Project description:Toxoplasma gondii, the causative agent of toxoplasmosis, infects roughly one-third of the global population, highlighting its remarkable adaptability and pathogenicity across a wide range of warm-blooded animals. A key aspect of T. gondii’s ability to interact with its hosts involves the secretion of extracellular vesicles (EVs), which play a crucial role in mediating host-parasite interactions. These EVs carry a variety of molecular cargoes, including proteins, lipids, and RNA, which can alter host cellular responses and promote parasite survival. This project focuses on understanding how T. gondii adapts its EVs to the host environment. To explore this, we analyzed the EV cargo of T. gondii cultured in four different host cell lines representing distinct species. Each cell type provides a unique environment that mimics the conditions encountered by T. gondii during infection. By comparing the EV compositions across these cell lines, we aim to identify the diverse adaptive strategies that T. gondii employs through its EVs. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we characterized the EVs and conducted a comprehensive quantitative analysis of their proteomic cargo. Our findings revealed notable differences in the abundance and composition of EVs produced by tachyzoites cultivated in distinct host environments, underscoring the significant influence of host cell type on the makeup of T. gondii EVs.
Project description:The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra-and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with its the/their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (100-1000 nm), a major EV-subtype remains limited. Our study provides critical insights into proteins operating at the large interactive platform of L-EVs, and molecular leads for future studies seeking to decipher relatively-understudied L-EV form, heterogeneity and function.
Project description:Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that Drosophila, like human cells release exosome-like EVs with diameter ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNA pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these extensive similarities suggest that EVs could also enable RNA-mediated intercellular communication in Drosophila. We performed RNA-seq on extracellular vesicles purified from of human and Drosophila cell line cultures. S2R+ and D17 Drosophila EVs were analyzed, along with human A431 and HepG2 EVs. No ribosomal RNA depletion or polyA selection was performed on EV samples. For comparative analyses, we also analyzed total cellular RNA from Drosophila D17 and human HepG2. Ribodepletion was performed on cellular samples.
Project description:As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation vs. apoptosis induction). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. The protein content of these vesicles was analyzed by bidimensional gel electrophoresis followed by mass spec and western blot.
Project description:Comparative RNA profiling between tumor cells and their secreted extracellular vesicles. Results revealed enrichment in genes involved in cellular migration and metastasis in extracellular vesicles, in agreement with their role as mediators of tumor progression. Mice were orthotoplically transplanted with MDA-MB-231 Breast Adenocarcinoma cells. Cells and extracellular vesicles (EVs) from the resulting tumors were isolated. EVs were characterized by electron microscopy and Nanoparticle Tracking Analysis before total RNA isolation for comparative analysis with cellular RNA. Three biological replicates were analyzed in (technical) duplicate.
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:We performed a proteomic analysis of EVs derived from human Sertoli cells, aiming to obtain the profiling of the proteins in these EVs to explore their possible roles in the development of testis.
Project description:Background: Biomarkers that reflect glioblastoma tumour activity and treatment response are urgently needed to help guide clinical management, particularly for recurrent disease. As the urinary system is a major clearance route of circulating extracellular vesicles (EVs; 30-1000 nm nanoparticles) we explored whether sampling urinary-EVs could serve as a simple and non-invasive liquid biopsy approach for measuring glioblastoma-associated biomarkers. Methods: Fifty urine specimens (15-60 ml) were collected from 24 catheterised glioblastoma patients immediately prior to primary (n=17) and recurrence (n=7) surgeries, following gross total resection (n=9), and from age/gender-matched healthy participants (n=14). EVs isolated by differential ultracentrifugation were characterised and extracted proteomes were analysed by high-resolution data-independent acquisition liquid chromatography tandem mass spectrometry (DIA-LC-MS/MS). Results: Overall, 6857 proteins were confidently identified in urinary-EVs (q-value≤0.01), including 94 EV marker proteins. Glioblastoma-specific proteomic signatures were determined, and putative urinary-EV biomarkers corresponding to tumour burden and recurrence were identified (FC≥|2|, adjust p-val≤0.05, AUC>0.9). Conclusion: In-depth DIA-LC-MS/MS characterisation of urinary-EVs substantiates urine as a viable source of glioblastoma biomarkers. The promising ‘liquid gold’ biomarker panels described here warrant further investigation.
Project description:The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of g-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting Microtubule Associated Protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.
Project description:Transcription profiling analysis was performed on purified CD34+ cell lines (Cord Blood CD34+) treated with ExtracellularVescicles (EVs) isolated from bone marrow mesenchymal stem cells (BM-MSC).