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BACKGROUND: In recent years, dental pulp stromal cells (DPSCs) have emerged as a promising therapeutic approach for Parkinson's disease (PD), owing to their inherent neurogenic potential and the lack of neuroprotective treatments for this condition. However, uncertainties persist regarding the efficacy of these cells in an undifferentiated state versus a neuronally-induced state. This study aims to delineate the distinct therapeutic potential of uninduced and neuronally-induced DPSCs in a rodent model of PD induced by 6-Hydroxydopamine (6-OHDA). METHODS: DPSCs were isolated from human teeth, characterized as mesenchymal stromal cells, and induced to neuronal differentiation. Neuronal markers were assessed before and after induction. DPSCs were transplanted into the substantia nigra pars compacta (SNpc) of rats 7 days following the 6-OHDA lesion. In vivo tracking of the cells, evaluation of locomotor behavior, dopaminergic neuron survival, and the expression of essential proteins within the dopaminergic system were conducted 7 days postgrafting. RESULTS: Isolated DPSCs exhibited typical characteristics of mesenchymal stromal cells and maintained a normal karyotype. DPSCs consistently expressed neuronal markers, exhibiting elevated expression of ßIII-tubulin following neuronal induction. Results from the animal model showed that both DPSC types promoted substantial recovery in dopaminergic neurons, correlating with enhanced locomotion. Additionally, neuronally-induced DPSCs prevented GFAP elevation, while altering DARPP-32 phosphorylation states. Conversely, uninduced DPSCs reduced JUN levels. Both DPSC types mitigated the elevation of glycosylated DAT. CONCLUSIONS: Our results suggested that uninduced DPSCs and neuronally-induced DPSCs exhibit potential in reducing dopaminergic neuron loss and improving locomotor behavior, but their underlying mechanisms differ.
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Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.
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Proliferação de Células , Vesículas Extracelulares , Macrófagos , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/citologia , Movimento Celular , Células THP-1 , Fibroblastos/metabolismo , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Queratinócitos/metabolismo , Queratinócitos/citologia , Citocinas/metabolismoRESUMO
Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 µg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.
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Vesículas Extracelulares , Cicatrização , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Proteômica , Secretoma , Células-Tronco/metabolismo , Queratinócitos , Vesículas Extracelulares/metabolismo , Dente DecíduoRESUMO
Mangroves are a challenging ecosystem for the microorganisms that inhabit them, considering they are subjected to stressful conditions such as high and fluctuating salinity. Metagenomic analysis of mangrove soils under contrasting salinity conditions was performed at the mouth of the Ranchera River to the Caribbean Sea in La Guajira, Colombia, using shotgun sequencing and the Illumina Hiseq 2500 platform. Functional gene analysis demonstrated that salinity could influence the abundance of microbial genes involved in osmoprotectant transport, DNA repair, heat shock proteins (HSP), and Quorum Sensing, among others. In total, 135 genes were discovered to be linked to 12 pathways. Thirty-four genes out of 10 pathways had statistical differences for a p-value and FDR < 0.05. UvrA and uvrB (nucleotide excision repair), groEL (HSP), and secA (bacterial secretion system) genes were the most abundant and were enriched by high salinity. The results of this study showed the prevalence of diverse genetic mechanisms that bacteria use as a response to survive in the challenging mangrove, as well as the presence of various genes that are recruited in order to maintain bacterial homeostasis under conditions of high salinity.
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Ecossistema , Salinidade , Solo , Metagenoma , Região do CaribeRESUMO
Cartilage repair has been a challenge in the medical field for many years. Although treatments that alleviate pain and injury are available, none can effectively regenerate the cartilage. Currently, regenerative medicine and tissue engineering are among the developed strategies to treat cartilage injury. The use of stem cells, associated or not with scaffolds, has shown potential in cartilage regeneration. However, it is currently known that the effect of stem cells occurs mainly through the secretion of paracrine factors that act on local cells. In this review, we will address the use of the secretome-a set of bioactive factors (soluble factors and extracellular vesicles) secreted by the cells-of mesenchymal stem cells as a treatment for cartilage regeneration. We will also discuss methodologies for priming the secretome to enhance the chondroregenerative potential. In addition, considering the difficulty of delivering therapies to the injured cartilage site, we will address works that use hydrogels functionalized with growth factors and secretome components. We aim to show that secretome-functionalized hydrogels can be an exciting approach to cell-free cartilage repair therapy.
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Cartilagem Articular , Células-Tronco Mesenquimais , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Secretoma , Engenharia TecidualRESUMO
Endothelial-like cells may be obtained from CD133+ mononuclear cells isolated from human umbilical cord blood (hUCB) and expanded using endothelial-inducing medium (E-CD133 cells). Their use in regenerative medicine has been explored by the potential not only to form vessels but also by the secretion of bioactive elements. Extracellular vesicles (EVs) are prominent messengers of this paracrine activity, transporting bioactive molecules that may guide cellular response under different conditions. Using RNA-Seq, we characterized the miRNA content of EVs derived from E-CD133 cells cultivated under normoxia (N-EVs) and hypoxia (H-EVs) and observed that changing the O2 status led to variations in the selective loading of miRNAs in the EVs. In silico analysis showed that among the targets of differentially loaded miRNAs, there are transcripts involved in pathways related to cell growth and survival, such as FoxO and HIF-1 pathways. The data obtained reinforce the pro-regenerative potential of EVs obtained from E-CD133 cells and shows that fine tuning of their properties may be regulated by culture conditions.
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Vesículas Extracelulares , MicroRNAs , Proliferação de Células , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Hipóxia/metabolismo , MicroRNAs/metabolismoRESUMO
Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133+ cells (eCD133+) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133+ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133+ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Insuficiência Renal Crônica , Animais , Células Cultivadas , Vesículas Extracelulares/metabolismo , Sangue Fetal , Células-Tronco Mesenquimais/metabolismo , Ratos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/terapiaRESUMO
Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.
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Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas Reguladoras de Apoptose , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Neoplasias Pulmonares/genética , Osteossarcoma/genética , Proteômica , Secretoma , Regulação para CimaRESUMO
DDX6 helicase is an RNA-binding protein involved in different aspects of gene expression regulation. The roles played by DDX6 depend on the complexes associated with it. Here, for the first time, we characterize the protein complexes associated with DDX6 in human adipose tissue-derived stem cells (hASCs) and analyze the dynamics of this helicase under different conditions of translational activity and differentiation. The results obtained demonstrated that the DDX6 helicase is associated with proteins involved in the control of mRNA localization, translation and metabolism in hASCs. DDX6 complexes may also assemble into more complex structures, such as RNA-dependent granules, the abundance and composition of which change upon inhibited translational activity. This finding supports the supposition that DDX6 is possibly involved in the regulation of the mRNA life cycle in hASCs. Although there was no significant variation in the protein composition of these complexes during early adipogenic or osteogenic induction, there was a change in the distribution pattern of DDX6: the number of DDX6 granules per cell was reduced during adipogenesis and was enhanced during osteogenesis.
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Adipogenia , Tecido Adiposo/citologia , Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/metabolismo , Osteogênese , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adipogenia/genética , Adolescente , Adulto , Proteínas de Transporte/genética , Biologia Computacional/métodos , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/genética , Feminino , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Osteogênese/genética , Ligação Proteica , Transporte Proteico , Proteômica , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
Mesenchymal stromal cells (MSCs) can self-renew, differentiate into specialised cells and have different embryonic origins-ectodermal for dental pulp-derived MSCs (DPSCs) and mesodermal for adipose tissue-derived MSCs (ADSCs). Data on DPSCs adipogenic differentiation potential and timing vary, and the lack of molecular and genetic information prompted us to gain a better understanding of DPSCs adipogenic differentiation potential and gene expression profile. While DPSCs differentiated readily along osteogenic and chondrogenic pathways, after 21 days in two different types of adipogenic induction media, DPSCs cultures did not contain lipid vacuoles and had low expression levels of the adipogenic genes proliferator-activated receptor gamma (PPARG), lipoprotein lipase (LPL) and CCAAT/enhancer-binding protein alpha (CEBPA). To better understand this limitation in adipogenesis, transcriptome analysis in undifferentiated DPSCs was carried out, with the ADSC transcriptome used as a positive control. In total, 14,871 transcripts were common to DPSCs and ADSCs, some were unique (DPSCs: 471, ADSCs: 1032), and 510 were differentially expressed genes. Detailed analyses of overrepresented transcripts showed that DPSCs express genes that inhibit adipogenic differentiation, revealing the possible mechanism for their limited adipogenesis.
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Adipogenia/genética , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Proteína Morfogenética Óssea 1/genética , Proteína Morfogenética Óssea 1/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Imunofenotipagem , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Família Multigênica , PPAR gama/genética , PPAR gama/metabolismo , RNA-Seq , Vacúolos/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Tissue-engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long-term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)-based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose-derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM-cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.
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Próteses Valvulares Cardíacas , Valvas Cardíacas/cirurgia , Valva Pulmonar/cirurgia , Transplante Heterólogo , Animais , Valva Aórtica/cirurgia , Fenômenos Biomecânicos/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Valvas Cardíacas/fisiologia , Humanos , Masculino , Suínos , Engenharia Tecidual/métodosRESUMO
Pericardial membrane derived from bovine heart tissues is a promising source of material for use in tissue-engineering applications. However, tissue processing is required for its use in humans due to the presence of animal antigens. Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the bovine pericardium (BP) after a soft decellularization process with a 0.1% sodium dodecyl sulfate (SDS) solution, with the aim to remove xenoantigens and preserve extracellular matrix (ECM) bioactivity. The decellularization process promoted a mean reduction of 77% of the amount of DNA in the samples in which cell nuclei staining was undetectable. The ECM content was maintained as mostly preserved after decellularization as well as its biomechanical properties. In addition, the decellularization protocol has proven to be efficient in removing the xenoantigen alpha-gal, which is responsible for immune rejection. The decellularized BP was noncytotoxic in vitro and allowed human adipose-derived stem cell (hASC) adhesion. Finally, after 7 days in culture, the tissue scaffold became repopulated by hASCs, and after 30 days, the ECM protein pro-collagen I was seen in the scaffold. Together, these characteristics indicated that soft BP decellularization with 0.1% SDS solution allows the acquirement of a bioactive scaffold suitable for cell repopulation and potentially useful for regenerative medicine.
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Matriz Extracelular/imunologia , Pericárdio/imunologia , Engenharia Tecidual , Alicerces Teciduais , Animais , Bovinos , Matriz Extracelular/metabolismo , Humanos , Dodecilsulfato de Sódio/metabolismo , Engenharia Tecidual/métodos , Transplante Heterólogo/métodosRESUMO
Mesenchymal stem cells (MSCs) represent an interesting population due to their capacity to release a variety of cytokines, chemokines, and growth factors, and due to their motile nature and homing ability. MSCs can be isolated from different sources, like adipose tissue or bone marrow, and have the capacity to differentiate, both in vivo and in vitro, into adipocytes, chondrocytes, and osteoblasts, making them even more interesting in the regenerative medicine field. Tumor associated stroma has been recognized as a key element in tumor progression, necessary for the biological success of the tumor, and MSCs represent a functionally fundamental part of this associated stroma. Exosomes represent one of the dominant signaling pathways within the tumor microenvironment. Their biology raises high interest, with implications in different biological processes involved in cancer progression, such as the formation of the pre-metastatic niche. This is critical during the metastatic cascade, given that it is the formation of a permissive context that would allow metastatic tumor cells survival within the new environment. In this context, we explored the role of exosomes, particularly MSCs-derived exosomes as direct or indirect modulators. All this points out a possible new tool useful for designing better treatment and detection strategies for metastatic progression, including the management of chemoresistance.
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Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Metástase Neoplásica/patologia , Animais , Humanos , Tropismo , Microambiente TumoralRESUMO
Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.
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Vesículas Extracelulares/metabolismo , Valvas Cardíacas/química , Células-Tronco Mesenquimais/citologia , Proteínas/metabolismo , Proteômica/métodos , Animais , Brasil , Adesão Celular , Proliferação de Células , Células Cultivadas , Ontologia Genética , Valvas Cardíacas/metabolismo , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Nanotecnologia , Neovascularização Fisiológica , Suínos , Bancos de TecidosRESUMO
Two essential oils (EO), thymol and carvacrol, were used in six ratio (100:00, 80:20, 60:40, 40:60, 20:80 and 00:100) combinations of both EO and in a dose of 0.2 g L-1 in bovine ruminal culture medium, 24-h cultures, to evaluate effects on total gas production (TGP), methane production, in vitro dry matter digestibility (IVDMD) and in vitro culture population dynamics of methanogenic and total bacteria. Total DNA extracted from ruminal microorganisms was subjected to denaturing gradient gel electrophoresis (DGGE)-polymerase chain reaction (PCR) to examine effects on bacterial populations. The effect of EO on TGP and IVDMD were assessed by comparison to untreated control cultures. In general, methane production by the microbial populations appeared to be higher with treatments containing the highest concentration of thymol than with treatments containing more carvacrol resulting in a tendency for greater methane-inhibiting activity achieved as the thymol concentration in the thymol:carvacrol mixtures decreased linearly. The population of total bacteria with a 74.5% Dice similarity coefficient for comparison of DGGE band patterns indicating shifts in bacterial constituents as EO ratios changed. No effects on TGP, IVDMD while only slight shifts in the methanogenic populations were seen with an overall 91.5% Dice similarity coefficient.
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Microbioma Gastrointestinal/efeitos dos fármacos , Metano/metabolismo , Monoterpenos/farmacologia , Rúmen/microbiologia , Timol/farmacologia , Animais , Técnicas Bacteriológicas , Bovinos , Cimenos , Eletroforese em Gel de Gradiente Desnaturante , Digestão , Fermentação , Microbioma Gastrointestinal/fisiologia , Óleos Voláteis/farmacologia , Reação em Cadeia da PolimeraseRESUMO
In the last decade, the interest in Indoor Location Based Services (ILBS) has increased stimulating the development of Indoor Positioning Systems (IPS). In particular, ILBS look for positioning systems that can be applied anywhere in the world for millions of users, that is, there is a need for developing IPS for mass market applications. Those systems must provide accurate position estimations with minimum infrastructure cost and easy scalability to different environments. This survey overviews the current state of the art of IPSs and classifies them in terms of the infrastructure and methodology employed. Finally, each group is reviewed analysing its advantages and disadvantages and its applicability to mass market applications.
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The increasing development of the automotive industry towards a fully autonomous car has motivated the design of new value-added services in Vehicular Sensor Networks (VSNs). Within the context of VSNs, the autonomous car, with an increasing number of on-board sensors, is a mobile node that exchanges sensed and state information within the VSN. Among all the value added services for VSNs, the design of new intelligent parking management architectures where the autonomous car will coexist with traditional cars is mandatory in order to profit from all the opportunities associated with the increasing intelligence of the new generation of cars. In this work, we design a new smart parking system on top of a VSN that takes into account the heterogeneity of cars and provides guidance to the best parking place for the autonomous car based on a collaborative approach that searches for the common good of all of them measured by the accessibility rate, which is the ratio of the free parking places accessible for an autonomous car. Then, we simulate a real parking lot and the results show that the performance of our system is close to the optimum considering different communication ranges and penetration rates for the autonomous car.
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In recent years, there has been an increasing interest in the development of pedestrian navigation systems for satellite-denied scenarios. The popularization of smartphones and smartwatches is an interesting opportunity for reducing the infrastructure cost of the positioning systems. Nowadays, smartphones include inertial sensors that can be used in pedestrian dead-reckoning (PDR) algorithms for the estimation of the user's position. Both smartphones and smartwatches include WiFi capabilities allowing the computation of the received signal strength (RSS). We develop a new method for the combination of RSS measurements from two different receivers using a Gaussian mixture model. We also analyze the implication of using a WiFi network designed for communication purposes in an indoor positioning system when the designer cannot control the network configuration. In this work, we design a hybrid positioning system that combines inertial measurements, from low-cost inertial sensors embedded in a smartphone, with RSS measurements through an extended Kalman filter. The system has been validated in a real scenario, and results show that our system improves the positioning accuracy of the PDR system thanks to the use of two WiFi receivers. The designed system obtains an accuracy up to 1.4 m in a scenario of 6000 m 2 .
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Pedestres , Smartphone , Algoritmos , Técnicas Biossensoriais/métodos , Humanos , Internet , Caminhada/fisiologiaRESUMO
BACKGROUND: DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. RESULTS: We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. CONCLUSIONS: Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Grânulos Citoplasmáticos/genética , Estresse Oxidativo/genética , Ribonucleoproteínas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células HeLa , Proteínas Hedgehog/genética , Humanos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genéticaRESUMO
Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs' prospective applications as PSC-derived cell-free therapy products.