Methods in molecular biology最新文献

The causative agent of "Surra", Trypanosoma brucei evansi (T. b. evansi), is thought to have originated from Trypanosoma brucei brucei (T. b. brucei) and primarily causes trypanosomosis in a wide range of wild and domesticated animals. The disease inflicts significant economic damage to farmers and the livestock industry. Additionally, T. b. evansi is considered a potential zoonotic pathogen, as atypical human infections have been reported. Unlike T. brucei, which requires the tsetse fly as a biological vector, T. b. evansi can be transmitted mechanically by various biting flies, leading to a broader and less predictable global distribution. The economic impact and the global presence of T. b. evansi increase the need for rapid, accurate, and field-deployable diagnostic tests. While polymerase chain reaction (PCR)-based tests are widely used for direct pathogen detection, they generally require skilled personnel and a laboratory environment to ensure proper protocol execution. In contrast, recombinase polymerase amplification (RPA) offers an alternative approach using isothermal nucleic acid amplification that is simple, fast, cost-effective, and well-suited for use in minimally equipped laboratories (and even in field settings). The results of RPA can be visualized using different methods, such as agarose gel electrophoresis (RPA-AGE), lateral flow assay (RPA-LFA), and real-time fluorescence (RPA-RT). In this chapter, we describe the procedures that are used for specifically detecting active T. b. evansi infections. The choice of procedure to be used is determined by several key factors, including the intended application, available resources, and the required sensitivity.

This chapter presents a comprehensive set of protocols for isolating chromatin and histones from Trypanosoma cruzi, tailored for applications ranging from gel-based analyses to high-resolution mass spectrometry. It outlines optimized workflows for extracting basic nuclear proteins, histones, linker histone H1, and chromatin-associated proteins, adapted to different parasite life stages and cell cycle phases. By integrating multiple approaches, these methods address challenges posed by the parasite's unique biology and provide flexible tools for studying histone post-translational modifications and chromatin proteins. The inclusion of protocols compatible with proteomic workflows supports broader investigations into epigenetic regulation and nuclear processes in T. cruzi.

Virus-induced gene silencing (VIGS) has been applied as a functional genomics tool across diverse plant species. Integrated with the Arabidopsis sequence-tagged T-DNA homozygous mutant library, VIGS enables an efficient screening approach that combines features of both forward and reverse genetics, facilitating the identification of novel regulators in plant immunity. Plant defense against pathogens relies on a two-layered immune system, classified as pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Dysregulation of key PTI or ETI components can lead to excessive or uncontrolled cell death. The cell death phenotype offers a unique avenue for genetic screens aimed at identifying suppressors of immune-related cell death. However, conventional genetic approaches face limitations due to seedling lethality and the consequent lack of viable seeds, restricting their efficiency. Here, we describe an Agrobacterium-mediated transient VIGS assay optimized for systematic gene silencing at seedling stages, leading to cell death phenotypes. This method enables high-throughput screening for cell death suppressors using T-DNA homozygous mutant collections. The platform provides a rapid, cost-efficient strategy for uncovering key regulators of plant immune signaling, offering new insights into mechanisms governing immune homeostasis and cell death suppression.

Plant adaptive responses to their environment are orchestrated by transcriptional reprogramming, regulated by sequence-specific transcription factors (TFs) recognizing specific TF-binding sites (TFBSs), and understanding their regulatory grammar is crucial for unravelling plant adaptation mechanisms. Empirical data have contributed to the elucidation of TFBS sequence motifs involved in biological processes, but comprehensive experimental approaches may not be feasible, especially in non-model species. Different computational algorithms have facilitated the elucidation of TFBS sequence motifs and the construction of predictive models, shedding light on plant gene regulatory networks. In this context, the development of straightforward computational pipelines and easy-to-use bioinformatics tools is of particular relevance to make gene expression analysis accessible to the research community. This chapter presents a methodology to infer TF regulators applicable to 60 plant species from RNA sequencing (RNA-seq) data as the starting point. It includes RNA-seq analysis and quantification, gene clustering using WGCNA to identify co-regulated gene modules, and searching for enriched TFBSs associated with these modules. The methodology is illustrated using Arabidopsis RNA-seq data related to abiotic stress. By providing a user-friendly pipeline, researchers are empowered to unravel the molecular basis of gene expression dynamics in plants.

Spiroplasma melliferum is a cultivable mollicute that can be used as a proxy model organism to test the inhibitory effects of filtrates, compounds, or substances against phytoplasma. In this protocol, we describe the use of the resazurin-based alamarBlue™ dye for the monitoring of Spiroplasma survival and growth in the presence of presumably inhibitory agents.

In this introductory chapter, we provide an overview of phytoplasma biology and outline the historical milestones that shaped the field, from their first discovery to their current taxonomic status. We also highlight how this third edition of "Phytoplasmas: Methods and Protocols" differs from previous volumes, reflecting both the adoption of new molecular and sequencing technologies and the revival of classical approaches. Taken together, these developments illustrate the evolving trajectory of phytoplasma research and set the stage for the detailed protocols presented in the following chapters.

Grapevine is among the most important economic plants that are susceptible to phytoplasma disease. Tools and procedures for genetic manipulation of grapevine are cumbersome and sometimes difficult to replicate; therefore, most of the research on plant-phytoplasma interactions focuses on species with more established transformation procedures instead, even though they might not be hosts of the studied phytoplasma. Studying the mechanisms of plant-phytoplasma interactions directly in the host plants would bring new and more accurate insights into the disease mechanisms. In this chapter, we present the protocols to obtain transformation-competent grapevine material from different tissues and the procedures for protoplast isolation and cultivation.

Immediately after birth, mammals are largely colonized by microorganisms, with the gastrointestinal tract being the most commonly colonized organ. Over the years, several studies have shown that the intestinal microbiota is important for various physiological functions of the host. Gnotobiotic animal models are frequently used to better understand how the microbiota influences health and disease scenarios. Among gnotobiotic models, germ-free (GF) animals were first used in 1895, but it was not until 60 years later that germ-free colonies were suitable for large-scale experiments. The use of GF mice is an interesting and rich tool for studying the microbiome. However, their maintenance is a complex process that needs to be done carefully. In this chapter, we describe step by step how to manage and manipulate the gut microbiota of GF mice.

Remarkable progress in basic, translational, and clinical cancer research has been observed during the last decade. This has opened possibilities for the development of novel diagnostics and therapeutic approaches and created opportunities for personalized medicine. Cancer biomarkers are key players in human cancer progression, both peripheral and at the site of tumor. Through reliable techniques detecting biomarkers, cancer can thus be predicted, diagnosed and progression and response to therapy can be followed. Multiplex analysis of biomarkers in small blood volumes allows for rapid quantification of large number of circulating analytes. The Luminex technique allows multiple biomarkers to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of large number of extracellular secreted biomarkers to be used in prediction and therapy prognosis in cancer. The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the biomarker of interest and allow simultaneous detection of up to hundreds of biomarkers in a dual-laser flow analyzer. Biomarkers can be detected in serum- and plasma samples as well as in cell culture supernatants from in vitro cultured and stimulated cells, e.g., peripheral blood mononuclear cells (PBMC) or cancer cell lines.The need for robust detection of biomarkers for prediction as well as outcome of cancer therapy progression is of great importance. This chapter describes the Luminex technique for detection of biomarkers associated with cancer by magnetic bead sandwich immunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMC that may affect the outcome of detection of biomarkers. The Luminex technique is thus one way to discover biomarkers to predict, prognose, and improve clinical outcome of cancer.

The Luminex multiplex bead immunoassay enables the detection of more than 100 analytes in a sample at a time. This method utilizes magnetic beads coated with antibodies of interest to measure multi-analytes simultaneously. Here, we describe a detailed protocol for multi-analyte detection of two proteins, fatty acid binding protein 1 (FABP1) and fibroblast growth factor 19 (FGF19), both related to fat metabolism, in cell lysates of HepG2 hepatocytes.