Current protocols最新文献

Living myocardial slices (LMS) are ultrathin sections of adult myocardium that can be maintained in culture. These slices provide a unique platform for studying interactions between cardiomyocytes (CMs), other cardiac cell types, and the extracellular matrix while maintaining the cytoarchitecture and electrical phenotype of CMs over extended periods. Despite their advantages over other cardiac models, LMS have limitations, particularly their reliance on slice quality. The primary factor influencing the quality of the slices is the method used to process the cardiac tissue block. Current methods typically require immediate slice preparation following the excision of the tissue block, which restricts the timing of experiments. To address this limitation, we developed a simple procedure for cryopreserving human adult myocardium, allowing the preparation of LMS at a later stage. The protocol provides a list of required equipment and reagents, as well as a detailed description of the methodology for processing the myocardium and slice preparation. We present typical results demonstrating that cryopreserved human cardiac tissue retains biomass and structural integrity comparable to freshly obtained myocardium. Furthermore, we assessed the LMS derived from both fresh and cryopreserved samples. Histological analysis confirmed the preservation of viability, normal cytomorphology, and gap junctions between CMs in all LMS after 24 h and up to 5 days of culture in the absence of electrical stimulation. Cryopreservation extends the interval between tissue collection and LMS preparation, facilitating longer-term and more complex experiments. Further research into the impact of cryopreservation on various cardiac cell types will promote better donor organ management and efficient banking of cardiac samples from a multitude of donors and disease states. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation and preservation of human adult myocardium

Basic Protocol 2: Preparation of adult living myocardial slices from cryopreserved blocks

Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using VSV as a platform for directed protease evolution Alternate Protocol: Dose response assay with TCID₅₀ readout Support Protocol 1: A pipeline for high-throughput VSV sequencing Support Protocol 2: Rescue of VSV.

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. Basic Protocol 1: Searching MobiDB query formats Basic Protocol 2: Searching MobiDB selected datasets and selected proteomes Basic Protocol 3: Performing a search on the Statistics page in MobiDB Support Protocol: Programmatic access with MobiDB REST API Basic Protocol 4: Visualizing and interpreting a MobiDB Entry: The GHR use case.

These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing in vitro fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo EDC treatment of rats and immunostaining of treated oocytes Basic Protocol 2: In vitro fertilization and immunostaining of early-stage embryos.

Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex

Basic Protocol 2: Deletion of specific genomic region using RNPs via nucleofection

Basic Protocol 3: Use of homology-directed repair system to introduce site-specific mutations

The cover image is based on the Article PhyKIT: A Multitool for Phylogenomics by Jacob L. Steenwyk et al., https://doi.org/10.1002/cpz1.70016.

While various methods exist for examining and visualizing the structures of RNA molecules, dimethyl sulfate-mutational profiling and sequencing (DMS-MaPseq) stands out for its simplicity and versatility. This technique has been proven effective for studying RNA structures both in vitro and in complex biological settings. We present an updated protocol of DMS-MaPseq, as well as methodology that enables it to be used for detection of antisense oligonucleotides (ASOs) binding to RNA. By applying this protocol, we successfully characterized the structural ensemble of the HIV1 Rev Response Element (RRE), along with its two alternative structures. The findings align with previously published research validating the accuracy of the method. We also demonstrate the utility of the DMS-MaPseq protocol by resolving and confirming ASO binding at the complementary sites of the P21-associated noncoding RNA DNA damage-activated (PANDA) long non-coding RNA via decreased DMS reactivity. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: DMS-MaPseq on HIV1-RRE

Basic Protocol 2: DMS-MaPseq on PANDA with ASO probing

Since their discovery, 3D cell cultures have emerged as powerful tools across various basic, translational research, and industrial discovery projects. One such application is in the physiological and pathophysiological modeling of pancreatic exocrine functions, which addresses critical clinical challenges, including acute and chronic pancreatitis. While several methods now exist for generating epithelial organoids (derived from induced pluripotent, embryonic, or adult stem cells), the advent of patient-derived organoids (PDOs) with controlled polarity has introduced a new frontier in pancreatic research. This advancement has significantly expanded the methodological arsenal available for studying human pancreatic epithelial secretion.

In this article, we present basic protocols and a troubleshooting guide for an advanced culture method that results in an apical-to-basal polarity switch. Alongside the protocols, we emphasize a comprehensive cost breakdown, an aspect often challenging to estimate when implementing new techniques. By sharing the technical nuances and financial implications of these protocols, we aim to encourage researchers to transition from rodent models to primary human epithelial cells wherever feasible. This aligns with the U.S. Environmental Protection Agency's efforts to accelerate the translation of significant scientific findings to address major clinical needs. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Establishment and maintenance of pancreatic PDOs

Basic Protocol 2: Cryopreservation and thawing of pancreatic PDOs

Basic Protocol 3: Inducing polarity switching in pancreatic PDOs

Lymph nodes (LNs) are specialized secondary lymphoid tissues essential to the priming and maintenance of adaptive immune responses, including the B cell germinal center response; thus, they are central to immunity. However, the anatomically restricted and time-resolved nature of immune priming means that sampling disease-relevant human LNs requires specialized techniques. This article describes the application of ultrasound-guided fine-needle aspiration (FNA) to sample LNs, using cervical LNs of the head and neck as an exemplar. This minimally invasive technique allows collection of both immune cells and cell-free material that are relevant to both neuroimmune diseases and basic lymphatic functions. Downstream use of cellular material can include multiplexed flow cytometry, single-cell transcriptome sequencing (RNA-seq), and B cell cultures. The cell-free supernatant can be used for proteomics or other similar ‘omics approaches. This unit describes collection of samples by FNA as well as processing and storage of samples for downstream assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Sampling of human cervical lymph nodes by ultrasound-guided fine-needle aspiration

Alternate Protocol: Sampling of human lymph nodes by ultrasound-guided fine-needle aspiration with negative pressure

Basic Protocol 2: Processing and storage of human lymph node samples

Oligosaccharidoses are a group of lysosomal storage disorders characterized by abnormal storage and excretion of incompletely processed glycan structures. As with other inherited metabolic disorders, early diagnosis and initiation of treatment are essential for optimizing outcomes. Biochemical investigation of suspected oligosaccharidoses has traditionally included thin layer chromatography to detect the presence of disease-specific free oligosaccharides in urine; however, this qualitative method has long been known to have limited sensitivity and specificity. In this unit, a quantitative technique for measuring oligosaccharides utilizing high-performance liquid chromatography–tandem mass spectrometry is described, which provides substantial improvements over other methods, in terms of sensitivity and specificity; moreover, it is relatively inexpensive, accessible, and requires significantly less time, effort, sample volume, and reagents to perform. © 2024 Wiley Periodicals LLC.

Basic Protocol: Analysis of urinary FOS by HPLC–MS/MS