Electrokinetic characterization of extracellular vesicles with capillary electrophoresis : A new tool for their identification and quantification

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 x 10(9) EV/mL, whereas the calibration curve was acquired from 1.22 x 10(10) to 1.20 x 10(11) EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method. (C) 2020 Elsevier B.V. All rights reserved.Peer reviewe

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.[br/] Results: The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.[br/] Conclusions: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery

Recent electrokinetic strategies for isolation, enrichment and separation of extracellular vesicles

International audienceExtracellular vesicles (EVs) are a family of cell-derived membrane vesicles that are present in almost all body fluids. EVs have gained significant interest over the last decades as mediators of key functions in numerous patho-physiological condition (clearance, signalling, trophic support, cargo delivery) and as potential prognostic or diagnostic biomarkers. The endogenous delivery capacities of these nanometric entities also hold a high potential as engineered drug nanocarriers for clinical and pharmaceutical ap- plications, especially for targeted therapies. Nevertheless, knowledge about the features of individual EVs (composition, physical and chemical characteristics) is still at the infancy because of the technical challenges to purify and analyze the various subpopulations of EVs. In this review, a comprehensive overview of electrokinetically driven methods for isolation, enrichment and characterization of EVs is presented. This review covers new trends of analytical science (over 7 years up till 2020), serving for high-quality EVs production, isolation, analysis and quality control, which are expected to provide powerful and complementary alternatives to the conventional and recently emerged approaches such as microfluidics. We critically discuss here the pros and cons of the different instrumental and methodo- logical developments for electrokinetic strategies applied to EVs

Qualitative Changes in the Proteome of Milk-derived Extracellular Vesicles in response to intra-mammary infection by Staphylococcus aureus

Qualitative Changes in the Proteome of Milk-derived Extracellular Vesicles in response to intra-mammary infection by Staphylococcus aureus . 6. annual meeting of the International Society for Extracellular Vesicle

Omics approach to characterize milk-derived extracellular vesicles/exosomes isolated from goats expressing or not alphas1-CN

International audienceBackgroundGoats homozygous for a null allele (O/O) at the CSN1S1 locus encoding αs1-casein display a chronic endoplasmic reticulum (ER) stress (Unfolded Protein Response) due to an accumulation of the other caseins in this compartment, thus triggering a general MEC dysfunction with a strong impact on milk composition. Milk-derived extracellular vesicles (MEVs) contain molecular information, which are thus recognized as mediators of intercellular communication. We made the assumption that the absence of CSN1S1 expression may influence MEVs cargos including miRNA, proteins, lipids and metabolites. The objective of this study was to assess the impact of αs1-casein deficit on MEVs cargos and to compare the biological material they convey.Material & MethodsWe have developed an improved method based on a density gradient ultracentrifugation to isolate MEVs. The quality of MEVs was analyzed morphologically by transmission electron microscopy (TEM) with negative staining (uranyl acetate), the specific “exosome” protein markers were detected by Western blot and ELISA and the size distribution and particle concentration were measured by NTA. The MEV’s proteome was acquired by LC-MS/MS and nucleic acid content (mRNA and miRNA) by NGS and qPCR. EV lipid content was extracted with the MTBE method. Data acquisition was performed by an Orbitrap-MS and analyzed using Lipid Data Analyzer. MEV metabolites were extracted using MPLEx protocol, data acquired by LC coupled with HRMS and analyzed using Metaboanalyst online workbenches. Differential statistical and bioinformatic analyses were performed using appropriate softwares.ResultsThe novel purification method gives MEV populations, free of contamination by other EVs and milk components, at sufficient concentrations to perform subsequent analyses. Nearly 280 proteins involved in the biogenesis of exosomes and MVB formation, their adhesion and internalization as well as proteins associated with membrane transport and enzymes involved in cellular metabolism were identified, among which 41 exosomal proteins differed between CSN1S1 O/O (null) and A/A (wildtype) genotypes. Ongoing profiling of RNA from MEVs has already identified over 230 miRNA and confirmed MEC origin due to the presence of mRNA encoding specific major milk proteins. The comparison of exosomal miRNomes of goat homozygous for A and O alleles at the CSN1S1 locus pointed out 15 miRNAs differentially abundant, potentially related to the MEC phenotype. Sphingomyelin and phosphatidylcholin were the major phospholipids observed in MEV populations. We have totally identified ca. 4,000 compounds using pHILIC and RPLC, 79 of which were significantly up or down-regulated in studied genotypes.ConclusionSeveral differences distinguishing goats according to the genotype at the CSN1S1 locus were found at each level of the omic analysis of MEVs. Differentially abundant miRNAs and transcriptome analyses are in agreement with UPR phenotype and confirmed their involvement in post-transcriptional regulatory mechanisms