Current protocols最新文献

{"title":"Diversification of Nucleosides Via Radical Mediated Deoxygenative Functionalization","authors":"Maochao Zhou,&nbsp;Gang He","doi":"10.1002/cpz1.70468","DOIUrl":"10.1002/cpz1.70468","url":null,"abstract":"<p>In this article we describe a general method for the synthesis of nucleoside derivatives bearing alkyl, cyano, and phosphate groups at the 3'-position or 2'-position. As exampled by thymidine and adenosine, this approach involves the installation of an activating reagent, 1,3-benzodithiol-2-ylium tetrafluoroborate (BDTF), at the 3'-hydroxy group of thymidine and 2'-hydroxy group of adenosine under mild conditions. Subsequent visible light promoted single-electron oxidation and β-scission generates the key radical intermediate and afford functionalized nucleosides by reaction with a broad range of radical acceptors. This method has the advantages of excellent yields and high diastereoselectivity, offering a versatile platform for the synthesis of complex, drug-relevant nucleoside derivatives. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of 3'-<i>O-</i>BDT-thymidine and 2'-<i>O-</i>BDT-adenosine</p><p><b>Basic Protocol 2</b>: Deoxygenative functionalization of 3'-<i>O-</i>BDT-thymidine and 2'-<i>O-</i>BDT-adenosine</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042266","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":"An Integrated Platform for the Comprehensive Analysis of Host-Fungal Interactions in the Murine Tongue","authors":"Julia Lagler,&nbsp;Mathieu Courivaud,&nbsp;Johnny Bonnardel,&nbsp;Salomé LeibundGut-Landmann","doi":"10.1002/cpz1.70473","DOIUrl":"10.1002/cpz1.70473","url":null,"abstract":"<p>This protocol describes a mouse model of <i>Candida albicans</i> oral colonization that establishes stable, long-term fungal persistence in the oral cavity, thereby recapitulating key features of human commensal colonization, including the induction of sustained adaptive antifungal immunity. The model enables the analysis of host-fungal interactions across multiple experimental readouts. Procedures are provided for isolation of single-cell suspensions from the whole tongue, the tongue epithelium, and the draining lymph nodes for flow cytometric analysis, supporting both enumeration and extended functional characterization of immune cell populations. Antigen-specific T cell responses can be assessed by re-stimulation of isolated T cells with <i>C. albicans</i>-pulsed dendritic cells prior to staining and analysis. Additional methods include quantification of fungal burden in tongue tissue, histological assessment of fungal localization and immune cell infiltration, and isolation of high-quality RNA using an optimized extraction workflow compatible with RT-qPCR and RNA sequencing. Collectively, these protocols provide a flexible and integrated platform for studying immune responses and host-fungal interactions during persistent oral colonization. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Oral colonization of mice with <i>Candida albicans</i></p><p><b>Basic Protocol 2</b>: Preparation of single-cell suspensions from the murine whole tongue, tongue epithelium, and draining lymph nodes</p><p><b>Basic Protocol 3</b>: Re-stimulation of cLNs and tongue T cells for qualitative and quantitative assessment of T cell effector functions</p><p><b>Basic Protocol 4</b>: Flow-cytometry-based analysis of tongue immune cell populations</p><p><b>Support Protocol 1</b>: Evaluation of fungal burden in the tongue of <i>C. albicans</i>-colonized mice</p><p><b>Support Protocol 2</b>: Visualization of tissue integrity, tissue inflammation, and fungal localization by histology</p><p><b>Support Protocol 3</b>: Extraction of total RNA from the tongue of <i>C. albicans</i>-colonized mice for RNA sequencing and RT-qPCR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70473","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042941","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":"PDBe PISA: Enhanced Analysis of Macromolecular Interactions","authors":"Grisell Díaz Leines,&nbsp;Paulyna Magaña,&nbsp;Sreenath Nair,&nbsp;Jennifer Fleming,&nbsp;Mihaly Varadi,&nbsp;Eugene Krissinel,&nbsp;Sameer Velankar","doi":"10.1002/cpz1.70446","DOIUrl":"10.1002/cpz1.70446","url":null,"abstract":"<p>Protein interactions play a pivotal role in determining the biological functions of living cells. Gaining structural insights into protein interfaces can illuminate their role in function and diseases and highlight their potential as therapeutic targets. In response, there has been a marked increase in efforts by the scientific community to decipher the nuances of macromolecular interfaces, aiming to predict interactions and binding specificity of different components within complexes. The “Proteins, Interfaces, Structures, and Assemblies” (PISA) software has become a standard tool for analyzing intermolecular interactions and elucidating macromolecular assemblies from molecular structures. This article presents detailed protocols for using Protein Data Bank in Europe (PDBe) PISA API, a new Application Programming Interface (API) for enhanced analysis of protein assembly interfaces, using an updated version of PISA software. Compared to its predecessor, the updated PISA software expands interface data, increasing the coverage and detail of interaction information for assemblies and introduces greater computational flexibility, allowing users to perform targeted interface analyses for selected assemblies efficiently. The PDBe team uses the PISA API to provide interaction data in a JavaScript Object Notation (JSON) format for weekly Protein Data Bank (PDB) releases to support the efforts of the broader scientific community. Our protocols are a comprehensive guide to harnessing macromolecular interaction data using PISA. We delve into practical examples, from querying interaction and interface data from JSON files to extracting interaction data for assemblies cataloged in the PDB archive using programmatic access and bulk download via File Transfer Protocol (FTP). © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Programmatic access to interaction data with PDBe PISA API</p><p><b>Basic Protocol 2</b>: FTP access to interaction data of assemblies</p><p><b>Basic Protocol 3</b>: Analysis of assembly interfaces and interactions</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70446","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042509","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":"Preparation and Infection of Bovine Precision-Cut Lung Slices (b-PCLS) as an Ex Vivo Model for Respiratory Pathogens","authors":"Asal Ahmadi,&nbsp;Ghazal Ahmadi,&nbsp;Vilde Galleberg-Wahlberg,&nbsp;Abdolrahman Khezri,&nbsp;Veslemøy Sunniva Oma,&nbsp;Jochen Meens,&nbsp;Mette Myrmel,&nbsp;Ann-Katrin Llarena","doi":"10.1002/cpz1.70459","DOIUrl":"10.1002/cpz1.70459","url":null,"abstract":"<p>Precision-cut lung slices (PCLS) represent a physiologically relevant <i>ex vivo</i> model that bridges the gap between simplified <i>in vitro</i> systems and <i>in vivo</i> animal studies by preserving native lung architecture, cellular diversity, and functional responses. This model provides a valuable platform for investigating respiratory tract infections and tissue-level host–pathogen interactions in bovine lung tissue, including those involving pathogens associated with bovine respiratory disease (BRD), a leading cause of morbidity, mortality, and economic loss in the global cattle industry. While PCLS protocols are well established for murine and human lungs, standardized and detailed methodologies optimized for bovine lung tissue remain scarce. Here, we present a comprehensive, reproducible protocol for generating, culturing, assessing viability, inoculating, and extracting RNA from bovine precision-cut lung slices (b-PCLS). The workflow includes lung inflation with 1.5% low-melting-point agarose, vibratome slicing, and culture conditions that preserve bronchiolar architecture and ciliary activity. Viability is assessed longitudinally using ciliary activity scoring and a metabolic assay, demonstrating that b-PCLS remain viable and highly active for at least 7 days in culture. We also describe inoculation procedures applicable to bacterial and viral pathogens relevant to BRD, exemplified by <i>Pasteurella multocida</i> and bovine coronavirus (BCoV), along with standardized sampling strategies for downstream analyses. Finally, we provide an optimized RNA extraction protocol that yields high-quality RNA from both control and inoculated b-PCLS, supporting downstream host and pathogen transcriptomic analyses. Collectively, these protocols expand the applicability of large-animal PCLS for research on respiratory infections, support the principles of replacement and reduction in animal experimentation, and provide a complementary platform for controlled investigation of tissue-level host–pathogen interactions in bovine lungs. Overall, this <i>ex vivo</i> system offers a valuable tool for bovine respiratory research, assay development, and molecular analysis. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation, maintenance, and viability assessment of b-PCLS</p><p><b>Basic Protocol 2</b>: Inoculation of b-PCLS with <i>Pasteurella multocida</i></p><p><b>Basic Protocol 3</b>: Inoculation of b-PCLS with bovine coronavirus</p><p><b>Basic Protocol 4</b>: Extraction of RNA from culture supernatants and b-PCLS</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70459","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042536","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":"Murine Model of Venous Bypass Grafting to Study Determinants of Graft Patency","authors":"Vanessa Heim,&nbsp;Maria Ioannou-Nikolaidou,&nbsp;Anh-Vu Nguyen,&nbsp;Lynn Muller,&nbsp;Veronika Niedrist,&nbsp;Jakob Hirsch,&nbsp;Jonas Eder,&nbsp;Dominik Hau,&nbsp;Leo Winter-Pölzl,&nbsp;Clemens Engler,&nbsp;Ronja Lohmann,&nbsp;Felix Nägele,&nbsp;Gerald Brandacher,&nbsp;Stefan Schneeberger,&nbsp;Nikolaos Bonaros,&nbsp;Michael Grimm,&nbsp;Can Gollmann-Tepeköylü,&nbsp;Michael Graber","doi":"10.1002/cpz1.70466","DOIUrl":"10.1002/cpz1.70466","url":null,"abstract":"<p>The investigation of patency and remodeling in venous grafts requires a reproducible animal model that enables the study of the underlying cellular and molecular processes. Here, we describe a simplified mouse model for venous bypass grafts, in which the inferior thoracic vena cava (IVC) of a donor mouse is interposed into the common carotid artery of a recipient mouse using a cuff technique. The procedure is divided into four basic protocols, which includes the harvesting of the inferior vena cava from donor mice (Basic Protocol 1), the preparation of the common carotid artery (Basic Protocol 2), the interposition of the inferior vena cava graft (Basic Protocol 3), and post-operative care (Basic Protocol 4). Detailed step-by-step instructions and an instructional video are provided to facilitate microsurgical training and to improve the reproducibility and accessibility of the procedure. This model enables the investigation of the patency and remodeling of venous grafts and provides a platform for researching the cellular and molecular mechanisms contributing to venous graft failure, as well as for evaluating potential therapeutic interventions. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Harvest of inferior caval vein from donor mice</p><p><b>Basic Protocol 2</b>: Preparation of the common carotid artery</p><p><b>Basic Protocol 3</b>: Inferior vena cava graft interposition</p><p><b>Basic Protocol 4</b>: Postoperative management</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600260/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031765","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":"Efficient, Capsule-Free Nymphal Ixodid Tick Placement for Site-Specific Attachment to Assess Anti-Tick Host Behavior and Immune Responses","authors":"Ronja A. Frigard,&nbsp;Serena Doh,&nbsp;Charles S. Grugan,&nbsp;Yanling Liu,&nbsp;Sara I. Mirbagheri,&nbsp;Luana A. Rogerio,&nbsp;Eva Iniguez,&nbsp;Tiago D. Serafim,&nbsp;Aline da Silva Moreira,&nbsp;Jack Collins,&nbsp;Daniel E. Sonenshine,&nbsp;Jesus G. Valenzuela,&nbsp;Johannes S. P. Doehl","doi":"10.1002/cpz1.70477","DOIUrl":"10.1002/cpz1.70477","url":null,"abstract":"<p>Ixodid ticks are among the most widespread and permissive groups of arthropod disease vectors in the world. They transmit various viral, bacterial, protozoan, and even nematode pathogens infective for humans and animals. Ticks are essential to studying pathogen transmission and infection in a new host; however, exposing in vivo models to ticks is very challenging. Most current tick placement protocols rely on capsules to increase attachment rates at a specific location. However, capsules interfere with hosts’ ability to engage with ticks by grooming/scratching. Conversely, available capsule-free tick infestation protocols, including free-roaming protocols, suffer from low and highly variable tick attachment efficacy. Here, we describe a highly efficient capsule-free nymphal tick placement protocol with tick attachment rates of ≥85% across various tick species and genera, offering the generation of highly reproducible data. This article also offers a set of protocols to the user primarily geared toward the study of itch-induced tick removal but are adaptable to other study settings. Published 2026. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Capsule-free, highly efficient multi-species nymphal tick placement for site-specific attachment</p><p><b>Basic Protocol 2</b>: Assessing efficacy of itch-induced tick removal by quantifying loss of ticks over time</p><p><b>Basic Protocol 3</b>: Assessment of health/ability of partially fed nymphal ticks to feed determined by scutal index</p><p><b>Basic Protocol 4</b>: Scratch bout quantification in guinea pigs</p><p><b>Support Protocol</b>: Development of a semi-supervised deep learning pipeline for automated detection and validation of scratching behavior</p><p><b>Basic Protocol 5</b>: Assessment of local skin inflammation due to immunity to tick bites using laser speckle contrast imaging</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600259/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031675","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":"Preparation of Acute Mouse Hippocampal Brain Sections for Transport to a Distant Electrophysiology Laboratory","authors":"Sofia Ioanna Nouka,&nbsp;Loukia Katsouri,&nbsp;Jonathan Rudge,&nbsp;John O'Keefe,&nbsp;Francesco Tamagnini","doi":"10.1002/cpz1.70461","DOIUrl":"10.1002/cpz1.70461","url":null,"abstract":"<p>This article describes a standardized method for preparing and transporting acute mouse hippocampal brain slices suitable for high-quality single-cell, CA1, pyramidal cells electrophysiological recordings between laboratories in departments or institutions located at distances up to 3 hr apart. The procedure emphasizes maintaining tissue viability and preserving physiological integrity during transport. Slices are prepared in sucrose-based protective solutions under ice-cold, oxygenated conditions, followed by a controlled rewarming period to restore metabolic activity. For transport, slices are submerged in oxygenated artificial cerebrospinal fluid (aCSF) contained in 250-ml parafilm-sealed Duran bottles, ensuring prolonged viability at near-ambient temperature. The method includes parameters for slice thickness, temperature control, oxygenation duration, and maximum transport time to ensure reproducibility across sites. Validation using patch-clamp recordings confirms that electrophysiological properties, such as resting membrane potential, input resistance, action potential firing, and spontaneous synaptic neurotransmission, remain comparable to those in freshly prepared slices. This protocol enables collaborative research where brain slice preparation and electrophysiological recordings occur in separate laboratories, while preserving the reliability and quality of single-cell functional data. This is particularly useful for laboratories located away from animal housing units. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Acute brain slice preparation available for transport</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13595899/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149019152","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":"Composable Visualization of High-Dimensional Biological Data with ggalign","authors":"Wanyi Liu,&nbsp;Jia Ding,&nbsp;Yujun Sun,&nbsp;Bin Yan,&nbsp;Zhangyu Wang,&nbsp;Chunyang Wang,&nbsp;Chenyang Shu,&nbsp;Jian-Guo Zhou,&nbsp;Guangchuang Yu,&nbsp;Yun Peng,&nbsp;Shixiang Wang","doi":"10.1002/cpz1.70453","DOIUrl":"10.1002/cpz1.70453","url":null,"abstract":"<p>ggalign is an R/CRAN package for creating flexible and composable multi-panel data visualizations. The package extends the ggplot2 grammar of graphics by introducing an integrative framework that supports both data-free and data-aware composition. After five years of continuous development, ggalign has evolved into a comprehensive solution that handles diverse data types and layout structures, including quad, circular, and stack layouts. It was originally designed for general-purpose composable visualization and has been expanded to support multi-omics data integration, extending the application of ggalign to pan-cancer analysis, single-cell transcriptomics, and microbiome studies. This article presents eight basic protocols for constructing complex visualizations using the declarative syntax of ggalign. Basic Protocol 1 describes data-free composition for flexible arrangement of multiple plots; Basic Protocol 2 describes data-aware quad layout for integrating a central plot with surrounding annotations; Basic Protocol 3 describes data-aware circular layout for visualizing ring-structured data; Basic Protocol 4 describes stack layout and nested composition for coordinated display of multi-track graphics; Basic Protocol 5 describes visualization of gene expression matrix heatmaps; Basic Protocol 6 describes visualization of somatic mutation landscapes using ggoncoplot(); Basic Protocol 7 describes circular visualization based on chromosome data, and Basic Protocol 8 describes cross-connection visualization between genes and pathways. The complete package reference is available at https://yunuuuu.github.io/ggalign/, with comprehensive documentation and tutorials at https://yunuuuu.github.io/ggalign-book/, and a gallery of example figures at https://yunuuuu.github.io/ggalign-gallery/. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Data-free composition</p><p><b>Basic Protocol 2</b>: Aligning data-aware with quad layouts</p><p><b>Basic Protocol 3</b>: Aligning data-aware with circular layouts</p><p><b>Basic Protocol 4</b>: Stack layouts and nested composition</p><p><b>Basic Protocol 5</b>: Visualizing heatmap of gene expression matrix</p><p><b>Basic Protocol 6</b>: Visualizing somatic mutation landscapes using ggoncoplot()</p><p><b>Basic Protocol 7</b>: Visualizing circos plots with ggalign</p><p><b>Basic Protocol 8</b>: Visualizing observational connections</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149013411","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":"Protoplasting and Transformation Using CRISPR/Cas9 in Coccidioides","authors":"Katrina M. Jackson,&nbsp;Matthew M. Morales,&nbsp;Edyta Szewczyk,&nbsp;Christina M. Homer,&nbsp;Ashley N. Itogawa,&nbsp;Rachel B. Brem,&nbsp;Anita Sil,&nbsp;Bridget M. Barker","doi":"10.1002/cpz1.70444","DOIUrl":"10.1002/cpz1.70444","url":null,"abstract":"<p><i>Coccidioides posadasii</i> and <i>C. immitis</i> are human fungal pathogens endemic to the American Southwest. <i>C. posadasii</i> and <i>C. immitis</i> are the causative agents of coccidioidomycosis, or Valley fever. They are pathogens of growing concern, as reported cases of coccidioidomycosis have increased 20-fold in the last two decades. Despite their importance as pathogens, <i>Coccidioides spp</i>. are understudied, especially compared to other human fungal pathogens with similar infectious burdens. The reasons <i>Coccidioides spp</i>. are understudied are multifactorial, including requirement of high biocontainment, technical difficulties in lab-based culture growth, and a historic lack of genetic tools. Previous methods of genetic manipulation have been technically challenging, time consuming, and inefficient. Here, we present protocols for designing gene deletion constructs, generating protoplasts from both genetically engineered biosafety level 2 (BSL2) and wildtype biosafety level 3 (BSL3) strains, and performing transformations with CRISPR/Cas9. The protoplasting protocol described here uses a cell wall digestion enzyme employed in the wine-making industry and results in high-quality protoplasts that have the potential to be used for applications beyond transformations. We also present a high-efficiency transformation method using CRISPR/Cas9. The protocols described here will allow for genetic manipulation of <i>Coccidioides</i>, using both BSL2 and BSL3 strains. This resource can be applied to expand research done in <i>Coccidioides spp</i>., build molecular tools, and expand overall knowledge of these important pathogens. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Construction of gene deletion construct</p><p><b>Alternate Protocol 1</b>: Construction of gene deletion construct</p><p><b>Basic Protocol 2</b>: Creation of protoplasts in BSL3</p><p><b>Alternate Protocol 2</b>: Creation of protoplasts in BSL2</p><p><b>Basic Protocol 3</b>: CRISPR/Cas9 transformation in BSL3</p><p><b>Alternate Protocol 3</b>: CRISPR/Cas9 transformation in BSL2</p><p><b>Support Protocol</b>: Passaging of mutants and PCR confirmation</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70444","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148993247","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":"Dataset Readiness Assessment With Large Language Model (DRAFT–LLM): A Multi-Axis Audit Guided by LLM","authors":"Guillaume Guerard,&nbsp;Sonia Djebali","doi":"10.1002/cpz1.70463","DOIUrl":"10.1002/cpz1.70463","url":null,"abstract":"<p>This article details the Dataset Readiness Assessment for Training (DRAFT), a systematic method for determining whether a high-dimensional biological dataset is suitable for developing reliable, equitable (i.e., the extent to which model performance, error patterns, and potential benefits or harms are evaluated and found to be acceptably distributed across relevant demographic, biological, clinical, and contextual subgroups), and scientifically meaningful machine-learning models, and DRAFT Large Language Model (DRAFT–LLM), its optional human-in-the-loop extension for calibrating study-specific audits through structured, critically reviewed LLM guidance. Standard model validation often fails to detect when apparent performance is driven by spurious correlations, technical artifacts, or hidden stratification, leading to irreproducible and inequitable findings. DRAFT–LLM addresses this gap by shifting the focus from model tuning to structured dataset auditing, organized around Support Protocols 1 to 4 that capture the scientific intent, data structure, and governance constraints of a given study. These Support Protocols: (1) elicit and formalize investigator input into a study intake and dataset card; (2) compute standardized dataset statistics and structural summaries suitable for downstream analysis and LLM context; (3) configure the language model using form-based responses, safety guardrails, and governance rules; and (4) generate personalized instructions, prompts, and code templates for running DRAFT audits. Basic Protocols 1 to 3 are instantiated from this support layer for generalization, equity, and stability: they are reusable execution patterns whose concrete behavior is determined by the cards, statistics, and configurations defined in the Support Protocols. DRAFT–LLM and DRAFT are demonstrated in this article through an end-to-end case study on The Cancer Genome Atlas (TCGA). © 2026 Wiley Periodicals LLC.</p><p><b>Support Protocol 1</b>: Study intake and dataset card construction</p><p><b>Support Protocol 2</b>: Dataset structure and advanced summary statistics for LLM context</p><p><b>Support Protocol 3</b>: LLM configuration using structured form responses</p><p><b>Support Protocol 4</b>: Generation of personalized instructions for DRAFT audits</p><p><b>Basic Protocol 1</b>: Generalization audit</p><p><b>Basic Protocol 2</b>: Equity audit</p><p><b>Basic Protocol 3</b>: Stability audit</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148993190","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}