Current protocols最新文献

{"title":"Development, Characterization, and Therapeutic Utility of Paclitaxel-Resistant Breast and Gastric Cancer In Vitro and In Vivo Models","authors":"Feng Tang,&nbsp;Hong Xu,&nbsp;Wen Cui,&nbsp;Xuelin Wang,&nbsp;Fangyi Ding,&nbsp;Yihan Zhang,&nbsp;Xiangnan Qiang,&nbsp;Qingyang Gu,&nbsp;Dong Wang,&nbsp;Zhixiang Zhang","doi":"10.1002/cpz1.70113","DOIUrl":"https://doi.org/10.1002/cpz1.70113","url":null,"abstract":"<p>Paclitaxel, one of the most commonly used anticancer agents, is employed in the treatment of a range of malignant tumors. However, resistance is one of the major barriers to successful therapy. Despite its clinical relevance, the molecular mechanisms underlying paclitaxel resistance remain poorly understood. In this protocol, we describe the methods for establishing paclitaxel-resistant tumor models both in vitro and in vivo, and how we investigated the underlying mechanisms of resistance. Additionally, we evaluated the potential of combination therapies to overcome paclitaxel resistance in these models. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of paclitaxel-resistant breast cancer model in vivo.</p><p><b>Basic Protocol 2</b>: Generation of paclitaxel-resistant gastric cancer model in vitro.</p><p><b>Basic Protocol 3</b>: Validation of drug resistance in vivo.</p><p><b>Basic Protocol 4</b>: In vitro and in vivo evaluation of combination therapy in paclitaxel-resistant models.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143534038","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":"Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)","authors":"Paolo Cadinu,&nbsp;Evan Yang,&nbsp;Rosalind J. Xu,&nbsp;Brianna R. Watson,&nbsp;Josh Luce,&nbsp;Jeffrey R. Moffitt","doi":"10.1002/cpz1.70111","DOIUrl":"https://doi.org/10.1002/cpz1.70111","url":null,"abstract":"<p>Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is a massively multiplexed single RNA–molecule imaging technique capable of spatially resolved single-cell transcriptomic profiling of thousands of genes in millions of cells within intact tissue slices. Initially introduced for brain tissues, MERFISH has since been extended to other tissues, where rapid RNA degradation during the preparation process can pose challenges. This protocol outlines the application of MERFISH in one such challenging tissue, the mammalian gastrointestinal tract. We describe two complementary protocols leveraging either fresh frozen or fixed frozen approaches and describe methods for combining RNA imaging with immunofluorescence. While these protocols were designed and validated in gut tissues, we anticipate that they will be useful resources for the application to other challenging tissue types. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Fixed-frozen sample preparation</p><p><b>Basic Protocol 2</b>: Fresh-frozen sample preparation</p><p><b>Basic Protocol 3</b>: Encoding probe construction</p><p><b>Basic Protocol 4</b>: MERFISH imaging</p><p><b>Basic Protocol 5</b>: Image decoding</p><p><b>Support Protocol 1</b>: Coverslip silanization</p><p><b>Support Protocol 2</b>: Poly-<span>d</span>-lysine (PDL) coating of the coverslips</p><p><b>Support Protocol 3</b>: Hybridization buffer preparation</p><p><b>Support Protocol 4</b>: Trolox quinone stock preparation</p><p><b>Support Protocol 5</b>: TCEP stock preparation</p><p><b>Alternate Protocol 1</b>: MERFISH-compatible immunofluorescent boundary stains in fresh frozen tissue</p><p><b>Alternate Protocol 2</b>: Immunofluorescent boundary stains with methacrylate-NHS-anchored antibodies for PFA-fixed samples</p><p><b>Alternate Protocol 3</b>: Guanidine-HCl tissue clearing</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143535951","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":"Visualization of Efavirenz-Induced Lipid Alterations in the Mouse Brain Using MALDI Mass Spectrometry Imaging","authors":"Nav Raj Phulara,&nbsp;Herana Kamal Seneviratne","doi":"10.1002/cpz1.70108","DOIUrl":"https://doi.org/10.1002/cpz1.70108","url":null,"abstract":"<p>This article highlights experimental procedures and troubleshooting tips for the utilization of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) methods for detecting and visualizing lipid alterations in the mouse brain tissue in response to efavirenz (EFV) treatment. To investigate drug-induced adverse effects, it is becoming increasingly important to understand the spatial alterations of lipid molecules in the target organs. EFV is a non-nucleoside reverse transcriptase inhibitor commonly used for HIV treatment in combination with other antiretrovirals. Importantly, EFV is a drug that is included in the World Health Organization's list of essential medications. However, EFV is known to be associated with neurotoxicity. To date, the mechanisms underlying EFV-induced neurotoxicity have not been fully elucidated. Therefore, it is important to gain understanding of the effect of EFV on the brain. It is known that the brain is composed of different neuroanatomical regions that are abundant in lipids. Described here is the use of a chemical imaging strategy, MALDI MSI, to detect, identify, and visualize the spatial localization of several lipid species across the brain tissue sections along with their alterations in response to EFV treatment. The set of protocols consists of three major parts: lipid detection, identification, and tissue imaging. Lipid detection includes testing different chemical matrices and how they facilitate the detection of analytes, which is then followed by identification. Collision-induced dissociation is employed to verify the identity of the lipid molecules. Lastly, tissue imaging experiments are performed to generate the spatial localization profiles of the lipids. The protocols described in this article can be employed to spatially visualize alterations in the lipid molecules in response to drug treatment. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: MALDI mass spectrometry (MALDI MS) profiling experiments for detection of lipids</p><p><b>Basic Protocol 2</b>: MALDI MS imaging of lipid molecules in mouse brain tissues</p><p><b>Basic Protocol 3</b>: MALDI MS data processing and analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143490037","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":"Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease","authors":"Marina Gertsenstein,&nbsp;Lauri G. Lintott,&nbsp;Lauryl M. J. Nutter","doi":"10.1002/cpz1.70109","DOIUrl":"https://doi.org/10.1002/cpz1.70109","url":null,"abstract":"<p>Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence—the orthologous sequence for base changes or the N or C terminus for epitope tags—appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Single guide RNA design and synthesis</p><p><b>Alternate Protocol 1</b>: Single guide RNA synthesis by primer extension and in vitro transcription</p><p><b>Basic Protocol 2</b>: Design of oligonucleotide repair template</p><p><b>Basic Protocol 3</b>: Preparation of RNA mixture for microinjection</p><p><b>Support Protocol 1</b>: Preparation of microinjection buffer</p><p><b>Alternate Protocol 2</b>: Preparation of RNP complexes for electroporation</p><p><b>Basic Protocol 4</b>: Collection and preparation of mouse zygotes for microinjection or electroporation</p><p><b>Basic Protocol 5</b>: Electroporation of Cas9 RNP into zygotes using cuvettes</p><p><b>Alternate Protocol 3</b>: Electroporation of Cas9 RNP into zygotes using electrode slides</p><p><b>Basic Protocol 6</b>: Screening and quality control of derived mice</p><p><b>Support Protocol 2</b>: Deconvoluting multiple sequence chromatograms with DECODR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143489743","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":"Development of Syngeneic Murine Glioma Models with Somatic Mismatch Repair Deficiency to Study Therapeutic Responses to Alkylating Agents and Immunotherapy","authors":"Deepti Bhatt,&nbsp;Ranjini K. Sundaram,&nbsp;Karla S. Lugo López,&nbsp;Teresa Lee,&nbsp;Susan E. Gueble,&nbsp;Juan C. Vasquez","doi":"10.1002/cpz1.70097","DOIUrl":"https://doi.org/10.1002/cpz1.70097","url":null,"abstract":"<p>Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA <i>O</i>⁶-methylguanine (O⁶MeG) lesions. Without O⁶MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited <i>in vitro</i> and <i>in vivo</i> resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Validation of mismatch repair knockouts and <i>in vitro</i> sensitivity to alkylating agents</p><p><b>Basic Protocol 2</b>: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and <i>in vivo</i> treatment</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481527","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":"Quantification of Glycosaminoglycans in Urine by Isotope Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry","authors":"Haoyue Zhang,&nbsp;James Beasley,&nbsp;Iskren Menkovic,&nbsp;Ashlee Stiles,&nbsp;David S. Millington,&nbsp;Sarah P. Young","doi":"10.1002/cpz1.70110","DOIUrl":"https://doi.org/10.1002/cpz1.70110","url":null,"abstract":"<p>Mucopolysaccharidoses (MPSs) are complex lysosomal diseases that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic/iduronic acid-<i>N</i>-acetylhexosamine or uronic/iduronic acid-<i>N</i>-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Urinary GAG analysis by ESI-MS/MS</p><p><b>Support Protocol 1</b>: Prepare calibration samples</p><p><b>Support Protocol 2</b>: Preparation of stable-isotope-labeled internal standards</p><p><b>Support Protocol 3</b>: Preparation of quality controls for GAG analysis in urine</p><p><b>Support Protocol 4</b>: Optimization of methanolysis time</p><p><b>Support Protocol 5</b>: Measurement of methanolic HCl concentration</p><p><b>Support Protocol 6</b>: Preparation of working methanolic HCl solution (1.1 M)</p><p><b>Support Protocol 7</b>: Dilution of prepared urine sample</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481528","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":"Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro","authors":"Maria A. Beamer,&nbsp;Saori Furuta","doi":"10.1002/cpz1.70107","DOIUrl":"https://doi.org/10.1002/cpz1.70107","url":null,"abstract":"<p>Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined “3D Flipwell system” using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH₄). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: 3D Flipwell construction, assembly, and collagen coating</p><p><b>Basic Protocol 2</b>: Flipwell cell seeding and cell culture</p><p><b>Basic Protocol 3</b>: Addition of bacterial culture to the Flipwell system</p><p><b>Basic Protocol 4</b>: Flipwell disassembly for scanning electron microscopy (SEM) studies</p><p><b>Basic Protocol 5</b>: Immunofluorescence antibody staining for confocal microscopy</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143431322","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":"Enhancing Reproducibility in Chemotaxis Assays for Caenorhabditis elegans","authors":"Leona Cesar,&nbsp;Julia Morud","doi":"10.1002/cpz1.70106","DOIUrl":"https://doi.org/10.1002/cpz1.70106","url":null,"abstract":"<p>The ability of <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) to navigate complex environments is essential for their survival. This natural behavior is commonly used in chemotaxis assays, which are important tools for studying the function of sensory neurons and neural circuits. Chemotaxis has been essential for discovering fundamental functions in neuronal signaling during the past decades. However, a lack of thoroughly optimized and standardized procedures can lead to variable results that can be difficult to interpret. To improve reproducibility, we optimized several aspects of chemotaxis protocols by testing different odorant concentrations, numbers of worms, and assay durations, as well as the preparation of chemotaxis plates and the washing procedures of worms. The usage of a 2-choice or a 4-choice assay was also evaluated. Our new protocol improves the clarity of results and simplifies worm counting. The protocol optimization is condensed into a 5-day step-by-step protocol that increases the reproducibility of chemotaxis in <i>C. elegans</i>. Compared to previously published chemotaxis protocols, the revised method reduces day-to-day variability using an improved and standardized assay design that ensures clear and reliable results. Several key components in the assay preparation and during the assay have been evaluated based on previous protocols, such as odor concentration, worm density, and assay length. By considering multiple factors that influence the worm's behavior, our optimized protocol enhances the reproducibility of chemotaxis assays in <i>C. elegans</i>, making them more reliable and accessible for studying phenotypes related to olfaction and neural circuit behavior. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Optimized Chemotaxis Assay for <i>C. elegans</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143431321","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":"Workflow4Metabolomics (W4M): A User-Friendly Metabolomics Platform for Analysis of Mass Spectrometry and Nuclear Magnetic Resonance Data","authors":"Cédric Delporte,&nbsp;Marie Tremblay-Franco,&nbsp;Yann Guitton,&nbsp;Cécile Canlet,&nbsp;Ralf J. M. Weber,&nbsp;Helge Hecht,&nbsp;Elliott James Price,&nbsp;Jana Klánová,&nbsp;Charlotte Joly,&nbsp;Céline Dalle,&nbsp;Julien Saint-Vanne,&nbsp;Etienne Thévenot,&nbsp;Isabelle Schmitz,&nbsp;Sylvain Chéreau,&nbsp;Sylvain Dechaumet,&nbsp;Binta Diémé,&nbsp;Franck Giacomoni,&nbsp;Gildas Le Corguillé,&nbsp;Mélanie Pétéra,&nbsp;Florence Souard","doi":"10.1002/cpz1.70095","DOIUrl":"10.1002/cpz1.70095","url":null,"abstract":"<p>Various spectrometric methods can be used to conduct metabolomics studies. Nuclear magnetic resonance (NMR) or mass spectrometry (MS) coupled with separation methods, such as liquid or gas chromatography (LC and GC, respectively), are the most commonly used techniques. Once the raw data have been obtained, the real challenge lies in the bioinformatics required to conduct: (i) data processing (including preprocessing, normalization, and quality control); (ii) statistical analysis for comparative studies (such as univariate and multivariate analyses, including PCA or PLS-DA/OPLS-DA); (iii) annotation of the metabolites of interest; and (iv) interpretation of the relationships between key metabolites and the relevant phenotypes or scientific questions to be addressed. Here, we will introduce and detail a stepwise protocol for use of the Workflow4Metabolomics platform (W4M), which provides user-friendly access to workflows for processing of LC–MS, GC–MS, and NMR data. Those modular and extensible workflows are composed of existing standalone components (e.g., XCMS and CAMERA packages) as well as a suite of complementary W4M-implemented modules. This tool suite is accessible worldwide through a web interface and is hosted on UseGalaxy France. The extensible Virtual Research Environment (VRE) provided offers pre-configured workflows for metabolomics communities (platforms, end users, etc.), as well as possibilities for sharing among users. By providing a consistent ecosystem of tools and workflows through Galaxy, W4M makes it possible to process MS and NMR data from hundreds of samples using an ordinary personal computer, after step-by-step workflow optimization. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: W4M account creation, working history preparation, and data upload</p><p><b>Support Protocol 1</b>: How to prepare an NMR zip file</p><p><b>Support Protocol 2</b>: How to convert MS data from proprietary format to open format</p><p><b>Support Protocol 3</b>: How to get help with W4M (IFB forum) and how to report a problem on the GitHub repository</p><p><b>Basic Protocol 2</b>: LC–MS data processing</p><p><b>Alternate Protocol 1</b>: GC–MS data processing</p><p><b>Alternate Protocol 2</b>: NMR data processing</p><p><b>Basic Protocol 3</b>: Statistical analysis</p><p><b>Basic Protocol 4</b>: Annotation of metabolites from LC–MS data</p><p><b>Alternate Protocol 3</b>: Annotation of metabolites from NMR data</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416477","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":"Monitoring Mouse Surface Temperature During Stress with a Thermal Camera: A Low-Cost Infrared Videography System for Evaluating Murine Metabolism","authors":"Breanna Page,&nbsp;Carolina Cora,&nbsp;James Reilly,&nbsp;Ryan Reno,&nbsp;Wadak Harbi,&nbsp;Maureen S. Lynes,&nbsp;Michael A. Lynes,&nbsp;Matthew D. Lynes","doi":"10.1002/cpz1.70098","DOIUrl":"https://doi.org/10.1002/cpz1.70098","url":null,"abstract":"<p>Energy is required for life, and organisms obtain their energy from fuel sources to enable both anabolic and catabolic processes. Some of this energy is radiated as heat, which can be quantified as a measure of metabolic rate. In some cases, environmental toxicants can alter metabolic energy in undesirable ways, and characterization of new pharmaceuticals can determine the efficacy of desirable metabolic rate manipulation or identify off-target adverse effects. Current methods to directly measure heat production in laboratory mice are expensive, can be laborious, and make it challenging to monitor animals in ways that are multiplexed, robust, and non-invasive. We present a set of protocols for assembling and deploying a simple, low-cost thermal camera to monitor and record thermogenic activity, modified from prior work. Parts used to build this system currently cost approximately $150 USD and, when assembled, can record mouse temperatures as well as ambient cage temperatures up to twice per second for extended periods. By using multiplexed cameras in a diurnal mouse incubator system, the thermogenic capacity of several mice can be simultaneously recorded and graphed. Exogenous agents and genotypes that alter metabolism can be readily identified with this technology. In this set of protocols, we describe the assembly of the thermal video camera device, its use, and related data capture and analysis methods. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Assembling thermal camera for thermogenic stress test</p><p><b>Basic Protocol 2</b>: <i>In vivo</i> measurement of mouse temperature under different ambient conditions</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70098","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143396822","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}