Current protocols最新文献

{"title":"Functional Clinical Flow Cytometry Assays to Assess Lymphocyte Proliferation, Neutrophil Oxidative Burst, and Leukocyte Adhesion Deficiency Immune Defects","authors":"Caleb Cornaby,&nbsp;Maurice R. O'Gorman","doi":"10.1002/cpz1.70462","DOIUrl":"10.1002/cpz1.70462","url":null,"abstract":"<p>Inborn errors of immunity (IEI) are a diverse group of inherited disorders caused by detrimental variants in the genome that compromise the immune system and lead to an increased susceptibility to infections, autoimmunity, and/or malignancy. Although the diagnostic process traditionally begins with a clinical workup and laboratory screening, such as complete blood counts, serum immunoglobulin measurements, and lymphocyte subset enumeration, functional assays serve as a vital bridge to diagnosis by providing a real-time assessment of immune system performance. Such assays include lymphocyte proliferation assays for evaluating T cell and B cell defects along with dihydrorhodamine-123 (DHR-123) and leukocyte adhesion deficiency testing to assess neutrophil function. These assays facilitate rapid screening and provide evidence for therapeutic intervention while helping to validate the pathogenicity of novel genetic variants and variants of unknown significance. Ultimately, functional assay results are indispensable in both the diagnosis and monitoring of treatment efficacy for patients with IEIs. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Evaluation of lymphocyte proliferation by EdU flow cytometry</p><p><b>Basic Protocol 2</b>: Dihydrorhodamine-123 (DHR-123) assessment of neutrophil oxidative burst</p><p><b>Basic Protocol 3</b>: Evaluation for leukocyte adhesion deficiency</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149187032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized Expression and Purification of Outer Membrane Protein 25 (Omp25) of Brucella melitensis","authors":"Harshita Mogaveera,&nbsp;Sudipta Patra,&nbsp;Niladri Bhusan Pati,&nbsp;Chiranjay Mukhopadhyay,&nbsp;Manjunath Joshi,&nbsp;Vandana Kalwaje Eshwara","doi":"10.1002/cpz1.70481","DOIUrl":"10.1002/cpz1.70481","url":null,"abstract":"<p>Our previous immunoproteomic studies on patients with acute brucellosis identified outer membrane protein 25 (Omp25) of <i>Brucella melitensis</i> as one of the immunogenic antigens, indicating its potential to be used as a diagnostic marker and a tool to differentiate acute and chronic human brucellosis. Expression and purification of Omp25 are technically challenging using conventional approaches owing to its membrane association. Existing expression and extraction protocols frequently lead to host cell toxicity, low protein yields, and poor reproducibility. To address these limitations, we developed a modified approach for the expression and purification of recombinant Omp25 protein that is standardized and reproducible and yields protein efficiently. The truncated <i>omp25</i> gene is cloned and expressed in <i>Escherichia coli</i> BL21 (DE3) cells, and induction is carried out at 28°C for 6 hr. The expressed recombinant Omp25 protein is predominantly found in the insoluble fraction, so it is extracted under denaturing conditions, purified by Ni-NTA affinity chromatography, refolded by stepwise dialysis, and desalted prior to characterization. In our hands, this set of protocols yielded 47.75 mg purified, high-quality recombinant Omp25 protein per liter of bacterial culture. SDS-PAGE analysis demonstrated a single predominant protein band at ∼24.3 kDa with no detectable contaminating protein bands, indicating successful purification. recombinant Omp25–based indirect ELISA confirmed that the refolded protein retained its antigenic properties, exhibiting significantly higher reactivity with sera from culture-confirmed brucellosis patients than with healthy control sera (p &lt; 0.001). This optimized set of protocols provides a reproducible workflow for the production of highly purified recombinant Omp25 protein and offers quantitative performance metrics that facilitate reproducibility and support this protein's application in immunological and diagnostic studies. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression and extraction of <i>Brucella melitensis</i> Omp25 protein from <i>Escherichia coli</i> BL21 (DE3) cells</p><p><b>Basic Protocol 2</b>: Purification of recombinant Omp25 protein</p><p><b>Basic Protocol 3</b>: Refolding and desalting of recombinant Omp25 protein</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13657259/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149172234","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":"Unmasking Hidden Seed Resistance to Storage Pests: Testing Antibiosis and Antixenosis","authors":"Sonja Gvozdenac,&nbsp;Aleksandra Ilić,&nbsp;Ivana Štajner Papuga,&nbsp;Julia Mikaela Parsons,&nbsp;Jordi Riudavets,&nbsp;Milica Škorić","doi":"10.1002/cpz1.70465","DOIUrl":"https://doi.org/10.1002/cpz1.70465","url":null,"abstract":"<p>Postharvest seed losses caused by stored-product insect pests can reach 90%, depending on the commodity, country, and storage technology, resulting in considerable economic losses and threats to food safety and security. Breeding genotypes (varieties, hybrids, landraces, inbred lines, etc.) resistant to insect pests is among the most effective pest management methods, although it has not yet been explored in detail or used to control stored-product pests. To help breeders develop resistant genotypes, it is a prerequisite to identify inherently resistant lines, identify seed defense traits, and define the underlying genetic basis of resistance. Resistance mechanisms include: i) antibiosis (assessed in “no-choice” tests), indicating seed suitability for insect development (measured as progeny production and % of consumed seed), and ii) antixenosis (assessed in “choice” olfactometric tests), analyzing specific genotypic attractiveness (i.e., insect preference) based on insects’ behavioral responses to different olfactory stimuli. There is an obvious lack of a joint protocol that integrates antibiosis and antixenosis test results and provides detailed information about the impact of unsuitable seed traits on insect pest bionomy. Moreover, no protocol exists for testing antixenosis across a large number of genotypes using olfactometric devices. The novelty of this work lies in: (i) the development of a choice test protocol for the assessment of seed attractiveness and insect preference across a large set of genotypes simultaneously; (ii) the integration of the results with those from no-choice tests; and (iii) a specifically designed mathematical model that indicates the level of susceptibility of each genotype. These tests were developed, optimized, and performed on two major crop groups—cereals and legumes—to assess their resistance to the main stored-product (seed) pests: the rice weevil, the bean beetle, and the lesser grain borer. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Antibiosis (no-choice) test</p><p><b>Basic Protocol 2</b>: Antixenosis (choice) test</p><p><b>Basic Protocol 3</b>: Choquet-based Attractiveness Ranking Protocol – CHARP</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Guide to Enzymatic Targeted Methylation Sequencing Bioinformatics Workflow: Best Practices","authors":"Vitória Rodrigues Guimarães Alves,&nbsp;Sheila T. Nagamatsu,&nbsp;Vanessa Kiyomi Ota,&nbsp;Cibele Edom Bandeira,&nbsp;Janitza L. Montalvo-Ortiz,&nbsp;Danilo Micali,&nbsp;Ana Carolina Coelho Milani,&nbsp;Ivaldo Silva,&nbsp;Cristiane Seixas Duarte,&nbsp;Andrea Parolin Jackowski,&nbsp;Jonathan Posner,&nbsp;Carolina Muniz Carvalho,&nbsp;Sintia Iole Belangero","doi":"10.1002/cpz1.70464","DOIUrl":"10.1002/cpz1.70464","url":null,"abstract":"<p>We present a best-practices protocol for targeted DNA methylation sequencing analysis that integrates both bisulfite- and enzymatic-conversion approaches using Twist Bioscience technology. Building on established tools and vendor-recommended workflows, this protocol provides a standardized, fully reproducible framework that addresses current gaps in the implementation, documentation, and downstream integration of targeted methylation sequencing analyses. The workflow incorporates widely used open-source tools across two main stages: preprocessing (adapter trimming with Trim Galore and quality control via FastQC/MultiQC) and methylation calling (alignment with BWA-meth, duplicate marking using Picard, and methylation extraction with MethylDackel). Importantly, we extend existing workflows by introducing a structured third stage for downstream statistical analysis in R, including β-value calculation, differential methylation analysis with methylKit, principal component analysis, and tiling-based regional profiling, all embedded in a unified, reproducible pipeline. A key contribution of this protocol is the integration of multi-stage quality control checkpoints, harmonized parameterization, and explicit reporting of intermediate outputs, enabling transparency and reproducibility across all analytical steps. The modular design allows tool substitution and adaptation to different experimental contexts, including enzyme-based methylation methods, while maintaining consistency in data processing and interpretation. This guide provides step-by-step instructions, troubleshooting strategies, and examples of expected outputs, along with built-in statistical routines that generate comprehensive results, including methylation matrices, correlation analyses, differentially methylated region annotations, and publication-ready visualizations. The complete workflow requires approximately 9–27 hr for 10 samples (approximately 134.2 million reads per sample) on Linux systems and assumes moderate proficiency in command-line environments and R. In summary, this protocol offers a practical, transparent, and reproducible framework that standardizes targeted DNA methylation analysis and facilitates its application across diverse biological and clinical research settings. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70464","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149140045","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":"Application of Liquid-Based Cytology Using the ThinPrep System to Thyroid Fine-Needle Aspiration Samples","authors":"Vargas-Castellanos Elizabeth,&nbsp;Arenas Avila Lina Alejandra,&nbsp;Rocha Franky","doi":"10.1002/cpz1.70484","DOIUrl":"10.1002/cpz1.70484","url":null,"abstract":"<p>Fine-needle aspiration (FNA) is the gold-standard diagnostic procedure for the initial evaluation of thyroid nodules because it is minimally invasive, cost-effective, and highly accurate for distinguishing benign from malignant lesions. However, conventional smear preparation is frequently affected by operator-dependent variability, uneven cell distribution, blood contamination, and air-drying artifacts, all of which may compromise specimen adequacy and diagnostic performance. Liquid-based cytology (LBC) has emerged as an effective alternative, offering standardized specimen processing, improved cellular preservation, and enhanced slide quality. Among the available LBC platforms, the ThinPrep system is one of the most extensively validated technologies for thyroid cytopathology and is increasingly integrated into routine clinical practice and molecular diagnostic workflows. This protocol provides a comprehensive, standardized workflow for processing thyroid FNA specimens using the ThinPrep liquid-based cytology system. The protocol describes specimen collection, transportation, fixation, processing with the ThinPrep 5000 processor, slide preparation, staining, and quality control procedures to ensure optimal cytomorphological evaluation. Recommendations for acceptable specimen preservation, including transport in PreservCyt solution or alternative fixation methods, are also provided. In addition, the protocol includes procedures for recovering the residual cellular material preserved in the ThinPrep vial for paraffin block processing, which allows for complementary studies, e.g., immunohistochemistry, fluorescence in situ hybridization (FISH), DNA and RNA extraction, and next-generation sequencing (NGS), thus expanding the diagnostic utility of thyroid cytology beyond conventional microscopic examination. When properly executed, this protocol generates high-quality monolayer cytology preparations suitable for standardized interpretation according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) while preserving residual material for downstream molecular analyses. Compared with conventional smear cytology, the ThinPrep system improves specimen reproducibility, reduces background artifacts and inadequate samples, facilitates laboratory standardization, and supports precision medicine applications. This protocol provides a practical and reproducible approach for both clinical diagnostic laboratories and translational research. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Simultaneous preparation of ThinPrep cytology slides and cell blocks from thyroid fine-needle aspiration specimens using liquid-based cytology</p><p><b>Alternate Protocol</b>: Alternative ThinPrep FNA processing using CytoLyt and PreservCyt</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13644887/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149129152","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":"Detection and Quantification of Spreading Depolarization in Drosophila melanogaster Using High-Throughput, Wide-Field Brain Imaging","authors":"Wolfgang Stein,&nbsp;Allison L. Harris","doi":"10.1002/cpz1.70475","DOIUrl":"10.1002/cpz1.70475","url":null,"abstract":"<p>Spreading depolarization (SD) is a ubiquitous neural phenomenon implicated in mammalian neurological disorders including cerebral ischemia, brain death, stroke, brain injury, and migraine, as well as in insect chill coma. During SD, a breakdown of ion homeostasis and a slowly spreading wave of neural depolarization leads to processing deficits, such as sensory disturbances and paralysis. Our protocols introduce high-throughput techniques using wide-field fluorescence brain imaging in <i>Drosophila melanogaster</i> larvae and adults to detect and quantify the spatial and temporal dynamics of SD and its initiation points. Two SD initiation techniques, rapid cooling and pharmacological manipulation, are detailed. The larval protocols describe how to elicit, detect, and quantify SD in up to 16 animals simultaneously in either intact or dissected preparations. The adult SD protocols describe the detailed steps to elicit, detect, and quantify SD in either fully intact animals or after microdissection that enables high resolution imaging of the brain. Detection, quantification, and analysis techniques are described in a separate protocol. Our protocols establish the fly as a high-throughput model for studying conserved cellular and circuit-level mechanisms of SD initiation and propagation. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: High-throughput SD detection in intact <i>Drosophila</i> larvae with fluorescent calcium reporters</p><p><b>Alternate Protocol 1</b>: High-resolution SD imaging in fillet larvae with fluorescent calcium reporters</p><p><b>Support Protocol 1</b>: SD imaging in larvae with fluorescent membrane voltage reporters</p><p><b>Basic Protocol 2</b>: High-throughput SD detection in adult <i>Drosophila</i> with fluorescent calcium reporters</p><p><b>Alternate Protocol 2</b>: High-resolution SD imaging in adult <i>Drosophila</i> with fluorescent calcium reporters</p><p><b>Support Protocol 2</b>: SD imaging in adults with fluorescent membrane voltage reporters</p><p><b>Basic Protocol 3</b>: High-throughput SD detection analysis in <i>Drosophila</i> with fluorescent calcium reporters</p><p><b>Alternate Protocol 3</b>: High-resolution SD imaging analysis in <i>Drosophila</i> with fluorescent calcium reporters</p><p><b>Support Protocol 3</b>: SD detection and spread analysis in <i>Drosophila</i> with fluorescent membrane voltage reporters</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13644547/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149129304","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":"Optimized Genome-Wide Profiling of Centromeric Chromatin Complexes by ChIP-seq","authors":"Alessia Daponte,&nbsp;Rachel Schwope,&nbsp;Caterina Manzari,&nbsp;Lucia Trisolini,&nbsp;Francesca Antonacci,&nbsp;Michele Morgante,&nbsp;Mario Ventura,&nbsp;Glennis A. Logsdon,&nbsp;Claudia R. Catacchio","doi":"10.1002/cpz1.70471","DOIUrl":"10.1002/cpz1.70471","url":null,"abstract":"<p>Centromeres are specialized chromosomal regions essential for faithful chromosome segregation and genome stability. Their highly repetitive DNA composition and epigenetically defined chromatin state present significant challenges for genome-wide profiling approaches such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Standard ChIP-seq workflows often perform poorly at centromeric regions, resulting in low signal-to-noise ratios and limited reproducibility. This article describes a ChIP-seq protocol optimized to enrich for and analyze centromeric chromatin from cell lines. The procedure builds on established ChIP-seq methodologies while incorporating centromere-focused modifications in nuclei isolation, chromatin fragmentation, immunoprecipitation, library preparation, and data analysis. Detailed step-by-step instructions, critical parameters, recommended controls, and troubleshooting guidance are provided to support reproducible implementation. When applied carefully, this protocol enables reliable profiling of centromere-associated chromatin and can be integrated into broader studies of chromosome organization, centromere function, and genome stability. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Sucrose gradient–based isolation of nuclei</p><p><b>Support Protocol 1</b>: Titration of micrococcal nuclease (MNase)</p><p><b>Basic Protocol 2</b>: MNase digestion and immunoprecipitation of CENP-A chromatin</p><p><b>Alternate Protocol 1</b>: Chromatin sonication and immunoprecipitation of CENP-C chromatin complexes</p><p><b>Support Protocol 2</b>: NET blocking and washing of Protein G Sepharose beads</p><p><b>Basic Protocol 3</b>: DNA extraction</p><p><b>Alternate Protocol 2</b>: Reversal of cross-links and DNA purification</p><p><b>Support Protocol 3</b>: Bead clean-up</p><p><b>Basic Protocol 4</b>: Library preparation and size selection</p><p><b>Support Protocol 4</b>: qPCR to assess α-satellite DNA enrichment</p><p><b>Basic Protocol 5</b>: ChIP-seq data processing and analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13644538/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149129354","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":"Purification and Thioflavin T Staining of Bacillus spp. Endospores for Fluorescence-Based Assays","authors":"Ephrem Debebe Zegeye,&nbsp;Marina Aspholm","doi":"10.1002/cpz1.70480","DOIUrl":"10.1002/cpz1.70480","url":null,"abstract":"<p>We present a simple, low-cost, and efficient protocol for fluorescent staining of <i>Bacillus</i> spp. endospores using the thioflavin T (ThT) dye. The article provides step-by-step procedures for culturing <i>Bacillus paranthracis</i>, <i>Bacillus thuringiensis</i> (2 strains), and <i>Bacillus subtilis</i>, purifying endospores from pure cultures, and staining them with ThT. ThT-stained endospores retain fluorescence for at least 2 years when stored at 4°C in the dark and remain fluorescent following formaldehyde fixation. Importantly, ThT staining does not impair spore viability, and stained endospores germinate normally. In contrast, vegetative <i>Bacillus</i> cells exhibit minimal to no ThT fluorescence. Overall, this robust and straightforward staining protocol is well suited for applications requiring fluorescently labeled endospores, including fluorescence microscopy, and provides a convenient plate-based readout for germinant screening and related assays. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Endospore purification</p><p><b>Basic Protocol 2</b>: Fluorescent staining of endospores</p><p><b>Support Protocol</b>: Fluorescence microscopy of ThT-stained spores</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13637824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149120657","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":"ProsperousPlus: A Bioinformatics Workflow for Protease Substrate Cleavage Site Prediction and Model Construction Utilizing Machine Learning.","authors":"Xudong Guo, Heyun Sun, Yi Hao, Lukasz Kurgan, Jiangning Song, Fuyi Li","doi":"10.1002/cpz1.70487","DOIUrl":"https://doi.org/10.1002/cpz1.70487","url":null,"abstract":"<p><p>Proteolytic cleavage is a fundamental biological process that regulates protein maturation, signaling pathways, immune responses, and disease progression. Accurate identification of protease substrate cleavage sites is critical for understanding protease function and substrate specificity. However, experimental determination of cleavage sites remains labor-intensive and time-consuming, motivating the development of efficient and accurate computational prediction methods. Here, we present a detailed protocol for using ProsperousPlus, a computational platform designed for large-scale prediction of protease-specific substrate cleavage sites and construction of customizable models for these predictions. ProsperousPlus integrates multiple sequence-derived scoring functions with an advanced machine learning framework that automates the construction of optimized predictive models. The framework's workflow includes three major functional modules: (1) prediction of cleavage sites using pre-trained models covering more than 100 proteases, (2) training of custom protease-specific models using user-provided datasets, and (3) deployment of trained models for large-scale prediction tasks. This protocol describes the required hardware and software environment, dataset preparation strategies, model training procedures, and interpretation of prediction results, including probability scores and multiple evaluation metrics such as accuracy and AUC. In addition, model interpretation using Shapley additive explanations (SHAP) enables identification of influential sequence features contributing to cleavage specificity. ProsperousPlus can be used either through a standalone software for large-scale, more frequent analyses or through a user-friendly web server for small-scale, ad hoc use. This protocol provides a practical and reproducible workflow for researchers seeking to analyze protease substrate specificity, construct customized prediction models, and perform high-throughput proteolytic analysis. © 2026 Wiley Periodicals LLC. Basic Protocol: Predicting protease-specific substrate cleavage sites and constructing and deploying protease-specific models using the standalone software version of ProsperousPlus Alternate Protocol: Using the web server version.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":"e70487"},"PeriodicalIF":2.5,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149221230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocol for the Preparation of Polyacrylamide Hydrogels with Tunable Stiffness and Their Application in Cell Culture, Live-Cell Imaging, and Fixed-Cell Immunofluorescence.","authors":"Shatruhan Singh Rajput, Osian W Jones, Laura M Machesky","doi":"10.1002/cpz1.70478","DOIUrl":"10.1002/cpz1.70478","url":null,"abstract":"<p><p>Tissue stiffness changes markedly in many pathological conditions, such as cancer and fibrosis, influencing cellular behavior and function. To investigate these effects in vitro, it is necessary to culture cells on substrates whose mechanical properties can be tuned to mimic both normal and diseased tissue environments. This protocol provides a detailed, reproducible procedure for preparing polyacrylamide (PAA) hydrogels with defined stiffness values that are functionalized with extracellular matrix (ECM) proteins and suitable for cell culture and imaging studies. It outlines the steps for cleaning and functionalizing glass coverslips, preparing acrylamide and bis-acrylamide mixtures of desired concentrations, initiating polymerization, and conjugating ECM proteins to support cell adhesion. The protocol further describes the process of seeding cells onto the hydrogels, followed by live-cell imaging, chemical fixation, and staining for visualization of cellular structures and protein localization. Practical notes and troubleshooting tips are also included for maintenance of hydrogel integrity and optimization of experimental reproducibility. The workflow enables systematic investigation of how substrate stiffness regulates cell morphology, migration, cytoskeletal organization, and other mechanosensitive responses. Overall, this protocol provides a reliable framework for preparing and using tunable PAA-based hydrogels in studies of cell-matrix interactions, mechanotransduction, and disease-related changes in tissue mechanics. It is adaptable to a wide range of cell types and compatible with various imaging modalities, offering a versatile platform for mechanobiology research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of polyacrylamide-based hydrogels of tunable rigidities and functionalization for cell culture Support Protocol 1: Preparation of reagents for hydrogel preparation Basic Protocol 2: Staining for live-cell imaging and visualization of cellular structures and protein localization Basic Protocol 3: Chemical fixation and immunofluorescence staining for visualization of cellular structures and protein localization Support Protocol 2: Preparation of reagents and materials for cell fixation and immunostaining.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 10","pages":"e70478"},"PeriodicalIF":2.5,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149221228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}