Biology Methods and Protocols最新文献

{"title":"Machine learning method for optimizing coding sequences in mammalian cells.","authors":"Elias Theodorou, Michael Stadler, Claes Gustafsson, Mark Welch","doi":"10.1093/biomethods/bpag057","DOIUrl":"10.1093/biomethods/bpag057","url":null,"abstract":"<p><p>Mammalian cell lines are the preferred hosts for producing commercially relevant therapeutic proteins such as antibodies, antibody derivatives, and cytokine fusion proteins. Even though significant investment is made to optimize upstream and downstream processes, the optimal gene design parameters for heterologous recombinant protein expression remain poorly understood. We describe a generic approach to gene optimization in which design features are systematically sampled and iteratively modulated using machine learning (ML). Synthetic gene variants encoding the Dasher fluorescent protein, differing only in synonymous codons, were used to interrogate the gene-sequence preferences of transient antibody-expressing HEK293 cells. Synonymous codon variations influenced expression by more than 2 orders of magnitude. This variation in protein yield was used to build ML models relating gene design features, which were then employed to design further-improved genes. The ML models were shown to be expression system-specific. Messenger RNA levels and ribosome occupancy were highly correlated with protein levels, suggesting that mRNA lifetime is influenced by coding bias. Our results illustrate a novel, generally applicable method to improve gene expression via synonymous re-coding for any protein target or host cell.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag057"},"PeriodicalIF":2.4,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13671747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149216911","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":"Optimization and evaluation of fluoride-functionalized nano-hydroxyapatite derived from <i>Labeo rohita</i> fish scales for remineralization of early enamel caries: Protocol for an <i>in vitro</i> materials-to-dentistry study.","authors":"Santosh R Patil, Mohmed Isaqali Karobari","doi":"10.1093/biomethods/bpag056","DOIUrl":"https://doi.org/10.1093/biomethods/bpag056","url":null,"abstract":"<p><p>Fish scales are a calcium-phosphate-rich by-product of food processing that can be converted to hydroxyapatite (HAp), offering a circular-bioeconomy pathway for the development of oral biomaterials. Although an established extraction and characterization basis exists for HAp derived from Rohu (<i>Labeo rohita</i>) scales, direct investigations of fish-scale nano-HAp on enamel remain limited. This protocol provides optimized calcination and fluoride-functionalization conditions for nano-HAp derived from <i>L. rohita</i> scales, comprehensively characterizes the optimized material, formulates pH-standardized gels (pH 7.0 ± 0.1 at 25°C) with an analytically determined final-gel total fluoride target of 1450 ± 75 ppm for the fluoride-containing groups, and compares its capacity to repair artificial early enamel lesions against fish-scale nHAp, commercial synthetic nHAp, fluoride-matched free-fluoride controls, and a vehicle control. Gel fluoride will be determined after validated complete digestion and confirmed against an independent ion-chromatography method; the fluoride controls will be matched to the measured fluoride content of the functionalized-gel batches. The study is structured across three linked phases. Phase I will prepare fish-scale HAp at calcination temperatures of 800, 900, and 1000°C, followed by wet planetary milling and controlled fluoride ion exchange at four nominal fluoride levels (0, 0.75, 1.45, and 2.15 wt%), with three independent synthesis batches per experimental condition. Phase II will select an optimized fluoride-functionalized nHAp using prespecified multi-response desirability criteria, followed by comprehensive characterization of phase composition, crystallinity, morphology, primary and hydrodynamic particle size, surface charge, Ca/P ratio, fluoride incorporation, trace element content, thermal behavior, surface area, and fluoride release profile. Phase III will employ 120 enamel specimens obtained from 60 sound permanent premolars, randomized to six treatment groups (<i>n</i> = 20 per group) within a 14-day pH-cycling model. The primary outcome measure is percentage surface microhardness recovery (%SMHR). Secondary outcomes include lesion mineral loss and depth, cross-sectional hardness, Ca/P ratio, surface roughness, mineral deposition, and resistance to a secondary acid challenge. This study evaluates the complete translational pathway from fish-processing waste to a chemically verified, fluoride-functionalized biomimetic material. By including a fluoride-matched physical-mixture control, the design is specifically configured to distinguish a genuine functionalization effect from the independent contributions of HAp and soluble fluoride.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag056"},"PeriodicalIF":2.4,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13659912/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149184688","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":"Optimization of an <i>in vitro</i> drug screening assay for anti-<i>Trypanosoma cruzi</i> activity using epimastigotes.","authors":"Jhindy-Tatiana Perez, María-Helena Arias, Giovanny Garavito","doi":"10.1093/biomethods/bpag051","DOIUrl":"https://doi.org/10.1093/biomethods/bpag051","url":null,"abstract":"<p><p>Chagas disease, caused by <i>Trypanosoma cruzi</i>, poses a significant global health burden. Current treatment relies solely on benznidazole and nifurtimox, both with limited efficacy and significant adverse effects. <i>In vitro</i> phenotype-based screening enables identification of alternative treatments. While epimastigote-based screening is not suitable for primary drug discovery, it provides valuable complementary data for compound evaluation. However, the lack of standardized experimental conditions across studies compromises reproducibility and inter-laboratory comparability. Here, we optimized experimental conditions for <i>T. cruzi</i> epimastigote drug susceptibility assays. <i>Trypanosoma cruzi</i> epimastigotes (strain Y) were cultured under systematically varied conditions to optimize medium composition, serum supplementation, medium replacement frequency, inoculum density, and treatment exposure time. Epimastigote growth peaked on day 8, declining thereafter. Low initial parasite inoculum showed reduced growth and high variability, while high parasite densities formed aggregates. Growth rates were similar across media, but Roswell Park Memorial Institute medium could promote trypomastigote-like forms. Fetal bovine serum (FBS) enhanced growth rates, with a positive correlation between serum concentration and proliferation. Three-day medium replacement maintained >98% epimastigotes. Benznidazole exhibited an IC₅₀ of 16.61 ± 0.38 µM under optimized conditions. Optimal conditions include: Liver Infusion Tryptose or Brain Heart Infusion medium with 10% FBS, medium replacement every 3 days, 1 × 10⁶ parasites/ml inoculum, and 72-h treatment at 28°C. The assay demonstrated robustness across microplate designs, producing consistent results with both U-bottom and flat-bottom 96-well plates. Under these optimized conditions, benznidazole serves as an effective positive control.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag051"},"PeriodicalIF":2.4,"publicationDate":"2026-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13659773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149184778","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":"Metal- and polymer-assisted SEM imaging of protein-bound DNA molecules: a protocol.","authors":"Chanyoung Noh, Taebin Yun, Minseo Kang, Kyubong Jo","doi":"10.1093/biomethods/bpag052","DOIUrl":"10.1093/biomethods/bpag052","url":null,"abstract":"<p><p>Direct mapping of protein positions along long, stretched DNA molecules requires simultaneous visualization of the DNA backbone and associated protein signals. We recently established a heavy-metal- and polymer-assisted scanning electron microscopy (SEM) approach for imaging stretched DNA molecules and associated protein signals on silicon wafers. Here, we present a practical protocol for implementing this workflow using λ DNA and streptavidin-fluorescent protein-labeled λ DNA as model DNA samples. The protocol encompasses silicon wafer preparation, microchannel-guided DNA deposition, heavy-metal staining with UranyLess, polyvinylpyrrolidone (PVP) treatment of the deposited DNA sample, SEM imaging, and image analysis. UranyLess provides heavy-metal staining to enhance SEM contrast, whereas the PVP concentration modulates the relative visibility of DNA backbones and protein-associated signals. Low-PVP conditions facilitate protein-signal visualization, while high-PVP conditions enhance DNA-backbone imaging. We also describe optional in-channel and droplet-based methods for delivering UranyLess and PVP. Finally, the protocol covers DNA backbone tracing, intensity-profile analysis, and machine-learning-based determination of protein positions from SEM images. Representative dCas9-bound DNA samples further demonstrate the applicability of the workflow to different protein-binding patterns, including closely spaced binding across a repetitive target array and localized binding on genomic DNA. This workflow provides a practical framework for preparing stretched DNA samples on silicon wafers and analyzing DNA backbones and associated protein signals by SEM.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag052"},"PeriodicalIF":2.4,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13613057/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149064948","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":"Comparison of cardiometabolic benefits and tolerability in patients on sustained low doses of semaglutide or tirzepatide.","authors":"Karthik Murugadoss, A J Venkatakrishnan, Venky Soundararajan","doi":"10.1093/biomethods/bpag048","DOIUrl":"https://doi.org/10.1093/biomethods/bpag048","url":null,"abstract":"<p><p>Semaglutide and tirzepatide are typically evaluated through dose escalation, yet many patients in routine care do not reach a recommended maintenance dose due to tolerability, access, cost, supply, or individualized goals. Here, we used repeated 0.25 mg only semaglutide and 2.5 mg only tirzepatide prescription records as real-world proxies for sustained treatment at the lowest approved dose, a prescribing pattern that may overlap with, but does not establish, intentional microdosing. We analyzed de-identified electronic health records (EHRs) from a federated system of 29 million patients. Among 490 072 semaglutide-treated patients, 814 met the stringent sustained low-dose criterion of at least three distinct 0.25-mg prescriptions spanning at least 6 months from the first to the last qualifying prescription and among 322 442 tirzepatide-treated patients, 1016 met the analogous criterion of at least three distinct 2.5-mg prescriptions spanning at least 6 months. In a matched head-to-head comparison aided by AI-enabled curation of clinical notes, sustained low-dose tirzepatide showed higher 24-month adverse event rates (<i>P </i>< .05) than sustained low-dose semaglutide for constipation (32.6% vs. 22.4%), acute kidney injury (2.7% vs. 0.4%), muscle cramps (8.3% vs. 4%), lumbar disc disease (3.3% vs. 0.3%), dyspnea on exertion (9.6% vs. 6.8%), lentigo (4.7% vs. 0.8%), and actinic keratosis (3.3% vs. 0.6%), whereas sustained low-dose semaglutide (vs. tirzepatide) had higher rates of including otitis (5.6% vs. 2.1%), diaphoresis (5.5% vs. 3.2%), and ankle swelling (6.9% vs. 3.7%). Sustained low-dose tirzepatide was associated with greater weight loss than semaglutide (mean 12-month percent weight change -5.5% vs. -2.2%; <i>P</i> < .05 at all timepoints). Analyses of prescription patterns supported the findings from clinical notes. For example, renal-supportive and hyperkalemia therapies increased after low-dose tirzepatide (5.6% to 9.5% at 12 months, <i>P = .</i>016) but not after low-dose semaglutide (8.2% to 6.6%, <i>P = .</i>382). Taken together, these observational associations motivate prospective studies to better characterize the outcomes and pharmacology of sustained initiation-dose semaglutide or tirzepatide, and related microdosing studies.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag048"},"PeriodicalIF":2.4,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13651699/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149168462","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":"Quantifying phenylhydrazine-induced Heinz body formation in normal human erythrocytes by flow cytometry versus microscopy or spectrophotometry.","authors":"Mewahyu Dewi, Sri Widia Azraki Jusman, Alida Roswita Harahap, John Kevin Baird","doi":"10.1093/biomethods/bpag047","DOIUrl":"10.1093/biomethods/bpag047","url":null,"abstract":"<p><p>Heinz bodies occur in patients experiencing specific drug-induced acute haemolytic crises, most often in those with an inherited deficiency in glucose-6-phosphate dehydrogenase activity. Laboratory methods for quantifying Heinz bodies in <i>in vitro</i> systems include direct microscopic counts from crystal violet-stained thin blood films or spectrophotometric measurement of free crystal violet in RBC suspensions treated for Heinz body induction. Each method is relatively laborious and prone to errors. In this study, we sought to evaluate and validate an improved flow cytometry-based method for detecting Heinz bodies in human red blood cells after <i>in vitro</i> exposure to phenylhydrazine using washed RBCs. We report good concordance between flow cytometric counts of Heinz bodies and microscopic and spectrophotometric analyses of the same suspensions. Flow cytometry offers a less laborious, more precise, and more accurate quantification of Heinz body formation, with strong potential to improve the ease of use in experimental applications.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag047"},"PeriodicalIF":2.4,"publicationDate":"2026-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13595877/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026250","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 novel WST-8-based colorimetric method for sensitive and interference-free determination of glutathione peroxidase activity.","authors":"Ayat A Abdulhassan, Mahmoud Hussein Hadwan","doi":"10.1093/biomethods/bpag049","DOIUrl":"10.1093/biomethods/bpag049","url":null,"abstract":"<p><p>Glutathione peroxidase (GPx) is a major antioxidant enzyme that protects cells from oxidative damage, and accurate quantification of GPx is important for biochemical, clinical, and diagnostic research. Current colorimetric and spectrophotometric GPx assays, however, are often limited by low sensitivity, unstable reagents, or interference from biological matrix components. We developed a colorimetric GPx assay based on the tetrazolium salt WST-8. GPx reduces peroxide substrates (ROOH) to water or the corresponding alcohols using reduced glutathione (GSH) as an electron donor, generating oxidized glutathione (glutathione disulfide, GSSG); glutathione reductase (GR) then regenerates GSH from GSSG at the expense of NADPH. Unreacted NADPH reduces WST-8 to a stable, water-soluble formazan dye measured at 460 nm, so GPx activity can be calculated indirectly from the extent of NADPH consumption. Assay performance was compared with established DTNB- and UV-based GPx methods. The WST-8-based assay showed high sensitivity, a wide linear range, good reagent stability, and strong resistance to interference from sugars, amino acids, and proteins. Activity values obtained with the new method correlated closely with those from the conventional DTNB assay, confirming its accuracy. The WST-8-GPx assay offers improved sensitivity and interference resistance while remaining operationally simple and compatible with routine microplate instrumentation, making it a practical option for both clinical diagnostics and oxidative stress research.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag049"},"PeriodicalIF":2.4,"publicationDate":"2026-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13595878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026219","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":"Whole-mount diffracted X-ray blinking analysis of intracellular amyloid-β-associated nanoscale dynamics in <i>Caenorhabditis elegans</i>.","authors":"Kotaro Ozaki, Yoichi Shinkai, Yuji C Sasaki, Masahiro Kuramochi","doi":"10.1093/biomethods/bpag044","DOIUrl":"10.1093/biomethods/bpag044","url":null,"abstract":"<p><p>Nanoscale protein dynamics are closely associated with molecular function and pathological structure formation, yet their measurement within animal tissues remains technically challenging. In this study, we developed a diffracted X-ray blinking (DXB) method to evaluate amyloid-β (Aβ)-associated nanoscale fluctuations in fixed and permeabilized whole-mount <i>Caenorhabditis elegans</i>. Intracellular Aβ expressed in neurons (nAβ) or body-wall muscle cells (mAβ) was labelled with gold nanoparticles using an antibody-mediated strategy, without dissection or sectioning. The labelled worms produced detectable Au(111) diffraction signals, and the resulting time-resolved diffraction intensity fluctuations were analysed by autocorrelation function (ACF) analysis. A comparison of the ACF decay constants obtained from nAβ and mAβ worms revealed that compared with nAβ, mAβ contained a greater fraction of fast relaxation components. Furthermore, in-vitro DXB measurements of Aβ revealed local fast relaxation components that changed over time in association with changes in Aβ assemblies, partly resembling the Aβ-associated motional features observed in worms. These findings suggest that the detected relaxation components may reflect structurally diverse Aβ states arising during aggregation. These results demonstrate the feasibility of applying DXB to detect Aβ-associated nanoscale fluctuation signatures in fixed whole-mount <i>C. elegans</i> and provide a methodological framework for analysing protein-associated dynamic states in animal tissue samples.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"11 1","pages":"bpag044"},"PeriodicalIF":2.4,"publicationDate":"2026-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13570790/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148956797","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":"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}