Methods in molecular biology最新文献

Although metalloproteases (MPs) have been studied for decades, their precise roles in various pathological processes remain to be fully elucidated. These enzymes often function within complex networks, and their activation states can vary over time and depending on the pathological context. In this dynamic landscape, there is a critical need for robust analytical tools capable of accurately identifying which MPs are present in their active forms and how their activation patterns evolve. Among the available technologies for profiling active MPs, affinity-based probes (AfBPs) have emerged as powerful chemical tools. These probes enable both the tracking and unambiguous identification of active MPs in complex biological systems. Here we attempt to provide guidelines for the design of AfBPs that could show selectivity for specific MP subclasses. We present the different alternatives for each structural element of a custom AfBP for MPs and the parameters that need to be considered in order to generate an effective AfBP for a target enzyme.

In this chapter, we present the possibility of using ultraviolet (UV) and circular dichroism (CD) spectroscopies to analyze RNA-RNA interactions as an alternative or complementary technique to the classical RNA gel mobility shift assay. We present relevant, detailed protocols and the results obtained for a specific biological model to investigate all three methods. In this model, we studied the binding of small noncoding sRNA molecules of bacteriophage origin to fragments of bacterial mRNAs that were selected based on in silico analysis. We conclude that spectroscopic methods can be especially useful in analyzing the interactions of small RNAs with target transcripts and in the experimental validation of their target sites of action.

Differential scanning fluorimetry (DSF) is a versatile and accessible technique for probing the thermal stability of peptide-major histocompatibility complexes (pMHCs). Understanding the thermal transition midpoint (T_m) between the folded and unfolded states of pMHCs provides critical insights into their structural stability, which is essential for studying antigen presentation and immune responses. pMHC stability is influenced by the inherent structural differences between MHC class I and class II molecules. Notably, class I MHCs require bound peptides for stability, while class II MHCs remain stable without them. Class I MHCs typically present peptides of 8-12 amino acids anchored at specific residues, whereas class II MHCs accommodate longer peptides with distinct anchoring positions. This manuscript outlines a comprehensive DSF protocol optimized for pMHCs, highlighting the method's advantages over other thermal stability techniques, such as differential scanning calorimetry (DSC) and circular dichroism (CD). DSF offers a simpler, cost-effective alternative, utilizing minimal sample volume and readily available real-time PCR (qPCR) instrumentation. We detail critical steps for sample preparation, including optimal buffer selection, dye addition, and degassing procedures, along with specific instrument setup guidelines for both qPCR-based systems and the NanoTemper Prometheus. Data analysis strategies using Microsoft Excel and Origin software are also discussed, including normalization, derivative calculation, and T_m determination. By providing a standardized DSF protocol tailored to pMHC analysis, this manuscript aims to support researchers in efficiently measuring thermal stability, thereby facilitating investigations into pMHC dynamics and immune function.

Pathogenic dimorphic fungi are endemic in certain regions and can cause from subclinical infections to systemic mycoses. These pathogens are associated with high mortality and morbidity rates and represent emerging infectious threats to human populations worldwide. Because most fungal pathogens cause lung infection or use the lung as a route to disseminate to other organs, mammalian pulmonary infection models are crucial tools in the advancement of medical mycology. Many different types of animal models of fungal infection have been developed, with murine models being hailed as the gold standard for studies of pathogenesis, to compare virulence between isolates, preclinical evaluation of vaccines and therapies, and host antifungal immune responses. The ability to control numerous variables in performing the model allows for mimicking human disease states and quantitatively monitoring the course of the disease. To model lung inflammation and injury, fungal infectious propagules can be inoculated via intranasal delivery, intratracheal, or aerosolization approaches. The protocol in this chapter details a method for intratracheal delivery of a fungal suspension, where fungal cells are administered directly into the lungs to initiate infection. The aim is to provide a comprehensive description of techniques required to perform mouse pulmonary infection, such that reproducible results are attained, allowing for the use of this in vivo approach for high-quality studies.

End-point PCR, followed by restriction fragment length polymorphism (RFLP) or sequencing and sequence analysis of the 16S rRNA gene, is a cornerstone method for the universal detection and identification of phytoplasmas, including previously undescribed strains. This protocol describes the use of end-point PCR in direct, nested, or semi-nested systems, coupled with RFLP analysis using 17 restriction enzymes, to classify phytoplasmas into distinct 16Sr ribosomal groups and subgroups while addressing challenges such as inter-operon heterogeneity and mixed infections. Alternatively, sequencing of the PCR-amplified 16S rRNA gene, followed by sequence analysis (e.g., virtual RFLP or sequence homology), enables classification into either 16Sr groups/subgroups or 'Candidatus Phytoplasma' species. The method's sensitivity, cost-effectiveness, and compatibility with established classification frameworks make it invaluable for epidemiological studies, quarantine measures, and the delineation of 'Candidatus Phytoplasma' species. By providing a clear framework for the precise diagnosis of phytoplasma-associated diseases in diverse plant and insect hosts, this protocol supports rapid responses to outbreaks and helps mitigate the economic impact of phytoplasmas.

Imp is a gene coding for the immunodominant membrane protein Imp that is supposed to be involved in host-pathogen interactions. It was identified in some 'Candidatus Phytoplasma' species and showed a considerable sequence variability. For this characteristic it can be exploited to complement conventional phytoplasma classification based on the conserved 16S rRNA gene, enhancing the differentiation of closely related phytoplasma strains. This protocol describes a method for amplification and sequence analysis of the imp gene of Flavescence dorée (FD) related phytoplasmas, that can be useful for detecting and genotyping the strains involved, for example, in the complex FD epidemiology.

CUT&Tag (Cleavage Under Targets and Tagmentation) is a powerful method for chromatin profiling based on an enzyme-tethering strategy. Compared with chromatin immunoprecipitation (ChIP) assay, CUT&Tag requires smaller amount of cell/nuclei input and generates data with higher signal-to-noise ratio, which has been widely applied in profiling histone modifications and protein-DNA interactions. Here, we describe a detailed protocol for bulk-cell CUT&Tag in plants and briefly introduced other derivative CUT&Tag methods.

Transient luciferase reporter assays are a widely used and powerful tool for investigating promoter activity and gene regulation in plant systems. These assays make use of the enzymatic conversion of a luciferin substrate into bioluminescence to provide a highly sensitive and reproducible measure of transcriptional activity. The dual-luciferase system, incorporating Firefly luciferase (LUC) as the primary reporter and Renilla luciferase (REN) as an internal control, enhances experimental accuracy by normalizing for variations in infiltration efficiency, tissue viability, and environmental conditions. Agroinfiltration-mediated transient expression in Nicotiana benthamiana leaves enables rapid and high-throughput analysis of transcriptional regulation, including the functional characterization of transcription factors (TFs), cis-regulatory elements, and enhancer/silencer activity. This approach facilitates systematic promoter dissection through site-directed mutagenesis, deletion mapping, and combinatorial TF studies to uncover complex regulatory interactions. Here, we present a detailed protocol for performing dual-luciferase transient expression assays, emphasizing best practices for ensuring robust and reproducible results.

Proximity labeling (PL) techniques have emerged as powerful approaches for the study of protein associations in vivo. In this chapter, we present a step-by-step TurboID-based PL protocol to define the "proxiome" of a protein of interest in plants, from the generation of the fusion protein to the analysis of the results, using the publicly available Ti-TAN plasmid collection.

The cistrome comprises genomic loci that regulate gene expression, playing a crucial role in defining cellular identity and function. Analyzing cistrome data reveals key molecular mechanisms underlying grapevine growth, development, and environmental responses. Identifying transcription factors that bind specific DNA sequences allows researchers to dissect the complex regulatory networks controlling gene expression. Moreover, this analysis can help pinpoint targets for crop improvement, as traits like fruit quality, disease resistance, and abiotic stress tolerance are often regulated by transcription factors. DNA affinity purification sequencing (DAP-seq) is a high-throughput, cost-effective method for mapping the cistrome, providing valuable insights into transcriptional regulation. This technique relies on the in vitro affinity purification of genomic DNA-protein complexes, followed by high-throughput sequencing of eluted DNA fragments. Unlike other in vitro DNA-binding assays, such as protein-binding microarrays (PBM) and systematic evolution of ligands by exponential enrichment (SELEX), DAP-seq allows transcription factors to interact directly with plant-derived genomic DNA, capturing all potential binding sites. The resulting data closely resemble those from chromatin immunoprecipitation sequencing (ChIP-seq) but are obtained much faster. Initially developed in Arabidopsis, DAP-seq has since been applied to several crops, including maize, tomato, and grapevine, generating extensive cistrome datasets and deepening our understanding of gene regulatory regions. However, despite its power in elucidating crop biology, DAP-seq faces certain limitations, particularly concerning the size and complexity of plant genomes. This chapter presents detailed protocols for DAP-seq studies aimed at the unbiased identification of transcription factor binding sites in crops. Additionally, we outline a standardized pipeline for DAP-seq data analysis, encompassing raw sequencing data processing (i.e., trimming, filtering, and read alignment), as well as peak calling and motif discovery analysis. This approach enables the efficient and scalable identification of transcription factor binding profiles in diverse crop species.