Month: June 2019

Atrophic myotubes presented molecular characteristics that are typically observed following DUX4 expression. Conversely, disorganized myotubes presented increased Lomitapide Mesylate levels of proteins involved in microtubule network organization and myofibrillar remodeling, which suggests a compensatory response to DUX4-mediated damage. Further studies are necessary to determine the relative contribution of other 4q35 genes leading to this phenotype. Moreover, our results suggest that FSHD pathogenesis could partially involve a defect of membrane microdomains as observed in other neuromuscular disorders. Finally, the study of a fraction enriched in nuclear proteins suggested a defect in RNA processing in FSHD myotubes. The temporo-mandibular joint disc is a fibrocartilaginous tissue that lies between the mandibular condyle and the temporal fossa-eminence. Several disorders may affect the TMJ disc, including intra-articular positional and structural abnormalities with high prevalence in adult populations, especially TMJ degenerative diseases, known as osteoarthrosis or osteoarthritis. Butenafine hydrochloride clinical management of the most prevalent TMJ disc disorders is very challenging due to the low regeneration capability of human cartilage, and emerging therapies based on cultured human TMJF and tissue engineering represent a novel treatment possibility. The TMJ disc is mainly composed by fibrochondrocytes, which have features of both chondrocytes and fibroblasts. Human TMJF are known to have the capability to synthetize different fibrillar extracellular matrix constituents, mainly collagen, and several non-fibrillar components, and to proliferate faster than hyaline chondrocytes. The distribution of TMJF into the disc appears to be heterogeneous, and cells tend to show a round morphology surrounded by pericellular matrix. Several efforts are currently ongoing in the field of TMJ disc tissue engineering using an immense variety of scaffolds and cell sources. Nevertheless, the scarce number of cells that can be obtained from small TMJ disc tissue biopsies and the drop of cell viability and cell differentiation levels caused by continuous cell passaging in order to obtain large amounts of cells, are significant limitations associated to TMJF culturing and TMJ disc tissue engineering. All these limitations can result in the failure of cell therapy and tissue engineering strategies of the human TMJ disc repair. For these reasons, a deep study of sequential cell passages of cultured human TMJF might be a useful tool for tissue engineers in order to select the most suitable cell passage in terms of cell viability and differentiation from a clinical standpoint. In fact, several previous studies previously demonstrated that cell viability may vary among several cell passages and that selection of the most adequate cell passage is very important for cell therapy success. In this study, we carried out a comprehensive analysis of cell proliferation, cell viability and cell function on 9 consecutive cell passages of human TMJF to determine which passage is the most adequate for future clinical use. A therapeutic advance in the treatment of TMJ pathological conditions could be the generation of biological substitutes of damaged discs generated by tissue engineering. Different models of engineered TMJ disc have been developed by using animal cells and different biomaterials and signaling. In most of these studies, the key importance of an accurate cell viability determination has been established, since only viable cells should be used for TMJ disc tissue engineering.

Similarly, our study highlights perturbations in the relative abundance of several caveolar proteins in FSHD myotubes. A defect in these membrane microdomain subtypes could also contribute to myotube deformation in FSHD. Because the alteration of caveolar proteins was also found in atrophic myotubes, further studies are necessary to precisely determine the contribution of each factor to the formation of a given phenotype. Because the predominantly atrophic or disorganized FSHD cultures that we have Albaspidin-AA analyzed are derived from comparable patients in terms of the number of D4Z4 units, sex and age, we assume that other factors could intervene to explain the emergence of a non-atrophic phenotype, despite the expression of DUX4. Other genes were suggested to be involved in FSHD, including FRG1, ANT1 and DUX4c, but further studies are necessary to explain the relative contribution of each 4q35 gene in FSHD. DUX4c is induced in FSHD muscles and could bind to DUX4-target promoters through its identical double homeodomain, as was described for PITX1. Because DUX4c overexpression is associated with increased myoblast proliferation and decreased differentiation, it is a good candidate to explain the emergence of a non-atrophic phenotype. DUX4-s, which is a putative protein derived from a short DUX4 mRNA variant that is often detected in control muscles and less frequently in FSHD muscles, was suggested to act as a dominant negative variant. DUX4-s may also take part in this process, but further studies are needed to determine whether this protein is endogenously expressed in FSHD or control muscle cells. The present data indicate that FSHD myotubes present clear changes in the relative abundance of proteins typical of caveolae, which are membrane lipid microdomains that are enriched in cholesterol and glycosphingolipids and are often considered to be a specialized lipid raft subtype. These membrane invaginations play a major role in signal transduction and appear to constitute signaling platforms that mediate the sequestration of certain receptors, transporters and signaling proteins. They are thus involved in numerous biological processes, e.g., membrane repair, redox signaling, immune response and lipid metabolism. Caveolin3 is the main caveolar protein in skeletal muscle and is a key factor in muscle cell fusion, and several mutations in the CAV3 gene cause heterogeneous neuromuscular diseases including caveolinopathies such as LGMD1. Caveolin-associated cavins, particularly PTRF/cavin-1, are crucial regulators of caveola formation. MURC/cavin-4 was first described as a cytosolic protein that is partly localized in the Z-line and associated with cardiac dysfunction through the modulation of the Rho/ROCK pathway. MURC expression is increased Lomitapide Mesylate during the differentiation of C2C12 myoblasts, and its RNAi-mediated knockdown impairs myogenic differentiation. In the present study, we reported a decreased level of MURC in FSHD myotubes. FSHD myoblasts fail to upregulate MURC during their differentiation, and this perturbation could also be linked to the general dampening of myogenic differentiation associated with FSHD as described in. Recently, MURC was found to be localized to sarcolemmal caveolae in normal muscle, with an impaired distribution in muscle from a patient with heterogeneous CAV3 expression, which suggests a potential role of MURC in caveolin-associated muscle disease. In conclusion, the use of an optimized proteomic approach has enabled us to define molecular differences between atrophic and disorganized FSHD.

Inversely to the 2-DE observation, the protein level of ENO1 showed a 1.5 fold reduction in 35.3% of GC samples compared to their paired controls by western blot. Only one sample presented a 1.5 fold increase. However, we only selected the spots differentially expressed with a 1.5 fold change between groups for the mass spectrometry analysis. These selection criteria may lead to the lack of correlation between western blot and proteomic analyses. Thus, other spots of ENO1 may present a slight reduced expression, but with a high impact in the mean of this protein expression. Our results show that different spots may be regulated differently inside a heterogeneous gastric sample. Our findings also highlight that the metabolic phenotype is not universal in tumor cells, especially considering that different cell clones are present inside a single 3,4,5-Trimethoxyphenylacetic acid cancer sample. Even in glycolytic tumors, oxidative phosphorylation is not completely shut down. Owing to the dynamic nature of the tumor microenvironment, it is suggested that the metabolic phenotype of tumor cells changes to adapt to the prevailing local conditions. The regulation of this metabolic flexibility is poorly understood. However, the feedback control between MYC and ENO1, as well as MBP1, may have a key role in this process since the MYC oncogene may stimulate both glycolysis and oxidative phosphorylation. However, these authors described that ENO1 immnuoreactivity seems to be significantly more intense in GC cells than non-neoplastic cells and its positive expression tends to be associated with poor prognosis. This in part corroborates our results that demonstrate that the level of ENO1 protein seems to be reduced more frequently in less invasive cancer samples. HSPB1 was selected for further investigation also due to its protective function against 4-(Benzyloxy)phenol infection and cellular stress. HSPB1 is one member of the family of heat shock proteins that is characterized as molecular chaperones. In addition to its chaperone function, HSPB1 also seems to be an important regulator of structural integrity and membrane stability, actin polymerization and intermediate filament cytoskeleton formation, cell migration, epithelial cell-cell adhesion, cell cycle progression, proinflammatory gene expression, muscle contraction, signal transduction pathways, mRNA stabilization, presentation of oxidized proteins to the proteasome, differentiation, and apoptosis. HSPB1 is highly induced by different stresses such as heat, oxidative stress, or anticancer drugs. In non stressed cells, HSPB1 is not expressed or at very low levels. Once induced, HSPB1 acts at multiple points in the apoptotic pathways to ensure that stressinduced damage does not inappropriately trigger cell death. Many cancer cells have markedly increased HSPB1 levels, and this protein expression contributes to the malignant properties of these cells, including increased tumorigenicity and treatment resistance, and apoptosis inhibition. Overexpression of HSPB1 has been described in several tumors and it has been reported as an indicator of poor prognosis. Elevated HSPB1 expression in neoplastic cells plays a key role in protection from spontaneous apoptosis in response to anticancer therapy and leading to tumor progression and resistance to treatment. In the present study, we observed one spot of the HSPB1 protein that presented a higher expression in GC compared to controls by 2-DE analysis, corroborating previously proteomic studies with GC patients from Asiatic countries. Here, no association was observed among HSPB1 expression and clinicopathological characteristics in the present study. However, HSPB1 was previously associated with gastric tumor size, distant metastasis, lymph node state and pStage in other populations. Despite the fact that HSPB1 expression was not associated with any clinicopathological characteristic in our population.

The possibility that miRs might modulate mRNA levels and subsequent toxicity by TCDD has not been fully explored. Recently, a few studies have begun exploring such mechanisms. One study reported that miR-27b related to AhR-regulated genes increased CYP1B1 levels. In another study, it was noted that treatment with TCDD in vivo caused few changes in miR levels in mouse or rat livers, and those changes that were statistically significant were of modest magnitude. These data are consistent with our studies where we noted that the magnitude of change in miR expression following TCDD treatment in most Tulathromycin B instances was 1.5 to 2 fold and only a few miRs showed 3�C8 fold change. The fact that liver may be more refractory was also indicated in another study in which it was noted that AhR activation by benzopyrene did not cause significant changes in miRs of the liver but altered the miR profiles in the lung. The miRs that were altered by BaP were involved in immune response, cell proliferation and cell cycle. Thus, it is likely that the AhR-agonist mediated changes in miRs may be organ-specific. While, the immunotoxic effects of prenatal exposure to TCDD on fetal thymocytes have been well characterized, there are no reports on such effects of TCDD on miR profiles. Understanding the role of various miRs in neonatal mice post-TCDD exposure may shed light on the “fetal basis of adult disease” hypothesis. This hypothesis proposes that many chronic diseases including autoimmune diseases during adult stage of life may be the result of prenatal exposure to nutritional, environmental or other forms of stress. In this study, therefore, we sought to examine miR profile in fetuses post-TCDD exposure. The cluster analysis data of miRs showed that TCDD caused significant changes in miR expression profile in fetal thymi when compared to vehicle-treated thymi. Of the miRs screened, 78 miRs were altered more than 1.5 fold and 28 miRs were altered two fold or more, post-TCDD exposure. We further validated the expression profile of some select miRs by performing Real-Time PCR. All the miRs that we analyzed by Real-Time PCR corroborated the data obtained from miR array analysis. Furthermore, the relationship of miRs and their target gene expression was also verified. For example, miRs that showed highly complementary sequence with 39UTR of AhR, CYP1A1, Fas, and FasL genes were downregulated by TCDD in fetal thymi and the data obtained from RT-PCR showed upregulated expression of the above genes in fetal thymi post-TCDD exposure. such as breast, cartilage, endothelial cells, embryonic tissues, etc. These downregulated miRs have been shown to control genes that are involved in various physiological functions in these tissues. Previous studies from our laboratory have demonstrated that TCDD-induced thymic atrophy in the adult and fetus may result, at least in part, from induction of apoptosis. We have also reported that such Folinic acid calcium salt pentahydrate apoptosis may be induced through the extrinsic pathway by the induction of Fas and FasL in thymocytes. Also miR200a has been shown to regulate apoptosis, whereas miR-491 has been shown to induce apoptosis by targeting Bcl-xL gene. Thus, these miRs may directly/indirectly be involved in apoptosis of thymic cells leading to thymic atrophy. TCDD has also been shown to cause cancer in various species and it is also considered to be a potential carcinogen in humans. There are reports demonstrating that TCDD exposure of mice triggers cutaneous papillomas and squamous cell carcinoma. In another report, prenatal TCDD exposure of rats was shown to make them susceptible to breast cancer. TCDD has also been shown to promote liver cancer. miRs have been shown to influence signaling pathways leading to development of various types of cancer.

Since sequences in the background set used for comparison also contain RCGTG motifs, this enrichment likely arises from the well known preference for A versus G in the first position of the HIF Orbifloxacin binding consensus. These results collectively suggest that several additional transcription factors could influence HIF transcriptional activity. Importantly, we noted that most of the enriched TFBSs corresponded to stress-responsive transcription factors. Varied stress-responsive TFs have been shown to coordinately regulate the same genes, and indeed several transcription factors are activated by the same stresses in mammalian cells. However, it is unclear whether this cooperation among stressresponsive pathways translates at the genomic level. In order to evaluate the functional significance of the TFBSs enriched in core HIF binding regions, we carried out an experimental validation by disrupting selected sequences in bona fide HIF-responsive promoters. Importantly, no experimental confirmation had been attempted on previously reported predictions, and therefore the biological significance of those findings remained unclear. In spite of being limited to three selected promoters, our results clearly indicate that, compared to control mutations, alteration of binding sequences of transcription factors enriched in HIF binding regions, and different from HIFs themselves, have a specific effect on the transcriptional activation of HIF-responsive promoters. In particular, we found negative effects on hypoxic induction of LDHA and GYS1 promoters upon disruption of CREB and CEBPB binding sites proximal to the HRE, whereas mutation of an AP1 site proximal to the CA9 HRE led to a slightly augmented hypoxic induction of the promoter. In agreement with our results, mutation of the same CREB binding site was been previously shown to alter LDHA hypoxic induction. Interestingly, USF binding to a palindrome CACGTG HRE in the LDHA promoter was suggested to complement HIF binding. However, our results do not allow us to corroborate these findings, as mutation of this HRE was not evaluated in our experiments. Furthermore, hypoxic CA9 Catharanthine sulfate expression has been linked to cooperation between AP1 family member ATF4 and HIF1a. In this study, ATF4 overexpression led to an augmented CA9 induction in hypoxia, with reduced hypoxic expression of CA9 being observed upon ATF4 knock-down. Chromatin immunoprecipitation experiments mapped ATF4 binding to the 21400/21000 region of the CA9 promoter, which falls outside of the promoter region employed in our experiments. Nevertheless, the apparent paradox with our results argues for careful interpretation of the role of AP1 in the HIF transcriptional response. In fact, both positive and negative effects of AP1 have been reported on hypoxic gene expression and, given the number of AP1 family members, these probably arise from compositional differences in AP1 complexes. Importantly, the effects observed upon mutation of CREB, CEBPB or AP1 binding sites were always moderate when compared to mutation of the HIF binding consensus RCGTG, suggesting that rather than being an absolute requirement for hypoxic induction, the integrity of these neighboring TFBSs fine-tunes the HIF-mediated transcriptional response. Thus, it is possible that multiple independent factors contribute, in an additive fashion, to HIF-mediated transcription. This model could also explain why we found a relatively large number of enriched TFBSs in HIF binding regions, but all of them sharing a modest statistical significance. On the whole, these observations indicate that several of the enriched TFBSs identified in our approach are of functional relevance for HIF-mediated transcription. Nevertheless, it should be noted that other TFs for which collaboration with HIFs has been previously suggested are not recovered as enriched in our approach.