, and a zinc-finger ubiquitin-binding domain. We have discovered BAS-2, a selective HDAC6 inhibitor with an isothiouronium core and no obvious zinc-binding group. To define its mechanism, we combine X-ray crystallography, structure-activity-relationships, molecular modeling and mutagenesis. BAS-2 potently inhibits human HDAC6 but it does not inhibit zebrafish HDAC6. Computational modeling highlighted Asp567 in human HDAC6 as critical for BAS-2 recognition and mutational analyses confirmed this. The corresponding zebrafish residue is Asn530 and the crystal structure of the N530D variant zHDAC6 revealed binding of a BAS-2-derived mercaptoacetamide that engages the catalytic zinc via strong thiolate-zinc coordination. Leveraging the orientation of BAS-2 binding, we designed a BAS-2-based proteolysis targeting chimera that induced proteasome-dependent HDAC6 degradation in cells, verified by global proteomics. Collectively, these insights clarify species selectivity and demonstrate that BAS-2 acts as a selective, mechanism-based inhibitor of human HDAC6. These discoveries will aid the development of the next generation of selective HDAC6 inhibitors and degraders.", "journal": "Nature communications", "year": "2026", "doi": "10.1038/s41467-026-73146-5", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39423707": { "pmid": "39423707", "title": "Novel cobalt-based aerogels for uric acid detection in fluids at physiological pH.", "abstract": "A sensor for uric acid (UA) based on the urate oxidase enzyme (UOx) immobilized in novel Co-based aerogels with transition metals synthesized by the sol-gel method was developed and evaluated. The Co-based aerogels: Co, Ni-Co and Pd-Co were physicochemically characterized by XRD and HR-TEM. The surface area values of 53, 57 and 66 m2 g-1 were determined for Co, Ni-Co and Pd-Co, respectively by N2 adsorption-desorption technique. Co-based aerogels were mixed by cross-linking with UOx enzymes and electrochemically characterized in buffers at pH 7.4 and 5.6 (pH values reported for biological fluids such as blood and sweat) in the presence of different uric acid concentrations. Co-based aerogels with UOx showed improved performance as a uric acid biosensor compared to using the enzyme alone. At a pH of 7.4, a higher sensitivity of 11 μA μM-1 was obtained with Pd-Co/UOx, 1.6 times higher than with UOx. At a pH value of 5.6, the highest sensitivity is achieved with Ni-Co/UOx. Stability and selectivity tests were performed in the presence of biological interferents without significant changes in the sensor. These results indicate a pleasing synergistic activity between Co-based aerogels and the enzyme.", "journal": "Biosensors & bioelectronics", "year": "2025", "doi": "10.1016/j.bios.2024.116850", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42455225": { "pmid": "42455225", "title": "Optimization of PCR amplification and sequencing of GC-rich protein-coding region of CEBPA (CCAAT/enhancer binding protein alpha) in acute myeloid leukemia patients using betaine.", "abstract": "CEBPA, a GC rich gene encodes for transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) which is essential for granulocyte development. Mutation(s) in CEBPA leads to block in myeloid cells differentiation which is characteristic of acute myeloid leukemia (AML). In addition, mutation(s) in CEBPA has been associated with prognosis in AML. Therefore, analysis of protein-coding sequence of CEBPA is critical in patients diagnosed with AML. This study aimed to optimize PCR amplification followed by Sanger sequencing of GC rich regions of CEBPA in AML patients using betaine which is a known PCR additive with relatively less inhibitory effect. PCR amplification using two sets of primer was performed with varying concentrations of betaine and MgCl2 at different annealing temperatures. Purified amplified PCR products were used for Sanger sequencing. PCR amplification of both 576 bp and 703 bp specific amplicons was achieved consistently with 1 M betaine and 2mM MgCl2. We found that increase in betaine inhibits PCR at higher annealing temperature which can be attributed to its ability to decrease the melting temperature of the DNA template. The optimized protocol did not interfere with the downstream applications such as PCR-product purification and Sanger sequencing in AML patients. These findings establish a potentially cost-effective and feasible method for sequencing protein-coding region of CEBPA in AML patients.", "journal": "Molecular biology reports", "year": "2026", "doi": "10.1007/s11033-026-12388-4", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41054873": { "pmid": "41054873", "title": "Advances in Isothermal Amplification for the Diagnosis of Tuberculosis.", "abstract": "Detecting tuberculosis (TB) remains challenging in low-resource settings due to limited access to advanced diagnostic tools. The emergence of isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), presents a promising solution for TB detection. Several isothermal amplification techniques, e.g., strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), and rolling cycle amplification (RCA), have been attracting attention for development as point-of-care testing (POCT) in many diagnostic fields. The present review summarizes the most common isothermal amplification techniques for the detection of TB. To collect the necessary information, we searched PubMed and Scopus for scientific evidence published from 2000 to September 2024, using the keywords loop-mediated isothermal amplification tuberculosis, strand displacement amplification tuberculosis, helicase-dependent amplification tuberculosis, recombinase polymerase amplification tuberculosis, rolling circle amplification tuberculosis, polymerase spiral reaction tuberculosis, cross-priming amplification tuberculosis, and multiple cross displacement amplification tuberculosis. The methodologies of the most usual isothermal amplification techniques are addressed, as well as the advantages and limitations of the technique, highlighting applications in TB diagnosis. Exploring these advances addresses the principles of innovation in isothermal amplification, including their performance and applicability across diverse sample types. Some techniques have already been launched into routine TB diagnosis, while other promising tests are still being researched or evaluated. These techniques are easy to perform and provide results rapidly, enabling prompt treatment within a few hours. Isothermal amplification techniques may be one of the key molecular tools to fight TB in pursuit of the goals of the End TB strategy.", "journal": "Journal of clinical laboratory analysis", "year": "2025", "doi": "10.1002/jcla.70113", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "41076558": { "pmid": "41076558", "title": "Targeting the PHD2/HIF-1α/HO-1 pathway: A key role of trimetazidine in hypertensive nephropathy.", "abstract": "This study seeks to unravel the effects of trimetazidine (TMZ) on hypertensive nephropathy (HN) in mice and its underlying mechanisms. Sixty male 129 mice (8-10 weeks old) were randomly categorized into six groups (n = 10 per group): control, model, TMZ, TMZ + small interfering RNA targeting prolyl hydroxylase domain protein 2 (si-PHD2), TMZ + zinc protoporphyrin [ZnPP, a heme oxygenase-1 (HO-1) inhibitor], and TMZ + KC7F2 [a hypoxia-inducible factor-1 alpha (HIF-1α) inhibitor]. All groups except the control group received angiotensin II to induce HN models. TMZ was administered by gavage for 28 days, while the other TMZ-based groups received additional si-PHD2, ZnPP, or KC7F2. Blood pressure, renal function, proinflammatory cytokines, and kidney pathology were measured. Protein/mRNA levels of PHD2, HO-1, HIF-1α, and Collagen I were analyzed via reverse transcription quantitative polymerase chain reaction/Western blot. The model group showed increased blood pressure, renal injury, fibrosis, and elevated levels of PHD2, HIF-1α, HO-1, Collagen I, and inflammatory markers compared to the control group (P < 0.05). TMZ treatment alleviated renal damage and downregulated PHD2, while upregulating HIF-1α and HO-1. These effects were further enhanced by PHD2 knockdown (TMZ + si-PHD2), but reversed by the inhibition of HO-1 or HIF-1α (TMZ + ZnPP, TMZ + KC7F2). TMZ improves HN in mice by modulating the PHD2/HIF-1α/HO-1 pathway.", "journal": "Clinical and experimental hypertension (New York, N.Y. : 1993)", "year": "2025", "doi": "10.1080/10641963.2025.2563033", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40811854": { "pmid": "40811854", "title": "Pooled CAR-T screening in pig-tailed macaques identifies designs with enhanced proliferation, trafficking, and persistence.", "abstract": "Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted, to our knowledge, the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study end point, unlike other CARs, which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T activity.", "journal": "Blood", "year": "2025", "doi": "10.1182/blood.2025028683", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42225468": { "pmid": "42225468", "title": "[Psychosocial rehabilitation applied to children].", "abstract": "Based on a developmental approach, psychosocial rehabilitation (PSR) for children assumes that a combination of factors points to problems and, as for adults, focuses on recovery. It does not depend solely on the child but on a process involving supportive adults. Recovery in children supports the hope of the parents and builds a new form of parenthood to cope with the child's disability. To optimize psychosocial abilities of a child with a neurodevelopmental disorder (NDD), a personalized assessment and early interventions are needed. Promoting parental skills is the key to preventing additional disabilities. Several stakeholders (education system, healthcare, social services, family support, child protection services) must be involved for inclusion and psychosocial risk management. Three psychological theories emphasized developmental needs which depend on chronological age, environmental context, and the brain's inhibitory capacity. A fourth theory suggests the need for a cultural-eco-social approach. For adults supporting a child with an NDD, psychoeducational approaches include therapeutic education; and peer support will foster empowerment. The recent presence of family peer caregivers included within the healthcare teams provides parents with complementary and different support, bringing hope. Through regional platforms, PSR brings together neuropsychologists, nurses, rehabilitation and socio-educational professionals, and school or work integration managers. The principles of a PSR day hospital for adults are well known, but specifications for the organization of child and adolescent PSR day hospitalization are needed. Day care programs need to use validated interventions whose effectiveness should be assessed. They must promote school inclusion and the autonomy of both parents and children. Psychoeducation and therapeutic patient education (TPE) sessions are applied. A neurocognitive assessment of pre- and post-treatment determines and assesses the objectives of the intervention. The interventions consist of workshops focusing on social skills, executive functions, and emotional regulation. A psychosocial intervention plan is developed collaboratively with the parents, and a review of the objectives is annually conducted. To conclude, psychosocial rehabilitation for children is not yet consensual, and its effectiveness still needs to be evaluated. It is a demanding model due to the assessments it requires, their repetition, and the need of parental support that must foster empowerment. The approaches take into account advances in scientific knowledge of children's cognitive development. These approaches must evolve rapidly and be highly personalized. They are aimed at both parents and healthcare partners. As with adults, health policy must promote the use of psychosocial rehabilitation and encourage the training of professionals in this area.", "journal": "L'Encephale", "year": "2026", "doi": "10.1016/j.encep.2026.02.019", "ptypes": [ "English Abstract", "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41742192": { "pmid": "41742192", "title": "RASGEF1B suppresses hepatocellular carcinoma through the ALDH7A1/Betaine/SNAI1 metabolic‒epigenetic axis.", "abstract": "BACKGROUND: Metabolic‒epigenetic crosstalk critically orchestrates hepatocellular carcinoma (HCC) pathogenesis. Deciphering the precise mechanism underlying epigenetic remodeling and metabolic reprogramming in HCC may lead to novel treatment paradigms, however, the key mechanisms remain elusive. METHODS: RT-qPCR, western blotting and tissue microarrary Immunohistochemistry were used to detect the expression of RasGEF domain family member 1B (RASGEF1B) in HCC and normal liver tissues. Transcriptome sequencing and high-resolution untargeted metabolomics were integrated to identify the downstream regulatory mechanism through which RASGEF1B inhibited the HCC progression. Epigenetic regulation was investigated using methylation-specific PCR and luciferase reporter assays. Bioinformatic prediction and molecular docking suggested a functional interplay among RASGEF1B, ALDH7A1, and BMI1, which was experimentally confirmed through coimmunoprecipitation, GST pull-down, and immunofluorescence assays. Protein stability and ubiquitination status of ALDH7A1 were examined using cycloheximide, immunoprecipitation assay, and an in vitro reconstituted ubiquitination system. RESULTS: In this study, the antitumor role of RASGEF1B was confirmed in vitro and in vivo. Transcriptomic profiling revealed that RASGEF1B overexpression significantly reduced the snail family transcriptional repressor 1 (SNAI1), a master regulator of the epithelial-mesenchymal transition. Untargeted metabolomics revealed that RASGEF1B promoted SNAI1 DNA methylation through Betaine-mediated methionine metabolic reprogramming. Further analysis confirmed that RASGEF1B competitively protected the ALDH7A1 protein from BMI1-dependent ubiquitination, thereby elevating cellular Betaine levels in HCC. CONCLUSIONS: This study revealed that RASGEF1B inhibited SNAI1 to suppress HCC through metabolite‒epigenetic crosstalk. Our findings potentially offer a new perspective on the classical RAS signaling framework, uncovering a metabolic‒epigenetic axis as an innovative therapeutic approach for improving clinical outcomes in patients with HCC.", "journal": "Journal of translational medicine", "year": "2026", "doi": "10.1186/s12967-026-07785-z", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "39556352": { "pmid": "39556352", "title": "Diagnosis and Treatment of Polycythemia Vera: A Review.", "abstract": "Polycythemia vera (PV), a myeloproliferative neoplasm characterized by an increased red blood cell mass and increased risk of thrombosis, affects approximately 65 000 people in the US, with an annual incidence of 0.5 to 4.0 cases per 100 000 persons. Erythrocytosis (hemoglobin >16.5 mg/dL in men or >16.0 mg/dL in women) is a required diagnostic criterion, although thrombocytosis (53%) and leukocytosis (49%) are common. Patients may have pruritus (33%), erythromelalgia (5.3%), transient visual changes (14%), and splenomegaly (36%) with abdominal discomfort. More than 95% of patients have a JAK2 gene variant, which helps distinguish PV from secondary causes of erythrocytosis, such as tobacco smoking or sleep apnea. Among 7 cohorts (1545 individuals), the median survival from diagnosis was 14.1 to 27.6 years. Prior to or at the time of PV diagnosis, arterial thrombosis occurred in 16% of patients and 7% had venous thrombotic events, which could involve unusual sites, such as splanchnic veins. PV is also associated with an increased bleeding risk, especially in patients with acquired von Willebrand disease, which can occur with extreme thrombocytosis (platelet count, ≥1000 × 109/L). All patients with PV should receive therapeutic phlebotomy (goal hematocrit, <45%) and low-dose aspirin (if no contraindications). Patients who are at higher risk of thrombosis include those aged 60 years or older or with a prior thrombosis. These patients and those with persistent PV symptoms may benefit from cytoreductive therapy with hydroxyurea or interferon to lower thrombosis risk and decrease symptoms. Ruxolitinib is a Janus kinase inhibitor that can alleviate pruritus and decrease splenomegaly in patients who are intolerant of or resistant to hydroxyurea. About 12.7% of patients with PV develop myelofibrosis and 6.8% develop acute myeloid leukemia. PV is a myeloproliferative neoplasm characterized by erythrocytosis and is almost universally associated with a JAK2 gene variant. PV is associated with an increased risk of arterial and venous thrombosis, hemorrhage, myelofibrosis, and acute myeloid leukemia. To decrease the risk of thrombosis, all patients with PV should be treated with aspirin and therapeutic phlebotomy to maintain a hematocrit of less than 45%. Cytoreductive therapies, such as hydroxyurea or interferon, are recommended for patients at high risk of thrombosis.", "journal": "JAMA", "year": "2025", "doi": "10.1001/jama.2024.20377", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Review" ], "first_author": "", "dirs": [ "B" ] }, "39832863": { "pmid": "39832863", "title": "A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.", "abstract": "Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2024.343593", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "39813459": { "pmid": "39813459", "title": "Molecular analysis of zoonotic pathogens in free-ranging six-banded armadillos (Euphractus sexcinctus) from the Brazilian semiarid region.", "abstract": "This study investigated infection by Leishmania spp., Leptospira spp., Toxoplasma gondii, and Trypanosoma cruzi in six-banded armadillos (Euphractus sexcinctus) from the semiarid region of northeastern Brazil. Twenty specimens of E. sexcinctus were captured alive by wildlife veterinarians from their natural habitats in different locations. The animals were euthanized following induction of anesthesia, and different biological samples were collected. Infection with four pathogens was subsequently evaluated: Leishmania infection was investigated by spleen and liver Polymerase Chain Reaction (PCR); Leptospira spp. infection was evaluated by kidney PCR and serologically by microscopic agglutination test; T. gondii infection was assessed by PCR of the heart, lung, and spleen; and T. cruzi infection was investigated by heart and whole blood PCR and hemoculture. All tests presented negative results apart from whole blood PCR to detect T. cruzi, which was positive in one of the 20 animals tested and confirmed by genetic sequencing. It is important to highlight that this is the first study comprising a molecular investigation of different zoonotic pathogens in six-banded armadillos, and the findings reported here bring new and important knowledge regarding zoonotic diseases in this species.", "journal": "Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria", "year": "2025", "doi": "10.1590/S1984-29612025002", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41791654": { "pmid": "41791654", "title": "Heparin-induced thrombocytopenia immune complexes activate the inflammasome pathway in a complement-dependent manner.", "abstract": "Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction to heparin caused by pathological immunoglobulin G (IgG) antibodies targeting platelet factor (PF) 4/heparin antigenic complexes. Immune complexes (ICs) consisting of IgG antibodies and PF4/heparin bind to cellular FcγRIIA receptors to initiate prothrombotic cell signaling, including cytokine production and release. This study sought to investigate the role of inflammasome cytokine production and release of interleukin (IL)-1β following HIT IC challenge. Inflammasome cytokine release of IL-1β was measured in a whole blood assay, where healthy donor whole blood was incubated with PF4/heparin antigen and ICs consisting of the monoclonal HIT-like antibody, KKO, or HIT patient plasma. Corresponding studies were conducted in peripheral blood mononuclear cells (PBMCs). Requirements for cellular FcγRIIA, complement pathway, and inflammasome were determined with respective inhibitors. FcγRIIA transgenic mice with or without a genetic deletion of Nlrp3 were subjected to an FcγRIIA-dependent thrombosis model. Whole blood or PBMCs incubated with HIT ICs or HIT patient plasma elicited a significant increase in IL-1β secretion, which was dependent on FcγRIIA. IL-1β secretion required complement, as heat-inactivated plasma or incubation with terminal complement inhibitors prevented cytokine release. Pretreatment of whole blood or PBMCs with the NLRP3 inhibitor MCC950 markedly reduced IL-1β secretion. Furthermore, transgenic mice given an FcγRIIA-dependent thrombotic challenge demonstrated a requirement for Nlrp3 to experience severe thrombocytopenia and thrombosis. These data demonstrate that HIT ICs activate the inflammasome pathway in an FcγR/complement-dependent manner leading to IL-1β secretion, which is a positive regulator of HIT pathology.", "journal": "Journal of thrombosis and haemostasis : JTH", "year": "2026", "doi": "10.1016/j.jtha.2026.02.009", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "39687989": { "pmid": "39687989", "title": "METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis.", "abstract": "Mitochondrial dysfunction is a key factor in the development of atherogenesis. METTL4 (methyltransferase-like protein 4) mediates N6- methyldeoxyadenosine (6mA) of mammalian mitochondrial DNA (mtDNA). However, the role of METTL4-mediated mitoepigenetic regulation in atherosclerosis is still unknown. This study aims to investigate the potential involvement of METTL4 in atherosclerosis, explore the underlying mechanism, and develop targeted strategies for treating atherosclerosis. Expression levels of mtDNA 6mA and METTL4 were determined in atherosclerotic lesions. We explored the mechanism of METTL4 involvement in atherosclerosis using Mettl4Mac-KO-Apoe-/- and Mettl4MUT-Apoe-/- mice and cell models, as well as bone marrow transplantation. Natural compound libraries were screened to identify potent METTL4 antagonists. In addition, bioinspired proteolysis targeting chimera technology targeting macrophages within plaques was used to increase the efficacy of the METTL4 antagonist. The expression levels of mtDNA 6mA and METTL4 were significantly increased in plaque macrophages. Mettl4Mac-KO-Apoe-/- mice displayed suppressed mtDNA 6mA levels and atherosclerotic progression, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Mechanistically, elevated METTL4 expression reduces mitochondrial ATP6 (MT-ATP6) expression by suppressing its transcription, thereby impairing the activity of mitochondrial respiration chain complex V. This disruption leads to the accumulation of excess protons in the mitochondrial intermembrane space, causing mitochondrial dysfunction. Consequently, mtDNA is released into the cytoplasm, ultimately triggering inflammasome activation. All results were reversed by the mutation in the METTL4 methyltransferase active site. Mettl4MUT-Apoe-/- mice showed suppressed mtDNA 6mA levels and atherosclerotic progression and repaired mitochondrial function of macrophage, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Pemetrexed was identified as the first METTL4 antagonist to effectively alleviate atherosclerotic progression. Furthermore, we generated a proteolysis targeting chimera drug based on pemetrexed that specifically targeted METTL4 in macrophages within plaques, showing a promising therapeutic effect on atherosclerosis. This study revealed a novel mechanism by which mtDNA 6mA orchestrated mitochondrial function-related gene expression in macrophages, thereby promoting atherosclerosis. Through various experimental techniques, such as gene manipulation, pharmacological inhibition, and proteolysis targeting chimera, this study demonstrated that mtDNA 6mA and its specific enzyme METTL4 hold potential as therapeutic targets for atherosclerosis.", "journal": "Circulation", "year": "2025", "doi": "10.1161/CIRCULATIONAHA.124.069574", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41316693": { "pmid": "41316693", "title": "Bimetallic Nanozymes: Structure-Activity, Regulatory Strategies, and Adaptive Applications.", "abstract": "Bimetallic nanozymes, an emerging class of artificial enzymes with dual metal active sites, have garnered significant attention due to their superior catalytic performance, enhanced stability, and tunable reactivity compared to monometallic counterparts. By leveraging synergistic electronic and geometric interactions between two distinct metals, these nanozymes address fundamental limitations in catalytic efficiency, selectivity, and functional versatility. This review systematically categorizes bimetallic nanozymes into five structural types-single-atom, cluster, nanoparticle, metal-organic framework (MOF)-based, and composite systems-and elucidates their enzyme-like activities, including oxidase-, peroxidase-, catalase-, and hydrolase-like behaviors. Furthermore, advanced strategies are discussed for regulating their activity through metal composition tuning, structural engineering, and application of external stimuli. The integration of bimetallic nanozymes in biomedical applications (e.g., tumor therapy, antibacterial and anti-inflammatory treatments, and biosensing), environmental remediation (pollutant detection and degradation), and food safety monitoring is thoroughly examined. Finally, current challenges and future directions are highlighted for the rational design and multifunctional application of bimetallic nanozymes, emphasizing interdisciplinary approaches to advance their translational potential.", "journal": "Small (Weinheim an der Bergstrasse, Germany)", "year": "2026", "doi": "10.1002/smll.202510317", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "42679539": { "pmid": "42679539", "title": "An energy-boosting hydrogel couples magnetoelectrical stimulation and metabolic support for enhanced spinal cord regeneration.", "abstract": "Spinal cord injury (SCI) disrupts both neural and vascular networks, leading to a dual deficiency of bioelectric and biochemical energy that severely impedes neuroregeneration. To address this challenge, we developed an energy-boosting hydrogel that integrates biosafe electrical stimulation with a sustained-release biochemical energy system, thereby alleviating energy deficits to promote neural repair. Biodegradable potassium sodium niobate/ferrosoferric oxide (KNN/FO) nanoparticles were synthesized to enable in situ magnetoelectric conversion, and their efficacy in promoting regeneration through electrical stimulation was confirmed in vitro. Transcriptomic and metabolomic analyses further elucidated the synergistic mechanisms between bioelectric and biochemical energy pathways. Based on these findings, phosphocreatine-grafted chitosan (PCr-C) was prepared for sustained biochemical energy release and integrated with KNN/FO nanoparticles into a 3D-printable gelatin-based composite hydrogel (PCr-CGNP). In a rat SCI model, PCr-CGNP successfully achieved dual-energy delivery under a magnetic field and significantly promoted neuroregeneration. This study elucidates the critical role of bioelectric and biochemical energy in post-SCI neuroregeneration and establishes a novel hydrogel-based dual-energy synergistic delivery strategy, providing a theoretical and therapeutic foundation for spinal cord repair.", "journal": "Biomaterials", "year": "2027", "doi": "10.1016/j.biomaterials.2026.124592", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41055316": { "pmid": "41055316", "title": "Circular RNA-Directed Therapeutic Strategy for Cold-Induced Diabetic Macrovascular Disease.", "abstract": "Aims: This study aims to elucidate the molecular mechanisms underlying the alleviation of cold-climate-induced diabetic macrovascular disease (DM-MVD) by targeting hsa_circ_0010154 with gold nanoparticles (AuNPs)-mediated antisense oligonucleotides (ASOs) delivery, combined with aerobic exercise, and to explore the therapeutic effects on glucose and lipid metabolism, inflammation, and oxidative stress. Results: Significant upregulation of hsa_circ_0010154 in DM-MVD was confirmed through bioinformatics analysis and qRT-PCR validation. The constructed gold nanoparticles-mediated antisense oligonucleotides delivery (AuNPs@ASO) complex exhibited efficient reactive oxygen species-responsive release and effective cellular uptake. Silencing hsa_circ_0010154 led to improved endothelial cell function, reduced inflammation markers, enhanced lipid metabolism, and reduced oxidative stress responses. In vivo studies demonstrated improved cardiac function, vascular remodeling, and enhanced antioxidant enzyme activity. Innovation: This study introduces a novel approach utilizing AuNPs@ASO targeting hsa_circ_0010154 in conjunction with aerobic exercise to address the complex pathophysiology of cold-climate-induced DM-MVD, presenting a targeted, low-toxicity therapeutic strategy with promising translational potential. Conclusion: The combined treatment of AuNPs@ASO and aerobic exercise, targeting hsa_circ_0010154, effectively modulates critical pathological pathways involved in DM-MVD, offering a precise and innovative approach for tackling this condition, with implications for clinical translation. Antioxid. Redox Signal. 00, 000-000.", "journal": "Antioxidants & redox signaling", "year": "2025", "doi": "10.1177/15230864251380269", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41299286": { "pmid": "41299286", "title": "Early pathological changes of coronary microvasculation in heart failure with preserved ejection fraction.", "abstract": "BACKGROUND: The majority of heart failure with preserved ejection fraction (HFpEF) patients present with coronary microvascular dysfunction (CMD). However, it is uncertain whether CMD is a primary factor in ventricular remodeling/diastolic dysfunction and HFpEF, or whether CMD is secondary to myocardial remodeling in HFpEF. METHODS: Eight-week-old male C57BL/6N mice were simultaneously administered with high-fat diet (HFD) and N(ω)-nitro-L-arginine methyl ester (L-NAME) via drinking water for 5 weeks. Body weight, blood pressure and glucose tolerance, athletic endurance were observed. Cardiac systolic and diastolic function were detected using trans-thoracic Doppler echocardiography. Heart weight/tibial length and wheat germ agglutinin staining (WGA) were performed to evaluate cardiomyocytes. To detect the degree of cardiac fibrosis, picrosirius red (PSR) staining was performed. The coronary microvascular and pericytes were investigated by immunofluorescence staining. Coronary flow reserve (CFR) was measured by echocardiography to assess the coronary microvascular function. RESULTS: The HFpEF mouse model was constructed using HFD and L-NAME. HFpEF was evident by preserved ejection fraction (EF), fractional shortening (FS) and significantly elevated E/e’, impaired glucose tolerance and reduced exercise capacity in mice treated with L-NAME and HFD. Cardiomyocyte hypertrophy and cardiac fibrosis were observed in the HFpEF group. The density of capillary was decreased in HFpEF group, accompanied by increased capillary diameter and tortuosity. Moreover, the number and coverage rate of cardiac pericytes were reduced in HFpEF group. No significant differences were found in CFR after modeling 5 weeks. But CFR was decreased in HFpEF group when extending modeling time to 12 weeks. CONCLUSIONS: Our findings have demonstrated that coronary microvascular pathological changes appeared during the early stages of HFpEF and appeared before coronary functional indicator changed.", "journal": "BMC cardiovascular disorders", "year": "2025", "doi": "10.1186/s12872-025-05354-8", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40955955": { "pmid": "40955955", "title": "The Associations Between Chronic Active Lesions and White Matter Disease: A 7 Tesla Imaging Study.", "abstract": "The relationship between paramagnetic rim lesions (PRLs) and surrounding normally appearing white matter (NAWM) disease, potentially contributory to the associations seen between PRLs and clinical impairment, is underexplored. To assess whether PRLs correlate with a greater degree of NAWM injury in early MS. PRLs were identified on susceptibility weighted imaging (SWI) of 68 newly diagnosed patients. Each PRL was paired with an anatomically matched contralateral non-PRL (nPRL) from the same participant. Quantitative magnetization transfer imaging derived macromolecular-to-free pool-size ratio (PSR) and relaxation rate of the free water pool (R1f) values were extracted and compared between PRLs and nPRLs, NAWM surrounding PRLs and nPRLs, and whole brain WM lesions and NAWM of PRL+ and PRL- people with MS (pwMS). PSR and R1f (p ≤ 0.028) values were lower in PRLs compared to nPRLs, but there were no differences in PSR and R1f (p ≥ 0.824) values between peri-PRLs and peri-nPRLs NAWM ROIs. PRL+ pwMS had similar PSR and R1f (p ≥ 0.267) of the whole brain NAWM, similar WM lesions PSR (p = 0.764) but lower R1f (p = 0.030) values. In the early stages of MS, there is no association between PRLs and surrounding NAWM degree of injury.", "journal": "Annals of clinical and translational neurology", "year": "2025", "doi": "10.1002/acn3.70101", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "41342874": { "pmid": "41342874", "title": "Chemobiocatalytic Production of 5-Hydroxymethyl-2-furancarboxylic Acid from Sucrose by a Choline Chloride-Lactic Acid Deep Eutectic Solvent and a Novel Gluconobacter oxidans Strain.", "abstract": "5-Hydroxymethyl-2-furancarboxylic acid (HMFCA), a critical polyester intermediate, faces an industrial scale-up challenge due to the high production cost using commercial 5-hydroxymethylfurfural (HMF) as a substrate. This study developed an efficient chemobiocatalytic route to synthesize HMFCA from inexpensive sucrose. A newly isolated Gluconobacter oxidans strain showed high HMF tolerance (246 mM) and completely converted HMF to HMFCA in 24 h with 99.3% yield (mol/mol). Meanwhile, the choline chloride-lactic acid deep eutectic solvent was found to efficiently convert sucrose to HMF, yielding 106.4 mM HMF (53.2% yield). The chemobiocatalytic potential of HMFCA production was evaluated using sucrose-derived crude HMF and G. oxidans whole-cell catalysis. Within 24 h, the crude HMF (64.8 mM) was completely consumed, and 63.5 mM HMFCA was produced. This integrated process achieved an overall HMFCA yield of approximately 52% based on the initial sucrose. These findings provide an efficient strategy for chemobiocatalytic conversion of inexpensive sucrose to high-value HMFCA.", "journal": "Journal of agricultural and food chemistry", "year": "2025", "doi": "10.1021/acs.jafc.5c08041", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41878847": { "pmid": "41878847", "title": "Metal Organic Framework-Based Biosensors for Healthcare and Agricultural Applications.", "abstract": "Metal-organic frameworks (MOFs), with tunable structures, high porosity, and catalytic activity, are promising platforms for next-generation biosensors. MOF-based nanozymes (MOFzymes) and enzyme-MOF composites combine biological specificity with synthetic robustness, overcoming limitations of natural enzymes in complex environments. This review highlights recent advances in MOF-enabled biosensors for agricultural and healthcare applications, focusing on enzyme immobilization, nanozyme activity, and signal amplification strategies. MOFzymes enable low-cost, portable, and ultrasensitive detection of biomarkers, pathogens, pesticides, and environmental contaminants, although challenges remain in reproducibility, stability, selectivity in complex matrices, and scalable integration.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.5c14374", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "41520404": { "pmid": "41520404", "title": "Piezoelectric printing-based droplet digital PCR for noninvasive and sensitive detection of Helicobacter pylori in stool samples.", "abstract": "Helicobacter pylori (H. pylori), classified by the WHO as a Group I carcinogen, is a major contributor to progressive gastric diseases, and its growing resistance to antibiotics has severely undermined first-line eradication therapies. Conventional stool-based diagnostics face three key limitations: limited ability to detect resistant strains, labor-intensive culture workflows, and insufficient sensitivity in inhibitor-rich matrices. These shortcomings underscore the urgent need for a noninvasive, highly sensitive, and inhibitor-tolerant platform that enables simultaneous pathogen detection and resistance assessment. To address this unmet need, we developed a piezoelectric printing-driven ddPCR platform. By optimizing piezoelectric parameters and oil-phase surfactant formulations, we achieved uniform and stable droplet generation, producing a high-density microarray of ∼30,000 droplets in 3 min. The droplets exhibited excellent thermal stability, enabling robust digital PCR amplification. Using stool samples, the platform simultaneously detected H. pylori infection and clarithromycin resistance mutations. For H. pylori detection, it exhibited a sensitivity of 1 copy/μL, a quantitative linear range of 100-105 copies/μL (R2 = 0.99), and 100 % concordance with commercial assays. This technology provides a scalable, cost-effective, and clinically practical solution for stool-based pathogen detection and antimicrobial resistance surveillance, advancing precision diagnostics in both clinical and public health settings.", "journal": "Talanta", "year": "2026", "doi": "10.1016/j.talanta.2026.129381", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40626715": { "pmid": "40626715", "title": "Tracking replicating HPV genomes in proliferating keratinocytes.", "abstract": "Human papillomavirus (HPV) genomes replicate and partition as minichromosomes alongside host chromatin during persistent infection. However, it is difficult to monitor genome dynamics in living cells because the small and compact genome will not easily tolerate expression cassettes. Here, we use ANCHOR technology to detect HPV18 genomes in living cells. We incorporated the cis-element from ANCHOR technology into the late region of the HPV18 genome and expressed the ParB-GFP protein from an HPV18-dependent replicon. The replicon contains the HPV18 replication origin and viral transcriptional enhancer element and can replicate stably in keratinocytes when complemented by the HPV18 genome. This small replicon expresses the neomycin resistance gene in both bacteria and eukaryotic cells and has minimal prokaryotic elements that could induce innate immunity. This molecular tool enables us to indirectly monitor the presence of the virus by detecting these fluorescent proteins in live cells and allows for real-time tracking of replicating HPV18-ANCH3 genomes in proliferating keratinocytes to inform on models of HPV genome maintenance, tethering, and amplification. Here, we visualize the partitioning of the viral DNA in dividing cells and show that HPV18-ANCH genomes are distributed somewhat equally to daughter cells by attachment to host chromosomes. In persistent human papillomavirus (HPV) infection, the viral genome is maintained at a constant copy number, replicates in synchrony with host DNA during S-phase, and is partitioned into daughter cells. The exact method by which HPVs partition to daughter cells is not well understood, and the elucidation of such mechanisms may reveal relevant pharmacological targets to combat persistent HPV infection.", "journal": "mBio", "year": "2025", "doi": "10.1128/mbio.01308-25", "ptypes": [ "Journal Article", "Research Support, N.I.H., Intramural" ], "first_author": "", "dirs": [ "C" ] }, "41590282": { "pmid": "41590282", "title": "Toothbrush-Driven Handheld Droplet Generator for Digital LAMP and Rapid CFU Assays.", "abstract": "Droplet microfluidics enables high-throughput, compartmentalized reactions using minimal reagent volumes, but most implementations rely on precision-fabricated chips and external pumping systems that limit portability and accessibility. Here, we present a handheld vibrational droplet generator that repurposes a consumer electric toothbrush and a modified disposable pipette tip to produce nearly monodisperse water-in-oil droplets without microfluidic channels or syringe pumps. The device is powered by the toothbrush's built-in motor and controlled by a simple 3D-printed adapter and adjustable counterweight that tune the vibration amplitude transmitted to the pipette tip. By varying the aperture of the pipette tip, droplets with diameters from ~100-300 µm were generated at rates of ~100 droplets s-1. Image analysis revealed narrow size distributions with coefficients of variation below 5% in typical operating conditions. We further demonstrate proof-of-concept applications in digital loop-mediated isothermal amplification (LAMP) and microbiological colony-forming unit (CFU) assays. A commercial feline parvovirus (FPV) kit manufactured by Beyotime Biotechnology Co., Ltd. (Shanghai, China), three template concentrations yielded emulsified reaction droplets that remained stable at 65 °C for 45 min and produced distinct fractions of fluorescent-positive droplets, allowing estimation of template concentration via a Poisson model. In a second set of experiments, the device was used as a droplet-based spreader to dispense diluted Escherichia coli suspensions onto LB agar plates, achieving uniform colony distributions across the plate at different dilution factors. The proposed handheld vibrational generator is inexpensive, easy to assemble from off-the-shelf components, and minimizes dead volume and cross-contamination because only the pipette tip contacts the sample. Although the current prototype still exhibits device-to-device variability and moving droplets in open containers complicate real-time imaging, these results indicate that toothbrush-based vibrational actuation can provide a practical and scalable route toward \"lab-in-hand\" droplet assays in resource-limited or educational settings.", "journal": "Biosensors", "year": "2026", "doi": "10.3390/bios16010030", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41390126": { "pmid": "41390126", "title": "Bitopertin shows efficacy in patients with erythropoietic protoporphyria: Results from the randomized, double-blind, placebo-controlled AURORA trial.", "abstract": "Erythropoietic protoporphyria is a rare genetic disorder of heme biosynthesis characterized by protoporphyrin-IX accumulation, painful phototoxic reactions, and hepatobiliary disease, for which no disease-modifying therapies are approved. To evaluate the efficacy and safety of bitopertin, an inhibitor of glycine transporter 1, in adults with erythropoietic protoporphyria. In this randomized, double-blind, phase 2 study, patients received once-daily oral bitopertin 20 mg, bitopertin 60 mg, or placebo for 17 weeks. The primary endpoint was percentage change from baseline in whole-blood metal-free protoporphyrin-IX levels at day 121. Patients received bitopertin 20 mg (n = 26), 60 mg (n = 25), or placebo (n = 24). At day 121, the percentage change from baseline in whole-blood metal-free protoporphyrin-IX versus placebo was -29.6% (P = .004) with bitopertin 20 mg and -49.8% (P < .001) with bitopertin 60 mg. Bitopertin was associated with a reduced incidence of phototoxic reactions. Bitopertin was well tolerated with no notable safety concerns identified. Small sample size, short follow-up period, and no formal adjustments for between-group multiple-pairwise comparisons. Bitopertin significantly reduced protoporphyrin-IX levels, showed improved measures of sunlight tolerance, and had a favorable safety profile in patients with erythropoietic protoporphyria.", "journal": "Journal of the American Academy of Dermatology", "year": "2026", "doi": "10.1016/j.jaad.2025.12.024", "ptypes": [ "Journal Article", "Randomized Controlled Trial", "Clinical Trial, Phase II", "Multicenter Study", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "41752493": { "pmid": "41752493", "title": "Advances in Active Materials of Enzymatic Electrochemical Sensors for Detecting Organophosphorus Pesticides.", "abstract": "Organophosphorus pesticides (OPs) have been widely employed to increase food production and alleviate the increasingly serious food crisis. However, excessive use of these pesticides has seriously affected human health and even caused death due to significant pesticide residues in food. Therefore, enzymatic electrochemical sensors have been developed to monitor OP residues in food. The electrochemical detection performance of these sensors is determined by the physicochemical properties of electrochemical active materials in their active layers. The definition and classification of OPs are first introduced in this review, then the components of enzymatic electrochemical sensors, including electrodes, electrochemical active layer and bioactive enzyme layer, are analyzed in detail. Furthermore, this review emphatically discusses the recent development of enzymatic electrochemical sensors based on various electrochemical active materials: carbon-based, polymer-based, metal-based, metallic compound-based, metal organic framework-based and covalent organic framework-based materials. Finally, probable research directions for developing enzymatic electrochemical sensors with high sensitivity, excellent stability and good reproducibility are outlined to accelerate rapid, effective and low-cost on-site detection OPs in food. This review is expected to provide inspiration for the design and preparation of the high-performance enzymatic electrochemical sensors.", "journal": "Molecules (Basel, Switzerland)", "year": "2026", "doi": "10.3390/molecules31040717", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "42227547": { "pmid": "42227547", "title": "Characterization of HIV-1 vpu Gene from Suppressed Viremic Older Individuals with HIV on Long-Term Antiretroviral Therapy.", "abstract": "Although advancements in antiretroviral therapy (ART) have been able to control HIV infection in people with HIV (HIV+) to achieve suppressed viral loads and recovered CD4 T cell counts, the virus persists in latent reservoirs. Characterizing the molecular, biological, and immunological features of HIV-1 genes from these HIV+ virologically controlled individuals is essential for the development of eradication strategies. The HIV-1 accessory gene, vpu, plays important roles in the release of progeny virions and modulation of innate immune signaling, contributing to viral pathogenesis and persistence in the host. In this study, we analyzed the vpu sequences from 21 predominantly older HIV+ individuals with long-term ART-mediated viral suppression (mostly undetectable viral load) and mostly restored CD4 T cell counts. Genomic DNA was isolated from peripheral blood mononuclear cells (PBMC) extracted from blood samples from 21 HIV+ individuals. Amplification of the vpu gene was performed by polymerase chain reaction (PCR), followed by cloning utilizing a TOPO vector. Bioinformatics analysis of the vpu nucleotide sequences was conducted. Phylogenetic analysis of 288 vpu sequences revealed that the vpu sequences from each of the 21 HIV+ individuals were distinctly separated from one another and formed individual clusters within their respective subtrees. These sequences showed a low degree of genetic variability, reduced genetic diversity estimates, and most patient sequences did not display evidence of positive selective pressure. In 82% of the deduced Vpu amino acid sequences, the open reading frames were intact, with most sequences demonstrating conservation of functional domains necessary for Vpu activity involving virion release, BST-2 degradation, oligomerization, and CD4 degradation. Deduced amino acid sequences exhibited increased variability in the previously identified CTL epitope within Vpu, suggesting the presence of escape mutants. Our findings demonstrate that the coding sequences and functional domains of the HIV-1 vpu gene from HIV+ PMBC DNA on long-term ART and improved CD4 counts were highly conserved with low genetic diversity and increased variability in the CTL epitope, sug-gesting a role for Vpu in viral persistence. The vpu sequences from 21 older HIV+ individuals on long-term ART and suppressed viremia exhibited a low degree of genetic diversity, conserved functional domains, and variability within the CTL epitope, suggesting a role in viral persistence in these HIV+ individuals. These findings may help develop new treatments and curative strategies.", "journal": "Current HIV research", "year": "2026", "doi": "10.2174/011570162X442062260513151551", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40193306": { "pmid": "40193306", "title": "Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle.", "abstract": "Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.", "journal": "Journal of visualized experiments : JoVE", "year": "2025", "doi": "10.3791/67748", "ptypes": [ "Journal Article", "Video-Audio Media" ], "first_author": "", "dirs": [ "A" ] }, "40997387": { "pmid": "40997387", "title": "Rhizosphere remediation of saline-alkali soils by Helianthus tuberosus: Linking microbial-metabolite synergies to carbon sequestration.", "abstract": "Saline-alkali soils (SAS) threaten global food security and climate resilience by degrading soil health and undermining carbon sequestration. While salt-tolerant crops offer nature-based solutions, their mechanisms of soil structure recovery and carbon stabilization remain unclear. This study across three typical Chinese SAS regions (Songnen, Hetao, and Jianghuai Plain) shows that cultivating Jerusalem artichoke (JA)-a high-value salt-tolerant crop-for 1-2 years drives SAS rehabilitation through rhizosphere engineering. JA improves aggregate stability (2.96-10.31-fold), reduces soil salinity (12.3-90.0 %), boosts soil organic carbon (SOC) stocks (9.21-12.78 Mg C ha-1 yr-1), and optimizes nitrogen cycling (NO₃/NH₄ decreased by 44.9-83.8 %). Key metabolites (e.g., trehalose 6-phosphate) mediate these improvements by selectively enriching key microbes identified via network and LEfSe analyses (Solirubrobacter, Sphingomonas, Nitrospira, Variovorax). These microbes drive region-specific carbon turnover through coordinated CAZyme activities: GH13 and CBM48 enhance microbial carbon use efficiency via labile carbon hydrolysis, GT51 promotes SOC accumulation through recalcitrant necromass production and aggregation, and CBM2/GH facilitates carbon accessibility, forming a degrade/rebuild cascade that integrates carbon retention with JA's adaptive growth. PLS-SEM analysis confirms that JA cultivation primarily enhances soil structure stability (total effect: +0.762) and SOC accumulation (+0.639) by stimulating a cohesive microbe-metabolite-enzyme network. This synergy directly improves soil structure, with Year 2 outperforming Year 1 as microbial functions shift from stress adaptation to nutrient cycling. JA thus balances agronomic productivity with ecological restoration, establishing a plant-soil feedback framework for sustainable SAS rehabilitation.", "journal": "The Science of the total environment", "year": "2025", "doi": "10.1016/j.scitotenv.2025.180533", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42773639": { "pmid": "42773639", "title": "Integrated metabolomic and physiological profiling reveals mechanisms of drought tolerance in soybean.", "abstract": "Drought is a major constraint to soybean productivity, yet the metabolic mechanisms underlying genotypic variation in drought tolerance remain incompletely understood. This study investigated metabolic and physiological responses to water deficit in three Egyptian soybean breeding lines (L07, L34, and L74). Gas chromatography-mass spectrometry profiling identified 132 metabolites in leaves and 86 in roots, including amino acids, organic acids, carbohydrates, polyphenols, and lipids. Principal component analysis clearly separated control and drought-stressed plants, with the first two components explaining 55.7% and 54.4% of the variance in leaves and roots, respectively. L74 exhibited greater metabolic stability and accumulated higher levels of protective polyphenols (vanillic and benzoic acids), compatible solutes (trehalose and mannitol), and stress-related amino acids (γ-aminobutyric acid and proline) than L07 and L34. Partial least squares discriminant analysis identified trehalose, quercetin, chlorogenic acid, and phosphorylated sugars as major drought-responsive metabolites (variable importance in projection = 2.65-3.14). Pathway analysis revealed coordinated changes in carbohydrate metabolism, tricarboxylic acid cycle intermediates, and antioxidant metabolism. Physiologically, L74 maintained higher relative water content (81%) than L07 (71%) and L34 (69%), together with greater shoot biomass and antioxidant enzyme activities. Integrated metabolic and physiological analyses indicate that L74 achieves superior drought tolerance through coordinated metabolic homeostasis involving osmotic adjustment, antioxidant protection, and maintenance of primary metabolism. The identified metabolic signatures provide potential biomarkers for screening and breeding drought-tolerant soybean germplasm. © 2026 Society of Chemical Industry.", "journal": "Journal of the science of food and agriculture", "year": "2026", "doi": "10.1002/jsfa.71086", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42287420": { "pmid": "42287420", "title": "Metabolic mechanisms underlying osmoprotectant-enhanced xylitol biosynthesis from glucose by Zygoascus hellenicus.", "abstract": "Xylitol, a high-value sugar alcohol, holds significant potential for applications in the food and pharmaceutical industries. Biomanufacturing xylitol from low-cost, green feedstocks provides a sustainable alternative to the energy-intensive and highly polluting chemical synthesis process. Current research primarily focuses on xylose-to-xylitol bioconversion, whereas native glucose-to-xylitol converters remain underexplored. In particular, a systematic understanding of their osmotic stress adaptation and metabolic regulation is lacking, which directly impedes the scale-up of xylitol production from low-cost glucose. To address this gap, this study investigated Zygoascus hellenicus, an osmotolerant yeast capable of directly converting glucose to xylitol, and proposed a novel strategy to enhance xylitol production by supplementing exogenous osmoprotectants. Results showed that adding 80 mL/L glycerol or 80 g/L trehalose increased xylitol titers to 28.44 g/L and 23.63 g/L, representing improvements of 41.99% and 17.99%, respectively, compared with the control group. Mechanistic investigations revealed the dual regulatory roles of these osmoprotectants. Physiologically, they enhanced osmotic tolerance, preserved membrane integrity, and stabilized enzyme activity. Metabolically, they significantly activated key enzymes of the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6PDH and 6PGDH) and inhibited glycolytic enzymes, thereby redirecting carbon flux toward xylitol biosynthesis. Dynamic metabolic flux analysis (dMFA) further confirmed that osmoprotectant treatment substantially increased the metabolic flux through the xylitol synthesis pathway mediated by G6PDH and 6PGDH, while reducing carbon flow in glycolysis. This study provides mechanism-based insights into xylitol production from glucose in a native producer, revealing a green and alternative glucose-based biomanufacturing route.", "journal": "Bioprocess and biosystems engineering", "year": "2026", "doi": "10.1007/s00449-026-03365-z", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41462712": { "pmid": "41462712", "title": "Protective Functions of β-Alanyl-L-Histidine and Glycyl-L-Histidyl-L-Lysine Glycoconjugates and Copper in Concert.", "abstract": "Two endogenous peptides, β-alanyl-L-histidine, named carnosine (Car), and glycyl-L-histidyl-L-lysine (GHK), derived from the matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), share many beneficial functions. The hydrolytic enzyme carnosinase for Car and the low stability for GHK can put into question their antioxidant, antiaggregating, and anti-inflammatory properties. The glycoconjugates of Car with a di- (trehalose, Tre) or polysaccharide (hyaluronan, HA) inhibit carnosinase, while the synthesis of HAGHK derivatives increases the tripeptide stability and protects/delays the biopolymer degradation. A synergic effect between the two components of the glycoconjugates is evident in their consequently preserved protective features. TreCar, HACar, and HAGHK maintain the copper-binding ability of the peptides alone, and the saccharides potentiate the Cu,Zn-superoxide dismutase-like ability of the copper(II) complexes with the glycoconjugates. These peptide derivatives behave as copper ionophores, utilizing Cu2+ present in the culture medium; also, an increase in the metal intracellular level occurs with a consequent stimulation of the copper-driven signaling pathways that produce the expression/release of trophic (Brain-Derived Neurotrophic Factor, BDNF, and Bone Morphogenetic Protein 2, BMP-2) and angiogenic (Vascular Endothelial Growth Factor, VEGF) proteins. Copper chaperons for SOD1, CCS, and Antioxidant 1 (Atox-1) are the copper chaperones that act as transcription factors.", "journal": "Antioxidants (Basel, Switzerland)", "year": "2025", "doi": "10.3390/antiox14121512", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "42334061": { "pmid": "42334061", "title": "The effect and mechanism of melatonin-trehalose-selenium nanoparticles in alleviating the phytotoxicity of butachlor in rice.", "abstract": "Nanoparticles show a great potential in enhancing crop stress tolerance, but their role in mitigating herbicide damage to rice remains unclear. This study synthesized melatonin-trehalose-selenium nanoparticles (MT-SeNPs) with 21 nm particle size, exhibited good stability and in vitro antioxidant activity. Under butachlor stress, compared with water priming, MT-SeNPs priming significantly alleviated the phytotoxic symptoms in rice and reduced butachlor residues by 21.72-50.55%. Integrated physiological and multi-omics analyses revealed that MT-SeNPs activated phenylpropanoid biosynthesis by upregulating the expression of lignin-biosynthetic genes, increasing the activity of phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, peroxidase enzymes (the enzyme activity increased by 22.94-49.57%, 56.02-56.60%, 26.20-37.08% and 41.30-152.66% at 3 to 5 days after butachlor stress, respectively), and promoting the accumulation of l-phenylalanine, trans-ferulic acid, sinapyl alcohol, thereby promoting deposition of lignin and cellulose. Additionally, MT-SeNPs enhanced the activities of detoxification enzyme (cytochrome P450, glutathione S-transferase, glycosyltransferase), activated glutathione metabolism. MT-SeNPs may mitigate butachlor phytotoxicity by reinforcing cell-wall barriers and accelerating herbicide metabolism. This study provides a new strategy for using nanomaterials to relieve herbicide damage. © 2026 Society of Chemical Industry.", "journal": "Pest management science", "year": "2026", "doi": "10.1002/ps.71004", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40944461": { "pmid": "40944461", "title": "Not only an inhibitor: Trehalose enhances the catalytic action exerted on oxaloacetate by rabbit lactate dehydrogenase.", "abstract": "Trehalose is an osmolyte featuring a prominent competence in stabilizing proteins and enzymes. In particular, in the presence of this peculiar disaccharide, quite a number of enzymes show improved stability against thermal denaturation and an enhanced ability to withstand exposure to freezing-thawing cycles. Moreover, it was recently reported that trehalose counteracts the acid-induced dissociation of oligomeric protein complexes. Concerning the catalytic action of enzymes, the addition of trehalose to assay mixtures was found to decrease the values of both Km and kcat, with the decrease in reaction velocity related to the increase in viscosity induced by the disaccharide. Here, we show that trehalose does not necessarily perform as a negative effector on reaction velocity. Using tetrameric rabbit muscle lactate dehydrogenase (rbLDH) as a model system, we report that trehalose does highly stimulate the catalytic action of this enzyme at the expense of oxaloacetate. In particular, stopped-flow assays revealed that trehalose slows down the binding of β-NADH to rbLDH, as well as its dissociation from the cofactor-enzyme complex. Conversely, the presence of the disaccharide does not alter the rate constant of the association to rbLDH of the substrate analogue oxamate. Furthermore, according to steady-state and stopped-flow assays, we present evidence that the increased velocity of oxaloacetate reduction triggered by trehalose is related to an improved occupancy by the substrate of the enzyme subunits, and by a favorable reciprocal orientation of β-NADH and oxaloacetate.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70304", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42380407": { "pmid": "42380407", "title": "Spray Dried Lysozyme Microspheres: Morphological Evolution and Enzymatic Activity Retention.", "abstract": "Conventional spray drying often produces heterogeneous enzyme powders because of broad droplet size distributions and complex turbulent flow fields, limiting mechanistic understanding of particle formation and quantitative structure-activity analysis. This study aimed to fabricate uniform lysozyme microspheres and clarify how formulation and drying conditions govern their microstructure and enzymatic activity. A micro-fluidic jet spray dryer was used to prepare highly uniform pure lysozyme and excipient-containing lysozyme microspheres. The effects of carbohydrate excipients and drying temperature on particle morphology, secondary structure, surface composition, and enzymatic activity were systematically evaluated. Pure spray-dried lysozyme microspheres showed a highly uniform single-hole bowl-like morphology with smooth surfaces. However, drying-induced thermal and shear stresses reduced α-helix content and caused activity loss. Carbohydrate excipients altered the structural evolution pathways. Surface elemental analysis indicated lysozyme enrichment in most formulations, except those containing dextran T40. Mannitol crystallization induced phase separation, lysozyme unfolding, and inactivation. Trehalose preserved conformational stability through a hydrogen-bonded glassy matrix, whereas dextran T40 mainly suppressed lysozyme aggregation through steric hindrance. This work establishes a formulation properties/process-microstructure-enzymatic activity relationship in a uniform microsphere system, supporting the rational design of performance-predictable spray-dried enzyme powders.", "journal": "Pharmaceutical research", "year": "2026", "doi": "10.1007/s11095-026-04144-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42554812": { "pmid": "42554812", "title": "Computationally Guided Engineering of Multi-Enzyme Cascades Enables Efficient Trehalose Biosynthesis.", "abstract": "Trehalose is a nonreducing disaccharide widely used for its biomolecule-protective properties. However, multienzyme cascade production remains limited by low enzyme expression and suboptimal catalytic performance. To address this, thermostable maltooligosyltrehalose synthase (TreY) and trehalohydrolase (TreZ) from Arthrobacter ramosus were individually expressed intracellularly in Bacillus subtilis, and the crude lysates were combined for trehalose biosynthesis, achieving 281.4 g/L trehalose and a yield of 0.7 g trehalose/g maltodextrin. Integrated computational screening identified MalQ-3 from Cyanobacterium stanieri as a suitable 4-α-glucanotransferase for soluble expression. Subsequent semirational engineering generated MalQ-3-M2 (S54P/V472F), with enhanced activity and stability associated with improved substrate-pocket dynamics, thereby facilitating glucan-chain rearrangement and short-chain reutilization. MalQ-3-M2 was separately expressed in B. subtilis and incorporated into the crude-lysate cascade, increasing the trehalose titer to 338 g/L and the yield to 0.85 g trehalose/g maltodextrin. Overall, this work establishes a scalable B. subtilis platform for efficient trehalose production.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.6c05712", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40554840": { "pmid": "40554840", "title": "Physio-biochemical and molecular analyses decipher distinct dehydration stress responses in contrasting genotypes of foxtail millet (Setaria italica L.).", "abstract": "Drought impairs plant growth and productivity by disrupting key physiological and biochemical processes. Foxtail millet (Setaria italica), a drought-resilient C4 crop, is well-suited for climate-smart agriculture, yet its stress adaptation mechanisms remain underexplored. This study deciphered dehydration responses in tolerant and sensitive genotypes, focusing on redox regulation, sugar metabolism, energy dynamics, and autophagy. For this, four drought-distinguished millet genotypes (2 tolerant and 2 sensitive) were subjected to dehydration stress (20 % PEG-6000) for different time points (0, 2, 6 and 12 h). Tolerant genotypes exhibited improved antioxidant enzyme activity and GSH:GSSG ratios, resulting in efficient detoxification of reactive oxygen species (ROS) and improved membrane stability. Sensitive genotypes, in contrast, accumulated ROS and showed elevated oxidative damage and electrolyte leakage. Tolerant genotypes also maintained higher trans-zeatin levels and suppressed chlorophyll degradation, thereby preserving photosynthesis and delaying senescence. Sugar metabolism was more efficient in tolerant types, with increased activities of sugar metabolism enzymes, enabling proper carbohydrate partitioning and osmotic adjustment. Contrastingly, sensitive genotypes showed sugar overaccumulation due to impaired mobilization. Also, tolerant genotypes retained higher ATP and pyruvate levels, indicating better energy homeostasis. Additionally, enhanced autophagy, marked by elevated ATG8 protein and ATG transcript levels, supported cellular recycling in tolerant genotypes. In contrast, repressed autophagy was observed despite increased abscisic acid in sensitive genotypes, likely due to sugar-mediated signalling and elevated trehalose-6-phosphate levels. These integrated responses highlight the roles of redox control, metabolic coordination, and autophagy in dehydration tolerance and offer multi-target strategies for breeding climate-resilient Setaria cultivars for drought-prone environments.", "journal": "Journal of plant physiology", "year": "2025", "doi": "10.1016/j.jplph.2025.154549", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41944838": { "pmid": "41944838", "title": "Investigation of regioselectivity and thermostability of free and immobilized Pleurotus citrinopileatus lipase.", "abstract": "A fungal lipase from the Basidiomycete Pleurotus citrinopileatus (PCI_Lip) has been investigated for its regioselectivity. Therefore, the conversion products of a triglyceride were analyzed using 1H-NMR to determine the position of hydrolysis within the substrate. PCI_Lip is a 1,3-selective lipase, as it preferentially hydrolyzes the fatty acids at the outer positions of the triglyceride. Furthermore, this study aimed to gain deeper insights into the stability of the lipase. The melting point of the enzyme was determined to be at 63 °C in ultrapure water, which could be increased to 71 °C by dissolving PCI_Lip in a 400 mM citrate buffer pH 6.0 with the addition of glucose and trehalose. To optimize the stability of the enzyme, PCI_Lip was immobilized via its affinity tag. Twenty-one supports were tested, with IB-HIS-19 being chosen as the most appropriate for PCI_Lip immobilization due to its high binding capacity and low leakage. This immobilized lipase displayed only minor loss of activity over 13 cycles of reuse. Immobilization of the wild-type preserved activity and selectivity, whereas it induced notable changes in the case of the double mutants. The increased stability of the immobilized PCI_Lip was further proven in a thermal inactivation experiment. Double mutants of PCI_Lip displayed even higher inactivation temperatures, while the immobilization raised this temperature by 5 °C. Overall, in this study, the 1,3-regioselective PCI_Lip identified from our group has been further characterized, focusing on the improvement of its thermal and kinetic stability. A major boost in stability can be achieved via immobilization of the enzyme to an appropriate support. KEY POINTS: IB-HIS-19 had the highest binding capacity and kept up enzyme activity over 13 cycles of the screened immobilization materials Immobilization increased the stability of PCI_Lip by 5 °C, protein engineering increased the stability of two double mutants up to 4 °C, and thermostability could be increased by 9 °C by using high molarity buffers PCI_Lip was found to be a 1,3-regioselective lipase by analyzing its products by 1H-NMR.", "journal": "Applied microbiology and biotechnology", "year": "2026", "doi": "10.1007/s00253-026-13801-5", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40277444": { "pmid": "40277444", "title": "Quaternary stabilization of a GH2 β-galactosidase from the psychrophile A. ikkensis, a flexible and unstable dimeric enzyme.", "abstract": "Studies of cold-active enzymes may elucidate the basis for low-temperature activity and contribute to their wider application in energy-efficient processes. Here we investigate the cold-active GH2 β-galactosidase from the psychrophilic bacterium Alkalilactibacillus ikkensis (AiLac). AiLac has a specific activity twice as high as its closest structural homolog (the mesophilic Escherichia coli GH2 β-galactosidase) toward the lactose analog ONPG at room temperature and neutral pH, and shows biphasic behavior in Michaelis-Menten plots. AiLac is activated by Mg2+ and Na+ and is most effective at pH 7.0 and 30°C. However, early unfolding events are observed already at room temperature. Stability studies using intrinsic fluorescence, circular dichroism, and small-angle x-ray scattering (SAXS), combined with activity assays, showed AiLac to be highly sensitive to heat and urea and to be stabilized, but also inhibited, by loss of structural flexibility induced by the osmolyte trehalose. AlphaFold structure prediction combined with SAXS and flow-induced dispersion analysis support a reversible monomer-dimer model, suggesting structural adaptation to cold temperatures on a quaternary level. The low amount of dimeric buried surface area, high flexibility, and remarkably low chemical and thermal stability present an extreme example of cold adaptation promoted by high levels of solvent interactions. To investigate the relationship between evolution and oligomerization, we trained a generative deep learning model to successfully engineer functional variants that form stabilized dimers and tetramers by introducing high evolutionary fitness mutations at the interface, demonstrating an efficient way to explore the local sequence fitness landscape to modulate the equilibrium of oligomerization.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70141", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "42162621": { "pmid": "42162621", "title": "From discovery to therapeutic development of monoclonal antibodies.", "abstract": "Monoclonal antibodies (mAbs) represent a powerful targeted therapeutic arsenal for treating diverse diseases, ranging from infectious and chronic conditions to cancer. However, successful development requires overcoming several complex stability and solubility challenges in the final stages. This review examines the discovery, characterization, and therapeutic development of monoclonal antibodies (mAbs), with particular emphasis on inherent stability challenges, including aggregation and degradation, that can compromise mAb efficacy and safety. We demonstrate how novel formulation strategies can address these intrinsic limitations to expand the therapeutic potential of an antibody. Our analysis emphasizes critical aspects of immunogenicity, antibody aggregation and degradation, and advanced characterization methods. We examine liquid, lyophilized, PEGylated, and spray-dried formulations, as well as methods to enhance stability through mutagenesis and chemical modifications. Additionally, we explore how antibody-drug conjugates (ADCs), controlled drug-delivery methods, and nanotechnology-based approaches can enhance bioavailability and therapeutic efficacy. We also cover initial antibody discovery, including high-throughput screening and advanced biophysical characterization techniques that ensure mAb functionality, specificity, and structural integrity. This comprehensive overview of current advances and challenges in biologics should serve as a valuable guide for academic researchers and biopharmaceutical professionals, providing essential knowledge and tools for navigating mAb development and advancing this therapeutics toward clinical success.", "journal": "Journal of controlled release : official journal of the Controlled Release Society", "year": "2026", "doi": "10.1016/j.jconrel.2026.115034", "ptypes": [ "Journal Article", "Review", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "A" ] }, "42814332": { "pmid": "42814332", "title": "High Immunophenotyping of Human PBMCs Using Lyophilized Antibodies.", "abstract": "Immunophenotyping of human peripheral blood mononuclear cells (PBMCs) is a critical technique for understanding immune responses in both health and disease. Traditionally, this process relies on the use of liquid antibody cocktails, which are time-consuming to prepare, prone to laboratory error, and have limited shelf lives. Recent advancements in lyophilization of antibodies and other reagents have addressed these challenges by enhancing reagent stability, extending shelf life, and simplifying transport and storage. Dried antibody cocktails have been shown to perform comparably to liquid cocktails in flow cytometry assays while reducing preparation time and minimizing handling errors. This review focuses on the use of lyophilized reagents for high-dimensional immunophenotyping of human PBMCs, discussing their advantages in large-scale studies, global health initiatives, and clinical research. By streamlining immune profiling workflows, lyophilized reagents have the potential to improve accessibility and facilitate the adoption of immunophenotyping in resource-limited settings.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2027", "doi": "10.1007/978-1-0716-5607-5_16", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "41226652": { "pmid": "41226652", "title": "Antiproliferative Evaluation of Dextran Polymer-Based Pomegranate Ethanolic Extract.", "abstract": "The pomegranate peel represents an important source of secondary metabolites such as hydrolysable ellagitannins, which are recognized for their antioxidant, anticancer and neuroprotective properties. In this work, the freeze-dried pomegranate peel was extracted by a combined mild maceration at room temperature and ultrasonication at 45 °C using ethanol and acetone as green solvents. The ethanol extract, with an extraction yield of 29%, and IC50 (mg/mL) 0.1067 and 0.0414 for DPPH and ABTS, respectively, was incorporated into a polymer based on dextran, using a grafting reaction, to improve its bioavailability and preserve the chemical integrity. In addition, the potential antitumor activity against breast cancer was evaluated based on the existing literature. In vitro studies have demonstrated the safety and biocompatibility of both free pomegranate peel extract (SSE2-L) and its dextran conjugate (SSPD), with no adverse effects on fibroblasts, erythrocytes, or immune cells. Both formulations inhibited the proliferation of breast cancer cell lines (MCF-7, MDA-MB-231) in a concentration- and time-dependent manner, with SSPD consistently showing superior efficacy. This enhanced activity was corroborated by reduced clonogenic growth, G1 cell-cycle arrest, and improved stability and bioactive retention conferred by polymer conjugation. Overall, these findings highlight dextran-conjugated pomegranate polyphenols as promising candidates for next-generation nutraceuticals and phytopharmaceuticals in cancer chemoprevention and adjunctive therapy, with potential applications extending to other biomedical fields and functional foods.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms262110618", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42590828": { "pmid": "42590828", "title": "Circular Logic: Engineering Next-Generation Circular RNA Therapeutics for Precision Oncology.", "abstract": "The topology of RNA therapeutics is emerging as a critical design dimension in precision oncology. Unlike linear mRNA, circular RNA (circRNA) lacks free ends, conferring exceptional resistance to exonuclease degradation and enabling sustained protein expression for days to weeks. Beyond their use as engineered therapeutics, endogenous circRNAs exhibit cancer-associated expression patterns and persistence in biofluids, supporting complementary roles in tumor biology and as candidate biomarkers for diagnosis and longitudinal disease monitoring. This review argues that circular topology should be viewed as an active pharmacologic variable, not merely a stability enhancement. We dissect recent advances in cap-independent translation initiation, including IRES elements and m6A-driven mechanisms, rolling-circle translation for multi-epitope vaccine design, and programmable stability circuits that integrate tumor-microenvironment cues such as miRNA signatures. Delivery innovations are equally transformative: antibody-guided lipid nanoparticles and engineered extracellular vesicles enable increasingly selective RNA delivery, while local depot formulations and organ-selective systemic routes expand therapeutic reach. Safety considerations are re-evaluated as double-edged tools-innate immunogenicity can serve as a self-adjuvant for cancer vaccines, whereas back-splice-junction neoantigens offer both vaccine opportunities and tolerance risks. Recent advances in scarless circularization, topology-sensitive purification, dsRNA depletion, and lyophilized formulations have begun to address key manufacturing bottlenecks, although clinical-scale recovery and process scalability remain insufficiently characterized. Key applications include circRNA cancer vaccines, transient CAR-T/NK cell engineering, tumor-suppressor replacement, and circRNA-encoded bispecific T-cell engagers. The field now requires real-time pharmacokinetic tracking, reproducible and scalable manufacturing, validated liquid-biopsy assays, and indication-specific regulatory pathways to translate circRNA from bench to bedside.", "journal": "Biotechnology and bioengineering", "year": "2026", "doi": "10.1002/bit.70344", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "42462918": { "pmid": "42462918", "title": "Lysine as a multifunctional excipient for protein-based biopharmaceutical formulations.", "abstract": "Lysine (Lys) is a nutritionally essential amino acid with a well-established parenteral safety record, yet its potential as a multifunctional excipient for protein-based biopharmaceutical formulation has not been the subject of a dedicated comprehensive review. The physicochemical properties of Lys include bifunctional amine structure, stable cationic charge under formulation relevant pH conditions, and low molecular weight suitable for free-volume anti-plasticization. These characteristics are discussed in accordance with protein formulation requirements. Thermal characterization reveals that the glass transition temperatures of frozen and dried Lys-based solids are markedly responsive to counter-ion selection, enabling rational process optimization. Stability data from liquid and freeze-dried protein systems demonstrate concentration-dependent aggregation suppression, viscosity reduction in concentrated monoclonal antibody formulations, and lyoprotective efficacy across multiple therapeutic protein classes. Four FDA-approved protein-based biologics currently incorporate Lys in their formulations. Protective mechanisms span ice nucleation inhibition, water replacement, vitrification, beta-relaxation suppression, and direct ion-dipole interactions with protein surfaces. Notably, Lys functions as an essential nutrient in upstream cell culture and as a process stabilizer throughout downstream purification, suggesting its potential applicability across multiple stages of the biopharmaceutical manufacturing lifecycle. Practical limitations including Maillard reactivity and thermal constraints are addressed. Future priorities include systematic counter-ion characterization, mechanistic studies using advanced spectroscopic methods, and integration of Lys derivatives into computationally guided formulation design.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127192", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40896582": { "pmid": "40896582", "title": "Thermostable unit solid dose formulations for subcutaneous administration of DNA vaccines.", "abstract": "The coronavirus disease 2019 pandemic has highlighted the critical need for thermostable vaccines to ensure equitable distribution and accessibility, particularly in regions lacking cold chain infrastructure. Here we present a thermostable, solid dose DNA vaccine (SDV) platform for subcutaneous delivery, based on a sugar-sugar alcohol-polymer formulation manufactured via lyophilization and compaction. Using luciferase-expressing plasmid as a model, we demonstrate that subcutaneous vaccination with SDV formulation of C57BL/6 mice results in efficient and durable transgene expression in vivo. In vitro stability assays confirmed that the SDV formulation maintained excellent thermostability after 30 days of storage at 4°C, 25°C, 37°C, and 42°C. We next applied the SDV platform to a Zika virus (ZIKV) NS1 DNA vaccine and immunized BALB/c mice. ZIKV-SDV vaccination elicited robust NS1-specific antibody and T cell responses, and conferred protection upon ZIKV challenge. These data establish the feasibility of lyophilized SDV DNA vaccines for needle-free thermostable delivery. By eliminating the need for reconstitution, refrigeration, and skilled administration, SDV formulation has the potential to enhance the deployment, cost effectiveness, and shelf-life of DNA vaccines in resource-limited settings.", "journal": "Molecular therapy. Nucleic acids", "year": "2025", "doi": "10.1016/j.omtn.2025.102628", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40177644": { "pmid": "40177644", "title": "Fundamental properties and principal areas of focus in antibody-drug conjugates formulation development.", "abstract": "Antibody-drug conjugates (ADCs) have emerged as a rapidly expanding class of therapeutics driven by their superior specificity and clinical efficacy. 14 out of 16 commercially approved ADCs are formulated as lyophilized forms because ADC is generally considered to be less stable than unmodified antibody. The formulation development for ADCs, particularly liquid formulation, presents unique challenges due to their intricate structural complexity, physicochemical properties, and degradation pathways. This review provides the first comprehensive analysis of formulation strategies employed in commercial ADCs. Furthermore, this review discusses the key areas of focus for ADCs throughout the formulation development workflow, spanning from the initial formulation development to the final stage of drug product manufacturing. In addition, we identify and analyze the distinctive technical challenges in ADC formulation development compared to unconjugated antibody, while proposing potential solutions to these challenges. Finally, we offer strategic perspectives on future directions in ADC formulation development to advance this promising therapeutic modality.", "journal": "Antibody therapeutics", "year": "2025", "doi": "10.1093/abt/tbaf005", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40733720": { "pmid": "40733720", "title": "Immunogenicity, Efficacy and Twelve-Month Storage Stability Studies of a Lyophilized Rabies mRNA Vaccine.", "abstract": "Background: Many new mRNA-based vaccine candidates in liquid mRNA-LNP formulations are under development; however, their stability limitations necessitate frozen storage, posing a significant challenge for long-term storage and transportation. Methods: In this study, an mRNA-LNP rabies vaccine, ABO1005, was prepared, freeze-dried and stored at 2-8 °C for 12-month storage stability evaluation. The immunogenicity, vaccine potency (the NIH method), and protective efficacy of ABO1005 were assessed in mice or dogs and compared to a commercialized inactivated vaccine. Results: Research conducted in mice indicated that the lyophilized vaccine exhibited comparable immunogenicity to its liquid form counterpart. Furthermore, the vaccine candidate elicited a robust humoral response lasting at least 175 days, and the specific antibody titers were not affected by the pre-administration of hyperimmune serum. In comparison to the commercialized inactivated vaccine (HDCV or PVRV), ABO1005 elicited significantly higher levels of humoral and cellular immunity. Vaccine potency testing (NIH) revealed that the potency of ABO1005 at 15 μg/dose was 8.85 IU/dose, which is substantially higher than the standard required for the lot release of rabies vaccines for current human use. In a post-exposure prophylaxis (PEP) study in Beagle dogs, the lyophilized vaccine provided 100% protection for dogs following a two-dose regimen (D0-D7), whereas commercially approved inactivated vaccine offered 83% protection. After storage at 2-8 °C for 12 months, no notable changes were observed in the particle size, encapsulation efficiency, and integrity of mRNA or in the immunogenicity of the lyophilized vaccine. Conclusions: This study successfully developed a formulation and process of freeze-drying for a rabies mRNA vaccine, paving the way for future lyophilized mRNA vaccine development.", "journal": "Vaccines", "year": "2025", "doi": "10.3390/vaccines13070743", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40573935": { "pmid": "40573935", "title": "Development and Preclinical Evaluation of a Lyophilized Vaccine Against Equine Herpesvirus Type 4 (EHV-4).", "abstract": "Equine rhinopneumonia, caused by equine herpesvirus types 1 and 4 (EHV-1 and EHV-4), continues to be a significant health and economic concern in the global equine industry, particularly in Kazakhstan. While vaccines targeting EHV-1 are available, there is currently no licensed monovalent vaccine for EHV-4, and existing formulations offer limited protection against this serotype. This study aimed to develop and evaluate a freeze-dried, live-attenuated EHV-4 vaccine with improved safety, stability, and immunogenicity. A field isolate of EHV-4 was attenuated through serial passaging in primary lamb testicle (LT-KK49) cell cultures. Viral biomass was concentrated and formulated with various stabilizers before freeze-drying. The most effective stabilizer composition-sucrose, gelatin, and lactalbumin hydrolysate-was selected based on viral titer retention. Safety and immunogenicity were assessed in mice, guinea pigs, rabbits, donkeys, and horses. A guinea pig reproductive challenge model was used to evaluate protective efficacy. The optimized lyophilized vaccine retained infectivity (>6.0 log10 TCID50/cm3) for at least six months at 4 °C. No adverse clinical signs were observed in any test species. Immunization induced robust neutralizing antibody responses in both small animals and equines. In the guinea pig model, vaccinated females demonstrated 100% pregnancy retention and fetal viability following challenge with a virulent EHV-4 strain. This freeze-dried, live-attenuated EHV-4 vaccine candidate is safe, immunogenic, and thermostable. It offers a promising platform for the targeted prevention of EHV-4 infection, particularly in young horses and in regions with limited cold-chain infrastructure.", "journal": "Vaccines", "year": "2025", "doi": "10.3390/vaccines13060604", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39799738": { "pmid": "39799738", "title": "A VZV-gE subunit vaccine decorated with mPLA elicits protective cellular immmune responses against varicella-zoster virus.", "abstract": "Herpes zoster is an acute infectious skin disease caused by the reactivation of latent varicella-zoster virus, vaccination, such as subunit vaccine with good safety, can effectively prevent shingles through increasing immunity of the body. However, protein antigens are prone to degradation and inactivation, which alone is generally not sufficient to induce potent immune effect. In this study, the liposomal vaccine platform modified with mPLA (TLR4 agonist) was developed to improve the immunogenicity of glycoprotein E (VZV-gE) derived from herpes zoster virus. The thin-film dispersion and freeze-drying methods were employed to encapsulate VZV-gE against degradation, enhance liposomal stability, and achieve better redissolution effects with an optimized cryoprotectant. The in vitro results presented that mPLA could effectively enhance the uptake of VZV-gE with DC2.4. In vivo immune effect evaluation showed that the prepared subunit vaccines could induce stronger IgG, IgG1, and IgG2a antibody levels in the mouse serum, improving humoral immune effects. And the secretion levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) in the splenocytes were significantly increased, inducing protective cellular immune responses. Overall, this work presented that combining immunomodulatory adjuvants decorated nanocarriers to develop subunit vaccine platforms was a promising strategy to prevent the occurrence of herpes zoster effectively.", "journal": "International immunopharmacology", "year": "2025", "doi": "10.1016/j.intimp.2025.114033", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42140132": { "pmid": "42140132", "title": "Antigen-specific IgY as an orally deliverable biomaterial for sustained immunomodulation in ulcerative colitis.", "abstract": "Over the past decades, antibodies have become indispensable tools due to their high specificity and versatility. Egg yolk antibody (IgY), a natural protein biomaterial noted for its biosafety, thermal stability, and cost-effective, is particularly suitable for long-term interventions in chronic diseases. Its intrinsic biocompatibility allows safe oral administration, while its structural robustness facilitates diverse delivery formats such as bioactive formulations or coatings. This makes IgY a promising candidate for managing conditions like inflammatory bowel disease (IBD), including ulcerative colitis (UC), which require prolonged, well-tolerated therapeutic strategies. However, the antigen-specific therapeutic potential of IgY in IBD remains underexplored. Here, we propose to use an engineered anti-TNFR1 IgY as an oral therapeutic agent for UC intervention as a TNFR1-selective strategy. Anti-TNFR1 IgY exhibited superior gastrointestinal stability to mammalian IgG and suppressed the production of key inflammatory mediators, such as IL-6 and MMP9, in stimulated RAW264.7 macrophages. In a DSS-induced murine colitis model, oral administration of anti-TNFR1 IgY significantly alleviated inflammation, evidenced by reduced biomarkers (calprotectin, IL-6, TNF-α) and elevated IL-10, alongside improved behavioral manifestations and attenuated histopathological staining. Moreover, it retained bioactivity following heating, freeze-drying, and hydrogel encapsulation, supporting its potential as a scalable robust bioactive building block. Our work highlights antigen-specific IgY as a biocompatible biomaterial-based strategy for long-term immunomodulation in intestinal inflammation. This platform may help connect orally administered targeted therapy with gastrointestinal health management.", "journal": "Colloids and surfaces. B, Biointerfaces", "year": "2026", "doi": "10.1016/j.colsurfb.2026.115813", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39579940": { "pmid": "39579940", "title": "Scaling up controlled nucleation in freeze drying: Translating vacuum-induced surface freezing from laboratory to GMP.", "abstract": "The freezing step often causes batch inhomogeneity and issues during freeze drying process transfer. The nucleation temperature at which the first ice is formed during freezing differs from vial to vial, and significantly between scales. To solve this issue, Controlled Ice Nucleation techniques can be applied to induce ice nucleation at a defined product temperature across the whole batch. This study describes the application of vacuum-induced surface freezing (VISF) for a therapeutic antibody formulation, including the process transfer from laboratory scale through pilot scale to a GMP line. The VISF method could be successfully implemented on all scales of freeze dryers without equipment adaptation. Some scale-dependent changes in pressure control and degassing were necessary to achieve nucleation in all vials and avoid defects. The resulting lyophilized products were characterized and further analyzed in a stability study. While most critical quality attributes were comparable for product manufactured with and without Controlled Nucleation, the appearance of cakes processed using VISF was much better, which could be linked to different product morphology due to freeze-concentration. The results of this study allow direct comparison of the application of controlled nucleation for an antibody formulation at different scales and confirm the applicability of the technology.", "journal": "European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences", "year": "2025", "doi": "10.1016/j.ejps.2024.106968", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40042247": { "pmid": "40042247", "title": "Nanourchin-like Uricase-Poly(L-proline) Conjugate with Retained Enzymatic Activity, Mitigated Immunogenicity, and Sustained Efficacy Upon Repeated Administrations.", "abstract": "The poor half-life and strong immunogenicity of proteins such as uricase (UOx), a therapeutic enzyme for chronic refractory gout and hyperuricemia, are pressing clinical challenges. Although conjugation of poly(ethylene glycol) (PEGylation) of UOx can improve the pharmacokinetics, preexisting or induced anti-PEG antibodies, which lead to accelerate blood clearance (ABC) and reduced response rate, have been a major clinical hurdle. Herein, we report the facile \"grafting-from\" preparation of a nanourchin-like uricase-poly(L-proline) conjugate, namely UOx-PLP, with high grafting-density, enhanced thermal, lyophilization, freeze-thaw, and proteolytic stability. Through a transient preblocking strategy in the synthesis, the UOx-PLP overcomes activity loss and retains ~82 % enzyme activity. In Sprague-Dawley rats, UOx-PLP stimulates minimum complement activation and anti-UOx antibodies. Unlike PEG-UOx gave a significantly reduced half-life after repetitive administrations, UOx-PLP shows no sign of ABC effect. Moreover, the half-life of UOx-PLP remain almost unchanged when cross-administrated to rats previously received PEG-UOx and with high titers of anti-UOx antibodies. Finally, UOx-PLP shows minimum loss of efficacy after five straight administrations in a UOx knock-out hyperuricemia mice model, whereas PEG-UOx experiences sharp loss of efficacy upon the same treatment. Overall, the simple preparation and outstanding nonclinical results highlight the enormous potential of UOx-PLP for future clinical translation.", "journal": "Angewandte Chemie (International ed. in English)", "year": "2025", "doi": "10.1002/anie.202425559", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "40817522": { "pmid": "40817522", "title": "Pectin/arabinoxylan dietary polysaccharide microcapsule encapsulated with Clostridium butyricum for targeted delivery in colitis treatment.", "abstract": "The gut microbiota plays a critical role in maintaining intestinal homeostasis, and its dysbiosis is a key factor in the development of inflammatory bowel diseases (IBD), such as colitis. This study developed an innovative dual-layer synbiotic microcapsule system for targeted delivery of Clostridium butyricum (C. butyricum) to treat dextran sulfate sodium (DSS) induced colitis. The microcapsules utilized pectin (PEC) and arabinoxylan (AX), both dietary polysaccharides with prebiotic properties, as encapsulating materials. The PEC@AX@C microcapsules enhanced the survival and colon-targeted release of C. butyricum under harsh gastrointestinal conditions, owing to their dual-layer structure and pH-sensitive release property. Key findings indicated that the microcapsules significantly improve the stability and viability of C. butyricum during gastrointestinal transit, long-term storage, and extreme temperature conditions. In vivo studies using DSS-induced colitis in mice demonstrated that PEC@AX@C microcapsules could mitigate weight loss, reduce inflammation, repair intestinal barrier function, and restore gut microbiota balance. The synergistic effects of probiotics, prebiotics, and postbiotics promoted the production of short-chain fatty acids, enhanced tight junction protein expression, and reduced pro-inflammatory cytokine levels. Additionally, the gut microbiota of treated mice exhibited increased α-diversity and a recovery in microbial profile similar to that of healthy control. These results highlight the potential of PEC@AX@C microcapsules as a promising strategy for precision IBD therapy, offering new insights into probiotic and prebiotic-based therapeutic systems.", "journal": "Carbohydrate polymers", "year": "2025", "doi": "10.1016/j.carbpol.2025.123971", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "40280286": { "pmid": "40280286", "title": "Spray drying for protein stabilization.", "abstract": "During formulation development, stabilizing buffers and excipients are added to therapeutic proteins to overcome their intrinsic instability. Another approach is their solidification using techniques like freeze drying or spray drying (SD). In this work, to enhance the stability of three proteins (α-chymotrypsin, catalase and Horseradish Peroxidase (HRP)), suitable buffers and excipients were selected in solution state for three formulations (referred to as concepts) for each protein using Differential Scanning Fluorimetry (DSF) combined with Static Light Scattering (SLS). Two of these concepts were supposed to stabilize the protein and one was less stabilizing. Then, SD was performed, and the protein stability was compared to that prior SD using dynamic light scattering, UV-VIS spectroscopy, far-UV circular dichroism, size-exclusion and reversed-phase chromatography. While the selected excipients did not differ much from the ones used during a previous study on Bovine Serum Albumin (BSA), Immunoglobulin G (IgG) and lysozyme, clear stability differences during solidification were observed. The measured recovery was partly 40 % lower than prior to SD and only one catalase concept was able to maintain its original concentration. While the stability of two catalase concepts was correctly predicted by DSF/SLS, it was not the case for the other proteins. This outcome led to the question whether it is sufficient to determine protein stability during solidification using DSF/SLS or if a more holistic approach is necessary.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125600", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39085576": { "pmid": "39085576", "title": "Dry powder formulations of hyperimmune serum.", "abstract": "Effective strategies against the spread of respiratory viruses are needed, as tragically demonstrated during the COVID-19 pandemic. Apart from vaccines, other preventive or protective measures are necessary: one promising strategy involves the nasal delivery of preventive or protective agents, targeting the site of initial infection. Harnessing the immune system's ability to produce specific antibodies, a hyperimmune serum, collected from an individual vaccinated against SARS-CoV-2, was formulated as a dry powder for nasal administration. The selection of adequate excipients and process are key to maintaining protein stability and modulating the aerodynamic properties of the powders for reaching the desired respiratory regions. To this end, a hyperimmune serum was formulated with trehalose and mannitol as bulking agents during spray drying, then the ability of the redissolved immunoglobulins to bind Spike protein was verified by ELISA; foetal bovine serum was formulated in the same conditions as a reference. Moreover, a seroneutralization assay against SARS-CoV-2 pseudoviruses generated from different variants of concern was performed. The neutralizing ability of the serum was slightly reduced with respect to the starting serum when trehalose was used as a bulking agent. The powders were loaded in hypromellose capsules and aerosolized employing a nasal insufflator in an in vitro model of the nasal cavity connected to a Next Generation Impactor. The analysis of the powder distribution confirmed that all powders were inhalable and could target, at the same time, the upper and the lower airways. This is a preliminary proof-of-concept that this approach can constitute an effective strategy to provide broad coverage and protection against SARS-CoV-2, and in general against viruses affecting the airway. According to blood availability from donors, pools of hyperimmune sera could be rapidly formulated and administered, providing a simultaneous and timely neutralization of emerging viral variants.", "journal": "Drug delivery and translational research", "year": "2025", "doi": "10.1007/s13346-024-01678-8", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "40732252": { "pmid": "40732252", "title": "Spray-Drying Microencapsulation of Natural Bioactives: Advances in Sustainable Wall Materials.", "abstract": "Background/Objectives: In recent years, increasing attention has been paid to the stabilization of natural biologically active compounds in order to expand their application in the food, pharmaceutical, and cosmetic industries. Such compounds, such as polyphenols, essential fatty acids, or vitamins, are extremely sensitive to environmental factors. This study aims to review the spray-drying-based microencapsulation technology and its application for stabilizing sensitive biologically active substances. Methods: This article systematically analyzes the main steps of the spray-drying microencapsulation process and discusses traditional and innovative wall materials, including natural polymers (polysaccharides and proteins), as well as new raw material sources (e.g., yeast cells, canola and pea protein isolates, and hemicelluloses). It also examines the potential of these systems for the stimulated release of active ingredients. Results: This review provides a comprehensive overview of the main stages of the spray-drying process and critically examines both conventional (e.g., maltodextrin and gum Arabic) and innovative wall materials (e.g., plant-based proteins and food industry by-products). Studies show that using different wall materials can achieve high encapsulation efficiency, improve the stability of biologically active substances, and control their release. Various compounds have been successfully microencapsulated-polyphenols, essential oils, carotenoids, fatty acids, and vitamins-protecting them from oxidation, light, and temperature. The review identifies key factors that can enhance product quality, increase encapsulation yield, and reduce processing costs and energy input-offering meaningful insights for optimizing the microencapsulation process. Conclusions: Spray-drying-based microencapsulation is an advanced technology that effectively protects sensitive active ingredients and allows for wider industrial food, pharmaceutical, and cosmetic applications. In the future, more attention is expected to be paid to personalized formulations, stimulated release systems, and sustainable wall materials from by-products.", "journal": "Pharmaceuticals (Basel, Switzerland)", "year": "2025", "doi": "10.3390/ph18070963", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40607021": { "pmid": "40607021", "title": "Low concentration press cake protein isolates preserve biological activity during lyophilization and spray drying.", "abstract": "A primary challenge in bringing cultivated meat to the market is the high cost of the cell culture media, largely due to their reliance on serum albumins. The production of these albumins is anticipated to become a major bottleneck of this industry. Recently, human serum albumin (HSA) was successfully substituted with seed protein isolates from press cakes enriched with plant albumins. However, these isolates require storage at -80°C to maintain activity, as long-term storage at 4°C or lyophilization leads to aggregation and loss of biological activity. Here, we show that concentrated protein isolates from Styrian oil pumpkin can effectively substitute for human serum albumin (HSA) and support higher proliferation rates in short term experiments as compared to HSA when stored at -80°C (p < 0.001), or even when lyophilized (p < 0.01). We also demonstrate that protein isolates from Styrian oil pumpkin and rapeseed press cakes perform comparably to HSA (no significant differences) when lyophilized or even spray-dried, provided that the concentration step is omitted. Furthermore, we report protein yields that are four times higher when a more thorough grinding method is utilized. These advancements eliminate the necessity for -80°C storage, thereby facilitating the utilization of locally available press cake protein isolates in media stabilization applications.", "journal": "Frontiers in nutrition", "year": "2025", "doi": "10.3389/fnut.2025.1602010", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42659960": { "pmid": "42659960", "title": "Spray dried β-carotene: Encapsulation, stabilization, and controlled-release.", "abstract": "Vitamin A deficiency remains a major public health challenge worldwide, and β-carotene, as a provitamin A carotenoid, is widely used in food fortification to improve vitamin A intake. However, its application in foods is constrained by poor water solubility, limited bioaccessibility, and high susceptibility to heat, light, and oxygen. This review evaluates spray drying as an encapsulation strategy for β-carotene, focusing on the effects of wall materials and operating conditions on powder formation, encapsulation efficiency, stability, and release behavior. It also summarizes the chemical structure, health benefits, and major degradation pathways of β-carotene relevant to food systems. Current studies indicate that spray drying encapsulation can greatly enhance β-carotene performance, with encapsulation efficiencies of up to 99%. Carbohydrate-based carriers, especially maltodextrin, are most commonly used, while proteins such as whey protein isolate are often combined to improve emulsion stability and matrix integrity. Encapsulation has been shown to improve stability during storage and thermal exposure, with some systems retaining more than 99% activity after four weeks at 60°C, while collagen peptide-based crosslinked protein-peptide matrices increased β-carotene bioaccessibility to as high as 82.95% through improved protection during gastric digestion and controlled release in the intestinal phase. Overall, spray drying is a promising and scalable approach for β-carotene delivery in foods, although further research focusing on large-scale continuous processing, cost-effective wall material selection, and long-term stability in complex food matrices is needed to bridge laboratory findings with industrial application.", "journal": "Food chemistry", "year": "2026", "doi": "10.1016/j.foodchem.2026.150682", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "A" ] }, "40057212": { "pmid": "40057212", "title": "The value of spray drying as stabilization process for proteins.", "abstract": "Protein stability in solution state is often poor due to the intrinsic instability of proteins. A solution is to solidify them by using techniques like freeze or spray drying (SD). To shield therapeutic proteins from stress (e.g., heat or shear stress) related to the solidification process, suitable buffers and excipients are added during formulation development. In this work, buffers and excipients were identified for the stabilization of three protein model compounds (BSA, IgG and lysozyme) in solution state using a design of experiments (DoE) approach based on screening results from differential scanning fluorimetry (DSF) combined with static light scattering (SLS). The aim was to investigate whether it is possible to predict protein stability in solid state using data from protein stabilization in solution state according to DSF/SLS. Therefore, three concepts per protein were analyzed after SD, two of which were expected to stabilize the protein, and one less stabilizing and compared these results to screening results obtained in solution state. Analytical techniques prior to and post SD were reversed-phase and size-exclusion chromatography (RPC and SEC, respectively), dynamic light scattering (DLS), UV and circular dichroism (CD). Furthermore, yield and residual moisture were analyzed. BSA and lysozyme showed high stability during SD and therefore only minor changes were observed. IgG was more affected by solidification which partly resulted in a loss of more than 15 % of the initial protein concentration in comparison to before SD. In future studies, the use of analytical techniques that do not require reconstitution would give additional value.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.125422", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39909221": { "pmid": "39909221", "title": "Developing ultra-high concentration formulations of human immune globulins for subcutaneous injectables.", "abstract": "This work describes the first development of high-concentration suspension formulations of human immune globulin. Colloidal-level dispersions of immune globulin were achieved by suspending a spray dried solid powder of protein in a protein solution made saturated by the addition of pharmaceutical excipients. The spray drying process was used to generate ∼90 % of particles below 20μ. The monomer and aggregates content of immunoglobulin were found to be 93 % and 0.3 %, respectively. The injection forces for the colloidal suspensions were characterized using a dynamic compression test. The concentrations of 300, 380, and 400 mg/mL formulations were injected at 3.8 N, 10 N, and 16.5 N of maximum injection forces, respectively, when a 24-gauge needle was used. The viscosity of a 300 mg/mL suspension was 128 cP. The viscosity of a 380 mg/mL suspension was 284 cP, and the viscosity was higher for the 400 mg/mL formulation; however, injectability was not an issue, which remains rare for non-Newtonian, shear-thickening systems. It is acknowledged that the 400 mg/mL suspension formulation remained relatively challenging as compared to other suspensions for injection because of its very high viscosity, and significant force was required to inject it. We show that where ultra-high-concentration immune globulin is being developed within reasonable constraints of pharmaceutical regulation, with an injectability parameter, formulations might make their way to the clinic when viscosity could say otherwise. However, further work should be conducted to assess chemical stability (using methods such as mass spectrometry) along with forced degradation studies prior any clinical use.", "journal": "Journal of pharmaceutical sciences", "year": "2025", "doi": "10.1016/j.xphs.2025.01.028", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "A" ] }, "41057059": { "pmid": "41057059", "title": "A Thermostable nasal spray dried COVID vaccine candidate.", "abstract": "A nasal dry powder adjuvanted subunit COVID vaccine candidate was manufactured via spray drying and evaluated for physicochemical stability and aerosol performance over the course of 10 months under accelerated conditions. A nanoliposomal adjuvant system containing synthetic TLR 4 agonist GLA and synthetic TLR 7/8 agonist 3 M-052 and a trimeric SARS-CoV-2 spike protein antigen were encapsulated using trehalose and varying amounts of trileucine as excipients. 1 % and 3 % trileucine batches as well as a trehalose-only control batch were spray dried to achieve varying levels of particle surface modification and to study the overall effects on stability and aerosol performance. All batches achieved good yields and low processing losses on drying. Samples were held at 25 °C and 40 °C and monitored for physical and chemical stability. All three batches showed excellent performance over the course of the study. Overall morphology; solid phase; moisture content; contents of GLA and 3 M-052; and aerosol performance were largely maintained after exposure to high temperatures for 10 months. Spike protein antigen remained present in all samples after exposure, and liposomal size distributions remained within acceptable ranges for all but one of the samples. Overall, this vaccine candidate showed performance suitable for distribution independent of the cold chain and would be able to withstand high-temperature conditions encountered during last-mile delivery.", "journal": "International journal of pharmaceutics", "year": "2025", "doi": "10.1016/j.ijpharm.2025.126240", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "42575384": { "pmid": "42575384", "title": "Inhalable spray dried human platelet lysate powders.", "abstract": "Human platelet lysate (PL) is a mixture of proteins containing growth factors, cytokines, and other biomolecules. PL shows promising effects on tissue regeneration and immunomodulation and may be applied to the lungs. Inhalation enables direct deposition of PL in the airways, maximising its therapeutic potential. This study aimed to develop inhalable spray dried PL powders with two excipients, trehalose and leucine. Spray dried PL alone contained 5% w/w residual solvent by mass and was amorphous, while powders containing excipients were partially crystalline with lower residual moisture content (2-4% w/w). Their particles were about 4 µm in volumetric size and exhibited hollow, rough-surfaced morphologies. Compared with spray dried pure PL, the excipient containing powders adsorbed significantly less moisture, and no recrystallisation was observed in the formulation with 60% w/w leucine. Our spray-drying process largely maintained protein content. Protein recovery was about 90% for spray dried PL alone, while the addition of trehalose further improved preservation. For aerosol performance, the fine particle fraction (FPF) of spray dried PL was 23%, with higher FPFs observed in the powders containing excipients. The highest FPF (34%) was achieved in the formulation with 60% w/w leucine. This formulation also significantly diminished the increased levels of nitric oxide and interleukine-6 (IL-6) in J774A.1 cells stimulated with lipopolysaccharide, indicating the preservation of immunomodulatory activity of PL in the inhalable powder. This study demonstrates the feasibility of producing inhalable and biological viable PL powders via spray drying and provides a proof of concept for developing platelet-based powders as potential treatments for lung conditions.", "journal": "International journal of pharmaceutics", "year": "2026", "doi": "10.1016/j.ijpharm.2026.127304", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41304729": { "pmid": "41304729", "title": "Leucine Protects Dry Powders for Inhalation Against Irreversible Moisture-Induced Aggregation.", "abstract": "Background: Pulmonary administration offers a promising route for certain biologics, particularly when treating respiratory diseases. Spray drying is widely employed to produce inhalable powders with the biologics incorporated in a stabilizing amorphous sugar. Hydrophobic amino acids such as leucine are frequently added to improve dispersibility. Objectives: While the aerodynamic benefits of leucine are well established, its influence on irreversible moisture-induced dry powder particle aggregation and protein stability during storage remains less evaluated. Methods: In this work, inulin-based powders with and without 4 wt-% leucine were spray dried and stored at 43%, 58%, 69%, and 75% relative humidity (RH) at 20 ± 2 °C. Results: Immediately after drying, both formulations displayed comparable physicochemical characteristics. However, during storage of inulin-only formulations, dry powder particles showed viscous flow and formed big irreversible aggregates after storage at an RH of 58% and above, whereas leucine-containing powders remained intact across all tested conditions up to 20 days. Protein stability was assessed using lactate dehydrogenase (LDH) and β-galactosidase (β-gal) as model proteins. At 43% RH, the Tg remained above the storage temperature, and both LDH and β-gal retained their enzymatic activity for up to 20 days. At 75% RH, however, the Tg dropped to below storage temperature, resulting in a loss of stability for LDH, consistent with its reliance on vitrification. In contrast, β-gal maintained its activity at 75% RH, indicative of stabilization through water replacement. Conclusions: Overall, these results demonstrate that leucine enhances the physical stability of inulin powders by preventing irreversible aggregation under humid conditions. However, this effect does not extend to protecting proteins reliant on vitrification. These findings highlight the potential of inulin and leucine to reduce the need for stringent storage conditions of biologics.", "journal": "Pharmaceutics", "year": "2025", "doi": "10.3390/pharmaceutics17111391", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "39856947": { "pmid": "39856947", "title": "Betalain-Chickpea Protein Particles Produced by Freeze Drying and Spray Drying: Physicochemical Aspects, Storage Stability, and In Vitro Digestion.", "abstract": "Beetroots are one of the primary sources of betalains, nitrogenous pigments with anti-inflammatory and antioxidant properties. However, due to their chemical instability, betalains have limited use in food applications. This work investigated whether betalains encapsulated in chickpea protein could be stabilized and delivered in a shelf-stable format. Freeze-dried (CB-FD) and spray-dried (CB-SD) protein-betalain particles encapsulated in chickpea protein isolate (6% w/v) were prepared. The encapsulation method affected particles' morphology, water activity, hygroscopicity, solubility, and color. Particles captured total betalains of 9.30 ± 0.61 and 4.40 ± 0.92 mg/g for CB-SD and CB-FD, respectively. LC-MS identified 12 betacyanins and 6 betaxanthins. The stability of betalains revealed that encapsulation efficiently preserved betalain integrity of over 6 weeks of storage at 4, 22, and 40 °C compared to dry beetroot extract. CB-SD particles were stable with no significant changes, while CB-FD showed slight degradation after 4 weeks due to increased Aw. Antioxidant activity correlated well with betalain concentration. In vitro digestion resulted in only 25% bioaccessibility of betacyanins, while betaxanthins were more stable with 100% recovery. Encapsulation with chickpea protein isolate is an efficient and straightforward strategy for expanding and diversifying applications of phytochemical-rich beetroot extracts for the food industry.", "journal": "Foods (Basel, Switzerland)", "year": "2025", "doi": "10.3390/foods14020281", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40865302": { "pmid": "40865302", "title": "Unraveling the loss of lysozyme concentration after tableting.", "abstract": "Therapeutic proteins are fragile compounds whose functions are dependent on their higher-order structure (HOS) formed by specific intramolecular interactions. Nevertheless, protein interactions can also impede the stability if they take place with surfaces or formulation components. Besides chemical and conformational alterations, it can lead to the reduction of free non-complexed protein concentration. These are all factors which can lower the protein's therapeutic effect and decrease therapeutic efficacy. During the minitablet development of lysozyme a loss of more than 30 % in protein recovery after tableting was observed using a reversed-phase chromatography (RPC) assay method. The aim of the current study was to unravel the cause for the loss in measured protein concentration, as prior to and post spray drying (SD) the recovery was 98.65 ± 0.76 %. It was shown that the loss was more linked to persistent protein-excipient interactions and not to the tableting process itself. It was possible to overcome these interactions with the selection of an optimized buffer system for the analytical sample preparation. For the selection of the buffer system, protein properties such as the isoelectric point (pI) and the charge had to be considered. In case of the minitablets containing lysozyme, it was a 100 mM arginine buffer pH 7.00 100 mM NaCl 0.1 % PS20 that allowed a protein recovery of almost 95.00 % after tableting. These results highlight the importance of analytical sample preparation and the selection of a suitable solvent for protein assay determinations.", "journal": "Journal of pharmaceutical and biomedical analysis", "year": "2026", "doi": "10.1016/j.jpba.2025.117116", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40611988": { "pmid": "40611988", "title": "Development of an inhalable dry powder formulation for inhibition of SARS-CoV-2.", "abstract": "Coronaviruses, including SARS-CoV-2, can cause significant lung damage and may result in multiple organ failure. The severity of COVID-19 is determined by the virus's entry into lung tissue and subsequent replication. This entry is facilitated by the angiotensin-converting enzyme 2 (ACE2) in combination with the serine protease TMPRSS2, which is a critical step. To reduce viral replication, it is necessary to prevent the uptake of the virus directly at the main route of transmission, which is the deposition of the virus as an aerosol in the respiratory tract. To reduce viral uptake into lung cells, an inhalable dry powder formulation was developed. The formulation contains camostat, a clinically proven serine protease inhibitor that inhibits the cellular uptake mechanisms on the lung surface. Camostat was spray-dried together with the mucolytic agent N-acetylcysteine to produce co-amorphous microparticles with sufficient solubility after deposition. Microparticles with properties suited for deposition in the deep part of the respiratory tract can be produced by using appropriate spray-drying parameters. The use of L-leucine enabled suitable aerodynamic properties and storage stability due to reduced interaction with environmental water. The geometric particle diameter, determined using laser light diffraction, decreased with L-leucine content which was found forming a partially crystalline L-leucine shell. The disintegration behavior of the microparticle formulation simulated under lung-like conditions indicated fast disintegration. A pseudo-viral in vitro assay demonstrated low acute toxicity in combination with a high activity. Cell viability and proliferation were not affected by camostat concentrations up to 11.1 μg/mL. The IC50 values of the two dry powder formulations tested on a HEK293T/ACE2-TMPRSS2 cell line were 0.008 μg/mL and 0.019 μg/mL, respectively, which is at least 100 times lower than the cytotoxic concentration. This dry powder formulation serves as a prototype microparticle matrix for incorporating nanoscale drug carriers in the future.", "journal": "International journal of pharmaceutics: X", "year": "2025", "doi": "10.1016/j.ijpx.2025.100346", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41855833": { "pmid": "41855833", "title": "Encapsulation of echium oil by electrospraying and mono- or coaxial spray-drying using plant protein hydrolysates as emulsifiers.", "abstract": "The development of echium oil-loaded nano-microcapsules by electrospraying, mono- and coaxial spray-drying was investigated. Brewers' spent grains (BT) or defatted grape seed flour (GT) hydrolysates were used as plant-based emulsifiers. The oxidative stability of the capsules was assessed by Electron Spin Resonance (ESR). Electrosprayed particles had narrow size distribution, mostly <1.5 μm, increased specific surface area and low encapsulation efficiency (EE =61%). On the contrary, capsules produced with mono- and coaxial spray-drying mostly ranged from 2 to 10 μm and had higher EE (>80%), showing higher oxidative stability. BT, exhibiting higher emulsifying capacity and leading to more viscoelastic interfacial films than GT, led to microcapsules with higher oxidative stability with no difference between mono- and coaxial processes. In contrast, coaxial spray-drying, resulting in capsules with core-shell structure, reduced oil leakage when using the less powerful emulsifier GT, enhancing oxidative stability when compared to the monoaxial process.", "journal": "Food chemistry", "year": "2026", "doi": "10.1016/j.foodchem.2026.148893", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "A" ] }, "42208274": { "pmid": "42208274", "title": "Whey protein isolate-procyanidin lutein microcapsules against blue light-induced retinal damage.", "abstract": "The increasing prevalence of prolonged exposure to electronic devices has made ocular health risks a major public health concern. Natural food pigments, such as procyanidins (PCs) and lutein, exhibit excellent antioxidant properties, but their poor stability greatly limits their bioavailability. To overcome these limitations, a noncovalent whey protein isolate-procyanidin complex (WPI-PC) was designed and its binding interactions were investigated using FTIR and fluorescence spectroscopy. After high-pressure homogenization and spray drying, the lutein retention rate in the resulting microcapsules reached 83.2%. Structural characterization showed that microcapsules prepared with 3 wt% WPI-PC had higher total oil content, lower surface oil content, lower hygroscopicity and water activity and good powder fluidity. Moreover, the lutein microcapsules remained stable under simulated gastric conditions and were efficiently digested in simulated intestinal fluid, supporting lutein release and potential intestinal absorption. In vitro and in vivo experiments further demonstrated that lutein microcapsules effectively reduced reactive oxygen species accumulation and inflammatory factor secretion and ultimately alleviated blue light-induced retinal damage in mice. Overall, these microcapsules represent promising nutraceutical formulations that synergistically enhance the stability, bioaccessibility and ocular protective efficacy of lutein and PCs, showing potential for dietary interventions related to eye health.", "journal": "Colloids and surfaces. B, Biointerfaces", "year": "2026", "doi": "10.1016/j.colsurfb.2026.115847", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "41655059": { "pmid": "41655059", "title": "Effect of Freeze-Drying and Spray-Drying on Emulsibility and Stability of Emulsions Stabilized by Covalent and Non-Covalent Whey Protein-Dextran Complexes.", "abstract": "This study focuses on the stability of whey protein isolate-dextran (WPI-D) complexes, exploring how their modification modes (covalent grafting/non-covalent mixing) and drying processes (spray-drying [SD]/freeze-drying [FD]) regulate structural properties and stability. Covalent WPI-D grafts (WPI-D-G) exhibit a higher degree of glycosylation, with significant fluorescence quenching and red shift. Fourier transform infrared (FTIR) spectral analysis reveals reduced absorbance of amide bands, collectively forming rigid cross-linked structures. Compared to non-covalent WPI-D mixtures (WPI-D-M), these structures exhibit superior emulsifying activity index (EAI), emulsion stability index (ESI), as well as stronger tolerance to environmental stresses (thermal stability: FD WPI-D-G (WPI-D-GF) maintain a particle size of 0.447 µm at 80°C, whereas SD WPI-D-G (WPI-D-GS) tend to aggregate; salt tolerance: stable at NaCl ≤ 20 mg/mL; pH stability: robust across pH 2-7). WPI-D-GF retain intact cross-linked structures, exhibiting enhanced emulsifying performance and colloidal stability (higher absolute zeta potential), whereas WPI-D-GS, despite forming smaller uniform spherical particles, undergo partial structural degradation. Regarding comparative stability under different conditions: WPI-D-GF outperforms WPI-D-GS in thermal stability, whereas covalent grafts from both drying methods demonstrate comparable salt and pH tolerance. These findings elucidate the structure-stability relationship of WPI-D complexes, thereby providing a theoretical foundation for regulating their stability through process optimization.", "journal": "Journal of food science", "year": "2026", "doi": "10.1111/1750-3841.70913", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40823141": { "pmid": "40823141", "title": "Effects of drying techniques on cowpea albumin as an egg replacement for meringue cookie applications.", "abstract": "In this study, we evaluated the effects of freeze-drying, spray-drying, and vacuum-drying on upcycled cowpea albumin powder and its functional performance in meringue cookie applications. Freeze-drying most effectively preserved protein structure and functionality, resulting in cookies with well-defined air cells, superior texture, and minimal color change. Conversely, spray-drying led to extensive protein denaturation and aggregation due to rapid thermal exposure, which compromised air incorporation and foam stability. Vacuum-drying exhibited intermediate performance, partially preserving protein integrity and exhibiting moderate foaming capacity. Structural and functional analyses revealed that freeze-dried albumin exhibited the highest foaming capacity (133.67 %) and foam stability (53.37 %), along with a significantly lower zeta potential (-6.79 mV) and higher surface hydrophobicity (H₀ = 363.38) compared with the other treatments. These findings highlight the significant role of the drying method in preserving the physicochemical properties of plant-based proteins.", "journal": "Food chemistry: X", "year": "2025", "doi": "10.1016/j.fochx.2025.102868", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "A" ] }, "40580283": { "pmid": "40580283", "title": "Digital PCR Genotyping of Pepino Mosaic Virus.", "abstract": "Pepino mosaic virus (PepMV) is a plant pathogen causing significant economic losses in tomato production. Sensitive, reliable, and robust detection methods are crucial for containing the spread of PepMV and reducing its damaging effects. Digital PCR (dPCR) presents several advantages to conventional real-time quantitative PCR (qPCR), including absolute quantification ability, robust quantitative multiplexing capabilities, and straightforward result analysis. Furthermore, dPCR is especially suitable for analysis of complex samples due to its remarkable tolerance to PCR inhibitors, which makes it a promising method for plant virus genotyping. In this chapter, we present two protocols for PepMV genotyping and quantification using one-step reverse transcription digital PCR (RT-dPCR). The first protocol outlines four simplex assays, while the second describes two duplex assays for precise and comprehensive genotyping of PepMV variants.", "journal": "Methods in molecular biology (Clifton, N.J.)", "year": "2025", "doi": "10.1007/978-1-0716-4642-7_3", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39721344": { "pmid": "39721344", "title": "An investigation of downstream processing methods for challenging skeletal samples.", "abstract": "While skeletal remains are known for their resilience and often serve as the final source of information for unidentified human remains (UHRs), the traditional downstream processing of these samples is challenging due to their low template nature, DNA degradation, and the presence of PCR inhibitors, typically resulting in limited probative information. To address this issue, advanced genotyping methods can be explored to retrieve additional genetic information from these challenging samples to maximize investigative leads. Therefore, this study investigated the effectiveness of three advanced genotyping methods and assessed their suitability with compromised skeletal samples: 1) targeted next generation sequencing (NGS) of both STRs and SNPs using the ForenSeq® DNA Signature Prep chemistry, 2) targeted NGS of SNPs using the ForenSeq® Kintelligence kit, and 3) SNP genotyping using a microarray via the Infinium Global Screening Array. The genotype recovery and added investigative leads were compared across all methods. All three approaches demonstrated success with the challenging skeletal samples used in this study. Specifically, the ForenSeq® DNA Signature Prep chemistry outperformed traditional STR typing by improving the recovery of CODIS core loci. Additionally, the ForenSeq® Kintelligence kit and Infinium Global Screening Array provided eligible results for forensic investigative genetic genealogy (FIGG) searching. Based on these successes, we have developed a proposed workflow for downstream processing of challenging skeletal samples. Following the guidelines of the US Department of Justice, the recovery of the CODIS core loci should be attempted through traditional CE-based methods or a NDIS-approved NGS chemistry, such as ForenSeq® DNA Signature Prep. Alternatively, a mitochondrial DNA profile may be uploaded to CODIS for comparisons in UHR cases. However, if no probative information is developed from the forensic profile uploaded to CODIS, then FIGG methods can be implemented using the Infinium Global Screening Array for high-quality skeletal samples (DNA concentrations ≥ 0.5 ng/µL) or the ForenSeq® Kintelligence chemistry for low-template skeletal remains (DNA concentration ≤ 0.5 ng/µL). These findings provide valuable insight into the suitability and efficacy of advanced genotyping methods, offering promising opportunities for enhancing the investigation of cases involving UHRs.", "journal": "Forensic science international. Genetics", "year": "2025", "doi": "10.1016/j.fsigen.2024.103209", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't", "Research Support, U.S. Gov't, Non-P.H.S." ], "first_author": "", "dirs": [ "B" ] }, "40489078": { "pmid": "40489078", "title": "Parallel dilution microfluidic device for enabling logarithmic concentration generation in molecular diagnostics.", "abstract": "In this study, we present a genetic diagnostic device with a four-stepwise logarithmic dilution capability for rapid and reliable detection of target nucleic acids in a single operation using the colorimetric loop-mediated isothermal amplification (LAMP) method. An innovative feature is the confluent point with differing microchannel heights ensuring the synchronized inflow of liquids while preventing backflow, even under large volumetric flow rate variations (10-10 000-fold). This enabled the independent generation of each dilution factor under constant pressure. Furthermore, an integrated asymmetric micromixer effectively mixed two liquids under laminar flow conditions, enabling simultaneous dispensing of the mixed solution at uniform concentrations into five microchambers for each dilution factor. Additionally, a permanent stop valve in the outlet of each microchamber prevented leakages, minimizing the waste of valuable samples and reagents. We demonstrate that diluted samples were accurately prepared at the intended logarithmic dilution factors in a single operation using purified cannabis seed DNA, achieving detection sensitivity similar to that of conventional turbidity-based LAMP assays. Moreover, we used crudely extracted cannabis resin DNA, which contains several gene amplification inhibitors, successfully detecting the target nucleic acids in a single test. Overall, this versatile device eliminates extensive manual sample preparation and has potential for on-site genetic testing in applications such as infectious disease detection, food safety, and illegal drug testing.", "journal": "Lab on a chip", "year": "2025", "doi": "10.1039/d5lc00356c", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "42634044": { "pmid": "42634044", "title": "Digital PCR in forensic genetics: principles, applications, and challenges.", "abstract": "Digital PCR (dPCR) has emerged as a transformative technology in forensic genetics, offering absolute quantification of nucleic acids without the need for standard curves. This review comprehensively summarizes the principles, analytical advantages, and diverse applications of dPCR in forensic medicine, while also addressing current challenges and future perspectives. By partitioning samples into thousands to millions of individual reactions, dPCR enables high-precision quantification, exceptional sensitivity for rare variants, and robust tolerance to PCR inhibitors and degraded DNA-characteristics particularly advantageous for analyzing challenging forensic specimens such as trace, degraded, and inhibitor-laden samples. Key applications discussed include DNA mixture deconvolution, differentiation of monozygotic twins through low-frequency variant detection, forensic age estimation based on DNA methylation and other molecular markers, body fluid and tissue identification using RNA and DNA methylation markers, and species identification of non-human biological samples. Despite its significant potential, the widespread implementation of dPCR in routine forensic practice is constrained by limited multiplexing capacity, higher per-sample costs, and the absence of standardized forensic guidelines. Future advancements in microfluidic integration, multiplexing technologies, and portable platform development are expected to further expand the utility of dPCR, positioning it as a powerful complementary tool in forensic casework, particularly for complex and compromised samples.", "journal": "International journal of legal medicine", "year": "2026", "doi": "10.1007/s00414-026-03973-0", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41373768": { "pmid": "41373768", "title": "Benchmarking Cost-Effective DNA Extraction Kits for Diverse Metagenomic Samples.", "abstract": "Extraction of high-quality microbial DNA remains a critical bottleneck in metagenomic research. Environmental samples often produce fragmented DNA and are prone to contaminations that interfere with downstream sequencing, while widely used commercial kits can be prohibitively expensive. Therefore, systematic evaluation of cost-effective alternatives is essential to support large-scale metagenomic studies. In this work, we benchmarked eight commercial DNA extraction kits from Magen, SkyGen, and Sileks against Qiagen reference kits. Four representative sample types were analyzed: freshwater, seafloor sediments, Pacific oyster (Magallana gigas) gut microbiome, and mammalian feces. DNA yield, integrity, purity, PCR inhibitor content, and eukaryotic DNA admixture were assessed. Microbial community composition, alpha diversity, reproducibility, and contamination (\"kitome\" and \"splashome\") were further evaluated using 16S rRNA amplicon sequencing. We revealed that several alternative kits performed comparably or better than the Qiagen reference standard. Magen Soil and Magen Bacterial provided high yields and reproducibility, though the latter produced more fragmented DNA. SkyGen Stool excelled with host-associated samples, while Sileks Soil and Metagenomic kits preserved higher diversity in sediments. Magen Microbiome consistently underperformed. This study identifies multiple cost-effective DNA extraction strategies and provides practical guidance for selecting balanced DNA purification methods for different sample types.", "journal": "International journal of molecular sciences", "year": "2025", "doi": "10.3390/ijms262311616", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40750203": { "pmid": "40750203", "title": "Rapid detection of NPY methylation in colorectal cancer using a singlet oxygen-based photoelectrochemical ratiometric bio-platform.", "abstract": "Aberrant methylation of Neuropeptide Y (NPY) gene has emerged as a promising biomarker for monitoring metastatic colorectal cancer and predicting its early recurrence. Current standard approaches for the detection of NPY hypermethylation levels rely on the use of enzymes and are associated with high costs, long times of analysis and interferences from PCR inhibitors, which limits their applicability in point-of-care or low-resource environments. Therefore, the need for an affordable, rapid, and sensitive technology for NPY methylation detection is clear. Herein we present a novel, rapid, sensitive, highly selective, and enzyme-free photoelectrochemical ratiometric bio-platform for the detection and quantification of NPY gene hypermethylation. At the core of this method lies the use of magnetic microbeads and singlet oxygen (1O2)-based photoelectrochemical detection. The use of bisulfite conversion and hybridization temperature allowed to achieve a 99 % discrimination efficiency of the methylated NPY target from the unmethylated one. Detection of 18 pM (18 fmol) of 94-nucleotide methylated NPY target sequence with 6 methylation sites in hybridization buffer and 45 pM (45 fmol) against the background of genomic DNA was achieved without any amplification. Ultimately, ratiometric quantitative sensing of methylated NPY gene over reference albumin gene was carried out and an LOD of 2 % methylation level was obtained. Overall, this technique demonstrated immense potential in overcoming the complexity, high costs, and need for enzymes of current strategies for hypermethylation detection and thus represents a great innovation in (photo)electrochemical sensing of aberrant DNA methylation. It, therefore, holds great promise for establishing in future a clinically relevant point-of-care testing device.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344381", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "41272443": { "pmid": "41272443", "title": "PCR optimization for buccal swab-derived samples: overcoming sporadic inhibition with bovine serum albumin.", "abstract": "Buccal swabs are widely used as a non-invasive method for genomic DNA collection in large-scale genotyping studies. However, the sporadic presence of PCR inhibitors within these samples may hinder PCR amplification and affect assay reliability. This study investigates the incorporation of bovine serum albumin (BSA) into the PCR reaction mixture as an additive to counteract PCR inhibition. In our high-throughput setting, the incorporation of BSA significantly improved robustness, lowering failure rates to 0.1% in subsequent routine operation across 1,000,000 buccal swab samples. Despite minor challenges with foaming during automated liquid handling, no detrimental effects on PCR performance were observed. These results underscore the efficacy of BSA in improving PCR robustness, enhancing the reliability of high-throughput molecular diagnostic assays.", "journal": "BMC genomics", "year": "2025", "doi": "10.1186/s12864-025-12350-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39506428": { "pmid": "39506428", "title": "Detection of equine parvovirus-hepatitis and efficacy of governmental regulation for equine biologics purity.", "abstract": "In 2018, a new virus, named equine parvovirus-hepatitis (EqPV-H), was discovered in a biologic product that had been administered to horses that subsequently developed clinical signs of equine serum hepatitis (Theiler disease). Further correlation of the virus with the disease sparked federal requirements that all equine biologics be free of EqPV-H. The initial quantitative real-time PCR (qPCR) test for EqPV-H has proved to be sensitive to co-extracted PCR inhibitors in template nucleic acids, causing false-negative results. We investigated the use of digital PCR (dPCR) as a more robust test. Examination of 227 equine biologic product lots available for purchase both before and after regulatory implementation using both detection methods indicated that dPCR is a more reliable platform. Nevertheless, use of the qPCR method for product screening had reduced the fraction of serials with EqPV-H detected from 39.6% prior to regulation to 6.8% after regulatory implementation. Adoption of dPCR testing is an opportunity to further decrease the prevalence of EqPV-H in equine biologics.", "journal": "Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc", "year": "2025", "doi": "10.1177/10406387241292343", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41220334": { "pmid": "41220334", "title": "Comparison of two digital PCR platforms for quantification of genetically modified soybean events.", "abstract": "In the European Union, the food and feed containing more than 0.9% of approved genetically modified organisms (GMOs) per ingredient must be labeled before placed on the market. In this legislative context, the official control laboratories have to perform validated PCR assays, according to the principles and requirements of ISO/IEC 17025 standard, regarding event-specific methods for the detection, identification and quantification of GMOs. In recent years, with the advent of digital PCR (dPCR) techniques, a growing number of laboratories have transferred the previously validated real-time PCR testings into a dPCR format. Compared to real-time PCR, the dPCR offers the advantage to provide accurate quantification without the need for external calibration samples, show less sensitivity to PCR inhibitors and is more suitable for multiplexing. In this study, an in-house validation of quantitative duplex dPCR methods was performed involving MON-04032-6 and MON89788 assays with the lectin reference gene, on the two different platforms Bio-Rad QX200 and Qiagen QIAcuity. All evaluated data and the validation parameters agree with the acceptance criteria validation performance parameters according to the JRC Guidance documents and technical reports in both platforms. The duplex PCR methods here investigated are equivalent in terms of performance to the singleplex real-time PCR method and suitable to perform a collaborative trial for a full validation.", "journal": "GM crops & food", "year": "2025", "doi": "10.1080/21645698.2025.2583781", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "", "dirs": [ "B" ] }, "40571259": { "pmid": "40571259", "title": "Optimized Molecular Detection of Cryptosporidium Within the Water-Soil-Plant-Food Nexus: Advancing Surveillance in Agricultural Systems.", "abstract": "Cryptosporidium, a protozoan parasite causing severe diarrheal illness in humans and animals, poses detection challenges due to low parasite concentrations, inhibitors, and inefficient DNA extraction. This study optimized DNA extraction and detection of Cryptosporidium in environmental samples and evaluated their practical use in agriculture. After evaluating 11 DNA extraction methods from spiked phosphate-buffered saline (PBS) samples, three methods for molecular detection of Cryptosporidium in water, soil, and fresh produce were selected and further tested using real-time PCR. A total of 188 artificially contaminated samples were prepared, consisting of distilled water (n = 36), environmental water (n = 44), soil (n = 36), and fresh produce (lettuce and spinach; n = 72). Each sample was inoculated with serial dilutions of 12,500 to 5 Cryptosporidium oocysts and tested using real-time PCR and droplet digital PCR (ddPCR) to evaluate detection sensitivity. Results demonstrated that extraction performance varied by matrix, with two spin-column kits excelling for water and another for soil and produce. DNA from as few as five oocysts was occasionally detectable, with ddPCR being less prone to be affected by PCR inhibitors than real-time PCR. These methods were then applied to detect Cryptosporidium in 210 environmental samples (water, soil, produce) from South African small-scale farms. None of the samples tested positive with real-time PCR, while ddPCR detected Cryptosporidium in 13.6% of water, 23.3% of soil, and 34.7% of fresh produce samples. Surface water showed the highest contamination at 28.6%. Soil amended with both fertilizer and manure had a 45% contamination rate. Among vegetables, roots were most affected (46.7%), followed by fruiting (40%) and leafy greens (30.15%). These findings highlight the health risks of Cryptosporidium in food systems and the need for improved detection methods to enhance surveillance and inform future outbreak prevention strategies.", "journal": "Journal of food protection", "year": "2025", "doi": "10.1016/j.jfp.2025.100568", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "39829744": { "pmid": "39829744", "title": "Metagenomic estimation of absolute bacterial biomass in the mammalian gut through host-derived read normalization.", "abstract": "Absolute bacterial biomass estimation in the human gut is crucial for understanding microbiome dynamics and host-microbe interactions. Current methods for quantifying bacterial biomass in stool, such as flow cytometry, qPCR, or spike-ins (i.e., adding cells or DNA from an organism not normally found in a sample), can be labor-intensive, costly, and confounded by factors like water content, DNA extraction efficiency, PCR inhibitors, and other technical challenges that add bias and noise. We propose a simple, cost-effective approach that circumvents some of these technical challenges: directly estimating bacterial biomass from metagenomes using bacterial-to-host (B:H) read ratios. We compare B:H ratios to the standard methods outlined above, demonstrating that B:H ratios are useful proxies for bacterial biomass in stool and possibly in other host-associated substrates. We show how B:H ratios can be used to track antibiotic treatment response and recovery in both mice and humans, which showed 403-fold and 45-fold reductions in bacterial biomass during antibiotic treatment, respectively. Our results indicate that host and bacterial metagenomic DNA fractions in human stool fluctuate longitudinally around a stable mean in healthy individuals, and the average host read fraction varies across healthy individuals by < 8-9 fold. B:H ratios offer a convenient alternative to other absolute biomass quantification methods, without the need for additional measurements, experimental design considerations, or machine learning algorithms, enabling retrospective absolute biomass estimates from existing stool metagenomic data.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.1101/2025.01.07.631807", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "B" ] }, "40424696": { "pmid": "40424696", "title": "Evaluation of direct-to-PCR (D2P) method for molecular diagnosis of infectious diseases.", "abstract": "This study evaluates the performance of the Direct-to-PCR (D2P) method as a streamlined, extraction-independent alternative to conventional nucleic acid extraction techniques for diagnosing urinary tract infections, sexually transmitted infections, and respiratory tract infections. The D2P approach employs proprietary antimicrobial peptide-based lysis buffers tailored for bacterial, fungal, and viral targets, enabling direct amplification from clinical and contrived specimens without column- or bead-based purification. Comparative analyses were conducted against silica column-based (QIAGEN) and magnetic bead-based (KingFisher) extraction methods using both microbial reference isolates and 116 residual clinical samples. Results demonstrate that the D2P method yields comparable sensitivity and specificity to conventional extraction workflows across a diverse panel of pathogens-including Gram-negative and Gram-positive bacteria, Candida species, ssRNA viruses (e.g., CoV-229E, Parainfluenza Virus 1 and 2), and dsDNA viruses (e.g., HSV, HAdV). Notably, D2P outperformed both QIAGEN and KingFisher in extracting nucleic acids from Candida auris, a multidrug-resistant fungal pathogen. Limit of detection and amplification efficiency remained within acceptable ranges across all platforms, with R2 values between 0.92 and 0.99, and slopes consistent with MIQE standards. The D2P protocol reduced total sample processing time from ∼120 min to ∼45 min, minimized hands-on steps, and demonstrated effective performance in turbid or hemolyzed samples-making it suitable for high-throughput and resource-limited settings. However, limitations were observed in samples with high PCR-inhibitor content or low target yield, and broader validation across additional matrices is recommended. These findings support D2P as a reliable, efficient, and scalable molecular diagnostic alternative with broad clinical utility. Integration of D2P into diagnostic workflows could enhance access to rapid, cost-effective pathogen detection in both centralized laboratories and decentralized or point-of-care environments.", "journal": "Experimental and molecular pathology", "year": "2025", "doi": "10.1016/j.yexmp.2025.104972", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "B" ] }, "39864397": { "pmid": "39864397", "title": "Methods of extraction of genetic material from hard tissues: A review of the 21st century advancements.", "abstract": "Skeletal remains are the only source of the genetic material of decomposed organisms or once-lived species. Unlike, soft tissues they are highly mineralized, and their anatomical and morphological structure prevents their deformation in the presence of adverse environmental factors. Therefore, bones and teeth protect the Deoxyribonucleic Acid (DNA) inside them. Obtaining DNA from hard tissues comes with challenges like contamination, degradation, PCR inhibitors, damage done by the environment on remains, etc. Traditional methods have been in use for a long time. To overcome the challenges in extracting DNA from hard tissues, researchers introduced various modifications, with time, to the standard procedures. We have reviewed the innovative approaches developed during the period ranging from 2000 to 2024, using the Google Scholar search engine. The last innovative method was discovered in 2021. Each method solves a particular challenge and makes it easier for future researchers to opt for a suitable protocol according to the specific requirements of their study. The methods were renamed based on their core specification, such as Carrier-Mediated Precipitation Method, CTAB Method, Buffered-Nondestructive Extraction Method, MDNAMI Method, Demineralization protocol, Rapid Column-based DNA Extraction, Bone Powdering and Bone Slicing, Short Fragmented DNA Extraction, Highly Degraded DNA Extraction, and Non-destructive Tooth DNA Extraction. A collaboration of researchers from forensic science, anthropology, archeology, evolutionary biology, molecular biology, etc. may develop more sophisticated techniques that ease extraction, increase yield, and reduce contamination of DNA from hard tissues.", "journal": "Forensic science international", "year": "2025", "doi": "10.1016/j.forsciint.2025.112382", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "40742180": { "pmid": "40742180", "title": "Metagenomic estimation of absolute bacterial biomass in the mammalian gut through host-derived read normalization.", "abstract": "Absolute bacterial biomass estimation in the human gut is crucial for understanding microbiome dynamics and host-microbe interactions. Current methods for quantifying bacterial biomass in stool, such as flow cytometry, quantitative polymerase chain reaction (qPCR), or spike-ins, can be labor-intensive, costly, and confounded by factors like water content, DNA extraction efficiency, PCR inhibitors, and other technical challenges that add bias and noise. We propose a simple, cost-effective approach that circumvents some of these technical challenges: directly estimating bacterial biomass from metagenomes using bacterial-to-host (B:H) read count ratios. We compared B:H ratios to the standard methods outlined above, demonstrating that B:H ratios are useful proxies for bacterial biomass in stool and possibly in other host-associated substrates. B:H ratios in stool were correlated with bacterial-to-diet (B:D) read count ratios, but B:D ratios exhibited a substantial number of outlier points. Host read depletion methods reduced the total number of human reads in a given sample, but B:H ratios were strongly correlated before and after host read depletion, indicating that host read depletion did not reduce the utility of B:H ratios. B:H ratios showed expected variation between health and disease states and were generally stable in healthy individuals over time. Finally, we showed how B:H and B:D ratios can be used to track antibiotic treatment response and recovery. B:H ratios offer a convenient alternative to other absolute biomass quantification methods, without the need for additional measurements, experimental design considerations, or machine learning, enabling robust absolute biomass estimates directly from stool metagenomic data.IMPORTANCEIn this study, we asked whether normalization by host reads alone was sufficient to estimate absolute bacterial biomass directly from stool metagenomic data, without the need for synthetic spike-ins, additional experimental biomass measurements, or training data. The approach assumes that the contribution of host DNA to stool is more constant or stable than biologically relevant fluctuations in bacterial biomass. We find that host read normalization is an effective method for detecting variation in gut bacterial biomass. Absolute bacterial biomass is a key metric that often gets left out of gut microbiome studies, and empowering researchers to include this measure more broadly in their metagenomic analyses should serve to improve our understanding of host-microbiota interactions.", "journal": "mSystems", "year": "2025", "doi": "10.1128/msystems.00984-25", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41148693": { "pmid": "41148693", "title": "Rapid Nanopore Sequencing of Positive Blood Cultures Using Automated Benzyl-Alcohol Extraction Improves Time-Critical Sepsis Management.", "abstract": "Background/Objective: Timely identification of bloodstream pathogens is critical for sepsis management; however, PCR inhibitors such as sodium polyanetholesulfonate (SPS) in blood culture broth compromise nucleic acid recovery and long read sequencing. We assessed whether coupling a benzyl alcohol SPS-removal step to the fully automated LabTurbo AIO extractor improves Oxford Nanopore-based pathogen detection. Methods: Thirteen positive blood culture broths were pre-treated with benzyl alcohol and divided: half volumes were purified on the LabTurbo AIO; paired aliquots underwent manual QIAamp extraction. DNA purity was evaluated by NanoDrop and Qubit. Barcoded libraries were sequenced on MinION R9.4.1 flow cells for 6 h. Results: Automated eluates showed a median A260/A280 of 1.92 and A260/A230 of 1.96, versus 1.80 and 1.48 for manual extracts. The automated workflow generated 1.69 × 106 total reads compared with 3.9 × 105 reads for manual extraction. The median N50 read length increased from 5.9 kb to 8.7 kb, and the median proportion of reads classified to species increased from 62% to 84%. The hands-on time was <5 min and the sample-to-answer turnaround was <8 h, compared with >9 h and 90 min for the manual protocol, respectively. Conclusions: Benzyl alcohol SPS removal integrated into the LabTurbo AIO extractor yielded purer, longer, and higher read counts, enhancing nanopore sequencing depth and accuracy while compressing diagnostic turnaround to a single working day. This represents a practical advance for rapid blood culture pathogen identification in critical care settings.", "journal": "Antibiotics (Basel, Switzerland)", "year": "2025", "doi": "10.3390/antibiotics14101001", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41423617": { "pmid": "41423617", "title": "Whole genome sequencing of hepatitis A virus: adapting Illumina protocols for foodborne investigation.", "abstract": "High-throughput sequencing-based whole-genome sequencing (WGS) is highly effective for identifying viral pathogens in microbial research. However, applying WGS directly to foodborne viruses remains challenging because food matrices contain PCR inhibitors and viral titers are typically much lower than those found in clinical specimens. This study aimed to develop a WGS method for analyzing the hepatitis A virus (HAV) genome in clams using the Illumina MiSeq platform. To enhance the HAV WGS method, we applied four approaches to HAV-positive clam field samples: size-exclusion chromatography for sample preparation, a specialized RNA extraction method, optimized cDNA synthesis, and the selection of DNA polymerase. Nine complete HAV genomes were obtained from clams. The obtained HAV genomes and their genetic characteristics were then compared based on phylogeny. Before optimization, only four clam samples yielded detectable amplification; however, following optimization, two additional samples became amplifiable, resulting in six samples suitable for downstream WGS analysis. The developed WGS method was able to sequence low contamination levels of 2.91-3.61 log10 genome copies/mL, achieving coverage of 97.5% and 92.6%. Notably, this study confirmed an average sequencing depth of up to 82.20× and a minimum depth of 25.19×. As a result of sequencing, one HAV-IA, and eight HAV-IB genotypes were identified from six clam samples including the multiple strains. The sequence identity between the strains from clams and serum was 97.80% for HAV-IA and 95.2-97.80% for HAV-IB. This method of viral WGS in food samples may contribute to rapid genotyping, understanding virus evolution, and enhancing epidemiological surveillance in foodborne virus outbreaks.", "journal": "NPJ science of food", "year": "2025", "doi": "10.1038/s41538-025-00659-6", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "39944917": { "pmid": "39944917", "title": "Droplet Digital PCR Provides Highly Sensitive and Accurate Opsin Gene SNP Detection From Wild Primate Fecal Samples.", "abstract": "Evaluating field-sourced samples with poor-quality and low-quantity DNA, like animal feces, presents significant challenges in the field of molecular biology. Nonetheless, recent innovations in PCR technology are promoted as effective tools to overcome many of these issues. Here, we evaluate the efficiency of droplet digital PCR (ddPCR) as a method for color vision assessment from feces of white-faced capuchins (Cebus imitator) and report frequencies of alleles and genotypes in a wild population. The sex-linked color vision polymorphism of monkeys in the Americas is driven by single nucleotide polymorphisms (SNPs) in opsin genes at up to three tuning sites. DNA was extracted from fecal samples collected from 211 wild capuchins (53.1% males) in Sector Santa Rosa, Costa Rica: 56 were evaluated with ddPCR, 24 with both ddPCR and Sanger sequencing, and 141 with Sanger sequencing (historical dataset). The same opsins and genotypes were derived for each monkey using Sanger and ddPCR; however, the latter method was far more sensitive and required far fewer samples to reach a definitive genotype. Overall, the most frequent phenotypes were red and green/red. The distribution of genotypes was: Females (N = 99): green/red (35.4%), red/red (33.3%), green/yellow (14.1%), yellow/red (12.1%), yellow/yellow (4.0%), and green/green (1.0%); Males (N = 112): red (60.7%), yellow (23.2%), and green (16.1%). Overall, ddPCR was a reliable method for evaluating color vision noninvasively in wild capuchins with the advantage of excellent sensitivity and high-throughput. ddPCR is highly robust to PCR inhibitors and can be potentially used to identify other disease-related SNP mutations noninvasively in wild animals.", "journal": "Ecology and evolution", "year": "2025", "doi": "10.1002/ece3.70996", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42761107": { "pmid": "42761107", "title": "Oral fluids for detection and characterization of Salmonella in commercial finisher pigs.", "abstract": "Salmonella remains an important pathogen in swine production due to its impact on animal health and its role in foodborne illnesses. However, routine surveillance in pigs relies largely on fecal sampling, which can be labor-intensive and difficult to scale across large production systems. This study aimed to develop and evaluate a novel approach for monitoring Salmonella in finisher pigs using oral fluids in combination with sequential enrichment, culture, and polymerase chain reaction (PCR). A cross-sectional study was conducted at three commercial finisher sites in Iowa between February and May 2025, where paired oral fluid and pooled fecal samples were collected from 51 pens. The samples were processed using sequential enrichment followed by bacteriological culture and multiplex polymerase chain reaction to detect and characterize Salmonella. Direct PCR testing of raw samples did not detect Salmonella in either sample type. However, tests performed after enrichment successfully detected the pathogen in both samples, demonstrating the importance of enrichment for molecular detection in oral fluids. The culture of enriched oral fluids yielded viable isolates suitable for serotyping, and molecular testing enabled the rapid identification of the two serotypes most relevant to swine and public health (Typhimurium and I 4,[5],12:i:-). Salmonella was detected in 86.3% of the pens using at least one method, and oral fluids demonstrated diagnostic performance comparable to pooled fecal samples. These findings establish that oral fluids, when combined with sequential enrichment, are a practical and scalable sample type for monitoring Salmonella in finisher pigs.", "journal": "Frontiers in veterinary science", "year": "2026", "doi": "10.3389/fvets.2026.1876360", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41526128": { "pmid": "41526128", "title": "Inner primer Blockers inhibit non-specific amplification in LAMP.", "abstract": "The frequent occurrence of non-specific amplification (NSA) in Loop-mediated Isothermal Amplification (LAMP) has significantly constrained its widespread adoption. When the amount of template is limited or absent, NSA products can be randomly generated, leading to unreliable and inaccurate detection results. The mechanisms underlying NSA remain poorly elucidated, resulting in mitigation strategies that inadequately address the fundamental causes and are therefore suboptimal. Non-template (primers-only) real-time LAMP assays were conducted with three published and three routinely designed primer sets. All reactions demonstrated detectable NSA, thereby supporting the hypothesis that primers constitute a principal source of NSA. Comprehensive analyses of two representative primer sets included single-primer reactions and various multi-primer combinations (ranging from two to six primers) under non-template conditions, a process referred to as primer \"SCAN\" analysis. These systematic investigations consistently revealed the existence of at least one initiator inner primer (IIP) within each set that was capable of independently initiating NSA. The IIP was consistently identified in the most efficient NSA-amplifying combinations and acted as the primary driver of unwanted amplification events. Importantly, the introduction of unamplified oligonucleotide Blockers specifically targeting F1c/B1c sequences of the IIP significantly inhibited NSA while preserving the original binding specificity, thereby demonstrating the effectiveness and selectivity of targeted inhibition strategies. Adding only one or two Blockers could significantly inhibit NSA. The -3 and -6 shortened Blocker achieve an optimal trade-off between inhibiting NSA and preserving the efficiency of the desired reaction via extending the NSA-free window. In some -6 Blocker groups, the limit of detection (LOD) was improved, greatly enhancing the accuracy of detecting low viral load samples in early stages of infection. This study confirms that certain primers, particularly IIP, play a key role in initiating NSA in LAMP. Blockers have the potential to serve as a universal strategy to effectively inhibit NSA in LAMP diagnostics. By postpone the times to positive (TTP) of primer-initiated NSA, the probabilities of false positives during a conventional detection time window (30-60 min) are reduced. In particular, some Blockers at suitable length achieved this at only minor cost of sensitivity. Blocker-LAMP approach represent a targeted strategy different from existing general additives or amplicon discrimination ones. It can be applied individually or jointly with other methods to mitigate NSA problem and improve the overall performance of LAMP assays.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2025.345020", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "40448616": { "pmid": "40448616", "title": "Enhanced recombinase polymerase amplification via UvsX engineering and reaction optimization for rapid detection of Vibrio parahaemolyticus.", "abstract": "Recombinase polymerase amplification (RPA) is a powerful isothermal nucleic acid amplification technique, yet its efficiency is critically dependent on the catalytic efficiency of the recombinase UvsX, a key enzyme mediating homologous DNA pairing and strand exchange. To address this limitation, in this study, we developed a specific, sensitive, and robust RPA detection method by optimizing the UvsX enzyme through protein engineering and refining the RPA reaction system. By conducting comparative structural and functional analysis of UvsX orthologs from 13 Myoviridae phages, we identified critical determinants of recombinase activity within the Loop 2 domain of T4 UvsX. Furthermore, we systematically optimized the stoichiometric ratios of core enzymes and crowding agents to establish a robust RPA system. This system was subsequently integrated with lateral flow strips for point-of-need detection of highly lethal Vibrio parahaemolyticus in shrimp. Our results demonstrated that the engineered UvsXv1 variant exhibited significantly improved strand displacement activity, leading to enhanced RPA amplification efficiency and stability.", "journal": "Letters in applied microbiology", "year": "2025", "doi": "10.1093/lambio/ovaf081", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41644930": { "pmid": "41644930", "title": "Advancements in somatic mutation detection through loop-mediated isothermal amplification (LAMP) in human tumors.", "abstract": "Loop-mediated isothermal amplification (LAMP) is a robust and specific isothermal nucleic acid assessment technique. Due to its practicality and diverse detection strategies, LAMP has gained new insights in the analysis of somatic mutations in various tumor types, where the proportion of mutated DNA is often markedly low compared to wild-type DNA. We conducted an evaluation of articles from PubMed and Google Scholar published up to May 2025, focusing on somatic mutation detection in human cancers using LAMP. In addition to reviewing the articles found using specific keywords, we also examined and evaluated the references cited in those articles. By employing various primer design strategies specific to single base substitutions such as mismatch and loop primers, the researchers addressed the challenge of distinguishing between wild-type and mutant alleles. Furthermore, the specificity and sensitivity of the LAMP assay were enhanced by incorporating Locked Nucleic Acid (LNA) and Peptide Nucleic Acid (PNA) probes. The use of additives and adjustments in reaction conditions has also been discussed as strategies to improve reactions efficiency. LAMP demonstrates the capability to detect mutations present at levels as low as 0.1% in tumor tissues, indicating its potential for high-sensitivity mutation detection. The studies contribute to refining LAMP as a reliable, rapid, and cost-effective method for somatic mutation detection. The versatile detection approaches further enhance the utility and accessibility of the LAMP technique in various research and clinical settings. This paves the way for its broader application in clinical diagnostics and personalized medicine.", "journal": "Journal of applied genetics", "year": "2026", "doi": "10.1007/s13353-025-01042-7", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "42052644": { "pmid": "42052644", "title": "From thermal cycling PCR to isothermal RPA: vibrational strong coupling as a new physical control axis for DNA amplification.", "abstract": "The emergence of vibrational strong coupling (VSC) strategies has reshaped how biochemical reactions are regulated-not by chemical additives or thermal cycling, but through vacuum-field-mediated restructuring of solvent dynamics and energy landscapes. This Perspective bridges fundamental developments in cavity-modified enzymatic catalysis with the unique biochemical architecture of recombinase polymerase amplification (RPA), a low-temperature, hydration-regulated nucleic acid amplification system. Earlier enzyme-specific VSC studies established that coupling the O-H stretching manifold of water to Fabry-Pérot (FP) modes reorganizes hydrogen bond topology, alters activation barriers, and selectively accelerates or suppresses catalytic turnover depending on vibrational mode alignment. These mechanistic principles translate directly to multi-enzyme amplification: recent experiments demonstrate that tuning the cavity length to the O-H stretching band of water modulates the RPA product yield, with on-resonance coupling suppressing amplification efficiency and off-resonance conditions restoring activity. Because RPA relies on hydration-assisted strand invasion, Mg2+-mediated recombinase filament formation, and solvent-regulated polymerase elongation, it represents a particularly responsive platform for cavity-controlled biochemical amplification. We further outline how dielectric engineering, microfluidic confinement, and multimode photonic architectures may enable deterministic control of amplification kinetics and selectivity. This work recasts optical cavities as active thermodynamic variables capable of sculpting biochemical free-energy landscapes, enabling enzyme systems that respond to field structure rather than bulk chemistry.", "journal": "Physical chemistry chemical physics : PCCP", "year": "2026", "doi": "10.1039/d5cp04753f", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41324273": { "pmid": "41324273", "title": "Senescence Reprogramming Unleashes Tumor Immune Surveillance via Coordinated Gene Modulation.", "abstract": "Cellular senescence can recruit immune cells for tumor therapy through the senescence-associated secretory phenotype (SASP). However, its therapeutic efficacy is limited by immune tolerance and the immunosuppressive tumor microenvironment (TME). Reprogramming tumor-specific senescence through coordinated modulation of P16INK4a and PD-L1 enhances tumor immunogenicity and alleviates immunosuppression. To achieve this, a target-enhanced gene delivery nanoparticle is engineered using the urokinase plasminogen activator receptor (uPAR) as a senescence-specific targeting ligand, combined with a telomerase reverse transcriptase (TERT) promoter and a nuclear localization signal-microtubule-associated sequence (NLS-MTAS) peptide. This system efficiently induces tumor-specific senescence through cell-cycle arrest and promotes the chemotactic recruitment of cytotoxic immune cells. In vivo, the nanoparticle induces a robust anti-tumor response without causing systemic toxicity and significantly enhances the therapeutic efficacy of αCTLA-4 immune checkpoint blockade in subcutaneous, lung metastasis, postoperative recurrence, and spontaneous tumor models. This study emphasizes the therapeutic potential of reprogramming tumor-specific senescence to improve targeted gene delivery and immunotherapy outcomes, offering a viable approach for the treatment of immunologically \"cold\" tumors.", "journal": "Advanced materials (Deerfield Beach, Fla.)", "year": "2026", "doi": "10.1002/adma.202516597", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41406950": { "pmid": "41406950", "title": "Potentiating immunotherapy in \"immune-cold\" solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering.", "abstract": "We engineer a tumor-targeted genetic plasmid vector (PαCD3&LIGHT) to systematically modulate T cell immunity. The tumor-specific telomerase reverse transcriptase (TERT) promoter drives simultaneous expression of tumor necrosis factor superfamily member 14 (LIGHT) and membrane-anchored anti-CD3 single-chain variable fragment (αCD3), which are important immunomodulators with closely clinical relevance. Secreted LIGHT induces high endothelial venule formation and chemokine secretion to recruit circulating lymphocytes, while remodeling extracellular matrix to facilitate immune cell penetration into tumor parenchyma. αCD3 establishes artificial immunological synapses between tumor cells and T lymphocytes. This dual mechanism synergistically establishes tertiary lymphoid structures de novo even within deep tumor regions, harboring stem cell-like CD8+ T cells and driving sustained immunity. Concurrently, αCD3-mediated T cell redirection not only amplifies TCR signaling but also reverses exhausted T cells. The orchestrated T cell immunity significantly potentiates checkpoint inhibitor and chimeric antigen receptor (CAR)-T cell therapies in \"immune-cold\" tumors without obvious side effects and also remarkably enhances the outcome of human CAR-T cells, demonstrating translational potential in solid tumor immunotherapy.", "journal": "Cell reports. Medicine", "year": "2025", "doi": "10.1016/j.xcrm.2025.102510", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41526614": { "pmid": "41526614", "title": "Modulation of the clonal burden in patients with lower-risk myelodysplastic neoplasms treated with imetelstat.", "abstract": "Existing treatments for lower-risk myelodysplastic syndromes/neoplasms (LR-MDS) focus on symptom relief. Until recently, altering the disease course was rarely considered a therapeutic objective. The first-in-class, direct, competitive telomerase inhibitor, imetelstat, demonstrated significantly higher rates of red blood cell (RBC) transfusion independence (TI) versus placebo in patients with non-del(5q), RBC transfusion-dependent LR-MDS who were relapsed/refractory to or ineligible for erythropoiesis-stimulating agents in the Phase 3 IMerge study (NCT02598661). In this exploratory analysis of IMerge, patients treated with imetelstat had greater sustained reductions in variant allele frequency of multiple mutations versus placebo recipients, which was positively associated with RBC-TI duration. Subsequent analyses showed that 70% of patients with a cytogenetic response with imetelstat achieved ≥1-year RBC-TI. Additionally, higher rates of ≥1-year RBC-TI were observed in patients with maximum variant allele frequency reduction of ≥50% in SF3B1 (58% vs. 7%), TET2 (90% vs. 9%), DNMT3A (100% vs. 13%), or ASXL1 (50% vs. 0%) and patients with ≥50% bone marrow ring sideroblast reduction (46% vs. 0%) versus patients who did not. Lastly, 60% of patients with ≥1-year RBC-TI had ≥50% reduction in telomerase activity/human telomerase reverse transcriptase RNA. These results suggest that imetelstat targets clonal progenitor cells and may modify LR-MDS biology.", "journal": "Leukemia", "year": "2026", "doi": "10.1038/s41375-025-02831-z", "ptypes": [ "Journal Article", "Clinical Trial, Phase III", "Randomized Controlled Trial" ], "first_author": "", "dirs": [ "B" ] }, "40897710": { "pmid": "40897710", "title": "Structural basis of the RNA-mediated Retron-Eco2 oligomerization.", "abstract": "In the evolutionary arms race between bacteria and viruses, retrons have emerged as distinctive antiphage defense systems. Here, we elucidate the structure and function of Retron-Eco2, which comprises a non-coding RNA (ncRNA) that encodes multicopy single-stranded DNA (msDNA, a DNA‒RNA hybrid) and a fusion protein containing a reverse transcriptase (RT) domain and a topoisomerase-primase-like (Toprim) effector domain. The Eco2 msDNA and RT-Toprim fusion protein form a 1:1 stoichiometric nucleoprotein complex that further assembles into a trimer (msDNA:RT-Toprim ratio of 3:3) with a distinctive triangular configuration. The RNA portion of the msDNA in one protomer closely intertwines around the RT domain of an adjacent protomer, mediating the formation of this self-inhibitory assembly. Upon activation, the Toprim effector domain exhibits RNase activity, degrading RNA to arrest phage replication. We further reveal that phage mutants evading Eco2-mediated defense harbor mutations in the endonuclease IV-like protein DenB, underscoring DenB's critical role in triggering the activation of this system. Together, these findings provide key structural and functional insights into Retron-Eco2, laying the groundwork for harnessing its potential in biotechnology and synthetic biology applications.", "journal": "Cell discovery", "year": "2025", "doi": "10.1038/s41421-025-00823-y", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41720267": { "pmid": "41720267", "title": "Telomere recapping via gene therapy as a beneficial strategy for cardio-renal syndrome type 4.", "abstract": "Cardio-renal syndrome type 4 (CRS4) is defined as heart failure driven by chronic kidney disease (CKD). Cardiovascular mortality is the leading cause of death in patients with advanced CKD with current medical treatments exhibiting limited efficacy. Short leukocyte telomere lengths in such patients are correlated with increased mortality, particularly cardiovascular mortality. Here, we examined telomere shortening in mouse and human cardiomyocyte models of CRS4 and its potential protective role by overexpression of telomerase reverse transcriptase. We engineered an adeno-associated virus 9 (AAV9) gene therapy JV101, which overexpressed catalytically inactive and nuclear localized human telomerase reverse transcriptase (modhTERT) under cardiac troponin T promoter control. Both in vivo and in vitro models were utilized to conduct a comprehensive evaluation of the therapeutic effects of modhTERT on CRS4. Overexpression of modhTERT recapped myocardial telomeres and reversed cardiac dysfunction, hypertrophy and fibrosis, and mitochondrial dysfunction in two CRS4 mouse models: 5/6 nephrectomy and Angiotensin II - high salt - uninephrectomy. The reversal of cardiac dysfunction by modhTERT was both long-term and persistent. In contrast, beta-blocker administration merely delayed the progression of heart failure in these models, without achieving functional reversal. Surprisingly, JV101 ameliorated kidney function as measured by blood urea nitrogen levels in CKD murine models. Experiments in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) demonstrated that modhTERT bound to myocardial telomeric ends. Transcriptomic analysis of isolated mouse cardiomyocytes revealed that modhTERT reversed differentially repressed genes that were highly enriched in mitochondrial metabolism. Coculture of hiPSC-CMs with serum from hemodialysis patients or indoxyl sulfate toxin induced myocardial contractile dysfunction. Overexpression of modhTERT also blocked further telomere shortening and reversed contractile dysfunction in hiPSC-CMs. Our results provide proof-of-concept evidence for treating CRS4 by telomere recapping gene therapy.", "journal": "Kidney international", "year": "2026", "doi": "10.1016/j.kint.2026.01.022", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "42186805": { "pmid": "42186805", "title": "Targeting Arrhythmogenic Late Sodium Current by FHF1A-Derivative FixR in Heart Failure Cardiomyocytes.", "abstract": "Enhanced late Na+ current (INa,L) in heart failure (HF) contributes to cardiomyocyte proarrhythmia. In addition to CaMKII (Ca2+/calmodulin-dependent protein kinase II), FGF (fibroblast growth factor) homologous factors 1-4 (FHF1-4) also modulate INa,L, and targeting these pathways may provide benefits in HF. Reverse-transcriptase quantitative polymerase chain reaction analysis of cardiac Na+ channel and FHF splice isoforms was performed in human arrhythmogenic HF with reduced ejection fraction (HFrEF) and in translational animal models of HFrEF and HF with preserved ejection fraction (HFpEF). We tested the effects of an engineered peptide, FixR (FHF inhibiting x region), on cardiomyocyte INa,L and electrophysiology in rabbit HFrEF and murine models of HFrEF and HFpEF. We also tested the in vivo electrophysiological effects of FixR via adenoviral delivery in transgenic mice with cardiomyocyte-specific overexpression of CaMKIIδC. Expression of key FHF long isoforms (FHF1A in humans, FHF2S in rabbits, and FHF2VY in mice) that have inhibitory effects on INa,L was reduced in human, rabbit, and murine failing hearts. INa,L was markedly enhanced in both HFrEF and HFpEF, and the FixR cell-penetrating peptide significantly reduced pathological INa,L in both forms of HF. FixR had no effect on transient peak Na+ current, L-type Ca2+ current, or major K+ currents in rabbit ventricular myocytes. FixR markedly attenuated proarrhythmogenic action potential changes (increased action potential duration, short-term variability, and alternans susceptibility) and delayed afterdepolarizations in both rabbit and murine HFrEF and HFpEF cardiomyocytes. These cellular antiarrhythmic effects were mimicked by the selective INa,L inhibitor GS967 and the CaMKII inhibitor AIP (autocamtide-2 inhibitory peptide). FixR also attenuated QT prolongation and in vivo arrhythmia susceptibility in transgenic mice with cardiomyocyte-specific overexpression of CaMKIIδC. In HF, INa,L is increased, and FHF splice isoform expression is altered, allowing a novel mechanism to therapeutically target INa,L. FixR, an FHF1A-derived peptide, is a potent and selective INa,L inhibitor and has antiarrhythmic properties in both HFrEF and HFpEF cardiomyocytes.", "journal": "Circulation research", "year": "2026", "doi": "10.1161/CIRCRESAHA.126.328204", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41817108": { "pmid": "41817108", "title": "DDX21 Links BRAFV600E and TERT to Promote Thyroid Cancer Progression.", "abstract": "The coexistence of BRAFV600E and telomerase reverse transcriptase (TERT) promoter alterations has been reported to cooperatively upregulate TERT expression and is well recognized as a biomarker for aggressive thyroid cancer. However, the mechanism underlying their synergistic effect remains elusive. TERT-interacting proteins were identified via immunoprecipitation (IP) and mass spectrometry and validated by Co-IP. DDX21 expression was detected in 60 paired thyroid cancer and adjacent normal tissues, and the function of DDX21 in thyroid cancer was explored in vitro and in vivo. Integrated analyses of RNA-seq, ChIP-seq, and Ribo-seq assays were conducted to elucidate the molecular mechanisms. BRAF/MEK inhibitors and BRAFV600E overexpression were used to investigate the regulation of DDX21 by BRAF mutation. In silico analysis, ChIP, and luciferase reporter assays were used to identify the transcription factors binding to DDX21 promoter. DDX21 interacted with TERT to regulate ribosomal RNA (rRNA) transcription by affecting the binding of Pol I to rDNA. DDX21 is upregulated in thyroid cancer, and DDX21 knockdown decreased the expression of multiple cancer hallmark genes and suppressed cell proliferation, colony formation, cell migration, and invasion of thyroid cancer cells. GABPA bound to the promoter of DDX21, and knockdown of GABPA or GABPB1 suppressed DDX21 promoter activity and transcription. Pharmacological inhibition of the MAPK pathway in BRAF-mutant cancer cells decreased the expression of GABPA, GABPB1, and DDX21. BRAFV600E upregulates expression of DDX21 through the MEK/ERK/GABP axis. DDX21, in turn, interacts with TERT to promote rRNA transcription and modulate the expression of multiple cancer hallmark genes, driving the malignant progression of thyroid cancer. These findings suggest that DDX21 acts as a mediator to bridge the crosstalk between BRAFV600E and TERT and may serve as a therapeutic target for patients with thyroid cancer harboring BRAF and TERT alterations.", "journal": "Thyroid : official journal of the American Thyroid Association", "year": "2026", "doi": "10.1177/10507256261417697", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "B" ] }, "40944426": { "pmid": "40944426", "title": "Identification of Variants in Four Families With Inherited Eye Disorders by Whole Exome Sequencing.", "abstract": "Inherited eye disorders are a significant cause of vision loss worldwide. According to the World Health Organization (WHO) estimates approximately 2.2 billion people have some degree of vision loss, but a significant proportion of these are blind since early childhood. Due to poor infrastructure for genetic diagnosis, many affected families remain genetically unexplained in underdeveloped countries, including Pakistan. In this study, we utilized homozygosity mapping and exome sequencing to identify the genetic basis of the vision loss in four Kashmiri families that were presented with different types of eye disorders. We also performed quantitative reverse transcriptase-PCR (qRT-PCR) and measured the relative mRNA abundance of ALMS1 in blood cells of patients, carrier parents, and a healthy control. Genetic analysis identified a novel homozygous 4 base pair frameshift deletion c.5747_5750del; p.(Ala1916Glufs*21) in ALMS1 in a family affected with Alstrom syndrome (AS), and already known variants were identified (CNGA3; c.1315C>T; p.(Arg439Trp) and FYCO1; c.2206C>T; p.(Gln736*) and c.3150 + 1G>T; p.(?)) in the remaining three families. The variant identified in the ALMS1 gene is predicted to activate nonsense mediated mRNA decay (NMD). Comparison of relative mRNA abundance of ALMS1 in patient-specific cells harboring c.5747_5750del negates the NMD activation. The results indicated the absence of NMD in patient-derived cells and therefore support the formation of a truncated ALMS1 protein in the patients with the c.5747_5750del variant. We expanded the mutation spectrum of ALMS1 but identified known variants in FYCO1 and CNGA3 genes. We also compiled the currently known mutations in these genes to establish genotype-phenotype correlation.", "journal": "Molecular genetics & genomic medicine", "year": "2025", "doi": "10.1002/mgg3.70141", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "41550658": { "pmid": "41550658", "title": "Conquering viral drug resistance: Structural and mechanistic paradigms for antiresistance drug design.", "abstract": "Viral drug resistance remains a critical challenge in antiviral therapy. This perspective highlights five studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus type 1 (HIV-1), monkeypox virus (MPXV), influenza A virus (IAV), and Hepatitis B virus (HBV), revealing novel resistance mechanisms and innovative strategies. For SARS-CoV-2, GC376's flexible benzyl group overcomes nirmatrelvir resistance. HIV-1's non-nucleoside reverse transcriptase inhibitors (NNRTIs) 5i3 adapts to resistant mutants via a quinazoline scaffold, while MPXV's tecovirimat acts as a \"molecular glue\" stabilizing F13 dimers. Expanding these paradigms, we present groundbreaking insights: An indazole-based IAV inhibitor (compound 24) disrupts the conserved PA-PB1 heterodimer, showing sub-micromolar potency against resistant strains. For HBV, a hydrophobic tagging degrader (HyT-S7) induces HBc degradation, bypassing resistance mutations impairing traditional capsid modulators. Key strategies include dynamic flexibility, multivalent interactions, and oligomerization control, integrated with AI-driven design and real-time surveillance. This perspective bridges structural insights with translational applications, offering a roadmap for next-generation, mutation-resilient antivirals.", "journal": "Pharmaceutical science advances", "year": "2025", "doi": "10.1016/j.pscia.2025.100094", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "40053490": { "pmid": "40053490", "title": "Noncanonical HIV drug resistance mutations: need to close existing gaps.", "abstract": "An increasing number of people with HIV (PWH) are failing treatment without HIV drug resistance in the drug target region. While sub-optimal adherence is likely the cause of treatment failure in many PWH, resistance emerging at noncanonical (HIV drug resistance mutations occurring outside the drug target site) drug target sites is also plausible. Noncanonical drug resistance mechanisms have been identified for integrase strand transfer inhibitors (INSTIs), protease inhibitors (PIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs) and NRTIs. Overall, they may act by restoring viral fitness caused by mutations in the drug target sites, enhance resistance when occurring with mutations at the drug target sites, independently cause resistance even in the absence of drug resistant mutations (DRMs) at the drug target site, and prime the emergence of resistant variants with DRMs at drug target sites. However, the clinical relevance of non-canonical HIV drug resistance mechanisms beyond in vitro and small in vivo studies is still needed and could include the assessment of such mechanisms in clinical trials and implementation studies. This information would be vital in guiding effective management of PWH with viral nonsuppression despite good adherence as well as informing public health surveillance strategies.", "journal": "AIDS (London, England)", "year": "2025", "doi": "10.1097/QAD.0000000000004170", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41140414": { "pmid": "41140414", "title": "ABCB1 Polymorphism in HIV-Infected Individuals Taking Antiretroviral Drugs.", "abstract": "ABC transporter P-glycoprotein (P-gp) and its expression enhance elimination and reduce drug exposure. The elimination of non-nucleoside reverse-transcriptase inhibitor (NNRTIs) drugs is associated with ABCB1 gene. Drug exposure is impacted by variants in the ABCB1 gene. Hence, the aim of the study was to investigate the association of ABCB1 1236 C/T and 3435 C/T polymorphisms with the modulation of antiretroviral (ARV)-associated hepatotoxicity. This is a cross-sectional study. Genotyping of the ABCB1 1236C/T and 3435C/T polymorphisms was performed in 165 HIV-infected individuals (34 with hepatotoxicity and 131 without hepatotoxicity) and 155 healthy controls using the PCR-RFLP method. The TC haplotype was likely to be associated with a higher risk of severe hepatotoxicity (OR = 1.96, p = 0.06), while CC and TT haplotypes were associated with a reduced risk of severe hepatotoxicity (OR = 0.34, p = 0.039; OR = 0.16, p = 0.006; OR = 0.09, p = 0.003). The ABCB1 3435CT genotype along with alcohol usage revealed a risk of HIV disease progression (OR = 2.47, p = 0.12). The ABCB1 1236TT genotype along with nevirapine (NVP) usage exhibited a risk for hepatotoxicity severity (OR = 2.11, p = 0.55). The ABCB1 3435CT genotype along with alcohol + NVP usage bared a risk of acquisition of hepatotoxicity (OR = 2.04, p = 0.23). In conclusion, ABCB1 haplotypes may influence the severity of ARV-induced hepatotoxicity. While individual polymorphisms and their interaction with alcohol or drug regimen showed no significant association, the 1236TT genotype with NVP use and the TC haplotype suggested a potential risk. Protective effects were observed for CC and TT haplotypes. Larger studies are warranted to confirm these findings and assess clinical relevance.", "journal": "AIDS research and treatment", "year": "2025", "doi": "10.1155/arat/9656615", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42202558": { "pmid": "42202558", "title": "A splice-altering intronic variant in two multiplex families refines autosomal recessive COG4-related congenital disorder of glycosylation.", "abstract": "Autosomal recessive COG4-related congenital disorder of glycosylation [COG4-CDG(ar)] is caused by biallelic deleterious variants in COG4, which encodes a component of the conserved oligomeric Golgi complex lobe A. COG4-CDG(ar) has been described in six individuals to date, and its clinical manifestations and disease mechanisms remain poorly understood. Exome sequencing identified the homozygous COG4 c.1647+5G>A variant in four affected individuals from two apparently unrelated Italian families. SNP-array genotyping revealed a common ancestral haplotype extending for 3.36 cM around COG4 and supports a founder effect for the identified variant. Whole transcriptome sequencing and reverse transcriptase PCR from peripheral blood detected an aberrant COG4 transcript which features skipping of exon 12 and results in a frameshift, predicted to introduce a premature termination codon. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling of serum N-glycans showed mildly deficient galactosylation and sialylation, consistent with an impaired Golgi-mediated glycosylation. As a novel finding, N-glycosylation study of serum IgG showed analogous N-glycan anomalies. These findings and literature review define COG4-CDG(ar) as a neurometabolic disorder with a progressive course leading to severe global disability, post-natal microcephaly with brain atrophy, seizures, coagulopathy, liver involvement, recurrent infections, and defective N-glycosylation of serum proteins.", "journal": "Molecular genetics and metabolism", "year": "2026", "doi": "10.1016/j.ymgme.2026.110155", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42118783": { "pmid": "42118783", "title": "Extensive diversity and impact of drug-resistant HIV-1 variants in individuals with prior virologic failure.", "abstract": "HIV-1 drug resistance remains a major challenge to treatment and control efforts, particularly in sub-Saharan Africa (sSA). However, standard resistance genotyping does not adequately capture linked drug resistance mutations within the viral quasispecies that may influence virologic failure (VF). We used a next-generation sequencing primer ID (NGS-Primer ID) assay to characterize linked HIV-1 drug resistance mutations in plasma from participants in the Resistance Testing to Improve Management of Virologic Failure (REVAMP) study who had detectable viremia on first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART) and were maintained on NNRTI-based regimens. For each participant, we calculated a weighted genotypic susceptibility score (wGSS) based on the GSS of each reported pattern and its sequence-supported frequency within the sample. Plasma specimens from 108 participants were sequenced. Sanger sequencing showed a median GSS of 1.0 (IQR, 1.0-2.0), whereas NGS-Primer ID identified a median of 10 distinct resistance patterns per participant (IQR, 5-17), with 67% (IQR, 8-92) of reported DRM-pattern frequency within participants corresponding to linked dual-class mutations. A broad spectrum of drug-susceptible and resistant- DRM patterns were observed, with the median difference between the highest and lowest pattern-specific GSS being 2.0 (2.0-2.75). Within each participant, a median of 10% of reported patterns showed less resistance, while 19% showed more resistance compared to that predicted by Sanger sequencing. Individuals with persistent VF had significantly lower wGSS at study entry than those who later achieved virologic re-suppression (median, 1.3 vs 2.1; p < 0.001). NGS-Primer ID revealed substantial intra-host diversity in linked HIV-1 drug resistance patterns that was not captured by conventional Sanger sequencing. Incorporating linked resistance patterns into susceptibility assessment may improve prediction of subsequent virologic failure.", "journal": "PLoS pathogens", "year": "2026", "doi": "10.1371/journal.ppat.1014118", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "42829380": { "pmid": "42829380", "title": "The therapeutic potential of transposable element activity modulation.", "abstract": "The reactivation of transposable elements (TEs) is a recurrent molecular event across cancer, neurodegeneration, autoimmune disease, and aging, yet its therapeutic implications remain largely unexplored. Normally repressed under strict epigenetic surveillance, reactivated transposable elements can generate immunostimulatory nucleic acids that engage cytosolic pattern-recognition receptors, converging on type I interferon signaling and driving chronic sterile inflammation. Yet this inflammatory potential is inherently dual: the same reactivation that drives pathology in some contexts can be harnessed to prime immunity in others. Here, we review two complementary strategies that exploit this duality. Suppressive approaches, including nucleoside reverse transcriptase inhibitors, antisense oligonucleotides, and epigenetic silencing agents, aim to quench pathological TE-driven inflammation in diseases and restore other adaptive mechanisms governing homeostasis. Conversely, controlled TE reactivation through epigenetic priming can induce a viral mimicry state in tumor cells, sensitizing them to immune checkpoint blockade. In parallel, TE-derived peptides presented on MHC class I molecules are emerging as a largely unexploited reservoir of shared tumor antigens for next-generation vaccines and engineered T cell therapies. Together, these observations reframe transposable elements not as mere viral relics to be tolerated, but as a tunable immunological switch whose therapeutic modulation holds promise across some of the most challenging diseases of our time.", "journal": "EMBO molecular medicine", "year": "2026", "doi": "10.1038/s44321-026-00528-z", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "42499346": { "pmid": "42499346", "title": "Prime editing in neuropsychiatric disorders: From mutation-specific target selection to clinical translation.", "abstract": "Prime editing, a novel clustered regularly interspaced short palindromic repeats (CRISPR)-based technology, fuses a reverse transcriptase (RT) to an engineered CRISPR-associated protein 9 (Cas9) and uses a prime editing guide RNA (pegRNA)-encoded template. It enables precise base substitutions, small insertions, and deletions without introducing double-strand breaks, thereby expanding the range of correctable mutations while reducing undesired repair outcomes. This technology offers a promising strategy for genomic correction in the nervous system. Here, we review the development of prime editing, its mechanistic rationale, and emerging preclinical evidence that supports its application in neuropsychiatric disorders. We discuss key biological and technological barriers, including limited editing efficiency in post-mitotic neurons, complex pegRNA design, reverse transcription-related errors, vector payload limitations, and blood-brain barrier (BBB) penetration. Nevertheless, in vitro and in vivo studies have demonstrated proof-of-concept correction and functional rescue in several monogenic neurodevelopmental disorders. Advances such as split-adeno-associated virus (AAV) systems, lipid nanoparticles, engineered peptides, and compact Cas variants are actively expanding their therapeutic potential. Further clinical translation will rely on improved editors with guide engineering, BBB-penetrant and neuron-targeted delivery platforms, transient or cell-type-specific expression strategies, and comprehensive genome-wide safety evaluations.", "journal": "Neuroprotection (Chichester, England)", "year": "2026", "doi": "10.1002/nep3.70041", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "42071158": { "pmid": "42071158", "title": "Viral Mimicry of Alzheimer's Disease: Innate Sensing of Self-Nucleic Acids as a Driver of Glial Senescence.", "abstract": "Alzheimer's disease (AD) is a devastating neurodegenerative disorder defined by progressive memory loss and synaptic failure. For decades, therapeutic development has focused on clearing amyloid-beta plaques, yet the repeated clinical failures of this approach necessitate a fundamental paradigm shift toward the brain's immunometabolic landscape. The \"Viral Mimicry\" hypothesis posits that AD represents a state of sterile autoimmunity where the innate immune system mistakenly identifies self-nucleic acids as viral pathogens. This \"ghost war\" is ignited by the convergence of metabolic dysfunction and genomic instability: specifically, the leakage of mitochondrial DNA into the cytosol and the epigenetic derepression of ancient retrotransposons (LINE-1, HERVs). These endogenous ligands activate the cGAS-STING cytosolic sensing axis, a pathway that drives a chronic interferon response. Consequently, microglia and astrocytes are transformed into senescent, pro-inflammatory phenotypes that release a toxic Senescence-Associated Secretory Phenotype (SASP), directly fueling synaptic elimination. Crucially, major genetic risk factors, including APOE4 and TREM2 variants, exacerbate this cascade by compromising mitochondrial integrity and lipid metabolism, thereby sensitizing the brain to innate surveillance failure. By reconceptualizing AD as an acquired interferopathy driven by the \"enemy within,\" this framework highlights novel therapeutic targets. Specifically, repurposing Nucleoside Reverse Transcriptase Inhibitors (NRTIs) to block retrotransposition and deploying senolytics to clear dysfunctional glia offer promising strategies to arrest the progression from healthy aging to cognitive decline. This review synthesizes current research on the molecular mechanisms of viral mimicry, detailing the impact of genetic risk factors and evaluating emerging therapeutic interventions targeting this innate immune axis.", "journal": "Journal of molecular neuroscience : MN", "year": "2026", "doi": "10.1007/s12031-026-02532-x", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41976152": { "pmid": "41976152", "title": "Biphenyl as a Privileged Structure in Medicinal Chemistry: Advances in Anti-Infective Drug Discovery.", "abstract": "The discovery of novel anti-infective agents is a continuous challenge in medicinal chemistry, particularly due to the rise in resistant fungal and viral strains. Within this context, the biphenyl subunit has been identified as a highly versatile privileged structure capable of interacting with diverse protein targets via hydrophobic and π-interactions. The purpose of this study is to review the pharmacological potential of biphenyl-based compounds, focusing on their application as anti-infective agents. We comprehensively analyzed recent literature and rational design strategies concerning biphenyl derivatives, examining structure-activity relationships, molecular docking insights, and structural optimizations aimed at enhancing both pharmacodynamics and pharmacokinetics. The reviewed studies demonstrate that incorporating biphenyl moieties yields compounds with potent antifungal and antiviral activities. Specifically, optimized biphenyl derivatives exhibit strong inhibitory effects against resistant Candida strains and crucial viral targets, including mutant variants of the HIV-1 reverse transcriptase and protease enzymes. Furthermore, strategic modifications, such as scaffold hopping and the introduction of specific substituents, successfully mitigated cytotoxicity and improved metabolic stability against cytochrome P450 enzymes. Biphenyl serves as a robust and adaptable scaffold for drug design. Its rational structural optimization provides a viable pathway to overcome drug resistance and develop effective, metabolically stable anti-infective therapeutics.", "journal": "Molecules (Basel, Switzerland)", "year": "2026", "doi": "10.3390/molecules31071109", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "B" ] }, "41472239": { "pmid": "41472239", "title": "An Assessment of Regional Genetic Diversity of HIV-1.", "abstract": "This study aimed to assess the genetic diversity of HIV-1 in the Far Eastern Federal District (Russia) to implement effective anti-epidemic measures, including the development of an anti-HIV vaccine and the selection of optimal antigens. The first stage of the study included an analysis of HIV-1 nucleotide sequences obtained in Khabarovsk city from 2022 to 2024. The second stage of the study included an additional download of nucleotide sequences from the Los Alamos HIV Sequence Database for phylogenetic cluster analysis. Additionally, an analysis of drug resistance mutations was conducted. The results showed the following distribution of HIV-1 genetic variants: A6-72.15%, CRF63-10.13%, URFs-7.59%, C-5.06%, B-3.8%, and CRF157-1.27%. The phylogenetic cluster analysis revealed a statistically significant difference in the number of clusters depending on the genetic variant. Among drug resistance mutations (DRMs), those associated with nucleoside reverse transcriptase inhibitors (NRTIs) were the most frequently observed, accounting for 55.7% (95% CI: 44.75%-66.65%). The most commonly detected NRTI DRMs were A62V (43.04%) and M184V (13.92%). The results of this study highlight several important indicators for public health, particularly in the development of vaccines aimed at combating HIV infection.", "journal": "Viruses", "year": "2025", "doi": "10.3390/v17121568", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "B" ] }, "40254182": { "pmid": "40254182", "title": "Pathogenic Variants and Prognosis in Meningiomas: A Systematic Review and Meta-Analysis.", "abstract": "Intracranial meningiomas are the most common primary tumors of the central nervous system. Although generally benign, some genetic alterations can induce aggressive behavior characterized by higher recurrence rates and reduced survival. A systematic review and meta-analysis were conducted following Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines, using PubMed, EMBASE, Web of Science, and Scopus databases to identify studies published until November 2024. Studies investigating genetic alterations in meningiomas with prognostic data (recurrence or survival) and a minimum sample size of five patients were included. Data were extracted and analyzed independently by two reviewers. Of 3032 studies identified, 20 met the inclusion criteria. The most frequently studied pathogenic variants were telomerase reverse transcriptase promoter (TERTp) and neurofibromatosis type 2 (NF2), both associated with shorter recurrence-free survival (RFS) and overall survival (OS), respectively TERTp RFS (hazard ratio [HR] 4.35, 95% confidence interval [CI] 2.87-6.60) and OS (HR 2.55, 95%CI 1.25-5.22), and NF2 RFS (HR 1.49, 95%CI 1.04-2.14) and OS (HR 2.98, 95% CI 1.37-6.49). Subgroup analysis suggested that the TERTp variant may be more predictive of lower survival for overall meningiomas (instead of World Health Organization III only), similar to NF2 variants. Additionally, Krüppel-like factor 4 was identified as a protective factor, while cyclin-dependent kinase inhibitor 2A/B was identified as a risk factor. This systematic review highlights the importance of pathogenic variants, particularly TERTp and NF2, as prognostic markers in intracranial meningiomas. These findings underscore the potential of integrating genetic profiling into clinical practice to refine risk stratification and guide personalized therapeutic strategies, ultimately improving patient outcomes and quality of life.", "journal": "World neurosurgery", "year": "2025", "doi": "10.1016/j.wneu.2025.123988", "ptypes": [ "Journal Article", "Systematic Review", "Meta-Analysis" ], "first_author": "", "dirs": [ "B" ] }, "41541812": { "pmid": "41541812", "title": "Advancing schistosomiasis monitoring through optimised environmental DNA detection.", "abstract": "Schistosomiasis, caused by parasites of the genus Schistosoma, remains a major public health burden in sub-Saharan Africa, particularly where access to clean water, sanitation, and hygiene is limited. Effective control requires large-scale surveillance, but traditional methods such as malacological surveys, and stool or urine microscopy often lack sensitivity and scalability. This study evaluated environmental DNA-based detection of Schistosoma mansoni in water samples from Lake Albert and Lake Victoria, Uganda. Three filtration techniques (open membrane, Waterra eDNA capsule, and Sylphium eDNA Dual filter capsule), were compared for eDNA yield and detection sensitivity. Quantitative PCR (qPCR) targeting the cytochrome c oxidase subunit 1 (COI) mitochondrial gene was used to quantify S. mansoni eDNA, following in silico and in vitro primer optimisation. Conventional malacological surveys were conducted in parallel for validation. Statistical analyses further examined associations between eDNA yield, detectability, and environmental factors. The qPCR assay had a practical limit of detection (LOD) of 100 DNA copies per reaction and a theoretical LOD/limit of quantification of 83 copies. Schistosoma mansoni eDNA was detected in 26 % (15/58) of samples from Lake Albert and 24 % (27/113) from Lake Victoria. Waterra filters yielded the most eDNA, and Sylphium purification produced significantly greater yields than column-based extraction kits. Both filter type and eDNA yield significantly influenced S. mansoni detection: Waterra and Sylphium-single filters had the highest amplification probabilities (∼40 %), while open membrane filters performed poorly (∼3 %). eDNA yield was a strong predictor of detection, with the odds of positivity increasing by ∼0.8 % per additional nanogram of eDNA. Among positive samples, Waterra filters produced the lowest mean Ct values, indicating greater recovery of amplifiable parasite DNA. Conversely, open membrane filters were the most affect by field contamination. Our findings highlight eDNA as a sensitive and scalable tool for surveillance of schistosomiasis and other water-borne parasitic diseases. While higher-capacity filters and two-phase extraction methods maximised eDNA yield, lower-yield methods still enabled detection in high-transmission settings. A comparative analysis of sampling effort, costs and contamination and infection risks is presented. Overall, our results support the adaptability of eDNA approaches across resource contexts and underscore the need for protocol standardisation, ecological validation, and field-deployable diagnostics such as LAMP.", "journal": "Food and waterborne parasitology", "year": "2026", "doi": "10.1016/j.fawpar.2025.e00313", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41918793": { "pmid": "41918793", "title": "Single-Tube Loop-Mediated Isothermal Amplification Assay Targeting the inlA Gene for Sensitive Detection of Listeria monocytogenes in Food.", "abstract": "Several loop-mediated isothermal amplification (LAMP) assays with good performance characteristics have been developed for the detection of Listeria monocytogenes in food; however, there are only a few cases in which DNA extraction, amplification and sensing have been performed in a single-tube system. The efficiency of DNA extraction by lysis buffers was tested using LAMP. New primer sets for LAMP assays were designed using PrimerExplorer V5 software. The sensitivity and specificity of the LAMP inner primers were determined by optimised PCR. The end-point detection involved gel electrophoresis, turbidity and eriochrome black T (EBT) colour reaction. The sensitivity, specificity and limit of detection (LOD) of the developed LAMP assays were then characterised using L. monocytogenes, non-monocytogenes Listeria and non-Listeria bacterial strains. Both the alkaline cell lysis-based sodium hydroxide and Tris-HCl (HotSHOT)+Tween buffer and the Triton X-100 and sodium azide-based TZ buffer generated amplifiable DNA templates under isothermal conditions for LAMP. However, the TZ buffer produced a significantly higher DNA yield than the HotSHOT+Tween buffer. LAMP primers were designed to target the hlyA and inlA virulence genes of L. monocytogenes. The sensitivity and specificity of the LAMP inner primers were 100 % for both genes; however, the PCR reaction targeting the inlA gene generated fewer non-specific PCR products than the hlyA-targeting PCR. The sensitivity of the InlA LAMP assay was 100 %, while its specificity was 96 %. The LOD was 500 fg per reaction, which corresponds to 157 genome copy numbers. The combination of DNA extraction, LAMP amplification, and colorimetric endpoint detection in a single tube resulted in a LAMP assay suitable for the detection of L. monocytogenes in food under laboratory conditions, with potential for further development for on-site detection with microfluidic platforms. To the best of our knowledge, this is the first report of a LAMP assay targeting the inlA gene of L. monocytogenes. The developed single-tube LAMP assay is well-suited for integration with microfluidic systems.", "journal": "Food technology and biotechnology", "year": "2026", "doi": "10.17113/ftb.64.01.26.9231", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42639043": { "pmid": "42639043", "title": "Development and validation of a LAMP assay for detection of genetically diverse Lactococcus c2-type bacteriophages in dairy environments.", "abstract": "Bacteriophages infecting Lactococcus spp. are a major cause of fermentation failures in the dairy industry. Among them, c2-type phages remain insufficiently studied, and rapid detection methods are limited. This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of genetically diverse c2 phages. As part of this work, 12 prevalent c2 phages isolated from Russian dairy plants and 22 additional c2 phages were sequenced and analyzed. Conserved genomic regions were identified, and two primer sets were designed to account for the high genetic variability of c2 phages. Together, these primer sets enabled detection of all 34 tested c2 isolates despite varying numbers of mismatches within primer-binding regions. The assay detected 34 c2 isolates with varying numbers of mismatches in primer-binding regions. The detection limit was 102 copies/μL for plasmid DNA and 102-103 PFU/mL for bacterial lysates. Validation using industrial cheese whey samples confirmed assay applicability. Addition of high-fidelity Tersus polymerase improved mismatch tolerance, and in silico analysis predicted potential detection of up to 98% of publicly available c2 phages genomes. The developed assay provides a rapid and cost-effective tool for phage monitoring in dairy fermentations and may help reduce the risk of fermentation failures. Furthermore, the proposed approach may serve as a framework for developing diagnostic assays targeting other genetically diverse bacteriophage groups.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1912103", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42346539": { "pmid": "42346539", "title": "Establishment of a Visual LAMP Technology and Detection of Cronartium ribicola Infecting Chinese White Pine in Southwestern China.", "abstract": "White pine blister rust disease (WPBR), caused by Cronartium ribicola, ranks among the most destructive pathogens of five-needle pines. We developed a hydroxynaphthol blue (HNB)-based Loop-mediated isothermal amplification (LAMP) assay enabling rapid, visual detection of C. ribicola directly following DNA extraction. LAMP primers targeting the internal transcribed spacer (ITS) region were designed and validated through in silico comparison with related Cronartium species and in vitro testing against sympatric forest fungi. The optimized 25 μL reaction contained 8.0 mM Mg2+, 1.0 mM dNTPs, and an inner-to-outer primer ratio of 8:1, with amplification conducted at 62 °C for 40 min. Positive amplification produced a distinctive color transition from purple to sky blue, enabling visual interpretation without instrumentation. Under the tested conditions, the assay achieved a detection limit of 460 ± 3.2 fg/μL genomic DNA-a 10-fold improvement over conventional PCR in concentration-based sensitivity. Assay applicability was evaluated using 211 field-collected Pinus armandii samples sourced from China. Detection efficiency varied significantly across tissue types. Symptomatic bark exhibited a substantially higher positive detection rate (68.97%, 95% CI: 49.2-84.7%) compared to needles from symptomatic trees (18.75%, 95% CI: 4.1-45.7%). Among asymptomatic samples, 3.75% of bark samples tested positive for C. ribicola DNA, whereas all needle samples were negative. Geographically, positive detections clustered at several discrete sampling sites in southwestern China, predominantly at elevated elevations. The established LAMP-HNB assay provides a rapid, visually interpretable diagnostic tool for early detection and quarantine monitoring of WPBR following DNA extraction. Beyond its practical utility, this assay establishes valuable baseline data for targeted disease surveillance in the context of evolving climate conditions.", "journal": "Journal of fungi (Basel, Switzerland)", "year": "2026", "doi": "10.3390/jof12060409", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42353430": { "pmid": "42353430", "title": "Rapid Point-of-Care Detection of Dirofilaria immitis and Dirofilaria repens in Canine Blood Using Two Direct Closed-Tube LAMP Assays.", "abstract": "Canine dirofilariasis, caused by Dirofilaria immitis and Dirofilaria repens, is an emerging vector-borne disease of increasing veterinary and zoonotic importance. Rapid and species-specific detection is essential for effective clinical management and epidemiological surveillance. This study aimed to develop and diagnostically evaluate two novel species-specific loop-mediated isothermal amplification (LAMP) assays for the direct detection of D. immitis and D. repens in canine whole blood, performed in parallel in separate reactions, with emphasis on simplified and potentially near-point-of-care applicability. Primers targeting mitochondrial COI and NADH gene regions were designed and validated. In silico specificity analysis against 13 filarioid species confirmed the absence of non-specific primer binding. A direct closed-tube LAMP protocol using sodium hydroxide-Chelex-100 lysis was optimized, enabling amplification without conventional DNA extraction while reducing contamination risk and processing time to under 60 min. Relative diagnostic performance was evaluated relative to quantitative real-time PCR (qPCR) results. Using purified DNA, the D. repens assay achieved 100% relative sensitivity and relative specificity, whereas the D. immitis assay showed 94.5% relative sensitivity and 100% specificity. In direct whole-blood assays, relative specificity remained 100% for both targets, while sensitivity decreased to 90.9% for D. immitis and 77.42% for D. repens, with most false-negative reactions associated with high qPCR Ct values (>30). These findings demonstrate that the proposed assays provide a rapid and practical molecular diagnostic approach with potential applicability for point-of-care veterinary testing.", "journal": "Animals : an open access journal from MDPI", "year": "2026", "doi": "10.3390/ani16121820", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41006776": { "pmid": "41006776", "title": "Development of loop mediated isothermal amplification assay for rapid detection and its comparison with PCR techniques for Ancylostoma duodenale.", "abstract": "Hookworm infections, primarily affecting children and pregnant women are major health concern. As part of global efforts to eradicate neglected tropical diseases (NTDs) through mass chemotherapy, reliable diagnostic methods are urgently needed as regions work towards elimination. In this study, we have developed a LAMP-based method to detect hookworm infection in faecal samples. The primers, tailored for the LAMP assay, were designed targeting ITS-1 region of Ancylostoma duodenale using Primer Explorer version 5. Five hundred (500) human stool samples were subjected to microscopy and DNA extraction simultaneously, followed by conventional PCR, real-time PCR and LAMP amplification. The specificity, sensitivity and inter-rater agreement of all assays were evaluated using MedCalc software taking real-time PCR as gold standard. The newly devised LAMP method successfully identified A. duodenale DNA in fecal samples in less than 40 min. It demonstrated perfect specificity (100%), with no instances of cross-reactivity. The LAMP assay achieved a sensitivity of 87.8%, 83.7% for conventional PCR and 59% for microscopy with 100% specificity for all methods. The newly developed LAMP assay represents a suitable diagnostic approach for identifying A. duodenale DNA in human stool samples due to its simplicity, affordability, sensitivity, specificity and potential for field applicability.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-93995-2", "ptypes": [ "Journal Article", "Comparative Study" ], "first_author": "", "dirs": [ "C" ] }, "41621553": { "pmid": "41621553", "title": "Species-specific isothermal nucleic acid amplification assay targeting Internal Transcribed Spacer (ITS) for rapid authentication of the medicinal crop Cirsium japonicum and Cirsium setosum in herbal markets.", "abstract": "Cirsium japonicum (CJ) and Cirsium setosum (CS) are two widely recognized medicinal crops in Chinese herbal medicine (CHM) that are listed as authentic herbal plants in the herbal pharmacopeia. Given their strikingly similar morphological characteristics, CJ and CS are particularly susceptible to adulteration in the herbal marketplace, raising significant concerns about product integrity and consumer safety. To ensure the safety and efficacy of CHM, proper authentication of these herbs is a critical step in maintaining high-quality pharmaceutical production. In this study, we developed a species-specific loop-mediated isothermal amplification (LAMP) method for the authentication of CJ and CS. We designed specific LAMP primer sets for both species using the selected DNA barcode, the internal transcribed spacer (ITS) sequence, through in silico analysis. After validating the specificity of the primers, we successfully authenticated CS and CJ using the LAMP primer sets and visualized the detection results through gel electrophoresis. The sensitivity of the LAMP method for CJ and CS authentication was established, with a limit of detection (LOD) of 100 pg for CJ and 50 pg for CS, using direct SYBR Green staining. When we applied the LAMP method to commercial Cirsium products, we found that only 25% (5 out of 20) of the samples contained CJ, while none contained CS. In conclusion, we successfully established a species-specific LAMP assay for authenticating CJ and CS. This method can be used for species verification and is applicable to commercial Cirsium products for authenticity testing, contributing to quality control in pharmaceutical manufacturing.", "journal": "Analytical biochemistry", "year": "2026", "doi": "10.1016/j.ab.2026.116063", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40029472": { "pmid": "40029472", "title": "Advances in porcine epidemic diarrhea virus research: genome, epidemiology, vaccines, and detection methods.", "abstract": "Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV). The economic impact of PEDV on the global pig industry has been significant, resulting in considerable losses. This paper presents a review of the latest research progress on PEDV genome, molecular epidemiology, vaccine development, and molecular detection methods. It was determined that the genetic diversity of the PEDV spike (S) gene was closely associated with the epidemiological trend of PEDV. The prevalence of S gene variants of different genotypes exhibited variability across regions and pig populations. Epidemiological analyses have demonstrated that PEDV can be transmitted via multiple routes, including direct contact, airborne aerosol, and water source contamination. With regard to vaccine research, the available vaccines can be classified into several categories, including live-attenuated vaccines, inactivated vaccines, subunit vaccines, bacterial vector vaccines, viral vector vaccines, mRNA vaccines, etc. Each of these has distinctive characteristics in terms of immunogenicity, protection efficiency, and safety. Molecular detection methods, including PCR-based methods, isothermal amplification techniques, immunological assays, and biosensors, play an important role in the diagnosis and monitoring of PEDV. Furthermore, this paper examines the current developments in PEDV research and identifies the key areas of future investigation. The objective of this paper is to establish a theoretical foundation for the prevention and control strategies of PED, and to provide a point of reference for further research on the genomics, epidemiology, vaccine development and detection methods of PEDV.", "journal": "Discover nano", "year": "2025", "doi": "10.1186/s11671-025-04220-y", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "40277382": { "pmid": "40277382", "title": "Fast-Flu: RT-RPA-CRISPR/Cas12a assisted one-step platform for rapid influenza B virus detection.", "abstract": "Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains a lack of rapid and accurate diagnostic methods for Flu B detection. In this study, we developed a novel Flu B detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization of reaction temperature and adjustment of Cas12a concentrations, we successfully balanced RPA amplification and CRISPR/Cas12a trans-cleavage activity, enabling the establishment of a one-step detection system. The one-step Fast-Flu system demonstrated the ability to specifically identify Flu B within 45 min, with a limit of detection of 58 copies per test. It eliminates the need for uncapping operations and minimizes the risk of cross-contamination, without cross-reactivity with other pathogens. When evaluated using 101 clinical throat swab samples, the one-step Fast-Flu system achieved a sensitivity of 56.25% and a specificity of 100% compared to the PCR-based method, with an overall concordance rate of 93.06% (94/101). The development of this one-step RT-RPA-CRISPR/Cas12a system represents a significant advancement in the rapid, convenient, and accurate detection of Flu B, highlighting its potential for clinical diagnosis. Furthermore, with future technical improvements to enhance sensitivity, this one-step RT-RPA-CRISPR assay holds promise as a versatile tool for the rapid nucleic acid detection of other RNA viruses. Influenza B virus (Flu B) is a significant global health concern, and rapid, accurate pathogen diagnosis is crucial for effective influenza prevention and control. The integration of isothermal amplification methods with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has achieved high sensitivity and specificity for nucleic acid detection. Although CRISPR/Cas-based systems have been developed for influenza detection, existing platforms require the transfer of amplified products into the CRISPR/Cas12a detection system through uncapping operations, which increases the risk of cross-contamination. In this study, we developed a one-step reverse transcription recombinase polymerase amplification-CRISPR/Cas12a Flu B detection method using a one-pot detection system. By optimizing the reaction temperature and Cas12a concentration, we achieved a streamlined and contamination-free workflow. This innovative approach not only improves Flu B detection but also serves as a valuable reference for constructing CRISPR/Cas systems for the detection of other pathogens and targets, paving the way for broader applications in molecular diagnostics.", "journal": "Microbiology spectrum", "year": "2025", "doi": "10.1128/spectrum.00365-25", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42075335": { "pmid": "42075335", "title": "Progress of Rapid Detection Technology for Aquatic Microorganisms: A Comprehensive Review.", "abstract": "Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020-2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption-from days to less than one hour-while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements.", "journal": "Microorganisms", "year": "2026", "doi": "10.3390/microorganisms14040939", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "40573432": { "pmid": "40573432", "title": "A Single-Tube Two-Step MIRA-CRISPR/Cas12b Assay for the Rapid Detection of Mpox Virus.", "abstract": "Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein-Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination.", "journal": "Viruses", "year": "2025", "doi": "10.3390/v17060841", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "40409910": { "pmid": "40409910", "title": "μLAMP-LFA: A nucleic acid analysis platform for the field diagnosis of pathogens in aquatic animals.", "abstract": "Aquatic animal diseases cause significant economic losses in aquaculture. Effective pathogen diagnostics enable targeted treatment measures. Traditional detection methods primarily rely on polymerase chain reaction. However, due to the bulky and expensive nature of the equipment, on-site testing is impractical. Furthermore, the process requires low-temperature preservation, transportation to centralized laboratories, and analysis by trained personnel. The combination of loop-mediated isothermal amplification (LAMP) and lateral flow assay (LFA) has promoted on-site detection with greater convenience. However, there are still some shortcomings with the current LAMP-LFA technology. A micro-volume LAMP-LFA platform (μLAMP-LFA) was developed for on-site diagnosis of aquatic animal pathogens. The platform comprised a microfluidic chip and dedicated equipment (mini LAMPer). The μLAMP-LFA chip integrated LAMP and LFA technologies. After rotating the chip, the amplification solution was automatically transferred into the LFA chamber. The hermetically sealed chip effectively prevented false-positive results caused by aerosols. Additionally, the chip included a dedicated compartment for separate storage of dilution solutions, facilitating micro-volume LAMP-LFA. The μLAMP-LFA chip demonstrated excellent flow control, fluidity, and mixing capabilities. The rapid heating rate and stable temperature of the mini LAMPer ensured optimal conditions for LAMP reactions. The platform detected white spot syndrome virus and Salmonella at concentrations as low as 28 copies/μL and 15 copies/μL, respectively. The dual pathogen detection capability of the platform was validated using real shrimp samples. Compared to conventional LAMP-LFA, μLAMP-LFA minimizes reagent usage, drastically lowering detection costs. More importantly, the pre-stored dilution solution in the chip plays a critical role in cooling the amplified products, preventing potential test strip failure. Its simple operation, portability and low cost grant the platform significant potential for field detection of pathogens in aquatic animals. In addition, it can also be applied to food safety, agricultural monitoring, environmental analysis, and other on-site detection applications.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344144", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "40480715": { "pmid": "40480715", "title": "A power-free microfluidic device for the detection of hepatitis B virus in resource-limited settings.", "abstract": "Rapid on-site nucleic acid testing plays a critical role in the prevention, diagnosis, and treatment of infectious diseases. Although real-time fluorescence polymerase chain reaction (PCR) technique, as the gold standard method, is widely used for nucleic acid quantification, its reliance on complex instrumentation and trained personnel in centralized laboratories presents significant challenges in applications such as rapid home-based self-testing or screening in remote areas. Therefore, there is an urgent need to develop miniaturized, low-cost, user-friendly, and high-performance point-of-care testing (POCT) devices for nucleic acid detection to meet the demands of infectious pathogen screening in resource-limited settings. In this study, a low-cost and power-free microfluidic device (PFMD), which realized contamination-free analysis with 'sample-in-visual-result-out' capability, was developed. The proposed system integrated rapid extraction and enrichment of nucleic acid from serum samples, isothermal nucleic acid amplification, and visual readout of lateral flow test strips. The entire detection process was completed within 40 min. Power-free isothermal amplification was enabled by a disposable warm patch. The detection performance of the PFMD was evaluated using nucleic acid standards of hepatitis B virus (HBV) and other pathogens, as well as clinical serum samples from suspected hepatitis B cases. The PFMD successfully accomplished rapid magnetic bead-based nucleic acid extraction and achieved a lower limit of detection of 3.04 copies/μL. The clinical experimental results of the device were consistent with those obtained by quantitative PCR and real-time fluorescent loop-mediated isothermal amplification (LAMP) methods, with 100 % diagnostic sensitivity and 100 % diagnostic specificity. This work presents an integrated, portable, low-cost, and power-free POCT method for nucleic acid detection, which demonstrates rapid diagnostic capabilities for complex clinical samples and exhibits outstanding sensitivity and specificity. Clinical validation confirms that the proposed method and device are well-suited for on-site rapid screening of infectious diseases such as hepatitis B, particularly in resource-constrained settings without access to electricity or trained professionals.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344154", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "39616759": { "pmid": "39616759", "title": "Both aerosol and primer dimer breakdown for straightforward genotyping based on an integrated immunochromatographic biosensor.", "abstract": "Straightforward genotyping can provide timely diagnostic information for diseases prevention and treatment. Taking advantages of speediness and convenience, although numerous genotyping strategies combined loop-mediated isothermal amplification (LAMP) and lateral flow have been reported to satisfy the demand of point-of-care test, the false positive result caused by aerosol and primer dimer as an innate conflict seriously limits their practical application. In this study, both aerosol and primer dimer as extrinsic and intrinsic inducements respectively are first broken through at one stroke based on an integrated immunochromatographic biosensor. By introducing digoxigenin labeled dUTP into LAMP, not only the amplicon can be analyzed through naked eye, but also the aerosol contamination can be eliminated thoroughly by uracil DNA glycosylase ignoring the open vessel. Primer dimer, the significant drawback in lateral flow-based strategies, has been overcome due to the bio-labeled deoxyribonucleotide and oligonucleotide cannot couple for signal generation even under the high primer concentration. Instead of colloidal gold, the gold magnetic nanoparticle is synthesized and assembled into this biosensor as a nanoprobe, which enables the result to be quantified by the magnetic signal for subjective bias elimination. The polymorphism of C677T in methylenetetrahydrofolate reductase, a crucial genetic code related to folate metabolism, is genotyped using saliva as noninvasive specimen dispense with DNA purification. Only 1 ng genomic DNA can provide accurate result within 25 min by a simple heater, which proves the potential of this biosensor to facilitate precision medicine.", "journal": "Talanta", "year": "2025", "doi": "10.1016/j.talanta.2024.127300", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42187759": { "pmid": "42187759", "title": "One-Pot LAMP-Coupled CRISPR/Cas12b Assay Enables Sensitive Detection of Helicobacter pylori.", "abstract": "Helicobacter pylori (H. pylori) infection is closely associated with the development of chronic gastritis, peptic ulcers, and gastric cancer, highlighting the importance of rapid and accurate detection for disease prevention and clinical management. In this study, a one-pot LAMP-CRISPR/Cas12b assay targeting the CagA gene was developed for H. pylori detection. First, the LAMP system was optimized by systematically screening key reaction components. Subsequently, a one-step LAMP-CRISPR/Cas12b detection platform was established through optimization of the ratio between the LAMP premix and CRISPR buffer, reaction temperature, Cas12b concentration, and ssDNA reporter concentration. Under optimal conditions, the assay achieved a detection limit of 3.14 × 101 copies/µL, representing a tenfold improvement in sensitivity compared with conventional LAMP and PCR assays (3.14 × 102 copies/µL). In addition, the entire detection process could be completed within 1 h. Validation using 17 culture-positive and 17 culture-negative samples demonstrated complete concordance with culture-based results, with no false-positive or false-negative detections observed. These findings indicate that the proposed platform possesses high sensitivity, excellent specificity, rapid turnaround, and operational simplicity, demonstrating strong potential for point-of-care testing and applications in resource-limited settings.", "journal": "Biology", "year": "2026", "doi": "10.3390/biology15100797", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "39205510": { "pmid": "39205510", "title": "Rapid detection of murine-derived ingredients in meat products using real-time enzymatic recombinase amplification.", "abstract": "Accurate identification of meat species is critical to prevent economic fraud and safeguard public health. The use of inappropriate meat sources, such as murine, poses significant health risks because of potential contamination with pathogens and allergens, leading to foodborne illnesses. The present study aimed to develop a novel real-time enzymatic recombinase amplification (ERA) method for the rapid and specific detection of murine DNA in meat products. A novel ERA primer and probe set was designed, targeting a murine-specific single-copy nuclear gene identified through bioinformatics analysis. The assay demonstrates high specificity, showing no amplification in commonly consumed meats, other animals or major crops. Additionally, it exhibits remarkable sensitivity, detecting as few as five copies of murine genomic DNA. For practical application, the ERA method could effectively identify mouse DNA in laboratory-prepared samples at concentrations as low as 0.5% and also quantify samples with mouse DNA content as low as 5%. It also accurately detects the presence of murine-derived ingredients in commercially available meat products. The detection process is straightforward, utilizing a simple isothermal device for incubation, blue light excitation and a smartphone camera for result interpretation. This rapid analysis can be completed within 20 min. The newly developed real-time ERA method provides a valuable tool for standardizing meat trade practices, promoting food safety and enhancing consumer confidence in the authenticity of meat products. © 2024 Society of Chemical Industry.", "journal": "Journal of the science of food and agriculture", "year": "2025", "doi": "10.1002/jsfa.13841", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "C" ] }, "39745462": { "pmid": "39745462", "title": "Validation of a simplified HPV genotyping assay designed for cervical screening in low-resource settings.", "abstract": "Human papillomavirus (HPV) genotype predicts cervical cancer risk, and genotyping could help guide the management of HPV positives as part of cervical screening. An isothermal amplification HPV extended genotyping test (ScreenFire HPV RS assay) can assay up to 96 samples/controls in 1 hour plus preparation time. A novel format with pre-aliquoted reagents and an anti-contamination component (Zebra BioDome) could simplify the HPV testing process and reduce the chances of post-amplification contamination. We validated the Zebra BioDome formulation prior to its clinical use. Residual provider-collected cervical samples (n = 450) from a population-based study in rural Nigeria were retested with ScreenFire, once using the standard assay version (liquid reagents combined onsite) and twice with Zebra BioDome. HPV results with adequate DNA (N = 427) were analyzed channel-by-channel and using the cervical cancer risk-based hierarchy of HPV type channels (HPV16, else 18/45, else 31/33/35/52/58, else 39/51/56/59/68, else high-risk HPV negative) to evaluate Zebra BioDome repeatability and accuracy against the standard version. Zebra BioDome reduced the number of pipetting steps to run the ScreenFire HPV assay. Following amplification, the BioDome material formed a sealant layer above the reaction components. Zebra BioDome had excellent repeatability and agreement with the standard version, both at the channel-specific analysis (positive percent agreement between 88.4% [HPV39/51/56/59/68] and 100% [HPV16]; negative percent agreement between 97.8% [HPV31/33/35/52/58] and 100% [HPV39/51/56/59/68]) and hierarchical analysis (overall agreement 97.2%). The assay version utilizing Zebra BioDome performed similarly to the previously validated standard version of the ScreenFire HPV assay and is now undergoing field evaluation. This solution has the potential to reduce assay preparation time and risk of contamination, providing a simpler, low-cost, near-point-of-care HPV testing and extended genotyping solution for cervical screening in lower-resource settings. The potential application of Zebra BioDome technology to other PCR assays should be considered. This work validates a novel pre-packed formulation for the ScreenFire human papillomavirus (HPV) assay, which has the potential to simplify the HPV testing process and to reduce the chances of post-amplification contamination, providing a simpler, low-cost, near-point-of-care HPV testing, and extended genotyping solution for cervical screening in resource-limited settings as part of the ultimate public health goal to accelerate cervical cancer prevention. This technology can also have broad applications for other DNA amplification assays beyond HPV.", "journal": "Journal of clinical microbiology", "year": "2025", "doi": "10.1128/jcm.01639-24", "ptypes": [ "Journal Article", "Validation Study" ], "first_author": "", "dirs": [ "C" ] }, "42068385": { "pmid": "42068385", "title": "Development of a one-tube RPA/Cas12a platform for dual-mode detection of Vibrio alginolyticus.", "abstract": "Vibrio alginolyticus is a halophilic marine bacterium that threatens aquaculture and food safety. Rapid and reliable detection is vital for early intervention and outbreak prevention. Recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a offers a sensitive, specific, and isothermal alternative suitable for portable detection. We developed a one-tube RPA/Cas12a assay for rapid and sensitive V. alginolyticus detection, which integrates fluorescence and lateral flow strip (LFS) readouts for flexible visualization. An optimal primer pair (F1/R1) and crRNA2 targeting the toxR gene enabled efficient amplification and Cas12a-mediated trans-cleavage. All key components—RPA mixture, Cas12a, and crRNA—were essential for signal generation. Reaction optimization achieved a detection limit of 0.747 copies/µL for fluorescence and 100 copies/µL for LFS. Specificity assays confirmed the exclusive detection of V. alginolyticus without cross-reactivity to other Vibrio spp. or marine bacteria. The method validation was carried out using multiple types of samples, including artificially contaminated clinical samples, aquatic products, as well as clinical isolated of V. alginolyticus, demonstrating robustness. This dual-mode system simplifies operation, minimizes contamination, and enables field-deployable, real-time monitoring of V. alginolyticus in aquaculture environments, offering a practical, rapid, and instrument-free tool for pathogen surveillance and coastal biosecurity.", "journal": "World journal of microbiology & biotechnology", "year": "2026", "doi": "10.1007/s11274-026-04925-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42762644": { "pmid": "42762644", "title": "Universal contamination-free nucleic acid biosensing enabled by guide-independent Argonaute amplicon elimination.", "abstract": "Amplicon contamination remains a major challenge in nucleic acid biosensing, frequently compromising diagnostic accuracy in both clinical testing and field-deployable sensing systems. Although strategies such as uracil-DNA glycosylase (UDG) treatment and CRISPR-based approaches have been developed to mitigate this problem, their broader implementation is often limited by incomplete decontamination, increased assay cost, and the complexity associated with exogenous guide design. Here we introduce CREDIT (chopping-based Argonaute-mediated testing), a molecular diagnostic biosensor platform that harnesses the intrinsic chopping activity of Pyrococcus furiosus Argonaute (PfAgo). Unlike conventional Argonaute-based biosensing assays that primarily use Argonaute as a signal-generating enzyme, CREDIT operates as a dual-function system: it degrades amplification products into short fragments that are inert to further amplification, while simultaneously repurposing these chopping-derived fragments as guides to direct sequence-specific cleavage of reporter probes for signal generation. We demonstrate that this integrated detect-and-destroy mechanism enables seamless coupling of CREDIT with both PCR and isothermal amplification for ultrasensitive nucleic acid detection. The platform preserves the attomolar sensitivity of the amplification-based assay without compromising assay speed. Notably, a stringent 20-round consecutive open-lid stress test confirms complete suppression of amplicon contamination, thereby enabling uncapped post-amplification handling for lateral flow strip (LFS) sensor readout-an operation previously considered unsafe in field-deployable molecular testing. Together, these results establish CREDIT as a contamination-resistant biosensing strategy for molecular diagnostics that enables reliable and consecutive nucleic acid detection, particularly in resource-limited settings.", "journal": "Biosensors & bioelectronics", "year": "2027", "doi": "10.1016/j.bios.2026.119251", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40253476": { "pmid": "40253476", "title": "Advances in the recombinase polymerase amplification platform for urogenital schistosomiasis diagnosis (ShDraI-RPA) towards implementation at the point-of-care.", "abstract": "Accurate diagnosis of schistosomiasis is crucial to achieve disease elimination as a public health problem. Rapid and highly sensitive diagnostic tools that can be used in decentralized environments and/or at the point-of-care are needed. This work optimises and simplifies an existing isothermal molecular diagnostic platform (recombinase polymerase amplification, RPA) for urogenital schistosomiasis, the ShDraI-RPA, with a focus on delivering a more accurate diagnosis in endemic settings. The standard ShDraI-RPA oligonucleotides were modified, incorporating a phosphorothioate backbone into the reverse primer and inverting the probe fluorophore and quencher, to prevent false positive results due to secondary structure formation. The sensitivity and specificity of the modified assay were evaluated on a donor urine spiked with one S. haematobium egg and an array of other schistosomes and human urinary tract pathogens. The stability of RPA reagents was assessed by storing them at ambient temperature (± 27 °C) in a dark environment for up to 90 days. Sample preparations were explored to develop a simple, rapid and low resource methodology that would complement the ShDraI-RPA platform when used in remote settings. The modified ShDraI-RPA assay was robust, sensitive and specific to S. haematobium group species, detecting down to 10 fg of gDNA and ten synthetic Dra I copies. DNA amplification was achieved at 42 °C within 20 min and results could easily be visualized using a portable fluorometer or under blue light. RPA reagents remained stable when stored in the absence of light at ± 27 °C for up to 30 days. A two-step DNA extraction method proved optimal for extracting DNA from single S. haematobium eggs in spiked urine. The optimized ShDraI-RPA platform shows improved specificity and sensitivity and has now reached several of the target product profile requirements set out by the WHO for the ideal diagnostic test for schistosomiasis.", "journal": "Scientific reports", "year": "2025", "doi": "10.1038/s41598-025-95887-x", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "40382111": { "pmid": "40382111", "title": "Target inhibition of rolling circle amplification generates G43 long nanowires for reverse signaling determination of alkaline phosphatase.", "abstract": "Alkaline phosphatase (ALP) can catalyze the hydrolysis of phosphate esters under alkaline conditions, mediate the dephosphorylation of substances such as proteins and nucleic acids. ALP can participate in physiological activities such as bone mineralization, cell division, and immune regulation. Abnormal ALP activity is associated with various diseases, making it an important clinical biomarker. In recent years, highly sensitive detection technologies based on isothermal amplification have developed rapidly. The Rolling Circle Amplification (RCA) technology uses T4 DNA ligase to connect the 5'-phosphate group and 3'-hydroxyl terminus of the padlock probe into a ring, achieving efficient amplification through continuous rolling circle replication. Leveraging this characteristic, we developed a highly sensitive sensing method (RCA-LG) based on RCA-generated G43 long nanowires for ALP detection. T4 DNA ligase circularizes the RCT into a template. Subsequently, Phi29 DNA polymerase amplifies the primer along the circular template, producing G43 long nanowires that bind with Thioflavin T (ThT) to generate fluorescent signals. When ALP is present, it dephosphorylates the 5' end of RCT, preventing circular template formation and thereby inhibiting the amplification reaction. The RCA-LG method can detect ALP concentrations as low as 1.0 × 10-6 U/mL within 2.5 h. We propose a novel one-step RCA approach that integrates ligation and amplification reactions into a single step, simplifying operations while enhancing amplification efficiency. Furthermore, we physically separated the ALP dephosphorylation and RCA processes to avoid potential contamination from repeated sample handling. This strategy takes advantage of the characteristics of RCA without the need for redundant phosphorylation modifications. Moreover, we have proposed an improved one-step RCA method combined with physical partitioning, enabling highly sensitive and specific detection of ALP and allowing the detection of this target in complex samples. Furthermore, the RCA-LG strategy can also play an important role in the screening of enzyme inhibitors.", "journal": "Analytica chimica acta", "year": "2025", "doi": "10.1016/j.aca.2025.344077", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "41661240": { "pmid": "41661240", "title": "Push-Button Microfluidic Platform with Dual-Signal Nanoprobes for Enhanced Sensitivity Detection of Healthcare-Associated Infection Pathogens at Point-of-Care.", "abstract": "Healthcare-associated infection (HAI) pathogens cause severe nosocomial outbreaks, jeopardizing patient safety and straining healthcare systems. Conventional loop-mediated isothermal amplification-lateral flow immunoassay (LAMP-ICA) allows rapid pathogen detection but is constrained by low sensitivity, a high false-positive rate, and an extended detection time. To address these limitations, we present a dual-mode (colorimetric/fluorescent) microfluidic biosensing platform based on silicon-gold/quantum dot core-shell nanoprobes (Si@Au/DQD NPs). The platform incorporates two key innovations: (1) The colorimetric/fluorescent dual-signal Si@Au/DQD nanoprobe enhances detection reliability and sensitivity through dual-signal complementary verification and multilayered QD design, halving the LAMP amplification time compared to traditional colloidal gold systems, and (2) a modular microfluidic chip integrates LAMP amplification and ICA detection within a closed system, effectively preventing leakage and contamination of amplification products. Performance evaluation showed that the fluorescence detection limit of this system for Staphylococcus aureus (S. aureus), Legionella pneumophila (L. pneumophila), and Klebsiella pneumoniae (K. pneumoniae) reaches 82-140 CFU/mL, with the entire process completed within 30 min. In addition, the detection of 25 clinical environmental samples verifies the practicality of the designed integrated detection platform. With high sensitivity, strong specificity, and dual-mode capability for qualitative colorimetric screening and quantitative fluorescence analysis, this technology offers an efficient solution for point-of-care testing (POCT) of HAI pathogens, particularly in resource-limited settings and in on-site emergency diagnostics.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c05437", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42715083": { "pmid": "42715083", "title": "Harmonizing amplification and cleavage kinetics through crRNA scaffold mutation enables robust one-pot CRISPR-Cas12 diagnostics.", "abstract": "CRISPR-based diagnostics (CRISPR-Dx) integrated with isothermal nucleic acid amplification have emerged as a promising strategy for point-of-care molecular testing. Their practical deployment, however, remains constrained by workflow-related limitations. Conventional two-step formats are laborious and highly susceptible to aerosol contamination during amplicon transfer, whereas simplified one-pot formats often suffer from reduced sensitivity because amplification and CRISPR-mediated cleavage compete kinetically within the same reaction vessel. In this work, we present a broadly applicable one-pot detection strategy termed mutant scaffold-mediated RPA-CRISPR/Cas12a (MS-CRISPR). This approach introduces rationally designed point mutations into the crRNA scaffold to moderately attenuate Cas12a activation and cleavage kinetics. Such kinetic tuning prevents the enzyme from prematurely depleting nascent RPA amplicons, a common source of signal loss in wild-type systems. By delaying cleavage until sufficient amplification products have accumulated, MS-CRISPR markedly enhances endpoint signal output. Using this kinetically balanced system, we achieved a limit of detection of 1 copy/μL for both SARS-CoV-2 and influenza A virus within 15 min, while preserving a fully closed-tube workflow that minimizes aerosol contamination and simplifies operation. We coupled the assay with an instrument-free lateral-flow readout and an ultraportable, multiplex, low-cost microfluidic platform (portable wireless isothermal nucleic acid detection). Clinical validation with patient samples showed 100% sensitivity and specificity for both viral targets. Collectively, MS-CRISPR establishes a sensitive, versatile, and field-deployable platform for rapid pathogen screening.", "journal": "Proceedings of the National Academy of Sciences of the United States of America", "year": "2026", "doi": "10.1073/pnas.2613134123", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "42133534": { "pmid": "42133534", "title": "CRISPR-Cas12a2-Based Multiplexed Diagnostic for Rapid and Highly Sensitive Detection of Respiratory Viruses.", "abstract": "Infectious diseases severely threaten global public health security, necessitating rapid and highly sensitive diagnosis. This study presents a novel multiplex diagnostic platform combining transcription-mediated amplification (TMA) with the CRISPR-Cas12a2 system for rapid and highly sensitive detection of respiratory viruses. The assay uses an integrated microfluidic chip, which can simultaneously identify influenza A/B and respiratory syncytial viruses (RSV-A/B) with optimized CRISPR RNAs and isothermal amplification, achieving detection limits as low as 102 copies/μL within 60 min. The detection system showed excellent specificity; nonspecific reactions were not observed in the presence of nucleic acids from other respiratory pathogens. Clinical validation using nasopharyngeal swabs demonstrated high concordance with real-time quantitative reverse transcription polymerase chain reaction, with most positive samples detected within 40 min. The system eliminates DNA amplification steps, reduces contamination risk, and simplifies the workflow. Using two-step reactions on a centrifugal microfluidic chip, the TMA-CRISPR-Cas12a2 platform offers a promising integrated platform for multiplex respiratory pathogen screening, thereby supporting timely diagnosis and outbreak management.", "journal": "Analytical chemistry", "year": "2026", "doi": "10.1021/acs.analchem.5c07791", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42024625": { "pmid": "42024625", "title": "Multicolor Bioluminescent Toolbox for Multiplex Glow-in-the-Dark Nucleic Acid Diagnostics.", "abstract": "Combining bioluminescent sensors and isothermal amplification enables fast and simple nucleic acid diagnostics, holding great potential for point-of-care applications. We previously developed the LUNAS (luminescent nucleic acid sensor) platform, which integrates recombinase polymerase amplification (RPA) with a clustered regularly interspaced short palindromic repeats (CRISPR) dCas9-mediated bioluminescent readout. Here, we extend LUNAS into a multiplex platform through the development of green- and red-light emitting LUNAS variants. These color variants can be integrated with duplex RPA for amplification and orthogonal detection of two DNA targets in a single reaction, without compromising sensitivity or reaction speed. Using this approach, we developed a duplex assay for the sexually transmitted infections gonorrhea and chlamydia, with attomolar sensitivity and a robust ratiometric readout. Using a simple digital camera, multicolor LUNAS enabled detection of bacterial DNA from clinical samples with loads down to ∼100 copies/μL and within ∼30 min, demonstrating its diagnostic performance for low-resource applications.", "journal": "ACS sensors", "year": "2026", "doi": "10.1021/acssensors.5c05012", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "C" ] }, "41893914": { "pmid": "41893914", "title": "Current and emerging molecular diagnostic approaches in the detection of human parasites.", "abstract": "Microscopy and morphological identification remain the gold standard for diagnosing most parasitic infections, yet their limited sensitivity in asymptomatic or low-burden cases, along with technical constraints, has accelerated the adoption of molecular diagnostics. Over the past three decades, advances in nucleic acid amplification and sequencing technologies have transformed parasite detection by improving sensitivity, specificity, and reproducibility, enabling earlier intervention and stronger surveillance. PCR remains the foundation of molecular diagnostics, with real-time PCR and digital PCR improving analytical performance and quantification. Multiplex qPCR supports simultaneous detection of multiple pathogens, while dPCR enables absolute quantification and rare variant detection, although broader implementation is limited by instrument cost. Isothermal amplification methods such as tHDA, NASBA, LAMP, and RPA offer rapid, low-cost amplification at constant temperature and are well suited for field diagnostics in resource-limited settings. Next-Generation Sequencing has advanced genotyping and epidemiological surveillance by resolving cryptic species, resistance mutations, and mixed infections through targeted panels, whole-genome sequencing, and metagenomics. CRISPR/Cas-based assays provide rapid and sensitive nucleic acid detection with strong potential for point-of-care deployment due to their simplicity and adaptability. Emerging biomarkers, including circulating cell-free DNA, non-coding RNAs, and microRNAs in extracellular vesicles, offer promising non-invasive diagnostic strategies, though further validation is required. This review offers a concise overview of these molecular approaches, emphasizing recent innovations such as dPCR, NGS, CRISPR/Cas systems, and biomarker-based detection. For each method, core technical principles, representative applications, and comparative strengths and limitations are presented to illustrate their diagnostic potential.", "journal": "Parasitology research", "year": "2026", "doi": "10.1007/s00436-026-08660-y", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "42465601": { "pmid": "42465601", "title": "CRISPR-Cas systems for plant virus management: detection, surveillance, and host resistance.", "abstract": "The CRISPR-Cas system has transformed genome manipulation by enabling precise and programmable modification of genetic material. Initially developed as a genome-editing tool, CRISPR technologies have expanded from fundamental research to applied use across plant, animal, and microbial systems due to their simplicity, accuracy, and versatility. In agriculture, CRISPR-Cas9 has progressed from crop improvement to host-directed strategies conferring resistance against a broad range of plant viruses. Concurrently, the discovery of additional Cas effector proteins, particularly Cas12a and Cas13a, has enabled highly sensitive nucleic acid-based diagnostic platforms supporting rapid, field-deployable pathogen detection. Here, we present a focused synthesis integrating CRISPR-mediated host resistance engineering with CRISPR-based diagnostic surveillance within a unified framework for plant virus management. Unlike previous reviews that treat these domains independently, we emphasize their convergence in enabling early detection, real-time surveillance, and targeted intervention across the disease cycle. Cas12a-based systems, currently the most widely implemented, have been coupled with isothermal amplification and visual readouts for rapid virus detection, whereas Cas13a-based platforms offer direct RNA targeting with potential for simplified workflows, although they remain less developed. We examine key design considerations, performance characteristics, and limitations of these platforms, including challenges related to sensitivity, multiplexing, and field deployment. Finally, we highlight future directions, including vector-based detection, multiplex diagnostics, and integration of CRISPR technologies into scalable surveillance systems. Collectively, this review positions CRISPR-based genome editing and diagnostics as complementary components of a next-generation strategy for plant virus detection, surveillance, and management.", "journal": "Frontiers in plant science", "year": "2026", "doi": "10.3389/fpls.2026.1804262", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "42208627": { "pmid": "42208627", "title": "A review of past, present and emerging techniques for detection of Mycoplasma pneumoniae.", "abstract": "Mycoplasma pneumoniae is a significant pathogen causing respiratory infections globally, with diagnosis complicated by the organism's unique biology and nonspecific clinical presentation. Its distinct biology, including the absence of a cell wall and ambiguous clinical presentation, challenges early and accurate diagnosis, which is critical for guiding optimal antimicrobial therapy and limiting antibiotic resistance. This study summarizes the progression of diagnostic procedures from traditional culture and serology to modern molecular techniques such as real-time PCR, multiplex PCR panels, isothermal amplification methods like SAT, LAMP, and emerging techniques such as NGS and CRISPR. The paper underlines the advantages of molecular approaches over culture and serology in terms of sensitivity, specificity, and turnaround time, focusing on FDA/CE-cleared multiplex PCR systems and quick antigen detection kits with silver amplification as valuable clinical tools. Emerging CRISPR-based diagnostics offer a wonderful opportunity for quick, ultrasensitive, and point-of-care diagnosis. Despite the variety of available assays, issues such as detecting asymptomatic carriage, distinguishing active infection, and ensuring accessibility in low-resource settings persist. The increased global incidence of macrolide-resistant M. pneumoniae strains, as well as the overlap of respiratory symptoms with other diseases, highlight the need for specific, quick, and reliable diagnostic tests. This review advocates for the incorporation of innovative molecular diagnostics into standard clinical workflows to improve early identification, optimize treatment methods, and improve patient outcomes.", "journal": "Journal of microbiological methods", "year": "2026", "doi": "10.1016/j.mimet.2026.107557", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "40400168": { "pmid": "40400168", "title": "Thermus thermophilus Argonaute-Mediated Single Particle Counting Platform for Multiplex Cancer-Related Biomarkers Detection.", "abstract": "The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system has achieved remarkable success in the field of nucleic acid detection, while its Achilles' heel lies in the difficulties encountered in flexibility regarding the multiplex detection. As a sister system of CRISPR-Cas, prokaryotic Argonautes (pAgos) have precise recognition, multiturnover, and more importantly multiple specific cleavage characteristics, which is a potential candidate for the next generation of multiplex detection. Herein, a single particle counting platform was developed for the simultaneous detection of three colorectal cancer-related miRNAs (miR-141, miR-31, and miR-21) by combining single particle inductively coupled plasma mass spectrometry (SP-ICPMS) with the Thermus thermophilus Argonaute protein (TtAgo), with nanoparticles as signal probes for cleavage. The platform demonstrated high sensitivity (aM level) and specificity due to the dual-cycle mechanism of exponential isothermal amplification (EXPAR) and TtAgo cleavage, as well as the combination of TtAgo's specific cleavage capability and the multiplex detection advantages of metal stable isotope tagging. Additionally, the platform showed good robustness in human serum and cell extracts, indicating significant potential in clinical applications.", "journal": "Analytical chemistry", "year": "2025", "doi": "10.1021/acs.analchem.5c02118", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42744140": { "pmid": "42744140", "title": "Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.", "abstract": "Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.", "journal": "Journal of microbiological methods", "year": "2026", "doi": "10.1016/j.mimet.2026.107710", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "40347696": { "pmid": "40347696", "title": "Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods.", "abstract": "In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.", "journal": "Forensic science international. Genetics", "year": "2025", "doi": "10.1016/j.fsigen.2025.103297", "ptypes": [ "Journal Article", "Comparative Study", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "C" ] }, "42587639": { "pmid": "42587639", "title": "SARS-CoV-2 Point-of-Care Testing Modalities: Integrating Molecular, Immunological, Biosensor, and AI Approaches.", "abstract": "The coronavirus disease 2019 (COVID-19) pandemic highlighted the critical need for rapid, accessible, and accurate diagnostic tools to support timely clinical decision-making, outbreak control, and public health surveillance. Point-of-care testing (POCT) has emerged as an essential component of decentralized healthcare by enabling diagnostic testing outside conventional laboratory settings. This review was based on a structured literature search of PubMed, Scopus, and Web of Science databases covering studies published between January 2020 and January 2026. The review evaluates current advances in COVID-19 POCT technologies, including molecular assays, antigen-based tests, antibody-based assays, biosensor platforms, and artificial intelligence (AI)-assisted diagnostic approaches. Molecular POCT methods, including rapid reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and CRISPR-based technologies, provide high analytical sensitivity and specificity and are increasingly suitable for decentralized diagnostic applications. Antigen-based assays offer rapid and cost-effective screening solutions, although diagnostic performance may vary depending on viral load, symptom onset, and circulating variants. Antibody-based POCT remains valuable for seroprevalence studies, retrospective diagnosis, and immune-response monitoring rather than acute infection detection. Emerging biosensor technologies and AI-enabled diagnostic systems demonstrate promising analytical capabilities and operational advantages; however, many remain at the prototype or early-validation stage and require further clinical evaluation before widespread implementation. The findings indicate that no single POCT modality is optimal for all clinical scenarios. Instead, molecular, antigen, antibody, biosensor, and AI-assisted approaches provide complementary strengths that support different diagnostic and public health objectives. Continued advances in assay design, digital connectivity, multiplex testing, and variant-resilient detection strategies are expected to further enhance the role of POCT in COVID-19 management and future infectious disease preparedness.", "journal": "Diagnostics (Basel, Switzerland)", "year": "2026", "doi": "10.3390/diagnostics16152402", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "42883035": { "pmid": "42883035", "title": "Emerging and Novel Ovine Viruses: Molecular Diagnostics, Genomic and Metagenomic Surveillance, and One Health Perspectives.", "abstract": "Emerging and novel ovine viruses pose increasing threats to animal health, livestock productivity, trade, food security, and public health preparedness. Their emergence is driven by complex interactions among animal movement, mixed-species production systems, wildlife-livestock interfaces, arthropod vectors, environmental changes, and viral evolution. This review critically compares current strategies for identifying major, emerging, re-emerging, zoonotic, and newly recognized ovine viruses, with emphasis on their analytical sensitivity, turnaround time, throughput, operational cost, accessibility, field applicability, validation status, and capacity for novel-virus detection. Conventional diagnostic approaches, including virus isolation, serology, antigen detection, histopathology, and immunohistochemistry, remain essential for confirmation and flock-level surveillance but may be limited by slow turnaround, dependence on specialized facilities, reduced sensitivity at low viral loads, and an inability to identify highly divergent or unknown viruses. Targeted molecular assays, including PCR, RT-PCR, qPCR, multiplex assays, digital PCR, isothermal amplification, and CRISPR-based diagnostics, have improved detection speed and sensitivity but generally require prior knowledge of viral genomic targets. Genomic and metagenomic approaches, including whole-genome sequencing, next-generation sequencing, nanopore sequencing, viral metagenomics, bioinformatics, and phylogenetic analysis, provide broader detection capabilities by enabling characterization of viral diversity, outbreak tracing, co-infection identification, and discovery of previously unrecognized viruses. However, their interpretation remains challenging due to low viral abundance, poor sample quality, host nucleic acid background, contamination, incomplete reference databases, limited computational capacity, and the inability of sequence detection alone to confirm disease causality. Therefore, future ovine virus surveillance requires integration of molecular diagnostics with active, passive, outbreak-based, risk-based, vector, wildlife, and animal-movement surveillance within a One Health framework. Linking genomic information with ecological, epidemiological, and environmental data will be essential for transforming ovine virus surveillance from reactive diagnosis toward proactive preparedness. Advances in standardized sampling, validated field diagnostics, affordable sequencing, curated databases, bioinformatics capacity, and cross-sector data sharing will strengthen early recognition, risk assessment, and preparedness against emerging viral threats in sheep.", "journal": "Animals : an open access journal from MDPI", "year": "2026", "doi": "10.3390/ani16193066", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "42182021": { "pmid": "42182021", "title": "Recent advances in rapid multiplex detection of nucleic acid markers using RPA and CRISPR-Cas.", "abstract": "The integration of recombinase polymerase amplification (RPA) with CRISPR-Cas systems has emerged as a powerful platform for rapid multiplex nucleic acid detection. Compared with quantitative polymerase chain reaction (qPCR) and Next-generation sequencing (NGS), RPA-CRISPR operates isothermally (37 °C-42 °C), requires minimal equipment, and achieves attomolar sensitivity in 20-90 min via collateral cleavage. Recent multiplex strategies, namely two-tube, spatial separation one-tube, and homogeneous one-pot, they have overcome crosstalk and enabled highly multiplexed detection in complex food matrices such as poultry, milk, and lettuce. These approaches are particularly suited for foodborne pathogen screening (e.g., Salmonella, Listeria), antimicrobial resistance profiling, and on-site surveillance, aligning with the scope of research at the frontier of food microbiology diagnostics. Despite advances, challenges persist in standardization, matrix inhibition, and regulatory approval. This mini-review summarizes recent advances (2020-2025) in RPA-CRISPR multiplex detection, outlines future directions for clinical implementation and food safety deployment, and provides guidance for subsequent research on its practical applications in these fields.", "journal": "Frontiers in microbiology", "year": "2026", "doi": "10.3389/fmicb.2026.1810544", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "C" ] }, "41497662": { "pmid": "41497662", "title": "Fitness Landscape for Antibodies 2: Benchmarking Reveals That Protein AI Models Cannot Yet Consistently Predict Developability Properties.", "abstract": "A prominent application of machine learning in therapeutic antibody design is the development of models that can generate or screen antibody candidates with a high probability of success in manufacturing and clinical trials. These models must accurately represent sequence-structure-function relationships, also known as the fitness landscape. Previous protein function benchmarks examine fitness landscapes across diverse protein families, but they exclude antibody data. Here, we introduce the second iteration of the Fitness Landscape for Antibodies (FLAb2), the largest public therapeutic antibody design benchmark to date. The datasets collected in FLAb2 contain developability assay data for over 4M antibodies across 32 studies, encompassing seven properties of therapeutic antibodies: thermostability, expression, aggregation, binding affinity, pharmacokinetics, polyreactivity, and immunogenicity. Using the curated data, we evaluate the performance of 30 artificial intelligence (AI) and biophysical models in learning these properties. Protein AI models on average do not produce statistically significant correlations for most (80%) of developability datasets. No models correlate with all properties or across multiple datasets of similar properties. Zero-shot predictions from pretrained models are incapable of accurately predicting all developability properties, although several models (IgLM, ProGen2, Chai-1, ESM2, ISM, IgFold) produce statistically significant correlations for multiple datasets for thermostability, expression, binding, or immunogenicity. Fine-tuning with at least 102 points improves performance on thermostability, aggregation, and binding, but polyreactivity and pharmacokinetics lack enough data for significance. Yet it is humbling to observe that given enough developability data (103 points), a fine-tuned one-hot encoding model can match the performance of fine-tuned billion-parameter pretrained models. Training data composition influences performance more than model architecture, and intrinsic biophysical properties (thermostability) are more readily learned than extrinsic properties (immunogenicity, pharmacokinetics). Controlling for germline distance with partial correlation reveals that protein language models draw substantially on evolutionary signal; on average, germline edit distance accounts for 40% of their apparent predictive power. FLAb2 data are accessible at https://github.com/Graylab/FLAb, together with scripts that allow researchers to benchmark, compare, and iteratively improve new AI-based developability prediction models.", "journal": "bioRxiv : the preprint server for biology", "year": "2025", "doi": "10.64898/2025.12.27.696706", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "D" ] }, "40151525": { "pmid": "40151525", "title": "Computational analysis reveals temperature-induced stabilization of FAST-PETase.", "abstract": "More than 10 % of global solid waste consists of poly(ethyleneterephthalate) (PET). Among other techniques, PET hydrolases (PETases) can be used to depolymerize this plastic. However, wildtype PETases exhibit poor specific activities and insufficient thermostability, limiting their use in depolymerization processes which require high temperatures. In 2022, machine learning-aided enzyme engineering of a PETase stemming from the bacterium Ideonella sakaiensis (IsPETase) resulted in a more functional, active, stable, and tolerant variant (FAST-PETase). To rationalize the molecular basis of FAST-PETase's improved thermal stability, we performed comparative Constraint Network Analysis (CNAnalysis) and Molecular Dynamics (MD) simulations of wildtype IsPETase (WT-PETase) and FAST-PETase at 30°C and 50°C identifying thermolabile sequence stretches in the wildtype enzyme. Further analysis of the backbone flexibility revealed that all mutations of FAST-PETase affected these critical regions. Counterintuitively, the in-silico analyses additionally highlighted that the flexibility of these regions decreased at 50°C in FAST-PETase, instead of exhibiting increased flexibility at higher temperature as would be expected from thermodynamic considerations. This effect was confirmed by physical energy calculations, which suggest that temperature-dependent conformational changes of FAST-PETase decrease the free energy of unfolding (ΔG(stability)) and rigidify the enzyme at elevated temperatures enhancing stability. Looking forward, these findings might help guide the rational engineering of protein thermostability and contribute to our understanding of the thermal adaptation of thermophilic enzymes.", "journal": "Computational and structural biotechnology journal", "year": "2025", "doi": "10.1016/j.csbj.2025.03.006", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41261139": { "pmid": "41261139", "title": "Enhancing functional proteins through multimodal inverse folding with ABACUS-T.", "abstract": "Structure-based sequence redesign or inverse folding can significantly enhance structural stability but often compromises functional activity when performed using existing models. Here, we introduce ABACUS-T, a multimodal inverse folding model that improves precision and minimizes functional loss. ABACUS-T unifies several important features into one framework: detailed atomic sidechains and ligand interactions, a pre-trained protein language model, multiple backbone conformational states, and evolutionary information from multiple sequence alignment (MSA). Redesigned proteins show notable improvements: an allose binding protein achieves 17-fold higher affinity while retaining conformational change; redesigned endo-1,4-β-xylanase and TEM β-lactamase maintain or surpass wild-type activity; and OXA β-lactamase gains altered substrate selectivity. All achieve substantially increase thermostability (∆Tm ≥ 10 °C). In each test case, these enhancements are achieved by testing only a few sequences, each containing dozens of simultaneously mutated residues. ABACUS-T thus offers a promising tool for reengineering functional proteins in biotechnological applications.", "journal": "Nature communications", "year": "2025", "doi": "10.1038/s41467-025-65175-3", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42090297": { "pmid": "42090297", "title": "PSMa: Learning Protein Surface Representations with Physicochemical Masked Autoencoders.", "abstract": "Protein modeling has largely centered on sequence representations, leaving the geometry and physicochemical context of molecular surfaces underused. We introduce PSMa (Protein Surface Masked autoencoder), a self-supervised framework that operates directly on molecular-surface point clouds with geometric and physicochemical attributes, reconstructing masked surface patches to learn transferable representations at scale from ∼220,000 PDB structures. On held-out proteins, PSMa outperforms sequence-only and structure-only baselines on two downstream tasks─(i) protein-protein interface (PPI) binding-site identification and (ii) protein thermal-stability prediction─matching state-of-the-art AUROC and AUPRC while substantially improving threshold-dependent classification performance. The learned representations generalize across protein-length ranges and across experimental as well as predicted structures, with decision thresholds closer to the neutral prior; these findings show that surface-centric pretraining provides a strong, generalizable foundation for structure-aware protein machine learning.", "journal": "Journal of chemical information and modeling", "year": "2026", "doi": "10.1021/acs.jcim.6c00288", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41456696": { "pmid": "41456696", "title": "Geometric deep learning assists protein engineering. Opportunities and Challenges.", "abstract": "Protein engineering is experiencing a paradigmatic transformation through the integration of geometric deep learning (GDL) into computational design workflows. While traditional approaches such as rational design and directed evolution have achieved significant progress, they remain constrained by the vastness of sequence space and the cost of experimental validation. GDL overcomes these limitations by operating on non-Euclidean domains and by capturing the spatial, topological, and physicochemical features that govern protein function. This perspective provides a comprehensive and critical overview of GDL applications in stability prediction, functional annotation, molecular interaction modeling, and de novo protein design. It consolidates methodological principles, architectural diversity, and performance trends across representative studies, emphasizing how GDL enhances interpretability and generalization in protein science. Aimed at both computational method developers and experimental protein engineers, the review bridges algorithmic concepts with practical design considerations, offering guidance on data representation, model selection, and evaluation strategies. By integrating explainable artificial intelligence and structure-based validation within a unified conceptual framework, this work highlights how GDL can serve as a foundation for transparent, interpretable, and autonomous protein design. As GDL converges with generative modeling, molecular simulation, and high-throughput experimentation, it is poised to become a cornerstone technology for next-generation protein engineering and synthetic biology.", "journal": "Biotechnology advances", "year": "2026", "doi": "10.1016/j.biotechadv.2025.108790", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "42601461": { "pmid": "42601461", "title": "Aligning protein-generative models to experimental fitness with ProteinDPO.", "abstract": "Biological generative models can predict biological functions without task-specific training data but often under-perform specialized models. This is due to a fundamental 'alignment gap', where the rules learned during unsupervised training are not related to the function of interest. Here we demonstrate how to provide task-specific information without losing the general knowledge learned during pretraining by using direct preference optimization to align a structure-conditioned protein language model to preferentially generate stable protein sequences. Our aligned model, ProteinDPO, achieves stability prediction competitive to task-specific models and consistently outperforms unsupervised and fine-tuned versions of the model. Notably, ProteinDPO generalizes beyond its training data to enable stabilization and improved binding affinity prediction of large multichain protein complexes. When applied to stabilization of the hemagglutinin trimer, a primary component of influenza vaccines, ~80% of designs achieve increased or similar stability compared with the native hemagglutinin and up to 32 °C improvements from recently emerged mammalian strains. Our results demonstrate how to augment generative models with biophysical information and, more broadly, provide a general framework for the alignment of biological foundation models.", "journal": "Nature methods", "year": "2026", "doi": "10.1038/s41592-026-03137-3", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42239239": { "pmid": "42239239", "title": "Accurate protein stability prediction for small domains using mega-scale experiments.", "abstract": "Predicting absolute protein folding stability is a long-standing challenge in biophysics, with broad applications in protein design and in understanding genetic variation and evolution. Physics-based simulations have shown limited success at predicting stability and are often computationally intractable, and machine learning methods have been constrained by the lack of sufficiently large experimental datasets. We recently introduced cDNA display proteolysis, a cell-free approach that can measure folding stability for nearly one million protein domains in parallel. Here, we applied this method to measure stability for 1.8 million diverse protein domains 60-80 amino acids in length primarily taken from the MGnify metagenomic database and spanning over 200,000 sequence families. Using this new \"MGnify Stability dataset\", we developed the predictive models SaProtΔG and ESM3ΔG, which accurately predict absolute folding stability for small domains with root mean squared error of 0.8 kcal/mol over a 6 kcal/mol range (Spearman rank correlation of 0.88). These predictors show high accuracy at predicting effects of substitutions, insertions, and deletions, successfully identify global trends toward higher stability in thermophilic organisms, and improve discrimination of stable and unstable computationally designed proteins. Our results illustrate how megascale biophysical measurements can complement existing evolutionary and structural data to enable accurate absolute stability prediction for small domains.", "journal": "bioRxiv : the preprint server for biology", "year": "2026", "doi": "10.64898/2026.05.19.726285", "ptypes": [ "Journal Article", "Preprint" ], "first_author": "", "dirs": [ "D" ] }, "40575548": { "pmid": "40575548", "title": "Reliable prediction of protein-protein binding affinity changes upon mutations with Pythia-PPI.", "abstract": "Protein-protein interactions (PPIs) are essential for numerous biological functions and predicting binding affinity changes caused by mutations is crucial for understanding the impact of genetic variation and advancing protein engineering. Although machine-learning-based methods show promise in improving prediction accuracy, limited experimental data remain a significant bottleneck. In this study, we employed multitask learning and self-distillation to overcome the data limitation and improve the accuracy of protein-protein binding affinity prediction. By incorporating a mutation stability prediction task, our model achieved state-of-the-art accuracy on the SKEMPI dataset and was subsequently used to predict binding affinity changes for millions of mutations, generating an expanded dataset for self-distillation. Compared with prevalent methods, Pythia-PPI increased the Pearson's correlation between predictions and experimental data from 0.6447 to 0.7850 on the SKEMPI dataset and from 0.3654 to 0.6050 on the viral-receptor dataset. Experimental validation further confirmed its ability to identify high-affinity mutations on the CB6 antibody in complex with the severe acute respiratory syndrome coronavirus 2 prototype receptor binding domain, with the best single-point mutant among the top 10 predictions showing a 2-fold increase in binding affinity. These findings demonstrate that Pythia-PPI is a valuable tool for analysing the fitness landscape of PPIs. A web server for Pythia-PPI is available at https://pythiappi.wulab.xyz for easy access.", "journal": "National science review", "year": "2025", "doi": "10.1093/nsr/nwaf231", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39950717": { "pmid": "39950717", "title": "Cloning, Characterization, and Computer-Aided Evolution of a Thermostable Laccase of the DUF152 Family From Klebsiella michiganensis.", "abstract": "Bacterial laccases exhibit relatively high optimal reaction temperatures and possess a broad substrate spectrum, thereby expanding the range of potential applications for laccase enzymes. This study aims to investigate the molecular evolution of bacterial laccases using computational 3D-structure prediction and molecular docking tools such as AlphaFold2, Metal3D, AutoDockVina, and Rosetta. We isolated a bacterium with laccase activities from fecal samples from a Hermann's tortoise (Testudo hermanni), identified it as Klebsiella michiganensis using 16S rRNA sequencing and nanopore genome sequencing, and then identified a sequence encoding a laccase with a predicted molecular weight of approximately 27.5 kDa. Expression of the corresponding, chemically synthesized DNA fragment resulted in the isolation of an active laccase. The enzyme showed considerable thermostability, retaining 21% of its activity after boiling for 30 min. Using state-of-the-art information technology and machine learning techniques, we conducted 3D-structure prediction on this sequence, predicted its copper-ion binding sites, and validated these predictions through site-directed mutagenesis and expression. Subsequently, we performed computer-aided evolution studies on this sequence and found that 90% of the results from the selected mutations exhibited improved performance. In summary, this study not only revealed a novel laccase but also demonstrated an efficient approach for advancing research on the molecular evolution of bacterial laccases using cutting-edge machine learning, next-generation sequencing, traditional bioinformatics approaches, and laboratory techniques, providing an effective strategy for discovering and design new bacterial laccases.", "journal": "Proteins", "year": "2025", "doi": "10.1002/prot.26784", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "41675712": { "pmid": "41675712", "title": "Hyena architecture enables fast and efficient protein language modeling.", "abstract": "The emergence of self-supervised deep language models has revolutionized natural language processing tasks and has recently extended its applications to biological sequence analysis. Traditional language models, primarily based on Transformer architectures, demonstrate substantial effectiveness in various applications. However, these models are inherently constrained by the attention mechanism's quadratic computational complexity, O ( L 2 ) , which limits their efficiency and leads to high computational costs. To address these limitations, we introduce ProtHyena, a novel approach that leverages the Hyena operator in protein language modeling. This innovative methodology alternates between subquadratic long convolutions and element-wise gating operations, which circumvents the constraints imposed by attention mechanisms and reduces computational complexity to subquadratic levels. This enables faster and more memory-efficient modeling of protein sequences. ProtHyena can achieve state-of-the-art results and comparable performance in 8 downstream tasks, including protein engineering (protein fluorescence and stability prediction), protein property prediction (neuropeptide cleavage, signal peptide, solubility, disorder, gene function prediction), protein structure prediction, with only 1.6 M parameters. The architecture of ProtHyena represents a highly efficient solution for protein language modeling, offering a promising avenue for fast and efficient analysis of protein sequences.", "journal": "iMetaOmics", "year": "2025", "doi": "10.1002/imo2.45", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42468708": { "pmid": "42468708", "title": "Artificial intelligence-guided engineering of a thermostable cellulase cocktail enables efficient biomass hydrolysis at elevated temperatures.", "abstract": "Efficient lignocellulosic biomass conversion under industrially relevant high temperatures is limited by the thermolability of commercial cellulases and a lack of synergistic, system-level thermostable cocktails. To address this gap, a thermostable cellulase cocktail comprising the endoglucanase TpEG, the cellobiohydrolase HmCel6A, and the β-glucosidase TnBglB was established, capable of synergistically hydrolyzing cellulose under high-temperature conditions. To further improve system performance, precise component optimization was performed using an advanced artificial intelligence framework. First, using the protein language model-guided enzyme mining pipeline VenusMine, EG5, an ultra-thermostable endoglucanase, was identified from a large sequence space and exhibited excellent thermostability at 90 °C. Concurrently, protein engineering of HmCel6A guided by the protein language model PRIME generated the variant S347P, yielding a 1.8-fold increase in catalytic activity. Both the wild-type (WT) and engineered cocktails showed excellent high-temperature hydrolytic activity. At 90 °C, DNS assay showed that the filter paper hydrolytic activity of the WT cocktail was approximately sevenfold higher than that of Cellic® CTec3 (Novonesis), while the engineered cocktail exhibited approximately 1.15-fold higher activity than the WT cocktail. HPLC analysis showed that the engineered cocktail released significantly more glucose from corn stover than both CTec3 and the WT cocktail. Furthermore, supplementation of CTec3 with these engineered enzymes led to increased reducing sugar release under the tested supplementation conditions. This study demonstrates the power of integrating artificial intelligence-driven enzyme discovery with protein engineering, expanding the design space for thermostable enzymes while delivering a high-performing cellulase cocktail for industrial-scale, high-temperature biomass saccharification.", "journal": "Bioresource technology", "year": "2026", "doi": "10.1016/j.biortech.2026.135430", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40277391": { "pmid": "40277391", "title": "Mass balance approximation of unfolding boosts potential-based protein stability predictions.", "abstract": "Predicting protein stability changes upon single-point mutations is crucial in computational biology, with applications in drug design, enzyme engineering, and understanding disease mechanisms. While deep-learning approaches have emerged, many remain inaccessible for routine use. In contrast, potential-like methods, including deep-learning-based ones, are faster, user-friendly, and effective in estimating stability changes. However, most of them approximate Gibbs free-energy differences without accounting for the free-energy changes of the unfolded state, violating mass balance and potentially reducing accuracy. Here, we show that incorporating mass balance as a first approximation of the unfolded state significantly improves potential-like methods. While many machine-learning models implicitly or explicitly use mass balance, our findings suggest that a more accurate unfolded-state representation could further enhance stability change predictions.", "journal": "Protein science : a publication of the Protein Society", "year": "2025", "doi": "10.1002/pro.70134", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "39786559": { "pmid": "39786559", "title": "Exploring Evolution to Uncover Insights Into Protein Mutational Stability.", "abstract": "Determining the impact of mutations on the thermodynamic stability of proteins is essential for a wide range of applications such as rational protein design and genetic variant interpretation. Since protein stability is a major driver of evolution, evolutionary data are often used to guide stability predictions. Many state-of-the-art stability predictors extract evolutionary information from multiple sequence alignments of proteins homologous to a query protein, and leverage it to predict the effects of mutations on protein stability. To evaluate the power and the limitations of such methods, we used the massive amount of stability data recently obtained by deep mutational scanning to study how best to construct multiple sequence alignments and optimally extract evolutionary information from them. We tested different evolutionary models and found that, unexpectedly, independent-site models achieve similar accuracy to more complex epistatic models. A detailed analysis of the latter models suggests that their inference often results in noisy couplings, which do not appear to add predictive power over the independent-site contribution, at least in the context of stability prediction. Interestingly, by combining any of the evolutionary features with a simple structural feature, the relative solvent accessibility of the mutated residue, we achieved similar prediction accuracy to supervised, machine learning-based, protein stability change predictors. Our results provide new insights into the relationship between protein evolution and stability, and show how evolutionary information can be exploited to improve the performance of mutational stability prediction.", "journal": "Molecular biology and evolution", "year": "2025", "doi": "10.1093/molbev/msae267", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40147097": { "pmid": "40147097", "title": "Alcalase immobilization in iota-carrageenan-matrix hydrogel beads derived from the macroalga Solieria filiformis.", "abstract": "This study aims to immobilize Bacillus licheniformis (Alcalase) protease in iota-carrageenan (ιCAR) matrix hydrogels via adsorption. CAR was extracted from macroalgae Solieria filiformis and used to produce hydrogels using Al3 + as the gelling agent. Subsequently, enzyme immobilization was performed at 25ºC, for 120 min using particles of ∼2.0 mm diameter, varying the medium pH values (7.0, 8.0, and 9.0). The immobilization at pH 8.0 resulted in the biocatalyst with the highest immobilization yield (100 %), expressed activity (88.9 %), and mass activity (10.4 U/g) for 1.0 mg/g of enzyme loading. When using particles with different diameters (1.0, 2.0, and 3.0 mm), the best results were obtained using 1.0 mm particles. This permitted a 100 % immobilization yield, 95.8 % expressed activity, and high mass activity (11.2 U/g). The lyophilized biocatalyst presented varying macro-pore diameters, ranging from 21 to 126 µm. The immobilized biocatalyst was 11 times more stable than the soluble enzyme at 60ºC and pH 8.0 and presented > 80 % retained activity in the pH range 6.0-9.0.", "journal": "Enzyme and microbial technology", "year": "2025", "doi": "10.1016/j.enzmictec.2025.110636", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39809391": { "pmid": "39809391", "title": "Immobilization and characterization of β-galactosidase from Aspergillus oryzae in PVA-CMC hydrogel.", "abstract": "Creating new formulations of immobilized enzymes has been a major focus of modern biotechnology. In this study, the industrially significant β-galactosidase was immobilized by being trapped in a polyvinyl alcohol and carboxymethyl cellulose (PVA-CMC) gel. The immobilized enzyme was optimized and characterized, and the results were compared with those obtained using free enzymes. The data show that 40 °C to 50 °C is the ideal temperature range for the enzyme after immobilization. The activity rose, the Vmax value increased from 1.94 U/mg to 6.01 U/mg, and the Km value fell from 4.86 mM to 3.35 mM at pH 5, the optimal pH. β-galactosidases immobilized on PVA-CMC gels exhibited 70 % activity at the end of the fifth week and 50 % activity at the end of the eighth week, depending on the storage stability of the immobilized enzyme. After three reuses, the initial activity of the enzymes decreased, yet the thermal stability of the immobilized enzyme remained superior to that of the free form, retaining 82 % of its initial activity. Thus, it might be claimed that immobilization amplifies the enzyme's catalytic impact. Consequently, it has been discovered that immobilized β-galactosidase exhibits stronger enzymatic characteristics than free β-galactosidase, making it potentially more useful in industrial operations.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.139816", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39978516": { "pmid": "39978516", "title": "Enhancing lipase enzymatic performance with dynamic covalent dextran-based hydrogels.", "abstract": "How to maintain high catalytic activity and stability in the process of biocatalysis is crucial, inspiring strategies to construct an appropriate immobilized hydrogel system. Inspired by biologically relevant interactions of natural polymers, we crafted a polysaccharide-based dynamic hydrogel through imine bonds among lipase, oxidized dextrans (ODex) and carboxymethylchitosan. The successful preparation of ODex and the in situ immobilization of lipase through a Schiff base reaction were verified by FT-IR, 1H NMR, XRD, and the hydroxylamine hydrochloride method. The resultant gel endows the lipase with improved storage stability, thermal stability, reusability, and a higher degree of triacylglycerol hydrolysis. Furthermore, the immobilized lipase in the gel exhibits superior activity under harsh conditions, including high temperatures, strong bases, and exposure to organic solvents. The polysaccharide-based dynamic hydrogel represents a promising platform for enzyme immobilization, offering versatile applications in industrial biocatalysis.", "journal": "International journal of biological macromolecules", "year": "2025", "doi": "10.1016/j.ijbiomac.2025.141254", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41002485": { "pmid": "41002485", "title": "Size-Controlled Fabrication of Alginate Hydrogel Microbeads Optimized for Lipase Entrapment.", "abstract": "Enzyme entrapment in alginate hydrogel microbeads is an effective method of immobilization for industrial applications, but many fabrication methods for alginate microbeads involve oil, organic solvents, or high temperatures that reduce enzymatic activity. In this study, we employed an oil- and solvent-free gas-shearing technique to prepare alginate microbeads for the entrapment of Candida rugosa lipase (CRL), thereby minimizing thermal- and solvent-induced inactivation. To enhance immobilization efficiency and reusability, the effects of gas flow rate, alginate concentration, and cross-linking metal ions were systematically investigated. CRL entrapped in Ba- and Fe-alginate microbeads showed superior immobilization yield, activity retention, and activity recovery compared with CRL entrapped in conventional Ca-alginate microbeads. Notably, both Ba- and Fe-alginate microbeads exhibited significantly enhanced stability, with half-lives up to 127-fold greater than that of free CRL at 60 °C, and maintained substantially higher pH stability across the tested range. Ba-alginate microbeads provided greater pH stability and substrate affinity, whereas Fe-alginate microbeads demonstrated enhanced thermal stability and catalytic turnover. These findings highlight gas-shearing as a scalable and gentle fabrication method for producing high-performance alginate microbeads with tunable properties, making them suitable for enzyme entrapment in diverse biocatalytic applications.", "journal": "Gels (Basel, Switzerland)", "year": "2025", "doi": "10.3390/gels11090710", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42351900": { "pmid": "42351900", "title": "pH-Responsive Inulin-Sodium Alginate Hydrogel Beads for Sustained Oral Delivery of Bacterial Serine Protease.", "abstract": "To overcome the limitations of conventional therapies, targeted delivery of therapeutic interventions is crucial. Specifically, novel hydrogels maximize efficacy while minimizing the premature degradation of therapeutic enzymes. Therefore, the present study aimed to evaluate a developed pH-responsive inulin-sodium alginate (Na+ alginate) hydrogel bead system for colon-specific release of serine protease. Scanning electron microscope (SEM) images revealed structural differences between blank and encapsulated hydrogel beads. Fourier transform infrared spectroscopy (FTIR) spectra further confirmed the successful encapsulation of the bacterial serine protease in inulin-Na+ alginate (IN-Na+ Alg-SP). Thermogravimetric analysis (TGA) confirmed the thermal stability of hydrogel beads over a wide temperature range. Fabricated IN-Na+ Alg hydrogel beads displayed an entrapment efficiency of 54 ± 0.99% with an apparent activity of 260 U/mL. In vitro studies confirmed pH-responsive release with minimal release at pH 1.2 and sustained release at pH 7.4 over 4 h and 30 min. The ex vivo intestinal study confirms that the developed hydrogel has excellent potential as an oral colon-targeted drug delivery system for therapeutic enzymes. The release data best fit the second-order and Korsmeyer-Peppas models (R2 > 0.96), indicating a combination of diffusion and erosion-controlled release mechanisms. These findings suggest that inulin-Na+ alginate hydrogels provide a promising carrier system for colon-specific delivery of serine protease with potential applications in targeted protein digestion and therapeutic interventions.", "journal": "Bioengineering (Basel, Switzerland)", "year": "2026", "doi": "10.3390/bioengineering13060655", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40763597": { "pmid": "40763597", "title": "Efficient immobilization of thermophilic rhamnosidase dual-network hybrid-gel with high enzymatic activity and stability.", "abstract": "Thermophilic enzymes possess numerous favorable properties for biotransformation, including high catalytic activity, exceptional thermal stability, and ease of purification and preparation. However, the challenge arises from the lack of suitable enzyme immobilization materials that exhibit both thermal tolerance and reusability, thereby hindering the effective and recurrent utilization of thermophilic enzymes. In this study, a specially designed dual-network hybrid-gel consisting of acrylamide (AM) and sodium alginate (SA) was developed to serve as a thermotolerant and reusable carrier for the efficient immobilization of a thermotolerant α-rhamnosidase IagRha. The optimal amounts of AM and SA were determined to be 5 % and 2 %, respectively. Following the step of acrylamide crosslinking to establish the skeleton of hybrid-gel, a subsequent immersion in a Ca2+ solution facilitated the formation of ionic bonds between SA and Ca2+, thereby adjusting the internal voids of the hydrogel, which effectively enhanced the thermal stability and enzyme immobilization efficiency of the hybrid-gel. This innovative approach resulted in the achievement of 93.65 % recovery of enzyme activity, along with remarkable enhancements in enzymatic properties. The immobilized α-rhamnosidase, AM-SA-IagRha, demonstrated a significant 1.34-fold increase in the Vmax value, rising from 5.28 U/mg to 7.06 U/mg, compared to free IagRha. Furthermore, AM-SA-IagRha exhibited excellent thermostability, with a half-life of 99.0 min at 90 °C, which was 1.57-fold higher than that of free IagRha. After four cycles of reuse, the productivities of rutin and epmdin C by the immobilized enzyme were 1.91-fold and 2.53-fold that of the free enzyme, respectively. Additionally, the enzyme activity of AM-SA-IagRha remained at 90.16 % after storage at 4 °C for 30 days, and retained at 85.63 % after 20 cycles of reuse, demonstrating exceptional storage stability and reusability. The optimal expression conditions for IagRha were identified in TB medium incubated at 37 °C without IPTG, resulting in an enzyme activity of 6.32 U/mL. These significant findings underscore the potential of AM-SA-IagRha for industrial applications, particularly in high-temperature biotransformation.", "journal": "Bioorganic chemistry", "year": "2025", "doi": "10.1016/j.bioorg.2025.108823", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "39003085": { "pmid": "39003085", "title": "Immobilization of laccase on magnetic PEGDA-CS inverse opal hydrogel for enhancement of bisphenol A degradation in aqueous solution.", "abstract": "Endocrine disruptors such as bisphenol A (BPA) adversely affect the environment and human health. Laccases are used for the efficient biodegradation of various persistent organic pollutants in an environmentally safe manner. However, the direct application of free laccases is generally hindered by short enzyme lifetimes, non-reusability, and the high cost of a single use. In this study, laccases were immobilized on a novel magnetic three-dimensional poly(ethylene glycol) diacrylate (PEGDA)-chitosan (CS) inverse opal hydrogel (LAC@MPEGDA@CS@IOH). The immobilized laccase showed significant improvement in the BPA degradation performance and superior storage stability compared with the free laccase. 91.1% of 100 mg/L BPA was removed by the LAC@MPEGDA@CS@IOH in 3 hr, whereas only 50.6% of BPA was removed by the same amount of the free laccase. Compared with the laccase, the outstanding BPA degradation efficiency of the LAC@MPEGDA@CS@IOH was maintained over a wider range of pH values and temperatures. Moreover, its relative activity of was maintained at 70.4% after 10 cycles, and the system performed well in actual water matrices. This efficient method for preparing immobilized laccases is simple and green, and it can be used to further develop ecofriendly biocatalysts to remove organic pollutants from wastewater.", "journal": "Journal of environmental sciences (China)", "year": "2025", "doi": "10.1016/j.jes.2023.10.017", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42081037": { "pmid": "42081037", "title": "Enhancing Enzymatic Activity and Stability of Crude Laccase Through Encapsulation in Gelatin/Chitosan Hydrogel.", "abstract": "Laccase is an environmentally friendly catalyst characterized by a wide substrate spectrum and clean reaction processes. It demonstrates the ability to efficiently oxidize various pollutants, with water being the sole by-product. Consequently, laccase holds significant potential for applications in diverse fields such as papermaking, environmental protection, food processing, and bioenergy production. In this study, crude laccase produced by Schizophyllum commune was immobilized within a composite material through chemical cross-linking to enhance the enzyme's activity and stability. Free laccase was immobilized using a gelatin/chitosan (GEL/CS) hydrogel, and the conditions for this process were optimized. Enzymatic properties of immobilized laccase (GEL/CS-laccase) were investigated to assess its potential applications. Results indicated that the maximum laccase activity by Schizophyllum commune reached 343.9 U·mL- 1 on day 6. Following optimization, GEL/CS-laccase achieved an activity recovery of 33.9% and an activity of 113.8 U·g- 1. Compared with free laccase, GEL/CS-laccase shows better adaptability to pH and temperature. Furthermore, GEL/CS-laccase demonstrates moderate reusability and excellent long-term storage stability: its activity remains above 34.7% after 5 cycles and exceeds 52.9% following 180 days of storage at -80 °C and vacuum. This study has developed an efficient enzyme immobilization method, enhancing its stability and operability, and laying a solid foundation for the practical application of laccase.", "journal": "Applied biochemistry and biotechnology", "year": "2026", "doi": "10.1007/s12010-026-05715-9", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42571606": { "pmid": "42571606", "title": "Gas-Shearing Microfluidic Fabrication of Alginate/Chitosan Hydrogel Microreactors for Genipin-Mediated Dual-Mode Lipase Immobilization.", "abstract": "Enzyme immobilization is an effective strategy to improve catalyst recovery and operational stability, but conventional approaches are often limited by diffusion resistance, steric hindrance, and insufficient control over the enzyme microenvironment. Hydrogel-based materials are attractive supports because their hydrated, tunable, and porous networks help preserve enzyme conformation while improving substrate diffusion and mass transfer. Here, a gas-shearing microfluidic strategy was developed to fabricate alginate/chitosan hydrogel microreactors for dual-mode lipase immobilization through entrapment and genipin-mediated covalent binding. By tuning gas-liquid shear, microspheres with controllable and narrow size distribution were generated without surfactants or UV-initiated polymerization. Importantly, genipin was introduced as a mild and biocompatible cross-linker to reinforce the alginate/chitosan network and create a stable interfacial microenvironment for enzyme immobilization. Compared with conventional cross-linkers, the milder and more controllable reactivity of genipin is advantageous in preserving enzyme conformation and catalytic performance. The resulting microreactors exhibited enhanced catalytic activity, improved storage stability, and good reusability. After five consecutive cycles, the entrapped and covalently immobilized systems retained 70% and 81% of their initial activity, respectively. These results demonstrate that gas-shearing microfluidics combined with genipin-mediated network reinforcement provides a robust and biocompatible platform for constructing hydrogel-based enzymatic microreactors with improved catalytic performance and operational durability.", "journal": "Macromolecular bioscience", "year": "2026", "doi": "10.1002/mabi.70238", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40209360": { "pmid": "40209360", "title": "Sodium alginate hydrogel beads loaded with clove essential oil liposomes: Preparation, characterization and fumigant activity.", "abstract": "This study successfully developed a stable and effective delivery system for clove essential oil (CEO) using combination of liposomes and hydrogel beads via thin-film hydration method. Chitosan (CH) was incorporated for protection, and β-cyclodextrin (β-CD) was used in combination with sodium alginate and calcium ions to form the hydrogel beads. The liposome encapsulation efficiency of CEO was 87.26 % and loading efficiency was 27.08 %, while the hydrogel beads (HB) were 71.78 % and 14.35 %, respectively. The incorporation of CH and β-CD into sodium alginate hydrogel beads improved the thermal stability of CEO. The HB displayed excellent slow-release properties, retaining 22.47 % of the essential oil after 30 days of storage. The hydrogel beads facilitated a gradual release of CEO over 24 h, minimizing direct cellular exposure and mitigating toxicity. CEO exhibited fumigant toxicity towards Sitophilus oryzae adults, and the slow-release HB maintained a lethality rate of 11.11 % to S. oryzae adults after 30 days, which inhibited the activities of detoxification enzymes, acetylcholinesterase, and antioxidant enzymes. This delivery system provides a foundation for developing eco-friendly and effective pest control solutions using essential oils encapsulated in liposomal hydrogel beads.", "journal": "Food chemistry", "year": "2025", "doi": "10.1016/j.foodchem.2025.144240", "ptypes": [ "Journal Article", "Evaluation Study" ], "first_author": "", "dirs": [ "D" ] }, "41905692": { "pmid": "41905692", "title": "Improved thermostability and catalytic performance of bacterial phytase via chitosan hydrogel immobilization.", "abstract": "Phytase enzymes are essential for enhancing phosphorus bioavailability and mitigating the impacts of phytic acid in agriculture. In this study, a phytase-producing bacterium was isolated from rhizospheric soil and poultry waste and identified via 16S rRNA sequencing as Bacillus pumilus (PersiPhytase1). The purified enzyme, classified as an alkaline phytase, exhibited a molecular weight of ∼39 kDa and was characterized by high thermal and proteolytic stability. The enzyme was biochemically characterized, followed by immobilization within a chitosan-based hydrogel to enhance its stability. Post-immobilization analyses revealed improvements in operational and thermal stability. The immobilized phytase demonstrated broad pH activity across pH 5.0-9.0, with maximum activity at pH 7.0 and retained over 90% activity at pH 9.0. Temperature-profile analysis showed optimal activity at 50 °C, maintaining over 85% activity even at 80 °C. Kinetic parameters indicated enhanced catalytic efficiency: Km decreased from 11.41 to 5.14 mM, and Vmax increased from 0.054 to 0.063 mM·min-1 upon immobilization. Furthermore, catalytic turnover (kcat) and efficiency (kcat/Km) increased from 0.108 to 0.127 min-1 and from 0.00954 to 0.024 mM-1·min-1, respectively. The immobilized enzyme retained 39.52% activity after 10 reuse cycles, with minimal leaching; even at 80 °C, enzyme release was 24.67%. Thermodynamic studies confirmed improved half-life and conformational stability, with a ΔG of 91.9 kJ/mol and ΔS of -0.157 kJ/mol·K at 70 °C. These findings indicate that chitosan-based hydrogel immobilization improves phytase stability and catalytic performance under laboratory conditions, highlighting its potential for feed-related applications pending further validation under processing and matrix conditions.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.151696", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41837769": { "pmid": "41837769", "title": "Synergistic Enhancement of Laccase-Cellulase via Co-Immobilization in a CMS/CPAM/PDA Hydrogel for Bamboo Pulp Fiber Flexibility.", "abstract": "Multienzyme complexes offer synergistic effects beyond individual enzymes, but their inherent instability and low activity severely limit the practical applications. Here, a sodium carboxymethyl starch/polyacrylamide/polydopamine (CMS/CPAM/PDA) hydrogel was developed to coimmobilize cellulase and laccase via Schiff-base formation between amino groups present on the enzymes and carbonyl groups of polydopamine. Immobilized enzyme complexes exhibit higher relative activity across pH values of 3-9, improved storage stability, and enhanced thermal tolerance. Furthermore, the Cu2+-activated immobilized enzyme complex for bamboo fiber treatment demonstrated a 7.54-fold improvement in flexibility, attributed to the synergistic laccase-mediated lignin degradation, which enhances cellulase accessibility to the fiber surface. The application of these flexibility-enhanced bamboo pulp fibers in tissue paper manufacturing yielded a 65.31% increase in softness. The enhanced flexibility of bamboo fibers achieved through complex enzyme immobilization within a CMS/CPAM/PDA hydrogel represents a key development for the utility of bamboo in tissue products.", "journal": "Journal of agricultural and food chemistry", "year": "2026", "doi": "10.1021/acs.jafc.5c15145", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42336021": { "pmid": "42336021", "title": "Amine-reactive hydrogels via nucleophilic thiol-yne reaction: A functional scaffold for laccase immobilization and dye degradation.", "abstract": "In this study, we synthesized a novel six-armed propiolic ester as a crosslinker to prepare PEG-based amine-reactive hydrogels via the thiol-yne 'click' reaction. By adjusting the molar ratios of components, three hydrogels with different compositions were obtained. All those hydrogels contained vinyl sulfide groups at junction points capable of nucleophilic addition, two of the formulations additionally retained residual propiolate functionality for post-modification through the amino-yne click reaction. Characterization of fabricated hydrogels was done by FTIR, TGA, swelling studies, and SEM. Covalent immobilization of Trametes versicolor laccase was achieved through reactions between amine groups of the enzyme and the reactive functionalities of the hydrogels. We observed that similar amounts of enzyme were immobilized on all hydrogel formulations, however there were significant differences in dye decolorization efficiency. The laccase-immobilized hydrogel with the highest density of accessible propiolate groups exhibited the greatest dye decolorization efficiency (89.71%), whereas the formulation lacking accessible propiolate groups showed only 25.59% decolorization. After subtracting the adsorption contribution of the bare hydrogels, the net enzymatic degradation efficiency increased from 17.03% in the hydrogel lacking accessible propiolate groups to 83.18% in the hydrogel containing the highest density of accessible propiolate functionalities. This difference was attributed to the increased accessibility of the active site of the enzyme and enhanced substrate diffusion due to lower crosslinking density. The laccase-hydrogel conjugate, showing the highest enzymatic activity displayed also good storage stability and reusability. These results demonstrate the potential of propiolic ester-based hydrogels as versatile platforms for enzyme immobilization and biocatalytic applications.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.153183", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42191289": { "pmid": "42191289", "title": "ZIF-8 functionalized alginate-chitosan hybrid microspheres as a versatile platform for α-glucosidase immobilization and high-throughput inhibitor screening.", "abstract": "Efficient immobilization of α-glucosidase (α-Glu) is crucial for its application in inhibitor screening, but conventional polysaccharide hydrogels suffer from limited mechanical strength and stability. We hypothesized that a hybrid hydrogel synergistically integrating covalent polymer cross-linking with metal-organic framework (MOF) nanoparticles would overcome these limitations. ZIF-8 reinforced alginate-chitosan (SC/ZIF-8) microspheres were fabricated via NHS/EDC-mediated covalent cross-linking and Ca2+-induced gelation. ZIF-8 incorporation dramatically enhanced mechanical strength (complex modulus increased >5-fold) and modulated swelling. The immobilized α-Glu@SC/ZIF-8 preserved intrinsic enzyme activity, exhibited superior pH stability (retaining ∼90% activity at pH 9.0), and demonstrated exceptional reusability (nearly 100% activity after 13 cycles) and storage stability (>95% after 28 days). Coupled with HPLC-MS/MS, the platform successfully identified batatasin I and 2,4-dimethoxy-6,7-dihydroxyphenanthrene as α-glucosidase inhibitors from Dioscorea opposita Thunb. Peel extracts. This work presents a robust, biocompatible, and reusable biocatalytic platform that addresses key limitations of existing immobilization systems. The SC/ZIF-8 hybrid strategy offers a versatile foundation for enzyme-based screening in natural product drug discovery.", "journal": "Analytica chimica acta", "year": "2026", "doi": "10.1016/j.aca.2026.345607", "ptypes": [ "Journal Article", "Research Support, Non-U.S. Gov't" ], "first_author": "", "dirs": [ "D" ] }, "40259074": { "pmid": "40259074", "title": "Development of a robust enzyme cascade system: co-immobilization of laccase and versatile peroxidase on polyacrylamide hydrogel for enhanced BPA degradation.", "abstract": "Biodegradation using a synergically integrated system of laccase (E.C. 1.10.3.2) and versatile peroxidase (EC 1.11.1.16) co-immobilized on the polyacrylamide (PAM) hydrogel presents a promising solution for removing endocrine disrupting chemicals (EDCs) like bisphenol A (BPA) from wastewater. In this study, we developed a tailored biocatalyst consisting of a fungal laccase from Pleurotus ostreatus IBL-02 and versatile peroxidase, enzyme cascade co-immobilized covalently on a 7% (w/v) PAM hydrogel, offering high catalytic potential across various pH and temperature ranges. The PAM-VP/Lac structure was analyzed using scanning electron microscopy and Fourier-transform infrared spectrophotometry, revealing improved characteristics compared to free counterparts (FLac and FVP). The optimal pH for FLac, FVP, Lac/VP, and PAM-VP/Lac was 4, 5, 6, and 7, respectively. PAM-VP/Lac exhibited optimal activity at 50-60 °C, higher than FLac, FVP, and Lac-VP. PAM-VP/Lac showed superior operational stability, retaining 99.2% of its activity after eight cycles, with an immobilization efficiency of 78.62 ± 1.15% and activity recovery of 33.71 ± 0.2%. It also demonstrated enhanced thermal stability, with a two-fold increase in half-life at 50-70 °C. Thermodynamic analysis showed significant improvements in stability parameters for PAM-VP/Lac. This system achieved complete BPA degradation within two and a half hr, highlighting its potential for industrial-scale environmental remediation.", "journal": "Biodegradation", "year": "2025", "doi": "10.1007/s10532-025-10129-1", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41116523": { "pmid": "41116523", "title": "Nanogel cage based on oxidized sodium alginate sacrificial template: Enhanced stability and protein crosslinking performance of immobilized TGase.", "abstract": "To address the challenges of natural enzyme instability and mass transfer limitations in traditional immobilization techniques, this study proposes a sacrificial template strategy to develop cage-like nanogels (GNI-TG) that integrate efficient substrate transport with enzyme conformation protection, significantly enhancing enzyme stability and catalytic efficiency. The study employed methacrylated/quaternized dual-modified dextrin (MQD) to construct the continuous matrix of the nanogel, while designing Ca2+-ethylenediaminetetraacetic acid (EDTA) dynamic-responsive oxidized sodium alginate (OSA) as the sacrificial template. Structural analysis confirmed that the OSA and MQD formed a stable TG-protected structure through synergistic cross-linking mechanisms involving covalent Schiff base bonding and non-covalent interactions. GNI-TG retained 70 % of initial enzyme activity while demonstrating exceptional thermal stability (>50 % activity after 70 °C treatment) and broad pH adaptability (>70 % activity within pH 4-9). Leveraging polysaccharide-protein synergistic effects, GNI-TG significantly enhanced the comprehensive performance of whey protein/gelatin hydrogels. Compared to physically crosslinked and free TG crosslinked systems, CNI-TG crosslinked hydrogels exhibited superior gel strength (536.4 g), compressive strength (28.12 kPa), water-holding capacity (>99 %), thermal stability (Tm = 138.4 °C), elastic behavior (G' > 1500 Pa), and network recovery (96.3 % recovery rate). This study establishes an enzyme immobilization paradigm through covalent anchoring, electrostatic assembly, and sacrificial templating strategies, demonstrating broad applicability in food processing, biocatalysis, and biomedical fields.", "journal": "Carbohydrate polymers", "year": "2025", "doi": "10.1016/j.carbpol.2025.124368", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42636925": { "pmid": "42636925", "title": "Construction of a chitosan/γ-poly(glutamic acid) composite hydrogel crosslinked by ZIF-8 for enhanced α-glucosidase immobilization and inhibitor screening.", "abstract": "Herein, a composite hydrogel based on two natural macromolecules-chitosan (CS) and γ-poly(glutamic acid) (γ-PGA)-was developed, crosslinked by zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, which serve as reactive crosslinking hubs to covalently connect the biopolymer chains for efficient immobilization of α-glucosidase (α-Glu). A carboxyl-terminated poly(ethylene glycol)-poly(glutamic acid) copolymer was first synthesized and its activated carboxyl groups underwent amidation with amino groups of CS to rapidly form a three-dimensional PC/ZIF-8 hydrogel. The effects of ZIF-8 content and CS concentration on micro-morphology, swelling behavior, and rheological properties were systematically investigated. ZIF-8-mediated crosslinking was essential for gel formation, enabling effective modulation of network density and pore architecture. The optimized hydrogel showed no inhibitory effect on α-Glu. Immobilized α-Glu@PC/ZIF-8 exhibited enhanced pH stability (retaining 53% and 70% activity at pH 4.0 and 9.0), outstanding storage stability (>90% after 4 weeks), excellent organic solvent tolerance (>90% in most solvents), and good reusability (near 100% activity over 6 cycles). Mass transfer studies confirmed that the designed crosslinking density provided efficient substrate/product diffusion channels, minimizing product inhibition. Hansen solubility parameter analysis revealed a synergistic \"thermodynamic exclusion + size-sieving\" mechanism for solvent tolerance. Coupled with HPLC, the immobilized enzyme successfully identified two α-glucosidase inhibitors-batatasin I and 2,4-dimethoxy-6,7-dihydroxyphenanthrene-from Dioscorea opposita Thunb. peel. This work demonstrates that rational chemical modification of natural macromolecular networks via MOF crosslinkers provides an effective strategy for constructing high-performance enzyme immobilization platforms.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.154147", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42632666": { "pmid": "42632666", "title": "Chemically crosslinked magnetic sunflower pollen microgels achieve stable protein glutaminase immobilization to enhance deamidation and structural modification of sunflower seed protein.", "abstract": "Protease immobilization is often faced with enzyme activity loss and mass transfer resistance. Here, we developed a composite carrier with sunflower pollen microgel (SPMG) and NH2-Fe3O4@SiO2 to immobilize protein glutaminase (PGase) for controlled deamidation of sunflower seed protein (SSP). Under optimal conditions (35 mg NH2-Fe3O4@SiO2, 0.15% EDC/NHS), the immobilized PGase exhibited markedly improved thermal, pH, and storage stability relative to free PGase, probably because the hierarchical pores and covalent/hydrogen bonding between SPMG and NH2-Fe3O4@SiO2 stabilize the enzyme microenvironment. At appropriate deamidation degrees, the SSP solubility, emulsifying properties, and foaming properties were increased by about 1.32, 3.84, and 0.65 folds, respectively, accompanied by enhanced umami and sweetness. The structural, functional, and flavor changes in SSP were characterized by SDS-PAGE, particle size, zeta potential, spectral, and interfacial analyses. This study elucidates how the carrier properties influence the performance of immobilized enzymes and proposes a novel strategy for designing enzyme immobilization systems.", "journal": "Food research international (Ottawa, Ont.)", "year": "2026", "doi": "10.1016/j.foodres.2026.119871", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42413674": { "pmid": "42413674", "title": "Tailored pea protein isolate-sodium alginate composite microgels as a function of transglutaminase concentration: Enhanced gel properties and delivery of Lactobacillus plantarum YTU 30007.", "abstract": "Plant proteins are gradually replacing animal proteins because of their health and environmental benefits. However, the poor gelation properties of pea protein isolate (PPI) limit its application in delivery systems. To address this limitation, this study developed PPI-sodium alginate (SA) composite microgels through double cross-linking using transglutaminase (TGase) and CaCl2 to encapsulate Lactobacillus plantarum (L. plantarum). With a constant Ca2+-induced single crosslinking system (2%, w/v), the influences of different TGase concentrations (0, 2.5, 5.0, 7.5, and 10.0 U/g protein) on the comprehensive properties of double-crosslinked PPI-SA microgels were explored. The results demonstrated that increasing TGase concentration enhanced the degree of covalent cross-linking within the microgels, significantly improving their structural stability. Gel strength, mechanical elasticity, and thermal stability exhibited an upward trend in groups with higher TGase concentrations. In simulated gastrointestinal fluid (enzyme-free) tests, both single- and double-crosslinked microgels displayed structural shrinkage, indicating structural shrinkage. TGase concentration significantly influenced the swelling behavior of the microgels, with double-crosslinked microgels exhibiting greater structural stability than single-crosslinked ones. When TGase was added at 10 U/g protein, the encapsulation efficiency of the microgels reached a maximum of 90.58%. In vitro digestion tests revealed a positive correlation between TGase concentration and the protective effect of microgels on probiotics. High TGase concentrations improved the structural integrity of the microgels in simulated gastric fluid and significantly increased the release of viable bacteria in simulated intestinal fluid, owing to the synergistic protective effects of CaCl2 and TGase. The research shows that the introduction of TGase to build a double crosslinking system based on the CaCl2 induced PPI-SA composite microgel can simultaneously improve the gel characteristics and the oral delivery effect of probiotics by enhancing the covalence of PPI. This double-crosslinking strategy demonstrates considerable potential as a macromolecular encapsulation platform for probiotic protection and targeted intestinal delivery.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.153372", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40985481": { "pmid": "40985481", "title": "Ionic liquid-based transdermal drug delivery systems for biopharmaceuticals.", "abstract": "The non-invasive transdermal delivery of biopharmaceuticals, including proteins, peptides, and nucleic acids, remains a considerable challenge because of the formidable barrier function of the stratum corneum. Ionic liquids (ILs) composed of tunable cations and anions have emerged as useful multifunctional materials in transdermal drug delivery systems (TDDS) because of their excellent physicochemical properties. By acting simultaneously as solvents and permeation enhancers, ILs can considerably improve the solubility and stability of labile biomolecules and facilitate their transport across the skin. Recent studies have demonstrated the successful integration of ILs into nanocarrier systems, including ethosomes, transethosomes, IL-in-oil micro-/nano-emulsion formulations, and solid-in-oil dispersions, enabling the effective transdermal delivery of insulin, siRNA, mRNA, and other biologics. Compared with conventional solvent-based transdermal systems, biocompatible IL-based formulations can confer high stability and enhanced drug bioavailability. This review surveys the most recent advances in IL-TDDS, with particular focus on lipid- and choline-derived IL-enabled TDDS that have demonstrated prolonged glycemic control in diabetic models and potent anti-tumor responses in nucleic-acid immunotherapy.", "journal": "Chemical communications (Cambridge, England)", "year": "2025", "doi": "10.1039/d5cc03935e", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "41645705": { "pmid": "41645705", "title": "The development of DNA delivery system based on ionic liquid and carboxylic acid modified polyethyleneimine.", "abstract": "Recombinant DNA vaccines offer significant potential for disease prevention and therapy, but their clinical success is often limited by poor immunogenicity, low cellular uptake, instability, and inefficient delivery without proper carriers. To address these challenges, we developed a series of novel cationic delivery systems by modifying branched polyethyleneimine (PEI, 25 kDa) with biocompatible carboxylic acids either lactic acid or glycolic acid and incorporating choline-based ionic liquids (choline glycolate [CG] and choline lactate [CL]) to create advanced mPEI/ionic liquid (IL) formulations. These systems were designed to enhance DNA complexation, protect against enzymatic degradation, improve nanoparticle stability, and fine-tune physicochemical properties for optimal cellular interaction. The resulting polyplexes formed stable nanoparticles with diameters 100 -125 nm and surface charges of +24 to +29 mV, supporting efficient cellular uptake. Compared to unmodified PEI, the modified formulations showed markedly reduced cytotoxicity and significantly improved transfection performance. Among all tested combinations, the combination of lactic acid-modified PEI with choline glycolate (LA-mPEI + CG) was the most effective, achieving a 76% increase in transfection efficiency, a 66% improvement in cellular uptake, and 66% enhanced in cell viability. These findings highlight the synergistic advantage of combining carboxylic acid modification PEI with ionic liquid incorporation, providing a promising strategy for safer and more effective non-viral DNA delivery. This platform may serve as a foundation for future advancements in gene therapy and DNA vaccine development.", "journal": "Drug delivery", "year": "2026", "doi": "10.1080/10717544.2026.2627021", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "39869109": { "pmid": "39869109", "title": "Exploring Biophysical and Chemoinformatics Approaches for Interactions of Ionic Liquids With Hemoglobin, DNA, BSA, and HSA.", "abstract": "This review article provides an inclusive overview of the intricate interactions amid ionic liquids (ILs) and essential biomacromolecules, mainly hemoglobin (Hb), bovine serum albumin (BSA), human serum albumin (HSA), and calf thymus-DNA (CT-DNA). ILs have recently become a topic of great attention because of their inimitable physicochemical properties and potential uses in different fields. The review systematically explores the binding mechanisms, thermodynamics, and structural changes induced by ILs on Hb, BSA, HSA, and CT-DNA using spectroscopic, thermodynamic, and computational techniques. The article highlighted various experimental and computational methodologies to explore the interactions between ILs and biomacromolecules. It offers an in-depth analysis of the techniques employed to decode the intricate nature of these molecular associations and the influence of ILs along with their structural characteristics on the conformational stability, activity, and functionality of biomacromolecules. This foundational understanding is essential for advancing research and developing strategies that exploit the distinctive properties of ILs to foster innovative and sustainable applications within the biomedical field.", "journal": "Chemistry & biodiversity", "year": "2025", "doi": "10.1002/cbdv.202402781", "ptypes": [ "Journal Article", "Review" ], "first_author": "", "dirs": [ "D" ] }, "39744967": { "pmid": "39744967", "title": "Bioinspired design of DNA in aqueous ionic liquid media for sustainable packaging of horseradish peroxidase under biotic stress.", "abstract": "We show that a combination of DNA and ionic liquid significantly increases the stability and activity of HRP and achieves a 4.8-fold higher peroxidase activity than PBS buffer. Also, HRP retains 84% of its activity in IL+DNA compared to 24% in PBS against trypsin digestion. Molecular modeling and spectroscopic studies reveal a protective microenvironment.", "journal": "Chemical communications (Cambridge, England)", "year": "2025", "doi": "10.1039/d4cc05803h", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41187335": { "pmid": "41187335", "title": "DNA Nanostructure Self-Assembly in an Aqueous Ionic Liquid Solution with Enhanced Stability and Target Binding Affinity.", "abstract": "DNA nanostructure-enabled functional constructs have shown potential to improve healthcare outcomes by offering advanced disease diagnostic and therapeutic strategies. Translating this potential of DNA nanostructure-based constructs to real life applications relies on maintaining and enhancing the structural integrity and functions of the surface-anchored moieties. In this study, we explored the possibility of utilizing choline dihydrogen phosphate (CDHP) solution, an aqueous solution of ionic liquid, to assemble DNA nanostructures of different sizes and complexities with enhanced biostability and ligand binding affinity. We show successful formation of the DNA nanostructures in aqueous CDHP solution using gel electrophoresis, atomic force microscopy (AFM), and circular dichroism (CD). Biostability assays reveal that the aqueous CDHP solution may provide passive protection to DNA nanostructures against DNase I and human serum for up to 48 h. We also demonstrate that this enhanced biostability arises both from the structural conformation imparted during CDHP-mediated folding and from the presence of free CDHP ions in the solution. Notably, removal of free ions reduced the passive protection effect, but did not eliminate it, indicating the contribution of both folding and surrounding free ions. Using flow cytometry and surface plasmon resonance assays, we show that the presence of aqueous CDHP solution can enhance the binding of aptamer-functionalized DNA nanostructures to specific receptors on acute myeloid leukemia (AML) cells. Our strategy of using ionic liquid solution for one-pot preparation with enhanced stability and functionality offers a robust, simpler and faster alternative for DNA nanostructure-based constructs.", "journal": "Journal of the American Chemical Society", "year": "2025", "doi": "10.1021/jacs.5c13969", "ptypes": [ "Journal Article", "Research Support, N.I.H., Extramural" ], "first_author": "", "dirs": [ "D" ] }, "42208827": { "pmid": "42208827", "title": "Interaction and structural stability of DNA in choline chloride and polyol-based deep eutectic solvents.", "abstract": "Deep eutectic solvents (DESs) have emerged as viable eco-friendly substitutes to traditional solvents for biomolecular applications, due to their biodegradability, less toxicity, and tunable physicochemical characteristics. Use of DESs as greener media for preserving DNA structure and stability is currently a topic of immense importance, as it provides a better potential in the domain of DNA biotechnology. Here, we explored the interaction and structural integrity of DNA within various choline chloride and polyol-based DES media. Synthesized DESs were characterized, and their key physicochemical properties were evaluated. CD-spectroscopic study suggests that DNA maintains its conformational structure, stacking, and preserves its native B-form in all studied DESs, even at higher DES concentrations. Results were additionally supported by fluorescence microscopic studies. DESs exhibited multiple interaction pathways with DNA. UV-Vis spectroscopic studies revealed that phosphate group of DNA interacts with the choline cation of DESs via electrostatic force of attraction. DNA melting study unveils stability of DNA even at higher temperatures in polyol-based DESs. The dye displacement assay suggests binding of DNA in minor grooves and intercalation as DESs displaced both Ethidium bromide and Hoechst dye as measured by Stern-Volmer constant. To understand the binding efficiency between DNA and DES constituents, molecular docking was performed. Results reveal that tetraethyleneglycol had the highest favorable binding energy, followed by PEG400 and glycerol. These results offer novel perspectives into the solvation behavior of DNA in DES environments and establish a fundamental framework for their potential application in nanobiotechnology, extraction, and preservation of DNA.", "journal": "International journal of biological macromolecules", "year": "2026", "doi": "10.1016/j.ijbiomac.2026.152773", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42018748": { "pmid": "42018748", "title": "Microscopic Understanding of DNA Groove Binding by Hydrophilic and Hydrophobic Ionic Liquids.", "abstract": "In addition to being biologically essential macromolecules, DNAs in duplex forms also have extensive biotechnological applications. DNA preservation is critical for any biotechnological use; however, long-term stability under ambient conditions remains challenging. Aqueous ionic liquid (IL) solutions have evolved as excellent storage media for nucleic acid samples in recent times. In this work, we have systematically investigated the interaction patterns of a series of glycinate-based ILs containing cations with varying hydrophilic and hydrophobic characteristics with duplex B-DNA using atomistic molecular dynamics (MD) simulations. The calculations revealed subtle local conformational modifications of the DNA in the presence of the ILs, though the overall DNA structure remained largely conserved. Besides, it is demonstrated that the presence of the ILs leads to nonuniform expulsion of water molecules from the minor and the major grooves of the DNA. Specifically, the propensity to exchange water molecules by the IL components is higher from the more constricted minor groove than that from the wider major groove. Furthermore, due to geometrical constraints and consequent stronger negative potential, a high degree of specificity in cation orientations and bindings has been observed within the minor groove as compared to random nonspecific cation orientations in the major groove. Importantly, an increased fraction of DNA-water hydrogen bond breaking within the minor groove in the presence of the IL cations, as compared to that in the major groove, is the microscopic origin behind such nonuniform effects on solvent properties in the two grooves.", "journal": "The journal of physical chemistry. B", "year": "2026", "doi": "10.1021/acs.jpcb.6c00668", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "40620022": { "pmid": "40620022", "title": "Molecular simulations of ssDNA/ssRNA in [BMIM][PF6] ionic liquid: a novel biopolymer electrolyte for rechargeable battery applications.", "abstract": "Single-stranded DNA (ssDNA) in ionic liquids (ILs) represents an unexplored class of polymer electrolytes with significant potential for rechargeable battery applications. This study investigates ssDNA + [BMIM][PF6] systems using all-atom molecular dynamics simulations with OL15/OL3 force fields for DNA/RNA and an optimized OPLS-AA force field for [BMIM][PF6]. Density profile analysis reveals that ssDNA + IL systems maintain an isotropic distribution of ions. Radial distribution functions and ion association probabilities indicate that the addition of ssDNA preserves the liquid structure of neatIL. Further, the negatively charged phosphate groups in the polymer backbone form a contracted solvation shell with the cations, which leads to smaller cation-anion clusters. A comprehensive analysis of ion diffusion, conductivity, ion-pair relaxation, and viscosity shows that while ssDNA-IL electrolytes exhibit an ionic conductivity 20-30 times lower than neat IL, their mechanical stability improves by a factor of 200-300. These findings establish ssDNA + IL systems as mechanically robust, biodegradable, and high-performing candidates for next-generation battery technologies.", "journal": "Physical chemistry chemical physics : PCCP", "year": "2025", "doi": "10.1039/d5cp01833a", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42723260": { "pmid": "42723260", "title": "Selective Extraction of Genomic DNA From Animal Tissues Using a Hydrophobic Magnetic Ionic Liquid.", "abstract": "The development of green and efficient methods for genomic DNA extraction from animal tissues is crucial for molecular diagnostics, food traceability, and genetic research. Conventional methods often involve toxic reagents, multiple centrifugation steps, and are time-consuming. In this study, a hydrophobic magnetic ionic liquid (MIL), N-octyl-4-dimethylaminopyridinium hexafluorophosphate MIL ([C8DMAP][PF6]‑Ni MIL), was synthesized and applied for the selective extraction of genomic DNA from various animal tissues. The material exhibited strong paramagnetic behavior, high thermal stability, and excellent hydrophobicity, enabling rapid phase separation under an external magnetic field. A mechanical shaking-assisted extraction method was developed, and key parameters including temperature, time, shaking speed, and [C8DMAP][PF6]-Ni MIL dosage were systematically optimized. The method demonstrated high selectivity for DNA over proteins, RNA, and amino acids, with a maximum recovery rate of 78.06 ± 1.91%. Compared to a commercial DNA extraction kit, the [C8DMAP][PF6]-Ni MIL-based approach provided higher yields from several tissues, including mouse liver, brain, and rabbit lung. Furthermore, the [C8DMAP][PF6]-Ni MIL could be reused for at least six cycles while maintaining extraction efficiency. This work not only provides a high-performance material for DNA extraction, but also demonstrates a sustainable and easily retrievable liquid-phase separation strategy, offering a generalizable platform for complex sample pretreatment.", "journal": "Journal of separation science", "year": "2026", "doi": "10.1002/jssc.70533", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42113245": { "pmid": "42113245", "title": "A high-yield, SPE-free DNA extraction method using a pyridinium-based ionic liquid compatible with downstream PCR analysis.", "abstract": "We describe a novel DNA extraction protocol based on the ionic liquid 1-octylpyridinium bromide ([C8Pyr]Br), offering a rapid and reproducible alternative to column-based commercial kits. The method involves a selective DNA precipitation via charge neutralization and groove binding, followed by a displacement of the ionic liquid using phosphate-buffered saline. This process results in high-quality DNA with A260/A280 and A260/A230 ratios within optimal analytical ranges. The method was tested on eukaryotic cell lines and Drosophila tissue, consistently achieving two- to fivefold higher DNA yields than QIAGEN kits. Extracted DNA was validated by UV spectrophotometry, gel electrophoresis, and PCR. Additionally, dye displacement assays and circular dichroism confirmed preservation of DNA structure and suggested a minor groove binding mechanism for [C8Pyr]Br. This efficient, low-cost protocol may be of particular interest in diagnostics, molecular biology workflows, and resource-limited laboratories.", "journal": "Analytical and bioanalytical chemistry", "year": "2026", "doi": "10.1007/s00216-026-06543-w", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "41849852": { "pmid": "41849852", "title": "A lysis-free, all-in-one platform based on oligonucleotide-functionalized magnetic ionic liquid for direct and sequence-specific DNA extraction from intact plant tissues.", "abstract": "The extraction of DNA from plant tissues is often hindered by the resilient cell wall, which necessitates cumbersome pretreatment steps in conventional methods. Furthermore, existing techniques typically co-extract total DNA without the capability for sequence-specific isolation. To address these limitations, this study developed a novel oligonucleotide-functionalized hydrophobic magnetic ionic liquid (oligo-HMIL). This material integrates high stability, strong magnetic responsiveness, and exceptional hydrophobicity, allowing it to be magnetically manipulated to directly penetrate plant cell walls and membranes for DNA extraction, thereby eliminating the need for a separate lysis step. More significantly, the material enables sequence-specific recognition and selectively isolates the target DNA from complex matrices containing substantial amounts of non-target DNA, proteins, and other interferents. Based on this material, a lysis-free, all-in-one platform for direct and sequence-specific extraction of DNA was established. Under optimized conditions, the method achieved efficient extraction and enrichment of the target DNA from various plant tissues, with an extraction efficiency of 78.25 ± 1.83%. Compared to a commercial kit, the proposed approach increased DNA yield by at least 3-fold while reducing the processing time from 4 h to 30 min and simplifying the workflow. The material can be recycled and reused, and the method demonstrated excellent stability (RSD = %, n = 3) and reproducibility (RSD = 2.34%, n = 3). This work provides a green, rapid, and reusable platform for targeted plant DNA extraction, holding broad application potential in genetic research and biotechnology.", "journal": "Journal of chromatography. A", "year": "2026", "doi": "10.1016/j.chroma.2026.466895", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] }, "42395794": { "pmid": "42395794", "title": "Comparative study of Pd-, Au-, and Cu-based nanostructured electrodes synthesized in deep eutectic solvents: linking nucleation mechanisms to electrochemical DNA sensing performance.", "abstract": "This work presents a systematic comparison of the nucleation mechanisms of Pd, Au, and Cu in deep eutectic solvents and their direct effect on electrochemical DNA sensing performance. Chronoamperometric analysis revealed progressive nucleation for Cu, mixed nucleation for Au, and instantaneous nucleation for Pd. The distinct nucleation behaviors significantly influenced the surface morphology and electrochemical characteristics of the deposited electrodes. Accordingly, the sensitivities of Cu-, Au-, and Pd-based sensors were determined to be 80.48, 163.35, and 318.54 µA nM-1 cm-2, respectively, and the corresponding limits of detection (LOD) were 1.97 × 10-12 M, 1.17 × 10-12 M, and 0.73 × 10-12 M. All sensors exhibited a wide linear detection range from 1.0 × 10-12 to 3.0 × 10-10 M, excellent reproducibility (RSD < 3.2%), good stability, and high selectivity toward complementary DNA sequences. Practical applicability was validated through the detection of PCR-amplified DNA extracted from the sputum samples of patients with tuberculosis, showing satisfactory recovery and reliability in complex biological matrices.", "journal": "RSC advances", "year": "2026", "doi": "10.1039/d6ra02774a", "ptypes": [ "Journal Article" ], "first_author": "", "dirs": [ "D" ] } }