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Whole cell responses involve multiple subcellular processes (SCPs). To understand how balance between SCPs controls the dynamics of whole cell responses we studied neurite outgrowth in rat primary cortical neurons in culture. We used a combination of dynamical models and experiments to understand the conditions that permitted growth at a specified velocity and when aberrant growth could lead to the formation of dystrophic bulbs. We hypothesized that dystrophic bulb formation is due to quantitative imbalances between SCPs. Simulations predict redundancies between lower level sibling SCPs within each type of high level SCP. In contrast, higher level SCPs, such as vesicle transport and exocytosis or microtubule growth characteristic of each type need to be strictly coordinated with each other and imbalances result in stalling of neurite outgrowth. From these simulations, we predicted the effect of changing the activities of SCPs involved in vesicle exocytosis or microtubule growth could lead to formation of dystrophic bulbs. siRNA ablation experiments verified these predictions. We conclude that whole cell dynamics requires balance between the higher-level SCPs involved and imbalances can terminate whole cell responses such as neurite outgrowth. [ABSTRACT FROM AUTHOR]

We study the crucial role of membrane fluctuations in maintaining a narrow gap between a fluid membrane tube and an enclosed solid particle. Solvent flows can occur in this gap, hence giving rise to a finite particle mobility along the tube. While our study has relevance for how cells are able to transport large organelles or other cargo along connecting membrane tubes, known as tunneling nanotubes, our calculations are also framed so that they can be tested by a specific in vitro experiment: A tubular membrane tether can be pulled from a membrane reservoir, such as an aspirated Giant Unilamellar Vesicle (GUV), e.g. using a conjugated bead that binds to the membrane and is held in a laser trap. We compute the subsequent mobility of colloidal particles trapped in the tube, focusing on the case when the particle is large compared to the equilibrium tube radius. We predict that the particle mobility should scale as ∼ σ−2/3, with σ the membrane tension. [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV) microRNAs are of major interest as potential diagnostic biomarkers in all cancer types. This study aims to identify miRNA profiles of shed microvesicles (sMVs) and exosomes (Exos) secreted from the isogenic colorectal cancer (CRC) cell lines SW480 and SW620 and evaluate their ability to predict CRC. Deep sequencing of miRNAs in parental cell lysates (CLs) and highly-purified sMVs and Exos was performed. We focused on miRNAs enriched in EVs and dysregulated miRNAs in metastatic cells (SW620) relative to primary cancer cells (SW480). We investigated the ability of EV miRNA signatures to predict CRC tumours using 594 tumours (representing different pathological stages) and 11 normal samples obtained from TCGA. In SW480 and SW620 cells we identified 345 miRNAs, of which 61 and 73 were upregulated and downregulated in SW620-CLs compared to SW480-CLs, respectively. Selective distribution of cellular miRNAs into EVs results in distinct miRNA signatures for sMVs and Exos in each cell line. Cross cell line comparisons of EV miRNA profiles reveal a subset of miRNAs critical in CRC progression from primary carcinoma to metastasis. Many miRNAs non-detectable (<5 TPM) in CLs were significantly enriched (>1000 TPM) in secreted EVs. Strikingly, miR-7641 which is non-detectable in SW480-CL but upregulated in SW620-CL is highly enriched in EVs secreted from both cell lines. Pearson correlation analysis demonstrated that EV miRNA profiles can be used to predict CRC tumours with ~96% accuracy. Our findings suggest that EV miRNA profiles from CRC cell lines may allow prediction of CRC tumours, and that miR-7641 may serve as an attractive candidate for the specific, non-invasive diagnosis and prognosis of CRC. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are important mediators of intercellular communication and have been implicated in myriad physiologic and pathologic processes within the hematopoietic system. Numerous factors influence the ability of EVs to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. In order to explore the effects of daily rhythms on EV-mediated intercellular communication, we used a well-established model of lung-derived EV modulation of the marrow cell transcriptome. In this model, co-culture of whole bone marrow cells (WBM) with lung-derived EVs induces expression of pulmonary specific mRNAs in the target WBM. To determine if daily rhythms play a role in this phenotype modulation, C57BL/6 mice were entrained in 12-hour light/12-hour dark boxes. Lungs harvested at discrete time-points throughout the 24-hour cycle were co-cultured across a cell-impermeable membrane with murine WBM. Alternatively, WBM harvested at discrete time-points was co-cultured with lung-derived EVs. Target WBM was collected 24hrs after co-culture and analyzed for the presence of pulmonary specific mRNA levels by RT-PCR. In both cases, there were clear time-dependent variations in the patterns of pulmonary specific mRNA levels when either the daily time-point of the lung donor or the daily time-point of the recipient marrow cells was altered. In general, WBM had peak pulmonary-specific mRNA levels when exposed to lung harvested at Zeitgeber time (ZT) 4 and ZT 16 (ZT 0 defined as the time of lights on, ZT 12 defined as the time of lights off), and was most susceptible to lung-derived EV modulation when target marrow itself was harvested at ZT 8- ZT 12. We found increased uptake of EVs when the time-point of the receptor WBM was between ZT 20 -ZT 24, suggesting that the time of day-dependent changes in transcriptome modulation by the EVs were not due simply to differential EV uptake. Based on these data, we conclude that circadian rhythms can modulate EV-mediated intercellular communication. [ABSTRACT FROM AUTHOR]

Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield “pure” vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium. [ABSTRACT FROM AUTHOR]

The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60–140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication and as possible therapeutic agents in inflammation-mediated demyelinating diseases, including multiple sclerosis (MS). In the present study, we investigated whether intravenously administered EVs derived from mesenchymal stem cells (MSCs) from human adipose tissue might mediate recovery in Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a progressive model of MS. SJL/J mice were subjected to EV treatment once the disease was established. We found that intravenous EV administration improved motor deficits, reduced brain atrophy, increased cell proliferation in the subventricular zone and decreased inflammatory infiltrates in the spinal cord in mice infected with TMEV. EV treatment was also capable of modulating neuroinflammation, given glial fibrillary acidic protein and Iba-1 staining were reduced in the brain, whereas myelin protein expression was increased. Changes in the morphology of microglial cells in the spinal cord suggest that EVs also modulate the activation state of microglia. The clear reduction in plasma cytokine levels, mainly in the Th1 and Th17 phenotypes, in TMEV mice treated with EVs confirms the immunomodulatory ability of intravenous EVs. According to our results, EV administration attenuates motor deficits through immunomodulatory actions, diminishing brain atrophy and promoting remyelination. Further studies are necessary to establish EV delivery as a possible therapy for the neurodegenerative phase of MS. [ABSTRACT FROM AUTHOR]

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells. [ABSTRACT FROM AUTHOR]

The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst. [ABSTRACT FROM AUTHOR]

Protocell multilevel selection models have been proposed to study the evolutionary dynamics of vesicles encapsulating a set of replicating, competing and mutating sequences. The frequency of the different sequence types determines protocell survival through a fitness function. One of the defining features of these models is the genetic load generated when the protocell divides and its sequences are assorted between the offspring vesicles. However, these stochastic assortment effects disappear when the redundancy of each sequence type is sufficiently high. The fitness dependence of the vesicle with its sequence content is usually defined without considering a realistic account on how the lower level dynamics would specify the protocell fitness. Here, we present a protocell model with a fitness function determined by the output flux of a simple metabolic network with the aim of understanding how the evolution of both kinetic and topological features of metabolism would have been constrained by the particularities of the protocell evolutionary dynamics. In our model, the sequences inside the vesicle are both the carriers of information and Michaelis-Menten catalysts exhibiting saturation. We found that the saturation of the catalysts controlling the metabolic fluxes, achievable by modifying the kinetic or stoichiometric parameters, provides a mechanism to ameliorate the assortment load by increasing the redundancy of the catalytic sequences required to achieve the maximum flux. Regarding the network architecture, we conclude that combinations of parallel network motifs and bimolecular catalysts are a robust way to increase the complexity of the metabolism enclosed by the protocell. [ABSTRACT FROM AUTHOR]

At parallel fibre terminals in the cerebellar cortex, glutamate released outside of the active zone can activate AMPA receptors on juxtaposed Bergmann glial cell processes. This process is termed “ectopic” release, and allows for directed transmission to astroglial cells that is functionally independent of synaptic transmission to postsynaptic Purkinje neurons. The location of ectopic sites in presynaptic terminals is uncertain. Functional evidence suggests that stimulation of parallel fibres at 1 Hz exhausts ectopic transmission due to a failure to rapidly recycle vesicles to the ectopic pool, and so would predict a loss of vesicles in the near vicinity of extrasynaptic glial processes. In this study we used this stimulation protocol to investigate whether the distribution of vesicles within the presynaptic terminal is altered after suppression of ectopic release. Stimulation at 1 Hz had only a minor impact on the distribution of vesicles in presynaptic terminals when analysed with electron microscopy. Vesicle number and terminal size were unaffected by 1 Hz stimulation, but the relative abundance of vesicles in close proximity to the active zone was marginally reduced. In contrast, the fraction of vesicles facing glial membranes was unchanged after suppression of ectopic transmission. 1 Hz stimulation also resulted in a small but statistically-significant increase in the distance between glial membrane and presynaptic terminal, suggesting withdrawal of glial membranes from synapses is detectable in ultrastructural anatomy within minutes. These results raise doubts about the location of ectopic release sites, but indicate that neuron-glial association varies on a dynamic time scale. [ABSTRACT FROM AUTHOR]

Exosomes are nanovesicles involved in intercellular communications. They are released by a variety of cell types; however, their presence in the inner ear has not been described in the literature. The aims of this study were to determine if exosomes are present in the inner ear and, if present, characterize the changes in their protein content in response to ototoxic stress. In this laboratory investigation, inner ear explants of 5-day-old Wistar rats were cultured and treated with either cisplatin or gentamicin. Hair cell damage was assessed by confocal microscopy. Exosomes were isolated using ExoQuick, serial centrifugation, and mini-column methods. Confirmation and characterization of exosomes was carried out using transmission electron microscopy (TEM), ZetaView, BCA protein analysis, and proteomics. Vesicles with a typical size distribution for exosomes were observed using TEM and ZetaView. Proteomic analysis detected typical exosome markers and markers for the organ of Corti. There was a statistically significant reduction in the exosome protein level and number of particles per cubic centimeter when the samples were exposed to ototoxic stress. Proteomic analysis also detected clear differences in protein expression when ototoxic medications were introduced. Significant changes in the proteomes of the exosomes were previously described in the context of hearing loss and ototoxic treatment. This is the first report describing exosomes derived from the inner ear. These findings may present an opportunity to conduct further studies with the hope of using exosomes as a biomarker to monitor inner ear function in the future. [ABSTRACT FROM AUTHOR]

Cancer cells secrete many exosomes, which facilitate metastasis and the later growth of cancer. For early cancer diagnosis, the detection of exosomes is a crucial step. Exosomes exist in biological fluid, such as blood, which contains various proteins. It is necessary to remove the proteins in the biological fluid to avoid test interference. This paper presented a novel method for exosome isolation using Fe3O4 magnetic nanoparticles (MNPs), which were synthesized using the chemical co-precipitation method and then coated with polyethylene glycol (PEG). The experimental results showed that the diameter of the PEG-coated Fe3O4 nanoparticles was about 20 nm, while an agglomerate of MNPs reached hundreds of nanometers in size. In the protein removal experiments, fetal bovine serum (FBS) was adopted as the analyte for bioassays of exosome purification. PEG-coated Fe3O4 MNPs reduced the protein concentration in FBS to 39.89% of the original solution. By observing a particle size distribution of 30~200 nm (the size range of various exosomes), the exosome concentrations were kept the same before and after purification. In the gel electrophoresis experiments, the bands of CD63 (~53 kDa) and CD9 (~22 kDa) revealed that exosomes existed in FBS as well as in the purified solution. However, the bands of the serum albumins (~66 kDa) and the various immunoglobulins (around 160 ~ 188 kDa) in the purified solution’s lane explained that most proteins in FBS were removed by PEG-coated Fe3O4 MNPs. When purifying exosomes from serum, protein removal is critical for further exosome investigation. The proposed technique provides a simple and effective method to remove proteins in the serum using the PEG-coated Fe3O4 MNPs. [ABSTRACT FROM AUTHOR]

This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0–2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10−7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates. [ABSTRACT FROM AUTHOR]

Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation. [ABSTRACT FROM AUTHOR]

Many cellular processes depend on a precise structural organization of molecular components. Here, we established that neurons grown in culture provide a suitable system for in situ structural investigations of cellular structures by cryo-electron tomography, a method that allows high resolution, three-dimensional imaging of fully hydrated, vitrified cellular samples. A higher level of detail of cellular components present in our images allowed us to quantitatively characterize presynaptic and cytoskeletal organization, as well as structures involved in axonal transport and endocytosis. In this way we provide a structural framework into which information from other methods need to fit. Importantly, we show that short pleomorphic linkers (tethers and connectors) extensively interconnect different types of spherical vesicles and other lipid membranes in neurons imaged in a close-to-native state. These linkers likely serve to organize and precisely position vesicles involved in endocytosis, axonal transport and synaptic release. Hence, structural interactions via short linkers may serve as ubiquitous vesicle organizers in neuronal cells. [ABSTRACT FROM AUTHOR]

Endothelial cells play an essential role in regulating an immune response through promoting leukocyte adhesion and cell migration and production of cytokines such as TNFα. Regulation of endothelial cell immune function is tightly regulated and recent studies suggest that extracellular vesicles (EVs) are prominently involved in this process. However, the importance of EVs in regulating endothelial activation in the context of a bacterial infection is poorly understood. To begin addressing this knowledge gap we characterized the endothelial cell response to EVs released from Mycobacterium tuberculosis (Mtb) infected macrophages. Our result showed increased macrophage migration through the monolayer when endothelial cells were pretreated with EVs isolated from Mtb-infected macrophages. Transcriptome analysis showed a significant upregulation of genes involved in cell adhesion and the inflammatory process in endothelial cells treated with EVs. These results were validated by quantitative PCR and flow cytometry. Pathway analysis of these differentially expressed genes indicated that several immune response-related pathways were up-regulated. Endothelial cells were also treated with EVs isolated from the serum of Mtb-infected mice. Interestingly, EVs isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation suggesting a change in EV function during the course of an Mtb infection. Immunofluorescence microscopy result indicated that NF-κB and the Type 1 interferon pathways were involved in endothelial activation by EVs. In summary, our data suggest that EVs can activate endothelial cells and thus may play an important role in modulating host immune responses during an Mtb infection. [ABSTRACT FROM AUTHOR]

Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst’s total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. [ABSTRACT FROM AUTHOR]

Analyzing extracellular vesicles (EVs) is an attractive approach to diagnosis of prostate diagnosis. However, existing methods of EVs isolation have low efficiency, purity, and long process time, and therefore have low diagnostic ability. To solve these the problems, a two-phase system is adapted to isolate EVs from a patient’s urine. Urine from 20 prostate cancer (PCA) patients and 10 benign prostate hyperplasia patients was used to quantify the EVs-isolation ability of an aqueous two-phase system (ATPS) and to compare the diagnostic ability of ATPS with that of the conventional diagnosis method. An optimized ATPS isolates EVs with ~100% efficiency within ~30 min, with 14 times as high as achieved by ultracentrifugation. Afterward, PCR and ELISA are used to detect EVs derived from PCA cells in urine. The results demonstrate that diagnostic ability based on ATPS is better than other conventional diagnostic methods. ATPS can obtain a high quality and quantity of EVs from patients’ urine. EVs contain cancer-related protein and genes, so these abundant sources enable diagnosis with high specificity and sensitivity. Therefore, ATPS is a useful tool to increase the specificity and sensitivity of diagnosis. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) released by virtually every cell of all organisms are involved in processes of intercellular communication through the delivery of their functional mRNAs, proteins and bioactive lipids. We previously demonstrated that mouse embryonic stem cell-released EVs (mESEVs) are able to transfer their content to different target retinal cells, inducing morphological and biochemical changes in them. The main objective of this paper is to characterize EVs derived from human embryonic stem cells (hESEVs) and investigate the effects that they have on cultured retinal glial, progenitor Müller cells, which are known to give rise to retinal neurons under specific conditions. This would allow us to establish if hESEVs have a pro-regenerative potential not yet described that could be used in the future for treatment of human retinal degenerative diseases. Initially, we showed that hESEVs are heterogeneous in size, contain mRNAs and proteins involved in the induction and maintenance of stem cell pluripotency and can be internalized by cultured Müller cells. After a single exposure to hESEVs these cells display changes in their gene expression profile, and with multiple exposures they de-differentiate and trans-differentiate into retinal neuronal precursors. hESEVs were then fractionated into microvesicles (MVs) and exosomes (EXOs), which were characterized by size, specific surface proteins and biochemical/molecular components. We demonstrate that despite the similar internalization of non-fractionated hESEVs, MVs and EXOs by Müller progenitor cells, in vitro, only the release of MVs’ cargo into the cells’ cytoplasm induces specific changes in their levels of pluripotency mRNAs and early retinal proteins. EXOs do not produce any detectable effect. Thus, we conclude that MVs and MVs-containing hESEVs are promising agents that possibly could promote the regeneration of diseased or damaged retinas in vivo through inducing glial Müller cells to become replacement neurons. [ABSTRACT FROM AUTHOR]

The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clinical applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resolution, and electron microscopy we demonstrate that rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, rhodomyrtone did not seem to be a typical membrane-inserting molecule. In fact, molecular dynamics simulations showed that instead of inserting into the bilayer, rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein-trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action. [ABSTRACT FROM AUTHOR]

Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution. A case study is reported to show the practicability of GV microfiltration. A clickable fluorescent probe was encapsulated inside GVs; more than 99.9% of the non-entrapped probe was easily and rapidly removed by multiple microfiltrations. This novel methodology is briefly discussed as a future tool for selection experiments on GV populations. [ABSTRACT FROM AUTHOR]

We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases. [ABSTRACT FROM AUTHOR]

Recently two approaches were reported that addressed a vitally important problem in regenerative medicine, i. e. the successful treatment of wounds even under diabetic conditions. Accordingly, these studies with diabetic rabbits [Sarojini et al. PLoS One 2017, 12(4):e0174899] and diabetic mice [Müller et al. Polymers 2017, 9, 300] identified a novel (potential) target for the acceleration of wound healing in diabetes. Both studies propose a raise of the intracellular metabolic energy status via exogenous administration either of ATP, encapsulated into lipid vesicles, or of polyphosphate (polyP) micro-/nanoparticles. Recently this physiological polymer, polyP, was found to release metabolic energy in form of ATP into both the extra- and also intra-cellular space. In the present work the uptake mechanism of the amorphous polyP microparticles “Ca-polyP-MP” has been described and found to be a clathrin-dependent endocytosis import, based on inhibition studies with the inhibitor trifluoperazine, which blocks the clathrin-dependent endocytosis import. The experiments had been performed with SaOS-2 cells, by studying the uptake and distribution of the electron-dense particles into the cells, and with HUVEC cells, for analysis of the intracellular accumulation of polyP, visualized by fluorescent staining of polyP. Concurrently with the uptake of particular polyP the intracellular ATP level increased as well. In contrast to “Ca-polyP-MP” the soluble polyP, administered as “Na-polyP[Ca2+]”, did not cause an increase in the intracellular Ca2+ level, suggesting a different mode of action of these two forms of polyP. Based on existing data on the effect of polyP and ATP on the induction of vascularization during wound repair, both groups (Sarojini et al. and Müller et al.) propose that the acceleration of wound repair is based on an increased metabolic energy supply directly to the regenerating wound area. [ABSTRACT FROM AUTHOR]

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane. [ABSTRACT FROM AUTHOR]

Cells release extracellular vesicles (EVs) in their environment and cellular lipids play an important role in their formation, secretion and uptake. Besides, there is also evidence that EV transferred lipids impact on recipient’s cell signaling. Cellular senescence is characterized by a state of permanent proliferation arrest and represents a barrier towards the development of neoplastic lesions. A peculiar feature of senescence is the release of many soluble factors, the so-called Senescence-Associated Secretory Phenotype, which play a key role in triggering paracrine senescence signals. Recently, evidences have suggested that this phenotype includes not only soluble factors, but also EVs. To identify lipid signatures associated with H-Ras-induced senescence in EVs, we expressed active H-Ras (H-RasV12) in human fibroblasts and investigated how it affects EV release and lipid composition. An enrichment of hydroxylated sphingomyelin, lyso- and ether-linked phospholipids and specific H-Ras-induced senescence signatures, e.g. sphingomyelin, lysophosphatidic acid and sulfatides, were found in EVs compared to cells. Furthermore, H-RasV12 expression in fibroblasts was associated with higher levels of tetraspanins involved in vesicle formation. [ABSTRACT FROM AUTHOR]

JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs) and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo. [ABSTRACT FROM AUTHOR]

It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications. [ABSTRACT FROM AUTHOR]

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind “non-cognate” Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone. [ABSTRACT FROM AUTHOR]

Autophagy is a physiological process for the recycling and degradation of cellular materials. Forming the autophagosome from the phagophore, a cup-shaped double-membrane vesicle, is a critical step in autophagy. The origin of the cup shape of the phagophore is poorly understood. In yeast, fusion of a small number of Atg9-containing vesicles is considered a key step in autophagosome biogenesis, aided by Atg1 complexes (ULK1 in mammals) localized at the preautophagosomal structure (PAS). In particular, the S-shaped Atg17-Atg31-Atg29 subcomplex of Atg1 is critical for phagophore nucleation at the PAS. To study this process, we simulated membrane remodeling processes in the presence and absence of membrane associated Atg17. We show that at least three vesicles need to fuse to induce the phagophore shape, consistent with experimental observations. However, fusion alone is not sufficient. Interactions with 34-nm long, S-shaped Atg17 complexes are required to overcome a substantial kinetic barrier in the transition to the cup-shaped phagophore. Our finding rationalizes the recruitment of Atg17 complexes to the yeast PAS, and their unusual shape. In control simulations without Atg17, with weakly binding Atg17, or with straight instead of S-shaped Atg17, the membrane shape transition did not occur. We confirm the critical role of Atg17-membrane interactions experimentally by showing that mutations of putative membrane interaction sites result in reduction or loss of autophagic activity in yeast. Fusion of a small number of vesicles followed by Atg17-guided membrane shape-remodeling thus emerges as a viable route to phagophore formation. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. Methods and findings: 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1–2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. Conclusions: EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes. [ABSTRACT FROM AUTHOR]

Respiratory secretions, such as saliva and bronchoalveolar fluid, contain anti-influenza activity. Multiple soluble factors have been described that exert anti-influenza activity and are believed to be responsible for the anti-influenza activity in respiratory secretions. It was previously shown that a bronchial epithelial cell culture could produce exosome-like particles with anti-influenza activity. Whether such extracellular vesicles in respiratory secretions have anti-influenza activity is unknown. Therefore, we characterized bronchoalveolar lavage fluid and found microparticles, which mostly stained positive for epithelial cell markers and both α2,3- and α2,6-linked sialic acid. Microparticles were purified from bronchoalveolar lavage fluid and shown to exhibit anti-influenza activity by a hemagglutination inhibition (HI) assay and a neutralization (NT) assay. In addition, physical binding between influenza virions and microparticles was demonstrated by electron microscopy. These findings indicate that respiratory microparticles containing viral receptors can exert anti-viral activity by probably trapping viral particles. This innate mechanism may play an important role in the defense against respiratory viruses. [ABSTRACT FROM AUTHOR]

A eukaryotic cell contains multiple membrane-bound compartments. Transport vesicles move cargo between these compartments, just as trucks move cargo between warehouses. These processes are regulated by specific molecular interactions, as summarized in the Rothman-Schekman-Sudhof model of vesicle traffic. The whole structure can be represented as a transport graph: each organelle is a node, and each vesicle route is a directed edge. What constraints must such a graph satisfy, if it is to represent a biologically realizable vesicle traffic network? Graph connectedness is an informative feature: 2-connectedness is necessary and sufficient for mass balance, but stronger conditions are required to ensure correct molecular specificity. Here we use Boolean satisfiability (SAT) and model checking as a framework to discover and verify graph constraints. The poor scalability of SAT model checkers often prevents their broad application. By exploiting the special structure of the problem, we scale our model checker to vesicle traffic systems with reasonably large numbers of molecules and compartments. This allows us to test a range of hypotheses about graph connectivity, which can later be proved in full generality by other methods. [ABSTRACT FROM AUTHOR]

Syncytiotrophoblast extracellular vesicles (STBEVs) are placenta derived particles that are released into the maternal circulation during pregnancy. Abnormal levels of STBEVs have been proposed to affect maternal vascular function. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a multi-ligand scavenger receptor. Increased LOX-1 expression and activation has been proposed to contribute to endothelial dysfunction. As LOX-1 has various ligands, we hypothesized that, being essentially packages of lipoproteins, STBEVs are able to activate the LOX-1 receptor thereby impairing vascular function via the production of superoxide and decreased nitric oxide bioavailability. Uterine arteries were obtained in late gestation from Sprague-Dawley rats and incubated for 24h with or without human STBEVs (derived from a normal pregnant placenta) in the absence or presence of a LOX-1 blocking antibody. Vascular function was assessed using wire myography. Endothelium-dependent maximal vasodilation to methylcholine was impaired by STBEVs (MCh Emax: 57.7±5.9% in STBEV-incubated arteries vs. 77.8±2.9% in controls, p<0.05). This was prevented by co-incubation of STBEV-incubated arteries with LOX-1 blocking antibodies (MCh Emax: 78.8±4.3%, p<0.05). Pre-incubation of the vessels with a nitric oxide synthase inhibitor (L-NAME) demonstrated that the STBEV-induced impairment in vasodilation was due to decreased nitric oxide contribution (ΔAUC 12.2±11.7 in STBEV-arteries vs. 86.5±20 in controls, p<0.05), which was abolished by LOX-1 blocking antibody (ΔAUC 98.9±17, p<0.05). In STBEV-incubated vessels, LOX-1 inhibition resulted in an increased endothelial nitric oxide synthase expression (p<0.05), to a level similar to control vessels. The oxidant scavenger, superoxide dismutase, did not improve this impairment, nor were vascular superoxide levels altered. Our data support an important role for STBEVs in impairment of vascular function via activation of LOX-1 and reduced nitric oxide mediated vasodilation. Moreover, we postulate that the LOX-1 pathway could be a potential therapeutic target in pathologies associated with vascular dysfunction during pregnancy. [ABSTRACT FROM AUTHOR]

Objectives: Hemoglobin vesicles (HbVs) function as a red blood cell (RBC) substitute and are composed of purified hemoglobin encapsulated in a phospholipid bilayer membrane. The performance of HbVs as a substitute for RBC transfusions was examined in a mouse model of pneumonectomy following acute 40% exchange-transfusion with HbVs. Methods: Before performing left pneumonectomies, 40% of the blood volume of mice was replaced with a) lactated Ringer’s solution (control), b) 5% recombinant human serum albumin (rHSA), c) mouse RBCs shed in rHSA (mRBCs/rHSA), or d) HbV suspended in rHSA (HbV/rHSA). We compared postoperative a) survival, b) functional recovery, and c) histopathological, immunohistochemical, and inflammatory responses among the study groups. Results: In the HbV/rHSA and mRBC/rHSA groups, all mice survived ≥7 days after pneumonectomy, whereas 100% of the control mice died within a few h and 50% of mice in the rHSA group died within 24 h after pneumonectomy. Immunohistochemical staining for hypoxia-inducible factor-1α showed that hepatic and renal hypoxic injuries were prominently mitigated by HbV and mRBCs. Conclusions: The oxygen-carrying performance of HbV was similar to that of mRBCs, even with impaired lung functions following pneumonectomy. HbV infusion did not interfere with the recovery from surgical injury. In the near future, HbVs could be used clinically as a substitute for the perioperative transfusion of RBCs, when or where donated RBCs are not immediately available. [ABSTRACT FROM AUTHOR]

Peritoneal dialysis inevitability results in activation of inflammatory processes and its efficiency is highly variable between patients. An improved method to isolate biomarkers and study pathophysiological mechanisms in peritoneal dialysis effluent (PDE) is expected to be of much benefit for the development of this treatment approach and help with patient management. Extracellular vesicles (EVs) are released as part of normal cellular processes. Their proteome is expected to reflect both type and health of their cell of origin. Although there is a significant interest in using EVs for “liquid biopsies”, little is reported of their presence or composition in plentiful dialysis waste fluids, including peritoneal dialysis effluent (PDE). Here we determined the presence of EVs in PDE and subsequently characterized their proteome. EVs were first isolated from PDE using differential centrifugation, then a further enrichment using size exclusion chromatography (SEC) was performed. The presence of EVs was demonstrated using transmission electron microscopy, and their particle counts were investigated using nanoparticle tracking analysis and dynamic light scattering. Using tandem mass spectrometry, marker proteins from three types of EVs i.e. apoptotic bodies, ectosomes, and exosomes were identified. The proteomic results demonstrated that the isolation of EVs by differential centrifugation helped enrich for over 2,000 proteins normally masked by abundant proteins in PDE such as albumin and SEC markedly further improved the isolation of low abundant proteins. Gene ontology analysis of all identified proteins showed the marked enrichment of exosome and membrane-associated proteins. Over 3,700 proteins were identified in total, including many proteins with known roles in peritoneal pathophysiology. This study demonstrated the prominence of EVs in PDE and their potential value as a source of biomarkers for peritoneal dialysis patients. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have been identified within different body fluids and cell culture media. However, there is very little information on the secretion of these vesicles during early embryonic development. The aims of this work were first to demonstrate the secretion of extracellular vesicles by pre-implantation bovine embryos and second to identify and characterize the population of EVs secreted by bovine blastocysts during the period from day seven to nine of embryo culture and its correlation with further embryo development up to day 11. Bovine embryos were produced by in vitro fertilization (IVF) or parthenogenetic activation (PA) and cultured until blastocyst stage. Blastocyst selection was performed at day 7 post IVF/PA considering two variables: stage of development and quality of embryos. Selected blastocysts were cultured in vitro for 48 hours in groups (exp. 1) or individually (exp. 2) in SOF media depleted of exosomes. At day 9 post IVF/PA the media was collected and EVs isolated by ultracentrifugation. Transmission electron microscopy revealed the presence of heterogeneous vesicles of different sizes and population: microvesicles (MVs) and exosomes (EXs) of rounded shape, enclosed by a lipid bi-layer and ranging from 30 to 385 nm of diameter. Flow cytometry analysis allowed identifying CD63 and CD9 proteins as exosome markers. Nanoparticle tracking analysis generated a large number of variables, which required the use of multivariate statistics. The results indicated that the concentration of vesicles is higher in those blastocysts with arrested development from day 9 up to day 11 of in vitro development (6.7 x 108 particles/ml) derived from IVF (p <0.05), compared to PA blastocysts (4.7 x 108 particles/ml). Likewise, the profile (concentration and diameter) of particles secreted by embryos derived from IVF were different from those secreted by PA embryos. In conclusion, we demonstrated that bovine blastocysts secrete MVs/EXs to the culture media. Data suggest that characteristics of the population of EVs vary depending on embryo competence. [ABSTRACT FROM AUTHOR]

Peritoneal Dialysis (PD) is considered the best option for a cost-effective mid-term dialysis in patients with Chronic Renal Failure. However, functional failure of the peritoneal membrane (PM) force many patients to stop PD treatment and start haemodialysis. Currently, PM functionality is monitored by the peritoneal equilibration test, a tedious technique that often show changes when the membrane damage is advanced. As in other pathologies, the identification and characterization of extracellular vesicles (EVs) in the peritoneal dialysis efflux (PDE) may represent a non-invasive alternative to identify biomarkers of membrane failure. Using size-exclusion chromatography, we isolated EVs from PDE in a group of patients. Vesicles were characterized by the presence of tetraspanin markers, nanoparticle tracking analysis profile, cryo-electron microscopy and mass spectrometry. Here, we report the isolation and characterization of PDE-EVs. Based on mass spectrometry, we have found a set of well-conserved proteins among patients. Interestingly, the peptide profile also revealed remarkable changes between newly enrolled and longer-treated PD patients. These results are the first step to the identification of PDE-EVs based new markers of PM damage, which could support clinicians in their decision-making in a non-invasive manner. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs. Methods: Simultaneous expression profiles of miRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and LC-MS/MS proteomic analysis in porcine MSCs and their daughter EVs (n = 3 each). TargetScan and ComiR were used to predict miRNA target genes. Functional annotation analysis was performed using DAVID 6.7 database to rank primary gene ontology categories for the enriched mRNAs, miRNA target genes, and proteins. STRING was used to predict associations between mRNA and miRNA target genes. Results: Differential expression analysis revealed 4 miRNAs, 255 mRNAs, and 277 proteins enriched in EVs versus MSCs (fold change >2, p<0.05). EV-enriched miRNAs target transcription factors (TFs) and EV-enriched mRNAs encode TFs, but TF proteins are not enriched in EVs. Rather, EVs are enriched for proteins that support extracellular matrix remodeling, blood coagulation, inflammation, and angiogenesis. Conclusions: Porcine MSC-derived EVs contain a genetic cargo of miRNAs and mRNAs that collectively control TF activity in EVs and recipient cells, as well as proteins capable of modulating cellular pathways linked to tissue repair. These properties provide the fundamental basis for considering therapeutic use of EVs in tissue regeneration. [ABSTRACT FROM AUTHOR]

Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the, endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. [ABSTRACT FROM AUTHOR]

Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response. [ABSTRACT FROM AUTHOR]

Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or -galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity. [ABSTRACT FROM AUTHOR]

Vesicle-inducing protein in plastids 1 (Vipp1) is thought to play an important role both in thylakoid biogenesis and chloroplast envelope maintenance during stress. Vipp1 is conserved in photosynthetic organisms and forms a high homo-oligomer complex structure that may help sustain the membrane integrity of chloroplasts. This study cloned two novel VIPP1 genes from Triticum urartu and named them TuVipp1 and TuVipp2. Both proteins shared high identity with the homologous proteins AtVipp1 and CrVipp1. TuVipp1 and TuVipp2 were expressed in various organs of common wheat, and both genes were induced by light and various stress treatments. Purified TuVipp1 and TuVipp2 proteins showed secondary and advanced structures similar to those of the homologous proteins. Similar to AtVipp1, TuVipp1 is a chloroplast target protein. Additionally, TuVipp1 was able to rescue the phenotypes of pale leaves, lethality, and disordered chloroplast structures of AtVipp1 (-/-) mutant lines. Collectively, our data demonstrate that TuVipp1 and TuVipp2 are functional proteins in chloroplasts in wheat and may be critical for maintaining the chloroplast envelope under stress and membrane biogenesis upon photosynthesis. [ABSTRACT FROM AUTHOR]

Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications. [ABSTRACT FROM AUTHOR]

In contrast to vertebrate immune systems, invertebrates lack an adaptive response and rely solely on innate immunity in which antimicrobial peptides (AMPs) play an essential role. Most of them are membrane active molecules that are typically unstructured in solution and adopt secondary/tertiary structures upon binding to phospholipid bilayers. This work presents the first characterization of a constitutive AMP from the hemolymph of an Opiliones order animal: the harvestman Acutisoma longipes. This peptide was named longipin. It presents 18 aminoacid residues (SGYLPGKEYVYKYKGKVF) and a positive net charge at neutral pH. No similarity with other AMPs was observed. However, high sequence similarity with heme-lipoproteins from ticks suggested that longipin might be a protein fragment. The synthetic peptide showed enhanced antifungal activity against Candida guilliermondii and C. tropicalis yeasts (MIC: 3.8–7.5 μM) and did not interfered with VERO cells line viability at all concentrations tested (200–0.1 μM). This selectivity against microbial cells is related to the highest affinity of longipin for anionic charged vesicles (POPG:POPC) compared to zwitterionic ones (POPC), once microbial plasma membrane are generally more negatively charged compared to mammalian cells membrane. Dye leakage from carboxyfluorescein-loaded POPG:POPC vesicles suggested that longipin is a membrane active antimicrobial peptide and FT-IR spectroscopy showed that the peptide chain is mainly unstructured in solution or in the presence of POPC vesicles. However, upon binding to POPG:POPC vesicles, the FT-IR spectrum showed bands related to β-sheet and amyloid-like fibril conformations in agreement with thioflavin-T binding assays, indicating that longipin is an amyloid antimicrobial peptide. [ABSTRACT FROM AUTHOR]

Microvesicles are cell-derived signaling particles emerging as important mediators and biomarkers of systemic inflammation, but their production in severe burn injury patients has not been described. In this pilot investigation, we measured circulating microvesicle levels following severe burns, with severe sepsis patients as a comparator group. We hypothesized that levels of circulating vascular cell-derived microvesicles are elevated acutely following burns injury, mirroring clinical severity due to the early onset and prevalence of systemic inflammatory response syndrome (SIRS) in these patients. Blood samples were obtained from patients with moderate to severe thermal injury burns, with severe sepsis, and from healthy volunteers. Circulating microvesicles derived from total leukocytes, granulocytes, monocytes, and endothelial cells were quantified in plasma by flow cytometry. All circulating microvesicle subpopulations were elevated in burns patients on day of admission (day 0) compared to healthy volunteers (leukocyte-microvesicles: 3.5-fold, p = 0.005; granulocyte-microvesicles: 12.8-fold, p<0.0001; monocyte-microvesicles: 20.4-fold, p<0.0001; endothelial- microvesicles: 9.6-fold, p = 0.01), but decreased significantly by day 2. Microvesicle levels were increased with severe sepsis, but less consistently between patients. Leukocyte- and granulocyte-derived microvesicles on day 0 correlated with clinical assessment scores and were higher in burns ICU non-survivors compared to survivors (leukocyte MVs 4.6 fold, p = 0.002; granulocyte MVs 4.8 fold, p = 0.003). Mortality prediction analysis of area under receiver operating characteristic curve was 0.92 (p = 0.01) for total leukocyte microvesicles and 0.85 (p = 0.04) for granulocyte microvesicles. These findings demonstrate, for the first time, acute increases in circulating microvesicles following burns injury in patients and point to their potential role in propagation of sterile SIRS-related pathophysiology. [ABSTRACT FROM AUTHOR]