Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

<p>Abstract</p> <p>Background</p> <p>Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.</p> <p>Method</p> <p>Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.</p> <p>Results</p> <p>RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.</p> <p>Conclusions</p> <p>Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.</p

Extracellular Vesicles: Evolving Factors in Stem Cell Biology

Stem cells are proposed to continuously secrete trophic factors that potentially serve as mediators of autocrine and paracrine activities, associated with reprogramming of the tumor microenvironment, tissue regeneration, and repair. Hitherto, significant efforts have been made to understand the level of underlying paracrine activities influenced by stem cell secreted trophic factors, as little is known about these interactions. Recent findings, however, elucidate this role by reporting the effects of stem cell derived extracellular vesicles (EVs) that mimic the phenotypes of the cells from which they originate. Exchange of genetic information utilizing persistent bidirectional communication mediated by stem cell-EVs could regulate stemness, self-renewal, and differentiation in stem cells and their subpopulations. This review therefore discusses stem cell-EVs as evolving communication factors in stem cell biology, focusing on how they regulate cell fates by inducing persistent and prolonged genetic reprogramming of resident cells in a paracrine fashion. In addition, we address the role of stem cell-secreted vesicles in shaping the tumor microenvironment and immunomodulation and in their ability to stimulate endogenous repair processes during tissue damage. Collectively, these functions ensure an enormous potential for future therapies

Exosomes Communicate Protective Messages during Oxidative Stress; Possible Role of Exosomal Shuttle RNA

BACKGROUND: Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress. CONCLUSIONS/SIGNIFICANCE: These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus

Vesiclepedia: A compendium for extracellular vesicles with continuous community annotation

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field

Кераміка «terra sigillata» з с. Зимне на Волині

Стаття присвячена публікації чотирьох керамічних посудин типу «terra sigillata», знайдених на дні р. Луги у с. Зимне Володимир-Волинського району Волинської області. Попередній аналіз цих знахідок дозволяє віднести їх до Понтійського центру виробництва такого посуду. Вірогідним шляхом потрапляння цієї колекції на Волинь була готська експансія у Північне Причорномор’я

Cell till cell signalering via exosomer (transport av genetiskt material mellan celler via exosomer) - Identifiering av RNA-bindande proteiner i exosomer som kan interagera med olika typer av RNA: RBP-medierad transport av RNA till exosomer

In the human body, communication between cells takes place in several different ways. Exosomes are a type of extracellular vesicles (30-200 nm in diameter) that also participate in this process. Exosomes can be released from most cell types in the body and exist naturally in different body fluids, such as blood. In 2007, we showed for the first time that exosomes also transport RNA molecules (both coding and non-coding RNA molecules) between cells (Valadi H. et al. Nature Cell Biology. 2007). The findings describe a new type of 'cell-cell' communication by which different cells send genetic messages to each other by secreting vesicles (exosomes) containing specific RNA molecules. Our current studies aim to (a) identify cellular actors involved in translocation (packaging) of RNA into exosomes during their biosynthesis, (b) identify the mechanism by which exosomes are taken up by recipient cells/ tissues, and (c) ) discover a method to introduce exogenous/ therapeutic RNA into exosomes, to be used for in vivo transport of therapeutic RNA to different organs. RNA-binding proteins (RBPs) present in exosomes derived from HTB177 cells (NCI-H460 [H460] (ATCC: HTB-177) were identified as described below. For more detailed description please see Statello L, Maugeri M, Garre E, Nawaz M, Wahlgren J, Papadimitriou A, et al. (2018) (doi: 10.1371/journal.pone.0195969. eCollection 2018). List of the identified proteins can be found in the section 'Data collection' as well as on the journal's website (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195969#sec025). Identification of RNA binding proteins in exosomes and their parent cells: To detect and isolate the RBPs in cells and exosomes, the biotinylated esRNAs, cellular miRNAs, and cellular mRNAs were attached to streptavidin-coated paramagnetic Dynabeads M-280 and were incubated with native protein extracts of exosomes in separate RNA species assays (total exosomal-protein extract + esRNA;, total exosomal-protein extract + cell-mRNA;, total exosomal-protein extract + cell-miRNA). The protein-RNA complexes were eluted from the beads. The RBPs bound to the different RNAs were then loaded onto an SDS-PAGE gel and target bands were excised, trypsinised and analyzed using LC-MS/MS. Samples without RNA were processed in parallel and were used as negative controls to identify proteins that bound non-specifically to the beads.I människokroppen sker kommunikationen mellan celler på flera olika sätt. Exosomer är en typ av extracellulär-vesiklar (30-200 nm i diameter) som också deltar i denna process. Exosomer kan frisättas från de flesta celltyper i kroppen och förekommer naturligt i olika kroppsvätskor så som blod. År 2007 visade vi för första gången att exosomer också transporterar RNA molekyler (både kodande och icke kodande RNA molekyler) mellan celler (Valadi H. et al. Nature Cell Biology. 2007). Resultaten beskriver en ny typ av ’cell-cell’ kommunikation med vilken olika celler skickar genetiska meddelande till varandra genom att frisätta vesiklar (exosomer) innehållande specifika RNA molekyler. Våra nuvarande studier syftar till att (a) identifiera cellulära aktörer som deltar i trans-lokaliseringen (packning) av RNA in i exosomer under deras biosyntes, (b) ta reda på med vilken mekanism tas upp exosomer av mottagarceller/ vävnader, och (c) upptäcka en metod för att introducera exogent/ terapeutiskt RNA in i exosomer, som ska nyttjas för in vivo transport av terapeutiskt RNA till olika vävnader. RNA-bindande proteiner (RBPs) närvarande i exosomer som härstammar från HTB177 celler (NCI-H460 [H460] (ATCC: HTB-177). För information om identifiering se den engelska katalogposten https://snd.gu.se/en/catalogue/study/SND1142. Mer detaljerad beskrivning finns i Statello L, Maugeri M, Garre E, Nawaz M, Wahlgren J, Papadimitriou A, et al. (2018) (doi: 10.1371/journal.pone.0195969. eCollection 2018). Lista över identifierade proteiner finns i avsnittet ’Datainsamling’ samt på tidskriftens hemsida (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195969#sec025)

Microaerobic glycerol formation in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD+-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae

Functional studies of the TDH and GPD gene-products in the glycolytic flux and glycerol metabolism of Saccharomyces cerevisiae

This thesis focuses on the physiology and control of the glycolysis and glycerol metabolism in the yeast Sacchromyces cerevisiae. The study is based on the relation ship between the first two reactions around the branch-point of the upper and lower part of glycolysis and the glycerol pathway. In these reactions, the oxidation of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphospho glycerate (1,3-BFG) is catalysed by the glyceraldehyde 3-phosphate dehydrogenases (encoded by the TDH-genes), while the reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) is catalysed by the glycerol 3-phosphate dehydrogenases (encoded by the GPD-genes). Both the Gpd- and Tdh-enzymes catalyse redox reactions using either part of the redox couple, NADH/NAD+, as cofactor. The products of the GPD and TDH genes are involved in many different aspects of yeast physiology such as; maintenance of redoxbalance, osmoregulation, lipid biosynthesis, and protection against reactive oxygen species (ROS). The GPD1 gene is mainly important when the cells are subjected to osmotic stress and deletion of this gene does not affect the growth rate aerobically or anaerobically under normal growth conditions. In contrast, deletion of the GPD2 gene causes a decrease of the growth rate and an increase of the TDH1 gene expression under anaerobic conditions. Deletion of the TDH1 gene (but not of TDH2 or TDH3) in a gpd2Δ strain can partially suppress the anaaerobic growth defect of the gpd2Δ strain. To understand the link between the Gpd- and the Tdh-reactions, we have made all combinatorial mutants between the GPD and TDH genes. In the present work, we have shown that the earlier reported lethal mutant of glycolysis, tdh2Δ tdh3Δ, is suppressed by disruption of the GPD genes. Our work has revealed that the glycolytic flow of the lethal tdh2Δ tdh3Δ mutant has a limiting step at the Tdh-reaction and that the Tdh1p is fully capable to perform this reaction if it expressed at sufficient levels. Further, the kinetic properties of Tdh-isoenzymes are elucidated and the pivotal role of the Tdh- and Gpd-enzymes in metabolism of S. cerevisiae are examined and discussed