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We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.
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Diatomáceas , Microalgas , Rhodospirillaceae , Bactérias/genética , Meios de Cultivo Condicionados , Diatomáceas/genética , Cromatografia Gasosa-Espectrometria de Massas , RNA Ribossômico 16S/genética , Espectrometria de Massas em TandemRESUMO
Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.
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Extracellular vesicles (EVs) are considered essential mediators of regenerative roles of autologous platelet- and extracellular vesicle-rich plasma (PVRP) and platelet- and extracellular vesicle-rich gel (PVRG). PVRP and PVRG are novel blood-derived products gaining attraction in regenerative medicine. However, despite their reported good efficacy, their preparation protocols are too time-consuming. Moreover, patient-tailored preparation protocols are desired to optimize platelet and EV count in PVRP and PVRG. This article presents the clinical implementation of one-step, patient-tailored erythrocyte sedimentation rate (ESR)-based, PVRP and PVRG preparation protocols through the presentation of three cases: (1) large chronic tympanic membrane (TM) perforation, (2) osteoradionecrosis of the lateral skull base, and (3) cerebrospinal fluid (CSF) leak in the sphenoid sinus. These were treated with PVRP and PVRG, prepared according to our preclinically constructed mathematical sedimentation model of cells and EVs based on the patient's ESR. (1) TM healed completely after the treatment with 3.6 mL of PVRP and PVRG (high platelet and EV protocol). The speech discrimination score and air conduction pure tone average improved from 75% to 95% and from 65 to 25 dB, respectively. (2) The osteoradionecrotic surface area decreased from 46 to 18 cm2, and infection was eradicated after six applications of 13-65 mL of PVRG ("half-volume" protocol). (3) No CSF leak recurrence was detected after surgical closure with 30 mL of PVRG postoperatively. Reproducible preparation protocols proved effective, safe, fast, and straightforward enough for the surgical staff to prepare PVRP and PVRG intraoperatively. To alleviate preparation, a calculator is provided. This pilot study presents a sound basis for further studies, which are needed to assess the therapeutic effect of PVRP and PVRG. Impact statement We introduce a clinical implementation of a patient-tailored, erythrocyte sedimentation rate-based platelet- and extracellular vesicle-rich plasma (PVRP) and gel (PVRG) preparation protocol based on a mathematical model. Products proved beneficial in wound healing and were, to our knowledge, used for the first time in the treatment of osteoradionecrosis of the lateral skull base. Furthermore, this reproducible preparation protocol is fast and straightforward to implement in clinical practice. A calculator is provided to alleviate PVRP and PVRG preparation for various clinical scenarios.
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Vesículas Extracelulares , Osteorradionecrose , Perfuração da Membrana Timpânica , Plaquetas , Humanos , Projetos PilotoRESUMO
In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = -0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.[Figure: see text].
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Plaquetas , Vesículas Extracelulares , Eritrócitos , Citometria de Fluxo/métodos , Humanos , PlasmaRESUMO
Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.
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Difusão Dinâmica da Luz/métodos , Eritrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Microscopia Eletrônica/métodos , Eritrócitos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , HumanosRESUMO
In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (Rh) and the radius of gyration (Rg) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. Rh and Rg of the predominant ENP population of OC patients were in the range 20-30 nm (diameter 40-60 nm). In thawed samples, larger particles (Rh mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The Rh and Rg of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1-2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.
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Ascite/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/metabolismo , Ascite/patologia , Estudos de Casos e Controles , Difusão Dinâmica da Luz , Detecção Precoce de Câncer , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Hidrodinâmica , Luz , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Neoplasias Ovarianas/diagnóstico , Tamanho da Partícula , Espalhamento de RadiaçãoRESUMO
Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC-MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry-based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.
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PURPOSE: To determine the efficacy of autologous platelet- and extracellular vesicle-rich plasma (PVRP) to treat chronic postoperative temporal bone cavity inflammation (CPTBCI) after exhausting surgical and standard conservative therapies. MATERIALS AND METHODS: Patients were randomly allocated to treatment with PVRP (PVRP group) or standard conservative methods (control group) in a setting of four once-monthly checkups and subsequent follow-up. The treatment outcome was measured with the Chronic Otitis Media Questionnaire-12 (COMQ-12), CPTBCI focus surface area, and CPTBCI symptom-free time after the fourth checkup. RESULTS: Eleven patients from each group completed the trial; 95% of patients suffered from chronically discharging mastoid cavity (the type of CPTBCI). Within four checkups, the COMQ-12 score decreased statistically significantly in the PVRP group (p < 0.001) but not in the control group (p = 0.339). The CPTBCI foci surface area decreased statistically significantly between the first and second checkups (p < 0.0005) but not between other checkups (p > 0.05) in the PVRP group. No statistically significant differences in CPTBCI foci surface area were detected between checkups in the control group (p = 0.152). Nine patients from the PVRP group and three patients from the control group were CPTBCI symptom-free at the fourth checkup. The median symptom-free time was 9.2 months (95% CI [7.4, 11.9]) in the PVRP group. Cumulatively, 49% of patients in the PVRP group remained CPTBCI symptom-free for 12.7 months after the fourth checkup. CONCLUSION: Autologous PVRP represents a novel additional and successful treatment modality for a chronically discharging radical mastoid cavity when the surgical and standard conservative treatment methods have been exhausted. TRIAL NUMBER: https://clinicaltrials.gov (NCT04281901).
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Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.
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Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/química , Microalgas/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Microalgas/genética , Ultracentrifugação/métodosRESUMO
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.
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Vesículas Extracelulares , Microalgas , Biomarcadores , Biotecnologia , Difusão Dinâmica da LuzRESUMO
Extracellular vesicles (EVs) isolated from biological samples are a promising material for use in medicine and technology. However, the assessment methods that would yield repeatable concentrations, sizes and compositions of the harvested material are missing. A plausible model for the description of EV isolates has not been developed. Furthermore, the identity and genesis of EVs are still obscure and the relevant parameters have not yet been identified. The purpose of this work is to better understand the mechanisms taking place during harvesting of EVs, in particular the role of viscosity of EV suspension. The EVs were harvested from blood plasma by repeated centrifugation and washing of samples. Their size and shape were assessed by using a combination of static and dynamic light scattering. The average shape parameter of the assessed particles was found to be ρ ~ 1 (0.94-1.1 in exosome standards and 0.7-1.2 in blood plasma and EV isolates), pertaining to spherical shells (spherical vesicles). This study has estimated the value of the viscosity coefficient of the medium in blood plasma to be 1.2 mPa/s. It can be concluded that light scattering could be a plausible method for the assessment of EVs upon considering that EVs are a dynamic material with a transient identity.