Formation of infectious EVs governs flavivirus infection in mosquitoes
Ontology highlight
ABSTRACT:
ORGANISM(S): Aedes Aegypti
SUBMITTER: 
Gong Cheng
PROVIDER: PXD069648 | iProX | Fri Oct 17 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description:With the goal to identify miRNAs and other small RNAs involved in EV-mediated oligodendrocyte-neuron communication, we performed deep sequencing (RNA-seq) of small RNAs derived from EVs isolated from culture supernatants of primary mouse oligodendrocytes by differential centrifugation. Oligodendrocytes were cultured and EVs harvested under serum-free conditions in presence of B27 supplement. We found that RNA isolated from EVs harvested from cells under serum-replacement conditions contains miRNA contaminants carried into the sample by defined media components.
2019-08-15 | GSE134096 | GEO
Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.
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Project description:Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several kinds of macromolecules implicated in cell communication. EVs have become an important topic in the study of pathogenic fungi, due to their relation with the fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EV cargo, because of plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, little is known about EVs of Botrytis cinerea, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the first isolation and characterization of B. cinerea EVs; and the description of their potential role during the infective process. All the experimental process was conducted in B. cinerea growing in mimimal salt medium supplemented with glucose as a constitutive stage, and deproteinized tomato cell walls (TCW) as virulence inductor. The isolation of EV was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EV fractions were visualised by TEM using negative staining. Proteomic analysis of EV cargo was addressed by LC-MS/MS. The used methodology had allowed, for the first time, the correct isolation of B. cinerea EVs, and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in the morphology under both assayed conditions. GO analysis of EV proteins showed an enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed difference in EV function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, between others. This is the first evidence and characterization of B. cinerea EVs production, showing that EVs are essential in the infection process of B. cinerea and share crucial functions with the conventional secretion pathway.
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Project description:Background & aims: MicroRNAs (miRNAs) encapsulated in EVs are potential diagnostic and prognostic biomarkers. However, discrepancies on miRNA patterns and their validation are still frequent due to differences in sample origin, EVs isolation, miRNA extraction and sequencing methods. Selecting appropriate EVs isolation methods is therefore a critical step for miRNA-based biomarker discovery. The aim of the present study is to find the most suitable EVs isolation method for miRNAs sequencing adequate for clinical application. Material & Methods EVs were isolated by Size Exclusion Chromatography (SEC), iodixanol gradients (GRAD) and the combination of both (SEC+GRAD), using the same plasma sample, in triplicate isolation assays. Isolated EVs were characterized and RNA was extracted. Three different protocols for miRNA library preparation were compared (NEBNext, NEXTFlex and SMARTer smRNA-seq) and miRNAs encapsulated on EVs were sequenced using NextSeq 500 system (Illumina). Finally, the yield, abundance and diversity of miRNAs using the three different EVs isolation protocols were analyzed and compared between them. Results The majority of lipoproteins, total cholesterol and plasma proteins were removed from the EVs-containing fractions by using SEC, GRAD, and SEC+GRAD. SEC method recovered a larger amount of EVs followed by GRAD and SEC+GRAD, while GRAD and SEC+GRAD yielded the purest vesicles. NEBNext was the library preparation kit that showed the highest reproducibility among replicas, higher number of reads corresponding to miRNAs and more different miRNAs, followed by NEXTFlex and SMARTer smRNA-seq. GRAD method showed the highest reproducibility among replicas, a higher number of reads corresponding to miRNAs and more different miRNAs, followed by SEC and SEC+GRAD methods. Conclusions These results render the GRAD method to isolate EVs as one of the most appropriate to detect miRNAs from Evs.
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