Presence Of Extracellular Vesicles Research Articles

Investigations on the Hemostatic Potential of Physiological Body Fluids.

Current blood coagulation models consider the interactions between blood, the vessel wall, and other tissues that expose tissue factor (TF), the main initiator of coagulation. A potential role of body fluids other than blood is generally not considered. In this review, we summarize the evidence that body fluids such as mother's milk saliva, urine, semen, and amniotic fluid trigger coagulation. The ability of these body fluids to trigger coagulation is explained by the presence of extracellular vesicles (EVs). These EVs expose extrinsic tenase complexes (i.e., complexes of TF and activated factor VII) that can trigger coagulation. Why these body fluids share this activity, however, is unknown. Possible explanations are that these body fluids contribute to hemostatic protection and/or to the regulation of the epithelial barrier function. Further investigations may help understand the underlying cellular and biochemical pathways regulating or contributing to coagulation and innate immunity, which may be directly relevant to medical conditions such as gastrointestinal bleeding and chronic inflammatory bowel disease.

Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis†.

Sperm maturation depends on exposure to microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the extracellular vesicles from different regions of the epididymis can ensure paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to extracellular vesicles collected from upstream (i.e., caput) segments. Impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that extracellular vesicles from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to extracellular vesicle supplementation). Of note, expressions of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were influenced by the presence of extracellular vesicles. Together, our findings comprise the first report in any species of paracrine control of segmental gene regulation by epididymal extracellular vesicles. These results contribute to a better understanding of epididymis biology and could lead to strategies to enhance or suppress male fertility.

Biophysical features of plant-derived nanovesicles: Focus on almonds

Almond is a traditional food with established beneficial effects on health. Nothing is known about the presence of extracellular vesicles (EVs), recently isolated from other plant material, ingested with food, or as engineered bioactive nanovectors. Aiming to develop and optimize a method to isolate almonds derived nano vesicles (ADNVs), we tested different protocols on pure, blanched and roasted almonds, and investigated the resulting biophysical features.The most feasible and valid method was the sucrose-based ultracentrifugation (DGUC). Size distribution ranged on average 100–200 nm. A negative zeta-potential in the range of –27 to –21 mV has been measured. Microscopy showed a spheroid morphology and preserved structural integrity of isolated ADNVs. Pure almond ADNVs showed the greater amount of TET8; PEN1 was also found, although cooking treatments resulted in a decrease.Attention should be paid to sample managing during DGUC steps and to sample deposition and drying for microscopy procedures. Our findings contribute to enriching the hot issue on EVs research from edible sources, giving reason to their possible role in interspecies communication, and their exploitation for the delivery of bioactive compounds or therapeutics molecules. The potentiality of almonds' industrial residuals should be investigated in further research.

Gene expression profile of human placental villous pericytes in the first trimester – An analysis by single-cell RNA sequencing

Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However, the markers and functions of placental villous pericytes (PVPs) as distinct subgroups of placental villous mesenchymal cells, remain unclear. Therefore, in this study, the markers and functions of PVPs were investigated. Single-cell sequencing data from the first-trimester placental villi was obtained and the Seurat tool was used to identify PVP markers. Gene Ontology (GO) analysis of specific genes was performed using the DAVID database. The Cellchat tool was employed to investigate the interaction signals between the PVPs and other cells. Expression of the PVP markers was confirmed using immunofluorescence. Presence of extracellular vesicles in the placental villous mesenchyme and PVPs was examined by transmission electron microscopy. Our findings revealed that renin (REN) and amphiregulin (AREG)-positive fibroblasts in the placental villi specifically expressed several classic pericyte markers. In the first trimester, certain conserved functions of pericytes were observed and they displayed tissue-specific functions such as in the integrin-mediated signaling pathway and extracellular exosomes. Moreover, the placental villous mesenchyme was found to be rich in extracellular vesicles. AREG is specifically transcribed in the first trimester PVPs, however, its protein was located in syncytiotrophoblasts. These insights contribute to a comprehensive understanding of early placental development and offer new therapeutic targets for placenta-derived pregnancy complications.

Anticancer platinum-drug delivered by mesenchymal stromal cells improves its activity on glioblastoma

BackgroundGlioblastoma multiforme (GBM) is nowadays the most aggressive tumor affecting brain in adults with a very poor prognosis due to the limited therapies and the systemic cytotoxicity. Among the different new drugs, recently has been reported the in vitro anti-glioma activity of a new cationic platinum(II) complex bearing 8-aminoquinoline as chelating ligand (Pt-8AQ). The purpose of this research work was to confirm the activity of Pt-8AQ on U87-GM spheroid and to investigate the ability of Mesenchymal Stromal Cells (MSCs) to incorporate and release Pt-8AQ in its active form. The MSCs were primed with Pt-8AQ under optimized conditions and the secretome was analyzed for evaluating the cytotoxic activity of Pt-8AQ and the presence of Extracellular Vesicles (Evs).ResultsThe principal results showed that Pt-8AQ incorporated by MSCs was released in the secretome and exerted a significant higher anticancer activity with respect to the free drug. The release of Pt-8AQ did not occur in Evs, as demonstrated for other drugs, but it could be delivered bound to some specific carriers able to enhance its bioavailability and efficacy. Some hypotheses are discussed to explain this surprisingly finding out that, however, it needs more investigations.ConclusionsThe major conclusions are that cell mediated drug delivery systems could provide a potential approach to facilitate the GBM therapy by intra-tumoral administration of cells loaded with Pt-8AQ, being MSCs able to integrate it into the tumor mass and exert high therapeutic efficacy in situ. The increased efficacy of Pt-8AQ delivered by MSCs even suggests to deeper investigate a possible direct use of MSCs secretome both in situ and/or by systemic administration, being secretome able to pass the blood–brain tumor.

Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum.

Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and modulators of fundamental biological processes including immunoregulation. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given their involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovary-enriched and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both endogenous and exogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length and endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we have previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect of ivermectin treatment on Ascaris circRNAs expression or secretion.

Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia.

Amniotic fluid surrounding the developing fetus is a complex biological fluid rich in metabolically active bio‐factors. The presence of extracellular vesicles (EVs) in amniotic fluid has been mainly related to foetal urine. We here characterized EVs from term amniotic fluid in terms of surface marker expression using different orthogonal techniques. EVs appeared to be a heterogeneous population expressing markers of renal, placental, epithelial and stem cells. Moreover, we compared amniotic fluid EVs from normal pregnancies with those of preeclampsia, a hypertensive disorder affecting up to 8% of pregnancies worldwide. An increase of CD105 (endoglin) expressing EVs was observed in preeclamptic amniotic fluid by bead‐based cytofluorimetric analysis, and further confirmed using a chip‐based analysis. HLA‐G, a typical placental marker, was not co‐expressed by the majority of CD105+ EVs, in analogy with amniotic fluid stromal cell derived‐EVs. At a functional level, preeclampsia‐derived EVs, but not normal pregnancy EVs, showed an antiangiogenic effect, possibly due to the decoy effect of endoglin. Our results provide a characterization of term amniotic fluid‐EVs, supporting their origin from foetal and placental cells. In preeclampsia, the observed antiangiogenic characteristics of amniotic fluid‐EVs may reflect the hypoxic and antiangiogenic microenvironment and could possibly impact on the developing fetus or on the surrounding foetal membranes.

Lactadherin: From a Well-Known Breast Tumor Marker to a Possible Player in Extracellular Vesicle-Mediated Cancer Progression.

Lactadherin is a secreted glycoprotein associated with the milk fat globule membrane, which is highly present in the blood and in the mammary tissue of lactating women. Several biological functions have been associated with this protein, mainly attributable to its immunomodulatory role promoting phagocyte-mediated clearance of apoptotic cells. It has been shown that lactadherin also plays important roles in cell adhesion, the promotion of angiogenesis, and tissue regeneration. On the other hand, this protein has been used as a marker of breast cancer and tumor progression. Recently, high levels of lactadherin has been associated with poor prognosis and decreased survival, not only in breast cancer, but also in melanoma, ovarian, colorectal, and other types of cancer. Although the mechanisms responsible for the tumor-promoting effects attributed to lactadherin have not been fully elucidated, a growing body of literature indicates that lactadherin could be a promising therapeutic target and/or biomarker for breast and other tumors. Moreover, recent studies have shown its presence in extracellular vesicles derived from cancer cell lines and cancer patients, which was associated with cancer aggressiveness and worse prognosis. Thus, this review will focus on the link between lactadherin and cancer development and progression, its possible use as a cancer biomarker and/or therapeutic target, concluding with a possible role of this protein in cellular communication mediated by extracellular vesicles

Acid Ceramidase (ASAH1) Mediates Intrinsic and Intercellular Transfer of Proteasome Inhibitor Resistance in Multiple Myeloma

INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells.METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models.RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls.CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. DisclosuresHampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Placental extracellular vesicles–associated microRNA-519c mediates endotoxin adaptation in pregnancy

Pregnancy represents a unique challenge for the maternal-fetal immune interface, requiring a balance between immunosuppression, which is essential for the maintenance of a semiallogeneic fetus, and proinflammatory host defense to protect the maternal-fetal interface from invading organisms. Adaptation to repeated inflammatory stimuli (endotoxin tolerance) may be critical in preventing inflammation-induced preterm birth caused by exaggerated maternal inflammatory responses to mild or moderate infections that are common during pregnancy. However, the exact mechanisms contributing to the maintenance of tolerance to repeated infections are not completely understood. MicroRNAs play important roles in pregnancy with several microRNAs implicated in gestational tissue function and in pathologic pregnancy conditions. MicroRNA-519c, a member of the chromosome 19 microRNA cluster, is a human-specific microRNA mainly expressed in the placenta. However, its role in pregnancy is largely unknown. This study aimed to explore the role of "endotoxin tolerance" failure in the pathogenesis of an exaggerated inflammatory response often seen in inflammation-mediated preterm birth. In this study, we investigated the role of microRNA-519c, a placenta-specific microRNA, as a key regulator of endotoxin tolerance at the maternal-fetal interface. Using a placental explant culture system, samples from term and second-trimester placentas were treated with lipopolysaccharide. After 24 hours, the conditioned media were collected for analysis, and the placental explants were re-exposed to repeated doses of lipopolysaccharide for 3 days. The supernatant was analyzed for inflammatory markers, the presence of extracellular vesicles, and microRNAs. To study the possible mechanism of action of the microRNAs, we evaluated the phosphodiesterase 3B pathway involved in tumor necrosis factor alpha production using a microRNA mimic and phosphodiesterase 3B small interfering RNA transfection. Finally, we analyzed human placental samples from different gestational ages and from women affected by inflammation-associated pregnancies. Our data showed that repeated exposure of the human placenta to endotoxin challenges induced a tolerant phenotype characterized by decreased tumor necrosis factor alpha and up-regulated interleukin-10 levels. This reaction was mediated by the placenta-specific microRNA-519c packaged within placental extracellular vesicles. Lipopolysaccharide treatment increased the extracellular vesicles that were positive for the exosome tetraspanin markers, namely CD9, CD63, and CD81, and secreted primarily by trophoblasts. Primary human trophoblast cells transfected with a microRNA-519c mimic decreased phosphodiesterase 3B, whereas a lack of phosphodiesterase 3B, achieved by small interfering RNA transfection, led to decreased tumor necrosis factor alpha production. These data support the hypothesis that the anti-inflammatory action of microRNA-519c was mediated by a down-regulation of the phosphodiesterase 3B pathway, leading to inhibition of tumor necrosis factor alpha production. Furthermore, human placentas from normal and inflammation-associated pregnancies demonstrated that a decreased placental microRNA-519c level was linked to infection-induced inflammatory pathologies during pregnancy. We identified microRNA-519c, a human placenta-specific microRNA, as a novel regulator of immune adaptation associated with infection-induced preterm birth at the maternal-fetal interface. Our study serves as a basis for future experiments to explore the potential use of microRNA-519c as a biomarker for infection-induced preterm birth.

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

Reply to Comments on Proteomic Investigations of Two Pakistani Naja Snake Venom Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles. Toxins 2020, 12, 669.

We appreciate the commentary on our article, and we would like to take the opportunity to address several points raised in the reviewers' commentary [...].

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

Specific and Non-Invasive Fluorescent Labelling of Extracellular Vesicles for Evaluation of Intracellular Processing by Intestinal Epithelial Cells.

The presence of extracellular vesicles (EVs) in milk has gained interest due to their capacity to modulate the infant’s intestinal and immune system. Studies suggest that milk EVs are enriched in immune-modulating proteins and miRNA, highlighting their possible health benefits to infants. To assess uptake of milk EVs by intestinal epithelial cells, a method was developed using labelling of isolated EVs with fluorophore-conjugated lactadherin. Lactadherin is a generic and validated EV marker, which enables an effective labelling of phosphatidylserine (PS) exposing EVs. Labelled EVs could effectively be used to describe a dose- and time-dependent uptake into the intestinal epithelial Caco-2 cell line. Additionally, fluorescence microscopy was employed to show that EVs colocalize with endosomal markers and lysosomes, indicating that EVs are taken up via general endocytotic mechanisms. Collectively, a method to specifically label isolated EVs is presented and employed to study the uptake of milk EVs by intestinal epithelial cells.

Impact of pyriproxyfen on virus behavior: implications for pesticide-induced virulence and mechanism of transmission

BackgroundMore than 3 years since the last Zika virus (ZIKV) outbreak in Brazil, researchers are still deciphering the molecular mechanisms of neurovirulence and vertical transmission, as well as the best way to control spread of ZIKV, a flavivirus. The use of pesticides was the main strategy of mosquito control during the last ZIKV outbreak.MethodsWe used vesicular stomatitis virus (VSV) tagged with green fluorescent protein (GFP) as our prototypical virus to study the impact of insecticide pyriproxyfen (PPF). VZV-GFP infected and uninfected Jurkat, HeLa and trophoblast cells were treated with PPF and compared to untreated cells (control). Cell viability was determined by the MTT assay. Cell morphology, presence of extracellular vesicles (EVs), virus infection/GFP expression as well as active mitochondrial levels/localization were examined by confocal microscopy.ResultsPPF, which was used to control mosquito populations in Brazil prior to the ZIKV outbreak, enhances VSV replication and has cell membrane-altering properties in the presence of virus. PPF causes enhanced viral replication and formation of large EVs, loaded with virus as well as mitochondria. Treatment of trophoblasts or HeLa cells with increasing concentrations of PPF does not alter cell viability, however, it proportionately increases Jurkat cell viability. Increasing concentrations of PPF followed by VSV infection does not interfere with HeLa cell viability. Both Jurkats and trophoblasts show proportionately increased cell death with increased concentrations of PPF in the presence of virus.ConclusionsWe hypothesize that PPF disrupts the lipid microenvironment of mammalian cells, thereby interfering with pathways of viral replication. PPF lowers viability of trophoblasts and Jurkats in the presence of VSV, implying that the combination renders immune system impairment in infected individuals as well as enhanced vulnerability of fetuses towards viral vertical transmission. We hypothesize that similar viruses such as ZIKV may be vertically transmitted via EV-to-cell contact when exposed to PPF, thereby bypassing immune detection. The impact of pesticides on viral replication must be fully investigated before large scale use in future outbreaks of mosquito borne viruses.

Argonautes in Extracellular Vesicles: Artifact or Selected Cargo?

Argonaute-2 (Ago2) is a key component of the RNA-induced silencing complex that mediates downregulation of mRNA by miRNAs. Its presence in extracellular vesicles (EV) has been postulated to be important for the activity of EV-carried miRNA in modulating gene expression in recipient cells. However, whether it is in fact contained within EVs or is instead an extravesicular contaminant is controversial. In this opinion piece, we argue that the ability to detect Ago2 in EVs is a result of multiple factors, including cell source, cell signaling control of Ago2 trafficking to EVs, experimental conditions, and detection methods.

Mass spectrometry-based investigation of measles and mumps virus proteome

BackgroundMeasles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs.MethodsMUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out.ResultsBy proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin β1 are part of the virions.ConclusionsAll HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations.

Blood manufacturing methods affect red blood cell product characteristics and immunomodulatory activity

AbstractTransfusion of red cell concentrates (RCCs) is associated with increased risk of adverse outcomes that may be affected by different blood manufacturing methods and the presence of extracellular vesicles (EVs). We investigated the effect of different manufacturing methods on hemolysis, residual cells, cell-derived EVs, and immunomodulatory effects on monocyte activity. Thirty-two RCC units produced using whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD), and whole blood–derived (WBD) methods were examined (n = 8 per method). Residual platelet and white blood cells (WBCs) and the concentration, cell of origin, and characterization of EVs in RCC supernatants were assessed in fresh and stored supernatants. Immunomodulatory activity of RCC supernatants was assessed by quantifying monocyte cytokine production capacity in an in vitro transfusion model. RCF units yielded the lowest number of platelet and WBC-derived EVs, whereas the highest number of platelet EVs was in AD (day 5) and in WBD (day 42). The number of small EVs (<200 nm) was greater than large EVs (≥200 nm) in all tested supernatants, and the highest level of small EVs were in AD units. Immunomodulatory activity was mixed, with evidence of both inflammatory and immunosuppressive effects. Monocytes produced more inflammatory interleukin-8 after exposure to fresh WBF or expired WBD supernatants. Exposure to supernatants from AD and WBD RCC suppressed monocyte lipopolysaccharide-induced cytokine production. Manufacturing methods significantly affect RCC unit EV characteristics and are associated with an immunomodulatory effect of RCC supernatants, which may affect the quality and safety of RCCs.

Cellular and extracellular vesicular origins of miRNAs within the bovine ovarian follicle.

The ovarian follicle components must provide an ideal environment to ensure the success of reproductive processes, and communication between follicular cells is crucial to support proper oocyte growth. Recently, it has been demonstrated that the presence of extracellular vesicles (EVs) carrying microRNAs (miRNAs) in follicular fluid represents an important autocrine and paracrine communication mechanism inside the ovarian follicle. In this study, we tested the hypothesis that the miRNA content of EVs isolated from ovarian follicular (granulosa and cumulus-oocyte complexes) cell-conditioned culture media is dependent upon cell type. We initially screened bovine granulosa cells (GCs) and cumulus-oocyte complexes (COCs), as well as their derived EVs for 348 miRNAs using real-time PCR, and detected 326 miRNAs in GCs and COCs cells and 62 miRNAs in GCs and COCs EVs. A bioinformatics analysis of the identified cell-specific and differentially expressed miRNAs predicted that they likely modulate important cellular processes, including signalling pathways such as the PI3K-Akt, MAPK and Wnt pathways. By investigating the origins of miRNAs within the follicular fluid, the results of this study provide novel insights into follicular miRNA content and intercellular communication that may be of invaluable use in the context of reproductive technologies, diagnostic of ovarian-related diseases and/or the identification of biomarkers for oocyte and embryo quality.