Current protocols最新文献

{"title":"A Novel NGS-Based Algorithm for Precise HPV Genotyping and Co-infection Detection in QCMD Samples and FFPE Tissues","authors":"Natalia Kuriata,&nbsp;Katarzyna Zofia Mazur,&nbsp;Magnús Máni Sæmundarson,&nbsp;Zarko Urosevic,&nbsp;Freyja Valsdóttir,&nbsp;Julia Yanzhong Xiao,&nbsp;Brynja Ármannsdóttir,&nbsp;Guðrún Erna Baldvinsdóttir,&nbsp;Arthur Löve,&nbsp;Ásgeir Erlendur Ásgeirsson,&nbsp;Huiping Chen","doi":"10.1002/cpz1.70231","DOIUrl":"https://doi.org/10.1002/cpz1.70231","url":null,"abstract":"<p>Accurate genotyping of Human papillomavirus (HPV) is essential for cervical cancer screening, prognostic risk assessment, and epidemiological surveillance. Conventional methods, such as Sanger sequencing and hybridization-based assays, often fail to detect mixed infections, particularly in cases involving DNA from formalin-fixed paraffin-embedded (FFPE) tissues. In this protocol, we present an optimized next-generation sequencing (NGS) based pipeline for comprehensive HPV genotyping. Our workflow includes a curated HPV reference database of 229 HPV genotypes and a proportional read-mapping algorithm. We validated our method using 30 samples from the Quality Control for Molecular Diagnostics (QCMD) proficiency testing panel and were able to fully replicate the expected outcomes—including detecting the defined co-infection with HPV16/18. We then applied our approach to 50 clinical FFPE samples, identifying 44 HPV-positive samples, including a co-infection scenario that traditional diagnostic assays might have missed. This low-cost, open-source NGS-based pipeline improves the sensitivity and accuracy of HPV detection from archival FFPE tissue specimens, cervical screening specimens, and other fresh tissue, and successfully closes the gap between research applications and clinical usage. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: DNA isolation</p><p><b>Basic Protocol 2</b>: PCR amplification</p><p><b>Basic Protocol 3</b>: NGS</p><p><b>Basic Protocol 4</b>: Data analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145272926","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":"Bilateral and Unilateral Renal Ischemia-Reperfusion Injury Model in Rats","authors":"Murat Çakır","doi":"10.1002/cpz1.70227","DOIUrl":"10.1002/cpz1.70227","url":null,"abstract":"<p>Renal ischemia-reperfusion injury (IRI), one of the most important causes of acute kidney injury, is a significant health problem that can result in acute kidney failure after surgical procedures in which blood flow to the kidney is temporarily interrupted. Although numerous studies have been conducted to prevent renal IRI damage, this problem remains unresolved due to its complex nature. During IRI, both ischemia and reperfusion contribute to damage, with oxidative stress, inflammation, and the activation of apoptotic mechanisms playing significant roles in exacerbating the damage. This article provides comprehensive protocols for inducing renal IRI in rats, including both bilateral and unilateral models. These protocols can be used in studies designed to understand the cellular and molecular mechanisms of renal IRI and exploring potential therapeutic strategies. When the described procedure is carefully applied in experimental IRI models, the mortality rate in animals will be lower. Furthermore, we outline opportunities for investigating oxidative stress, inflammatory and apoptotic pathways, and highlight how these analyses may contribute to preclinical therapeutic studies. We also provide recommendations on which analyses should be performed in such studies. Specifically, information is provided on which kidney damage biomarkers should be examined in samples obtained from animals. Additionally, we explain which investigations can be performed on apoptotic and inflammatory pathways involved in kidney damage. In summary, this study integrates bilateral and unilateral approaches and provides a detailed methodological framework for mechanistic and therapeutic research on renal ischemia-reperfusion injury. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Bilateral kidney ischemia-reperfusion in rats</p><p><b>Alternate Protocol</b>: Unilateral kidney ischemia-reperfusion in rats</p><p><b>Basic Protocol 2</b>: Analyses performed on kidney tissues and serum of rats after ischemia-reperfusion</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145254044","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":"µMap Photoproximity Labeling on the Cell Surface","authors":"Hong Kai Ng,&nbsp;Cameron J. Douglas,&nbsp;Ciaran P. Seath","doi":"10.1002/cpz1.70216","DOIUrl":"10.1002/cpz1.70216","url":null,"abstract":"<p>Understanding a protein's interactome provides crucial insight into its function and its contribution to disease. Traditional methods such as co-immunoprecipitations often fail to capture interactions that are dependent on native cellular architecture such as those found on the cellular membrane. While enzyme-based proximity labeling utilizing peroxidases or biotin ligases can achieve in situ interactome mapping, these approaches are limited by labeling radii, cellular engineering, and amino acid labeling biases. Antibody-guided µMap photoproximity labeling addresses the constraints of these alternative platforms. Here, we apply µMap photoproximity labeling to study the interactome of HER2 by using antibody-guided proximity labeling to target the endogenous protein, ablating the need for cellular engineering, and leveraging the advantages of µMap's short 4-nm labeling radius. The protocols presented here describe the preparation of iridium-antibody conjugates and its application in studying protein interactomes through mass-spectrometry based analysis. While HER2 was used as a model in this article, this method is broadly applicable and can be used to study any cell surface protein with an appropriate commercially available antibody. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation and validation of iridium-antibody conjugates</p><p><b>Basic Protocol 2</b>: Proximity labeling and streptavidin enrichment for mass spectrometry</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12507076/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145254021","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":"Integration of Primary Murine Osteocytes for In Vitro Mechanobiology Studies","authors":"Kimberly Seaman,&nbsp;Chun-Yu Lin,&nbsp;Xin Song,&nbsp;Amel Sassi,&nbsp;William W. Du,&nbsp;Yu Sun,&nbsp;Burton Yang,&nbsp;Lidan You","doi":"10.1002/cpz1.70217","DOIUrl":"10.1002/cpz1.70217","url":null,"abstract":"<p>This article describes the isolation, culture, and integration of primary murine osteocytes on a microfluidic platform for in vitro mechanobiology studies. Osteocytes are mechanosensitive cells embedded within the bone matrix and regulate bone remodeling. The MLO-Y4 osteocyte-like cell line typically used for mechanobiology studies can provide a good physiological response to mechanical stimulation but has been shown to have missing or low levels of key osteocyte markers found in vivo, such as sclerostin. However, primary osteocytes are difficult to isolate and study in vitro due to their location within the bone matrix, and the scope of existing protocols for osteocyte extraction only includes the isolation of primary cells using collagenase. Here, optimized protocols for the isolation, culture, and real-time calcium imaging of primary osteocytes in response to oscillatory fluid flow are outlined. Moreover, we discuss how to incorporate and maintain primary osteocytes on a physiologically relevant microfluidic platform for in vitro bone metastasis studies. In calcium imaging and microfluidic experiments, primary osteocytes are compared to the MLO-Y4 cell line as a control or reference group. Primary osteocytes can be extracted in 6 to 8 hr and typically require 1 week to recover before use in experiments. Calcium imaging of primary osteocytes can be completed in 24 hr. The time required for microfluidic culture varies on the platform used; the microfluidic experiment described here takes 6 days to complete. We anticipate that the information from this protocol will aid other researchers with incorporating primary osteocytes to enhance the biological relevance of in vitro studies on bone mechanobiology and disease. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of primary murine osteocytes using Liberase TM</p><p><b>Basic Protocol 2</b>: Primary osteocyte subculture and E11/podoplanin staining</p><p><b>Basic Protocol 3</b>: Real-time calcium imaging of primary osteocytes</p><p><b>Basic Protocol 4</b>: Integration of primary murine osteocytes onto a microfluidic platform for observing bone metastasis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12507077/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145254149","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":"Computational Tools to Analyze the Pathogenicity and Drug Sensitivity of Oncogenic Mutants.","authors":"Sai Charitha Mullaguri, Sravani Akula, Rama Krishna Kancha","doi":"10.1002/cpz1.70233","DOIUrl":"https://doi.org/10.1002/cpz1.70233","url":null,"abstract":"<p><p>Large-scale genomics efforts led to the identification of an increasing number of mutations in various cancers. However, the functional role of a vast majority of these mutations in disease pathogenesis remains unknown. For enzymes whose activity can be blocked by approved drugs, knowledge regarding the effect of novel or uncommon mutations on inhibitor sensitivity helps in opting for effective treatment strategies. However, it is impossible to experimentally evaluate pathogenic effect and drug sensitivity for all mutations that are being identified in multiple diseases. Therefore, computational predictions of pathogenicity and drug sensitivity can potentially help in the design of an individualized treatment approach. This article includes computational methods to: (a) predict the pathogenicity of mutations based on primary and tertiary structures of the target enzyme, (b) study the effect of mutations on protein conformation, and (c) predict the binding affinity of mutant structures towards targeted therapeutics. All the methods utilize freely available computational tools and have considerable translational value in improving patient outcomes with targeted therapy. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Pathogenicity prediction of mutations based on primary and tertiary structures Basic Protocol 2: Homology modeling of mutant protein structures Basic Protocol 3: Understanding the effect of mutations on protein conformation Basic Protocol 4: Predicting the binding affinities of mutant proteins towards specific inhibitors.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":"e70233"},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282299","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":"Use of a Dihydroxyacetone Derivative as Protecting Reagent to Phosphorylate Oligonucleotides","authors":"Rémy Lartia","doi":"10.1002/cpz1.70215","DOIUrl":"https://doi.org/10.1002/cpz1.70215","url":null,"abstract":"<p>We present here a new reagent enabling the supported synthesis of oligodeoxynucleotides (ODNs) and oligoribonucleotides (ORNs) containing a phosphate group at the 5′-terminal position after complete deprotection. This reagent, derived from a dihydroxyacetone core, contains a dimethoxytrityl (DMTr) group. The procedure for final deprotection is very similar to that routinely used in the synthesis of unmodified ODNs or ORNs. In particular, it preserves the advantages of the “trityl-on” method, namely facile purification by reverse-phase high-performance liquid chromatography (RP-HPLC), short treatment with an acetic acid solution in water, the possibility of on-resin monitoring by trityl cation assay, and the use of equipment commonly founded in chemical laboratories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Phosphorylation of oligodeoxyribonucleotides (ODNs)</p><p><b>Alternate Protocol</b>: Phosphorylation of oligoribonucleotides (ORNs)</p><p><b>Support Protocol</b>: Preparation of phosphorylation reagent <b>1</b></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70215","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145196472","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":"Autoantigen-Humanized Mouse Models of Bullous Pemphigoid.","authors":"Takuya Kawamura, Hideyuki Ujiie","doi":"10.1002/cpz1.70225","DOIUrl":"https://doi.org/10.1002/cpz1.70225","url":null,"abstract":"<p><p>Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by tense blisters and itchy erythema, predominantly affecting elderly individuals. The pathogenic autoantibodies mainly target collagen XVII (COL17), leading to subepidermal blister formation and infiltrations of immune cells, including eosinophils and neutrophils. Although systemic oral corticosteroids remain the mainstay of treatment, their use in elderly patients is often limited by serious complications and adverse effects, highlighting the unmet need for novel therapeutic targets. Several mouse models for BP have been reported; however, the inconsistency of disease induction has hindered therapeutic investigations. The active BP mouse model has emerged as a reliable system that recapitulates key disease features, making it valuable for both preclinical therapeutic studies and elucidation of BP pathophysiology. Furthermore, autoantigen-humanized mouse models, including the active BP model and the neonatal passive IgG transfer model, provide significant advantages for the development of antigen-specific therapies. Here, we describe detailed materials and methods for mouse models for BP using the COL17-humanized mouse, including the active BP mouse model and the neonatal passive IgG transfer model. Protocol modifications may be necessary when using different donor mice and antibodies. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Active BP mouse model Basic Protocol 2: Passive IgG transfer mouse model using neonatal mice.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":"e70225"},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282294","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":"Design and Analysis of Untargeted Metabolomics Experiments.","authors":"Zane G Long, Rachel W Martin","doi":"10.1002/cpz1.70232","DOIUrl":"https://doi.org/10.1002/cpz1.70232","url":null,"abstract":"<p><p>Untargeted metabolomics is a powerful approach for identifying small molecules from highly complex mixtures, such as biological tissues or environmental samples. This technology enables the relatively fast and inexpensive identification of metabolites in situations where many or most of the chemical species are unknown before the experiment begins. This situation often arises in biomedical and environmental research, as well as in the case described here, the discovery of metabolites from plants. The objective of this paper is to provide practical and technical knowledge about untargeted metabolomics using mass spectrometry as the detection method. Specifically, we focus on liquid chromatography tandem mass spectrometry (LC-MS/MS). We provide a consolidated protocol for new users, serving as a starting point for experimental design, data collection, and data analysis. We explain the terminology and technical details in the context of real experiments and samples. In addition to general background information, step-by-step protocols are provided for sample preparation, liquid chromatography-tandem mass spectrometry data collection, and data analysis, utilizing readily available and widely used software. The chosen example data set is based on plant metabolites with varying chemical properties; however, the approach is applicable to essentially any complex biological sample. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for LC-MS/MS Support Protocol 1: Preparing a 'master mix' sample for assessment of liquid chromatography and sensitivity Basic Protocol 2: LC-MS/MS data collection Basic Protocol 3: Data analysis using the software MSConvert, MZMine, and SIRIUS Support Protocol 2: Using the MZMine batch file.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":"e70232"},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282348","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":"Automated Deep Learning-Based Pipelines for Multi-Objective De Novo Protein Design.","authors":"Amrita Nallathambi, Brian Kuhlman","doi":"10.1002/cpz1.70208","DOIUrl":"https://doi.org/10.1002/cpz1.70208","url":null,"abstract":"<p><p>Computational protein design has been transformed by deep learning models that can accurately predict protein structure and generate sequences compatible with desired folds. Here we present a detailed protocol for EvoPro, an automated platform that uses a genetic algorithm along with iterative structure prediction (AlphaFold2/AlphaFold3) and sequence design (ProteinMPNN/LigandMPNN) to engineer protein-protein interactions with customizable properties. The protocol describes how to implement multistate design objectives to simultaneously optimize positive and negative design goals. We provide step-by-step instructions for setting up the genetic algorithm, configuring scoring functions for different design challenges, and analyzing results. The method builds on our previously validated approach, which successfully generated high-affinity binding domains without requiring experimental optimization. We describe key considerations for adapting the protocol to diverse protein engineering objectives, including binding site targeting, conformational specificity, and symmetric assembly. The complete computational protocol can be installed and executed in a week by a new user and provides a framework for leveraging deep learning models to address challenging protein design problems. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing protein binders Basic Protocol 2: Engineering conformational switches Basic Protocol 3: Designing de novo homo-oligomers Support Protocol 1: Setting up the EvoPro code and environment Support Protocol 2: Input preparation for different design scenarios Support Protocol 3: Optimizing the scoring function and other parameters.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":"e70208"},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282346","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":"Humanized Mouse Models for Type 1 Diabetes.","authors":"David V Serreze, Marissa Tousey-Pfarrer, Jeremy J Racine","doi":"10.1002/cpz1.70224","DOIUrl":"https://doi.org/10.1002/cpz1.70224","url":null,"abstract":"<p><p>T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 10","pages":"e70224"},"PeriodicalIF":2.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282367","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}