Methods in molecular biology最新文献

Laser microdissection (LMD) combined with high-resolution mass spectrometry (MS) has become a powerful approach for analyzing targeted regions of fixed tissues, enabling detailed investigation of their heterogeneity. Here, we present two protocols optimized for low-input samples: a solid-phase enhanced preparation method (SP3) and a detergent/organic solvent-based approach (DDM/ACN). Both workflows are designed to minimize sample loss and are suitable for manual processing when automation platforms are not available. We provide practical guidance to reduce input requirements down to 1500 μm² of 10 μm thick FFPE tissue while maintaining reproducibility and proteome depth. These protocols, when coupled with fast instruments such as the Orbitrap Astral mass spectrometer working in DIA acquisition mode, enable sensitive, high-resolution proteomic analysis of microdissected tissue regions. Together, they establish a robust approach for investigating spatial heterogeneity in clinical samples with high coverage and precision.

Laser-capture microdissection (LCM) enables the collection of regions of interest from target tissues, making it compatible with a number of spatially resolved downstream omics applications. Here, we provide a detailed protocol for LCM coupled to ATAC-seq (LCM-ATAC-seq) for the spatial analysis of chromatin landscape based on the method published by Carraro et al. (Cell Rep Methods 3:100598, 2023). LCM-ATAC-seq allows the efficient capture down to single nuclei and can be combined with the morphological staining of target cell populations to characterize chromatin states in fresh-frozen tissue samples.

Identification of similarity between protein sequences is an important component for the assignment of function. With ever-growing databases of genome sequence, this becomes an increasing challenge, and especially in the detection of relationships between distantly related sequences, which is frequently an issue with euglenids. The introduction of artificial intelligence tools to the prediction of protein structure has been, without exaggeration, revolutionary. In particular, AlphaFold3 (AF3), the latest iteration of the AI predictor from DeepMind, a Google subsidiary, offers a potent combination of speed, accuracy, and ease-of-use, all free of charge. Here I will describe a basic workflow for the detection of low similarity between proteins, that is otherwise cryptic, using AF3, discuss how to interpret the predictions, and highlight examples of bizarre predictions or hallucinations.

Proximity labeling (PL) with TurboID provides a powerful alternative to traditional protein interaction mapping methods, allowing the capture of both stable and transient interactions in living cells. Here, we describe a standardized and optimized protocol for generating high-confidence proximity proteomes in Trypanosoma cruzi using a novel vector system, pTcTurboID. This vector allows the stable and regulatable expression of TurboID fusion proteins, ensuring minimal bait expression to preserve physiological function while maintaining sufficient biotinylation activity for protein detection. The protocol comprises eight steps, including the generation of spatial control and bait lineages, optimization of biotinylation conditions, and refinement of the purification process with magnetic streptavidin beads. Key features include the use of compartment-specific spatial controls to minimize nonspecific background and statistical analysis to identify true interactors. Our methodology has been validated with nuclear and cytoplasmic baits, yielding reproducible proximity interactomes. This protocol provides an efficient, reproducible, and robust framework for proximity proteomics in T. cruzi.

Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.

Expansion microscopy is a powerful technique, which increases the effective resolution of a fluorescence microscope by physically enlarging the specimen within a swellable polymer matrix. When combined with confocal microscopy, it enables affordable and easy-to-implement super-resolution imaging of protein localization in an entire cellular volume. Here, we describe in detail a protocol for the expansion of Euglenozoa, such as Trypanosoma brucei and Euglena gracilis, which achieves an average expansion factor of 4.6 in the case of T. brucei and 3.5 in the case of E. gracilis. This method is compatible with a broad range of available antibodies targeting endogenous proteins or small epitope tags. Additionally, we demonstrate the use of a fluorescent NHS ester for whole-proteome staining, BODIPY lipid stain, and the SYTOX nucleic acid stain for reproducible DNA labeling in expanded cells of these organisms. These reagents further increase the versatility of this simple yet robust super-resolution approach for studies of the cell biology of Euglenozoa.

To accompany a new collection of methods and protocols, we discuss the relevance of the microbial eukaryotes belonging to the protist phylum Euglenozoa. For those interested by Euglena, applied biology is often relevant: as a starting point for useful natural products including biofuels, nutritional supplements, and metabolites with biomedical potential, or as an environmental agent for bioremediation. Arguably the most widely studied euglenozoans are the parasitic trypanosomatids. Collectively, trypanosomatids cause several serious neglected tropical diseases and economically important diseases of animals and plants; since the early 1900s, drug discovery and disease intervention have been prominent research areas. Yet for those interested in evolution, trypanosomatids and Euglena are host to all sorts of extreme biology either not seen or so pronounced in other eukaryotes. Euglenozoans are also relevant in an ecology context: free-living relatives of the trypanosomatids are abundant in freshwater environments. Moreover, the other major euglenozoan group, the diplonemids, are recently recognized as the most abundant heterotrophic protists in the world's oceans, their diversity and abundance at least comparable to major algal groups. Finally, the long history of euglenozoan study illustrates nicely the evolving nature of scientific discovery and reporting since Van Leeuwenhoek first saw Euglena in the pioneering days of microscopy.

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.