Current protocols最新文献

High-quality DNA with sufficient yield is the goal of DNA extraction protocols. We present an optimized, cost-effective method for extracting next-generation sequencing (NGS)-quality genomic DNA from Bacillus and Clostridium species using the chloroform-isoamyl approach. The protocol involves two main procedures: cultivation of the bacteria under appropriate conditions, followed by DNA extraction through cell lysis, phase separation, DNA precipitation, and cleanup. This method has been successfully applied to several hundred strains of Bacillus and Clostridium species in our laboratory, including B. cereus, B. licheniformis, C. sporogenes, and C. tyrobutyricum, demonstrating its efficacy and reliability for producing high-quality DNA that meets NGS quality control standards. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Culturing Bacillus and Clostridium species

Basic Protocol 2: DNA extraction © 2024 by John Wiley & Sons, Inc.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC₅₀ values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities

Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity

Basic Protocol 3: Image acquisition, processing, and data analysis

Conventional live virus research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease-19 (COVID-19), requires Biosafety Level 3 (BSL-3) facilities. SARS-CoV-2 pseudotyped viruses have emerged as valuable tools in virology, mimicking the entry process of the SARS-CoV-2 virus into human cells by expressing its spike glycoprotein in a surrogate system using recombinant plasmids. One significant application of this tool is in functional assays for the evaluation of neutralizing antibodies. Pseudotyped viruses have the advantage of being competent for only a single cycle of infection, providing better safety and versatility and allowing them to be studied in BSL-2 laboratories. Here, we describe three protocols for the detection of SARS-CoV-2 neutralizing antibodies through a pseudotyped virus assay. First, SARS-CoV-2 S pseudotyped viruses (PV SARS-CoV-2 S) are produced using a Moloney murine leukemia virus (MuLV) three-plasmid system. The plasmids are designed to express the GagPol packing proteins, enhanced green fluorescent protein (eGFP) as a readout system, and the SARS-CoV-2 S protein modified to remove the endoplasmic reticulum retention domain and to improve infection. Next, the internalization of PV SARS-CoV-2 S protein in human embryonic kidney 293T (HEK-293T) cells overexpressing angiotensin-converting enzyme 2 (HEK-293T-ACE2) is confirmed by fluorescence microscopy and quantified using flow cytometry. Finally, PV SARS-CoV-2 S is used to screen neutralizing antibodies in serum samples from convalescent COVID-19 patients; it can also be used for studying the cell entry mechanisms of different SARS-CoV-2 variants, evaluating antiviral agents, and designing vaccines. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Generation of PV SARS-CoV-2 S pseudotyped virus

Basic Protocol 2: Assay of PV SARS-CoV-2 S internalization in target cells.

Basic Protocol 3: Detection of neutralizing antibodies in serum samples.

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the Proflow^TM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A₂₆₀/A₂₈₀) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC.

Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples

Support Protocol 1: The GenElute extraction method

Support Protocol 2: Hybrid 1 extraction method

Single-cell genomic analysis enables researchers to gain novel insights across diverse research areas, including developmental biology, tumor heterogeneity, and disease pathogenesis. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging as the amount of genomic DNA present in a single cell is limited. Advancements in multiple displacement amplification (MDA) technologies allow the unbiased amplification of limited quantities of DNA under conditions that maintain its integrity. This method of amplification results in high yield and facilitates the generation of high-complexity NGS libraries that ensure the highest coverage to effectively allow variant calling.

With the introduction of new sequencing platforms and chemistry, whole genome sequencing became a more cost-effective application, but enrichment of specific regions of interest further reduces the amount of required sequencing output and associated costs. There are two enrichment methods, polymerase chain reaction (PCR)–based and hybrid-capture-based methods. PCR-based methods are very flexible and highly effective but focus on specific loci, typically known to be associated with disease. Inherited diseases of unknown genetic origin require a more comprehensive approach to capture the genetic variation that is not yet associated with a specific disease. Hybrid capture enrichment methods require considerable amounts of DNA such that exome enrichment from single cells is only possible after preamplification of this limited material.

This article describes the complete workflow from single cells and small quantities of DNA to exome-NGS libraries for Illumina sequencing instruments and includes the following protocols: © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Whole genome amplification from single cells or small amounts of gDNA

Basic Protocol 2: NGS library generation of MDA-amplified material

Basic Protocol 3: Exome enrichment

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni²⁺ and the target protein's His₆-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His₆-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification

Basic Protocol 2: Purification of His₆-tagged proteins using the VFD

Alternate Protocol: VFD-mediated His₆-tagged protein purification from non-clarified lysate

Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification

LipidOne 2.0 (https://lipidone.eu) is a new web bioinformatic tool for the analysis of lipidomic data. It facilitates the exploration of the three structural levels of lipids: classes, molecular species, and lipid building blocks (acyl, alkyl, or alkenes chains). The tool's flexibility empowers users to seamlessly include or exclude experimental groups and lipid classes at any stage of the analysis. LipidOne 2.0 offers a range of mono- and multivariate statistical analyses, specifically tailored to each structural level. This includes a novel lipid biomarker identification function, integrating four diverse statistical parameters. LipidOne 2.0 incorporates Lipid Pathway analysis across all three structural levels of lipids. Users can identify lipid-involved reactions through case-control comparisons, generating lists of genes/enzymes and their activation states based on Z scores. Accessible without the need for registration, LipidOne 2.0 provides a user-friendly and efficient platform for exploring and analyzing lipidomic data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Dataset preparation for LipidOne 2.0

Support Protocol: Lipid nomenclature from spectrometric experiments

Basic Protocol 2: Uploading a dataset into LipidOne 2.0

Basic Protocol 3: Data mining of lipidomic dataset by LipidOne 2.0

Primary human hepatocytes (PHHs) are recognized as the "gold standard" for evaluating toxicity of various drugs or chemicals in vitro. However, due to their limited availability, primary hepatocytes isolated from rodents are more commonly used in various experimental studies than PHHs. However, bigger differences in drug metabolism were seen between humans and rats compared to those between human and non-human primates. Here, we describe a method to isolate primary hepatocytes from the liver of rhesus macaques (Macaca mulatta, a species of Old-World monkey) after in situ whole liver perfusion. Techniques for cryopreserving and recovering primary macaque hepatocytes (PMHs) are also described. Given the remarkable physiological and genetic similarity of non-human primates to humans, PMHs isolated using this protocol may serve as a reliable surrogate of PHHs in toxicological research and preclinical studies. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

Basic Protocol 1: In situ whole liver perfusion

Basic Protocol 2: Primary macaque hepatocyte isolation and cell plating

Basic Protocol 3: Cryopreservation and recovery of primary macaque hepatocytes

The study of human intestinal physiology and host-microbe interactions is crucial for understanding gastrointestinal health and disease. Traditional two-dimensional cell culture models lack the complexity of the native intestinal environment, limiting their utility in studying intestinal biology. Here, we present a detailed protocol for the set up and utilization of a three-dimensional (3D) in vitro bioreactor system for human intestinal studies and bacterial co-culture. This article outlines the design and assembly of the bioreactor system, scaffold fabrication, bacterial culture techniques, analysis methods, and troubleshooting tips. By providing step-by-step instructions, the goal is to enable other laboratories to utilize physiologically relevant tissue models of the human intestine, incorporating key features, such as nutrient flow, multiple human cell types, 3D architecture, and microbial communities. The incorporation of commensal bacteria into the bioreactor system allows for the investigation of complex host-microbe interactions, providing insight into gastrointestinal health and pathology. This article serves as a comprehensive resource for scientists seeking to advance their understanding of intestinal biology toward the development of novel therapeutic strategies for gastrointestinal disorders. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Scaffold design

Basic Protocol 2: Intestinal cell culture: Caco2 cells

Basic Protocol 3: Intestinal cell culture: organoids

Basic Protocol 4: Bioreactor design and set up

Basic Protocol 5: Bacteria in 3D bioreactor set up

Basic Protocol 6: Bacteria and drug dosing

Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence-activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS-based methods have recently been deployed in single-cell purification for single-cell RNA sequencing–based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off-target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of transfected canola protoplasts for sorting

Basic Protocol 2: Fluorescence-activated protoplast sorting

Basic Protocol 3: Bead culture of sorted protoplasts and recovery of plantlets