Methods in molecular biology最新文献

Transcriptomic profiling of plant-bacterial interactions provides critical insights into the molecular mechanisms underlying parasitism, commensalism, and mutualism. RNA sequencing (RNA-seq) enables the simultaneous analysis of plant and bacterial transcriptomes during colonization; however, integrated computational workflows specifically tailored for co-transcriptome analysis remain limited. Here, we present a step-by-step bioinformatics pipeline for analyzing co-transcriptome landscapes in plant-bacterial interactions. This workflow includes: (1) quality control and processing of raw RNA-seq data from both plant host and in-planta bacterial populations; (2) statistical analyses for differential gene expression; (3) prediction of orthologous bacterial genes and functional annotation of bacterial transcripts using the KEGG database; (4) integration and comparative analysis across multiple bacterial strains; and (5) correlation-based analysis of transcriptional dynamics between plants and bacteria. Designed for researchers with basic familiarity with command-line tools and R programming, this pipeline enables comprehensive analysis of plant-bacterial transcriptional interplay and facilitates hypothesis generation in both pathogenic and symbiotic contexts.

BN-PAGE (Blue Native-Polyacrylamide Gel Electrophoresis) is a non-denaturing electrophoretic technique used to analyze the molecular weight and oligomeric states of protein complexes under near-native conditions. NLRs (nucleotide-binding leucine-rich repeat proteins), which function as intracellular immune receptors in plants, form oligomeric higher-order complexes known as resistosomes upon activation by recognition of pathogen effectors-a mechanism elucidated through BN-PAGE and structural analyses. Here, we describe a method combining BN-PAGE with Agrobacterium-mediated complementation assay to investigate the resistosome formation of the NLR protein ZAR1 in Nicotiana benthamiana.

The histone H1 family comprises of essential components of chromatin, which bind to the linker DNA connecting individual nucleosomes and contribute to the formation of higher-order chromatin structures. Multiple histone H1 exist, each with distinct interactions with the nucleosome that specifically influence chromatin organization and nuclear functions. Histone H1 variants have been shown to play a role as drivers in cancer and may serve as biomarkers for patient stratification. To overcome the limitations associated with antibody- and RNA-based methods for analyzing histone H1, we developed a mass spectrometry (MS)-based label-free approach to simultaneously analyze all somatic histone H1 variants in patient-derived samples. Here, we describe how this method can be used for the analysis of low-amount clinical samples obtained through laser capture microdissection of tissue sections.

Subcellular fractionation of Euglena gracilis has been conducted for over 50 years in various forms by numerous research groups. The development of this technique is closely tied to the specific organelle or fraction required for specific purposes. In this chapter, we describe our approach to this process and discuss the insights we gain from it. Sucrose and iodixanol gradients are employed to separate the main organelles of interest; however, these methods alone do not lead to the complete purification of the organelles.

Post-transcriptional control is well established as a key mechanism of gene regulation in trypanosomatids. However, recent studies suggest that transcriptional regulation may also play a role, challenging long-standing dogma. The Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) provides a genome-wide overview of chromatin accessibility (i.e., whether chromatin is more open or closed), without requiring prior knowledge of chromatin markers which may be absent or poorly understood in trypanosomatids. Here, we present an optimized ATAC-seq protocol for use in Trypanosoma brucei, which has been used in both bloodstream and procyclic forms, and can be used to inform application of the method to other Euglenozoa. We also provide guidance on bioinformatic analysis, including integration of output files with established differential accessibility and RNA-seq data analysis pipelines.

Camelid single-domain antibodies (sdAbs), commercially known as Nanobodies™, possess remarkable properties that render them highly suitable as versatile tools for target discovery and product development. Interestingly, despite their successful and broad deployment in life sciences, sdAbs remain heavily underutilized in the field of molecular parasitology. In this chapter, we describe how we have employed an unbiased camelid immunization strategy to discover novel diagnostic biomarkers with sdAbs. This protocol shows the potential of camelid sdAbs as powerful tools for novel target discovery in kinetoplastid research.

Analysis of the cell cycle in kinetoplastid parasites involves the assessment of the replication of single copy organelles, such as the nucleus, kinetoplast, and flagellum, alongside the observation of cell cycle stage-associated morphological changes, e.g., cell shape changes and the appearance of a mitotic spindle or cytokinesis furrow, which together allow the cell cycle stage of individual parasites to be determined. To date, most kinetoplastid cell cycle analysis has been performed using light microscopy and/or flow cytometry of fixed cells, but while these methods have proven highly valuable, microscopy can be time-consuming and flow cytometry can lack resolution. We have previously shown that imaging flow cytometry offers significant benefits for depth and speed of analysis. This is due to its ability to directly link the high-throughput and quantitative nature of standard flow cytometry with the visual and spatial data of microscopy, over an extensive array of morphological and fluorescence parameters, which can be calculated for both brightfield and fluorescence images of each cell. Furthermore, the ability to automate image analysis ensures high throughput. Here, we provide a step-by-step guide to analyzing the cell cycle of live promastigote Leishmania mexicana using imaging flow cytometry. We outline a method for quantitative DNA staining in live L. mexicana promastigotes using Vybrant™ DyeCycle™ Orange and provide protocols, guidance, and example analysis templates for using an ImageStream^®X MkII imaging flow cytometer (Cytek) to acquire and analyze brightfield and fluorescence images of the parasite to determine cell cycle stage. We also detail how to employ mNeonGreen tagging of the orphan spindle kinesin, KINF, to provide greater resolution of cell cycle position. Our automated masking and gating pipeline enables rapid, high-throughput and semi-automated analysis of the L. mexicana cell cycle in live cells, in near real time, offering many advantages over conventional analysis methods. In addition, we envisage that this pipeline could be adapted to allow similar high-throughput analysis of the cell cycle of other kinetoplastid species and outline the approaches that could be taken to achieve this.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.

Biosensors are devices leveraging biological modules for the detection of molecules of interest, including biomarkers of inflammation and cancer. Using elements capable of detecting small molecules, such as transcription factors, aptamers, and antibodies, they specifically and sensitively interact with a target analyte, producing a detectable response. By applying traditional and synthetic biology approaches, these modules can be engineered to detect different molecules. A whole-cell biosensor (WCB) is a subtype of biosensor in which a whole living organism with an active metabolism is used to detect a compound. WCBs based on synthetic transcription factors are adaptable and accessible sensing tools for the detection of small soluble molecules. In this chapter, we describe a pipeline for the construction of a prokaryotic biosensor strain bearing a genetic circuit that integrates a custom-made transcription factor. As an example, we use the strain Escherichia coli MG1655 (pCKT-Sphnx, pHC_DYO-LacI-R), which has been engineered to detect neuraminic acid, an oral cancer biomarker present in saliva.

Enzyme assays are used to measure the activity or concentration of enzymes in biochemical or cell-based systems. Most enzyme assays are based on the detection of fluorescent, luminescent, or spectrophotometric endpoint signals. In recent years, they have been developed and widely used for diagnostics, mechanisms of action, and inflammatory activities. An enzyme assay essentially works by the conversion of a substrate into a product by the enzyme of interest. In this case, it is extremely important to know the optimal conditions for enzyme activity, as these affect the specificity and efficacy of the assay. For optimal reaction conditions, temperature, pH, and the presence of ions should be considered. In this chapter, the enzymatic assays for the detection of the enzymes N-acetylglucosaminidase (NAG), myeloperoxidase (MPO), and eosinophil peroxidase (EPO) are addressed. These assays are used to assess inflammatory parameters, for example, at the peripheral level in models of viral disease. They are based on an index of neutrophil, macrophage, or eosinophil accumulation in inflammatory tissues from animals by measuring the specific activity of the marker enzymes. The enzyme activity assays discussed here are based on colorimetric reactions compatible with any experimental model in which the respective cells has an active role. The advantage of using these enzymatic assays in inflammation response models is that they are simpler and less expensive compared to techniques such as Western blot or quantitative PCR.