Characterization of antiviral extracellular vesicle cargo in epithelial cells
Ontology highlight
ABSTRACT: We set out to identify the contents of extracellular vesicles in epithelial cells and how they differ in vesicles with enhanced antiviral activity.
Project description:The aim of this experiment was to highlight differences in expression of microRNAs transported by extracellular vesicles between two strains of the HL-60 cell line (acute promyelocytic leukemia cell line): the chemo-sensitive strain (HL-60) and an anthracyclin-resistant strain (HL-60/AR).
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: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.
Project description:Several transcription factors are known to be expressed in discrete regions of the otic vesicle and Dlx5 is one of those that is expressed highly in the presumptive dorsal vestibular region. Mice lacking Dlx5 have vestibular defects. Specifically, they fail to form the endolymphatic duct (a defect visible as early as E10) as well as the anterior and posterior semi-circular canals. The lateral canal does form but is smaller, whereas the saccule, the utricle and the cochlea appear relatively normal. The goal of this study was to use microarrays to identify differentially expressed genes between wild-type and Dlx5-null otic vesicles microdissected from E10 and 10.5 and identify downstream targets of Dlx5 by searching the immediate 3kb promoter regions of the differentially expressed genes for homeodomain binding sites followed by chromatin immunoprecipitation in an otic vesicle-derived cell line over-expressing Dlx5. Normal vestibular morphogenesis is compromised in mice lacking Dlx5, a member of the Distal-less family of homeobox transcription factors. We identified its direct downstream targets in the developing mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed in mutants when compared to wild-type in at least one of the two stages. To further constrain the list of likely direct targets of Dlx5, we examined the genomic DNA sequences in the 3kb promoter regions immediately proximal to the transcriptional start sites of these genes. We searched for (i) one or more previously described binding site for Dlx5, (ii) one or more novel 12bp-long motifs with a canonical homeodomain response element (HDRE) shared by promoters of two or more genes, and (iii) 100% conservation of the 12bp-long HDRE-containing motifs in promoter regions of human orthologs. Forty genes passed one or more of these filters, 12 of which are known to be expressed in the developing otic vesicle in domains that at least partially overlap with that of Dlx5 in one or both stages that we examined. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoter regions of seven of these genes (Atbf1, Bmper, Large, Lrrtm1, and Msx1, all of which were down-regulated in mutants, and Ebf1 and Lhx1, both of which were up-regulated in mutants) in an otic vesicle-derived cell line over-expressing Dlx5. Gene expression profiling of this cell line showed that Bmper and Lrrtm1 transcripts were up-regulated, further supporting their identification as direct targets of Dlx5 activity. Otic vesicles from wild-type and Dlx5-null embryos were microdissected from mouse developmental stages E10 and E10.5 and expression profiled in duplicate for each stage and genotype. Dlx5-transfected and empty vector-transfected 2B1 cell line samples were also profiled in duplicate (independent cell cultures).
Project description:The fungal pathogen Candida albicans can form biofilms that protect it from drugs and the immune system. The biofilm cells release extracellular vesicles (EVs) that promote extracellular matrix formation and resistance to antifungal drugs. Here, we define functions for numerous EV cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination. We use a machine-learning analysis of cargo proteomic data from mutants with EV production defects to identify 63 candidate gene products for which we construct mutant and complemented strains for study. Among these, 17 mutants display reduced biofilm matrix accumulation and antifungal drug resistance. An additional subset of 8 cargo mutants exhibit defects in adhesion and/or dispersion. Representative cargo proteins are shown to function as EV cargo through the ability of exogenous wild-type EVs to complement mutant phenotypic defects. Most functionally assigned cargo proteins have roles in two or more of the biofilm phases. Our results support that EVs provide community coordination throughout biofilm development in C. albicans.
Project description:The present dataset contains proteomics data from extracellular vesicles steadily released by donor matched, cultured human CD4+ and CD8+ T cells and from extracellular vesicles released within the immunological synapse.
Project description:Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Extracellular vesicle RNA was pooled (n=4 per status) and analyzed for small RNAs by sequencing on the Ion Torrent PGM platform and analysis with CLC Genomics Workbench small RNA workflow based on the miRBase (Release 19) Bos taurus database. Small RNA analysis of day 14 uterine luminal fluid extracellular vesicles isolated from pregnant and cyclic ewes.
Project description:Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA (miRNA) and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
Project description:In this study, we address mRNA composition of hepatocyte-like derived extracellular vesicles (EVs), using as cellular model the mouse liver derived cell line MLP29, and primary cell culture of rat hepatocyte (RH) obtained by in vivo liver perfusion. The study shows qualitative characterization of RNA, identification of transcripts and its functional characterization through gene expression array technique. To reach a deeper nowledge in the biology of EVs, we perform RNase protection assay, density gradients matching RNA with typical exosomal protein markers, and capture assays to probe that mRNA was internalized. Aim of the project: To identify transcripts present in extracellular vesicles secreted by Rat hepatocytes primary cell culture and to identify extracellular vesicles secreted by mouse hepatocyte cell line MLP29, and in this case, compare the enrichment of transcripts respect to the cell, to know if the composition in the extracellular vesicles is similar to the cell, or if their composition is not directly determined by the abundance of transcripts in the cell.
Project description:The Gram-negative photoheterotrophic bacterium Dinoroseobacter shibae is a member of the high abundant marine Roseobacter group. In the ocean, OMVs have about the same abundance as bacteria, a distinct depth distribution, and contain DNA from a variety of marine bacterial taxa. In the present study, we determined the abundance, size, and ultrastructure of membrane vesicles of D. shibae and analysed the protein inventory of the soluble and membrane fractions of cells and vesicles in order to study the origin of OMV membranes and content. The proteomic analyses were complemented by fatty acid analyses.