The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

SummaryBackgroundAlthough CT scans are very useful clinically, potential cancer risks exist from associated ionising radiation, in particular for children who are more radiosensitive than adults. We aimed to assess the excess risk of leukaemia and brain tumours after CT scans in a cohort of children and young adults.MethodsIn our retrospective cohort study, we included patients without previous cancer diagnoses who were first examined with CT in National Health Service (NHS) centres in England, Wales, or Scotland (Great Britain) between 1985 and 2002, when they were younger than 22 years of age. We obtained data for cancer incidence, mortality, and loss to follow-up from the NHS Central Registry from Jan 1, 1985, to Dec 31, 2008. We estimated absorbed brain and red bone marrow doses per CT scan in mGy and assessed excess incidence of leukaemia and brain tumours cancer with Poisson relative risk models. To avoid inclusion of CT scans related to cancer diagnosis, follow-up for leukaemia began 2 years after the first CT and for brain tumours 5 years after the first CT.FindingsDuring follow-up, 74 of 178 604 patients were diagnosed with leukaemia and 135 of 176 587 patients were diagnosed with brain tumours. We noted a positive association between radiation dose from CT scans and leukaemia (excess relative risk [ERR] per mGy 0·036, 95% CI 0·005–0·120; p=0·0097) and brain tumours (0·023, 0·010–0·049; p<0·0001). Compared with patients who received a dose of less than 5 mGy, the relative risk of leukaemia for patients who received a cumulative dose of at least 30 mGy (mean dose 51·13 mGy) was 3·18 (95% CI 1·46–6·94) and the relative risk of brain cancer for patients who received a cumulative dose of 50–74 mGy (mean dose 60·42 mGy) was 2·82 (1·33–6·03).InterpretationUse of CT scans in children to deliver cumulative doses of about 50 mGy might almost triple the risk of leukaemia and doses of about 60 mGy might triple the risk of brain cancer. Because these cancers are relatively rare, the cumulative absolute risks are small: in the 10 years after the first scan for patients younger than 10 years, one excess case of leukaemia and one excess case of brain tumour per 10 000 head CT scans is estimated to occur. Nevertheless, although clinical benefits should outweigh the small absolute risks, radiation doses from CT scans ought to be kept as low as possible and alternative procedures, which do not involve ionising radiation, should be considered if appropriate.FundingUS National Cancer Institute and UK Department of Health.

Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.

It is generally assumed that a specific ubiquitin ligase (E3)linksIn eukaryotic cells, ubiquitination serves to target regulatory and misfolded proteins for rapid degradation by proteasomes (1-3), to trigger endocytosis of membrane proteins (4), and also to allow specific protein-protein associations important in signal transduction, DNA repair, and gene transcription (5-8). Protein ubiquitination involves formation of isopeptide linkages between the C-terminal carboxyl group of a ubiquitin (Ub) 4 and an ⑀-amino group on a lysine on the protein substrate or a preceding Ub to form a polyUb chain. To synthesize such linkages, the C-terminal carboxyl group of a Ub is first activated by formation of a thioester bond with a cysteine on the Ubactivating enzyme (E1), and the activated Ub is then transferred as a thioester to one of the 20 -40 Ub-conjugating enzymes (E2) of the cell. The formation of a Ub chain on the substrate is then catalyzed by a Ub ligase (E3), which binds the substrate and an E2. Several families of E3s exist that differ in structure and mechanism. If ubiquitination is catalyzed by a member of the Ring finger or the U-box E3 family, the activated Ub is transferred from the E2 directly to a lysine on the protein substrate or to a preceding Ub. The abundant Ring finger and the related U-box families are small monomeric proteins that bind the substrate at one end and then in that vicinity release the reactive Ub from the E2-Ub thioester (9, 10). If ubiquitination is catalyzed by an E3 of the HECT domain family, the activated Ub is transferred from the E2 first to a cysteine on the E3 to form another thioester bond and then to the substrate or to a preceding Ub * This work was supported by grants from the NIGMS, the High Q Foundation, the Fund for Innovation from Elan Corp. (to A. L. G.), the National Institutes of Health (to S. P. G.), and National Institutes of Health R01 Grant GM65267 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Ub, ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ubactivating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring.

Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria) to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids) present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info) might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.

Gram-negative bacteria constitutively secrete outer membrane vesicles (OMVs) into the extracellular milieu. Recent research in this area has revealed that OMVs may act as intercellular communicasomes in polyspecies communities by enhancing bacterial survival and pathogenesis in hosts. However, the mechanisms of vesicle formation and the pathophysiological roles of OMVs have not been clearly defined. While it is obvious that mass spectrometry-based proteomics offers great opportunities for improving our knowledge of bacterial OMVs, limited proteomic data are available for OMVs. The present review aims to give an overview of the previous biochemical, biological, and proteomic studies in the emerging field of bacterial OMVs, and to give future directions for high-throughput and comparative proteomic studies of OMVs that originate from diverse Gram-negative bacteria under various environmental conditions. This article will hopefully stimulate further efforts to construct a comprehensive proteome database of bacterial OMVs that will help us not only to elucidate the biogenesis and functions of OMVs but also to develop diagnostic tools, vaccines, and antibiotics effective against pathogenic bacteria.

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.

Background-Use of computed tomography (CT) for diagnostic evaluation has increased dramatically over the past two decades. Even though CT is associated with substantially higher radiation exposure than conventional x-rays, typical clinical doses are not known. We sought to estimate the radiation dose associated with common CT studies in clinical practice; assess variation in dose across types of studies, patients, and institutions; and quantify the potential cancer risk associated with these examinations. Methods-Retrospective cross-sectional study describing radiation dose associated with the 11 most common types of diagnostic CT studies performed on 1,119 consecutive adult patients at four San Francisco Bay Area institutions between January 1 and May 30, 2008. We estimated lifetime attributable risks of cancer by study type from these measured doses.