High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types

A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy

We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming

Cell-derived extracellular vesicles can be used as a biomarker reservoir for glioblastoma tumor subtyping

Glioblastoma (GBM) is one of the most aggressive solid tumors for which treatment options and biomarkers are limited. Small extracellular vesicles (sEVs) produced by both GBM and stromal cells are central in the inter-cellular communication that is taking place in the tumor bulk. As tumor sEVs are accessible in biofluids, recent reports have suggested that sEVs contain valuable biomarkers for GBM patient diagnosis and follow-up. The aim of the current study was to describe the protein content of sEVs produced by different GBM cell lines and patient-derived stem cells. Our results reveal that the content of the sEVs mirrors the phenotypic signature of the respective GBM cells, leading to the description of potential informative sEV-associated biomarkers for GBM subtyping, such as CD44. Overall, these data could assist future GBM in vitro studies and provide insights for the development of new diagnostic and therapeutic methods as well as personalized treatment strategies

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery

Thermally Responsive Amphiphilic Conetworks and Gels Based on Poly(N‑isopropylacrylamide) and Polyisobutylene

Novel amphiphilic conetworks (APCN) consisting of thermoresponsive poly(N-isoproplyacrylamide) (PNiPAAm) cross-linked by hydrophobic methacrylate-telechelic polyisobutylene (MA-PIB-MA) were successfully synthesized in a broad composition range. The resulting PNiPAAm-l-PIB conetworks (“l” stands for “linked by”) were obtained by radical copolymerization of NiPAAm with MA-PIB-MA in tetrahydrofuran, a cosolvent for all the components. Low amounts of extractables substantiated efficient network formation. The composition dependent two glass transition temperatures (Tg) by DSC analysis indicate microphase separation of the cross-linked components without mixed phases. It was found that the PNiPAAm-l-PIB conetworks are uniformly swellable in both water and n-hexane; i.e., these new materials behave either as hydrogels or as hydrophobic gels in aqueous or nonpolar media, respectively. The uniform swelling in both polar and nonpolar solutes indicates cocontinuous (bicontinuous) phase morphology. The equilibrium swelling degrees (R) depend on composition, that is, the higher the PIB content, the lower the R in water and the higher in n-hexane. The PNiPAAm phase keeps its thermoresponsive behavior in the conetworks as shown by significant decrease of the swelling degree in water between 20 and 35 °C. The lower critical solubility temperature (LCST) values determined by DSC are found to decrease from 34.1 °C (for the pure PNiPAAm homopolymer) to the range of 25–28 °C in the conetworks, and the extent of the LCST decrease is proportional with the PIB content. Deswelling-swelling, i.e., heating–cooling, cycle indicates insignificant hysteresis in these new thermoresponsive materials. This indicates that PNiPAAm-l-PIB conetworks with predetermined and thermoresponsive swelling behavior can be designed and utilized in several advanced applications on the basis of results obtained in the course of this study

Effectiveness and safety of opicapone in Parkinson's disease patients with motor fluctuations: The OPTIPARK open-label study

BACKGROUND: The efficacy and safety of opicapone, a once-daily catechol-O-methyltransferase inhibitor, have been established in two large randomized, placebo-controlled, multinational pivotal trials. Still, clinical evidence from routine practice is needed to complement the data from the pivotal trials. METHODS: OPTIPARK (NCT02847442) was a prospective, open-label, single-arm trial conducted in Germany and the UK under clinical practice conditions. Patients with Parkinson’s disease and motor fluctuations were treated with opicapone 50 mg for 3 (Germany) or 6 (UK) months in addition to their current levodopa and other antiparkinsonian treatments. The primary endpoint was the Clinician’s Global Impression of Change (CGI-C) after 3 months. Secondary assessments included Patient Global Impressions of Change (PGI-C), the Unified Parkinson’s Disease Rating Scale (UPDRS), Parkinson’s Disease Questionnaire (PDQ-8), and the Non-Motor Symptoms Scale (NMSS). Safety assessments included evaluation of treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs). RESULTS: Of the 506 patients enrolled, 495 (97.8%) took at least one dose of opicapone. Of these, 393 (79.4%) patients completed 3 months of treatment. Overall, 71.3 and 76.9% of patients experienced any improvement on CGI-C and PGI-C after 3 months, respectively (full analysis set). At 6 months, for UK subgroup only (n = 95), 85.3% of patients were judged by investigators as improved since commencing treatment. UPDRS scores at 3 months showed statistically significant improvements in activities of daily living during OFF (mean ± SD change from baseline: − 3.0 ± 4.6, p < 0.0001) and motor scores during ON (− 4.6 ± 8.1, p < 0.0001). The mean ± SD improvements of − 3.4 ± 12.8 points for PDQ-8 and -6.8 ± 19.7 points for NMSS were statistically significant versus baseline (both p < 0.0001). Most of TEAEs (94.8% of events) were of mild or moderate intensity. TEAEs considered to be at least possibly related to opicapone were reported for 45.1% of patients, with dyskinesia (11.5%) and dry mouth (6.5%) being the most frequently reported. Serious TEAEs considered at least possibly related to opicapone were reported for 1.4% of patients. CONCLUSIONS: Opicapone 50 mg was effective and generally well-tolerated in PD patients with motor fluctuations treated in clinical practice. TRIAL REGISTRATION: Registered in July 2016 at clinicaltrials.gov (NCT02847442)

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

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

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

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

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