Current protocols最新文献

{"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}

{"title":"Efficient Site-Directed Mutagenesis Mediated by Primer Pairs with 3’-Overhangs","authors":"Negar Mousavi,&nbsp;Ethan Zhou,&nbsp;Arezousadat Razavi,&nbsp;Elham Ebrahimi,&nbsp;Paulina Varela-Castillo,&nbsp;Xiang-Jiao Yang","doi":"10.1002/cpz1.70104","DOIUrl":"https://doi.org/10.1002/cpz1.70104","url":null,"abstract":"<p>Site-directed mutagenesis is an essential tool in molecular biology, protein engineering, plasmid engineering and synthetic biology. While the QuickChange method has been one of the most employed methods for site-directed mutagenesis, it is hindered by low efficiency and frequent introduction of unwanted mutations at the primer sites, raising the urgent need for new, more efficient, and reliable methods. Here, we present an optimized site-directed mutagenesis protocol that leverages partially complementary primer pairs with 3’-overhangs to improve mutagenesis efficiency and reduce error rates. Our method significantly enhances success rates, achieving an average efficiency of ∼50% with some instances approaching the ideal threshold of 100%, while also minimizing the time required for mutant generation. Typically, only 3 colonies need to be analyzed per mutagenesis reaction, and a skillful trainee can engineer 1 to 2 dozen mutant plasmids within a week. In addition, with an in-house protocol for preparing highly competent bacterial cells, we have further increased the reliability and cost-effectiveness of the method. Notably, such competent cells have been kept in a liquid nitrogen tank for &gt;12 years with minimal loss of competency. Thus, this refined method offers a robust, efficient, and scalable solution for high-precision gene modification in vitro, with broad applications in protein and plasmid engineering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: In vitro site-directed mutagenesis using an optimized primer design strategy</p><p><b>Basic Protocol 2</b>: Preparation of high-efficiency chemocompetent DH5α cells for transformation of mutagenized plasmid products</p><p><b>Basic Protocol 3</b>: Transformation of chemocompetent DH5α cells and obtaining bacterial colonies with correctly mutagenized plasmid products</p><p><b>Alternate Protocol</b>: Transformation if glycerol stocks are unavailable</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/cpz1.70104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143404606","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":"Minimal Component, Protein-Free, and Cost-effective Human Pluripotent Stem Cell Cardiomyocyte Differentiation","authors":"Jessika B. Iwanski,&nbsp;Odunayo S. Lawal,&nbsp;William T. Kwon,&nbsp;Isabella Vazquez,&nbsp;Jared M. Churko","doi":"10.1002/cpz1.70099","DOIUrl":"https://doi.org/10.1002/cpz1.70099","url":null,"abstract":"<p>Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the <i>in vitro</i> modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and <span>l</span>-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: hPSC cell culture</p><p><b>Basic Protocol 2</b>: hPSC-CM differentiation</p><p><b>Basic Protocol 3</b>: Characterization of hPSC-CMs by immunofluorescence (IF) imaging</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143388900","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":"High-sensitivity Epimutations Testing with MS-HRM","authors":"Olga Taryma-Leśniak,&nbsp;Katarzyna E. Sokolowska,&nbsp;Magdalena Liput,&nbsp;Tomasz K. Wojdacz","doi":"10.1002/cpz1.70094","DOIUrl":"https://doi.org/10.1002/cpz1.70094","url":null,"abstract":"<p>Epimutation is defined as any heritable change in gene activity that does not affect the actual sequence of DNA. One of the most researched epimutations is promoter methylation of breast cancer susceptibility gene <i>BRCA1</i>. This epimutation may arise de novo in somatic tissues, such as breast or ovarian cancer tissue, but has also been shown to be widely distributed throughout tissues. <i>BRCA1</i> methylation detectable in peripheral blood has been associated with the risk of both breast and ovarian cancer in patients without a germline pathogenic variant status of this gene. The strength of this association suggests that diagnostic screening for the <i>BRCA1</i> epimutation should be considered with potential implications in cancer risk assessment. However, the detection of <i>BRCA1</i> epimutation may be challenging, as it is generally detectable at a very low level in blood. Methylation-sensitive high-resolution melting (MS-HRM) was shown to provide a high sensitivity of methylation detection, and as it is a PCR-based method, epimutation screening based on MS-HRM is labor- and cost-effective. Here we describe the MS-HRM protocol for <i>BRCA1</i> epimutation screening. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: High sensitivity constitutional epimutations testing with MS-HRM</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143380118","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":"Porphyria Diagnostics Part 2: Essential Biochemical Testing for Diagnosis of the Porphyrias","authors":"Vaithamanithi-Mudumbai Sadagopa Ramanujam,&nbsp;Akshata Moghe,&nbsp;Ruksana Huda,&nbsp;Shalonda B. Turner,&nbsp;Karl E. Anderson","doi":"10.1002/cpz1.70092","DOIUrl":"https://doi.org/10.1002/cpz1.70092","url":null,"abstract":"<p>Porphyrins and porphyrin precursors are normally detected in small amounts in healthy individuals but are found in large quantities in the urine, feces, blood, plasma, bone marrow, and liver in patients with various types of porphyrias. These are intermediates, or are derived from intermediates, in the pathway for heme biosynthesis. Heme is synthesized in all body tissues but in the largest amounts in the bone marrow and liver. Accurately measuring these compounds is important for diagnosis and monitoring of porphyrias. In addition, measurement of enzyme activities and mutation analyses by DNA sequencing enables confirmation of a porphyria diagnosis and genetic counseling. Biochemical approaches described here include measurements of porphyrin precursors and porphyrins in the urine, feces, plasma, erythrocytes, and liver, and determination of specific enzyme activities in erythrocytes and other cells. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143380117","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}