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Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease characterised by the deposition of aggregated proteins including TAR DNA-binding protein 43 (TDP-43) in vulnerable motor neurons and the brain. Extracellular vesicles (EVs) facilitate the spread of neurodegenerative diseases and can be easily accessed in the bloodstream. This study aimed to identify a panel of EV miRNAs that can capture the pathology occurring in the brain and peripheral circulation. EVs were isolated from the cortex (BDEVs) and serum (serum EVs) of 3 month-old and 6-month-old TDP-43*Q331K and TDP-43*WT mice. Following characterisation and miRNA isolation, the EVs underwent next-generation sequencing where 24 differentially packaged miRNAs were identified in the TDP-43*Q331K BDEVs and 7 in the TDP-43*Q331K serum EVs. Several miRNAs, including miR-183-5p, were linked to ALS. Additionally, miR-122-5p and miR-486b-5p were identified in both panels, demonstrating the ability of the serum EVs to capture the dysregulation occurring in the brain. This is the first study to identify miRNAs common to both the serum EVs and BDEVs in a mouse model of ALS., (© 2024. Crown.)

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Small extracellular vesicles (EVs) are signalling messengers that regulate inter-tissue communication through delivery of their molecular cargo. Here, we show that liver-derived EVs are acute regulators of whole-body glycaemic control in mice. Liver EV secretion into the circulation is increased in response to hyperglycaemia, resulting in increased glucose effectiveness and insulin secretion through direct inter-organ EV signalling to skeletal muscle and the pancreas, respectively. This acute blood glucose lowering effect occurs in healthy and obese mice with non-alcoholic fatty liver disease, despite marked remodelling of the liver-derived EV proteome in obese mice. The EV-mediated blood glucose lowering effects were recapitulated by administration of liver EVs derived from humans with or without progressive non-alcoholic fatty liver disease, suggesting broad functional conservation of liver EV signalling and potential therapeutic utility. Taken together, this work reveals a mechanism whereby liver EVs act on peripheral tissues via endocrine signalling to restore euglycaemia in the postprandial state., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Extracellular vesicles (EVs) and non-coding RNAs (ncRNAs) are emerging contributors to Alzheimer's disease (AD) pathophysiology. Differential abundance of ncRNAs carried by EVs may provide valuable insights into underlying disease mechanisms. Brain tissue-derived EVs (bdEVs) are particularly relevant, as they may offer valuable insights about the tissue of origin. However, there is limited research on diverse ncRNA species in bdEVs in AD., Objective: This study explored whether the non-coding RNA composition of EVs isolated from post-mortem brain tissue is related to AD pathogenesis., Methods: bdEVs from age-matched late-stage AD patients (n = 23) and controls (n = 10) that had been separated and characterized in our previous study were used for RNA extraction, small RNA sequencing, and qPCR verification., Results: Significant differences of non-coding RNAs between AD and controls were found, especially for miRNAs and tRNAs. AD pathology-related miRNA and tRNA differences of bdEVs partially matched expression differences in source brain tissues. AD pathology had a more prominent association than biological sex with bdEV miRNA and tRNA components in late-stage AD brains., Conclusions: Our study provides further evidence that EV non-coding RNAs from human brain tissue, including but not limited to miRNAs, may be altered and contribute to AD pathogenesis.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs., (© 2023 New York Academy of Sciences.)

Prion diseases (PrD) or transmissible spongiform encephalopathies (TSE) are invariably fatal and pathogenic neurodegenerative disorders caused by the self-propagated misfolding of cellular prion protein (PrP C ) to the neurotoxic pathogenic form (PrP TSE ) via a yet undefined but profoundly complex mechanism. Despite several decades of research on PrD, the basic understanding of where and how PrP C is transformed to the misfolded, aggregation-prone and pathogenic PrP TSE remains elusive. The primary clinical hallmarks of PrD include vacuolation-associated spongiform changes and PrP TSE accumulation in neural tissue together with astrogliosis. The difficulty in unravelling the disease mechanisms has been related to the rare occurrence and long incubation period (over decades) followed by a very short clinical phase (few months). Additional challenge in unravelling the disease is implicated to the unique nature of the agent, its complexity and strain diversity, resulting in the heterogeneity of the clinical manifestations and potentially diverse disease mechanisms. Recent advances in tissue isolation and processing techniques have identified novel means of intercellular communication through extracellular vesicles (EVs) that contribute to PrP TSE transmission in PrD. This review will comprehensively discuss PrP TSE transmission and neurotoxicity, focusing on the role of EVs in disease progression, biomarker discovery and potential therapeutic agents for the treatment of PrD., (© 2022. The Author(s).)

Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Increasing evidence suggests neuroinflammation is a highly coordinated response involving multiple cell types and utilising several different forms of cellular communication. In addition to the well documented cytokine and chemokine messengers, extracellular vesicles (EVs) have emerged as key regulators of the inflammatory response. EVs act as vectors of intercellular communication, capable of travelling between different cells and tissues to deliver selectively packaged protein, miRNA, and lipids from the parent cell. During neuroinflammation, EVs transmit specific inflammatory mediators, particularly from microglia, to promote inflammatory resolution. This mini-review will highlight the novel neuroinflammatory mechanisms contributing to the biogenesis and selective packaging of EVs., (© 2022. The Author(s).)

Parkinson's disease (PD) is a common neurodegenerative condition affecting a significant number of individuals globally, resulting in the presentation of debilitating motor and non-motor symptoms, including bradykinesia, resting tremor, as well as mood and sleep disorders. The pathology of PD has been observed to spread through the central nervous system resulting in progressive brain degeneration and a poor prognosis. Aggregated forms of the protein α-synuclein, particularly intermediary aggregates, referred to as oligomers, or preformed fibrils, have been implicated as the causative agent in the degeneration of neuronal processes, including the dysfunction of axonal transport, mitochondrial activity, and ultimately cellular death. Extracellular vesicles (EVs) have been strongly implicated in the propagation of PD pathology. Current observations suggest that aggregated α-synuclein is transported between neurons via small EVs in a series of exocytosis and endocytosis cellular processes leading to the observed spread of neurotoxicity and cellular death. Despite some understanding of the role of EVs in neurodegeneration, the exact mechanism by which these lipidic particles participate in the progression of Parkinson's pathology is not entirely understood. Here we review the current understanding of the role of EVs in the propagation of PD and explore their potential as a therapeutic target., (© 2022 The Author(s).)

The field of extracellular vesicle (EV) research has developed rapidly over the last decade from the study of fundamental biology to a subject of significant clinical relevance. The potential of harnessing EVs in the diagnosis and treatment of diseases - including cancer and neurological and cardiovascular disorders - is now being recognized. Accordingly, the applications of EVs as therapeutic targets, biomarkers, novel drug delivery agents and standalone therapeutics are being actively explored. This Review provides a brief overview of the characteristics and physiological functions of the various classes of EV, focusing on their association with disease and emerging strategies for their therapeutic exploitation., (© 2022. Springer Nature Limited.)

The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil ( Sarcophilus harrisii ). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis; 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Espejo, Wilson, Pye, Ratcliffe, Ruiz-Aravena, Willms, Wolfe, Hamede, Hill, Jones, Woods and Lyons.)

Background: Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare., Objective: We aimed to reveal the pathological mechanisms inside the brain by profiling the tissue and bdEV proteomes in AD patients. In addition, to indicate targets for capturing and molecular profiling of bdEVs in the periphery, CNS cell-specific markers were profiled on the intact bdEV surface., Methods: bdEVs were separated and followed by EV counting and sizing. Brain tissue and bdEVs from age-matched AD patients and controls were then proteomically profiled. Total tau (t-tau), phosphorylated tau (p-tau), and antioxidant peroxiredoxins (PRDX) 1 and 6 were measured by immunoassay in an independent bdEV separation. Neuron, microglia, astrocyte, and endothelia markers were detected on intact EVs by multiplexed ELISA., Results: Overall, concentration of recovered bdEVs was not affected by AD. Proteome differences between AD and control were more pronounced for bdEVs than for brain tissue. Levels of t-tau, p-tau, PRDX1, and PRDX6 were significantly elevated in AD bdEVs compared with controls. Release of certain cell-specific bdEV markers was increased in AD., Conclusion: Several bdEV proteins are involved in AD mechanisms and may be used for disease monitoring. The identified CNS cell markers may be useful tools for peripheral bdEV capture.

Bacterial membrane vesicles (BMVs) are produced by all bacteria and facilitate a range of functions in host-microbe interactions and pathogenesis. Quantification of BMVs is a critical first step in the analysis of their biological and immunological functions. Historically, BMVs have been quantified by protein assay, which remains the preferred method of BMV quantification. However, recent studies have shown that BMV protein content can vary significantly between bacterial strains, growth conditions, and stages of bacterial growth, suggesting that protein concentration may not correlate directly with BMV quantity. Here, we show that the method used to quantify BMVs can alter experimental outcomes. We compared the enumeration of BMVs using different protein assays and nanoparticle tracking analysis (NTA). We show that different protein assays vary significantly in their quantification of BMVs and that their sensitivity varies when quantifying BMVs produced by different species. Moreover, stimulation of epithelial cells with an equivalent amount of BMV protein quantified using different protein assays resulted in significant differences in interleukin 8 (IL-8) responses. Quantification of Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus BMVs by NTA and normalization of BMV cargo to particle number revealed that BMV protein, DNA, and RNA contents were variable between strains and species and throughout bacterial growth. Differences in BMV-mediated activation of Toll-like receptors, NF-κB, and IL-8 responses were observed when stimulations were performed with equivalent BMV particle number but not equivalent protein amount. These findings reveal that the method of BMV quantification can significantly affect experimental outcomes, thereby potentially altering the observed biological functions of BMVs. IMPORTANCE Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration. Here, we reveal that the method used to enumerate BMVs can significantly alter experimental outcomes. Specifically, standardization of BMVs by protein amount reduced the ability to distinguish strain differences in the immunological functions of BMVs. In contrast, species-, strain-, and growth stage-dependent differences in BMV cargo content were evident when BMVs were enumerated by particle number, and this was reflected in differences in their ability to induce immune responses. These findings indicate that parameters critical to BMV function, including bacterial species, strain, growth conditions, and sample purity, should form the basis of standard reporting in BMV studies. This will ultimately bring uniformity to the field to advance our understanding of BMV functions.

The iconic Tasmanian devil (Sarcophilus harrisii) is endangered due to the transmissible cancer Devil Facial Tumour Disease (DFTD), of which there are two genetically independent subtypes (DFT1 and DFT2). While DFT1 and DFT2 can be differentially diagnosed using tumour biopsies, there is an urgent need to develop less-invasive biomarkers that can detect DFTD and distinguish between subtypes. Extracellular vesicles (EVs), the nano-sized membrane-enclosed vesicles present in most biofluids, represent a valuable resource for biomarker discovery. Here, we characterized the proteome of EVs from cultured DFTD cells using data-independent acquisition-mass spectrometry and an in-house spectral library of > 1500 proteins. EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions. Furthermore, hallmark proteins of epithelial-mesenchymal transition were enriched in DFT2 EVs relative to DFT1 EVs. These findings were validated in EVs derived from serum samples, revealing that the mesenchymal marker tenascin-C was also enriched in EVs derived from the serum of devils infected with DFT2 relative to those infected with DFT1 and healthy controls. This first EV-based investigation of DFTD increases our understanding of the cancers' EVs and their possible involvement in DFTD progression, such as metastasis. Finally, we demonstrated the potential of EVs to differentiate between DFT1 and DFT2, highlighting their potential use as less-invasive liquid biopsies for the Tasmanian devil., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Extracellular vesicles (EVs) are small cargo-bearing vesicles released by cells into the extracellular space. The field of EVs has grown exponentially over the past two decades; this growth follows the realisation that EVs are not simply a waste disposal system as had originally been suggested by some, but also a complex cell-to-cell communication mechanism. Indeed, EVs have been shown to transfer functional cargo between cells and can influence several biological processes. These small biological particles are also deregulated in disease. As we approach the 75th anniversary of the first experiments in which EVs were unknowingly isolated, it seems right to take stock and look back on how the field started, and has since exploded into its current state. Here we review the early experiments, summarise key findings that have propelled the field, describe the growth of an organised EV community, discuss the current state of the field, and identify key challenges that need to be addressed., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed nanoparticles released by cells. They play a role in intercellular communication and are involved in numerous physiological and pathological processes. Cells release subpopulations of EVs with distinct composition and inherent biological function which overlap in size. Current size-based isolation methods are, therefore, not optimal to discriminate between functional EV subpopulations. In addition, EVs overlap in size with several other biological nanoparticles, such as lipoproteins and viruses. Proteomic analysis has allowed for more detailed study of EV composition, and EV isolation approaches based on this could provide a promising alternative for purification based on size. Elucidating EV heterogeneity and the characteristics and role of EV subpopulations will advance our understanding of EV biology and the role of EVs in health and disease. Here, we discuss current knowledge of EV composition, EV heterogeneity and advances in affinity based EV isolation tools., (© 2021 The Authors. Proteomics published by Wiley-VCH GmbH.)

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high ( n = 7)- or low ( n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid ( p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region.  AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Neurodegenerative diseases are characterised by the irreversible degeneration of neurons in the central or peripheral nervous systems. These include amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases. Small extracellular vesicles (sEVs), a type of EV involved in cellular communication, have been well documented as propagating neurodegenerative diseases. These sEVs carry cargo, such as proteins and RNA, to recipient cells but are also capable of promoting protein misfolding, thus actively contributing to the progression of these diseases. sEV secretion is also a compensatory process for lysosomal dysfunction in the affected cells, despite inadvertently propagating disease to recipient cells. Despite this, sEV miRNAs have biomarker potential for the early diagnosis of these diseases, while stem cell-derived sEVs and those generated through exogenous assistance demonstrate the greatest therapeutic potential. This Review will highlight novel advancements in the involvement of sEVs as propagators of neuropathology, biomarkers and potential therapeutics in neurodegenerative diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The misfolding and fibrillization of the protein, α-synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically-derived vesicles. In this study, we comprehensively characterised the ability of lipid-rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Statement: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the deposition of misfolded proteins in the motor cortex and motor neurons. Although a multitude of ALS-associated mutated proteins have been identified, several have been linked to small extracellular vesicles such as exosomes involved in cell-cell communication. This study aims to determine the proteome of extracellular vesicles isolated from the motor cortex of ALS subjects and to identify novel ALS-associated deregulated proteins. Motor cortex extracellular vesicles (MCEVs) were isolated from human postmortem ALS (n = 10) and neurological control (NC, n = 5) motor cortex brain tissues and the MCEVs protein content subsequently underwent mass spectrometry analysis, allowing for a panel of ALS-associated proteins to be identified. This panel consists of 16 statistically significant differentially packaged proteins identified in the ALS MCEVs. This includes several upregulated RNA-binding proteins which were determined through pathway analysis to be associated with stress granule dynamics. The identification of these RNA-binding proteins in the ALS MCEVs suggests there may be a relationship between ALS-associated stress granules and ALS MCEV packaging, highlighting a potential role for small extracellular vesicles such as exosomes in the pathogenesis of ALS and as potential peripheral biomarkers for ALS.

Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles., (Copyright © 2020. Published by Elsevier Inc.)

Extracellular vesicles (EVs) include exosomes and microvesicles and have been shown to have roles in the CNS ranging from the removal of unwanted biomolecules to intercellular communication to the spread of pathogenic proteins associated with neurodegenerative diseases. EVs carry protein, lipid, and genetic cargo, and research over more than a decade has shown that they contain the misfolded forms of proteins associated with Alzheimer's, Parkinson's, and the prion diseases. Altered genetic cargo, usually in the form of miRNAs, have also been identified in EVs patients with these diseases, suggesting that EVs may be a source of disease biomarkers. Whether EVs play a key role in the pathogenesis of neurological diseases remains to be firmly established because most current research is performed using cell culture and transgenic animal models. If EVs are identified as a key pathological contributor to neurological conditions, they will form a novel target for therapeutic intervention. This Dual Perspectives article will discuss the current understanding of the role of EVs in neurological diseases and raise some of the limitations of our current understandings of this field., (Copyright © 2019 the authors.)

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2 + cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2 + CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2 + CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2 + CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2 + CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.

Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse. A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression. Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment. They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically. Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear. Here we investigated if extracellular vesicles from BRAF inhibitor-resistant melanoma could influence drug sensitivity in recipient melanoma cells. We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition. These data suggest that the BRAF inhibitor-sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in V600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF -mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression., Competing Interests: Conflict of interest statement: J.L. has written several patents in the field of extracellular vesicles as therapeutics and is currently an employee of Codiak BioSciences, in parallel with his position at the University of Gothenburg. R.O.B. has received honoraria from Roche for lectures.

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research., (© 2017 American Heart Association, Inc.)

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Extracellular vesicles, including exosomes, are small membranous vesicles released from many biotypes, contributing to the disease progression and spreading. These extracellular vesicles provide an important mode of cell-to-cell communication by delivering proteins, lipids and RNA to target cells. Exosomes are found associated with neurodegenerative diseases, which are characterised by progressive degeneration of neurons and often associated with misfolded protein. The common diseases include Parkinson's disease (PD), Alzheimer's diseases (AD), amyotrophic lateral sclerosis (ALS), and the prion diseases. Of all neurodegenerative diseases, prion diseases are classified as the distinctive group owing to its transmissible and infectious nature of misfolded prion protein. The infectious prion particles have been demonstrated to be present in exosomes to spread prion infectivity within cells. Similarly, misfolded proteins involved in other neurodegenerative diseases such as Amyloid-β and tau in AD, α-synuclein in PD, and superoxide dismutase 1 in ALS have been demonstrated to exploit exosomes for induced spreading of misfolded proteins in a prion-like mechanism. Furthermore, RNA molecules can be taken up by the recipient cells as cargo in exosomes. These RNAs can module the expression of the target genes by repressing or inhibiting protein translation. Here we review the role of exosomes in prion diseases and other common neurodegenerative diseases, and discuss the potential of these vesicles for disease pathogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A core function of neural cells is the exchange and integration of information. Extracellular vesicles such as exosomes and microvesicles recently entered the scene of neuroscience as novel vehicles transmitting complex signals between neural cells. Carrying a defined but mixed cargo of biomolecules, extracellular vesicles possess versatile biological activities with the ability to profoundly modulate the molecular configuration and behaviour of target cells. Extracellular vesicles are suggested to carry out functions during neural development and maintenance, they appear to spread neuropathology and furthermore, convey neuroprotection and regeneration. Understanding the molecular mechanisms of this sophisticated cellular crosstalk will fundamentally improve our insight in complex intercellular processes in the healthy and diseased nervous system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: To address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research., Methods: By following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations., Results: We have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: "extracellular vesicle", "extracellular exosome", "apoptotic body", and "microvesicle" were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO., Conclusions: As an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search.

Growing evidence indicates that small extracellular vesicles, called exosomes, are prominent mediators of neurodegenerative diseases such as prion, Alzheimer's and Parkinson's disease. Exosomes contain neurodegenerative disease associated proteins such as the prion protein, β-amyloid and α-synuclein. Only demonstrated so far in vivo with prion disease, exosomes are hypothesised to also facilitate the spread of β-amyloid and α-synuclein from their cells of origin to the extracellular environment. In the current review, we will discuss the role of exosomes in Alzheimer's and Parkinson's disease including their possible contribution to disease propagation and pathology and highlight their utility as a diagnostic in neurodegenerative disease.

Biologically active metals such as copper, zinc and iron are fundamental for sustaining life in different organisms with the regulation of cellular metal homeostasis tightly controlled through proteins that coordinate metal uptake, efflux and detoxification. Many of the proteins involved in either uptake or efflux of metals are localised and function on the plasma membrane, traffic between intracellular compartments depending upon the cellular metal environment and can undergo recycling via the endosomal pathway. The biogenesis of exosomes also occurs within the endosomal system, with several major neurodegenerative disease proteins shown to be released in association with these vesicles, including the amyloid-β (Aβ) peptide in Alzheimer's disease and the infectious prion protein involved in Prion diseases. Aβ peptide and the prion protein also bind biologically active metals and are postulated to play important roles in metal homeostasis. In this review, we will discuss the role of extracellular vesicles in Alzheimer's and Prion diseases and explore their potential contribution to metal homeostasis., (© 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Many cell types, including neurons, are known to release small membranous vesicles known as exosomes. In addition to their protein content these vesicles have recently been shown to contain messenger RNA (mRNA) and micro RNA (miRNA) species. Roles for these vesicles include cell-cell signalling, removal of unwanted proteins, and transfer of pathogens (including prion-like misfolded proteins) between cells, such as infectious prions. Prions are the infectious particles that are responsible for transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Exosomes are also involved in processing the amyloid precursor protein (APP), which is associated with Alzheimer's disease (AD). As exosomes can be isolated from circulating fluids such as serum, urine, and cerebrospinal fluid (CSF), they provide a potential source of biomarkers for neurological conditions. Here, we review the roles these vesicles play in neurodegenerative disease and highlight their potential in diagnosing these disorders through analysis of their RNA content., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.