Current protocols最新文献

In rodents, the first weeks of postnatal life feature remarkable changes in fear memory acquisition, retention, extinction, and discrimination. Early development is also marked by profound changes in brain circuits underlying fear memory processing, with heightened sensitivity to environmental influences and stress, providing a powerful model to study the intersection between brain structure, function, and the impacts of stress. Nevertheless, difficulties related to breeding and housing young rodents, preweaning manipulations, and potential increased variability within that population pose considerable challenges to developmental fear research. Here we discuss several factors that may promote variability in studies examining fear conditioning in young rodents and provide recommendations to increase replicability. We focus primarily on experimental conditions, design, and analysis of rodent fear data, with an emphasis on mouse studies. The convergence of anatomical, synaptic, physiological, and behavioral changes during early life may increase variability, but careful practice and transparency in reporting may improve rigor and consensus in the field. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Identifying impacted pathways is important because it provides insights into the biology underlying conditions beyond the detection of differentially expressed genes. Because of the importance of such analysis, more than 100 pathway analysis methods have been developed thus far. Despite the availability of many methods, it is challenging for biomedical researchers to learn and properly perform pathway analysis. First, the sheer number of methods makes it challenging to learn and choose the correct method for a given experiment. Second, computational methods require users to be savvy with coding syntax, and comfortable with command-line environments, areas that are unfamiliar to most life scientists. Third, as learning tools and computational methods are typically implemented only for a few species (i.e., human and some model organisms), it is difficult to perform pathway analysis on other species that are not included in many of the current pathway analysis tools. Finally, existing pathway tools do not allow researchers to combine, compare, and contrast the results of different methods and experiments for both hypothesis testing and analysis purposes. To address these challenges, we developed an open-source R package for Consensus Pathway Analysis (RCPA) that allows researchers to conveniently: (1) download and process data from NCBI GEO; (2) perform differential analysis using established techniques developed for both microarray and sequencing data; (3) perform both gene set enrichment, as well as topology-based pathway analysis using different methods that seek to answer different research hypotheses; (4) combine methods and datasets to find consensus results; and (5) visualize analysis results and explore significantly impacted pathways across multiple analyses. This protocol provides many example code snippets with detailed explanations and supports the analysis of more than 1000 species, two pathway databases, three differential analysis techniques, eight pathway analysis tools, six meta-analysis methods, and two consensus analysis techniques. The package is freely available on the CRAN repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Processing Affymetrix microarrays

Basic Protocol 2: Processing Agilent microarrays

Support Protocol: Processing RNA sequencing (RNA-Seq) data

Basic Protocol 3: Differential analysis of microarray data (Affymetrix and Agilent)

Basic Protocol 4: Differential analysis of RNA-Seq data

Basic Protocol 5: Gene set enrichment analysis

Basic Protocol 6: Topology-based (TB) pathway analysis

Basic Protocol 7: Data integration and visualization

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species’ development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids

Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer

Support Protocol 2: Preparation of in vitro–transcribed EBNA1 mRNA

Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines

Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR

Support Protocol 5: PiPSC characterization: Immunostaining

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites’ physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites

Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei

Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei

R-loops are nucleic acid structures composed of a DNA:RNA hybrid with a displaced non-template single-stranded DNA. Current approaches to identify and map R-loop formation across the genome employ either an antibody targeted against R-loops (S9.6) or a catalytically inactivated form of RNase H1 (dRNH1), a nuclease that can bind and resolve DNA:RNA hybrids via RNA exonuclease activity. This overview article outlines several ways to map R-loops using either methodology, explaining the differences and similarities among the approaches. Bioinformatic analysis of R-loops involves several layers of quality control and processing before visualizing the data. This article provides resources and tools that can be used to accurately process R-loop mapping data and explains the advantages and disadvantages of the resources as compared to one another. © 2024 Wiley Periodicals LLC.

In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay—the GTPase Glo assay by Promega—for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Conducting GTPase Glo assays

Support Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTP

Support Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates

Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancies, leading to disorders such as uterine placenta previa, placenta accreta spectrum (PAS), and Cesarean scar pregnancy. With rates of C-section at ∼30% of deliveries in the US and projected to continue to climb, an understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are sorely needed. However, there are currently very few animal models of uterine injury and its subsequent impacts on maternal as well as in utero and postnatal fetal outcomes. Here, we describe a procedure for a novel model of surgically induced uterine injury in the genetically tractable laboratory mouse (Mus musculus). We describe preparatory steps for surgery, the induction of uterine injury itself, and post-surgical recovery. We then provide supporting information regarding downstream dissection of pregnant mice. Lastly, we include additional information regarding estrous cycle staging in order to perform surgeries and dissections at the relevant phase in non-pregnant mice. This procedure for incurring uterine injury in a mouse model presents an important step forward in understanding uterine damage and its associated pregnancy disorders. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Preparation for surgery

Basic Protocol 2: Surgery and induction of uterine injury

Basic Protocol 3: Mating and dissection of pregnant mice as endpoint analyses

Support Protocol: Estrous staging of animals

The loading (i.e., substitution) of solid supports for oligonucleotide synthesis is an important parameter in large-scale manufacturing of oligonucleotides. Several key process parameters are dependent on the substitution of the solid support, including the number of phosphoramidite nucleoside equivalents used in the coupling step. For dimethoxytrityl (DMTr)–loaded solid supports, the substitution of the resin is determined by quantitatively cleaving the DMTr protecting group from the resin under acidic conditions and then analyzing the DMTr cation extinction by UV/vis spectroscopy. The spectrometric measurement can be performed at 409 nm or the global extinction maximum of 510 nm. The substitution is then calculated based on the Lambert-Beer law analogously to the substitution determination of Fmoc-substituted resins. Below, the determination of the molar extinction coefficient at 510 nm in a solution of 10% dichloroacetic acid in toluene and subsequent determination of the DMTr loading of DMTr-substituted resins is reported. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Determination of the molar extinction coefficient at 510 nm in DCA Deblock solution

Basic Protocol 2: Substitution determination of DMTr-substituted resins by cleavage of the DMTr cation

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation

Support Protocol 1: Evans blue staining to identify resting spore viability

Support Protocol 2: Storage of Plasmodiophora brassicae

Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates

Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates

Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores

Basic Protocol 5: Molecular detection of Plasmodiophora brassicae

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology

Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay

Support Protocol: Testing of sgRNA efficiency in HEK 293 cells

Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo