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Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by both healthy and cancerous cells. They are considered as biomarkers for early cancer diagnosis and are involved in many pathologies and physiological pathways. They could serve as diagnostic tools in liquid biopsies, as therapeutics in regenerative medicine, or as drug delivery vehicles. Our aim is here to encapsulate luminescent nanoprobes in the aqueous compartment of human EVs extracted from reproductive fluids. The analysis and labeling of the EVs content with easily detectable luminescent nanoparticles could enable a powerful tool for early diagnosis of specific diseases and also for the design of new therapeutics. In this view, gold nanoclusters (AuNCs) appear as an attractive alternative as nontoxic fluorophore probes because of their luminescence properties, large window of fluorescence lifetimes (1 ns-1 µs), ultrasmall size (<2 nm), good biocompatibility, and specific ability as X-ray photosensitizers. Here, we investigated an attractive method that uses fusogenic liposomes to deliver gold nanoclusters into EVs. This approach guarantees the preservation of the EVs membrane without any breakage, thus maintaining compartmental integrity. Different lipid compositions of liposomes preloaded with AuNCs were selected to interact electrostatically with human EVs and compared in terms of fusion efficiency. The mixture of liposomes and EVs results in membrane mixing as demonstrated by FRET experiments and fusion revealed by flux cytometry and cryo-TEM. The resulting fused EVs exhibit typical fluorescence of the AuNCs together with an increased size in agreement with fusion. Moreover, the fusion events in mixtures of EVs and AuNCs preloaded liposomes were analyzed by using cryo-electron microscopy. Finally, the ratio of released AuNCs during the fusion between the fusogenic liposomes and the EVs was estimated to be less than 20 mol % by Au titration using ICP spectroscopy.
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Vesículas Extracelulares , Oro , Liposomas , Nanopartículas del Metal , Oro/química , Humanos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Nanopartículas del Metal/química , Liposomas/químicaRESUMEN
Essential oils are well known for their biological properties, making them useful for the treatment of various diseases. However, because of their poor stability and high volatility, their potential cannot be fully exploited. The use of nanoformulations to deliver essential oils can solve these critical issues and amplify their biological activities. We characterized an essential oil from Satureja thymbra via GC-MS and HPLC-DAD to provide qualitative and quantitative data. The essential oil was formulated in phospholipid vesicles which were characterized for size, surface charge, and storage stability. The entrapment efficiency was evaluated as the quantification of the major monoterpenoid phenols via HPLC-DAD. The morphological characterization of the vesicles was carried out via cryo-TEM and SAXS analyses. The essential oil's antioxidant potential was assayed via two colorimetric tests (DPPH⢠and FRAP) and its cytocompatibility was evaluated in HaCaT skin cell cultures. The results showed that the nanoformulations developed for the loading of S. thymbra essential oil were below 100 nm in size, predominantly unilamellar, stable in storage, and had high entrapment efficiencies. The vesicles also displayed antioxidant properties and high cytocompatibility. These promising findings pave the way for further investigation of the therapeutic potential of S. thymbra nanoformulations upon skin application.
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Lamiaceae , Aceites Volátiles , Satureja , Aceites Volátiles/análisis , Antioxidantes , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43 mM ethanol and 25 nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/genética , Etanol/toxicidad , Pez Cebra , Benzo(a)pireno/toxicidad , Larva , Transcriptoma , Mitocondrias , HemoRESUMEN
Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs' encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.
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Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells.
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The oocyte microenvironment constituted by the follicular fluid (FF) is a key for the optimal development of female gametes. Its composition reflects the physiological state of the ovarian follicle. The particularity of FF is to contain a huge diversity of extracellular vesicles specific to women, in the same way as seminal plasma in men. Here, we described and compared morphological aspects of broad subcategories of human FF-related Extracellular Vesicles (EVs). EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. EVs isolated from FF are involved in different biological functions related to follicular growth, oocyte maturation, and embryo development. However, knowledge on the morphology of FF-derived EVs is limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. The aim of this study was to provide a comprehensive morphological description of EVs from FF of healthy subjects and quantification. EVs separation was realized by centrifugation, with comparison of the EV yield obtained from differential centrifugation and one-step ultracentrifugation. Cryo-Transmission Electron Microscopy was used to reveal the morphology, size, and phenotype of EVs. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) were used to quantify and analyze the size distribution for each centrifugation step. We performed a comprehensive inventory of human follicular fluid EVs. We show that human FF contains a huge diversity of EVs. This study brings novel insights on EVs from normal FF and provides a reference for further studies of EVs in ovarian diseases.
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Vesículas Extracelulares , Líquido Folicular , Vesículas Extracelulares/fisiología , Femenino , Humanos , Masculino , Oocitos , Oogénesis , Folículo OváricoRESUMEN
According to their high electron density and ultrasmall size, gold nanoclusters (AuNCs) have unique luminescence and photoelectrochemical properties that make them very attractive for various biomedical fields. These applications require a clear understanding of their interaction with biological membranes. Here we demonstrate the ability of the AuNCs as markers for lipidic bilayer structures such as synthetic liposomes and biological extracellular vesicles (EVs). The AuNCs can selectively interact with liposomes or EVs through an attractive electrostatic interaction as demonstrated by zetametry and fluorescence microscopy. According to the ratio of nanoclusters to vesicles, the lipidic membranes can be fluorescently labeled without altering their thickness until charge reversion, the AuNCs being located at the level of the phosphate headgroups. In presence of an excess of AuNCs, the vesicles tend to adhere and aggregate. The strong adsorption of AuNCs results in the formation of a lamellar phase as demonstrated by cryo-transmission electron microscopy and small-angle X-ray scattering techniques.
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Oro , Nanopartículas del Metal , Oro/química , Membrana Dobles de Lípidos , Liposomas , Luminiscencia , Nanopartículas del Metal/químicaRESUMEN
Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness and resistance, as they carry and disseminate oncogenic material in the local tumor microenvironment and at distance. However, whether their quality and quantity reflect individual health status and changes in homeostasis is still not fully elucidated. Here, we separated EVs from plasma collected at different time points alongside with the clinical management of GBM patients. Our findings confirm that plasmatic EVs could be separated and characterized with standardized protocols, thereby ensuring the reliability of measuring vesiclemia, i.e. extracellular vesicle concentration in plasma. This unveils that vesiclemia is a dynamic parameter, which could be reflecting tumor burden and/or response to treatments. Further label-free liquid chromatography tandem mass spectrometry unmasks the von Willebrand Factor (VWF) as a selective protein hallmark for GBM-patient EVs. Our data thus support the notion that EVs from GBM patients showed differential protein cargos that can be further surveyed in circulating EVs, together with vesiclemia.
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Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/patología , Vesículas Extracelulares/metabolismo , Glioblastoma/patología , Proteoma/metabolismo , Microambiente Tumoral/inmunología , Factor de von Willebrand/metabolismo , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Glioblastoma/sangre , Glioblastoma/inmunología , Glioblastoma/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Proteoma/análisisRESUMEN
The incorporation of lipophilic phosphonodithioesters in phospholipid formulations generates clickable liposomes that react with amines. The kinetics of this metal free phosphonodithioester-amine coupling (PAC) on liposomes in water is reported and can be classified as a fast reaction with a second order rate constant of k ≈ 8 × 102 M-1 s-1. The PAC reaction represents a versatile strategy to functionalize liposomes.
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LiposomasRESUMEN
PURPOSE: Current preclinical therapeutic strategies involving nanomedicine require increasingly sophisticated nanosystems and the characterization of the complexity of such nanoassemblies is becoming a major issue. Accurate characterization is often the factor that can accelerate the translational approaches of nanomedicines and their pharmaceutical development to reach the clinic faster. We conducted a case study involving the adsorption of the NFL-TBS.40-63 (NFL) peptide (derived from neurofilaments) to the surface of lipid nanocapsules (LNCs) (a combined nanosystem used to target glioblastoma cells) to develop an analytical approach combining the separation and the quantification in a single step, leading to the characterization of the proportion of free peptide and thus the proportion of peptide adsorbed to the lipid nanocapsule surface. METHODS: LNC suspensions, NFL peptide solution and LNC/NFL peptide mixtures were characterized using a Size-Exclusion Chromatography method (with a chromatographic apparatus). In addition, this method was compared to centrifugal-filtration devices, currently used in literature for this case study. RESULTS: Combining the steps for separation and characterization in one single sequence improved the accuracy and robustness of the data and led to reproducible results. Moreover the data deviation observed for the centrifugal-filtration devices demonstrated the limits for this increasingly used characterization approach, explained by the poor separation quality and highlighting the importance for the method optimization. The high potential of the technique was shown, proving that H-bond and/or electrostatic interactions mediate adsorption of the NFL peptide to the surface of LNCs. CONCLUSIONS: Used only as a characterization tool, the process using chromatographic apparatus is less time and solvent consuming than classical Size-Exclusion Chromatography columns only used for separation. It could be a promising tool for the scientific community for characterizing the interactions of other combinations of nanosystems and active biological agents.
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Neoplasias Encefálicas/tratamiento farmacológico , Portadores de Fármacos/química , Glioblastoma/tratamiento farmacológico , Nanocápsulas/química , Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Adsorción , Línea Celular Tumoral , Química Farmacéutica , Humanos , Lípidos/química , Proteínas de Neurofilamentos/administración & dosificación , Fragmentos de Péptidos/administración & dosificaciónRESUMEN
The structures of fed state intestinal assemblies containing bile components, dietary fat, and fat-soluble vitamins are not well known, although they are involved in lipid transport. In this study, several methods were used to investigate structural transitions upon various dietary lipids or various fat-soluble vitamins incorporation in bile intestinal assemblies. In particular, DLS and turbidimetry were used to study transition points as a function of component concentration, and cryo-TEM and SAXS were used to resolve assembly structures at microscopic and supramolecular scales, respectively. Results showed that increasing the concentration of dietary lipids in bile assembly induced a transition from core-shell micelles to unilamellar vesicles (except with caprylate lipids, always yielding micelles). In these specific assemblies, increasing the concentration of a fat-soluble vitamin either induced a systematic structural transition, defining a solubilization capacity (α-tocopherol or phylloquinone), or induced a structural transition only in micelles (retinol), or did not induce any structural transition up to very high concentrations (cholecalciferol). Using SAXS data, ideal molecular organizations are proposed for assemblies in the absence or presence of α-tocopherol.
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Vitamina A , Vitaminas , Grasas de la Dieta , Micelas , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Lutein is a xanthophyll carotenoid provided exclusively by the diet, that has protective functions and beneficial effects on human health. Supplementation in lutein is necessary to reach the recommended daily dietary intake. However, the introduction of lutein into foods and beverages is a real challenge since this lipophilic nutrient has a poor aqueous solubility and a low bioavailability. In this study, we investigated the capacity of egg-sphingomyelin (ESM) vesicles called sphingosomes to solubilise lutein into the bilayers. The thermal and structural properties of ESM bilayers were examined in presence of various amounts of lutein by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the structures of sphingosomes and lutein crystals were observed by microscopic techniques. ESM bilayers were in the fluid Lα phase above the phase transition temperature Tm = 39.6 °C and in the lamellar ripple Pß' phase below Tm where ESM sphingosomes exhibited ondulations and were facetted. Lutein molecules were successfully incorporated into the ESM bilayers where they induced a structural disorganisation. For ESM/lutein 90/10 %mol (91.8/8.2 %wt; 89 mg lutein / g ESM), lutein partitioning occured with the formation of lutein crystals in the aqueous phase together with lutein-loaded ESM vesicles. This study highlighted the capacity of new lipid carriers such as egg-sphingosomes to solubilise lutein and opens perspectives for the formulation of effective lutein-fortified functionnal foods and beverages providing health benefits.
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Luteína , Esfingomielinas , Rastreo Diferencial de Calorimetría , Humanos , Membrana Dobles de Lípidos , Difracción de Rayos XRESUMEN
Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.
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Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Inflamación/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Apoptosis , Hipoxia de la Célula , Línea Celular Tumoral , Daño del ADN , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/ultraestructura , Femenino , Inflamación/inmunología , Inflamación/patología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/ultraestructura , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos NOD , MicroARNs/metabolismo , Fenotipo , Células RAW 264.7 , Vías Secretoras , Transducción de SeñalRESUMEN
A growing body of evidences indicate the major role of extracellular vesicles (EVs) as players of cell communication in the pathogenesis of liver diseases. EVs are membrane-enclosed vesicles released by cells into the extracellular environment. Oxidative stress is also a key component of liver disease pathogenesis, but no role for hepatocyte-derived EVs has yet been described in the development of this process. Recently, some polycyclic aromatic hydrocarbons (PAHs), widespread environmental contaminants, were demonstrated to induce EV release from hepatocytes. They are also well-known to trigger oxidative stress leading to cell death. Therefore, the aim of this work was to investigate the involvement of EVs derived from PAHs-treated hepatocytes (PAH-EVs) in possible oxidative damages of healthy recipient hepatocytes, using both WIF-B9 and primary rat hepatocytes. We first showed that the release of EVs from PAHs -treated hepatocytes depended on oxidative stress. PAH-EVs were enriched in proteins related to oxidative stress such as NADPH oxidase and ferritin. They were also demonstrated to contain more iron. PAH-EVs could then induce oxidative stress in recipient hepatocytes, thereby leading to apoptosis. Mitochondria and lysosomes of recipient hepatocytes exhibited significant structural alterations. All those damages were dependent on internalization of EVs that reached lysosomes with their cargoes. Lysosomes thus appeared as critical organelles for EVs to induce apoptosis. In addition, pro-oxidant components of PAH-EVs, e.g. NADPH oxidase and iron, were revealed to be necessary for this cell death.
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Vesículas Extracelulares , Hidrocarburos Policíclicos Aromáticos , Animales , Vesículas Extracelulares/metabolismo , Hepatocitos , Hierro/metabolismo , Estrés Oxidativo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , RatasRESUMEN
The design of a simple platform to target the delivery of notably hydrophobic drugs into cancer cells is an ultimate goal. Here, three strategies were combined in the same nanovector, in limiting the use of excipients: cell-penetrating peptides, an amphiphilic prodrug, and self-assembly. Light scattering and cryogenic transmission electron microscopy revealed one size population of objects around 100 nm with a narrow size distribution. However, in-depth analysis of the suspension by nanoparticle tracking analysis, small-angle X-ray scattering, and nuclear magnetic resonance (NMR) diffusometry demonstrated the presence of another population of small objects (<2 nm). It has been shown that these small self-assemblies represented >99% of the matter! This presence was clearly and unambiguously demonstrated by NMR diffusometry experiments. The study highlights the importance and the complementary contribution of each characterization method to reflect the reality of the studied nanoassembly.
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Péptidos de Penetración Celular/química , Compuestos Ferrosos/química , Células A549 , Péptidos de Penetración Celular/metabolismo , Microscopía por Crioelectrón , Compuestos Ferrosos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Nanoestructuras/química , Tamaño de la Partícula , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.
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Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogénicas/metabolismo , Multimerización de Proteína , Proteostasis , Animales , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Masculino , Ratones , Mucoproteínas/genética , Proteínas Oncogénicas/genéticaRESUMEN
In many cases, it takes several minutes after an earthquake to publish online a seismic location with confidence. Via monitoring for specific types of increased website, app, or Twitter usage, crowdsourced detection of seismic activity can be used to "seed" the search in the seismic data for an earthquake and reduce the risk of false detections, thereby accelerating the publication of locations for felt earthquakes. We demonstrate that this low-cost approach can work at the global scale to produce reliable and rapid results. The system was retroactively tested on a set of real crowdsourced detections of earthquakes made during 2016 and 2017, with 50% of successful locations found within 103 s, 76 s faster than GEOFON and 271 s faster than the European-Mediterranean Seismological Centre's publication times, and 90% of successful locations found within 54 km of the final accepted epicenter.
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Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in vitro with a designated protein content. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live S. aureus induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by LTA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined in vitro and in vivo data suggest that EVs are worth to be investigated to better understand the S. aureus pathogenesis and are relevant tools to develop strategies against bovine S. aureus mastitis.