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  • 1
    Online Resource
    Online Resource
    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
    Wiley ; 2018
    In:  Journal of Extracellular Vesicles Vol. 7, No. 1 ( 2018-12)
    Details
    In: Journal of Extracellular Vesicles, Wiley, Vol. 7, No. 1 ( 2018-12)
    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.
    Type of Medium: Online Resource
    ISSN: 2001-3078 , 2001-3078
    URL: Article
    DOI: 10.1080/20013078.2018.1535750
    Language: English
    Publisher: Wiley
    Publication Date: 2018
    detail.hit.zdb_id: 2683797-3
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  • 2
    Online Resource
    Online Resource
    Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper
    Wiley ; 2017
    In:  Journal of Extracellular Vesicles Vol. 6, No. 1 ( 2017-12)
    Details
    In: Journal of Extracellular Vesicles, Wiley, Vol. 6, No. 1 ( 2017-12)
    Abstract: The release of RNA‐containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA‐containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV‐RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA‐encoded messages affect signalling processes in EV‐targeted cells. Moreover, EV‐RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV‐RNA in vivo . These topics were discussed at the 2015 EV‐RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(‐RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV‐RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV‐RNA and calls for caution in interpretation of the obtained data.
    Type of Medium: Online Resource
    ISSN: 2001-3078 , 2001-3078
    URL: Article
    DOI: 10.1080/20013078.2017.1286095
    Language: English
    Publisher: Wiley
    Publication Date: 2017
    detail.hit.zdb_id: 2683797-3
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  • 3
    Online Resource
    Online Resource
    DataSheet_1.docx
    Frontiers Media SA ; 2022
    In:  Frontiers in Oncology Vol. 12 ( 2022-3-1)
    Details
    In: Frontiers in Oncology, Frontiers Media SA, Vol. 12 ( 2022-3-1)
    Abstract: Increased levels of the chaperone protein GRP78 have been implicated in poorer outcomes of cancer therapy. We have therefore explored the functional connection between the expression of GRP78 and the development of radioresistance and metastatic behavior in HNSCC. Material and Methods The association between gene expression of GRP78 and survival in HNSCC patients was examined using the TCGA database. The influence of ionizing radiation on the GRP78 levels in HNSCC cell lines, their secreted extracellular vesicles (EV) and non-irradiated EV-recipient cells was investigated by Western Blot and FACS. The consequences of chemical inhibition or experimental overexpression of GRP78 on radioresistance and migration of HNSCC cells were analyzed by clonogenic survival and gap closure assays. Results Elevated levels of GRP78 RNA in HNSCC correlated with poorer overall survival. Radiation increased GRP78 protein expression on the surface of HNSCC cell lines. Experimental overexpression of GRP78 increased both radioresistance and migratory potential. Chemical inhibition of GRP78 impaired cell migration. EVs were identified as a potential source of increased GRP78 content as elevated levels of surface GRP78 were found in EVs released by irradiated cells. These vesicles transferred GRP78 to non-irradiated recipient cells during co-cultivation. Conclusions We have identified the chaperone protein GRP78 as a potential driver of increased radioresistance and motility in HNSCC. The uptake of GRP78-rich EVs originating from irradiated cells may contribute to a poorer prognosis through bystander effects mediated by the transfer of GRP78 to non-irradiated cells. Therefore, we consider the chaperone protein GRP78 to be an attractive target for improving radiotherapy strategies.
    Type of Medium: Online Resource
    ISSN: 2234-943X
    URL: Article
    URL: Article
    DOI: 10.3389/fonc.2022.842418
    DOI: 10.3389/fonc.2022.842418.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2649216-7
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  • 4
    Online Resource
    Online Resource
    Radiation Exposure of Peripheral Mononuclear Blood Cells Alters the Composition and Function of Secreted Extracellular Vesicles
    MDPI AG ; 2020
    In:  International Journal of Molecular Sciences Vol. 21, No. 7 ( 2020-03-27), p. 2336-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 21, No. 7 ( 2020-03-27), p. 2336-
    Abstract: Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms21072336
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by Next‐Generation Sequencing
    Wiley ; 2019
    In:  Journal of Extracellular Vesicles Vol. 8, No. 1 ( 2019-12)
    Details
    In: Journal of Extracellular Vesicles, Wiley, Vol. 8, No. 1 ( 2019-12)
    Type of Medium: Online Resource
    ISSN: 2001-3078 , 2001-3078
    URL: Article
    DOI: 10.1080/20013078.2019.1581487
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2683797-3
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  • 6
    Online Resource
    Online Resource
    Table2.xlsx
    Frontiers Media SA ; 2024
    In:  Frontiers in Cardiovascular Medicine Vol. 11 ( 2024-4-25)
    Details
    In: Frontiers in Cardiovascular Medicine, Frontiers Media SA, Vol. 11 ( 2024-4-25)
    Abstract: Atherosclerosis is a widespread disorder of the cardiovascular system. The early detection of plaques by circulating biomarkers is highly clinically relevant to prevent the occurrence of major complications such as stroke or heart attacks. It is known that extracellular vesicles (EVs) are important in intercellular communication in atherosclerotic disorders and carry many components of their cells of origin, including microRNAs (miRNAs). In this study, we test the assumption that miRNAs present in material acquired from plaques in patients undergoing surgery for atherosclerotic carotid artery stenosis are also expressed in circulating EVs obtained from the identical patients. This would allow the adoption of a liquid biopsy approach for the detection of plaques. Methods We studied 22 surgical patients with atherosclerotic carotid arterial stenosis and 28 healthy controls. EVs were isolated from serum by precipitation. miRNA expression profiles of serum-derived EVs were obtained by small RNA sequencing and in plaque material simultaneously acquired from patients. A comparative analysis was performed to identify circulating atherosclerosis-associated miRNAs that are also detectable in plaques. Results Seven miRNAs were found to be differentially regulated in patient serum compared with the serum of healthy controls. Of these, miR-193b-5p, miR-193a-5p, and miR-125a-3p were significantly upregulated in patients compared with that in healthy controls and present in both, circulating EVs and plaque material. An overrepresentation analysis of experimentally validated mRNA targets revealed an increased regulation of inflammation and vascular growth factors, key players in atherosclerosis and plaque formation. Conclusion Our findings suggest that circulating EVs reflect plaque development in patients with symptomatic carotid artery stenosis, which can serve as biomarker candidates for detecting the presence of atherosclerotic plaques.
    Type of Medium: Online Resource
    ISSN: 2297-055X
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fcvm.2024.1307832
    DOI: 10.3389/fcvm.2024.1307832.s001
    DOI: 10.3389/fcvm.2024.1307832.s002
    DOI: 10.3389/fcvm.2024.1307832.s003
    DOI: 10.3389/fcvm.2024.1307832.s004
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2024
    detail.hit.zdb_id: 2781496-8
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  • 7
    Online Resource
    Online Resource
    Transcriptomic profiling of cell‐free and vesicular microRNAs from matched arterial and venous sera
    Wiley ; 2019
    In:  Journal of Extracellular Vesicles Vol. 8, No. 1 ( 2019-12)
    Details
    In: Journal of Extracellular Vesicles, Wiley, Vol. 8, No. 1 ( 2019-12)
    Abstract: Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellular communication. Biomarker studies addressing disorders such as cardiovascular diseases often focus on circulating microRNAs (miRNAs) and may, depending on the type of disease and clinic routine, utilise patient specimens sampled from arterial or venous blood vessels. Thus, it is essential to test whether circulating miRNA profiles depend on the respective sampling site. We assessed potential differences in arterial and venous cell‐free miRNA profiles in a cohort of 20 patients scheduled for cardiac surgery. Prior to surgery, blood was simultaneously sampled from the radial artery and the internal jugular vein. After precipitating crude EVs, we performed small RNA Sequencing, which failed to detect significantly regulated miRNAs using stringent filtering criteria for differential expression analysis. Filtering with less strict criteria, we detected four miRNAs slightly upregulated in arterial samples, one of which could be validated by reverse transcription real‐time PCR. The applicability of these findings to purified arterial and venous EVs was subsequently tested in a subset of the initial study population. While an additional clean‐up step using size‐exclusion chromatography seemed to reduce overall miRNA yield compared to crude EV samples, no miRNAs with differential arteriovenous expression were detected. Unsupervised clustering approaches were unable to correctly classify samples drawn from arteries or veins based on miRNAs in either crude or purified preparations. Particle characterisation of crude preparations as well as characterisation of EV markers in purified EVs resulted in highly similar characteristics for arterial and venous samples. With the exception of specific pathologies (e.g. severe pulmonary disorders), arterial versus venous blood sampling should therefore not represent a likely confounder when studying differentially expressed circulating miRNAs. The use of either arterial or venous serum EV samples should result in highly similar data on miRNA expression profiles for the majority of biomarker studies. Abbreviations ACE inhibitors: Angiotensin‐converting‐enzyme inhibitors; ApoA1: Apolipoprotein A1; CNX: Calnexin; Cv: Coefficient of variation; cDNA: Complementary DNA; CABG: Coronary artery bypass graft; DGE: Differential gene expression; DPBS: Dulbecco's Phosphate Buffered Saline; EVs: Extracellular vesicles; log2FC: Log2 fold change; baseMean: Mean miRNA expression; miRNA: MicroRNA; NTA: Nanoparticle Tracking Analysis; NGS: Next‐Generation Sequencing; RT‐qPCR: Reverse transcription quantitative real‐time PCR; rRNA: Ribosomal RNA; RT: Room temperature; SEC: Size‐exclusion chromatography; snoRNA: Small nucleolar RNA; snRNA: Small nuclear RNA; small RNA‐Seq: Small RNA Sequencing; SD: Standard deviation; tRNA: Transfer RNA; TEM: Transmission electron microscopy; UA: Uranyl acetate.
    Type of Medium: Online Resource
    ISSN: 2001-3078 , 2001-3078
    URL: Article
    DOI: 10.1080/20013078.2019.1670935
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2683797-3
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  • 8
    Online Resource
    Online Resource
    Diagnostic potential of circulating cell‐free microRNAs for community‐acquired pneumonia and pneumonia‐related sepsis
    Wiley ; 2020
    In:  Journal of Cellular and Molecular Medicine Vol. 24, No. 20 ( 2020-10), p. 12054-12064
    Details
    In: Journal of Cellular and Molecular Medicine, Wiley, Vol. 24, No. 20 ( 2020-10), p. 12054-12064
    Abstract: Cell‐free microRNAs (miRNAs) are transferred in disease state including inflammatory lung diseases and are often packed into extracellular vesicles (EVs). To assess their suitability as biomarkers for community‐acquired pneumonia (CAP) and severe secondary complications such as sepsis, we studied patients with CAP (n = 30), sepsis (n = 65) and healthy volunteers (n = 47) subdivided into a training (n = 67) and a validation (n = 75) cohort. After precipitating crude EVs from sera, associated small RNA was profiled by next‐generation sequencing (NGS) and evaluated in multivariate analyses. A subset of the thereby identified biomarker candidates was validated both technically and additionally by reverse transcription quantitative real‐time PCR (RT‐qPCR). Differential gene expression (DGE) analysis revealed 29 differentially expressed miRNAs in CAP patients when compared to volunteers, and 25 miRNAs in patients with CAP, compared to those with sepsis. Sparse partial‐least discriminant analysis separated groups based on 12 miRNAs. Three miRNAs proved as a significant biomarker signature. While expression levels of miR‐1246 showed significant changes with an increase in overall disease severity from volunteers to CAP and to sepsis, miR‐193a‐5p and miR‐542‐3p differentiated patients with an infectious disease (CAP or sepsis) from volunteers. Cell‐free miRNAs are potentially novel biomarkers for CAP and may help to identify patients at risk for progress to sepsis, facilitating early intervention and treatment.
    Type of Medium: Online Resource
    ISSN: 1582-1838 , 1582-4934
    URL: Issue
    URL: Article
    DOI: 10.1111/jcmm.v24.20
    DOI: 10.1111/jcmm.15837
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 2076114-4
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  • 9
    Online Resource
    Online Resource
    The need for transparency and good practices in the qPCR literature
    Springer Science and Business Media LLC ; 2013
    In:  Nature Methods Vol. 10, No. 11 ( 2013-11), p. 1063-1067
    Details
    In: Nature Methods, Springer Science and Business Media LLC, Vol. 10, No. 11 ( 2013-11), p. 1063-1067
    Type of Medium: Online Resource
    ISSN: 1548-7091 , 1548-7105
    URL: Article
    DOI: 10.1038/nmeth.2697
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2013
    detail.hit.zdb_id: 2163081-1
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing
    Wiley ; 2018
    In:  Journal of Extracellular Vesicles Vol. 7, No. 1 ( 2018-12)
    Details
    In: Journal of Extracellular Vesicles, Wiley, Vol. 7, No. 1 ( 2018-12)
    Abstract: Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery‐focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method‐specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA‐based biomarker discovery, methods based on size‐exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV‐miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.
    Type of Medium: Online Resource
    ISSN: 2001-3078 , 2001-3078
    URL: Article
    DOI: 10.1080/20013078.2018.1481321
    Language: English
    Publisher: Wiley
    Publication Date: 2018
    detail.hit.zdb_id: 2683797-3
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