Methods in molecular biology最新文献

Peroxisomal or glycosomal membrane proteins (PMPs) depend on specific signal sequences, known as membrane peroxisomal targeting signals (mPTS), which are recognized by the cytosolic chaperone and import receptor PEX19. Computational prediction of PEX19-binding sites is a critical tool for identifying both known and novel PMPs and for advancing our understanding of the biogenesis of peroxisomes or glycosomes across diverse organisms, including humans, plants, fungi, and members of the Euglenozoa. PEX19-binding site prediction, including mPTS, is a computationally intensive process with broad applications, from identifying PMPs involved in essential cellular functions to uncovering pathogenic proteins in infectious agents, such as viruses, which interfere with the biogenesis or function of host cell peroxisomes. A web-based tool developed by Rottensteiner et al. previously enabled the prediction of PEX19-binding sites (PEX19BS), but it has become inaccessible, creating a significant gap for researchers aiming to identify and analyze these critical targeting motifs efficiently. To address this limitation, we have restored accessibility to a previously developed matrix-based prediction tool, now available as an open-access Google Colab notebook. This implementation enables efficient analysis and export of results, reinstating a valuable resource for studying membrane targeting in the biogenesis of peroxisomes and glycosomes.

Mitochondrial membrane potential (ΔΨ_m) is a critical component of the protonmotive force that drives ATP synthesis and supports essential mitochondrial functions, including metabolite transport, ion homeostasis, and protein import. In the parasitic protist Trypanosoma brucei, ΔΨ_m regulation is uniquely adapted across life cycle stages to meet changing metabolic demands. In the insect-stage procyclic form (PF), ΔΨ_m is generated by a canonical electron transport system (ETS), while in the bloodstream form (BF), where complexes III and IV are absent, ΔΨ_m is maintained by the reverse operation of ATP synthase, consuming ATP to pump protons. In T. brucei evansi, which lacks mitochondrial DNA, the ATP synthase is unable to translocate protons, and ΔΨ_m is sustained solely by electrogenic ADP/ATP exchange through the mitochondrial carrier. This chapter presents three complementary fluorescence-based methods to evaluate ΔΨ_m in T. brucei and T. b. evansi cells, highlighting their applicability to both intact and permeabilized parasites. We detail the use of two ΔΨ_m-sensitive dyes-TMRE, a cell-permeable dye suited for live-cell assays, and Safranine O, used in permeabilized preparations-and describe protocols for flow cytometry and fluorescence spectroscopy, respectively. These approaches allow robust, qualitative and semi-quantitative analysis of ΔΨ_m under different metabolic and experimental conditions. We address specific challenges associated with using fluorescent dyes to measure ΔΨ_m including issues of dye concentration, cellular permeability and potential artifacts that can affect interpretation of ΔΨ_m measurements.

Immunoprecipitation enables the isolation of protein complexes. Here we describe a method to purify YFP-tagged proteins from Trypanosoma brucei. This method has been optimized for kinetochore proteins but can readily be adapted to other proteins.

Ribosomal RNA (rRNA) constitutes a large proportion of total RNA, often making it necessary to deplete rRNA to enrich other RNA species for downstream applications. Ribodepletion is particularly challenging in Euglena gracilis, as its large subunit (LSU) rRNA is inherently fragmented into 14 stable pieces, rendering standard depletion methods ineffective. To address this limitation, we developed a targeted depletion strategy employing sequence-specific oligonucleotides and streptavidin beads to selectively remove rRNA while preserving other RNA species. Furthermore, the modular design of our oligonucleotide probe system facilitates straightforward adaptation to other euglenids and euglenozoans, thereby advancing transcriptomic research in this evolutionarily significant protist group.

The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.

Here, we describe a method to detect the oxidation of the pool of low molecular weight thiols (LMWT) in the infective stage of Trypanosoma brucei brucei in situ and non-invasively. The redox reporter cell line was generated by transfecting the parasites with a DNA construct coding for a redox-sensitive green fluorescent protein fused to human glutaredoxin 1 (hGrx1-roGFP2). The reporter gene is expressed in a tetracycline-inducible manner in the parasite's cytosol. roGFP2 displays a ratiometric and reversible change in fluorescence emission at 510 nm when excited at 405 and 488 nm, which is proportional to the changes in the ratio of oxidized vs. reduced LMWT trypanothione and glutathione. The role of hGrx1 is to catalyze a rapid equilibration of the LMWT pool with roGFP2 redox state, thereby allowing a biosensor response within seconds. The dynamic response of the biosensor enables the monitoring of cellular events in response to drugs or other stimuli in real time. The assay was adapted to a 96-well plate format for flow cytometry-based analysis. The fluorescent readout can be intensiometric or ratiometric, depending on the flow cytometer features, and the use of calibration controls is recommended for quantitative analysis. This bioassay can be applied to study fundamental questions of trypanosomatids' redox biology, as go/no-go criteria in drug discovery campaigns, and to investigate drug mode of action.

Identification of key genes regulating critical cellular functions, such as cancer cell proliferation, invasion, and immune escape, is essential for the development of novel cancer therapeutic targets and patient stratification. Our studies have shown that the expression of the proto-oncogene Bcl3 and the pro-angiogenic chemokine IL-8 is increased in ovarian cancer (OC) tissues and correlates with the expression of immune checkpoint PD-L1, resulting in increased proliferation of OC cells. Here, we describe a protocol to analyze the gene co-expression of Bcl3, IL-8, and PD-L1 by using the Xena platform and show that Bcl3, IL-8, and PD-L1 are co-expressed in glioblastoma as well as in pan-cancer samples.

Periodontal disease (PD) is a chronic inflammatory process of infectious etiology that affects the periodontal tissues. PD is caused by the subgingival biofilm, which, in dysbiosis, leads to an uncontrolled response of the immunological system in the periodontal tissues. To further understand the mechanisms involved in PD and how it is linked to other diseases, several animal models have been developed. These models allow researchers to study the different aspects of PD in a controlled setting, such as its pathogenesis and treatment options. Oral inoculation of periodontal bacteria, such as Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis, is one of the most commonly used models for studying PD. In these methods, the bacteria are inoculated directly into the oral cavity, allowing for rapid colonization and development of the disease. Another widely used mouse model for PD involves the application of a silk ligature around the second molar, the ligature triggers oral micro-organisms accumulation inducing an inflammatory response in the surrounding tissues, leading to gingival inflammation and pocket formation. The application of mouse models of PD has several advantages, such as relatively low cost, fast results, and the possibility of performing more accurate studies. In this chapter, we will describe bacteria- and ligature-induced periodontal disease models in detailed steps.

Nucleotide sequence analyses provide insights into changes that might have an impact on proteins and their function. With the rapid accumulation of sequence data, it is now possible to recover the evolutionary history of most genes at the population level to species and beyond. Those sequences can be compared for substitutions that might change or not change the encoded protein and its function, but they can also help to estimate evolutionary relationships. These hypotheses, as phylogenetic trees, provide visual and statistical guidance for characterizing the degree of relatedness among biological entities. In a phylogenetic tree, ancestor-descendant relationships are represented by connections, and closely related entities share most of these links, which represent their evolutionary closeness. In this chapter, I outlined a method to retrieve and label nucleotide sequences of the cytokine IL17A gene, align them to identify substitutions in homologous sites, estimate phylogenetic trees with support values, and visualize these trees as images. The methodology outlined here uses free software packages in the R environment and the Python language.

The whole genome amplification (WGA) allows new clinical applications with minimal genetic material, such as in the genetic diagnosis of Mendelian diseases in embryos before implantation (Preimplantation Genetic Test for Mendelian Abnormalities, PGT-M). This approach allows couples to avoid the transmission of Mendelian disease by undergoing assisted reproduction treatment through in vitro fertilization (IVF). First, Preimplantation Genetic Testing for Aneuploidy (PGT-A) is used to identify chromosomal aneuploidies in IVF-generated embryos. Then, or in parallel, euploid embryos can be screened for specific diseases caused by variants in a single gene to achieve the conception of offspring free of a specific monogenic disease.Here, we detail the WGA preparation and two downstream usages: (1) preparation of PCR fragments for Sanger sequencing, exemplifying the diseases we detected for healthy embryo selection and transfer in IVF, and (2) detection of chromosome Y for embryo sex diagnosis.