Dataset Information

Extracellular vesicules proteomic profile of P. brasiliensis yeasts recovered from granulomatous lesions of C57BL/6 WT mice

ABSTRACT: C57BL/6 WT mice were infected with 1x10^6 P. brasiliensis yeasts. After 8 weeks and 12 weeks of infection the animals were euthanized and the granulomatous lesions were recovered. Then, the yeasts were recovered and cultivated in a rich medium. After cultivation the yeasts extracellular vesicles were isolated and their proteins extracted, digested with trypsin and analyzed by LC-MS/MS. The data show an induced metabolism by the recovered yeasts, with a higher abundance of proteins related to energy metabolism, lipid metabolism, nucleotide metabolism, amino acid metabolism. Additionally there is a higher abundance of some virulence factor.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Paracoccidioides Brasiliensis (strain Pb18)

SUBMITTER: Bruno Borges

LAB HEAD: Flávio Vieira Loures

PROVIDER: PXD046549 | Pride | 2024-05-23

REPOSITORIES: Pride

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			Action	DRS
	16fev23_BrunoBorges_amostra1_1ug.raw	Raw
	16fev23_BrunoBorges_amostra2_1ug.raw	Raw
	16fev23_BrunoBorges_amostra3_1ug.raw	Raw
	23fev23_BrunoBorges_amostra4_1ug.raw	Raw
	23fev23_BrunoBorges_amostra5_1ug.raw	Raw

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Publications

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

Montanari Borges Bruno B Gama de Santana Monique M Willian Preite Nycolas N de Lima Kaminski Valéria V Trentin Gabriel G Almeida Fausto F Vieira Loures Flávio F

Virulence 20240321 1

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immun ...[more]

PMID: 38511558

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Project description:There is a ever widening gap between the availability of donor livers suitable for transplantation and liver transplant demand of a growing waiting list. To expand the pool of liver grafts, donation after circulatory death is increasingly being used. Livers procured after circulatory death are exposed to a harmful period of warm Ischemia (WI) during the donation process, reducing post-transplant results significantly. Additionally, the conventional technique of static cold storage does not protect grafts that suffered WI. New dynamic preservation strategies by means of Normothermic Machine Perfusion (NMP) showed enhanced preservation of these type of grafts, but it is unknown how NMP influences liver cell biology during perfusion. Hepatocytes and cholnagiocytes have been shown to release Extracellular Vesicles (EVs) in the systemic circulation and bile, respectively. We hypothesized that EVs are released during liver NMP as well, in perfusate and bile. EVs have been separated from perfusate and bile during NMP of porcine livers, and the RNA cargo of the EVs has been sequenced to gain insights on liver cell biology during machine perfusion. Pigs were randomly allocated to undergo liver procurement either immediately after the start of surgery (no-WI, n=5) or after a period of WI of 60-minutes (clamping of thoracic aorta; WI-60, n=5). All livers (n=10) were cooled down and flushed out with ice-cold preservation solution, according to standard clinical practice. Porcine blood was collected and washed with a cell saver to obtain packed red blood cells for NMP. The hepatic artery, portal vein, and inferior vena cava were cannulated and after a period of about 2-hours of cold storage, NMP was started. All liver underwent NMP for 6-hours. Packed red blood-based perfusate samples were taken at 1, 3, and 6h of NMP; whereas, the bile produced the first 3-hours and the last 3-hours was pooled in 2 samples. Perfusate and bile samples were processed immediately for EVs separation with charge-based precipitation in a mixture of protamine and polyethylene glycol. The total RNA was extracted from EVs. Libraries of mRNA and of small RNA were prepared and analyzed for quality and size range, and sequenced on an Illumina HiSeq4000 instrument. Count-based differential expression analysis was done with R-based Bioconductor package DESeq2, and the following comparisons were performed: - Unpaired (between groups) comparison - perfusate samples WI-60 vs. no-WI - bile samples WI-60 vs. no-WI - Paired (withing group) comparison - no-WI - perfusate sample 1h vs. perfusate sample 3h - perfusate sample 1-3h vs. perfusate sample 6h - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples - WI-60 - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples Results were adjusted for multiple testing with the Benjamini-Hochberg procedure, to control for false discovery rate. Additionally, the function of the transcripts identified in the EVs of both groups and of the differentially expressed transcripts was investigated with gene ontology enrichment analysis for the domains \\"biological process\\" and \\"pathways\\".

Dataset Information

Extracellular vesicules proteomic profile of P. brasiliensis yeasts recovered from granulomatous lesions of C57BL/6 WT mice

Dataset's files

Publications

Extracellular vesicles from virulent <i>P. brasiliensis</i> induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

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