Current protocols最新文献

{"title":"Quantifying Competitive Fitness in Yeast with High-Throughput Fluorescence Microscopy Imaging","authors":"Aruni S. Sumanarathne,&nbsp;Aleeza C. Gerstein","doi":"10.1002/cpz1.70093","DOIUrl":"10.1002/cpz1.70093","url":null,"abstract":"<p>Competitive fitness is a fundamental concept in evolutionary biology that captures the ability of organisms to survive, reproduce, and compete for resources in their environment. Competitive fitness is typically assessed in the lab by growing two or more competitors together and measuring the frequency of each at multiple time points. Traditional microbial competitive fitness assays are labor intensive and involve plating on solid medium and counting colonies. Here, we describe a method to quantitatively measure competitive fitness using fluorescence microscopic imaging and machine-learning-enabled image analysis to directly count the number of cells from each competitor in the mixed population. This high-throughput, primarily automated, and efficient process gives accurate and reproducible results for competitive fitness. Here, we describe the entire process, from sample preparation through microscopy to quantification, and provide instructions and scripts for the image analysis, fitness calculations, and sample data visualizations. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Sample preparation</p><p><b>Basic Protocol 2</b>: Photographing fluorescing and non-fluorescing cells using an EVOS microscope</p><p><b>Basic Protocol 3</b>: Counting fluorescing and non-fluorescing cells with Orbit Image Analysis</p><p><b>Basic Protocol 4</b>: Getting the average cell counts per well and changing the file names</p><p><b>Basic Protocol 5</b>: Calculating competitive fitness using R</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11771579/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143048612","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":"Plasmid Library Construction From Genomic DNA","authors":"Valeria Florez-Cardona,&nbsp;Jessica Khani,&nbsp;Emily McNutt,&nbsp;Bruno Manta,&nbsp;Mehmet Berkmen","doi":"10.1002/cpz1.70088","DOIUrl":"10.1002/cpz1.70088","url":null,"abstract":"<p>Functional genomic approaches have been effective at uncovering the function of uncharacterized genes and identifying new functions for known genes. Often these approaches rely on an <i>in vivo</i> screen or selection to associate genes with a phenotype of interest. These selections and screens are dependent upon the expression of proteins encoded in genomic DNA from an expression vector, such as a plasmid. Despite the utility of genomic DNA plasmid libraries, the protocols for their construction have remained unchanged in the past 40 years. Here, we present a procedure for constructing plasmid libraries from genomic DNA. This procedure is scalable and relies on simple techniques and common laboratory equipment and reagents. Briefly, the genomic DNA is extracted and then physically fragmented with a g-TUBE, overhangs are repaired, and fragments are selectively purified with magnetic beads to obtain an average fragment size of 2.5 kb. Blunted fragments are ligated into a blunt-end-digested and dephosphorylated vector. Finally, the library is amplified by electroporating the ligation into a high-transformation-efficiency <i>Escherichia coli</i> strain and extracting the plasmid DNA from the transformants. As a proof of concept, we built and sequenced three genomic libraries from different genomes and calculated their coverage using a next-generation sequencing (NGS) workflow. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Plasmid library construction</p><p><b>Alternate Protocol</b>: Selection of gDNA fragments using SageELF gel fractionator</p><p><b>Support Protocol 1</b>: Extraction of gDNA with phenol/chloroform</p><p><b>Support Protocol 2</b>: Vector preparation</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11752411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143018604","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":"Modeling Malignant Mesothelioma in Genetically Engineered Mice","authors":"Yuwaraj Kadariya,&nbsp;Eleonora Sementino,&nbsp;Xiang Hua,&nbsp;Dietmar J. Kappes,&nbsp;Joseph R. Testa","doi":"10.1002/cpz1.70086","DOIUrl":"10.1002/cpz1.70086","url":null,"abstract":"<p>Mesothelioma is a lethal cancer of the serosal lining of the body cavities. Risk factors include environmental and genetic factors. Asbestos exposure is considered the principal environmental risk factor, but other carcinogenic mineral fibers, such as erionite, also have a causal role. Pathogenic germline (heritable) mutations of specific genes, especially <i>BAP1</i>, are thought to predispose the individual to mesothelioma in about 10% of cases. Somatic mutations and deletions of specific tumor suppressor genes, particularly <i>BAP1</i>, <i>CDKN2A/B</i>, and <i>NF2</i>, occur frequently in human mesothelioma, and asbestos-exposed mice with heterozygous deletions of any one of these genes have been shown to develop mesothelioma more often and at an accelerated rate than in control animals. Autochthonous mesothelioma mouse models, which are genetically engineered to carry multiple genetic lesions matching those observed in the human disease counterpart, closely resemble the disease phenotype and the extensive inflammatory responses that characterize human mesothelioma. Because autochthonous mice do not require asbestos exposure and form tumors rapidly, these models are invaluable for assessing novel therapeutic strategies in an immunocompetent setting. The overlapping genetic, epigenetic, and immune environments of the tumors observed in these genetically engineered mouse models (GEMMs) and human primary mesothelioma specimens support the clinical relevance of these preclinical models. This article presents protocols for studies of asbestos-induced mesothelioma in GEMMs and non-carcinogenic conditional knockout models of mesothelioma, including an example of a preclinical application. These models are invaluable for understanding the biological underpinnings of mesothelioma and for testing new therapeutics and chemoprevention or interception agents. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of a genetically engineered mouse model (GEMM) with a germline <i>Bap1</i> knockout allele</p><p><b>Basic Protocol 2</b>: Generation of GEMMs with germline <i>Bap1</i> knock-in alleles</p><p><b>Basic Protocol 3</b>: Asbestos carcinogenicity investigations with GEMMs</p><p><b>Basic Protocol 4</b>: Preclinical chemoprevention and chemotherapy studies using a GEMM with asbestos-induced mesothelioma</p><p><b>Basic Protocol 5</b>: Generation of a GEMM with conditional knockout of <i>Bap1</i></p><p><b>Basic Protocol 6</b>: Generation of a conditional knockout model of mesothelioma</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960348","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":"A Simple Protocol for Visualization of RNA–Protein Complexes by Atomic Force Microscopy","authors":"Andrea Tripepi,&nbsp;Huma Shakoor,&nbsp;Petr Klapetek","doi":"10.1002/cpz1.70084","DOIUrl":"10.1002/cpz1.70084","url":null,"abstract":"<p>Atomic force microscopy (AFM) has recently received increasing interest in molecular biology. This technique allows quick and reliable detection of biomolecules. However, studying RNA–protein complexes using AFM poses significant challenges. Here, we describe a simple and reliable method to visualize positively charged proteins bound to RNA that does not require metallic cations. This method allowed us to effectively detect and visualize Staufen–RNA complexes by height or logarithmic stiffness. The study of the mechanical properties is particularly important in the case of protein-coated RNA complexes, where RNA cannot be detected by height channel. In any case, it is necessary to compare AFM data with the data derived from other techniques like nuclear magnetic resonance, X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Preparation and visualization of RNA–protein complex</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713223/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960698","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":"Miniscope Imaging of Nucleus Accumbens Neural Activity in Freely Behaving Rats: Virus Injection, Gradient Index Lens Implantation, Recording Strategies, and Analytical Methods","authors":"Nicholas J. Beacher,&nbsp;Michael W. Wang,&nbsp;Matthew C. Broomer,&nbsp;Jessica Y. Kuo,&nbsp;Kayden A. Washington,&nbsp;Miranda Targum,&nbsp;Yan Zhang,&nbsp;Giovanni Barbera,&nbsp;Da-Ting Lin","doi":"10.1002/cpz1.70090","DOIUrl":"10.1002/cpz1.70090","url":null,"abstract":"<p><i>In vivo</i> calcium imaging in freely moving rats using miniscopes provides valuable information about the neural mechanisms of behavior in real time. A gradient index (GRIN) lens can be implanted in deep brain structures to relay activity from single neurons. While such procedures have been successful in mice, few reports provide detailed procedures for successful surgery and long-term imaging in rats, which are better suited for studying complex human behaviors. We present a robotic surgical protocol for same-day virus injection and GRIN lens implantation into the rat nucleus accumbens core. Our procedure utilizes a direct lens insertion without tissue aspiration and produces quality image retention for months of recording. We also describe daily recording strategies to minimize damage and promote long-term imaging. Finally, we present custom protective strategies to eliminate the need to remove miniscopes between sessions. This methodology protects rats from repeated isoflurane exposure and ensures a consistent focal plane for the entirety of the experiment. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Craniotomy</p><p><b>Basic Protocol 2</b>: Virus injection</p><p><b>Basic Protocol 3</b>: GRIN lens implantation</p><p><b>Basic Protocol 4</b>: Baseplate mounting and assessment of the anesthetized rat</p><p><b>Basic Protocol 5</b>: Assessment of the awake, behaving rat</p><p><b>Support Protocol 1</b>: Protective miniscope cone fabrication</p><p><b>Support Protocol 2</b>: Miniscope cable fabrication</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11718238/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960329","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":"Generation of Retinal Ganglion Cells from Reprogrammed Keratocytes of Non-Glaucoma and Glaucoma Donors","authors":"Shahna S. Hameed,&nbsp;Tasneem P. Sharma","doi":"10.1002/cpz1.70091","DOIUrl":"10.1002/cpz1.70091","url":null,"abstract":"<p>Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Culturing of keratocytes from human cadaveric donors</p><p><b>Basic Protocol 2</b>: Reprogramming donor keratocytes into iPSCs</p><p><b>Basic Protocol 3</b>: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping</p><p><b>Basic Protocol 4</b>: Generation of 3D ROs</p><p><b>Basic Protocol 5</b>: Dissociation and culturing of RGCs from 3D ROs</p><p><b>Support Protocol 1</b>: Immunostaining for phenotypic characterization of cells</p><p><b>Support Protocol 2</b>: Sectioning of 3D ROs and immunostaining</p><p><b>Support Protocol 3</b>: Western blotting for cleaved CASP3 and THY1</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960699","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":"Genetically Engineered and Implantable Mouse Brain Tumor Models: Characterization by Immunohistochemistry and Flow Cytometry","authors":"Apoorva Mirji,&nbsp;Gurveer Singh,&nbsp;Anzar A. Mujeeb,&nbsp;Brandon L. McClellan,&nbsp;YingXiang Li,&nbsp;Makayla Perez,&nbsp;Maria G. Castro","doi":"10.1002/cpz1.70080","DOIUrl":"10.1002/cpz1.70080","url":null,"abstract":"<p>Gliomas are aggressive tumors with a poor prognosis. The protocols presented here outline the methods used to study tumor progression, the tumor microenvironment (TME), and the effects of experimental treatments. The Sleeping Beauty (SB) transposase system induces tumors <i>de novo</i> to generate mouse models that recapitulate human gliomas. Plasmids are constructed with oncogenic drivers and other genetic alterations of interest. which are recognized by their unique position in between inverted/direct repeat (IR/DR) sequences. Luciferase enzyme is used to monitor the uptake of the plasmid, tumor growth, and response to experimental therapies. The genes of interest are tracked using fluorescent markers. Tumors will arise in immunocompetent hosts, which provides a relevant preclinical platform for analysis of tumor initiation, progression, survival, immune microenvironment, and histopathological features. Once the tumor grows within the desired brain location, it can be harvested to generate cell cultures of neurospheres for future experimentation. The benefit of implantable models generated from SB tumors is that they provide specific anatomical and genetic context, in which specific genetic characteristics can be tracked, as they are co-expressed with fluorescent markers. Post glioma cell implantation, additional analysis of the TME and tumor growth can be performed through immunohistochemistry (IHC) and flow cytometry. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Creation of mouse glioma models by Sleeping-Beauty-mediated transposition</p><p><b>Basic Protocol 2</b>: Generation of orthotopic implantable brain tumors and neurospheres</p><p><b>Basic Protocol 3</b>: Hematoxylin and eosin staining of glioma tissue samples</p><p><b>Basic Protocol 4</b>: Immunohistochemistry of glioma tissue samples</p><p><b>Basic Protocol 5</b>: Flow cytometry for immune cell analysis of the tumor microenvironment</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960050","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":"Targeted C-to-T Base Editing in the Arabidopsis Plastid Genome","authors":"Issei Nakazato,&nbsp;Shin-ichi Arimura","doi":"10.1002/cpz1.70075","DOIUrl":"10.1002/cpz1.70075","url":null,"abstract":"<p><i>Arabidopsis thaliana</i>, particularly the ecotype Columbia-0 (Col-0), has been extensively employed in the study of genetics of the nuclear genome. However, the difficulty of modifying the plastid genome of Col-0, the most widely used ecotype, has hindered investigation of the functional interactions between nuclear-encoded and plastid-encoded genes in this ecotype. Recently, we achieved targeted base editing, substituting a specific C:G pair with a T:A pair in the plastid genome of Col-0 through the application of genome-editing technology. This article introduces the method employed to accomplish this targeted base editing. The process involves four steps: (i) designing and constructing a binary vector encoding the genome-editing enzyme, (ii) introducing the binary vector into the nuclear genome of Col-0 via floral dipping, (iii) identifying base-edited plants, and (iv) verifying inheritance of the edited base(s) by the next generation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Design and construction of a binary vector encoding ptpTALECD or ptpTALECD_v2</p><p><b>Basic Protocol 2</b>: <i>Agrobacterium</i>-mediated introduction of a binary vector into the <i>Arabidopsis</i> nuclear genome</p><p><b>Basic Protocol 3</b>: Selection of plants harboring T-DNA in the nucleus and detection of base editing in the plastid genome</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70075","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142934111","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":"Simple Isolation of Human Bone Marrow Adipose Tissue–Derived Mesenchymal Stem/Stromal Cells","authors":"Gülsena Tonyalı,&nbsp;Emine Kiliç,&nbsp;Bihter Muratoğlu,&nbsp;Esin Alpdündar-Bulut,&nbsp;Cansu Özdemir,&nbsp;Duygu Uçkan-Çetinkaya","doi":"10.1002/cpz1.70081","DOIUrl":"10.1002/cpz1.70081","url":null,"abstract":"<p>Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell &amp; Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow</p><p><b>Basic Protocol 2</b>: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs</p><p><b>Support Protocol 1</b>: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry</p><p><b>Support Protocol 2</b>: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs</p><p><b>Support Protocol 3</b>: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142924299","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":"Searching and Using MobiDB Resource 6 to Explore Predictions and Annotations for Intrinsically Disordered Proteins","authors":"Maria Cristina Aspromonte,&nbsp;Federica Quaglia,&nbsp;Alexander Miguel Monzon,&nbsp;Damiano Clementel,&nbsp;Alessio Del Conte,&nbsp;Damiano Piovesan,&nbsp;Silvio C. E. Tosatto","doi":"10.1002/cpz1.70077","DOIUrl":"10.1002/cpz1.70077","url":null,"abstract":"<p>Intrinsically disordered proteins (IDPs) make up around 30% of eukaryotic proteomes and play a crucial role in cellular processes and in pathological conditions such as neurodegenerative disorders and cancers. However, IDPs exhibit dynamic conformational ensembles and are often involved in the formation of biomolecular condensates. Understanding the function of IDPs is critical to research in many areas of science. MobiDB is a unique resource that serves as a comprehensive knowledgebase of IDPs and intrinsically disordered regions (IDRs), combining disorder annotations from experimental evidence and predictions for a broad range of protein sequences. Over the past decade, MobiDB has evolved with a focus on expanding annotation coverage, standardizing annotation provenance, and enhancing database accessibility. The latest MobiDB, version 6, released in July 2024, includes significant improvements, such as the integration of AlphaFoldDB predictions and a new homology transfer pipeline that has substantially increased the number of entries with high-quality annotations. The user interface has also been updated, highlighting annotation features, clarifying the entry page, and providing an immediate overview of disorder, binding, and disorder functions information in the protein sequence. This protocol guides the user through applications of the MobiDB, including disorder prediction, curated data analysis, and exploration of interaction data. This guide covers how to perform a search in MobiDB annotations using the web interface and the MobiDB REST API for programmatic access. The protocols use a step-by-step walkthrough using the human growth hormone receptor to demonstrate MobiDB's functions for visualization and interpretation of protein disorder data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Searching MobiDB query formats</p><p><b>Basic Protocol 2</b>: Searching MobiDB selected datasets and selected proteomes</p><p><b>Basic Protocol 3</b>: Performing a search on the Statistics page in MobiDB</p><p><b>Support Protocol</b>: Programmatic access with MobiDB REST API</p><p><b>Basic Protocol 4</b>: Visualizing and interpreting a MobiDB Entry: The GHR use case</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11668231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883994","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}