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.

Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.

A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (~48.5%) and 100-300 nm (~51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV.

e Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ؎ 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ؎ 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ؎ 0.03 g of CaCO 3 g of Ca(CHOOH) 2 ؊1 calcium carbonate precipitate yield was obtained when a culture of 10 9 cells ml ؊1 and 5 g of calcium formate liter ؊1 were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry. Microbially induced carbonate precipitation (MICP) is a wellknown process and has been extensively described in the past (1-3). In short, MICP produces carbonate minerals, e.g., calcium carbonate, as a result of alterations in environmental conditions. In nature, examples of MICP include calcite formation in soils (4), limestone caves (5), seas (6), and soda lakes (7). Four different key parameters that govern microbially induced calcium carbonate precipitation are the: (i) concentration of nonprecipitated calcium, (ii) concentration of the total inorganic carbon, (iii) pH, and (iv) availability of nucleation sites for calcium carbonate crystal formation (3). Among the four parameters, bacterial activities mainly influence the pH of the environment (1).MICP is the basis for several biotechnological applications in the construction sector (for a review, see reference 1 and references therein). These include the use of calcium carbonate precipitate to protect concrete surface against the ingress of deleterious substances (e.g., chloride ions) (8) or to heal cracks in aging concrete (9, 10). Among the bacterial activities that can induce calcium carbonate precipitation, urea degradation by heterotrophic bacteria is typically used for applications on building materials. In biogenic urea degradation, urea is transformed to ammonia and carbonate ions to initiate precipitation (11). Bacillus spp. (e.g., B. sphaericus) is the most commonly applied urea degrader for MICP in the construction sector due to several advantages such as the high initial urea degradation rate by the strain and a highly negative potential ...