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.

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Cerebral deposition of B-amyloid peptide @A) is a hallmark of Alzheimer's disease. Concentration of /IA could play a critical role in the rate of amyloid deposition. It is therefore of considerable importance to identify proteases involved in processing ofpA. '251-labeled synthetic /IA specifically cross-linked to a single protein with M, = 110,000 in cytosol fractions from rat brain and liver. This protein was identified as insulin degrading enzyme (IDE) since the labeling of the 110 kDa protein was completely blocked by an excess of insulin, and anti-IDE monoclonal antibodies precipitated the labeled protein. Purified rat IDE effectively degraded /IA.Key wor& Alzheirner's disease; p-Amyloid;Insulin degrading enzyme; Oxidative modification; Protein degradation; Brain; Rat IntrodwtionAlzheimer's disease amyloid is composed of a 39-43 residue /I-amyloid protein (BA) [l-3] that is derived from a set of larger transmembrane precursor proteins @4PPs) [4,5]. G rowing evidence is accumulating that processing of jL4PP may occur intracellularly [6-81 and that /IA is a normal product of cell metabolism [9-l 11. A recent finding that cultured human neurons generate /IA intracellularly before it is released into the medium [12] suggests the possibility that PA accumulation in Alzheimer's brain could result from defects in intracellular proteolytic machinery responsible for its breakdown. Thus, protease involved in processing ofBA is a potential target for pharmacological treatment of the disease.We considered that among intracellular proteases which could participate in degradation of PA, the insulin degrading enzyme (IDE; EC 3.4.22.11) is a very likely candidate. IDE is a highly selective protease involved in the breakdown of certain peptide hormones [ 13-151. IDE does not have a strict specificity for any amino acid residue [16]. It has been suggested, however, that its specificity is based on recognition of specific secondary structures rather than specific peptide bonds [16]. It is interesting to note that although the IDE substrates, insulin, atria1 natriuretic peptide and glucagon, are greatly different in primary structure, they probably share a common structural motif allowing them to form amyloid fibrils under certain conditions in vitro [ 171 and in vivo [18,19]. In amyloid fibrils, different proteins have been shown to be organized in a cross-j? conformation [ 171. Therefore, IDE specificity could be based on recognition of/3-structures either present on the surface of the substrate molecules or adopted upon binding to the protease. This possibility suggests that IDE might also degrade PA.Here we show that PA is a substrate for rat liver IDE. Furthermore, we demonstrate that IDE is the only protein that can be specifically cross-linked to PA in crude extracts of rat brain and liver, suggesting for a role for IDE in the cellular processing of /3A. Materials and methods MaterialsThe following materials were obtained: ['2SI]insulin (1.2 kCiimmo1) from Dainabott (Tokyo, Japan), /JA,,, and PAi, from Bachem ...