Current protocols最新文献

{"title":"A Pocket Guide for the Experimenter Studying Daily Rhythms in Memory-Related Behavior","authors":"Matthew J. Hartsock,&nbsp;Ilia N. Karatsoreos,&nbsp;Robert L. Spencer","doi":"10.1002/cpz1.70157","DOIUrl":"10.1002/cpz1.70157","url":null,"abstract":"<p>The study of circadian rhythms requires a deep understanding of how biological systems shift on a daily timescale. Similarly, the study of memory requires an appreciation of how memory mechanisms change across time. These complexities create serious challenges for examining circadian rhythms in memory-related behavior, an undertaking in which both sets of considerations must be integrated and assessed simultaneously. In this article, we present a pocket guide with an experimental design optimized to distinguish circadian influences on different stages of the memory process. Our approach distills ideas from a complex body of literature, offering a simple path forward in circadian memory research. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144308852","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":"Production of Functional miRNA Mimics Using In Vitro Transcription","authors":"Teja N. Sata,&nbsp;Md Ismail,&nbsp;Amrendra K. Sah,&nbsp;Senthil K. Venugopal","doi":"10.1002/cpz1.70163","DOIUrl":"10.1002/cpz1.70163","url":null,"abstract":"<p>MicroRNAs (miRNAs) are nearly 22 nucleotide RNA species involved in modulating gene expression via post-transcriptional regulation. In almost all in vitro studies, miRNA mimics are used to overexpress them to understand their role in various cellular processes. These mimics are also utilized as therapeutics for various diseases, such as scleroderma, mesothelioma, and multiple solid tumors. Commercial miRNA mimics are chemically synthesized, followed by HPLC purification. This article describes a simple in vitro transcription (IVT) procedure to generate miRNA mimics from DNA templates using RNA polymerase, followed by purification using silica-based columns and annealing. The procedure is economical and quick. Produced miRNA mimics can be overexpressed in mammalian cells using transfection agents. A comparison between chemically synthesized miRNA mimics and IVT-synthesized miRNA mimics demonstrates similar efficiencies in post-transcriptional regulation. After poly(A) polymerase-mediated cDNA synthesis, validation is performed by qPCR expression analysis of target genes. Alternatively, miRNA mimics can be validated by immunoblotting target proteins. We present efficient, quick protocols to synthesize functional miRNA mimics using IVT, whose function can be validated by qPCR or immunoblotting. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Design of in vitro transcription templates, in vitro transcription, RNA purification, and RNA strand annealing</p><p><b>Basic Protocol 2</b>: Transfection of miRNA mimics, total RNA isolation, poly(A) polymerase-mediated cDNA synthesis, validation of miRNA mimic expression by qPCR, and functional validation by immunoblotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144308853","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":"Epigenetic Histone Deacetylases Activity Assay in the Brain and Peripheral Organ Tissues","authors":"Doodipala Samba Reddy,&nbsp;Hudson Boyd,&nbsp;Vinitha Karnati,&nbsp;Joon Kim,&nbsp;Sreevidhya Ramakrishnan,&nbsp;Xin Wu","doi":"10.1002/cpz1.70143","DOIUrl":"10.1002/cpz1.70143","url":null,"abstract":"<p>Histone deacetylases (HDACs) are crucial epigenetic regulators involved in the modulation of gene expression and are promising therapeutic targets for treating various diseases, including central nervous system disorders and cancer. HDAC inhibitors exhibit neuroprotective, antiepileptogenic, and antidepressant properties in animal models, underscoring their clinical relevance. Quantifying HDAC activity is essential for identifying inhibitors and evaluating their effects under physiological or pathological conditions. This article outlines a comprehensive, sensitive, and robust assay to quantify HDAC activity in tissue lysates, with specific application to brain tissue. The assay is based on the catalytic removal of an acetyl group from the Boc-Lys(Ac)-AMC substrate by HDAC enzymes. Following nuclear protein extraction, tissue HDAC activity can be quantitatively assessed using a fluorometric Boc-Lys(Ac) HDAC activity kit. Adding a developer containing trypsin then converts the deacetylated product into a measurable fluorophore. This enables precise quantification of HDAC activity levels across different tissues, making the method suitable for screening putative HDAC inhibitors and assessing their effects on epigenetically modulated phenotypes. We validated these protocols using brain tissue samples from mice subjected to traumatic brain injury, a condition known to elevate HDAC activity levels. This assay provides an efficient, scalable tool for exploring HDAC function, evaluating therapeutic interventions, and advancing the understanding of HDAC-mediated mechanisms in animal models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of nuclear protein from brain and other tissues</p><p><b>Support Protocol 1</b>: Harvesting and microdissection of brain and other tissues</p><p><b>Support Protocol 2</b>: Estimation of extracted protein using the Pierce bicinchoninic acid (BCA) assay</p><p><b>Basic Protocol 2</b>: HDAC activity fluorometric assay in the brain and other tissues</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70143","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144264612","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":"Methods to Observe Plant Tissue Colonization by Fusarium oxysporum","authors":"Domingo Martínez-Soto,&nbsp;Gengtan Li,&nbsp;Li-Jun Ma","doi":"10.1002/cpz1.70152","DOIUrl":"10.1002/cpz1.70152","url":null,"abstract":"<p><i>Fusarium oxysporum</i>, an important soil-borne pathogen, causes vascular wilts in more than 100 plant species, leading to billions of dollars in annual yield losses. Controlling <i>Fusarium</i> wilt diseases is challenging due to the persistence of pathogen spores in infested fields and the growing resistance to available fungicides. Understanding the molecular interactions between <i>F. oxysporum</i> and its host plants is crucial for developing novel control strategies, but studying these interactions is difficult because <i>F. oxysporum</i> invades plant roots long before wilt symptoms can be detected in above-ground tissues. To illuminate the hidden interactions between <i>F. oxysporum</i> and its plant hosts, we present three confocal microscopy protocols for visualizing fungal colonization in plant tissues and the associated plant responses. The first protocol employs wheat germ agglutinin–Alexa Fluor 488 and propidium iodide to stain fungal cells and plant host tissues, respectively. The second uses sirofluor to detect deposition of callose, a (1,3)-β-glucan polymer found in plant cell walls that plays a significant role in plant defense. The third utilizes fluorescent protein–tagged fungal isolates and a stable transgenic <i>Arabidopsis thaliana</i> line, providing a clean and easily accessible system for visualizing early infection stages. The protocols described here will shed light on underground plant-pathogen interactions, aiding researchers in unraveling the complex dynamics between diverse <i>F. oxysporum</i> pathotypes and their plant hosts.© 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Observation of <i>F. oxysporum</i> cells in the tomato stem vasculature</p><p><b>Basic Protocol 2</b>: Observation of callose deposition in <i>F. oxysporum</i>–colonized tomato plant roots</p><p><b>Basic Protocol 3</b>: Observation of fungal colonization in an <i>F. oxysporum</i>–<i>A. thaliana</i> model system</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244811","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":"From Genetic Association to Therapeutic Target: A Pipeline for Pleiotropic Gene Prioritization","authors":"Morgan Ewald,&nbsp;Erin Young,&nbsp;Michael Kuehn,&nbsp;Olivia Veatch","doi":"10.1002/cpz1.70148","DOIUrl":"10.1002/cpz1.70148","url":null,"abstract":"<p>As our understanding of human health grows, we often see that similar biological dysfunction underlies the co-occurrence of various complex diseases. It remains difficult to determine if there are common genetic mechanisms contributing to clinically distinct conditions or if expression of both conditions relates to other shared risk factors. For example, in some situations, genetic variation may increase risk for one condition, and expression of this condition then increases risk for another disease. Identifying potentially pleiotropic genes is crucial for advancing the development of more effective treatment options, especially in instances where current therapies are insufficient. Genome-wide association studies (GWAS) provide cross-trait associations but do not provide the full functionality of how dysfunction in genes being tagged by GWAS hits are contributing to two or more distinct phenotypes. Fortunately, as other types of available data continue to grow exponentially (e.g., RNA-seq, mass spectrometry, mouse knock-out phenotype associations), these can be leveraged to help process GWAS results into meaningful information. The aim of this protocol is to provide clear instructions for using various databases and available software tools to identify key pleiotropic genes contributing to two distinct phenotypes of interest. The protocol uses information from various publicly available databases, including GWAS Catalog, Functional Mapping and Annotation (FUMA), Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT), International Mouse Phenotype Consortium (IMPC), STRINGdb, Pharos, and Cytoscape for network visualization. This pipeline, with code written in R and RStudio software, helps the user identify and generate hypotheses about shared genetic mechanisms contributing to their selected phenotypes of interest as well as prioritize genes of interest to functionally follow up in model systems that are more likely to be clinically relevant. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Pleiotropic gene prioritization pipeline for studies in model systems</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244596","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":"Quantitative Assessment of Mitochondrial Volumetric Transitions in Arabidopsis thaliana","authors":"Bryan Ocampo-Hernández,&nbsp;Delia Luisa Rodríguez-Bustos,&nbsp;Javier Plasencia,&nbsp;Manuel Gutiérrez-Aguilar","doi":"10.1002/cpz1.70156","DOIUrl":"10.1002/cpz1.70156","url":null,"abstract":"<p>Mitochondria in plants typically appear as discrete spherical or slightly tubular organelles, with their morphology and volume serving as indicators of metabolic state and dysfunction. Measuring changes in mitochondrial volume is relatively straightforward in organisms lacking plastids. However, in chlorophyll-rich tissues, such assessments often require purification protocols that may compromise accuracy. Here, we present protocols for the quantitative assessment of mitochondrial volume transitions in leaf mesophyll cells of <i>Arabidopsis thaliana</i>. The methods are simple and highly sensitive and offer a reliable approach for studying mitochondrial morphology transitions under both physiological and stress conditions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Leaf mesophyll treatment and mitochondrial imaging</p><p><b>Basic Protocol 2</b>: Leaf mesophyll mitochondrial volume assessment</p><p><b>Basic Protocol 3</b>: Mitochondrial volume statistics</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70156","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144206343","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":"Peptide–Oligonucleotide Conjugates: Catalytic Preparation in Aqueous Solution or On-Column","authors":"Marion Gras,&nbsp;Michael Smietana,&nbsp;Pauline Adler","doi":"10.1002/cpz1.70154","DOIUrl":"10.1002/cpz1.70154","url":null,"abstract":"<p>Peptide–oligonucleotide conjugates (POCs) are synthesized through a novel catalytic and sustainable approach. The amide coupling between peptide and oligonucleotide is facilitated by 1,4-diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) as a stoichiometric activating agent. Two distinct methods are described for the POC synthesis: the first involves an on-column conjugation in an anhydrous environment, while the second enables a post-synthetic conjugation in an aqueous buffer. This last approach has been successfully extended to an iterative process, offering significant potential for the development of DNA-encoded libraries.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: On-column conjugation of an amino acid or of a peptide to an oligonucleotide</p><p><b>Support Protocol</b>: Deprotection of the Boc-protected amine of the lysine</p><p><b>Basic Protocol 2</b>: In-solution conjugation of an amino acid or of a peptide on an oligonucleotide</p><p><b>Basic Protocol 3</b>: Iterative process for the preparation of a peptide–oligonucleotide conjugate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70154","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144197140","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":"Cover Image, Volume 5, Issue 5","authors":"","doi":"10.1002/cpz1.70159","DOIUrl":"10.1002/cpz1.70159","url":null,"abstract":"<p>The cover image is based on the article Purification of recombinant histones and mononucleosome assembly by Jingjun Hong et al., https://doi.org/10.1002/cpz1.70155.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70159","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140410","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":"Co-Expression and Purification of the Complex of Single-Chain H2B-H2A.Z and Histone Chaperone YL1","authors":"Yue Liu,&nbsp;Wansen Tan,&nbsp;Jingjun Hong","doi":"10.1002/cpz1.70151","DOIUrl":"10.1002/cpz1.70151","url":null,"abstract":"<p>Chromatin remodeling serves as a critical mechanism for gene regulation, enabling the replacement of canonical histones with their variants through the assistance of histone chaperones. Our study focused on the process by which the histone variant H2A.Z replaces H2A, mediated by the mammalian SRCAP chromatin remodeling complex subunit hYL1. Previous approaches typically involved separately expressing the two proteins and validating their interaction <i>in vitro</i>. In contrast, we designed an artificial single-chain hH2B-hH2A.Z (hTBZ) construct, co-transformed the hYL1 gene with hTBZ, and co-expressed the proteins in E. coli BL21(DE3). We then performed purification, fast protein liquid chromatography (FPLC), and SDS-PAGE analysis. This approach successfully yielded the complexed proteins, confirming the <i>in vivo</i> interaction between hYL1 and hTBZ. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Co-transformation of hTBZ/YL1 into BL21 (DE3) competent cells, followed by induction and acquisition of protein</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140419","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":"Simple and Efficient Transformation and Gene Editing of Marchantia polymorpha Spores","authors":"Rebecca M. Yorker,&nbsp;Simon C. Deroles,&nbsp;Yanfei Zhou,&nbsp;Jennifer A. Tate,&nbsp;Kevin M. Davies,&nbsp;Nick W. Albert","doi":"10.1002/cpz1.70149","DOIUrl":"10.1002/cpz1.70149","url":null,"abstract":"<p><i>Marchantia polymorpha</i> (Marchantia) has become a model species for liverwort studies, owing to its rapid growth <i>in vitro</i>, ease of propagation, simple genetics, small genome, haploid-dominant life cycle, and because it is amenable to genetic transformation. Efficient transformation provides a foundation for many molecular and genetic analyses. The protocol described here is a simple and robust procedure for transforming Marchantia for a variety of applications, including gene overexpression and CRISPR genome editing. This simplified <i>Agrobacterium tumefaciens</i>-mediated transformation protocol targets spores, using common <i>Agrobacterium</i> strains GV3101 or EHA105, and overcomes challenges experienced in other methods. Spores are sterilized and distributed over sterile filter papers, which effectively retain spores and regenerating spores (known as sporelings). This approach enables the sporelings to be transferred to different agar growth media at different stages of transformation. A critical feature is preculturing the spores with acetosyringone (AS) prior to co-cultivation with <i>Agrobacterium</i>. This treatment profoundly enhances the transformation rate, particularly for <i>Agrobacterium</i> strain GV3101. GV3101 is preferred for its rapid growth rate, simple transformation, and lack of a recombinase (<i>recA</i>), stabilizing plasmids. The protocol is suitable for transforming Marchantia with constructs for CRISPR gene editing. Editing efficiency can be increased by introducing a heat-shock treatment during the transformation procedure, which increases the proportion of plants with larger edited sectors, facilitating mutant identification and propagation. Constructs and strategies for both overexpression and multiplex genome editing with sgRNA arrays using new and existing vectors are described. Using this spore transformation protocol for CRISPR gene editing, we routinely achieve 60% to 70% mutation rates, significantly reducing the effort required to generate and isolate mutants for functional analyses. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: <i>Agrobacterium</i>-mediated transformation of <i>Marchantia polymorpha</i> spores</p><p><b>Support Protocol</b>: Induction of spores</p><p><b>APPENDIX 1</b>: Overexpression Vectors</p><p><b>APPENDIX 2</b>: Multiplex CRISPR using tRNA arrays</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140418","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}