International Society for Extracellular Vesicles

🧑🎓 Introducing the #ISEV2025 Keynote Speakers! 🫧 Harald Stenmark will discuss 'The ESCRT machinery in biogenesis of intraluminal and extracellular vesicles'. 📖 Bio:

❓ Have you registered already for #ISEV2025? 🐤 Don't wait, early bird discount rates last until March 7th! https://buff.ly/3CyMjEE

New in #JEV Mechanism of Centrosomal Protein 55 (CEP55) Loading Into Exosomes Christian Dahlstroem, Johanna Barezani, Jing Li, Kostiantyn Sopelniak, Stefanie Muhs, Carola Schneider, Roland Thünauer, Rudolph Reimer, Sabine Windhorst Up-regulation of Centrosomal Protein 55 (CEP55) in cancer cells increases malignancy, and the protein can be transferred via exosomes. However, the mechanism of how CEP55 is delivered to exosomes is unknown. In this study, we addressed this issue and analysed trafficking of EGFP-CEP55 from early to late endosomes by using high-resolution microscopy. Our data show that endogenous as well as EGFP-CEP55 appeared as dot-like structures in cancer cells. However, we did not find an internalization of CEP55 into early Rab5- and late Rab7-positive endosomes but only into secretory late CD63-positive endosomes. In addition, an association of the CEP55 dots with the endoplasmic reticulum and with ALG-2-interacting protein X (Alix) dots was detected. Moreover, mutation of the CEP55-Alix interaction site strongly reduced the formation of CEP55 dots as well as CEP55 localization in extracellular vesicles. In summary, our data indicate that delivery of CEP55 into exosomes does not occur by the canonical early-to-late endosome pathway but by Alix-mediated recruitment to secretory late secretory CD63 endosomes. https://lnkd.in/enJwhedH

🆕New in #JExBio 🆕 Establishment of an experimental system to analyse extracellular vesicles during apoplastic fungal pathogenesis Nathaniel Hearfield, Dominik Brotherton, Zedi Gao, Jameel Inal, Henrik U. Stotz Phoma stem canker disease of oilseed rape (Brassica napus) is caused by the extracellular fungal pathogen Leptosphaeria maculans. Although this pathogen resides exclusively in apoplastic spaces surrounding plant cells, the significance of extracellular vesicles (EVs) has not been assessed. Here, we show a method to collect apoplastic fluids (AFs) from infected leaves or cotyledons for collection of EVs during the process of host colonisation. The 15,000 × g supernatants of AFs were shown to contain ribulose-bisphosphate carboxylase (RuBisCO) at 7 days post-inoculation with L. maculans, a protein that was absent from unchallenged cotyledons. RuBisCO release coincided with the switch from biotrophy to necrotrophy, suggesting the involvement of host cell death. However, RuBisCO release did not differ between compatible and incompatible interactions, suggesting necrotrophic host cell death might not be the only process involved. EVs were also collected from axenic fungal cultures and characterised for their particle size distribution using nanoparticle tracking analysis and transmission electron microscopy. The protein composition of EV-enriched fractions was analysed using SDS-PAGE and proteomics. Enrichment analysis of gene ontology terms provided evidence for involvement of glucan and chitin metabolism as well as catalase and peptidase activities. Most of the proteins identified have previously been found in EV studies and/or EV databases, and for most of the proteins evidence was found for an involvement in pathogenicity/virulence. https://lnkd.in/eZGp5a_b

👩🎓 Introducing the #ISEV2025 Keynote Speakers! 🧬 nicole meisner-kober will discuss her work 'Converging EV, LNP and virus biology: towards oral RNA drugs'. 📖 Read more about her work:

📆 SAVE THE DATE: 2nd conference on EVs in Nervous Systems October, 29-31, 2025 🌎 Location: Montreal, Quebec, Canada 🧠 Expect a world-class lineup of cutting-edge topics, inspiring speakers, meaningful discussions and interactions (2–2.5 days) More info: https://buff.ly/4bkmwwH #EViNS

New in #JEV Extracellular Histones as Exosome Membrane Proteins Regulated by Cell Stress Birendra Singh, Marcus Fredriksson Sundbom, Uma Muthukrishnan, Balasubramanian Natarajan, Stephanie Stransky, André Görgens, Joel Z. Nordin, Oscar P. B. Wiklander, Linda Sandblad, Simone Sidoli, Samir El Andaloussi, Michael Haney, Jonathan D. Gilthorpe Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of EV histone secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones and histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. Histones colocalized to tetraspanin positive EVs but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease. https://lnkd.in/eADVQSfU

New in #JExBio Comparison of Methods for Isolation and Characterization of Total and Astrocyte-Enriched Extracellular Vesicles From Human Serum and Plasma Leandra K. Figueroa-Hall, Kaiping Burrows, Ahlam M. Alarbi, Bethany N. Hannafon, Cole Hladik, Chibing Tan, Rajagopal Ramesh, Jennifer L. Stewart, Victoria B. Risbrough, Martin P. Paulus, T. Kent Teague Extracellular vesicles (EV) which play critical roles in intercellular communication, have garnered interest as biomarkers with researchers studying brain-related disease processes due to their ability to be isolated from various biofluids. Astrocytes, a type of glial cell, play a critical role in neuronal regulation and function. As such, EV enriched from astrocytes can be used to interrogate cargo and identify mechanisms by which astrocytes communicate with other cells of the central nervous system or shed light on pathophysiological conditions. This manuscript compared five EV isolation methods (differential ultracentrifugation [dUC], precipitation, precipitation + purification, silicon carbon resin and size exclusion chromatography [SEC]) using small volumes of human plasma and serum with a focus on immunocapture of astrocyte-enriched EV (AEEV), with the excitatory amino acid transporter 1, or GLAST. Methods were evaluated on yield, purity, recovery and downstream application to include immunoassays for tetraspanin, immune and astrocyte markers. Results revealed that whilst precipitation-based methods such as ExoQuick yielded higher EV concentrations, size exclusion (SmartSEC, qEV) provided greater purity, emphasizing a trade-off between yield and purity. This study provides a comprehensive resource for researchers in selecting EV isolation methods tailored to small biobanked clinical samples, with the goal of advancing biomarker discovery in Neuroscience. https://lnkd.in/e4dxcC75

New in #JEV Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure Yen-Ting Lu, Tzu-Yu Chen, Hsin-Hung Lin, Ya-Wen Chen, Yu-Xiu Lin, Duy‑Cuong Le, Yen-Hua Huang, Andrew H.-J. Wang, Cheng-Chung Lee, Thai-Yen Ling Acetaminophen (APAP) overdose can cause severe liver injury and life-threatening conditions that may lead to multiple organ failure without proper treatment. N-acetylcysteine (NAC) is the accepted and prescribed treatment for detoxification in cases of APAP overdose. Nonetheless, in acute liver failure (ALF), particularly when the ingestion is substantial, NAC may not fully restore liver function. NAC administration in ALF has limitations and potential adverse effects, including nausea, vomiting, diarrhoea, flatus, gastroesophageal reflux, and anaphylactoid reactions. Mesenchymal stromal cell (MSC)-based therapies using paracrine activity show promise for treating ALF, with preclinical studies demonstrating improvement. Recently, MSC-derived extracellular vesicles (EVs) have emerged as a new therapeutic option for liver injury. MSC-derived EVs can contain various therapeutic cargos depending on the cell of origin, participate in physiological processes, and respond to abnormalities. However, most therapeutic EVs lack a distinct orientation upon entering the body, resulting in a lack of targeting specificity. Therefore, enhancing the precision of natural EV delivery systems is urgently needed. Thus, we developed an advanced targeting technique to deliver modified EVs within the body. Our strategy aims to employ bioorthogonal click chemistry to attach a targeting molecule to the surface of small extracellular vesicles (sEVs), creating exogenous chimeric antigen receptor-modified sEVs (CAR-sEVs) for the treatment. First, we engineered azido-modified sEVs (N3-sEVs) through metabolic glycoengineering by treating MSCs with the azide-containing monosaccharide N-azidoacetyl-mannosamine (Ac4ManNAz). Next, we conjugated N3-sEVs with a dibenzocyclooctyne (DBCO)-tagged single-chain variable fragment (DBCO-scFv) that targets the asialoglycoprotein receptor (ASGR1), thus producing CAR-sEVs for precise liver targeting. The efficacy of CAR-sEV therapy in ALF models by targeting ASGR1 was validated. MSC-derived CAR-sEVs reduced serum liver enzymes, mitigated liver damage, and promoted hepatocyte proliferation in APAP-induced injury. Overall, CAR-sEVs exhibited enhanced hepatocyte specificity and efficacy in ameliorating liver injury, highlighting the significant advancements achievable with cell-free targeted therapy. https://lnkd.in/eHbSqXtP

New in #JEV Stoichiometric constraints for detection of EV-borne biomarkers in blood Nataša Zarovni, Danilo Mladenović, Dario Brambilla, Federica Panico, Marcella Chiari Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumour or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumour-specific EV detection, or more precisely, for tumour identification via EV based assays. Our endeavour involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on ‘low prevalence—low concentration’ scenario. Finally, we evaluate the advantages versus limitations of single-particle analysers over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution. https://lnkd.in/grBDnqkc