1,235 research outputs found

    Impaired receptivity of thin endometrium: therapeutic potential of mesenchymal stem cells

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    The endometrium is a resilient and highly dynamic tissue, undergoing cyclic renewal in preparation for embryo implantation. Cyclic endometrial regeneration depends on the intact function of several cell types, including parenchymal, endothelial, and immune cells, as well as adult stem cells that can arise from endometrial or extrauterine sources. The ability of the endometrium to undergo rapid, repeated regeneration without scarring is unique to this tissue. However, if this tissue renewal process is disrupted or dysfunctional, women may present clinically with infertility due to endometrial scarring or persistent atrophic/thin endometrium. Such disorders are rate-limiting in the treatment of female infertility and in the success of in vitro fertilization because of a dearth of treatment options specifically targeting the endometrium. A growing number of studies have explored the potential of adult stem cells, including mesenchymal stem cells (MSCs), to treat women with disorders of endometrial regeneration. MSCs are multipotent adult stem cells with capacity to differentiate into cells such as adipocytes, chondrocytes, and osteoblasts. In addition to their differentiation capacity, MSCs migrate toward injured sites where they secrete bioactive factors (e.g. cytokines, chemokines, growth factors, proteins and extracellular vesicles) to aid in tissue repair. These factors modulate biological processes critical for tissue regeneration, such as angiogenesis, cell migration and immunomodulation. The MSC secretome has therefore attracted significant attention for its therapeutic potential. In the uterus, studies utilizing rodent models and limited human trials have shown a potential benefit of MSCs and the MSC secretome in treatment of endometrial infertility. This review will explore the potential of MSCs to treat women with impaired endometrial receptivity due to a thin endometrium or endometrial scarring. We will provide context supporting leveraging MSCs for this purpose by including a review of mechanisms by which the MSC secretome promotes regeneration and repair of nonreproductive tissues

    Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca2+-dependent gap junction intercellular communication

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    Abstract. Background:. Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Methods:. Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca2+-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Results:. Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Conclusions:. Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy

    Development of in vitro blood-brain barrier models: from traditional 2D models to microfluidics

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    Ingles. The blood-brain barrier (BBB) is a highly specialised complex structure, and is directly responsible for substances and drugs entering the central nervous system from the blood. Traditional in vitro models have problems to mimic the properties of the barrier in vivo. Therefore, the aim of this project is to work on the development of an in vitro model of the blood-brain barrier based on a microfluidic chip device. By studying the transition from a traditional model using transwell inserts to a three-dimensional model using a microfluidic chip device, which has features that better mimic the barrier in vivo. Progress was made in the development of the two-dimensional model in the inserts and the first steps for implementation in the three-dimensional model were taken

    Two-compartment neuronal spiking model expressing brain-state specific apical-amplification, -isolation and -drive regimes

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    There is mounting experimental evidence that brain-state specific neural mechanisms supported by connectomic architectures serve to combine past and contextual knowledge with current, incoming flow of evidence (e.g. from sensory systems). Such mechanisms are distributed across multiple spatial and temporal scales and require dedicated support at the levels of individual neurons and synapses. A prominent feature in the neocortex is the structure of large, deep pyramidal neurons which show a peculiar separation between an apical dendritic compartment and a basal dentritic/peri-somatic compartment, with distinctive patterns of incoming connections and brain-state specific activation mechanisms, namely apical-amplification, -isolation and -drive associated to the wakefulness, deeper NREM sleep stages and REM sleep. The cognitive roles of apical mechanisms have been demonstrated in behaving animals. In contrast, classical models of learning spiking networks are based on single compartment neurons that miss the description of mechanisms to combine apical and basal/somatic information. This work aims to provide the computational community with a two-compartment spiking neuron model which includes features that are essential for supporting brain-state specific learning and with a piece-wise linear transfer function (ThetaPlanes) at highest abstraction level to be used in large scale bio-inspired artificial intelligence systems. A machine learning algorithm, constrained by a set of fitness functions, selected the parameters defining neurons expressing the desired apical mechanisms.Comment: 19 pages, 38 figures, pape

    In vitro models of breast cancer bone metastasis: analyzing drug resistance through the lens of the microenvironment

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    Even though breast cancers usually have a good outcome compared to other tumors, the cancer can progress and create metastases in different parts of the organism, the bone being a predilection locus. These metastases are usually the cause of death, as they are mostly resistant to treatments. This resistance can be caused by intrinsic properties of the tumor, such as its heterogeneity, but it can also be due to the protective role of the microenvironment. By activating signaling pathways protecting cancer cells when exposed to chemotherapy, contributing to their ability to reach dormancy, or even reducing the amount of drug able to reach the metastases, among other mechanisms, the specificities of the bone tissue are being investigated as important players of drug resistance. To this date, most mechanisms of this resistance are yet to be discovered, and many researchers are implementing in vitro models to study the interaction between the tumor cells and their microenvironment. Here, we will review what is known about breast cancer drug resistance in bone metastasis due to the microenvironment and we will use those observations to highlight which features in vitro models should include to properly recapitulate these biological aspects in vitro. We will also detail which elements advanced in vitro models should implement in order to better recapitulate in vivo physiopathology and drug resistance

    Pan-cancer analysis of the prognostic and immunological role of GJB2: a potential target for survival and immunotherapy

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    BackgroundGJB2 plays an essential role in the growth and progression of several cancers. However, asystematic pan-cancer analysis of GJB2 is lacking. Therefore, in this study, we performed a comprehensive pan-cancer analysis to determine the potential role of GJB2 in prognostic prediction and cancer immunotherapy response.MethodsThe differential expression of GJB2 in the tumor and adjacent normal tissues of various cancer types was analyzed using the TIMER, GEPIA, and Sangerbox databases. GEPIA and Kaplan–Meier plotter databases were used to analyze the survival outcomes based on GJB2 expression levels in pan-cancer. Furthermore, the association of GJB2 expression with the immune checkpoint (ICP) genes, tumor mutational load (TMB), microsatellite instability (MSI), neoantigens, and tumor infiltration of immune cells was analyzed using via the Sangerbox database. The cBioPortal database was used to determine the characteristics of GJB2 gene alterations in the cancer tissues. The STRING database was used to identify the GJB2-binding proteins. GEPIA database was used to identify the GJB2 co-expressed genes. DAVID was used to perform the functional enrichment analysis of gene ontology (GO) terms and KEGG pathways associated with GJB2. Finally, the mechanistic role of GJB2 in pancreatic adenocarcinoma (PAAD) was analyzed using the LinkedOmics database.ResultsThe GJB2 gene was highly expressed in a variety of tumors. Furthermore, GJB2 expression levels showed significant positive or negative association with the survival outcomes in various cancers. GJB2 expression levels cor related with tumor mutational burden, microsatellite instability, neoantigens, and tumor infiltration of immune cells in multiple cancers. This suggested that GJB2 played a critical role in the tumor microenvironment. Functional enrichment analysis showed that the biological role of GJB2 in tumors included modulation of gap junction-mediated intercellular transport, regulation of cell communication by electrical coupling, ion transmembrane transport, autocrine signaling, apoptotic signaling pathway, NOD-like receptor signaling pathway, p53 signaling pathway, and PI3K-Akt signaling pathway.ConclusionsOur study demonstrated that GJB2 played a significant role in tumorigenesis and tumor immunity in multiple cancers. Furthermore, GJB2 is a potential prognostic biomarker and a promising therapeutic target in multiple types of cancers

    Interactive mechanism between connexin43 and Cd-induced autophagic flux blockage and gap junctional intercellular communication dysfunction in rat hepatocytes

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    Cadmium (Cd) is a significant environmental contaminant known for its potential hepatotoxic effects. However, the precise mechanisms underlying Cd-induced hepatotoxicity have yet to be fully understood. Therefore, the purpose of this study was to investigate the dynamic role of connexin 43 (Cx43) in response to Cd exposure, particularly its impact on gap junctional intercellular communication (GJIC) and autophagy in hepatocytes. To establish an in vitro model of Cd-induced hepatocyte injury, the Buffalo rat liver 3A cell line (BRL3A) was utilized.In order to elucidate the mechanism by which Cx43 influences Cd-induced hepatocyte toxic injury, inhibitors of Cx43 (Dynasore) and P-Cx43 (Ro318220) were employed in the model. The findings revealed that inhibiting Cx43 and its phosphorylation further compromised GJIC function, exacerbating the impairment, while also intensifying the blockage of autophagic flux. To gain further insight into the role of Cx43, siRNA was utilized to knock down Cx43 expression, yielding similar results. The down-regulation of Cx43 expression was found to worsen the morphological damage induced by cadmium exposure, diminish the cell proliferation capacity of BRL3A cells, and exacerbate the disruption of GJIC and autophagic flow caused by Cd.These findings suggest that Cx43 may serve as a potential therapeutic target for the treatment of liver damage resulting from Cd exposure. By targeting Cx43, it may be possible to mitigate the adverse effects of Cd on hepatocytes

    Radiation-Induced Bystander Effect via GRID Radiotherapy and Medium Transfer in the A-375 Human Melanoma Cancer Cell Line: An In-vitro Study

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    Purpose: The goal of this research was to investigate the bystander effect in the A-375 cell line under the spatially fractionated radiation therapy (GRID therapy technique). In GRID therapy, due to direct and indirect cell damage after high-dose radiation, evaluation of Radiation-Induced Bystander Effects (RIBE) is of the most importance for investigating the risk of therapy. Materials and Methods: The potential role of RIBE was evaluated with different doses of 6 MeV electron radiation and different incubation times after irradiation using two methods; GRID therapy and medium transfer. Colony Formation Assay (CFA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test were used to detect the mentioned effects. Alpha and beta parameters were calculated from the cell survival curve by the quadratic-linear model. Results: The result showed that the survival fraction significantly decreases by increasing the radiation dose for both bystander and irradiated cells. However, a decrease in the number of colony-forming cells caused by electron radiation greater than 4MeV to target cells was significantly increased compared with bystander cells (P 0.05). Furthermore, the RIBE level in non-target cells increased up to a dose of 4Gy, but decreased significantly at doses higher than 4Gy. This result in high doses confirmed that a negative feedback mechanism was responsible for reducing the RIBE response. Conclusion: Based on the results, we can state there are classic radiation-induced bystander effects in A-375 monolayer exposed by GRID therapy and medium transfer technique, which can play an important role in pre-clinical and clinical studies

    Regulation of Cx43 Gap Junction Intercellular Communication by Bruton’s Tyrosine Kinase and Interleukin-2-Inducible T-Cell Kinase

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    T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca2+ and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43. Mass spectroscopy revealed that BTK and ITK phosphorylate Cx43 residues Y247, Y265, and Y313, which are identical to the residues phosphorylated by Src. Overexpression of BTK or ITK in the HEK-293T cells led to increased Cx43 tyrosine phosphorylation as well as decreased gap junction intercellular communication (GJIC) and Cx43 membrane localization. In the lymphocytes, activation of the B cell receptor (Daudi cells) or T cell receptor (Jurkat cells) increased the BTK and ITK activity, respectively. While this led to increased tyrosine phosphorylation of Cx43 and decreased GJIC, the cellular localization of Cx43 changed little. We have previously identified that Pyk2 and Tyk2 also phosphorylate Cx43 at residues Y247, Y265, and Y313 with a similar cellular fate to that of Src. With phosphorylation critical to Cx43 assembly and turnover, and kinase expression varying between different cell types, there would be a need for different kinases to achieve the same regulation of Cx43. The work presented herein suggests that in the immune system, ITK and BTK have the capacity for the tyrosine phosphorylation of Cx43 to alter the gap junction function in a similar manner as Pyk2, Tyk2, and Src

    Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

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    Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression
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