Biology Methods and Protocols最新文献

{"title":"Reverse vaccinology and immunoinformatics identify multi-epitope vaccine candidates against molluscum contagiosum virus.","authors":"Shahrazad A Khalaf, Musaab M Jasim, Younus Jasim, Murtada Naser","doi":"10.1093/biomethods/bpag045","DOIUrl":"10.1093/biomethods/bpag045","url":null,"abstract":"<p><p>Molluscum contagiosum virus (MCV) is a human-specific poxvirus responsible for a common contagious skin infection, yet no licensed prophylactic vaccine is available. Therefore, this study aimed to identify potential vaccine candidates against MCV using integrated reverse vaccinology and immunoinformatics approach. Candidate proteins involved in immune evasion, viral entry, and virion assembly were assessed for physicochemical properties, transmembrane topology, antigenicity, allergenicity, and toxicity. Linear B-cell, cytotoxic T-lymphocyte (CTL), and helper T-lymphocyte (HTL) epitopes were predicted and prioritized based on antigenicity, safety, HLA-binding affinity, and worldwide population coverage. The selected epitopes were assembled into a multi-epitope vaccine construct, followed by physicochemical characterization, secondary and tertiary structure prediction, and molecular docking analysis with Toll-like receptor 4 (TLR4). Eight candidate proteins were selected for immunoinformatics analysis, of which six were predicted to be antigenic. Five HTL epitopes, five CTL epitopes, and three non-toxic antigenic B-cell epitopes were selected, providing an estimated worldwide population coverage of 80.69%. The final 351-amino acid construct had a predicted molecular weight of 37.48 kDa, an instability index of 33.29, and a hydrophilic GRAVY value of -0.201. AlphaFold 3 produced a structurally organized model, while docking predicted a favorable interaction with TLR4 (score, -190.46) supported by hydrogen bonds, salt bridges, and electrostatic interactions. The vaccine construct exhibited favorable physicochemical properties, broad population coverage, structural stability, and strong interaction with TLR4, suggesting its potential as a prophylactic vaccine candidate. Nevertheless, further <i>in vitro</i> and <i>in vivo</i> experimental validation is required to confirm its safety and protective efficacy.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag045"},"PeriodicalIF":2.4,"publicationDate":"2026-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13577158/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148978243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GLP-1-based incretin therapy is associated with lower incident Alzheimer's disease and cardiorenal events in adults with documented neuropsychiatric, cognitive, or sensory risk.","authors":"Karthik Murugadoss, A J Venkatakrishnan, Venky Soundararajan","doi":"10.1093/biomethods/bpag043","DOIUrl":"https://doi.org/10.1093/biomethods/bpag043","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Alzheimer's disease (AD) develops over years, creating an opportunity for interventions that target modifiable risk factors before the onset of clinical dementia. Here we applied a federated electronic health record (EHR) system encompassing over 29 million de-identified patients toward a target-trial emulation framework involving 153,412 adults aged 50 years or older with at least one documented AD risk factor. New users of glucagon-like peptide-1 (GLP-1)-based incretin therapy were compared with new users of non-GLP-1 antidiabetic medications after a 12-month washout, with 1:1 propensity-score matching on 30 baseline variables and additional exact matching on index year of therapy initiation and 5-year age band. In the primary matched cohort, 28,901 patients per arm, incretin initiation was associated with lower first recorded AD diagnosis (hazard ratio [HR] 0.46, 95% confidence interval [CI] 0.29-0.73, &lt;i&gt;q&lt;/i&gt; = 0.003), all-cause dementia (HR 0.66 [0.55-0.79], &lt;i&gt;q&lt;/i&gt; &lt; 0.001), and all-cause mortality (HR 0.46 [0.37-0.58], &lt;i&gt;q&lt;/i&gt; &lt; 0.001), with directionally-lower Mild Cognitive Impairment (HR 0.76 [0.61-0.94], &lt;i&gt;q&lt;/i&gt; = 0.135) and Alzheimer's-related medication initiation (HR 0.82 [0.65-1.03], &lt;i&gt;q&lt;/i&gt; = 0.217). The AD diagnosis mitigation signal was independently reproduced for semaglutide (HR 0.56; &lt;i&gt;N&lt;/i&gt; = 23,675 per arm; &lt;i&gt;q&lt;/i&gt; = 0.02), with the directionally consistent tirzepatide point estimate (HR 0.60) not reaching significance. GLP-1 RA initiation was also associated with substantially lower (all &lt;i&gt;q&lt;/i&gt; &lt; 0.001) incidence of heart failure (HR 0.50 [0.45-0.55]), cardiomyopathy (HR 0.38 [0.31-0.47]), major adverse cardiovascular events (HR 0.74 [0.67-0.82]), acute kidney injury (HR 0.67 [0.67-0.74]), and chronic kidney disease (HR 0.68 [0.62-0.75]). Negative-control outcomes showed no significant separation (all &lt;i&gt;q&lt;/i&gt; &gt; 0.45), including allergic rhinitis (HR 0.96 [0.87-1.07]), hemorrhoids (HR 1.07 [0.95-1.21]), and inguinal hernia (HR 1.39 [0.89-2.18]). Stricter two-code ICD definitions supported lower first recorded AD diagnosis in the incretin arm (HR 0.51 [0.29-0.89], &lt;i&gt;q&lt;/i&gt; = 0.018) and lower first recorded all-cause dementia diagnosis (HR 0.68 [0.54-0.85], &lt;i&gt;q&lt;/i&gt; = 0.003). In 12-month landmark analyses among semaglutide initiators, ≥5% weight-loss responders had lower 3-year AD cumulative incidence than non-responders after matching (0.07% vs. 0.40%; incidence ratio 5.76; &lt;i&gt;P&lt;/i&gt; = .036), although this was not significant in the hazard-ratio model (HR 0.54, &lt;i&gt;P&lt;/i&gt; = .39). Sustained-dose stratification showed no comparable gradient (high-dose vs. low-dose 0.30% vs. 0.14%; HR 2.77; &lt;i&gt;P&lt;/i&gt; = .38), with ≤12 AD events per group. The weight-loss-specific pattern did not extend to all-cause dementia: weight-loss responders and non-responders had similar 3-year cumulative incidence (1.54% vs. 1.70%; HR 0.87, &lt;i&gt;P&lt;/i&gt; = .53). Initiation of GLP-1 receptor agonist therapy in adults with documented AD risk factors","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag043"},"PeriodicalIF":2.4,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13475380/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148762616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Semaglutide use linked to lower COVID-19 and influenza severity with better renal outcomes after pandemic or seasonal infection.","authors":"Robert Matson, A J Venkatakrishnan, Karthik Murugadoss, Venky Soundararajan","doi":"10.1093/biomethods/bpag042","DOIUrl":"10.1093/biomethods/bpag042","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Respiratory viral infections continue to impose a large burden of hospitalization, organ failure and death, particularly among older adults and people with cardiometabolic disease. Whether prior semaglutide exposure is associated with lower post-infection severity across distinct respiratory viruses remains uncertain. We analyzed de-identified longitudinal electronic health record data from a federated U.S. network spanning more than 29 million patients. Adults with documented COVID-19 or influenza infection were classified by pre-infection semaglutide use versus metformin use without prior GLP-1 receptor agonist exposure and one-to-one propensity matched on demographics, BMI, HbA1c, vaccination status, prior infection status, antiviral use, healthcare utilization extent, and baseline comorbidities, yielding matched cohorts of &lt;i&gt;N&lt;/i&gt; = 7092 for COVID-19 and &lt;i&gt;N&lt;/i&gt; = 1920 for influenza (flu). In the COVID-19 cohort, semaglutide exposure was associated with significantly lower 30-day mortality (0.3% vs 0.8%, RR 0.39, &lt;i&gt;P&lt;/i&gt; &lt; .001), hospitalization (5.3% vs 7.4%, RR 0.72, &lt;i&gt;P&lt;/i&gt; &lt; .001), and the composite disease severity outcome (8.2% vs 10.6%, RR 0.77, &lt;i&gt;P&lt;/i&gt; &lt; .001). Differences in rates of intensive care admission (1.4% vs 1.8%, RR 0.79, &lt;i&gt;P&lt;/i&gt; = .07) and mechanical ventilation (0.7% vs 0.9%, RR 0.76, &lt;i&gt;P&lt;/i&gt; = .14) were not statistically significant. In the flu cohort, semaglutide use was also associated with significantly lower mortality (0.2% vs 0.7%, RR 0.29, &lt;i&gt;P&lt;/i&gt; = .02), hospitalization (6.5% vs 9.6%, RR 0.68, &lt;i&gt;P&lt;/i&gt; &lt; .001), and the composite outcome (10.3% vs 14.6%, RR 0.70, &lt;i&gt;P&lt;/i&gt; &lt; .001); ICU admissions (1.9% vs 2.7%, RR 0.71, &lt;i&gt;P&lt;/i&gt; = .11) and mechanical ventilation (0.7% vs 0.9%, RR 0.78, &lt;i&gt;P&lt;/i&gt; = .48) were not statistically significant. Among organ-specific outcomes, for which &lt;i&gt;P&lt;/i&gt; values were adjusted for multiple comparisons, a significant risk reduction was observed for acute kidney injury (COVID-19 RR 0.73, &lt;i&gt;P&lt;/i&gt; = .02; flu RR 0.58, &lt;i&gt;P&lt;/i&gt; = .03) in the 30-day observation period, whereas reductions in acute coronary syndrome (COVID-19 RR 0.67, &lt;i&gt;P&lt;/i&gt; = .14; flu RR 1.00, &lt;i&gt;P&lt;/i&gt; = 1.00) and acute respiratory failure (COVID-19 RR 0.84, &lt;i&gt;P&lt;/i&gt; = .17; flu RR 0.74, &lt;i&gt;P&lt;/i&gt; = .27) were not statistically significant against the metformin comparator. Within the COVID-19 cohort, prior semaglutide exposure was associated with lower composite COVID-19 severity across the full study period, reaching significance in both vaccinated (RR 0.81, &lt;i&gt;P&lt;/i&gt; = .04) and unvaccinated (RR 0.76, &lt;i&gt;P&lt;/i&gt; &lt; .001) patients, and during the pandemic period ending May 11, 2023, reaching significance in unvaccinated patients (RR 0.76, &lt;i&gt;P&lt;/i&gt; = .001) but not vaccinated patients (RR 0.85, &lt;i&gt;P&lt;/i&gt; = .20). When restricting to those treated with standard-of-care therapeutics during the COVID-19 pandemic era, semaglutide users had significantly lower 30-day composite risk among those receiving Paxlovid within ±7 days","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag042"},"PeriodicalIF":2.4,"publicationDate":"2026-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13425107/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148654743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Co-culture of bone marrow-derived macrophages with aged primary myoblasts.","authors":"Ryan Arsenault, Anthony Ziadeh, Alexander Green, Junio Dort","doi":"10.1093/biomethods/bpag041","DOIUrl":"10.1093/biomethods/bpag041","url":null,"abstract":"<p><p>Skeletal muscle regeneration depends on the coordinated activity of multiple cell populations within the muscle stem cell niche, most prominently macrophages, which undergo dynamic phenotypic transitions essential to tissue repair. Aging disrupts this process, impairing macrophage signaling and muscle stem cell function in ways that are not yet fully understood. Existing approaches (e.g. conditioned media transfer and indirect transwell co-culture) fail to recapitulate the direct cell-to-cell contact required for continuous, reciprocal regulation throughout the regenerative cascade. Here, we present a protocol for the isolation of bone marrow-derived and tissue-resident macrophages from skeletal muscle, together with a direct co-culture methodology designed to interrogate macrophage-myoblast interactions in an age-relevant context.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag041"},"PeriodicalIF":2.4,"publicationDate":"2026-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13412162/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148621585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the efficacy of skin swabbing as a minimally invasive method of genetic sampling from Indian amphibians.","authors":"G Vaibhavi, C K Aravind, Shashank K Borkar, Sona M Sebastian, Pavan Kumar Thunga, G Ravikanth, K V Gururaja, Priti Hebbar","doi":"10.1093/biomethods/bpag040","DOIUrl":"10.1093/biomethods/bpag040","url":null,"abstract":"<p><p>Amphibians are facing an unprecedented extinction crisis; 41% of the species are currently on the verge of extinction, making them among the most threatened vertebrates. One of the integral components in amphibian conservation is genetic research, which provides vital insights into their fitness and long-term persistence. Molecular studies on amphibians most often use invasive methods that are detrimental to them. Instead, minimally invasive methods such as cloacal, buccal, and skin swabbing can be effective. In this study, we collected skin swab samples from multiple anuran species in the Western Ghats biodiversity hotspot and the urban areas of Bengaluru, India, to assess the efficacy of skin swabs for generating genetic data without compromising animal welfare. Two extraction methods (salt vs Qiagen™ DNeasy kit) and two swab storage conditions (dry vs wet) were assessed. There was no significant difference in DNA yield between the two DNA extraction methods, whereas kit-based extractions consistently yielded DNA within the ideal purity range (A260/280 = 1.8-2.0). Swab storage did not significantly affect DNA concentration, but dry swabs provided significantly higher purity. Samples showed 86.36% amplification success on the gel, and 67.05% yielded high-quality DNA sequences suitable for downstream analyses. Our results demonstrate that minimally invasive skin swabs are a non-lethal technique that can be replicated in anurans using less DNA. This is the first study from the Indian tropical region to utilise multiple species of varying body sizes to demonstrate the feasibility of minimally invasive skin swabbing as a method for anuran research and conservation.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag040"},"PeriodicalIF":2.4,"publicationDate":"2026-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13415466/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148621635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Selective elimination of donor bacteria to analyze plasmid reactions during conjugative transfer.","authors":"Meng Wen, Emma Mettouchi, Alejandra López Sánchez, Yoshiharu Yamaichi","doi":"10.1093/biomethods/bpag038","DOIUrl":"10.1093/biomethods/bpag038","url":null,"abstract":"<p><p>Bacterial conjugation is an important means of horizontal gene transfer in which DNA is transferred from a donor to a recipient cell by direct cell-to-cell contact. Dissemination of antibiotic resistance in bacteria is very often driven by conjugative plasmids harboring antibiotic resistance genes. Recent research highlighted the temporal cascade of DNA reactions that are critical for the establishment of a plasmid in the new host, which include prompt and robust induction of anti-defense genes as well as re-formation of double-stranded circular form of the plasmid after transfer as single-stranded linear DNA. The nature of conjugation which occurs in a mixture of cell populations, constrained the research on plasmid establishment. Since plasmid molecules before and after transfer are indistinguishable, distinction between donor and recipient/transconjugant cells is required, and physical separation is particularly necessary for the genomic approaches. We established a novel method exploiting a mutant donor which can be expeditiously eliminated from the conjugation, mixture with simple manipulation. This method, dubbed <i>ED-TA</i> for elimination of donor cells for transconjugant analysis, was shown to be a powerful tool to unveil plasmid gene expression profile at the early stages of conjugation (Wen et al. 2025, <i>Nucleic Acids Res</i>, doi: 10.1093/nar/gkaf1299). Here we present an optimized protocol for the <i>ED-TA</i> method which will help our study on plasmid actions during establishment in new host cell, including emerging interests between host defense and plasmid anti-defense systems.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag038"},"PeriodicalIF":2.4,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13375375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148473288","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"EEG-based AI-BCI wheelchair advancement: Transformer-based learning with motor imagery for brain computer interface.","authors":"Bipul Thapa, Biplov Paneru, Bishwash Paneru, Khem N Poudyal","doi":"10.1093/biomethods/bpag039","DOIUrl":"10.1093/biomethods/bpag039","url":null,"abstract":"<p><p>This article presents an artificial intelligence integrated approach to brain-computer interface-based wheelchair development, utilizing a motor imagery right-left-hand movement mechanism for control. The system is designed to simulate wheelchair navigation based on motor imagery right- and left-hand movements using electroencephalogram (EEG) data. A pre-filtered dataset, obtained from an open-source EEG repository, was segmented into arrays of 19 × 200 to capture the onset of hand movements. The data were acquired at a sampling frequency of 200 Hz. The system integrates a Tkinter-based interface for simulating wheelchair movements, offering users a functional and intuitive control system. We propose TFormerEEG, a Transformer-driven deep learning architecture, for motor imagery EEG classification. The model achieves a test accuracy of 93.04% compared with various machine learning baseline models, including XGBoost, EEGNet, and an EEG-Deformer model. The TFormerEEG achieved a mean accuracy of 91.18% through stratified cross-validation, showcasing the effectiveness of this model.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag039"},"PeriodicalIF":2.4,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13401820/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148593735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced drug-disease association prediction through representation learning on similarity networks.","authors":"Duc-Hau Le","doi":"10.1093/biomethods/bpag037","DOIUrl":"10.1093/biomethods/bpag037","url":null,"abstract":"<p><p>Drug repositioning has emerged as a promising strategy for accelerating therapeutic discovery by identifying novel indications for existing drugs. Recent graph representation learning methods have shown encouraging performance for drug-disease association prediction; however, many existing approaches directly utilize heterogeneous drug-disease networks during representation learning, potentially introducing label leakage and limiting generalizability. In this study, we propose similarity network-based representation learning for drug repositioning (SimNetRLDR), a similarity network-based representation learning framework for drug repositioning. The proposed method independently learns drug and disease embeddings from homogeneous similarity networks using a weighted graph attention network encoder. The learned representations are subsequently integrated and used for downstream drug-disease association prediction through an extreme gradient boosting (XGBoost) classifier. Comprehensive experiments were conducted on benchmark datasets under single and integrated/multiplex disease similarity network settings. SimNetRLDR consistently outperformed existing methods, achieving superior area under the receiver operating characteristic curve, area under the precision-recall curve, F1-score, and accuracy with strong robustness across cross-validation folds. Additional robustness evaluations using external dataset, drug-wise and disease-wise cold-start settings further demonstrated the generalizability of the proposed framework, particularly for unseen drugs. Hyperparameter sensitivity analysis demonstrated stable performance across different neighborhood sizes and attention head numbers. Component-wise ablation studies further confirmed the effectiveness of the weighted graph attention encoder and the decoupled XGBoost classifier design. To evaluate biological and clinical relevance, we analyzed predicted associations supported by shared Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and manually curated evidence from ClinicalTrials.gov. After rigorous evidence filtering, 12 predicted drug-disease associations showed plausible clinical support, including Sulindac-Breast Neoplasms, Methotrexate-Schizophrenia, and Liothyronine-Breast Neoplasms. Overall, these findings demonstrate that SimNetRLDR provides an effective, robust, and biologically meaningful framework for computational drug repositioning.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag037"},"PeriodicalIF":2.4,"publicationDate":"2026-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13344851/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148413090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A simple suspension culture method for generating human iPSC-derived liver organoids.","authors":"Ryosuke Morozumi, Mawo Kinoshita, Ryo Takahashi, Masayuki Mishima, Kazuki Izawa, Kei-Ichi Sugiyama, Masataka Tsuda","doi":"10.1093/biomethods/bpag036","DOIUrl":"10.1093/biomethods/bpag036","url":null,"abstract":"<p><p>Human liver <i>in vitro</i> models are indispensable for toxicity studies requiring metabolic competence. However, widely used two-dimensional hepatocyte-derived cell lines exhibit limited drug-metabolizing capacity, whereas primary human hepatocytes are constrained by limited availability, donor variability, dedifferentiation, and poor long-term functional stability. Although three-dimensional liver models offer greater physiological relevance, many existing methods depend on exogenous extracellular matrix (ECM) scaffolds or specialized equipment, thereby limiting standardization, scalability, and broader implementation. Here, we developed a simple, scalable suspension culture method for generating human induced pluripotent stem cell (iPSC)-derived liver organoids (HLOs) without exogenous ECM scaffolds or specialized equipment. The resulting HLOs secreted albumin and exhibited hepatic gene expression profiles by RNA-seq analysis. Notably, cytochrome P450 3A4 activity was maintained more stably over an extended culture period than under the ECM-embedding condition. Immunostaining further confirmed the presence of hepatocyte-like populations together with CK19-positive epithelial structures exhibiting E-cadherin-positive cell-cell adhesion and ZO-1-positive tight junction-associated regions. These findings indicate that the HLOs recapitulate key structural and functional features of human liver tissue. Collectively, our results demonstrate that this simple suspension culture method provides a practical and accessible strategy for generating HLOs with sustained hepatic functionality. This platform should facilitate broader application of HLOs in drug screening, toxicity assessment, disease modeling, and other <i>in vitro</i> settings requiring reproducible and scalable human liver models.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag036"},"PeriodicalIF":2.4,"publicationDate":"2026-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13354523/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148425056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Auditable large language model curation of clinical notes refines glucagon-like peptide-1 initiation, persistence ascertainment, and compounding use beyond prescription records.","authors":"Gowtham Varma, Karthik Murugadoss, Mathews E Kurian, Jerin Varghese, A J Venkatakrishnan, Venky Soundararajan","doi":"10.1093/biomethods/bpag035","DOIUrl":"10.1093/biomethods/bpag035","url":null,"abstract":"<p><p>Real-world benefit from incretin-based therapies depends on actual treatment initiation and sustained exposure, yet structured prescription records may capture prescribing intent rather than confirmed use. Using a large federated, de-identified US EHR platform spanning over 29 million patients, we analyzed patterns of glucagon-like peptide-1 receptor agonists (GLP-1RA) initiation, persistence, and compounding from clinical notes using an evidence-linked large language model (LLM) pipeline evaluated against an independent physician-adjudicated extracted-event reference standard, achieving 98.4% accuracy for structural chunk triage and 88.2% accuracy for medication-status adjudication. Among 553 073 adults with at least one semaglutide (<i>n</i> = 376 697) or tirzepatide (<i>n</i> = 176 376) prescription and baseline weight availability, documented initiation within ±3 months of the first structured prescription was identified in 141 189 semaglutide patients and 62 040 tirzepatide patients. Among these patients with documented initiation, frictionless starts accounted for 70.1% of semaglutide and 77.9% of tirzepatide starts, initiation after documented friction accounted for 17.2% and 15.9%, and early interruption after initiation within 3 months accounted for 12.7% and 6.2%, respectively. We analyzed treatment persistence over an 18-month period in a subset of 69 976 patients with \"frictionless initiation.\" Clinical note-based and structured prescription-derived persistence yielded materially different trajectories within each drug (log-rank <i>P</i> <b><</b> .001). A note-derived ascertainment approach showed that 42.3% and 43.1% of semaglutide and tirzepatide patients, respectively, continued therapy, while structured prescription-based ascertainment yielded higher persistence estimates of 55.4% for semaglutide and 58.3% for tirzepatide. Among the patients with a documented 3-18-month barrier or discontinuation event, insurance, cost barriers, perioperative holds and gastrointestinal adverse events were the top documented reasons. Compounded exposure was detected in 1696 frictionless initiators, with the first compounding signal occurring on or before the first structured prescription in 50.8% of semaglutide and 57.9% of tirzepatide patients. These findings show that AI curation of clinical notes can distinguish prescription intent from verified exposure. With the potential time-to-market confounding, semaglutide shows higher initiation and lower non-initiation than tirzepatide, with comparable note-confirmed persistence at 18 months. More broadly, these findings support integrative exposure ascertainment to strengthen real-world GLP-1 evidence generation, payer decision-making, safety surveillance, and strategies to improve sustained access and treatment continuity.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag035"},"PeriodicalIF":2.4,"publicationDate":"2026-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13353216/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148425011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}