Current protocols最新文献

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

In this article, we describe the synthesis of N³-methyluridine (m³U) and 2′-O-alkyl/2′-fluoro-N³-methyluridine (2′-O-alkyl/2′-F-m³U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2′-O-alkyl-m³U phosphoramidite starts with commercially available uridine to achieve a tritylated m³U intermediate, followed by 2′-O-alkylation and finally phosphitylation. Synthesis of the 2′-F-m³U phosphoramidite is obtained from a commercially available 2′-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2′-position of m³U that can be employed in numerous nucleic acid–based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of 2′-O-alkyl-N³-methyluridine analogs and their corresponding phosphoramidites

Alternate Protocol 1: Synthesis of 2′-O-TBDMS-N³-methyluridine and its phosphoramidite

Alternate Protocol 2: Synthesis of 2′-fluoro-N³-methyluridine and its phosphoramidite

Basic Protocol 2: Solid-phase synthesis of N³-methyluridine-modified DNA and RNA oligonucleotides

Understanding the genetic basis of antimicrobial resistance is crucial for developing effective mitigation strategies. One necessary step is to identify the antimicrobial resistance genes (ARGs) within a microbial population, referred to as the resistome, as well as the mobile genetic elements (MGEs) harboring ARGs. Although shotgun metagenomics has been successful in detecting ARGs and MGEs within a microbiome, it is limited by low sensitivity. Enrichment using cRNA biotinylated probes has been applied to address this limitation, enhancing the detection of rare ARGs and MGEs, especially when combined with long-read sequencing. Here, we present the TELCoMB protocol, a Snakemake workflow that elucidates resistome and mobilome composition and diversity and uncovers ARG-MGE colocalizations. The protocol supports both short- and long-read sequencing and does not require enrichment, making it versatile for various genomic data types. TELCoMB generates publication-ready figures and CSV files for comprehensive analysis, improving our understanding of antimicrobial resistance mechanisms and spread. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Installing TELCOMB Locally

Alternate Protocol: Installing TELCOMB on a SLURM Cluster

Basic Protocol 2: Data Preprocessing

Basic Protocol 3: Calculation of Resistome Distribution and Composition

Basic Protocol 4: Identification of ARG-MGE Colocalizations

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs

Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies

Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs

Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization

Current Protocols is issuing corrections for the following protocol article:

Andreou, I., Storbeck, M., Hahn, P., Rulli, S., & Lader, E. (2024). Exome sequencing starting from single cells. Current Protocols, 4, e70017. doi: 10.1002/cpz1.70017

In the above-referenced article:

A new Table 10 has been added and the existing Table 10 has been renumbered as Table 12.

The existing Tables 12 to 14 have been renumbered as Tables 13 to 15.

The current version online now includes these corrections and may be considered the authoritative version of record.