Current protocols最新文献

Extracellular vesicles (EVs) play an important role in cell-cell communication, carrying bioactive molecules including DNA. EV-associated DNA (EV-DNA) has created enormous interest in the field of biomarkers, particularly related to liquid biopsy. However, its analysis is challenging due to the nanoscale structure of EVs, the low abundance of EV-DNA, and surrounding biogenetic debate. Therefore, novel protocols to enhance the accurate detection of EV-DNA are essential to study its role in normal physiology and disease states. Here, we provide two protocols for confirming the presence of EV-DNA from biological samples. In the first protocol, ultrathin sectioning of EVs is combined with immunogold labeling to detect the presence of double-stranded (ds) DNA within the EV lumen using transmission electron microscopy (TEM). In the second protocol, whole-mount EV immunogold labeling allows detailed morphological analysis of EVs and their surface-associated DNA. Using TEM imaging, we have demonstrated that cancer-cell-derived individual EVs exhibit simultaneous positivity for dsDNA and the EV surface protein tetraspanin 9. We believe that this method can be used to label any proteins of interest inside as well as on the surface of EVs. This can aid in the characterization of single EVs and in the identification and verification of EV-associated biomarkers. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: EV isolation from cell-culture-conditioned medium, EV embedding, ultrathin sectioning, labeling, and imaging

Basic Protocol 2: Whole-mount immunolabeling of EV-DNA

NeuroClick is a software tool designed for the in silico execution of azide-alkyne cycloaddition reactions, commonly known as click reactions. We developed this graphical user interface application to expedite the drug discovery process by generating libraries of 1,2,3-triazole compounds. NeuroClick enables users to input reagent SMILES strings, rapidly generating and screening extensive combinatorial libraries at a pace of 10,000 molecules per minute. The software applies stringent criteria to ensure the relevance and accuracy of the generated compounds, excluding molecules without azide groups or those with multiple reactive functional groups to maintain dataset integrity. NeuroClick incorporates advanced filtering options based on Lipinski's rule of five and blood–brain barrier (BBB) permeability predictors, allowing researchers to identify drug-like molecules with potential central nervous system activity. The software's high-throughput and user-friendly interface significantly enhance the efficiency of early-stage drug development by facilitating the exploration of vast chemical spaces and identifying promising lead compounds for further development. This article provides comprehensive guidance on the installation, usage, and features of NeuroClick, ensuring that users can leverage its full potential in their research endeavors. © 2024 Wiley Periodicals LLC.

Basic Protocol: NeuroClick workflow for generating BBB-permeating drugs

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Genotyping known pathogenic tandem repeat loci

Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy

Support Protocol 1: Installation of tools and ExpansionHunter catalog modification

Basic Protocol 2: Performing genome-wide genotyping of tandem repeats

Basic Protocol 3: Discovering de novo tandem repeat expansions

Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)

Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells

Support Protocol 1: Plasmid amplification

Support Protocol 2: Lentivirus preparation

Support Protocol 3: Lentivirus titration

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca²⁺]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca²⁺] by releasing Ca²⁺ from the sarcoplasmic reticulum (SR). Timely Ca²⁺ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca²⁺ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca²⁺ release events across a range of [Ca²⁺]. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Cardiomyocyte isolation from mouse

Basic Protocol 2: Preparation of cardiomyocytes for Ca²⁺ imaging

Basic Protocol 3: Confocal microscopy and quantitative Ca²⁺ analysis using SparkMaster 2

Multiple sequence alignments and phylogenetic trees are rich in biological information and are fundamental to research in biology. PhyKIT is a tool for processing and analyzing the information content of multiple sequence alignments and phylogenetic trees. Here, we describe how to use PhyKIT for diverse analyses, including (i) constructing a phylogenomic supermatrix, (ii) detecting errors in orthology inference, (iii) quantifying biases in phylogenomic data sets, (iv) identifying radiation events or lack of resolution using gene support frequencies, and (v) conducting evolution-based screens to facilitate gene function prediction. Several PhyKIT functions that streamline multiple sequence alignment and phylogenetic processing—such as renaming FASTA entries or tree tips—are also discussed. These protocols demonstrate how simple command-line operations in the unified framework of PhyKIT facilitate diverse phylogenomic data analysis and processing, from supermatrix construction and diagnosis to gaining clues about gene function. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Installing PhyKIT and syntax for usage

Basic Protocol 2: Constructing a phylogenomic supermatrix

Basic Protocol 3: Detecting anomalies in orthology relationships

Basic Protocol 4: Quantifying biases in phylogenomic data matrices and related measures

Basic Protocol 5: Identifying polytomies

Basic Protocol 6: Assessing gene-gene coevolution as a genetic screen

Coral reefs are invaluable ecosystems that are under threat from various anthropogenic stressors. There has been a recent increase in the diagnostic tools utilized to understand how these threats impact coral reef health. Unfortunately, the application of diagnostic tools like transmission electron microscopy (TEM) is not as standardized or developed in coral research as in other research fields. Utilizing TEM in conjunction with other diagnostic methods can aid in understanding the impact of these stressors on the cellular level because TEM offers valuable insight into the structures and microsymbionts associated with coral tissue that cannot be obtained with a conventional light microscope. Additionally, a significant amount of coral tissue ultrastructure has not yet been extensively described, causing a considerable gap in our understanding of cellular structures that could relate to the immune response, cellular function, or symbioses. Moreover, additional standardization is needed for TEM in coral research to increase comparability and reproducibility of findings across studies. Here, we present standardized TEM sample fixation, embedding, and sectioning techniques for coral studies that ensure consistent ultrastructural preservation and minimize artifacts, enhancing the reliability and accuracy of TEM observations. We also demonstrate that these TEM protocols allow for the observation and quantification of bacterial and viral-like particles within the coral tissue as well as the endosymbiotic microalgae, potentially providing insight into their interactions within coral cells and how they relate to overall coral health and resilience. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Primary fixation

Basic Protocol 2: Decalcification

Basic Protocol 3: Sample dissection, secondary fixation, dehydration, and embedding

Basic Protocol 4: Sectioning and grid staining

Basic Protocol 5: Imaging

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Production of the erm-bar DNA

Basic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeri

Alternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strains

Basic Protocol 3: Setting up mixed populations of barcode-tagged strains

Basic Protocol 4: Performing a competitive growth assay

Basic Protocol 5: Amplicon library preparation and equimolar pooling

Basic Protocol 6: Sequencing on Illumina iSeq 100

Basic Protocol 7: BarSeq data analysis

Transcriptomic data is often expensive and difficult to generate in large cohorts relative to genomic data; therefore, it is often important to integrate multiple transcriptomic datasets from both microarray- and next generation sequencing (NGS)-based transcriptomic data across similar experiments or clinical trials to improve analytical power and discovery of novel transcripts and genes. However, transcriptomic data integration presents a few challenges including reannotation and batch effect removal. We developed the Gene Expression Data Integration (GEDI) R package to enable transcriptomic data integration by combining existing R packages. With just four functions, the GEDI R package makes constructing a transcriptomic data integration pipeline straightforward. Together, the functions overcome the complications in transcriptomic data integration by automatically reannotating the data and removing the batch effect. The removal of the batch effect is verified with principal component analysis and the data integration is verified using a logistic regression model with forward stepwise feature selection. To demonstrate the functionalities of the GEDI package, we integrated five bovine endometrial transcriptomic datasets from the NCBI Gene Expression Omnibus. These transcriptomic datasets were from multiple high-throughput platforms, namely, array-based Affymetrix and Agilent platforms, and NGS-based Illumina paired-end RNA-seq platform. Furthermore, we compared the GEDI package to existing tools and found that GEDI is the only tool that provides a full transcriptomic data integration pipeline including verification of both batch effect removal and data integration for downstream genomic and bioinformatics applications. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: ReadGE, a function to import gene expression datasets

Basic Protocol 2: GEDI, a function to reannotate and merge gene expression datasets

Basic Protocol 3: BatchCorrection, a function to remove batch effects from gene expression data

Basic Protocol 4: VerifyGEDI, a function to confirm successful integration of gene expression data