Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.Peer reviewe

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

May Measurement Month 2018: a pragmatic global screening campaign to raise awareness of blood pressure by the International Society of Hypertension

Aims Raised blood pressure (BP) is the biggest contributor to mortality and disease burden worldwide and fewer than half of those with hypertension are aware of it. May Measurement Month (MMM) is a global campaign set up in 2017, to raise awareness of high BP and as a pragmatic solution to a lack of formal screening worldwide. The 2018 campaign was expanded, aiming to include more participants and countries. Methods and results Eighty-nine countries participated in MMM 2018. Volunteers (≥18 years) were recruited through opportunistic sampling at a variety of screening sites. Each participant had three BP measurements and completed a questionnaire on demographic, lifestyle, and environmental factors. Hypertension was defined as a systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, or taking antihypertensive medication. In total, 74.9% of screenees provided three BP readings. Multiple imputation using chained equations was used to impute missing readings. 1 504 963 individuals (mean age 45.3 years; 52.4% female) were screened. After multiple imputation, 502 079 (33.4%) individuals had hypertension, of whom 59.5% were aware of their diagnosis and 55.3% were taking antihypertensive medication. Of those on medication, 60.0% were controlled and of all hypertensives, 33.2% were controlled. We detected 224 285 individuals with untreated hypertension and 111 214 individuals with inadequately treated (systolic BP ≥ 140 mmHg or diastolic BP ≥ 90 mmHg) hypertension. Conclusion May Measurement Month expanded significantly compared with 2017, including more participants in more countries. The campaign identified over 335 000 adults with untreated or inadequately treated hypertension. In the absence of systematic screening programmes, MMM was effective at raising awareness at least among these individuals at risk

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

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

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Purification and characterisation of extracellular vesicles released from pluripotent stem cells

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into biological fluids such as blood, plasma, saliva and urine. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. Hence, EVs are postulated to be an alternative source of cell-cell communication in both normal physiological systems and pathological situations. Recently, EVs from mesenchymal stem cells have been shown to promote regeneration of injured cells. Induced pluripotent stem cells (iPSCs) are a relative newer type of stem cells that is emerging as useful tools for re-modeling diseases. However, knowledge about EVs from iPSCs is relatively sparse. In initial experiments, we successfully purified EVs from mouse iPSCs using the differential ultracentrifugation (UC) method. However, we noticed a discrepancy between total particle counts and expression of EV markers, across different EV batches. One crucial prerequisite for EV research is the availability of a standardized workflow for collection and purification of EVs from biological sources. Increasing evidence from recent studies has suggested that the original UC method is limited by several shortcomings such as low EV yield, purity and altered biophysical properties. Hence, this has led to a new wave in the development of alternative EV purification strategies. In this thesis, we start with a systematic comparison study between UC and an alternative purification protocol, size-exclusion liquid chromatography (LC) of EVs from serum-free conditions. We found that LC is better than UC in terms of overall EV yields, purity and vesicle integrity. Subsequently, we demonstrate that LC allowed for the derivation of pure EVs from complex media sources used for growing stem cells like iPSCs, which was previously impossible with UC. Lastly, we describe novel data on the characterization of EVs from pluripotent stem cell sources and discuss the possible roles of these EVs in stem cell biology.</p

Purification and characterisation of extracellular vesicles released from pluripotent stem cells

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into biological fluids such as blood, plasma, saliva and urine. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. Hence, EVs are postulated to be an alternative source of cell-cell communication in both normal physiological systems and pathological situations. Recently, EVs from mesenchymal stem cells have been shown to promote regeneration of injured cells. Induced pluripotent stem cells (iPSCs) are a relative newer type of stem cells that is emerging as useful tools for re-modeling diseases. However, knowledge about EVs from iPSCs is relatively sparse. In initial experiments, we successfully purified EVs from mouse iPSCs using the differential ultracentrifugation (UC) method. However, we noticed a discrepancy between total particle counts and expression of EV markers, across different EV batches. One crucial prerequisite for EV research is the availability of a standardized workflow for collection and purification of EVs from biological sources. Increasing evidence from recent studies has suggested that the original UC method is limited by several shortcomings such as low EV yield, purity and altered biophysical properties. Hence, this has led to a new wave in the development of alternative EV purification strategies. In this thesis, we start with a systematic comparison study between UC and an alternative purification protocol, size-exclusion liquid chromatography (LC) of EVs from serum-free conditions. We found that LC is better than UC in terms of overall EV yields, purity and vesicle integrity. Subsequently, we demonstrate that LC allowed for the derivation of pure EVs from complex media sources used for growing stem cells like iPSCs, which was previously impossible with UC. Lastly, we describe novel data on the characterization of EVs from pluripotent stem cell sources and discuss the possible roles of these EVs in stem cell biology.</p

DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

10.1038/s41467-021-22638-7NATURE COMMUNICATIONS12