Current protocols最新文献

{"title":"Preparation of Acute Mouse Hippocampal Brain Sections for Transport to a Distant Electrophysiology Laboratory","authors":"Sofia Ioanna Nouka,&nbsp;Loukia Katsouri,&nbsp;Jonathan Rudge,&nbsp;John O'Keefe,&nbsp;Francesco Tamagnini","doi":"10.1002/cpz1.70461","DOIUrl":"10.1002/cpz1.70461","url":null,"abstract":"<p>This article describes a standardized method for preparing and transporting acute mouse hippocampal brain slices suitable for high-quality single-cell, CA1, pyramidal cells electrophysiological recordings between laboratories in departments or institutions located at distances up to 3 hr apart. The procedure emphasizes maintaining tissue viability and preserving physiological integrity during transport. Slices are prepared in sucrose-based protective solutions under ice-cold, oxygenated conditions, followed by a controlled rewarming period to restore metabolic activity. For transport, slices are submerged in oxygenated artificial cerebrospinal fluid (aCSF) contained in 250-ml parafilm-sealed Duran bottles, ensuring prolonged viability at near-ambient temperature. The method includes parameters for slice thickness, temperature control, oxygenation duration, and maximum transport time to ensure reproducibility across sites. Validation using patch-clamp recordings confirms that electrophysiological properties, such as resting membrane potential, input resistance, action potential firing, and spontaneous synaptic neurotransmission, remain comparable to those in freshly prepared slices. This protocol enables collaborative research where brain slice preparation and electrophysiological recordings occur in separate laboratories, while preserving the reliability and quality of single-cell functional data. This is particularly useful for laboratories located away from animal housing units. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Acute brain slice preparation available for transport</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13595899/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149019152","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":"Composable Visualization of High-Dimensional Biological Data with ggalign","authors":"Wanyi Liu,&nbsp;Jia Ding,&nbsp;Yujun Sun,&nbsp;Bin Yan,&nbsp;Zhangyu Wang,&nbsp;Chunyang Wang,&nbsp;Chenyang Shu,&nbsp;Jian-Guo Zhou,&nbsp;Guangchuang Yu,&nbsp;Yun Peng,&nbsp;Shixiang Wang","doi":"10.1002/cpz1.70453","DOIUrl":"10.1002/cpz1.70453","url":null,"abstract":"<p>ggalign is an R/CRAN package for creating flexible and composable multi-panel data visualizations. The package extends the ggplot2 grammar of graphics by introducing an integrative framework that supports both data-free and data-aware composition. After five years of continuous development, ggalign has evolved into a comprehensive solution that handles diverse data types and layout structures, including quad, circular, and stack layouts. It was originally designed for general-purpose composable visualization and has been expanded to support multi-omics data integration, extending the application of ggalign to pan-cancer analysis, single-cell transcriptomics, and microbiome studies. This article presents eight basic protocols for constructing complex visualizations using the declarative syntax of ggalign. Basic Protocol 1 describes data-free composition for flexible arrangement of multiple plots; Basic Protocol 2 describes data-aware quad layout for integrating a central plot with surrounding annotations; Basic Protocol 3 describes data-aware circular layout for visualizing ring-structured data; Basic Protocol 4 describes stack layout and nested composition for coordinated display of multi-track graphics; Basic Protocol 5 describes visualization of gene expression matrix heatmaps; Basic Protocol 6 describes visualization of somatic mutation landscapes using ggoncoplot(); Basic Protocol 7 describes circular visualization based on chromosome data, and Basic Protocol 8 describes cross-connection visualization between genes and pathways. The complete package reference is available at https://yunuuuu.github.io/ggalign/, with comprehensive documentation and tutorials at https://yunuuuu.github.io/ggalign-book/, and a gallery of example figures at https://yunuuuu.github.io/ggalign-gallery/. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Data-free composition</p><p><b>Basic Protocol 2</b>: Aligning data-aware with quad layouts</p><p><b>Basic Protocol 3</b>: Aligning data-aware with circular layouts</p><p><b>Basic Protocol 4</b>: Stack layouts and nested composition</p><p><b>Basic Protocol 5</b>: Visualizing heatmap of gene expression matrix</p><p><b>Basic Protocol 6</b>: Visualizing somatic mutation landscapes using ggoncoplot()</p><p><b>Basic Protocol 7</b>: Visualizing circos plots with ggalign</p><p><b>Basic Protocol 8</b>: Visualizing observational connections</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149013411","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":"Protoplasting and Transformation Using CRISPR/Cas9 in Coccidioides","authors":"Katrina M. Jackson,&nbsp;Matthew M. Morales,&nbsp;Edyta Szewczyk,&nbsp;Christina M. Homer,&nbsp;Ashley N. Itogawa,&nbsp;Rachel B. Brem,&nbsp;Anita Sil,&nbsp;Bridget M. Barker","doi":"10.1002/cpz1.70444","DOIUrl":"10.1002/cpz1.70444","url":null,"abstract":"<p><i>Coccidioides posadasii</i> and <i>C. immitis</i> are human fungal pathogens endemic to the American Southwest. <i>C. posadasii</i> and <i>C. immitis</i> are the causative agents of coccidioidomycosis, or Valley fever. They are pathogens of growing concern, as reported cases of coccidioidomycosis have increased 20-fold in the last two decades. Despite their importance as pathogens, <i>Coccidioides spp</i>. are understudied, especially compared to other human fungal pathogens with similar infectious burdens. The reasons <i>Coccidioides spp</i>. are understudied are multifactorial, including requirement of high biocontainment, technical difficulties in lab-based culture growth, and a historic lack of genetic tools. Previous methods of genetic manipulation have been technically challenging, time consuming, and inefficient. Here, we present protocols for designing gene deletion constructs, generating protoplasts from both genetically engineered biosafety level 2 (BSL2) and wildtype biosafety level 3 (BSL3) strains, and performing transformations with CRISPR/Cas9. The protoplasting protocol described here uses a cell wall digestion enzyme employed in the wine-making industry and results in high-quality protoplasts that have the potential to be used for applications beyond transformations. We also present a high-efficiency transformation method using CRISPR/Cas9. The protocols described here will allow for genetic manipulation of <i>Coccidioides</i>, using both BSL2 and BSL3 strains. This resource can be applied to expand research done in <i>Coccidioides spp</i>., build molecular tools, and expand overall knowledge of these important pathogens. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Construction of gene deletion construct</p><p><b>Alternate Protocol 1</b>: Construction of gene deletion construct</p><p><b>Basic Protocol 2</b>: Creation of protoplasts in BSL3</p><p><b>Alternate Protocol 2</b>: Creation of protoplasts in BSL2</p><p><b>Basic Protocol 3</b>: CRISPR/Cas9 transformation in BSL3</p><p><b>Alternate Protocol 3</b>: CRISPR/Cas9 transformation in BSL2</p><p><b>Support Protocol</b>: Passaging of mutants and PCR confirmation</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70444","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148993247","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":"Dataset Readiness Assessment With Large Language Model (DRAFT–LLM): A Multi-Axis Audit Guided by LLM","authors":"Guillaume Guerard,&nbsp;Sonia Djebali","doi":"10.1002/cpz1.70463","DOIUrl":"10.1002/cpz1.70463","url":null,"abstract":"<p>This article details the Dataset Readiness Assessment for Training (DRAFT), a systematic method for determining whether a high-dimensional biological dataset is suitable for developing reliable, equitable (i.e., the extent to which model performance, error patterns, and potential benefits or harms are evaluated and found to be acceptably distributed across relevant demographic, biological, clinical, and contextual subgroups), and scientifically meaningful machine-learning models, and DRAFT Large Language Model (DRAFT–LLM), its optional human-in-the-loop extension for calibrating study-specific audits through structured, critically reviewed LLM guidance. Standard model validation often fails to detect when apparent performance is driven by spurious correlations, technical artifacts, or hidden stratification, leading to irreproducible and inequitable findings. DRAFT–LLM addresses this gap by shifting the focus from model tuning to structured dataset auditing, organized around Support Protocols 1 to 4 that capture the scientific intent, data structure, and governance constraints of a given study. These Support Protocols: (1) elicit and formalize investigator input into a study intake and dataset card; (2) compute standardized dataset statistics and structural summaries suitable for downstream analysis and LLM context; (3) configure the language model using form-based responses, safety guardrails, and governance rules; and (4) generate personalized instructions, prompts, and code templates for running DRAFT audits. Basic Protocols 1 to 3 are instantiated from this support layer for generalization, equity, and stability: they are reusable execution patterns whose concrete behavior is determined by the cards, statistics, and configurations defined in the Support Protocols. DRAFT–LLM and DRAFT are demonstrated in this article through an end-to-end case study on The Cancer Genome Atlas (TCGA). © 2026 Wiley Periodicals LLC.</p><p><b>Support Protocol 1</b>: Study intake and dataset card construction</p><p><b>Support Protocol 2</b>: Dataset structure and advanced summary statistics for LLM context</p><p><b>Support Protocol 3</b>: LLM configuration using structured form responses</p><p><b>Support Protocol 4</b>: Generation of personalized instructions for DRAFT audits</p><p><b>Basic Protocol 1</b>: Generalization audit</p><p><b>Basic Protocol 2</b>: Equity audit</p><p><b>Basic Protocol 3</b>: Stability audit</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148993190","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":"Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro Phenotyping","authors":"Dosh Whye,&nbsp;Erika M. Norabuena,&nbsp;Wardiya Afshar-Saber,&nbsp;Becca Lewis,&nbsp;Taryn J. Polanco,&nbsp;Delaney Wood,&nbsp;Mustafa Sahin,&nbsp;Elizabeth D. Buttermore","doi":"10.1002/cpz1.70449","DOIUrl":"10.1002/cpz1.70449","url":null,"abstract":"<p>Pluripotent stem cell (PSC)–derived telencephalic organoids have long been established as an <i>in vitro</i> cell model to study the molecular and cellular features of the developing cerebral cortex and associated brain structures. Numerous directed organoid differentiation protocols have been developed that recapitulate cell subtype specification, cytoarchitectural organization, and cellular function with a high degree of fidelity. Protocol development has focused on the scalability of this <i>in vitro</i> system, and most organoid culture platforms involve a population-based approach in large vessel sizes. This makes comparative study with multiple cell lines both costly and time-consuming, and lowers the throughput for phenotypic screening. We have established a high-throughput approach for <i>in vitro</i> generation of hiPSC-derived telencephalic organoids that can be used to develop comparative phenotypic assays involving high-content imaging platforms. This article first highlights the use of a scaled-down, miniaturized cell culture volume that is amenable to high-throughput production of multiple 384-well plates. Then, using controlled morphogen cues, 3D hiPSC aggregates are directed into an anterior telencephalic identity and further specified into dorsal pallium or ventral subpallium cell fates, which can additionally be combined into assembloids for modeling excitatory and inhibitory networks or left to develop separately as single organoid subtypes. Finally, we introduce a Support Protocol detailing an <i>in vitro</i> 3D cell painting assay for organoid morpho-phenotypic characterization. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: High-throughput 3D aggregation of hiPSCs in morphogen-rich media</p><p><b>Basic Protocol 2</b>: Recombinant inhibitory protein–directed induction of telencephalic organoids</p><p><b>Basic Protocol 3</b>: Morphogen-guided induction of dorsal telencephalic/pallial organoids</p><p><b>Basic Protocol 4</b>: Morphogen-guided induction of ventral telencephalic/subpallial organoids</p><p><b>Support Protocol</b>: 3D live cell painting for organoid morphometric phenotyping</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148984157","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":"Embryotoxicity and Cardiotoxicity Assessment of Chemical Compounds Using Eggs of Lymnaea stagnalis","authors":"Kesavi Sri Malika Maddila,&nbsp;Meghana Korikani,&nbsp;Rumana Ansari,&nbsp;Rida Fatima,&nbsp;Preetha Kemphu,&nbsp;Rama Krishna Kancha","doi":"10.1002/cpz1.70457","DOIUrl":"https://doi.org/10.1002/cpz1.70457","url":null,"abstract":"<p>There is a paucity of invertebrate models for the assessment of general and organ-specific toxicity caused by chemicals, such as chemotherapeutics and environmental pollutants. To address this, we have established the embryo of <i>Lymnaea stagnalis</i> (great pond snail) as a simple and rapid model to assay toxicity in a medium-throughput manner. For measuring general toxicity, we have successfully repurposed Alamar Blue dye to assay physiological activity of the embryos. Due to the transparent nature of the <i>L. stagnalis</i> eggs, we have employed a microscope-based phenotypic assay to determine cardiotoxicity. Furthermore, we have previously shown that this model can be used to test cardioprotective strategies to overcome toxicity indued by cancer chemotherapeutics. Due to similarities of the <i>L. stagnalis</i> heart with that of a vertebrate heart, combined with the advantages of easy handling, <i>L. stagnalis</i> eggs serve as an emerging model for embryotoxicity and cardiotoxicity screening of pharmaceutical agents and environmental pollutants. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of clusters and separation of individual eggs of <i>L. stagnalis</i></p><p><b>Basic Protocol 2</b>: Assessment of cardiotoxicity and evaluation of cardioprotective strategies</p><p><b>Basic Protocol 3</b>: Alamar Blue dye–based embryotoxicity test</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148943206","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":"Light-Activated Molecular Purification (LAMP): A Photo-Responsive Affinity-Chromatography Protocol for Recombinant Protein Purification","authors":"Krishna Agrawal,&nbsp;Aishwarya Satyanarayana,&nbsp;Yashwant Kumar,&nbsp;Karthik Pushpavanam","doi":"10.1002/cpz1.70440","DOIUrl":"https://doi.org/10.1002/cpz1.70440","url":null,"abstract":"<p>Recombinant protein purification is a critical step prior to their use in therapeutics and diagnostics. Affinity and ion-exchange chromatography are among the most widely used purification strategies; however, both rely on extensive trial-and-error optimization and require chemical additives or high salt concentrations for protein elution. These conditions necessitate additional, time-consuming downstream dialysis steps to remove residual chemicals. To address these limitations, we propose a light-triggered protein elution strategy that eliminates the need for chemical elution agents and simplifies downstream processing. This approach exploits PhoCl1, a photocleavable protein that undergoes light-induced cleavage into an N-terminal empty barrel and a short C-terminal peptide fragment (Zhang et al., 2017; Lu et al., 2021). We engineered a fusion construct in which a 6×His tag is positioned at the N-terminus of PhoCl1, while the protein of interest (POI) is fused to its C-terminus (6×His–PhoCl1–POI). Following immobilization of the whole-cell lysate on Ni-NTA resin and removal of nonspecifically bound proteins, light exposure induces PhoCl1 cleavage. The His-tagged N-terminal fragment remains bound to the resin, while the POI is selectively released into the supernatant. Using this method, we have achieved protein purities exceeding 90%, demonstrating a rapid, reagent-free, and highly efficient alternative to conventional protein purification workflows. Here, for demonstration purposes, we have included a POI, riboflavin kinase (<i>Mj</i>RibK) found in <i>Methanocaldococcus jannaschii</i>. In this protocol, we describe the design, expression, and characterization of PhoCl1 fusion proteins using an intracellular expression system in <i>Escherichia coli</i>, with all steps optimized for cytosolic production. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Light-Activated Affinity-Based Recombinant Protein Purification</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148943023","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 Step-by-Step Guide to Successful Multiparametric Microbiota Flow Cytometry","authors":"Toni Sempert,&nbsp;Lisa Budzinski,&nbsp;Robin Kempkens,&nbsp;Gi-Ung Kang,&nbsp;René Maier,&nbsp;Hyun-Dong Chang","doi":"10.1002/cpz1.70460","DOIUrl":"10.1002/cpz1.70460","url":null,"abstract":"<p>The role of the human microbiota in host health is an important area of research. Conventional characterization of the complex microbial communities colonizing the human body is primarily performed using culture-based or high-throughput sequencing approaches, which have improved general understanding yet lack cellular features relevant for interaction and function at the single-cell level. Flow cytometry is an established tool for single-cell analysis, and recent improvements, such as increased resolution of small cells and particles, offer possibilities for characterizing complex microbial communities. Here, we describe an updated protocol to characterize complex microbial communities derived from human stool samples at the single-cell level by multiparametric microbiota flow cytometry. Our protocol covers the process from the isolation of bacteria from stool, preparation of cryo-stocks, staining procedure for phenotypic features of the bacteria, and flow cytometric analysis. Our phenotypic characterization includes light scattering properties, quantitative DNA staining, isotype-specific staining of host–antibody coating, and profiling of bacterial cell surface sugar moieties. In addition, we highlight the importance of determining the bacterial load for improved biological interpretation. External staining controls ensure reproducibility and quality controls. Additionally, we suggest a downstream analysis pipeline involving segmentation of multivariate data by a self-organized map (SOM) and machine learning to generate specific microbiota fingerprints. Optionally, bacteria with specific phenotypic characteristics can be isolated by fluorescence-activated cell sorting (FACS) for further investigation. Our protocol can be applied to any microbial community or sample source and offers the flexibility to be expanded with additional phenotypic markers. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of cryo-preserved bacterial stocks from a single-cell microbiota suspension derived from native human stool sample</p><p><b>Basic Protocol 2</b>: Staining protocol for cryo-preserved microbiota stocks and acquisition using a flow cytometer</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13544355/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148893170","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":"Optimized Extraction Protocol of Chloroplast DNA from Chenopodium album (Amaranthaceae) Enabling Whole Plastid Genome Sequencing.","authors":"Rajkumari Jashmi Devi, Merinashwari Konjengbam, Biseshwori Thongam","doi":"10.1002/cpz1.70482","DOIUrl":"https://doi.org/10.1002/cpz1.70482","url":null,"abstract":"<p><p>Chenopodium is one of the most complex genera in the family Amaranthaceae, exhibiting ploidy levels ranging from diploid to hexaploid. The lack of distinct morphological characters and phenotypic plasticity has complicated species identification in this genus. The advent of next-generation sequencing technology has enabled in-depth genomic studies of Chenopodium species. To date, the chloroplast genomes of six Chenopodium species-Chenopodium acuminatum, C. ficifolium, C. berlandieri, C. petiolare, C. quinoa, and C. album-have been reported using genomic DNA as templates. On the other hand, high-quality chloroplast DNA (cpDNA) is recommended for effective chloroplast genome sequencing. The present study reports an optimized high-salt extraction method for extracting cpDNA from C. album. The quality of isolated cpDNA was validated by determining the complete chloroplast genome of C. album using the Illumina platform with a 150 × 2-pair-end (PE) library and cpDNA as the template. The chloroplast genome of C. album is 152,028 base pairs and contains 129 genes. Of these, 84 are protein-coding genes, 8 are rRNA genes, and 37 are tRNA genes. This study lays the foundation for isolating high-quality cpDNA using a simple yet efficient method for extracting cpDNA from C. album, enabling efficient sequencing of the whole plastid genome for further understanding of the evolution, genetics, and taxonomy of this complex plant. © 2026 Wiley Periodicals LLC Basic Protocol: An optimized high-salt extraction protocol for chloroplast DNA isolation from Chenopodium album (Amaranthaceae), enabling whole plastid genome sequencing.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":"e70482"},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149066907","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 Stepwise Method to Derive Functional Pancreatic Islet-like Clusters from Human Pluripotent Stem Cells.","authors":"Manuj Bandral, Jimena Alvarez-Castanon, David S Lorberbaum","doi":"10.1002/cpz1.70467","DOIUrl":"10.1002/cpz1.70467","url":null,"abstract":"<p><p>The most common treatment for people with people with type 1 diabetes (T1D) requires injections of exogenous insulin to regulate blood glucose homeostasis. A longer-term therapeutic solution for T1D has emerged whereby patients may regain insulin independence through transplantation of donated human pancreatic islets, which contain insulin-secreting beta (β)-cells. Donated islets, however, are in short supply. Much effort has been dedicated to using human pluripotent stem cell (hPSC)-derived islets (SC-islets) as an unlimited source of insulin-secreting β-cells. Here, we describe a robust and reproducible 40-day, seven-stage stepwise process for generating hPSC-derived SC-islets. Our differentiation protocol aims to mimic in vivo pancreas development using stage-specific signals to promote the generation of SC-islets. The initial maintenance and the first four stages are performed in two-dimensional monolayer culture; the subsequent three stages require three-dimensional culture in microwells to induce the formation of islet-like clusters. The SC-islets generated at the end of the procedure respond to glucose challenges, reliably express appropriate endocrine markers, contain intracellular insulin, and display an islet-like architecture. Most SC-islets also partially express maturity markers that are often challenging to activate in vitro. These protocols thus provide a scalable platform for generating SC-islets for disease modeling and cell replacement studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Thawing, recovery, and passaging of cryopreserved hPSCs Support Protocol 1: Preparation of solutions and media for hPSC maintenance and expansion Support Protocol 2: Preparation of Matrigel-coated plates for passaging hSPCs Basic Protocol 2: Differentiation of hPSCs to SC-islets.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":"e70467"},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13614477/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149066839","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}