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1. The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Author

Artuyants A, Guo G, Flinterman M, Middleditch M, Jacob B, Lee K, Vella L, Su H, Wilson M, Eva L, Shelling AN, and Blenkiron C

Subjects
Humans, Female, Proteomics methods, Body Mass Index, Middle Aged, Endometrial Neoplasms metabolism, Endometrial Neoplasms pathology, Extracellular Vesicles metabolism, Proteome metabolism, Proteome analysis, Obesity metabolism, Obesity pathology, Biomarkers, Tumor metabolism
Abstract

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0 µg protein: 1.38 × 10 11 -1.10 × 10 12 particles), characterised by their size (∼150 nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2)., (© 2024 The Authors. Proteomics published by Wiley‐VCH GmbH.)

Published
2024
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2. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Author

Welsh JA, Goberdhan DCI, O'Driscoll L, Buzas EI, Blenkiron C, Bussolati B, Cai H, Di Vizio D, Driedonks TAP, Erdbrügger U, Falcon-Perez JM, Fu QL, Hill AF, Lenassi M, Lim SK, Mahoney MG, Mohanty S, Möller A, Nieuwland R, Ochiya T, Sahoo S, Torrecilhas AC, Zheng L, Zijlstra A, Abuelreich S, Bagabas R, Bergese P, Bridges EM, Brucale M, Burger D, Carney RP, Cocucci E, Crescitelli R, Hanser E, Harris AL, Haughey NJ, Hendrix A, Ivanov AR, Jovanovic-Talisman T, Kruh-Garcia NA, Ku'ulei-Lyn Faustino V, Kyburz D, Lässer C, Lennon KM, Lötvall J, Maddox AL, Martens-Uzunova ES, Mizenko RR, Newman LA, Ridolfi A, Rohde E, Rojalin T, Rowland A, Saftics A, Sandau US, Saugstad JA, Shekari F, Swift S, Ter-Ovanesyan D, Tosar JP, Useckaite Z, Valle F, Varga Z, van der Pol E, van Herwijnen MJC, Wauben MHM, Wehman AM, Williams S, Zendrini A, Zimmerman AJ, Théry C, and Witwer KW

Subjects
Biological Transport, Biomarkers metabolism, Phenotype, Extracellular Vesicles metabolism, Exosomes metabolism
Abstract

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.)

Published
2024
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3. Placental Extracellular Vesicles Can Be Loaded with Plasmid DNA.

Author

Kang M, Hisey C, Tsai B, Nursalim Y, Blenkiron C, and Chamley LW

Subjects
Pregnancy, Female, Humans, DNA, Drug Delivery Systems, Plasmids genetics, Placenta, Extracellular Vesicles
Abstract

Recently, extracellular vesicles (EVs) have garnered considerable interest as potential vehicles for drug delivery, including gene therapy. Although EVs from diverse sources have been investigated, current techniques used in the field for EV generation limit large-scale EV production. The placenta is essentially a tissue transplant and has unique properties that allow it to avoid the maternal immune system making it likely that placental EVs will not generate inflammatory responses and will avoid clearance by the immune system. We propose that placental EVs produced from explant cultures are an efficient method to produce considerable quantities of EVs that would be safe to administer, and we hypothesize that placental EVs can be loaded with large exogenous plasmids. To this end, we trialed three strategies to load plasmid DNA into placental EVs, including loading via electroporation of placental tissue prior to EV isolation and loading directly into placental EVs via electroporation or direct incubation of the EVs in plasmid solution. We report that the placenta releases vast quantities of EVs compared to placental cells in monolayer cultures. We show successful loading of plasmid DNA into both large- and small-EVs following both exogenous loading strategies with more plasmid encapsulated in large-EVs. Importantly, direct incubation did not alter EV size nor quantity. Further, we showed that the loading efficiency into EVs was dependent on the exogenous plasmid DNA dose and the DNA size. These results provide realistic estimates of plasmid loading capacity into placental EVs using current technologies and showcase the potential of placental EVs as DNA delivery vehicles.

Published
2023
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4. The biodistribution of placental and fetal extracellular vesicles during pregnancy following placentation.

Author

Kang M, Blenkiron C, and Chamley LW

Subjects
Animals, Pregnancy, Female, Humans, Placentation, Tissue Distribution, Fetus, Placenta metabolism, Extracellular Vesicles
Abstract

Human pregnancy is a highly orchestrated process requiring extensive cross-talk between the mother and the fetus. Extracellular vesicles released by the fetal tissue, particularly the placenta, are recognized as important mediators of this process. More recently, the importance of placental extracellular vesicle biodistribution studies in animal models has received increasing attention as identifying the organs to which extracellular vesicles are targeted to helps us understand more about this communication system. Placental extracellular vesicles are categorized based on their size into macro-, large-, and small-extracellular vesicles, and their biodistribution is dependent on the extracellular vesicle's particle size, the direction of blood flow, the recirculation of blood, as well as the retention capacity in organs. Macro-extracellular vesicles are exclusively localized to the lungs, while large- and small-extracellular vesicles show high levels of distribution to the lungs and liver, while there is inconsistency in the reporting of distribution to the spleen and kidneys. This inconsistency may be due to the differences in the methodologies employed between studies and their limitations. Future studies should incorporate analysis of placental extracellular vesicle biodistribution at the macroscopic level on whole animals and organs/tissues, as well as the microscopic cellular level., (© 2023 The Author(s).)

Published
2023
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5. Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask.

Author

Artuyants A, Chang V, Reshef G, Blenkiron C, Chamley LW, Leung E, and Hisey CL

Subjects
Bioreactors, Culture Media, Conditioned metabolism, Serum metabolism, Extracellular Vesicles metabolism
Abstract

The efficient production of extracellular vesicles (EVs) from adherent cells in vitro can be challenging when using conventional culture flasks. Issues such as low cell density leading to low EV yield, and the inability to completely remove bovine serum EVs without starvation contribute to this challenge. By comparison, the two-chamber CELLine adherent bioreactor can produce significantly more EVs with improved time, space, and resource efficiency. Furthermore, it is highly accessible and can continually produce EVs using long term cultures without the need for passaging. Lastly, the 10 kDa semipermeable, cellulose acetate membrane separating the cell and media chambers allows for the continual use of bovine serum in the media chamber while preventing bovine EVs from contaminating the conditioned media., (© 2021. Springer Science+Business Media, LLC.)

Published
2022
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6. Updating MISEV: Evolving the minimal requirements for studies of extracellular vesicles.

Author

Witwer KW, Goberdhan DC, O'Driscoll L, Théry C, Welsh JA, Blenkiron C, Buzás EI, Di Vizio D, Erdbrügger U, Falcón-Pérez JM, Fu QL, Hill AF, Lenassi M, Lötvall J, Nieuwland R, Ochiya T, Rome S, Sahoo S, and Zheng L

Subjects
Humans, Extracellular Vesicles metabolism, Reference Standards
Abstract

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field-consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Published
2021
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7. Ruminant Milk-Derived Extracellular Vesicles: A Nutritional and Therapeutic Opportunity?

Author

Ong SL, Blenkiron C, Haines S, Acevedo-Fani A, Leite JAS, Zempleni J, Anderson RC, and McCann MJ

Subjects
Animals, Cattle, Humans, Infant, Infant Nutritional Physiological Phenomena, Infant, Newborn, Nutritional Status, Species Specificity, Bottle Feeding, Extracellular Vesicles metabolism, Milk metabolism, Nutritive Value, Ruminants
Abstract

Milk has been shown to contain a specific fraction of extracellular particles that are reported to resist digestion and are purposefully packaged with lipids, proteins, and nucleic acids to exert specific biological effects. These findings suggest that these particles may have a role in the quality of infant nutrition, particularly in the early phase of life when many of the foundations of an infant's potential for health and overall wellness are established. However, much of the current research focuses on human or cow milk only, and there is a knowledge gap in how milk from other species, which may be more commonly consumed in different regions, could also have these reported biological effects. Our review provides a summary of the studies into the extracellular particle fraction of milk from a wider range of ruminants and pseudo-ruminants, focusing on how this fraction is isolated and characterised, the stability and uptake of the fraction, and the reported biological effects of these fractions in a range of model systems. As the individual composition of milk from different species is known to differ, we propose that the extracellular particle fraction of milk from non-traditional and minority species may also have important and distinct biological properties that warrant further study.

Published
2021
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8. Micropatterned growth surface topography affects extracellular vesicle production.

Author

Hisey CL, Hearn JI, Hansford DJ, Blenkiron C, and Chamley LW

Subjects
Bioreactors, Cell Line, Cell Movement, Extracellular Vesicles
Abstract

Extracellular vesicles (EVs) are micro and nanoscale packages that circulate in all bodily fluids and play an important role in intercellular communication by shuttling biomolecules to nearby and distant cells. However, producing sufficient amounts of EVs for many types of in vitro studies using standard culture methods can be challenging, and despite the success of some bioreactors in increasing EV-production, it is still largely unknown how individual culture conditions can alter the production and content of EVs. In this study, we demonstrate a simple and inexpensive micropatterning technique that can be used to produce polystyrene microtracks over a 100 mm diameter growth surface area. We then demonstrate that these microtracks can play a significant role in increasing EV production using a triple-negative breast cancer cell line (MDA-MB-231) and that these changes in EV production correlate with increases in cellular aspect ratio, alignment of the cells' long axes to the microtracks, and single-cell migration rates. These findings have implications in both biomanufacturing of EVs and potentially in enhancing the biomimicry of EVs produced in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Published
2021
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9. Biodistribution of extracellular vesicles following administration into animals: A systematic review.

Author

Kang M, Jordan V, Blenkiron C, and Chamley LW

Subjects
Animals, Biological Availability, Drug Delivery Systems methods, Humans, Organ Specificity drug effects, Organ Specificity physiology, Extracellular Vesicles metabolism, Extracellular Vesicles transplantation, Tissue Distribution physiology
Abstract

In recent years, attention has turned to examining the biodistribution of EVs in recipient animals to bridge between knowledge of EV function in vitro and in vivo. We undertook a systematic review of the literature to summarize the biodistribution of EVs following administration into animals. There were time-dependent changes in the biodistribution of small-EVs which were most abundant in the liver. Detection peaked in the liver and kidney in the first hour after administration, while distribution to the lungs and spleen peaked between 2-12 h. Large-EVs were most abundant in the lungs with localization peaking in the first hour following administration and decreased between 2-12 h. In contrast, large-EV localization to the liver increased as the levels in the lungs decreased. There was moderate to low localization of large-EVs to the kidneys while localization to the spleen was typically low. Regardless of the origin or size of the EVs or the recipient species into which the EVs were administered, the biodistribution of the EVs was largely to the liver, lungs, kidneys, and spleen. There was extreme variability in the methodology between studies and we recommend that guidelines should be developed to promote standardization where possible of future EV biodistribution studies., Competing Interests: There is no conflict of interest for any of the authors., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Published
2021
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10. Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study.

Author

Heatlie J, Chang V, Fitzgerald S, Nursalim Y, Parker K, Lawrence B, Print CG, and Blenkiron C

Subjects
Cell-Free Nucleic Acids, Edetic Acid, Humans, Liquid Biopsy, Pilot Projects, Extracellular Vesicles
Abstract

Background: Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Methods: Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. Results: MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Conclusions: Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells.

Published
2020
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11. Novel Electrochemically Switchable, Flexible, Microporous Cloth that Selectively Captures, Releases, and Concentrates Intact Extracellular Vesicles.

Author

Akbarinejad A, Hisey CL, Brewster D, Ashraf J, Chang V, Sabet S, Nursalim Y, Lucarelli V, Blenkiron C, Chamley L, Barker D, Williams DE, Evans CW, and Travas-Sejdic J

Subjects
Aptamers, Nucleotide chemistry, Aptamers, Nucleotide metabolism, Bridged Bicyclo Compounds, Heterocyclic chemistry, Cell Line, Extracellular Vesicles metabolism, Gold chemistry, Humans, MCF-7 Cells, Polymers chemistry, Porosity, Sulfur chemistry, Tetraspanin 30 metabolism, Electrochemical Techniques methods, Extracellular Vesicles chemistry
Abstract

There is a significant and growing research interest in the isolation of extracellular vesicles (EVs) from large volumes of biological samples and their subsequent concentration into clean and small volumes of buffers, especially for applications in medical diagnostics. Materials that are easily incorporated into simple sampling devices and which allow the release of EVs without the need for auxiliary and hence contaminating reagents are particularly in demand. Herein, we report on the design and fabrication of a flexible, microporous, electrochemically switchable cloth that addresses the key challenges in diagnostic applications of EVs. We demonstrate the utility of our electrochemically switchable substrate for the fast, selective, nondestructive, and efficient capture and subsequent release of EVs. The substrate consists of an electrospun cloth, infused with a conducting polymer and decorated with gold particles. Utilizing gold-sulfur covalent bonding, the electrospun substrates may be functionalized with SH-terminated aptamer probes selective to EV surface proteins. We demonstrate that EVs derived from primary human dermal fibroblast (HDFa) and breast cancer (MCF-7) cell lines are selectively captured with low nonspecific adsorption using an aptamer specific to the CD63 protein expressed on the EV membranes. The specific aptamer-EV interactions enable easy removal of the nonspecifically bound material through washing steps. The conducting polymer component of the cloth provides a means for efficient (>92%) and fast (<5 min) electrochemical release of clean and intact captured EVs by cathodic cleavage of the Au-S bond. We demonstrate successful capture of diluted EVs from a large volume sample and their release into a small volume of clean phosphate-buffered saline buffer. The developed cloth can easily be incorporated into different designs for separation systems and would be adaptable to other biological entities including cells and other EVs. Furthermore, the capture/release capability holds great promise for liquid biopsies if used to targeted disease-specific markers.

Published
2020
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12. Estimation of the burden of human placental micro- and nano-vesicles extruded into the maternal blood from 8 to 12 weeks of gestation.

Author

Liu H, Kang M, Wang J, Blenkiron C, Lee A, Wise M, Chamley L, and Chen Q

Subjects
Adaptation, Physiological, Cardiovascular Physiological Phenomena, DNA analysis, Extracellular Vesicles chemistry, Extracellular Vesicles immunology, Female, Humans, Immune System physiology, Pregnancy, Pregnancy Trimester, First, Proteins analysis, Tissue Culture Techniques, Extracellular Vesicles physiology, Gestational Age, Placenta ultrastructure
Abstract

Background: The human placenta extrudes a variety of extracellular vesicles (EVs) into the maternal blood daily. These vesicles may be crucial to the adaptation of the maternal cardiovascular and immune systems to pregnancy. Quantifying the EVs that are released in early gestation is important to our understanding of how placental EVs may contribute to the regulation of maternal physiology., Methods: EVs were isolated from first trimester placental explants and separated into micro- and nano-vesicles by differential centrifugation. The numbers of each type of EVs extruded from each milligram of placentae between gestational weeks 8 and 12 was determined by Nanoparticle Tracking Analysis. The total protein or DNA content of the vesicles was determined by BCA assay or Qubit ® 2.0., Results: Neither the number of micro- nor nano-EVs/mg explant (n = 49), nor the total protein (n = 19) and DNA content (n = 29) of these EVs changed significantly between 8 and 12 weeks of gestation. When the increasing placental weight with gestation was accounted for, the daily number of placental EVs extruded into the maternal blood increased by more than 100 fold between 8 and 12 weeks (micro-EVs 6.23 X 10 14 and nano-EVs 1.84 X 10 14  at 12 weeks, p = 0.0003)., Discussion: Constant production of micro- and nano-EVs per-milligram placenta, regardless of gestational age, and the increased daily burden of EVs across gestational age indicate these EVs have the potential to regulate maternal physiology from early pregnancy. Since total EV protein content, like EV numbers was, constant, this is a potentially reliable surrogate for quantifying EVs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Published
2018
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13. The functional RNA cargo of bacterial membrane vesicles.

Author

Dauros-Singorenko P, Blenkiron C, Phillips A, and Swift S

Subjects
Bacteria metabolism, Bacterial Infections microbiology, Biological Transport, Eukaryotic Cells microbiology, Extracellular Vesicles metabolism, RNA, Bacterial genetics, RNA, Untranslated genetics, Signal Transduction, Bacteria cytology, Bacteria genetics, Extracellular Vesicles chemistry, RNA, Bacterial metabolism, RNA, Untranslated metabolism
Abstract

Bacteria secrete RNAs, some of which have effects on other cells and on other species as signalling RNAs. Prokaryotic membrane vesicles (MVs) contain a range of RNA types. The MV structure offers protection from degradation as well as receptors to facilitate delivery to target cells. Microscopic imaging and molecular biology analyses have provided evidence to demonstrate that bacterial MVs deliver their RNA into eukaryotic cells. Moreover, in some cases the RNA cargo is demonstrably functional and phenotypic changes can be identified in MV-RNA treated target cells. The challenge now is to dissect the effect of MV-RNA on target cells away from the effects of non-RNA components of the MV such as lipopolysaccharide that can co-purify with RNA.

Published
2018
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14. Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA.

Author

Blenkiron C, Simonov D, Muthukaruppan A, Tsai P, Dauros P, Green S, Hong J, Print CG, Swift S, and Phillips AR

Subjects
Cell Line, Tumor, Centrifugation, Density Gradient, Epithelial Cells microbiology, Epithelial Cells pathology, Escherichia coli Infections microbiology, Humans, RNA, Bacterial genetics, RNA, Bacterial metabolism, RNA, Ribosomal genetics, RNA, Ribosomal metabolism, RNA, Transfer genetics, RNA, Transfer metabolism, Urinary Bladder Neoplasms microbiology, Uropathogenic Escherichia coli cytology, Uropathogenic Escherichia coli pathogenicity, Extracellular Vesicles genetics, Uropathogenic Escherichia coli genetics
Abstract

Background: Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection., Methods and Results: Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different 'size' RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells., Conclusions: These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.

Published
2016
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15. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Author

Théry, Clotilde, Witwer, Kenneth W, Aikawa, Elena, Alcaraz, Maria Jose, Anderson, Johnathon D, Andriantsitohaina, Ramaroson, Antoniou, Anna, Arab, Tanina, Archer, Fabienne, Atkin‐Smith, Georgia K, Ayre, D Craig, Bach, Jean‐Marie, Bachurski, Daniel, Baharvand, Hossein, Balaj, Leonora, Baldacchino, Shawn, Bauer, Natalie N, Baxter, Amy A, Bebawy, Mary, Beckham, Carla, Zavec, Apolonija Bedina, Benmoussa, Abderrahim, Berardi, Anna C, Bergese, Paolo, Bielska, Ewa, Blenkiron, Cherie, Bobis‐Wozowicz, Sylwia, Boilard, Eric, Boireau, Wilfrid, Bongiovanni, Antonella, Borràs, Francesc E, Bosch, Steffi, Boulanger, Chantal M, Breakefield, Xandra, Breglio, Andrew M, Brennan, Meadhbh Á, Brigstock, David R, Brisson, Alain, Broekman, Marike LD, Bromberg, Jacqueline F, Bryl‐Górecka, Paulina, Buch, Shilpa, Buck, Amy H, Burger, Dylan, Busatto, Sara, Buschmann, Dominik, Bussolati, Benedetta, Buzás, Edit I, Byrd, James Bryan, Camussi, Giovanni, Carter, David RF, Caruso, Sarah, Chamley, Lawrence W, Chang, Yu‐Ting, Chen, Chihchen, Chen, Shuai, Cheng, Lesley, Chin, Andrew R, Clayton, Aled, Clerici, Stefano P, Cocks, Alex, Cocucci, Emanuele, Coffey, Robert J, Cordeiro‐da‐Silva, Anabela, Couch, Yvonne, Coumans, Frank AW, Coyle, Beth, Crescitelli, Rossella, Criado, Miria Ferreira, D'Souza‐Schorey, Crislyn, Das, Saumya, Chaudhuri, Amrita Datta, de Candia, Paola, De Santana, Eliezer F, De Wever, Olivier, del Portillo, Hernando A, Demaret, Tanguy, Deville, Sarah, Devitt, Andrew, Dhondt, Bert, Di Vizio, Dolores, Dieterich, Lothar C, Dolo, Vincenza, Rubio, Ana Paula Dominguez, Dominici, Massimo, Dourado, Mauricio R, Driedonks, Tom AP, Duarte, Filipe V, Duncan, Heather M, Eichenberger, Ramon M, Ekström, Karin, Andaloussi, Samir EL, Elie‐Caille, Celine, Erdbrügger, Uta, Falcón‐Pérez, Juan M, Fatima, Farah, Fish, Jason E, Flores‐Bellver, Miguel, Försönits, András, and Frelet‐Barrand, Annie

Subjects
Biochemistry and Cell Biology, Biological Sciences, extracellular vesicles, exosomes, ectosomes, microvesicles, minimal information requirements, guidelines, standardization, microparticles, rigor, reproducibility, Biochemistry and cell biology
Abstract

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.

Published
2018

16. Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Author

Théry, Clotilde, Witwer, Kenneth W, Aikawa, Elena, Alcaraz, Maria Jose, Anderson, Johnathon D, Andriantsitohaina, Ramaroson, Antoniou, Anna, Arab, Tanina, Archer, Fabienne, Atkin-Smith, Georgia K, Ayre, D Craig, Bach, Jean-Marie, Bachurski, Daniel, Baharvand, Hossein, Balaj, Leonora, Baldacchino, Shawn, Bauer, Natalie N, Baxter, Amy A, Bebawy, Mary, Beckham, Carla, Bedina Zavec, Apolonija, Benmoussa, Abderrahim, Berardi, Anna C, Bergese, Paolo, Bielska, Ewa, Blenkiron, Cherie, Bobis-Wozowicz, Sylwia, Boilard, Eric, Boireau, Wilfrid, Bongiovanni, Antonella, Borràs, Francesc E, Bosch, Steffi, Boulanger, Chantal M, Breakefield, Xandra, Breglio, Andrew M, Brennan, Meadhbh Á, Brigstock, David R, Brisson, Alain, Broekman, Marike Ld, Bromberg, Jacqueline F, Bryl-Górecka, Paulina, Buch, Shilpa, Buck, Amy H, Burger, Dylan, Busatto, Sara, Buschmann, Dominik, Bussolati, Benedetta, Buzás, Edit I, Byrd, James Bryan, Camussi, Giovanni, Carter, David Rf, Caruso, Sarah, Chamley, Lawrence W, Chang, Yu-Ting, Chen, Chihchen, Chen, Shuai, Cheng, Lesley, Chin, Andrew R, Clayton, Aled, Clerici, Stefano P, Cocks, Alex, Cocucci, Emanuele, Coffey, Robert J, Cordeiro-da-Silva, Anabela, Couch, Yvonne, Coumans, Frank Aw, Coyle, Beth, Crescitelli, Rossella, Criado, Miria Ferreira, D'Souza-Schorey, Crislyn, Das, Saumya, Datta Chaudhuri, Amrita, de Candia, Paola, De Santana, Eliezer F, De Wever, Olivier, Del Portillo, Hernando A, Demaret, Tanguy, Deville, Sarah, Devitt, Andrew, Dhondt, Bert, Di Vizio, Dolores, Dieterich, Lothar C, Dolo, Vincenza, Dominguez Rubio, Ana Paula, Dominici, Massimo, Dourado, Mauricio R, Driedonks, Tom Ap, Duarte, Filipe V, Duncan, Heather M, Eichenberger, Ramon M, Ekström, Karin, El Andaloussi, Samir, Elie-Caille, Celine, Erdbrügger, Uta, Falcón-Pérez, Juan M, Fatima, Farah, Fish, Jason E, Flores-Bellver, Miguel, Försönits, András, Frelet-Barrand, Annie, Fricke, Fabia, Fuhrmann, Gregor, Gabrielsson, Susanne, Gámez-Valero, Ana, Gardiner, Chris, Gärtner, Kathrin, Gaudin, Raphael, Gho, Yong Song, Giebel, Bernd, Gilbert, Caroline, Gimona, Mario, Giusti, Ilaria, Goberdhan, Deborah Ci, Görgens, André, Gorski, Sharon M, Greening, David W, Gross, Julia Christina, Gualerzi, Alice, Gupta, Gopal N, Gustafson, Dakota, Handberg, Aase, Haraszti, Reka A, Harrison, Paul, Hegyesi, Hargita, Hendrix, An, Hill, Andrew F, Hochberg, Fred H, Hoffmann, Karl F, Holder, Beth, Holthofer, Harry, Hosseinkhani, Baharak, Hu, Guoku, Huang, Yiyao, Huber, Veronica, Hunt, Stuart, Ibrahim, Ahmed Gamal-Eldin, Ikezu, Tsuneya, Inal, Jameel M, Isin, Mustafa, Ivanova, Alena, Jackson, Hannah K, Jacobsen, Soren, Jay, Steven M, Jayachandran, Muthuvel, Jenster, Guido, Jiang, Lanzhou, Johnson, Suzanne M, Jones, Jennifer C, Jong, Ambrose, Jovanovic-Talisman, Tijana, Jung, Stephanie, Kalluri, Raghu, Kano, Shin-Ichi, Kaur, Sukhbir, Kawamura, Yumi, Keller, Evan T, Khamari, Delaram, Khomyakova, Elena, Khvorova, Anastasia, Kierulf, Peter, Kim, Kwang Pyo, Kislinger, Thomas, Klingeborn, Mikael, Klinke, David J, Kornek, Miroslaw, Kosanović, Maja M, Kovács, Árpád Ferenc, Krämer-Albers, Eva-Maria, Krasemann, Susanne, Krause, Mirja, Kurochkin, Igor V, Kusuma, Gina D, Kuypers, Sören, Laitinen, Saara, Langevin, Scott M, Languino, Lucia R, Lannigan, Joanne, Lässer, Cecilia, Laurent, Louise C, Lavieu, Gregory, Lázaro-Ibáñez, Elisa, Le Lay, Soazig, Lee, Myung-Shin, Lee, Yi Xin Fiona, Lemos, Debora S, Lenassi, Metka, Leszczynska, Aleksandra, Li, Isaac Ts, Liao, Ke, Libregts, Sten F, Ligeti, Erzsebet, Lim, Rebecca, Lim, Sai Kiang, Linē, Aija, Linnemannstöns, Karen, Llorente, Alicia, Lombard, Catherine A, Lorenowicz, Magdalena J, Lörincz, Ákos M, Lötvall, Jan, Lovett, Jason, Lowry, Michelle C, Loyer, Xavier, Lu, Quan, Lukomska, Barbara, Lunavat, Taral R, Maas, Sybren Ln, Malhi, Harmeet, Marcilla, Antonio, Mariani, Jacopo, Mariscal, Javier, Martens-Uzunova, Elena S, Martin-Jaular, Lorena, Martinez, M Carmen, Martins, Vilma Regina, Mathieu, Mathilde, Mathivanan, Suresh, Maugeri, Marco, McGinnis, Lynda K, McVey, Mark J, Meckes, David G, Meehan, Katie L, Mertens, Inge, Minciacchi, Valentina R, Möller, Andreas, Møller Jørgensen, Malene, Morales-Kastresana, Aizea, Morhayim, Jess, Mullier, François, Muraca, Maurizio, Musante, Luca, Mussack, Veronika, Muth, Dillon C, Myburgh, Kathryn H, Najrana, Tanbir, Nawaz, Muhammad, Nazarenko, Irina, Nejsum, Peter, Neri, Christian, Neri, Tommaso, Nieuwland, Rienk, Nimrichter, Leonardo, Nolan, John P, Nolte-'t Hoen, Esther NM, Noren Hooten, Nicole, O'Driscoll, Lorraine, O'Grady, Tina, O'Loghlen, Ana, Ochiya, Takahiro, Olivier, Martin, Ortiz, Alberto, Ortiz, Luis A, Osteikoetxea, Xabier, Østergaard, Ole, Ostrowski, Matias, Park, Jaesung, Pegtel, D Michiel, Peinado, Hector, Perut, Francesca, Pfaffl, Michael W, Phinney, Donald G, Pieters, Bartijn Ch, Pink, Ryan C, Pisetsky, David S, Pogge von Strandmann, Elke, Polakovicova, Iva, Poon, Ivan Kh, Powell, Bonita H, Prada, Ilaria, Pulliam, Lynn, Quesenberry, Peter, Radeghieri, Annalisa, Raffai, Robert L, Raimondo, Stefania, Rak, Janusz, Ramirez, Marcel I, Raposo, Graça, Rayyan, Morsi S, Regev-Rudzki, Neta, Ricklefs, Franz L, Robbins, Paul D, Roberts, David D, Rodrigues, Silvia C, Rohde, Eva, Rome, Sophie, Rouschop, Kasper Ma, Rughetti, Aurelia, Russell, Ashley E, Saá, Paula, Sahoo, Susmita, Salas-Huenuleo, Edison, Sánchez, Catherine, Saugstad, Julie A, Saul, Meike J, Schiffelers, Raymond M, Schneider, Raphael, Schøyen, Tine Hiorth, Scott, Aaron, Shahaj, Eriomina, Sharma, Shivani, Shatnyeva, Olga, Shekari, Faezeh, Shelke, Ganesh Vilas, Shetty, Ashok K, Shiba, Kiyotaka, Siljander, Pia R-M, Silva, Andreia M, Skowronek, Agata, Snyder, Orman L, Soares, Rodrigo Pedro, Sódar, Barbara W, Soekmadji, Carolina, Sotillo, Javier, Stahl, Philip D, Stoorvogel, Willem, Stott, Shannon L, Strasser, Erwin F, Swift, Simon, Tahara, Hidetoshi, Tewari, Muneesh, Timms, Kate, Tiwari, Swasti, Tixeira, Rochelle, Tkach, Mercedes, Toh, Wei Seong, Tomasini, Richard, Torrecilhas, Ana Claudia, Tosar, Juan Pablo, Toxavidis, Vasilis, Urbanelli, Lorena, Vader, Pieter, van Balkom, Bas Wm, van der Grein, Susanne G, Van Deun, Jan, van Herwijnen, Martijn Jc, Van Keuren-Jensen, Kendall, van Niel, Guillaume, van Royen, Martin E, van Wijnen, Andre J, Vasconcelos, M Helena, Vechetti, Ivan J, Veit, Tiago D, Vella, Laura J, Velot, Émilie, Verweij, Frederik J, Vestad, Beate, Viñas, Jose L, Visnovitz, Tamás, Vukman, Krisztina V, Wahlgren, Jessica, Watson, Dionysios C, Wauben, Marca Hm, Weaver, Alissa, Webber, Jason P, Weber, Viktoria, Wehman, Ann M, Weiss, Daniel J, Welsh, Joshua A, Wendt, Sebastian, Wheelock, Asa M, Wiener, Zoltán, Witte, Leonie, Wolfram, Joy, Xagorari, Angeliki, Xander, Patricia, Xu, Jing, Yan, Xiaomei, Yáñez-Mó, María, Yin, Hang, Yuana, Yuana, Zappulli, Valentina, Zarubova, Jana, Žėkas, Vytautas, Zhang, Jian-Ye, Zhao, Zezhou, Zheng, Lei, Zheutlin, Alexander R, Zickler, Antje M, Zimmermann, Pascale, Zivkovic, Angela M, Zocco, Davide, Zuba-Surma, Ewa K, dB&C I&I, LS Celbiologie-Algemeen, Celbiologie, Afd Pharmaceutics, Sub General Pharmaceutics, Sub Biomol.Mass Spect. and Proteomics, Afd Pharmacology, Urology, Pathology, Medical Oncology, Immunité et cancer, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Johns Hopkins University School of Medicine [Baltimore], Stress Oxydant et Pathologies Métaboliques (SOPAM), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM) - U 1192 (PRISM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Infections Virales et Pathologie Comparée - UMR 754 (IVPC), Institut National de la Recherche Agronomique (INRA)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Immuno-Endocrinologie Cellulaire et Moléculaire [Nantes] (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Department for Molecular Biology and Nanobiotechnology, National Institute of chemitry, Slovenia, Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie des Adaptations Nutritionnelles (PhAN), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Imagerie Moléculaire et Nanobiotechnologies - Institut Européen de Chimie et Biologie (IECB), Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), Molecular Biotechnology Center, Università degli studi di Torino = University of Turin (UNITO), Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University (JCU), Department of Oncology - Pathology, Cancer Center Karolinska [Karolinska Institutet] (CCK), Karolinska Institutet [Stockholm]-Karolinska Institutet [Stockholm], Departamento de Ciências Biológicas, Universidade do Porto = University of Porto, Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Cancer Research Institute Ghent (CRIG), Universiteit Gent = Ghent University [Belgium] (UGENT), Department of Medical and Surgical Sciences for Children and Adults [Modena, Italy] (Laboratory of Cellular Therapy), Università degli Studi di Modena e Reggio Emilia (UNIMORE), Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden, Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm], Center for Cooperative Research in Biosciences (CIC bioGUNE), Partner site Munich, German Centre for Infection Research (DZIF), Institute for Transfusion Medicine, University Hospital Essen, Universität Duisburg-Essen [Essen], Mécanismes Adaptatifs et Evolution (MECADEV), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Psychiatry, Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Department of Bacteriology and Immunology [Helsinki], Haartman Institute [Helsinki], Faculty of Medecine [Helsinki], Helsingin yliopisto = Helsingfors universitet = University of Helsinki-Helsingin yliopisto = Helsingfors universitet = University of Helsinki-Faculty of Medecine [Helsinki], Helsingin yliopisto = Helsingfors universitet = University of Helsinki-Helsingin yliopisto = Helsingfors universitet = University of Helsinki, Rigshospitalet [Copenhagen], Copenhagen University Hospital, Dalhousie University [Halifax], Department of Biology, Molecular Cell Biology, University of Mainz, Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Glycobiologie et signalisation cellulaire, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, University of Gothenburg (GU), Universidad de Alicante, École supérieure du professorat et de l'éducation - Académie de Créteil (UPEC ESPE Créteil), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), University of Antwerp (UA), Université Catholique de Louvain = Catholic University of Louvain (UCL), Research Institute, IRCCS Ospedale Pediatrico Bambino Gesù [Roma], Department of Veterinary Disease Biology [Copenhagen], Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU), Biologie et Pathologie du Neurone (Brain-C), Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Mathematics and Statistics, American University, University of Pretoria [South Africa], Ecole des Ingénieurs de la Ville de Paris (EIVP), Universitat Pompeu Fabra [Barcelona] (UPF), Instituto de Investigaciones Biomedicas, Universidad Nacional Autónoma de México (UNAM), Istituto Ortopedico Rizzoli, Department of Molecular Therapeutics, The Scripps Research Institute, Laboratoire d'Informatique de Grenoble (LIG ), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Montreal Children's Hospital, McGill University Health Center [Montreal] (MUHC), Compartimentation et dynamique cellulaires (CDC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cardiovascular Research Center, Massachusetts General Hospital [Boston], University Medical Center [Utrecht], University of Toronto, Fiocruz Minas - René Rachou Research Center / Instituto René Rachou [Belo Horizonte, Brésil], Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Federal University of Sao Paulo (Unifesp), Functional Genomics / Genómica Funcional [Montevideo], Institut Pasteur de Montevideo, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università degli Studi di Perugia (UNIPG), Hospital Santa Cristina Instituto de Investigación Sanitaria Princesa C, Unidad de Investigación, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Department of Physiology, University of California [San Francisco] (UCSF), University of California-University of California, University of Vermont [Burlington], Peking University [Beijing], Shandong Agricultural University (SDAU), State Key Laboratory of Quality Research in Chinese Medicine Taipa, Macau SAR, (Institute of Chinese Medical Sciences), Human Genetics, Universität Ulm - Ulm University [Ulm, Allemagne], INSERM, Institut Curie, INCa [INCA-11548], French National Research Agency [ANR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043], SIDACTION [17-1-AAE-1138], Fondation ARC [PGA1 RF20180206962, PJA 20171206453], NIDA [DA040385, DA047807], Ministry of Education, NIA [AG057430], NIMH [MH118164], Institut National de la Recherche Agronomique (INRA)-École Pratique des Hautes Études (EPHE), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN), Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University (UGENT), Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Universität Duisburg-Essen = University of Duisburg-Essen [Essen], Biotechnology and Biological Sciences Research Council (BBSRC)-Aberystwyth University, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), Universidad Nacional Autónoma de México = National Autonomous University of Mexico (UNAM), The Scripps Research Institute [La Jolla, San Diego], Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ), Università degli Studi di Perugia = University of Perugia (UNIPG), Instituto de Investigacion Sanitaria del Hospital de la Princesa, Hospital Universitario de La Princesa, University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), ANR-17-CE09-0025,MADNESS,Une approche microfluidique générique pour la qualification des nanoparticules biologiques(2017), Institut National de la Recherche Agronomique (INRA)-École pratique des hautes études (EPHE)-Université Claude Bernard Lyon 1 (UCBL), Biomedical Engineering and Physics, ACS - Atherosclerosis & ischemic syndromes, ACS - Microcirculation, Laboratory Specialized Diagnostics & Research, Radiotherapie, RS: GROW - R2 - Basic and Translational Cancer Biology, Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Université Nice Sophia Antipolis (... - 2019) (UNS), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), Università degli studi di Torino (UNITO), Universidade do Porto, University of Helsinki-University of Helsinki-Faculty of Medecine [Helsinki], University of Helsinki-University of Helsinki, Johannes Gutenberg - Universität Mainz (JGU), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Université de Toronto [Canada], Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM) - U1192 (PRISM), Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Université de Franche-Comté (UFC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Technologie de Belfort-Montbeliard (UTBM), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Universidade do Porto [Porto], Ghent University [Belgium] (UGENT), FEMTO-ST Institute, Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Franche-Comté (UFC)-CNRS : UMR6174, Mécanismes adaptatifs : des organismes aux communautés (MECADEV), Centre National de la Recherche Scientifique (CNRS)-Muséum national d'Histoire naturelle (MNHN), Johannes Gutenberg - University of Mainz (JGU), Université Catholique de Louvain (UCL), Universitat Pompeu Fabra [Barcelona], Laboratoire d'Informatique de Grenoble (LIG), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Laboratoire Réactions et Génie des Procédés (LRGP), Fiocruz Minas - René Rachou Research Center / Instituto René Rachou, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Functional Genomics Unit, Institut Curie-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), University of Vermont College of Medicine [Burlington, VT, USA], Extracellular Vesicles, Molecular and Integrative Biosciences Research Programme, Thery, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. D., Andriantsitohaina, R., Antoniou, A., Arab, T., Archer, F., Atkin-Smith, G. K., Ayre, D. C., Bach, J. -M., Bachurski, D., Baharvand, H., Balaj, L., Baldacchino, S., Bauer, N. N., Baxter, A. A., Bebawy, M., Beckham, C., Bedina Zavec, A., Benmoussa, A., Berardi, A. C., Bergese, P., Bielska, E., Blenkiron, C., Bobis-Wozowicz, S., Boilard, E., Boireau, W., Bongiovanni, A., Borras, F. E., Bosch, S., Boulanger, C. M., Breakefield, X., Breglio, A. M., Brennan, M. A., Brigstock, D. R., Brisson, A., Broekman, M. L. D., Bromberg, J. F., Bryl-Gorecka, P., Buch, S., Buck, A. H., Burger, D., Busatto, S., Buschmann, D., Bussolati, B., Buzas, E. I., Byrd, J. B., Camussi, G., Carter, D. R. F., Caruso, S., Chamley, L. W., Chang, Y. -T., Chaudhuri, A. D., Chen, C., Chen, S., Cheng, L., Chin, A. R., Clayton, A., Clerici, S. P., Cocks, A., Cocucci, E., Coffey, R. J., Cordeiro-da-Silva, A., Couch, Y., Coumans, F. A. W., Coyle, B., Crescitelli, R., Criado, M. F., D'Souza-Schorey, C., Das, S., de Candia, P., De Santana, E. F., De Wever, O., del Portillo, H. A., Demaret, T., Deville, S., Devitt, A., Dhondt, B., Di Vizio, D., Dieterich, L. C., Dolo, V., Dominguez Rubio, A. P., Dominici, M., Dourado, M. R., Driedonks, T. A. P., Duarte, F. V., Duncan, H. M., Eichenberger, R. M., Ekstrom, K., EL Andaloussi, S., Elie-Caille, C., Erdbrugger, U., Falcon-Perez, J. M., Fatima, F., Fish, J. E., Flores-Bellver, M., Forsonits, A., Frelet-Barrand, A., Fricke, F., Fuhrmann, G., Gabrielsson, S., Gamez-Valero, A., Gardiner, C., Gartner, K., Gaudin, R., Gho, Y. S., Giebel, B., Gilbert, C., Gimona, M., Giusti, I., Goberdhan, D. C. I., Gorgens, A., Gorski, S. M., Greening, D. W., Gross, J. C., Gualerzi, A., Gupta, G. N., Gustafson, D., Handberg, A., Haraszti, R. A., Harrison, P., Hegyesi, H., Hendrix, A., Hill, A. F., Hochberg, F. H., Hoffmann, K. F., Holder, B., Holthofer, H., Hosseinkhani, B., Hu, G., Huang, Y., Huber, V., Hunt, S., Ibrahim, A. G. -E., Ikezu, T., Inal, J. M., Isin, M., Ivanova, A., Jackson, H. K., Jacobsen, S., Jay, S. M., Jayachandran, M., Jenster, G., Jiang, L., Johnson, S. M., Jones, J. C., Jong, A., Jovanovic-Talisman, T., Jung, S., Kalluri, R., Kano, S. -I., Kaur, S., Kawamura, Y., Keller, E. T., Khamari, D., Khomyakova, E., Khvorova, A., Kierulf, P., Kim, K. P., Kislinger, T., Klingeborn, M., Klinke, D. J., Kornek, M., Kosanovic, M. M., Kovacs, A. F., Kramer-Albers, E. -M., Krasemann, S., Krause, M., Kurochkin, I. V., Kusuma, G. D., Kuypers, S., Laitinen, S., Langevin, S. M., Languino, L. R., Lannigan, J., Lasser, C., Laurent, L. C., Lavieu, G., Lazaro-Ibanez, E., Le Lay, S., Lee, M. -S., Lee, Y. X. F., Lemos, D. S., Lenassi, M., Leszczynska, A., Li, I. T. S., Liao, K., Libregts, S. F., Ligeti, E., Lim, R., Lim, S. K., Line, A., Linnemannstons, K., Llorente, A., Lombard, C. A., Lorenowicz, M. J., Lorincz, A. M., Lotvall, J., Lovett, J., Lowry, M. C., Loyer, X., Lu, Q., Lukomska, B., Lunavat, T. R., Maas, S. L. N., Malhi, H., Marcilla, A., Mariani, J., Mariscal, J., Martens-Uzunova, E. S., Martin-Jaular, L., Martinez, M. C., Martins, V. R., Mathieu, M., Mathivanan, S., Maugeri, M., Mcginnis, L. K., Mcvey, M. J., Meckes, D. G., Meehan, K. L., Mertens, I., Minciacchi, V. R., Moller, A., Moller Jorgensen, M., Morales-Kastresana, A., Morhayim, J., Mullier, F., Muraca, M., Musante, L., Mussack, V., Muth, D. C., Myburgh, K. H., Najrana, T., Nawaz, M., Nazarenko, I., Nejsum, P., Neri, C., Neri, T., Nieuwland, R., Nimrichter, L., Nolan, J. P., Nolte-'t Hoen, E. N. M., Noren Hooten, N., O'Driscoll, L., O'Grady, T., O'Loghlen, A., Ochiya, T., Olivier, M., Ortiz, A., Ortiz, L. A., Osteikoetxea, X., Ostegaard, O., Ostrowski, M., Park, J., Pegtel, D. M., Peinado, H., Perut, F., Pfaffl, M. W., Phinney, D. G., Pieters, B. C. H., Pink, R. C., Pisetsky, D. S., Pogge von Strandmann, E., Polakovicova, I., Poon, I. K. H., Powell, B. H., Prada, I., Pulliam, L., Quesenberry, P., Radeghieri, A., Raffai, R. L., Raimondo, S., Rak, J., Ramirez, M. I., Raposo, G., Rayyan, M. S., Regev-Rudzki, N., Ricklefs, F. L., Robbins, P. D., Roberts, D. D., Rodrigues, S. C., Rohde, E., Rome, S., Rouschop, K. M. A., Rughetti, A., Russell, A. E., Saa, P., Sahoo, S., Salas-Huenuleo, E., Sanchez, C., Saugstad, J. A., Saul, M. J., Schiffelers, R. M., Schneider, R., Schoyen, T. H., Scott, A., Shahaj, E., Sharma, S., Shatnyeva, O., Shekari, F., Shelke, G. V., Shetty, A. K., Shiba, K., Siljander, P. R. -M., Silva, A. M., Skowronek, A., Snyder, O. L., Soares, R. P., Sodar, B. W., Soekmadji, C., Sotillo, J., Stahl, P. D., Stoorvogel, W., Stott, S. L., Strasser, E. F., Swift, S., Tahara, H., Tewari, M., Timms, K., Tiwari, S., Tixeira, R., Tkach, M., Toh, W. S., Tomasini, R., Torrecilhas, A. C., Tosar, J. P., Toxavidis, V., Urbanelli, L., Vader, P., van Balkom, B. W. M., van der Grein, S. G., Van Deun, J., van Herwijnen, M. J. C., Van Keuren-Jensen, K., van Niel, G., van Royen, M. E., van Wijnen, A. J., Vasconcelos, M. H., Vechetti, I. J., Veit, T. D., Vella, L. J., Velot, E., Verweij, F. J., Vestad, B., Vinas, J. L., Visnovitz, T., Vukman, K. V., Wahlgren, J., Watson, D. C., Wauben, M. H. M., Weaver, A., Webber, J. P., Weber, V., Wehman, A. M., Weiss, D. J., Welsh, J. A., Wendt, S., Wheelock, A. M., Wiener, Z., Witte, L., Wolfram, J., Xagorari, A., Xander, P., Xu, J., Yan, X., Yanez-Mo, M., Yin, H., Yuana, Y., Zappulli, V., Zarubova, J., Zekas, V., Zhang, J. -Y., Zhao, Z., Zheng, L., Zheutlin, A. R., Zickler, A. M., Zimmermann, P., Zivkovic, A. M., Zocco, D., Zuba-Surma, E. K., dB&C I&I, LS Celbiologie-Algemeen, Celbiologie, Afd Pharmaceutics, Sub General Pharmaceutics, Sub Biomol.Mass Spect. and Proteomics, Afd Pharmacology, CCA - Imaging and biomarkers, Amsterdam Neuroscience - Neuroinfection & -inflammation, and Amsterdam Neuroscience - Cellular & Molecular Mechanisms

Subjects
ectosome, ectosomes, exosomes, extracellular vesicles, guidelines, microparticles, microvesicles, minimal information requirements, reproducibility, rigor, standardization, Histology, Cell Biology, [SDV]Life Sciences [q-bio], size-exclusion, Medicine and Health Sciences, CELL-DERIVED MICROPARTICLES, FIELD-FLOW FRACTIONATION, requirements, circulating, ComputingMilieux_MISCELLANEOUS, Manchester Cancer Research Centre, lcsh:Cytology, PROSTATE-CANCER, microparticle, Cell interaction, microvesicle, chromatography, Position Paper, guideline, Life Sciences & Biomedicine, ectosomes, exosomes, extracellular vesicles, guidelines, microparticles, microvesicles, minimal information requirements, reproducibility, rigor, standardization, MEMBRANE-VESICLES, FETAL BOVINE, Ectosomes, Exosomes, Extracellular Vesicles, Guidelines, Microparticles, Microvesicles, Minimal Information Requirements, Reproducibility, Rigor, Standardization, CIRCULATING MICROPARTICLES, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, ddc:570, exosome, SURFACE-PLASMON RESONANCE, ddc:610, lcsh:QH573-671, Biology, Interacció cel·lular, Science & Technology, ResearchInstitutes_Networks_Beacons/mcrc, Cell membranes, HUMAN URINARY EXOSOMES, PREANALYTICAL PARAMETERS, minimal information requirement, SIZE-EXCLUSION CHROMATOGRAPHY, 1182 Biochemistry, cell and molecular biology, extracellular vesicle, Human medicine, Membranes cel·lulars
Abstract

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.

Published
2018

17. Analysis of the Escherichia coli extracellular vesicle proteome identifies markers of purity and culture conditions.

Author

Hong, Jiwon, Dauros-Singorenko, Priscila, Whitcombe, Alana, Payne, Leo, Blenkiron, Cherie, Phillips, Anthony, and Swift, Simon

Subjects
DENSITY gradient centrifugation, GEL permeation chromatography, FALSE discovery rate, MASS spectrometry, BACTERIAL cultures, MOLECULAR chaperones
Abstract

Bacteria release nano-sized extracellular vesicles (EVs) into the extracellular milieu. Bacterial EVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the EV proteome of two Escherichia coli strains, uropathogenic (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the proteome of crude input EVs prepared by ultracentrifugation alone with EVs purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC). We further compared the proteome of EVs from bacterial cultures that were grown in iron-restricted (R) and iron-supplemented (RF) conditions. Overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle EVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC EVs and proteins involved in glycolytic processes and ligase activity in Nissle EVs. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified EV samples in comparison to their crude input EV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input EVs for both UPEC and Nissle in R condition. Such proteins may have utility as technical markers for assessing the purity of E. coli EV preparations. Several proteins were changed in their abundance depending on the iron availability in the media. Data are available via ProteomeXchange with identifier PXD011345. In summary, we have undertaken a comprehensive characterization of the protein content of E. coli EVs and found evidence of specific EV cargos for physiological activity and conserved protein cargo that may find utility as markers in the future. Abbreviation: DGC: density gradient centrifugation; DTT: 1,4-dithiothreitol; EV: extracellular vesicles; FDR: false discovery rate; GO: Gene Ontology; R: iron-restricted; RF: iron-supplemented; iTRAQ: isobaric tags for relative and absolute quantitation; OMV: outer membrane vesicle; SWATH-MS: sequential window acquisition of all theoretical mass spectra; SEC: size exclusion chromatography. [ABSTRACT FROM AUTHOR]

Published
2019
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18. Circulatory exosomal miRNA following intense exercise is unrelated to muscle and plasma miRNA abundances.

Author

D’Souza, Randall F., Woodhead, Jonathan S. T., Zeng, Nina, Blenkiron, Cherie, Merry, Troy L., Cameron-Smith, David, and Mitchell, Cameron. J.

Abstract

MicroRNAs (miRNAs) regulate gene expression via transcript degradation and translational inhibition, and they may also function as long distance signaling molecules. Circulatory miRNAs are either protein-bound or packaged within vesicles (exosomes). Ten young men (24.6 ± 4.0 yr) underwent a single bout of high-intensity interval cycling exercise. Vastus lateralis biopsies and plasma were collected immediately before and after exercise, as well as 4 h following the exercise bout. Twenty-nine miRNAs previously reported to be regulated by acute exercise were assessed within muscle, venous plasma, and enriched circulatory exosomes via qRT-PCR. Of the 29 targeted miRNAs, 11 were altered in muscle, 8 in plasma, and 9 in the exosome fraction. Although changes in muscle and plasma expression were bidirectional, all regulated exosomal miRNAs increased following exercise. Three miRNAs were altered in all three sample pools (miR-1-3p, -16-5p, and -222-3p), three in both muscle and plasma (miR-21-5p, -134-3p, and -107), three in both muscle and exosomes (miR-23a-3p, -208a-3p, and -150-5p), and three in both plasma and exosomes (miR-486-5p, -126-3p, and -378a-5p). There was a marked discrepancy between the observed alterations between sample pools. A subset of exosomal miRNAs increased in abundance following exercise, suggesting an exercise-induced release of exosomes enriched in specific miRNAs. The uniqueness of the exosomal miRNA response suggests its relevance as a sample pool that needs to be further explored in better understanding biological functions. [ABSTRACT FROM AUTHOR]

Published
2018
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19. Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity.

Author

Dauros Singorenko, Priscila, Chang, Vanessa, Whitcombe, Alana, Simonov, Denis, Hong, Jiwon, Phillips, Anthony, Swift, Simon, and Blenkiron, Cherie

Subjects
VESICLES (Cytology), PROKARYOTES, HETEROGENEITY, DENSITY gradient centrifugation, ENDOTOXINS
Abstract

Prokaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs forMycobacterium smegmatisandEscherichia coliand compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation. [ABSTRACT FROM PUBLISHER]

Published
2017
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20. Specialized cell-free DNA blood collection tubes can be repurposed for extracellular vesicle isolation: a pilot study

Author

Cherie Blenkiron, Yohanes Nursalim, Ben Lawrence, Jessica K. Heatlie, Cristin G. Print, Kate Parker, Vanessa Chang, Sandra Fitzgerald, Heatlie, Jessica, Chang, Vanessa, Fitzgerald, Sandra, Nursalim, Yohanes, Parker, Kate, Lawrence, Ben, Print, Cristin G., and Blenkiron, Cherie

Subjects
Medicine (miscellaneous), Nanoparticle tracking analysis, Pilot Projects, exosomes, General Biochemistry, Genetics and Molecular Biology, cancer biomarker, Blood cell, cell-free DNA, Extracellular Vesicles, medicine, Humans, Bicinchoninic acid assay, Centrifugation, Edetic Acid, Chemistry, Liquid Biopsy, Cell Biology, General Medicine, Extracellular vesicle, Molecular biology, Microvesicles, medicine.anatomical_structure, Cell-free fetal DNA, Blood Collection Tube, extracellular vesicles, Cell-Free Nucleic Acids
Abstract

Background: Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Methods: Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. Results: MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Conclusions: Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells. Refereed/Peer-reviewed

Published
2020

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