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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [7 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1577 journals]
  • Heparan Sulfate Proteoglycan Deficiency Up-Regulates the Intracellular
           Production of Nitric Oxide in Chinese Hamster Ovary Cell Lines
    • Abstract: We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2-) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1 and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/ oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells. This article is protected by copyright. All rights reserved
  • Progress and Prospects of Circular RNAs in Hepatocellular Carcinoma: Novel
           insights into their function
    • Abstract: Hepatocellular carcinoma (HCC) is one of the most predominant subjects of liver malignancies, which arouses global concern in the recent years. Advanced studies have found that Circular RNAs (circRNAs) are differentially expressed in HCC, with its regulatory capacity in HCC pathogenesis and metastasis. However, the underlying mechanism remains largely unknown. In this review, we summarized the functions and mechanisms of those aberrantly expressed circRNAs in HCC tissues. We hope to enlighten more comprehensive studies on the detailed mechanisms of circRNAs and explore their potential values in clinic applications. It revealed that hsa_circ_0004018 can be used as a potential biomarker in HCC diagnosis, with its superior sensitivity to alpha-fetoprotein (AFP). Notably, the correlation of circRNA abundance in the proliferation of liver regeneration (LR) has recently been clarified and different circRNA profiles served as candidates for nonalcoholic steatohepatitis (NASH) diagnosis also be discussed. Therefore, the improved understanding of circRNAs in HCC pathogenesis and metastasis proposed a novel basis for the early diagnosis in HCC patients, which provides a useful resource to explore the pathogenesis of HCC. This article is protected by copyright. All rights reserved
  • Development of the LYVE-1 Gene with An Acidic-amino-acid-rich (AAAR)
           Domain in Evolution Is Associated with Acquisition of Lymph Nodes and
           Efficient Adaptive Immunity
    • Abstract: CRSBP-1 (mammalian LYVE-1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF-BB and VEGF-A) containing CRS motifs (clusters of basic amino-acid residues). The ligand binding activities are mediated by Link module and acidic-amino-acid-rich (AAAR) domains, respectively. These CRSBP-1/LYVE-1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild-type mice. We hypothesize that CRSBP-1/LYVE-1 ligands, particularly CRS-containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE-1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE-1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE-1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP-1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF-BB and VEGF-A, respectively, specifically bind to CRSBP-1 but do not interact with either PDGFβR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus vaccine in pigs. These results support the notion that LYVE-1 is involved in adaptive immunity in mammals. This article is protected by copyright. All rights reserved
  • Tetrahydrocurcumin Ameliorates Homocysteine Mediated Mitochondrial
           Remodeling in Brain Endothelial Cells
    • Abstract: Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated of fission marker (DRP-1), fusion markers (Mfn2) and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) was co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15µM) ameliorated Hcy induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction. This article is protected by copyright. All rights reserved
  • Multifaceted role of IL-21 in rheumatoid arthritis: Current understanding
           and future perspectives
    • Abstract: Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder designated with hyperplastic synovium, bone destruction and cartilage degradation. Current therapies involve targeting major cytokines and inflammatory mediators involved in RA to alleviate the pain and provide a temporary relief. Interleukin 21 (IL-21), a recently identified cytokine is known to possess a versatile role in modulating the cells of the RA synovium. Over the past decade, the pleiotropic role of IL-21 in RA pathogenesis has been implicated in several aspects. T helper 17 (Th17) and follicular T helper cells (Tfh), being the key immunomodulators of the RA synovium secrete high amounts of IL-21 during disease progression. Several studies have provided experimental evidences elucidating the multifaceted role of IL-21 in RA disease progression. IL-21 has the potential to activate T cells, B cells, monocytes/macrophages and synovial fibroblasts in RA pathogenesis through activation of JAK-STAT, MAPK and PI3K/Akt signaling pathways. Till date, therapies targeting Th17 cells and its inflammatory cytokines have been under investigation and are subjected to various clinical trials. This review showcases the function of IL-21 in RA pathogenesis and recent reports implicating its function in various immune cells, major signaling pathways and in promoting osteoclastogenesis. This article is protected by copyright. All rights reserved
  • Simvastatin induces G1 arrest by up-regulating GSK3β and down-regulating
           CDK4/cyclin D1 and CDK2/cyclin E1 in human primary colorectal cancer cells
    • Abstract: Simvastatin (SIM), a widely used cholesterol-lowering drug, also exhibits tumor-suppressive potentials in several types of malignancy. Colorectal cancer (CRC), the third most common malignant neoplasm, accounts for the second most leading cause of cancer-related deaths worldwide. In the present study, we investigated the anticancer effects of SIM on CRC using primary cancer cells lines (CPs: CP1 to CP5) isolated from five Taiwanese colorectal cancer patients as a model for colorectal cancer. We treated all five CPs with SIM for 24 to 72 hours and observed the respective cell viability by an MTT assay. SIM increased DNA content of the G1 phase, but did not induce apoptosis/necrosis in CPs as shown by flow cytometry with propidium iodide (PI)/annexin V double staining and PI staining. The expression of G1 phase-related proteins was analyzed by RT-PCR and Western blotting. SIM suppressed cell growth and induced cell cycle G1-arrest by suppressing the expression of CDK4/cyclin D1 and CDK2/cyclin E1, but elevating the expression of glycogen synthase kinase 3β in CPs. Our findings indicate that SIM may have antitumor activity in established colorectal cancer. This article is protected by copyright. All rights reserved
  • Transition metal dependent regulation of the signal transduction cascade
           driving oocyte meiosis
    • Abstract: The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis. This article is protected by copyright. All rights reserved
  • The silent enemy: Celiac disease goes viral
    • Abstract: Celiac disease is a multifactorial autoimmune chronic inflammatory disorder affecting approximately one percent of the worldwide population. In such patients, ingestion of gluten proteins from cereals like wheat, barley and rye causes damage of the small intestine mucosa, with potentially severe consequences. Onset of the disease in predisposed individuals is believed to require a still not clearly identified external trigger, such as viral infections. A very recent study has begun to shed light on a possible mechanistic basis for this hypothesis, and surprisingly linked intestinal infections caused by common reoviruses to the onset of celiac disease. This article is protected by copyright. All rights reserved
  • TRPC3-mediated Ca2+ signals as a promising strategy to boost therapeutic
           angiogenesis in failing hearts: the role of autologous endothelial colony
           forming cells
    • Abstract: Endothelial progenitor cells (EPCs) are a sub-population of bone marrow-derived mononuclear cells that are released in circulation to restore damaged endothelium during its physiological turnover or rescue blood perfusion after an ischemic insult. Additionally, they may be mobilized from perivascular niches located within larger arteries' wall in response to hypoxic conditions. For this reason, EPCs have been regarded as an effective tool to promote revascularization and functional recovery of ischemic hearts, but clinical application failed to exploit the full potential of patients-derived cells. Indeed, the frequency and biological activity of EPCs are compromised in aging individuals or in subjects suffering from severe cardiovascular risk factors. Rejuvenating the reparative phenotype of autologous EPCs through a gene transfer approach has, therefore, been put forward as an alternative approach to enhance their therapeutic potential in cardiovascular patients. An increase in intracellular Ca2+ concentration constitutes a pivotal signal for the activation of the so-called endothelial colony forming cells (ECFCs), the only known truly endothelial EPC subset. Studies from our group showed that the Ca2+ toolkit differs between peripheral blood- and umbilical cord blood (UCB)-derived ECFCs. In the present article, we first discuss how VEGF uses repetitive Ca2+ spikes to regulate angiogenesis in ECFCs and outline how VEGF-induced intracellular Ca2+ oscillations differ between the two ECFC subtypes. We then hypothesize about the possibility to rejuvenate the biological activity of autologous ECFCs by transfecting the cell with the Ca2+-permeable channel Transient Receptor Potential Canonical 3, which selectively drives the Ca2+ response to VEGF in UCB-derived ECFCs. This article is protected by copyright. All rights reserved
  • C-C Motif Chemokine Ligand 2 Regulates LPS-Induced Inflammation and ER
           Stress to Enhance Proliferation of Bovine Endometrial Epithelial Cells
    • Abstract: Chemokines play an important role in regulating the complex immune system at the maternal-fetal interface during pregnancy. Among various chemokines, C-C motif chemokine ligand 2 (CCL2) plays a role in the recruitment of immune regulatory cells to implantation sites within the endometrium. In cattle, CCL2 is abundantly expressed in the uterine endometrium. However, its intracellular signaling has not been identified. In this study, we examined the effects of CCL2 on bovine endometrial (BEND) cell proliferation. CCL2 stimulated BEND cell proliferation by abundant expression of PCNA, accumulation of cells in the G2/M phase, and activation of the PI3K/AKT and MAPK signaling pathways. Moreover, CCL2 reduced endoplasmic reticulum stress and restored the inflammation-induced reduction in BEND cell proliferation by regulating the unfolded protein response genes and cytokines. Collectively, these results demonstrated that CCL2 plays a pivotal role in reproductive tissues and may support maternal-fetal interface to improve efficiency of pregnancy. This article is protected by copyright. All rights reserved
  • mSEL-1L deficiency affects vasculogenesis and neural stem cell lineage
    • Abstract: mSEL-1L is a highly conserved ER-resident type I protein, involved in the degradation of misfolded peptides through the ubiquitin–proteasome system (UPS), a pathway known to control the plasticity of the vascular smooth muscle cells (VSMC) phenotype and survival. In this article we demonstrate that mSEL-1L deficiency interferes with the murine embryonic vascular network, showing particular irregularities in the intracranic and intersomitic neurovascular units and in the cerebral capillary microcirculation. During murine embryogenesis, mSEL-1L is expressed in cerebral areas known to harbor progenitor neural cells, while in the adult brain the protein is specifically restricted to the stem cell niches, co-localizing with Sox2 and Nestin. Null mice are characterized by important defects in the development of telenchephalic regions, revealing conspicuous aberration in neural stem cell lineage commitment. Moreover, mSEL-1L depletion in vitro and in vivo appears to affect the harmonic differentiation of the NSCs, by negatively influencing the corticogenesis processes. Overall, the data presented suggests that the drastic phenotypic characteristics exhibited in mSEL-1L null mice can, in part, be explained by the negative influence it plays on Notch1 signaling pathway. This article is protected by copyright. All rights reserved
  • Chrysin Attenuates Progression of Ovarian Cancer Cells by Regulating
           Signaling Cascades and Mitochondrial Dysfunction
    • Abstract: Chrysin is mainly found in passion flowers, honey, and propolis acts as a potential therapeutic and preventive agent to inhibit proliferation and invasion of various human cancer cells. Although chrysin has anti-carcinogenic effects in several cancers, little is known about its functional roles in ovarian cancer which shows poor prognosis and chemoresistance to traditional therapeutic agents. In the present study, we investigated functional roles of chrysin in progression of ovarian cancer cells using ES2 and OV90 (clear cell and serous carcinoma, respectively) cell lines. Results of the current study demonstrated that chrysin inhibited ovarian cancer cell proliferation and induced cell death by increasing reactive oxygen species (ROS) production and cytoplasmic Ca2+ levels as well as inducing loss of mitochondrial membrane potential (MMP). Moreover, chrysin activated mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in ES2 and OV90 cells in concentration-response experiments. Collectively, our results led us to propose that chrysin-induced apoptotic events are mediated by the activation of PI3K and MAPK pathways in human ovarian cancer cells. This article is protected by copyright. All rights reserved
  • Involvement of sperm acetylated histones and the nuclear isoform of
           Glutathione Peroxidase 4 in fertilization
    • Abstract: We previously demonstrated that the nuclear form of Glutathione Peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida-deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization. This article is protected by copyright. All rights reserved
  • Pmepa1 induced by RANKL-p38 MAPK pathway has a novel role in
    • Abstract: Osteoclasts are multinucleated cells formed by fusion of preosteoclasts (POCs) derived from cells of the monocyte/macrophage lineage. We have reported a culture system that supports the formation of POCs from stroma-depleted rat bone marrow cells. Global gene expression analysis of this culture system identified genes highly expressed in POCs. Here, we have analyzed the expression and function of one of these highly expressed genes, prostate transmembrane protein androgen induced 1 (Pmepa1), a target of TGF-β and binds Nedd4 ubiquitin ligase, which plays a role in intracellular trafficking. We show here that the expression of Pmepa1 was strongly induced by RANKL in mouse bone marrow macrophage and in the osteoclast precursor cell line RAW-D. The expression of Pmepa1 was increased at 24 h of culture, but was decreased at 72 h. Pmepa1 protein was localized to intracellular vesicle membrnane of mononuclear cells, some of which were cathepsin-K positive. RANKL-induced expression of Pmepa1 was significantly reduced by inhibitors of p38 MAPK signaling. Pmepa1 siRNA suppressed the formation of osteoclasts in RAW-D cells, and inhibited the expression of cathepsin K and c-fos but not RANK. In addition, inhibition of Pmepa1 expression reduced the surface expression of RANK in RAW-D cells induced by RANKL. These results demonstrate that Pmepa1 is induced by RANK-p38 MAPK pathway signaling, and upregulates cell surface expression of RANK, suggesting that Pmepa1 plays a role in osteoclastogenesis and osteoclast signaling. This article is protected by copyright. All rights reserved
  • Apelin/APJ system: a novel potential therapy target for kidney disease
    • Abstract: Apelin is an endogenous ligand of seven-transmembrane G protein-coupled receptor APJ. Apelin and APJ are distributed in various tissues, including the heart, lung, kidney, and even in tumor tissues. Studies show that apelin mRNA is highly expressed in the inner stripe of kidney outer medulla, which plays an important role in process of water and sodium balance. Additionally, more studies also indicate that apelin/APJ system exerts a broad range of activities in kidney. Therefore, we review the role of apelin/APJ system in kidney diseases such as renal fibrosis, renal ischemia/reperfusion injury, diabetic nephropathy, polycystic kidney disease and hemodialysis. Apelin/APJ system can improve renal interstitial fibrosis by reducing the deposition of extracellular matrix. Apelin/APJ system significantly reduces renal ischemia/reperfusion injury by inhibiting renal cell death. Apelin/APJ system involves the progression of diabetic nephropathy. Apelin/APJ system also predicts the process of polycystic kidney disease. Besides, apelin/APJ system prevents some dialysis complications in hemodialysis patients. And apelin/APJ system alleviates chronic kidney disease by inhibiting vascular calcification. Overall, apelin/APJ system plays diversified roles in kidney disease and may be a potential target for the treatment of kidney disease. This article is protected by copyright. All rights reserved
  • Spleen tyrosine kinase influences the early stages of multilineage
           differentiation of bone marrow stromal cell lines by regulating
           phospholipase C gamma activities
    • Abstract: Bone marrow stromal cells (BMSCs) are multipotent cells that can differentiate into adipocytes and osteoblasts. Inadequate BMSC differentiation is occasionally implicated in chronic bone metabolic disorders. However, specific signaling pathways directing BMSC differentiation have not been elucidated. Here, we explored the roles of spleen tyrosine kinase (Syk) in BMSC differentiation into adipocytes and osteoblasts. We found that Syk phosphorylation was increased in the early stage, whereas its protein expression was gradually decreased during the adipogenic and osteogenic differentiation of two mouse mesenchymal stromal cell lines, ST2 and 10T(1/2), and a human BMSC line, UE6E-7-16. Syk inactivation with either a pharmacological inhibitor or Syk-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the gene expression of fatty acid protein 4 (Fabp4), peroxisome proliferator-activated receptor γ2 (Pparg2), CCAAT/enhancer binding proteins α(C/EBPα) and C/EBPβ . In contrast, Syk inhibition promoted osteogenic differentiation, represented by increase in matrix mineralization and alkaline phosphatase (ALP) activity, as well as the expression levels of osteocalcin, runt-related transcription factor 2 (Runx2) and distal-less homeobox 5 (Dlx5) mRNAs. We also found that Syk-induced signals are mediated by phospholipase Cγ1 (PLCγ1) in osteogenesis and PLCγ2 in adipogenesis. Notably, Syk-activated PLC 2 signaling was partly modulated through B-cell linker protein(BLNK) in adipogenic differentiation. On the other hand, growth factor receptor-binding protein 2 (Grb2) was involved in Syk-PLCγ1 axis in osteogenic differentiation. Taken together, these results indicate that Syk-PLCγ signaling has a dual role in regulating the initial stage of adipogenic and osteogenic differentiation of BMSCs. This article is protected by copyright. All rights reserved
  • CRISPR Editing in Biological and Biomedical Investigation
    • Abstract: Recently, clustered regularly interspaced short palindromic repeats (CRISPR) based genomic editing technologies have armed researchers with powerful new tools to biological and biomedical investigations. To further improve and expand its functionality, natural and engineered CRISPR associated 9 proteins (Cas9s) have been investigated, various CRISPR delivery strategies have been tested and optimized, and multiple schemes have been developed to ensure precise mammalian genome editing. Benefiting from those in-depth understanding and further development of CRISPR, versatile CRISPR-based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine. In biological research area, CRISPR has been widely adopted in both fundamental and applied research fields, such as accurate base editing, transcriptional regulation and genome-wide screening. In biomedical research area, CRISPR has also shown its extensive applicability in the establishment of animal models for genetic disorders especially those large animals and non-human primates models, and gene therapy to combat virus infectious diseases, to correct monogenic disorders in vivo or in pluripotent cells. In this prospect article, after highlighting recent developments of CRISPR systems, we outline different applications and current limitations of CRISPR use in biological and biomedical investigation. Finally, we provide a perspective for future development and potential risks of this multifunctional technology. This article is protected by copyright. All rights reserved
  • Novel role for the testis-enriched HSPA2 protein in regulating epidermal
           keratinocyte differentiation
    • Abstract: HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis. This article is protected by copyright. All rights reserved
  • Effects of pulsed electromagnetic fields and platelet rich plasma in
           preventing osteoclastogenesis in an in vitro model of osteolysis
    • Abstract: Osteolysis is the main limiting cause for the survival of an orthopedic prosthesis and is accompanied by an enhancement in osteoclastogenesis and inflammation, due by wear debris formation. Unfortunately therapeutic treatments, besides revision surgery, are not available.The aim of the present study was to evaluate the effects of Pulsed ElectroMagnetic Fields (PEMFs) and platelet rich plasma (PRP), alone or in combination, in an in vitro model of osteolysis.Rats peripheral blood mononuclear cells were cultured on Ultra High Molecular Weight Polyethylene particles and divided into 4 groups of treatments: 1) PEMF stimulation (12 hours/day, 2.5 mT, 75Hz, 1.3 ms pulse duration); 2) 10% PRP; 3) combination of PEMFs and PRP; 4) no treatment. Treatments were performed for 3 days and cell viability, osteoclast number, expression of genes related to osteoclastogenesis and inflammation and production of pro-inflammatory cytokines were assessed up to 14 days.PEMF stimulation exerted best results because it increased cell viability at early time points and counteracted osteoclastogenesis at 14 days. On the contrary, PRP increased osteoclastogenesis and reduced cell viability in comparison to PEMFs alone. The combination of PEMFs and PRP increased cell viability over time and reduced osteoclastogenesis in comparison to PRP alone. However, these positive results did not exceed the level achieved by PEMF alone. At longer time points PEMF could not counteract osteoclastogenesis increased by PRP.Regarding inflammation, all treatments maintained the production of pro-inflammatory cytokines at low level, although PRP increased the level of interleukin 1 beta. This article is protected by copyright. All rights reserved
  • Artesunate inhibits RANKL-induced osteoclastogenesis and bone resorption
           in vitro and prevents LPS-induced bone loss in vivo
    • Abstract: Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side-effects caused by the currently available drugs, a continuous search for novel bone-protective therapies is essential. Artesunate (Art), the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, the mRNA expression of osteoclastic-specific genes, and resorption pit formation in a dose-dependent manner in primary bone marrow-derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL-induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF-κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)-induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL-induced osteoclastogenesis by suppressing the NF-κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.Artesunate (Art), the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties. However, its effects on osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis and lipopolysaccharide (LPS)-induced bone resorption.
  • TGF-β and Th17 cells related injuries in patients with sulfur mustard
    • Abstract: Sulfur mustard (SM) is a vesicating agent that has been employed as a chemical warfare agent. High-dose exposure to sulfur mustard may lead to the damage of rapidly proliferating cells of bone marrow and, therefore, suppression of the immune system. This may be continued as dysfunction of the immune system, and ultimately result in secondary immune disorders. Studies have suggested a role for T cells in SM-induced lung injury. Moreover, observations from animal studies indicate a delayed-type hypersensitivity (DTH) response after skin exposure to SM, providing an understanding that SM can stimulate specific T cell-mediated immune responses. On the other hand, T helper (Th) 17 cells, which are a subset of CD4+ T cells, have recently been reported to be involved in a number of inflammatory, autoimmune, and chronic fibrotic lung diseases. Furthermore, a strong association has been established between the overproduction of profibrotic cytokines like transforming growth factor (TGF)-β and Th17 cell number. In this review, we aimed to go through the new findings about the involvement and interactions of TGF-β and Th17 in SM-related injuries.In this review, we aimed to go through the new findings about the involvement and interactions of TGF-β and Th17 in SM-related injuries.
  • IFN-gamma priming of adipose-derived stromal cells at
           “physiological” hypoxia
    • Abstract: Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2) were assessed as alterations of ASCs’ CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2-adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in “wound closure.” Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.IFN-gamma-mediated priming may provide the enhancement of tissue O2-adapted ASC “reparative” functions, due to on site retention, the attenuation of ASC differentiating capacity and the enhancement of soluble and surface-associated molecules, and regulation cell-to-cell and cell-matrix interactions. Acute O2 deprivation may partly attenuate inflammatory activation, thus suppressing remodelling-requested functions of ASCs.
  • Reprogramming mouse embryo fibroblasts to functional islets without
           genetic manipulation
    • Abstract: The constant quest for generation of large number of islets aimed us to explore the differentiation potential of mouse embryo fibroblast cells. Mouse embryo fibroblast cells isolated from 12- to 14-day-old pregnant mice were characterized for their surface markers and tri-lineage differentiation potential. They were subjected to serum-free media containing a cocktail of islet differentiating reagents and analyzed for the expression of pancreatic lineage transcripts. The islet-like cell aggregates (ICAs) was confirmed for their pancreatic properties via immunofluorecence for C-peptide, glucagon, and somatostain. They were positive for CD markers—Sca1, CD44, CD73, and CD90 and negative for hematopoietic markers—CD34 and CD45 at both transcription and translational levels. The transcriptional analysis of the ICAs at different day points exhibited up-regulation of islet markers (Insulin, PDX1, HNF3, Glucagon, and Somatostatin) and down-regulation of MSC-markers (Vimentin and Nestin). They positively stained for dithizone, C-peptide, insulin, glucagon, and somatostatin indicating intact insulin producing machinery. In vitro glucose stimulation assay revealed three-fold increase in insulin secretion as compared to basal glucose with insulin content being the same in both the conditions. The preliminary in vivo data on ICA transplantation showed reversal of diabetes in streptozotocin induced diabetic mice. Our results demonstrate for the first time that mouse embryo fibroblast cells contain a population of MSC-like cells which could differentiate into insulin producing cell aggregates. Hence, our study could be extrapolated for isolation of MSC-like cells from human, medically terminated pregnancies to generate ICAs for treating type 1 diabetic patients.
  • Lipid composition of membrane microdomains isolated detergent-free from
           PUFA supplemented RAW264.7 macrophages
    • Abstract: Profound alterations in the lipid profile of raft and non-raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. For the first time lipids in the detergent-free isolated membrane domains of phagocytic immune cells were characterized by mass spectrometry. The extent of remodeling of the membrane lipids differed with different n3 and n6 PUFA supplements. The mildest effects were detected for -linolenic acid (LNA) and linoleic acid (LA), the C18 precursors of the n3 and n6 families, respectively. When the effects of highly unsaturated PUFAs were compared, eicosapentaenoic acid (EPA) caused more extensive restructuring of membrane lipids than docosahexaenoic acid (DHA) or arachidonic acid (AA). The supplements altered the lipid species composition of both the raft and non-raft membrane fractions. The rafts containing elevated proportions of highly unsaturated lipid species may relocate sterically incompatible lipids and proteins originally belonging to this microdomain. Such effect was evident for sphingomyelin, which favored non-rafts instead of rafts after EPA supplementation. The current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture. This article is protected by copyright. All rights reserved
    • Abstract: A series of 2, 3-dihydroquinazolinone derivatives were synthesized, characterized and their anticancer activity was determined. Among the compounds synthesized and screened, one compound (17) showed potent anticancer activity against human head and neck squamous cell carcinoma cell line, SCC131 and was non-toxic to normal cells. The compound inhibited the growth of SCC131 cells, with an IC50 of 1.75 μM, triggered apoptotic mode of cell death and caused tumor regression of SCC131 tumor xenografts in athymic mice. To decipher the target for the lead compound, ahigh throughput qPCR array was performed. Results showed that the compound 17, inhibited the expression of a vital transcription factor HNF4A,involved in regulation of metabolic pathways. Thus, the present work has identified a lead compound 17, with potent anticancer activity, minimal normal cell toxicity and a plausible target and hence definitely holds future prospects as an anticancer agent. This article is protected by copyright. All rights reserved
  • Double sex and mab-3 related transcription factor 1 regulates
           differentiation and proliferation in dairy goat male germline stem cells
    • Abstract: The protein encoded by double sex and mab-3 related transcription factor 1 (Dmrt1)gene contains a double sex/mab-3 domain, which was considered as one of the most conservative structures in sex determination. However, its effect on spermatogenesis of dairy goat spermatogonial stem cells (SSCs) remains to be clarified. For the first time, the roles of Dmrt1 in spermatogenesis of livestock are highlighted. Here, we investigated the expression pattern of Dmrt1 in the testes of dairy goats. Dmrt1 primarily located in undifferentiated SSCs. Moreover,Dmrt1 enhanced differentiation and proliferation of mGSCs. On the contrary, the level of meiosis was down-regulated, as Dmrt1 determines whether SSCs undergo mitosis and spermatogonial differentiation or meiosis. In the busulfan-treated mice testes, Dmrt1 repair germ cell damage was emphasized as well. Our results exposed that Dmrt1 maintenance mGSCs in two ways: facilitating proliferation and self-renewal of SSCs; and reducing the inflammatory response caused by reproductive injury. These findings identify a central role for Dmrt1 in controlling population stability and injury restoring of SSCs. This article is protected by copyright. All rights reserved
  • Crosstalk of ER stress-mediated autophagy and ER-phagy: involvement of UPR
           and the core autophagy machinery
    • Abstract: Endoplasmic reticulum (ER) stress, a common cellular stress response, is closely related to the activation of autophagy that is an important and evolutionarily conserved mechanism for maintaining cellular homeostasis. Autophagy induced by ER stress mainly includes the ER stress-mediated autophagy and ER-phagy. The ER stress-mediated autophagy is characterized by the generation of autophagosomes that include worn-out proteins, protein aggregates, and damaged organelles. While the autophagosomes of ER-phagy selectively include ER membranes, and the double membranes also derive, at least in part, from the ER. The signaling pathways of IRE1α, PERK, ATF6 and Ca2+ are necessary for the activation of ER stress-mediated autophagy, while the receptor-mediated selective ER-phagy degrades the ER is Atg40/FAM134B. The ER stress-mediated autophagy and ER-phagy not only have differences, but also have connections. The activation of ER-phagy requires the core autophagy machinery, and the ER-phagy may be a branch of ER stress-mediated autophagy that selectively targets the ER. However, the determined factors that control the changeover switch between ER stress-mediated autophagy and ER-phagy are largely obscure, which may be associated with the type of cells and the extent of stimulation. This review summarized the crosstalk between ER stress-mediated autophagy and ER-phagy and their signaling networks. Additionally, we discussed the possible factors that influence the type of autophagy induced by ER stress. This article is protected by copyright. All rights reserved
  • Characterization of a novel EB1 acetylation site important for the
           regulation of microtubule dynamics and cargo recruitment
    • Abstract: Microtubule plus ends undergo highly dynamic modifications to regulate different aspects of cellular activities. Most microtubule plus-end tracking proteins (+TIPs) are recruited to the microtubule ends by the master loading factor, end-binding protein 1 (EB1). These proteins coordinately regulate microtubule dynamics and cellular plasticity. Acetylation is known to modulate EB1 function; however, the molecular details of EB1 acetylation remain largely unclear. We mapped the acetylation pattern of EB1 and identified several previously uncharacterized sites of EB1 acetylation. We examined the effects of lysine-212 (K212) acetylation and found that acetylation of this site accelerates autophagy-mediated EB1 degradation. By time-lapse microscopy, we found that the acetylation-deficient K212R mutant increased the percentage of fast-growing and long-lived microtubules. Although K212 acetylation did not affect microtubule stability in vitro and the association of EB1 with microtubules, the K212R mutant significantly promoted microtubule regrowth in cells. Coimmunoprecipitation assays further revealed that the K212 site was critical for the recruitment of different +TIP cargoes. These data thus uncover a critical role for a novel EB1 acetylation site in regulating the dynamic structure of microtubules. This article is protected by copyright. All rights reserved
  • Increased Expression of PD-L1 and PD-L2 in Dermal Fibroblasts from
           Alopecia Areata Mice
    • Abstract: Alopecia areata (AA) is a common autoimmune disorder affecting millions of people worldwide, which manifests as a sudden, non-scarring hair loss. The expression of a pro-inflammatory cytokine, interferon-gamma (INF-γ), has been well established to be involved in the development of AA. As IFN-γ and other cytokines are also known to up-regulate programmed cell death ligand 1 and 2 (PD-L1 and PD-L2), which both negatively control immune responses, we asked whether or not a high number of infiltrated T cells, seen in AA lesions, can modulate the expression of PD-L1 and PD-L2 in skin cells. From a series of experiments, we showed that a significantly higher number of PD-L1 or PD-L2 positive cells affect the skin in AA mice, compared to the skin of non-AA mice. The number of PD-L1 positive cells was well correlated with the number of infiltrated T cells, especially CD8+ T cells. We also found that the expression of PD-L1 and PD-L2 was co-localized with type 1 pro-collagen, CD90 and vimentin, which are biomarkers for dermal fibroblasts. Further studies revealed that releasable factors from activated, but not inactivated, lymphocytes significantly increase the expressions of both PD-L1 and PD-L2 in cultured dermal fibroblasts. In conclusion, our findings suggest that the expression of PD-L1 and PD-L2 in dermal fibroblasts is up-regulated by activated T cells in AA-affected skin, and as such, these regulatory molecules may not exert a negative control of the immune activation seen in AA lesions. This article is protected by copyright. All rights reserved
    • Abstract: Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance. This article is protected by copyright. All rights reserved
  • Basal progenitor cells bridge the development, malignant cancers and
           multiple diseases of esophagus
    • Abstract: The esophagus is a pivotal organ originating from anterior foregut that links the mouth and stomach. Moreover, its development involves precise regulation of multiple signal molecules and signal transduction pathways. After abnormal regulation of these molecules in the basal cells of the esophagus occurs, multiple diseases, including esophageal atresia with or without tracheoesophageal fistula, Barrett esophagus, gastroesophageal reflux, and eosinophilic esophagitis, will take place as a result. Furthermore, expression changes of signal molecules or signal pathways in basal cells and the microenvironment around basal cells both can initiate the switch of malignant transformation. In this review, we highlight the molecular events underlying the transition of normal development to multiple esophageal diseases. Additionally, the animal models of esophageal development and related diseases, challenges, and strategies are extensively discussed. This article is protected by copyright. All rights reserved
  • Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling
    • Abstract: Extracellular nucleotides can regulate cell proliferation in both normal and tumorigenic tissues. Here, we studied how extracellular nucleotides regulate the proliferation of ZL55 cells, a mesothelioma-derived cell line obtained from bioptic samples of asbestos-exposed patients. ADP and 2-MeS-ADP inhibited ZL55 cell proliferation, whereas ATP, UTP and UDP were inactive. The nucleotide potency profile and the blockade of the ADP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 suggest that P2Y1 receptor controls ZL55 cell proliferation. The activation of P2Y1 receptor by ADP leads to activation of intracellular transduction pathways involving [Ca2+]i, PKC-δ/PKC-α, and MAPKs ERK1/2 and JNK1/2. Cell treatment with ADP or 2-MeS-ADP also provokes the activation of p53, causing an accumulation of the G1 cyclin-dependent kinase inhibitors p21WAF1 and p27Kip. Inhibition of ZL55 cell proliferation by ADP was completely reversed by inhibiting MEK1/2 or JNK1/2 or PKC-δ and PKC-α. Through the inhibition of ADP-activated transductional kinases it was found that PKC-δ was responsible for JNK1/2 activation. JNK1/2 has a role in transcriptional up-regulation of p53, p21WAF1/CIP1 and p27kip1. Conversely, the ADP-activated PKC-α provoked ERK1/2 phosphorylation. ERK1/2 increased p53 stabilization, required to G1 arrest of ZL55 cells. Concluding, the importance of the study is twofold: first, results shed light on the mechanism of cell cycle inhibition by ADP; secondly, results suggest that extracellular ADP may inhibit mesothelioma progression. This article is protected by copyright. All rights reserved
  • Bifunctional Role of Ephrin A1-Eph System in Stimulating Cell
           Proliferation and Protecting Cells from Cell Death through the Attenuation
           of ER Stress and Inflammatory Responses in Bovine Mammary Epithelial Cells
    • Abstract: Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals. This article is protected by copyright. All rights reserved
  • Iodixanol vs iopromide in cancer patients: evidence from a randomized
           clinical trial
    • Abstract: Purpose: To assess the safety profile of iso-osmolar contrast medium (CM) vs low osmolar CM in cancer patients with an estimated glomerular filtration rate (eGFR) > 60 ml/min.Materials and Methods: In this multicentre, blind trial of patients seeking a chest-abdomen-pelvis CT with iodated CM, participants were centrally randomized to iodixanol or iopromide. Contrast induced nephropathy (CIN) at 24 and/or 72 hours were our primary outcomes. We further considered irreversible CIN, average eGFR percentage variation (%Δ), and adverse events (AEs).Results: Overall, 607 patients were enrolled. Among them, 497 eligible patients were randomized to iodixanol (N:247) or iopromide (N:250). No differences emerged by descriptive characteristics. Seven and 3 CIN at 24 hours (p = 0.34) and 8 and 2 CIN at 72 hours (p = 0.11) occurred in the iopromide and iodixanol group, respectively. Within the subgroup of individual patients who developed CIN (N:17), the event rate was higher in the iopromide arm (p = 0.045). No cases of permanent CIN or significant differences in terms of AEs or GFR %Δ were observed.Conclusions: Our results suggest a more favourable safety profile of iodixanol vs iopromide. Adequately sized trials with similar design are warranted to confirm our findings and clarify the underlying biological mechanisms. This article is protected by copyright. All rights reserved
  • Hypomethylation-mediated H19 overexpression increases the risk of disease
           evolution through the association with BCR-ABL transcript in chronic
           myeloid leukemia
    • Abstract: Previous study has revealed that H19 expression is required for efficient tumor growth induced by BCR-ABL in chronic myeloid leukemia (CML). Herein, we further determined H19 expression and its clinical implication in patients with CML. H19 expression and methylation were detected by real-time quantitative PCR and real-time quantitative methylation-specific PCR, and then clinical implication of H19 expression was further analyzed. H19 expression was significantly up-regulated in CML patients (P 
  • Mesenchymal stem cell differentiation: Control by calcium-activated
           potassium channels
    • Abstract: Mesenchymal stem cells (MSCs) are widely used in modern medicine for which understanding the mechanisms controlling their differentiation is fundamental. Ion channels offer novel insights to this process because of their role in modulating membrane potential and intracellular milieu. Here, we evaluate the contribution of calcium-activated potassium (KCa) channels to the three main components of MSC differentiation: initiation, proliferation and migration. First, we demonstrate the importance of the membrane potential (Vm) and the apparent association of hyperpolarization with differentiation. Of KCa subtypes, most evidence points to activity of big-conductance channels in inducing initiation. On the other hand, intermediate-conductance currents have been shown to promote progression through the cell cycle. Whilst there is no information on the role of KCa channels in migration of MSCs, work from other stem cells and cancer cells suggest that intermediate-conductance and to a lesser extent big-conductance channels drive migration. In all cases, these effects depend on species, tissue origin and lineage. Finally, we present a conceptual model that demonstrates how KCa activity could influence differentiation by regulating Vm and intracellular Ca2+ oscillations. We conclude that KCa channels have significant involvement in MSC differentiation and could potentially enable novel tissue engineering approaches and therapies. This article is protected by copyright. All rights reserved
  • β-asarone inhibited cell growth and promoted autophagy via
           P53/Bcl-2/Bclin-1 and P53/AMPK/mTOR pathways in Human Glioma U251 cells
    • Abstract: Glioma is the most common type of primary brain tumor and has an undesirable prognosis. Autophagy plays an important role in cancer therapy, but it's effect is still not definite. P53 is an important tumor suppressor gene and protein that is closely to autophagy. Our aim was to study the effect of β-asarone on inhibiting cell proliferation in human glioma U251 cells and to detect the effect of the inhibition on autophagy through the P53 signal pathway. For cell growth, the cells were divided into four groups: the model, β-asarone, temozolomide (TMZ) and co-administration groups. For cell autoghapy and the P53 pathway, the cells were divided into six groups: the model, β-asarone, 3MA, Rapa, Pifithrin-µ and NSC groups. The counting Kit-8 assay and flow cytometry (FCM) were then used to measure the cell proliferation and cycle. Electron microscopy was used to observe autophagosome formation. Cell immunohistochemistry/-immunofluorescence, FCM and western blot (WB) were used to examine the expression of Beclin-1 and P53. The levels of P53 and GAPDH mRNA were detected by RT-PCR. Using WB, we determined autophagy-related proteins Beclin-1, LC3-aaa/aaa and P62 and those of the P53 pathway-related proteins P53, Bcl-2, mTOR, P-mTOR, AMPK, P-AMPK and GAPDH. We got the results that β-asarone changed the cellular morphology, inhibited cell proliferation and enhanced the expression of P53, LC3-aaa/aaa, Beclin-1, AMPK and pAMPK while inhibiting the expression of P62, Bcl-2, mTOR and pmTOR. All the data suggested that β-asarone could reduce the cell proliferation and promote autophagy possible via the P53 pathway in U251 cells. This article is protected by copyright. All rights reserved
  • Characterization and assessment of potential microRNAs involved in
           phosphate-induced aortic calcification
    • Abstract: Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague-Dawley rat aortic explants to high inorganic phosphate level (Pi, 6 mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de-differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22α) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans-differentiation into osteo-chondrocyte-like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real-time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT-PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post Pi treatment versus control untreated aortas. At day 3, miR-200c, -155, 322 were upregulated and miR-708 and 331 were downregulated. After 6 days of treatment, miR-328, -546, -301a were upregulated whilst miR-409 and miR-542 were downregulated. Our results indicate that high Pi levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies. This article is protected by copyright. All rights reserved
  • Potential Role of the Jagged1/Notch1 Signaling Pathway in the
           Endothelial-Myofibroblast Transition during BLM-induced Pulmonary Fibrosis
    • Abstract: Endothelial cell myofibroblast transition (EndoMT) is found during the process of bleomycin (BLM)-induced pulmonary fibrosis in rats, and plays a very important role in sustaining inflammation and collagen secretion. Moreover, some studies have suggested that the Notch1 signaling pathway may be involved in the expression of Î ± -smooth muscle actin (Î ± -SMA) in pulmonary microvascular endothelial cells (PMVECs), a protein marker of EndoMT. Therefore, we aimed to investigate the expression level of Î ± -SMA and Notch1-related signaling molecules in PMVECs from BLM-induced rats and determine the relationship between the Notch1 signaling pathway and the expression of Î ± -SMA in PMVECs. We found that the expression levels of Î ± -SMA, Notch1, and Jagged1 were upregulated, while the expression levels of Dll4 were downregulated. Furthermore, there was a positive correlation between the expression of Jagged1 and the Î ± -SMA proteins in PMVECs, and NF-ΰB was downregulated by decreasing the expression of Jagged1. In conclusion, the Jagged1/Notch1 signaling pathway is activated in PMVECs during the pathogenesis of BLM-induced pulmonary fibrosis in rats, and it may induce Î ± -SMA expression via a non-canonical pathway involving NF-ΰB as the target molecule. The precise mechanism and the molecules involved in this signaling pathway need to be further elucidated. This article is protected by copyright. All rights reserved
  • Tannic acid Attenuates TGF-β1-induced Epithelial-to-mesenchymal
           Transition by Effectively Intervening TGF-β Signaling in Lung Epithelial
    • Abstract: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-β1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-β1 with or without TA. Results showed that TA addition, markedly inhibited TGF-β1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin and vimentin. Furthermore, TA inhibited TGF-β1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-β1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2 and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-β1-induced increase in TGF-β receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-β1. Finally, we conclude that TA might directly interact with TGF-β1, thereby repressing TGF-β signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis. This article is protected by copyright. All rights reserved
  • Cyanidin Chloride Inhibits Ovariectomy-Induced Osteoporosis by Suppressing
           RANKL-mediated Osteoclastogenesis and Associated Signaling Pathways
    • Abstract: Over-production and activation of osteoclasts is a common feature of osteolytic conditions such as osteoporosis, tumor-associated osteolysis, and inflammatory bone erosion. Cyanidin Chloride, a subclass of anthocyanin, displays antioxidant and anti-carcinogenesis properties, but its role in osteoclastic bone resorption and osteoporosis is not well understood. In this study, we showed that Cyanidin Chloride inhibits osteoclast formation, hydroxyapatite resorption, and receptor activator of NF-κB ligand (RANKL)-induced osteoclast marker gene expression; including ctr, ctsk and trap. Further investigation revealed that Cyanidin Chloride inhibits RANKL-induced NF-κB activation, suppresses the degradation of IκB-α and attenuates the phosphorylation of extracellular signal-regulated kinases (ERK). In addition, Cyanidin Chloride abrogated RANKL-induced calcium oscillations, the activation of nuclear factor of activated T cells calcineurin-dependent 1 (NFATc1), and the expression of c-Fos. Further, we showed that Cyanidin Chloride protects against ovariectomy-induced bone loss in vivo. Together our findings suggest that Cyanidin Chloride is capable of inhibiting osteoclast formation, hydroxyapatite resorption and RANKL-induced signal pathways in vitro and OVX-induced bone loss in vivo, and thus might have therapeutic potential for osteolytic diseases. This article is protected by copyright. All rights reserved
  • Annexin A2 Positively Regulates Milk Synthesis and Proliferation of Bovine
           Mammary Epithelial Cells through the mTOR Signaling Pathway
    • Abstract: Annexin A2 (AnxA2) has been shown to play multiple roles in growth, development and metabolism, but the functions of AnxA2 and the signaling pathways associated with AnxA2 are still not fully understood. In this study, we aim to reveal whether and how AnxA2 could be involved in milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Using gene function study approaches, we found that AnxA2 positively regulates PIP3 level, phosphorylation of mTOR and protein levels of SREBP-1c and Cyclin D1 leading to milk synthesis and cell proliferation. We further observed that both AnxA2-36 kD phosphorylated form and AnxA2-33 kD protein could be induced from AnxA2-36 kD protein in BMECs under methionine, leucine, estrogen or prolactin stimulation. These above results strongly demonstrate that AnxA2 functions as a critical regulator for amino acid or hormone-induced milk synthesis and cell proliferation via the PI3K-mTOR-SREBP-1c/Cyclin D1 signaling pathway. This article is protected by copyright. All rights reserved
  • TLR4 and NFκB signaling is critical for taxol resistance in ovarian
           carcinoma cells
    • Abstract: We report here that toll-like receptor 4 (TLR4) and ABCB1 are upregulated in SKOV3 ovarian carcinoma cells that acquired resistance to the anticancer drug taxol. Silencing of TLR4 using short-hairpin RNA sensitized taxol-resistant SKOV3 cells to taxol (4.6 fold), whereas ectopic expression of TLR4 in parental, taxol-sensitive SKOV3 cells or TLR4-null HEK293 cells induced taxol resistance (∼2 fold). A sub-lethal dose of taxol induced ABCB1 protein expression in taxol-resistant SKOV3 cells. Inactivation of TLR4 using chemical inhibitors (CLI-095 and AO-I) downregulated ABCB1 protein expression and enhanced the cytotoxic activity of taxol in taxol-resistant SKOV3 cells. While the sensitization effect of TLR4 inactivation was also detected in TOV21G ovarian cancer cells, which express moderate level of TLR4, ectopic expression of ABCB1 prevented the sensitization effect in these cells. Notably, the NFκB pathway was significantly activated by taxol, and inhibition of this pathway suppressed TLR4-regulated ABCB1 expression. Furthermore, taxol-induced NFκB signaling was reduced following TLR4 silencing in taxol-resistant SKOV3 cells. Consistent with these results, ectopic expression of TLR4 in taxol-sensitive SKOV3 cells enhanced ABCB1 expression and conferred resistance to taxol. The protective effect of exogenous TLR4 expression against taxol was reduced by treatment with NFκB inhibitor in these cells. These results demonstrate that taxol activates the TLR4-NFκB pathway which in turn induces ABCB1 gene expression. This cellular pathway thus represents a novel target to limit resistance to taxol in ovarian cancer cells. This article is protected by copyright. All rights reserved
  • miR-146a protects small intestine against ischemia/reperfusion injury by
           down-regulating TLR4/TRAF6/NF-κB pathway
    • Abstract: Previous studies reported that miR-146a was involved in small intestine ischemia-reperfusion (I/R) injury, but the mechanism is largely vague. Here we aimed to identify the change of miR-146a in patients with mesenteric ischemia and explore the potential regulatory mechanism of miR-146a in intestine epithelial cells survival under ischemia and I/R injury. The plasma of 20 patients with mesenteric ischemia and 25 controls was collected to examine the miR-146a expression by qPCR. Rat intestinal epithelial cells (IEC-6) and 24 male Sprague-Dawley rats were included to build ischemia and I/R model in vitro and in vivo. The qPCR results showed that miR-146a decreased both in the plasma of patients with mesenteric ischemia and in IEC-6 cells and rat small intestine tissues in ischemia and I/R model compared to controls. Both the vitro and vivo results showed that I/R resulted in more severe apoptotic injury than ischemia. Cleaved-caspase 3, TLR4, TRAF6 and nuclear NF-κB p65 were up-regulated accompanying reduced XIAP and SOCS3 expression in intestinal ischemia and I/R injury. After up-regulation of miR-146a in IEC-6 cells, increased cell survival and decreased cell apoptosis were observed, concomitant with decreased cleaved-caspase 3 and down-regulated TLR4/TRAF6/NF-κB pathway. What's more, this protective effect was blocked by TRAF6 overexpression and increased nuclear NF-κB p65 nuclear. Taken together, this study revealed that miR-146a expression was decreased in small intestine ischemia and I/R injury. And miR-146a improves intestine epithelial cells survival under ischemia and I/R injury through inhibition TLR4, TRAF6 and p-IκBα, subsequently leading to decreased NF-κB p65 nuclear translocation. This article is protected by copyright. All rights reserved
  • The ubiquitin ligase TRIM56 inhibits ovarian cancer progression by
           targeting vimentin
    • Abstract: Tumor metastasis is responsible for 90% of all cancer-related deaths. Epithelial to mesenchymal transition (EMT) is an important prerequisite for tumor metastasis. One of the important mediators of EMT and cancer progression in ovarian cancer is the vimentin protein. The objective of the current study was to evaluate the molecular mechanism that regulates vimentin expression in ovarian cancer cells. Vimentin was robustly induced in the ovarian cancer cell line SKOV-3 compared to normal ovarian epithelial cell line Moody and the induction was not due to transcriptional upregulation. Treatment with the proteasomal inhibitor MG-132 revealed that vimentin is actively degraded by the proteasome in Moody cells and stabilized in the SKOV-3 cell line. Mass spectrometric analysis of vimentin immunoprecipitate of MG-132 treated Moody cells revealed candidate ubiquitin ligases associated with vimentin. RNAi mediated silencing of the candidate ubiquitin in Moody cells and concurrent overexpression of the candidate ubiquitin ligases in SKOV-3 confirmed that TRIM56 is the ubiquitin ligase that is degrading vimentin in Moody cells. RNAi mediated silencing of TRIM56 in Moody cells and ectopic overexpression of TRIM56 in SKOV-3 cells, respectively, significantly up- and down-regulated in vitro migration and invasion in these cells. Analysis of TRIM56 transcript level and vimentin protein expression in 25 patients with ovarian carcinoma confirmed an inverse correlation between TRIM56 and vimentin expression. Cumulatively, our data reveals for the first time a novel post-translational regulatory mechanism of regulating vimentin expression, EMT, and metastatic progression in ovarian cancer cells. This article is protected by copyright. All rights reserved
  • The role of mitochondrial ROS in antibacterial immunity
    • Abstract: Reactive oxygen species (ROS) are essential participants of various innate immune cell responses against microorganisms and are also involved in many cellular regulatory pathways. It was believed that the main pool of ROS in the innate immune cells is generated by the NADPH oxidase enzymatic complex. However, it was discovered recently that mitochondrial ROS (mtROS) are equally important for the functioning of the immune system. mtROS play an important role in the development of the antimicrobial innate immune responses. The present mini-review summarizes the most recent data on the role of mtROS in the antibacterial immunity. The principles of mtROS formation and possible mechanisms of their generation under the activation of innate immunity are highlighted in this review. We also speculate on the possibilities of using activators of mtROS production in clinical practice. This article is protected by copyright. All rights reserved
  • Aberrantly miRNA promoter methylation and EMT-involving miRNAs in breast
           cancer metastasis: diagnosis and therapeutic implications
    • Abstract: Breast cancer (BC) is the most prevalent cancer in women worldwide. Although extensive studies are ongoing concerning its intricate molecular mechanisms, development of novel therapies and more accurate diagnostic and prognostic approaches is still a challenge. Epithelial-mesenchymal transition (EMT) enables the invasion of metastatic cancer cells and has recently been highlighted in a cancer stem cell model of BC. Epigenetic events as well as miRNA expression are the master regulators of tumorigenesis and add a further layer to the complexity of BC pathogenesis. The miRNAs are related to epigenetic event and additionally affect epigenetic pathways. Recent evidence demonstrates that epigenetic mechanisms such as DNA methylation may control miRNA expression. Because each miRNA may regulate several target genes, dysregulation of miRNA caused by aberrant DNA methylation patterns of the locus may influence important downstream pathways. Some miRNA is believed to regulate important DNA methylator factors. Any disruption or modification of this intricate network can contribute to the disease process; thus, it is essential to understand these changes. Advancements in new sequencing technologies to detect DNA methylation patterns has provided the opportunity to determine differentially methylated regions (DMRs) of the miRNA locus and their effect on expression profiles to improve BC diagnosis and treatment. The current review examines the interplay of DNA methylation mechanisms and miRNA function in invasive tumorigenesis, specifically EMT of BC, to highlight its potential for advancements on BC etiology, diagnosis and therapy.
  • EPA blocks TNF-α-induced inhibition of sugar uptake in Caco-2 cells
           via GPR120 and AMPK
    • Abstract: The aim of the present work was to investigate in Caco-2 cells whether eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, could block the inhibitory effect of tumor necrosis factor-α (TNF-α) on sugar transport, and identify the intracellular signaling pathways involved.After pre-incubation of the Caco-2 cells with TNF-α and EPA for 1 h, EPA prevented the inhibitory effect of the cytokine on α-methyl-D-glucose (αMG) uptake (15 min) and on SGLT1 expression at the brush border membrane, measured by Western blot. The ERK1/2 inhibitor PD98059 and the AMPK activator AICAR also prevented the inhibitory effect of TNF-α on both αMG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-α-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-α-induced decrease of αMG uptake and AMPK phosphorylation.In summary, TNF-α inhibits αMG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-α through the involvement of GPR120 and AMPK activation. This article is protected by copyright. All rights reserved
  • Dysregulation of mitochondrial bioenergetics and quality control by HIV-1
           Tat in cardiomyocytes
    • Abstract: Cardiovascular disease remains a leading cause of morbidity and mortality in HIV-positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV-encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV-infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study, we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+]m) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis.Tat expression increases mitochondrial mass and impairs mitophagy. ICC using an antibody againse Tom20 showed that Tat altered mitochondrial morphology.
  • The nucleus is irreversibly shaped by motion of cell boundaries in cancer
           and non-cancer cells
    • Abstract: Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume.In many models of nuclear shaping in adherent cells, nuclear deformations (flattened or elongated shapes) are proposed to store elastic energy. However, in this work, we found that there was no elastic relaxation after removing deformed nuclear shapes out of cells, implying no storage of elastic energy. We propose that motion of nearby cell boundaries exert irreversible stresses on the nucleus to change its shape.
  • Electric fields accelerate cell polarization and bypass myosin action in
           motility initiation
    • Abstract: Stationary symmetrical fish keratocyte cells break symmetry and become motile spontaneously but slowly. We found that applying electric field (EF) accelerates the polarization by an order of magnitude. While spontaneously polarized cells move persistently for hours, the EF-induced polarity is lost in a majority of cells when the EF is switched off. However, if the EF is applied for a long time and then switched off, the majority of cell move stably. Myosin inhibition abolishes spontaneous polarization, but does not slow down EF-induced polarization, and after the EF is turned off, motility does not stop; however, the cell movements are erratic. Our results suggest that the EF rapidly polarizes the cells, but that resulting polarization becomes stable slowly, and that the EF bypasses the requirement for myosin action in motility initiation. This article is protected by copyright. All rights reserved
  • Mineral trioxide aggregate enhances the osteogenic capacity of periodontal
           ligament stem cells via NF-κB and MAPK signaling pathways
    • Abstract: IntroductionMineral trioxide aggregate (MTA), as a bioactive material, has a widespread application in clinical practice. To date, the effects of MTA on the proliferation and differentiation of human periodontal ligament stem cells (hPDLSCs) remain unclear.Materials and methodshPDLSCs were isolated from human periodontal ligament tissues and cultured with MTA conditioned media. Cell counting kit-8 (CCK-8) assay was performed to assess the proliferation capacity of MTA-treated hPDLSCs. Immunofluorescence assay, alkaline phosphatase (ALP) activity, alizarin red staining, real-time RT-PCR, and western blot analyses were used to investigate the odonto/osteogenic capacity of hPDLSCs as well as the involvement of NF-κB and MAPK pathways.ResultsALP activity assay revealed that 2 mg/mL was the optimal concentration for the induction of hPDLSCs by MTA. The protein expression of DSP, RUNX2, OCN, OSX, OPN, DMP1, ALP, and COL-I in MTA-treated hPDLSCs was significantly higher than those in control group (P 
  • Microtubules Regulate Brush Border Formation
    • Abstract: Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation towards the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity. This article is protected by copyright. All rights reserved
  • Cover Image, Volume 232, Number 11, November 2017
    • Abstract: Cover: The cover image, by Yi Sheng et al., is based on the Mini-Review Article Membrane Ion Channels and Receptors in Animal Lifespan Modulation,
      DOI : 10.1002/jcp.25824.
  • Table of Contents, Editor's Choice, Highlights
  • Mammary alveolar epithelial cells convert to brown adipocytes in
           post-lactating mice
    • Abstract: During pregnancy and lactation, subcutaneous white adipocytes in the mouse mammary gland transdifferentiate reversibly to milk-secreting epithelial cells. In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes, highlighting a hitherto unappreciated feature of mouse adipose organ plasticity.In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes.
  • Dysbiosis and Disease: Many Unknown Ends, Is It Time to Formulate
           Guidelines for Dysbiosis Research'
    • Abstract: Dysbiosis has been implicated in modulation of disease and treatment outcome. It also has been linked to the reproducibility concerns. However, research community needs guidelines on animal models and dysbiosis research to tackle the complexities associated with it. There is a necessity for multi-disciplinary collaborative approach in setting up certain guidelines to hasten the dysbiosis research in a hassle-free manner. J. Cell. Physiol. 232: 2929–2930, 2017. © 2016 Wiley Periodicals, Inc.Dysbiosis has been implicated in modulation of disease and treatment outcome. It also has been linked to the reproducibility concerns. However, research community needs guidelines on animal models and dysbiosis research to tackle the complexities associated with it. There is a necessity for multi-disciplinary collaborative approach in setting up certain guidelines to hasten the dysbiosis research in a hassle-free manner.
  • Targeting stroma in pancreatic cancer: Promises and failures of targeted
    • Abstract: Desmoplasia or abundant fibrotic stroma is a typical property of most malignancies, which has a great effect on tumorigenesis, angiogenesis, and resistance to therapy. The activated stroma cells comprises several cell types including endothelial cells, nerve cells, inflammatory/macrophages cells, stellate cells, and extracellular matrix. In other word, the interactions of cancer-stroma modulate tumorigenesis, therapy resistance, and poor delivery of drugs. Therefore, targeting the tumor stroma in combination with conventional chemotherapeutic agents could provide a promising approach in the treatment of pancreatic cancer. This review summarizes the current knowledge about pancreatic stellate cells, targeting stroma compartments with particular emphasis on preclinical, and clinical trials on targeting of stroma as an option in pancreatic cancer treatment.
  • Interplay between the miRNome and the epigenetic machinery: Implications
           in health and disease
    • Abstract: Epigenetics refers to functionally relevant genomic changes that do not involve changes in the basic nucleotide sequence. Majorly, these are of two types: DNA methylation and histone modifications. Small RNA molecules called miRNAs are often thought to mediate post-transcriptional epigenetic changes by mRNA degradation or translational attenuation. While DNA methylation and histone modifications have their own independent effects on various cellular events, several reports are suggestive of an obvious interplay between these phenomena and the miRNA regulatory program within the cell. Several miRNAs like miR-375, members of miR-29 family, miR-34, miR-200, and others are regulated by DNA methylation and histone modifications in various types of cancers and metabolic diseases. On the other hand, miRNAs like miR-449a, miR-148, miR-101, miR-214, and miR-128 target members of the epigenetic machinery and their dysregulation leads to diverse cellular aberrations. In spite of being independent cellular events, emergence of such reports that suggest a connection between DNA methylation, histone modification, and miRNA function in several diseases indicate that this connecting axis offers a valuable target with great therapeutic potential that might be exploited for disease management. We review the current status of crosstalk between the major epigenetic modifications and the miRNA machinery and discuss this in the context of health and disease.DNA methylation and histone modifications are the two main epigenetic events within the cell. miRNAs regulate cellular pathways by transcriptional attenuation and/or mRNA degradation. Interestingly, both of these phenomena demonstrate significant crosstalk between themselves. Such a connection offers a valuable composite target with great therapeutic potential.
  • Membrane ion Channels and Receptors in Animal lifespan Modulation
    • Abstract: Acting in the interfaces between environment and membrane compartments, membrane ion channels, and receptors transduce various physical and chemical cues into downstream signaling events. Not surprisingly, these membrane proteins play essential roles in a wide range of cellular processes such as sensory perception, synaptic transmission, cellular growth and development, fate determination, and apoptosis. However, except insulin and insulin-like growth factor receptors, the functions of membrane receptors in animal lifespan modulation have not been well appreciated. On the other hand, although ion channels are popular therapeutic targets for many age-related diseases, their potential roles in aging itself are largely neglected. In this review, we will discuss our current understanding of the conserved functions and mechanisms of membrane ion channels and receptors in the modulation of lifespan across multiple species including Caenorhabditis elegans, Drosophila, mouse, and human.Acting in the interfaces between environment and membrane compartments, membrane ion channels and receptors transduce various physical and chemical cues into downstream signaling events. In this review, we will discuss our current understanding of the conserved functions and mechanisms of membrane ion channels, and receptors in the modulation of lifespan across multiple species including Caenorhabditis elegans, Drosophila, mouse, and human.
  • Osteoblast role in osteoarthritis pathogenesis
    • Abstract: Even if osteoarthritis pathogenesis is still poorly understood, numerous evidences suggest that osteoblasts dysregulation plays a key role in osteoarthritis pathogenesis. An abnormal expression of OPG and RANKL has been described in osteoarthritis osteoblasts, which is responsible for abnormal bone remodeling and decreased mineralization. Alterations in genes expression are involved in dysregulation of osteoblast function, bone remodeling, and mineralization, leading to osteoarthritis development. Moreover, osteoblasts produce numerous transcription factors, growth factors, and other proteic molecules which are involved in osteoarthritis pathogenesis.(1) Osteoarthritis pathogenesis is still poorly understood. (2) Osteoblasts dysregulation plays a key role in osteoarthritis pathogenesis.
  • Roles of Notch1 Signaling in Regulating Satellite Cell Fates Choices and
           Postnatal Skeletal Myogenesis
    • Abstract: Adult skeletal muscle stem cells, also called satellite cells, are indispensable for the growth, maintenance, and regeneration of the postnatal skeletal muscle. Satellite cells, predominantly quiescent in mature resting muscles, are activated after skeletal muscle injury or degeneration. Notch1 signaling is an evolutionarily conserved pathway that plays crucial roles in satellite cells homeostasis and postnatal skeletal myogenesis and regeneration. Activation of Notch1 signaling promotes the muscle satellite cells quiescence and proliferation, but inhibits differentiation of muscle satellite cells. Notably, the new roles of Notch1 signaling during late-stage of skeletal myogenesis including in post-differentiation myocytes and post-fusion myotubes have been recently reported. Here, we mainly review and discuss the regulatory roles of Notch1 in regulating satellite cell fates choices and skeletal myogenesis. J. Cell. Physiol. 232: 2964–2967, 2017. © 2016 Wiley Periodicals, Inc.Notch1 signaling is an evolutionarily conserved pathway that plays crucial roles in satellite cells homeostasis and postnatal skeletal myogenesis. Recently, the new roles of Notch1 signaling during late-stage of skeletal myogenesis including in post-differentiation myocytes and post-fusion myotubes have been reported. Here, we mainly review and discuss the regulatory roles of Notch1 in regulating satellite cell fates choices and skeletal myogenesis.
  • Anti-Atherosclerotic Effects of Vitamins D and E in Suppression of
    • Abstract: Atherosclerosis is a progressive and multifactorial disease which occurs under the influence of various risk factors including endothelial dysfunction (ED), oxidative stress, and low-density lipoprotein (LDL) oxidation. In contract to the initial hypotheses on the usefulness of vitamin E supplementation for cardiovascular disease prevention, large outcome trials showed consumption of vitamin E has no obvious effect on cardiovascular disease and, in some cases, it may even increase the rate of mortality. This seemingly unexpected finding may be due to the opposite effects of vitamin E compounds. Vitamin E is a group of compounds which have different and even opposing effects, yet in most of the studies, the exact consumed component of vitamin E is not determined. It appears that the combined consumption of gamma-tocopherol, vitamin C, D, and tetrahydrobiopterin (BH4) may be extremely effective in both preventing atherogenesis and suppressing plaque development. In this regard, one of main issues is effect of vitamins E and D deficiency on microRNAs network in atherosclerosis. Various studies have indicated that miRNAs have key roles in atherosclerosis pathogenesis. The deficiency of vitamins E and D could provide a deregulation for miRNAs network and these events could lead to progression of atherosclerosis. Here, we highlighted a variety of mechanisms involve in the progression of atherosclerosis and effects of vitamins D and E on these mechanisms. Moreover, we summarized miRNAs involve in atherosclerosis and their regulation by vitamins E and D deficiency. J. Cell. Physiol. 232: 2968–2976, 2017. © 2016 Wiley Periodicals, Inc.
  • Crosstalk of autophagy and apoptosis: Involvement of the dual role of
           autophagy under ER stress
    • Abstract: Endoplasmic reticulum (ER) stress is a common cellular stress response that is triggered by a variety of conditions that disturb cellular homeostasis, and induces cell apoptosis. Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is closely related to the apoptosis induced by ER stress. There are common upstream signaling pathways between autophagy and apoptosis induced by ER stress, including PERK/ATF4, IRE1α, ATF6, and Ca2+. Autophagy can not only block the induction of apoptosis by inhibiting the activation of apoptosis-associated caspase which could reduce cellular injury, but also help to induce apoptosis. In addition, the activation of apoptosis-related proteins can also inhibit autophagy by degrading autophagy-related proteins, such as Beclin-1, Atg4D, Atg3, and Atg5. Although the interactions of different autophagy- and apoptosis-related proteins, and also common upstream signaling pathways have been found, the potential regulatory mechanisms have not been clearly understood. In this review, we summarize the dual role of autophagy, and the interplay and potential regulatory mechanisms between autophagy and apoptosis under ER stress condition.The dura role of autophagy, pro-survival, and -death, may be dependent on the extent of ER stress, and it implements by the regulation of apoptosis. Autophagy can not only block the induction of apoptosis by inhibiting the activation of apoptosis-associated caspase which could reduce cellular injury, but also help to induce apoptosis. In addition, the activation of apoptosis-related proteins can also inhibit autophagy by degrading autophagy-related proteins, such as Beclin-1, Atg4D, Atg3, and Atg5.
  • End stage renal disease-induced hypercalcemia may promote aortic valve
           calcification via Annexin VI enrichment of valve interstitial cell
           derived-matrix vesicles
    • Abstract: Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p 
  • Stimulation of oral fibroblast chemokine receptors identifies CCR3 and
           CCR4 as potential wound healing targets
    • Abstract: The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.Gingiva fibroblasts express 12 chemokine receptors. Fourteen chemokines were used to study proliferation (top), migration (middle), and secretion of wound healing mediators by gingiva fibroblasts (bottom). Predominantly CCR3 and CCR4 stimulation influenced these oral wound healing parameters.
  • 2-methoxyestradiol impacts on amino acids-mediated metabolic reprogramming
           in osteosarcoma cells by its interaction with NMDA receptor
    • Abstract: Deregulation of serine and glycine metabolism, have been identified to function as metabolic regulators in supporting tumor cell growth. The role of serine and glycine in regulation of cancer cell proliferation is complicated, dependent on concentrations of amino acids and tissue-specific. D-serine and glycine are coagonists of N-methyl-D-aspartate (NMDA) receptor subunit GRIN1. Importantly, NMDA receptors are widely expressed in cancer cells and play an important role in regulation of cell death, proliferation, and metabolism of numerous malignancies. The aim of the present work was to associate the metabolism of glycine and D-serine with the anticancer activity of 2-methoxyestradiol. 2-methoxyestradiol is a potent anticancer agent but also a physiological 17β- estradiol metabolite. In the study we have chosen two malignant cell lines expressing functional NMDA receptors, that is osteosarcoma 143B and breast cancer MCF7. We used MTS assay, migration assay, flow cytometric analyses, Western blotting and immunoprecipitation techniques as well as molecular modeling studies. We have demonstrated the extensive crosstalk between the deregulated metabolic network and cancer cell signaling. Herein, we observed an anticancer effect of high concentrations of glycine and D-serine in osteosarcoma cells. In contrast, the amino acids when used at low, physiological concentrations induced the proliferation and migration of osteosarcoma cells. Importantly, the pro-cancergogenic effects of both glycine and D-serine where abrogated by the usage of 2-methoxyestradiol at both physiological and pharmacological relevant concentrations. The obtained data confirmed that 2-methoxyestradiol may be a physiological anticancer molecule.Interaction of 2-methoxyestradiol with NMDA receptor. GluN1/GluN2A Ligand Binding Domain containing 2-metoxyestradiol and agonists-Glycine or D-Serine.
  • DJ-1/Park7 Sensitive Na+/H+ Exchanger 1 (NHE1) in CD4+ T Cells
    • Abstract: DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+/H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pHi) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2′,7′ −dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ-1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4+ T cells, and blunted the difference between DJ-1−/− and DJ-1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1−/− CD4+ T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 232: 3050–3059, 2017. © 2016 Wiley Periodicals, Inc.Our study described that DJ-1 deficient CD4+ T cells leads to upregulation of NHE1 and enhanced ROS production, therefore results in increase in NHE1 activity. NHE1 regulation in DJ-1 deficient CD4+ T cells is dependent on ROS and PTK signaling. Thus, our data first time described that link between DJ-1 proteins with responses on ROS production and NHE1 functions.
  • SV40 Infection of Mesenchymal Stromal Cells From Wharton's Jelly Drives
           the Production of Inflammatory and Tumoral Mediators
    • Abstract: The Mesenchymal Stromal Cells from umbilical cord Wharton's jelly (WJSCs) are a source of cells with high potentiality for the treatment of human immunological disorders. Footprints of the oncogenic viruses Simian Virus 40 (SV40) and JC Virus (JCPyV) have been recently detected in human WJSCs specimens. The aim of this study is to evaluate if WJSCs can be efficiently infected by these Polyomaviruses and if they can potentially exert tumoral activity. Cell culture experiments indicated that WJSCs could sustain both SV40 and JCPyV infections. A transient and lytic replication was observed for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing a viral progeny at low titer without evident cytopathic effect (CPE). Considering the association between SV40 and human tumors and the reported ability of the oncogenic viruses to drive the host innate immune response to cell transformation, the expression profile of a large panel of immune mediators was evaluated in supernatants by the Bioplex platform. RANTES, IL-3, MIG, and IL-12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly measured at high concentration comparing to control. These findings represent a new aspect of SV40 biological activity in the humans, highlighting its interaction with specific host cellular pathways. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source. J. Cell. Physiol. 232: 3060–3066, 2017. © 2016 Wiley Periodicals, Inc.This study aims to evaluate if WJSCs can be efficiently infected by the Polyomaviruses SV40 and JCPyV and if they can exert their potential tumoral activity. Results reported a transient and lytic replication for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing viral progeny. Specific factors, including RANTES, IL-3, MIG, and IL-12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly produced at high concentration. These findings represent a new aspect of SV40 biological activity in human host. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source.
  • Deregulated ALG-2/HEBP2 axis alters microtubule dynamics and mitotic
           spindle behavior to stimulate cancer development
    • Abstract: Cancer cells are characterized by genomic instability, resulting in the accumulation of mutations that promote cancer progression. One way that genomic instability can arise is through improper regulation of the microtubule cytoskeleton that impacts the function of the mitotic spindle. In this study, we have identified a critical role for the interaction between apoptosis-linked gene 2 (ALG-2) and heme-binding protein 2 (HEBP2) in the above processes. Our data show that the gene copy numbers and mRNA levels for both ALG-2 and HEBP2 are significantly upregulated in breast and lung cancer. Coexpression of ALG-2 and HEBP2 markedly increases the cytoplasmic pool of ALG-2 and alters the subcellular distribution of HEBP2. Our data further reveal that abnormality in the ALG-2/HEBP2 interaction impairs spindle orientation and positioning during mitosis. In addition, this complex appears to modulate the dynamic properties of microtubules in cancer cells. These finding thus uncover an important function for deregulated ALG-2/HEBP2 axis in cancer development by influencing microtubule dynamics and spindle behavior, providing novel insight into the etiology and pathogenesis of cancer.In this study, we uncover a previously unrecognized role for deregulated ALG-2/HEBP2 axis in cancer development. Mechanistically, this is mediated by altered microtubule dynamics and impaired spindle orientation and positioning.
  • LCN2 overexpression in bone enhances the hematopoietic compartment via
           modulation of the bone marrow microenvironment
    • Abstract: Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg). In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild-type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF, and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice.The overexpression of LCN2 in bone stimulates the expansion of the HSC by inducing pro-retention factors and inhibiting pro-egress signals for hematopoietic stem cells.
  • Pyk2 inhibition promotes contractile differentiation in arterial smooth
    • Abstract: Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24–96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.The Pyk2 inhibitor PF-4594755 potently inhibits Pyk2 phosphorylation of vascular smooth muscle cells stimulated by FBS or PDGF-BB. This is accompanied by increased expression of smooth muscle marker proteins without concomitant effects on cell proliferation. Pyk2 inhibition upregulates SERCA2a expression and may preserve the contractile phenotype by lowering basal intracellular calcium and promoting voltage-dependent control of calcium influx over the plasma membrane.
  • Smoothened-antagonists reverse homogentisic acid-induced alterations of
           Hedgehog signaling and primary cilium length in alkaptonuria
    • Abstract: Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway.Primary cilia of alkaptonuric (AKU) chondrocytes are shorter as compared to healthy cells and HGA treatment leads to the same phenotype. Primary cilia alterations are correlated to an aberrant Hedgehog (Hh) signaling activation. Treatment of HGA-treated chondrocytes with antagonists of Smoothened reported cilia lengths and Hh activation to the normal levels.
  • Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and
           fibroblast growth factor receptor signaling
    • Abstract: Receptor tyrosine kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and fibroblast growth factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila insulin receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glia.Receptor tyrosine kinase signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Glycosphingolipids modulate signal transduction, including that mediated by the receptor tyrosine kinases. We report that an early product in glycosphingolipid biosynthesis, mactosylceramide, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glial cells.
  • HMGB1 down-regulation mediates terameprocol vascular anti-proliferative
           effect in experimental pulmonary hypertension
    • Abstract: Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative, and apoptotic-resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well-established pre-clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi-ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT-treated rats, using an iTRAQ-based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti-proliferative effect of TMP seems to be explained, at least in part, by the down-regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH.TMP-reduced pulmonary and cardiac remodeling and hemodynamic features of PAH. Its vascular anti-proliferative effect seems to be mediated by the protein HMGB1. TMP may be an effective therapeutic option in the clinical management of PAH.
  • Optimized method for isolating highly purified and functional porcine
           aortic endothelial and smooth muscle cells
    • Abstract: Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.Isolating pure populations of endothelial and smooth muscle cells from large elastic mammalian vessels remains a challenge in vascular biology. A sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells has been established. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.
  • A critical role for adiponectin-mediated development of endometrial
    • Abstract: Adiponectin is one of the adipokines in the collagen superfamily. It is secreted primarily by white adipocytes and influences reproductive processes including ovarian and uterine functions. Adiponectin regulates energy homeostasis, insulin sensitivity, and is anti-inflammatory in various tissues. Its receptors (ADIPOR1 and ADIPOR2) are widely expressed in mammalian tissues, including porcine conceptuses and endometrial during the estrous cycle and peri-implantation period of pregnancy. However, regulatory effects of adiponectin on endometrial epithelial cells are unknown. Therefore, we investigated the effects of parity on expression of ADIPOR1 and ADIPOR2 and the effects of adiponectin in the porcine endometrium during early pregnancy. Results of this study revealed robust expression of ADIPOR1 and ADIPOR2 in uterine luminal (LE) and glandular (GE) epithelia during early pregnancy and expression decreased as with increasing parity. For porcine luminal epithelial (pLE) cells, adiponectin enhanced proliferation, and increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time-dependent manner. Moreover, the abundance of adiponectin-activated signaling molecules were suppressed by pharmacological inhibitors including wortmannin, U0126, SP600125, and SB203580, respectively, in pLE cells. Furthermore, inhibition of each targeted signal transduction molecule influenced proliferation of adiponectin-stimulated pLE cells. In addition, adiponectin inhibited tunicamycin-induced endoplasmic reticulum (ER)-stress through effects on ER stress regulated proteins in pLE cells. Collectively, these results suggest that adiponectin affects development of porcine uterine epithelia and reproductive performance through modulation of PI3K/AKT and MAPK cell signaling pathways.Adiponectin enhance uterine receptivity to implantation and placentation through PI3K/AKT and MAPK pathways for development of the porcine uterus and reproductive performance.
  • TRIM33 is essential for osteoblast proliferation and differentiation via
           BMP pathway
    • Abstract: Tripartite motif containing 33 (TRIM33) functions both as a positive and negative regulator of the TGF-β/BMP pathway in tumors; however, its effect and mechanism during osteoblast proliferation and differentiation, which involves the TGF-β/BMP pathway is not defined. In this study, we used mouse C3H10T1/2 mesenchymal stem cell line and MC3T3-E1 preosteoblasts to investigate the role of TRIM33 during this process. The results demonstrated that the expression of TRIM33 increased during the differentiation. Moreover, the overexpression or knockdown of TRIM33 resulted in both an augmentation or decrease in osteoblast differentiation, which were measured by the expression of alkaline phosphatase (ALP) at the mRNA level, both Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) at the protein level, and the formation of mineral modules. To further demonstrate the mechanism of TRIM33 in this process, we found that TRIM33 could positively mediate the BMP pathway by forming TRIM33-Smad1/5 complex. This interaction between TRIM33 and Smad1/5 triggered the phosphorylation of Smad1/5. In addition, the essential role of TRIM33 in osteoblast proliferation was determined in this study by CellCounting Kit (CCK) −8 and cell cycle assays. In summary, we establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway.We establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway.
  • Regulation of chondrocyte functions by transient receptor potential cation
           channel V6 in osteoarthritis
    • Abstract: Transient receptor potential vanilloid (TRPV) channels function to maintain the dynamic balance of calcium signaling and calcium metabolism in bones. The goal of this study was to determine the potential role of TRPV6 in regulation of chondrocytes. The level of TRPV6 expression was analyzed by western blot in articular cartilage derived from the knee joints of osteoarthritis (OA) rat models and OA patients. Bone structure and osteoarthritic changes in the knee joints of TRPV6 knockout mice were examined using micro-computed and histological analysis at the age of 6 and 12 months old. Furthermore, to investigate the effects of TRPV6 on chondrocyte extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis, we decreased and increased TRPV6 expression in chondrocytes with lentiviral constructs encoding shRNA targeting TRPV6 and encoding TRPV6, respectively. The results showed that the level of TRPV6 expression in an OA rat model was markedly down-regulated. TRPV6 knockout mice showed severe osteoarthritis changes, including cartilage fibrillation, eburnation, and loss of proteoglycans. In addition, deficiency of TRPV6 clearly affected chondrocyte function, such as extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis. Taken together, our results implicated that TRPV6 channel, as a chondro-protective factor, was involved in the pathogenesis of OA.The level of TRPV6 expression in an OA rat model was markedly down-regulated. TRPV6 knockout mice showed severe osteoarthritic changes. Deficiency of TRPV6 clearly affected chondrocyte function.
  • Biological roles of glial fibrillary acidic protein as a biomarker in
           cartilage regenerative medicine
    • Abstract: Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology.We provide a rational that GFAP of the auricular chondrocytes can be used as a matrix production marker in culture auricular chondrocytes.
  • Cover Image, Volume 232, Number 1, January 2017 (page i)
  • Atrogin-1 increases smooth muscle contractility through myocardin
           degradation. Pavneet Singh, Dong Li, Yu Gui, Xi-Long Zheng
  • Retraction: “Identification of Novel Biomarkers for Pancreatic Cancer
           Using Integrated Transcriptomics With Functional Pathways Analysis” by
           Zhang, X., Tong, P., Chen, J., Pei, Z., Zhang, X., Chen, W., Xu, J. and
           Wang, J.
    • Abstract: The above article from the Journal of Cellular Physiology, published online on 10 March 2016 in Wiley Online Library as Early View (, has been retracted by agreement between Gary Stein, the journal's Editor-in-Chief, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation at the University of Texas, MD Anderson Cancer Center, which confirmed that the article was submitted and approved for publication by Dr. Jin Wang without acknowledgement of NIH funding received or the consent and authorship of Dr. Ann Killary and Dr. Subrata Sen, with whom the manuscript was originally drafted.
  • GSK-3β Inhibition Suppresses Instability-induced Osteolysis by a Dual
           Action on Osteoblast and Osteoclast Differentiation
    • Abstract: Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/β-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/β–catenin signaling by inhibiting glycogen synthase kinase-3β (GSK-3β) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3β inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3β inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of β-catenin, Runx2, Osterix, Col1α1 and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3β inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3β inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3β inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis. This article is protected by copyright. All rights reserved
  • TNF-α-induced NF-κB activation promotes myofibroblast differentiation of
           LR-MSCs and exacerbates bleomycin-induced pulmonary fibrosis
    • Abstract: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease of unknown cause. It has been reported that both lung resident mesenchymal stem cells (LR-MSCs) and tumor necrosis factor-α (TNF-α) play important roles in the development of pulmonary fibrosis. However, the underlying connections between LR-MSCs and TNF-α in the pathogenesis of pulmonary fibrosis are still elusive. In this study, we found that the pro-inflammatory cytokine TNF-α and the transcription factor nuclear factor kappa B (NF-κB) p65 subunit were both upregulated in bleomycin-induced fibrotic lung tissue. In addition, we discovered that TNF-α promotes myofibroblast differentiation of LR-MSCs through activating NF-κB signaling. Interestingly, we also found that TNF-α promotes the expression of β-catenin. Moreover, we demonstrated that suppression of the NF-κB signaling could attenuate myofibroblast differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis which were accompanied with decreased expression of β-catenin. Our data implicates that inhibition of the NF-κB signaling pathway may provide a therapeutic strategy for pulmonary fibrosis, a disease that warrants more effective treatment approaches. This article is protected by copyright. All rights reserved
  • Primary familial brain calcification with a novel SLC20A2 mutation:
           Analysis of PiT-2 expression and localization
    • Abstract: Primary Familial Brain Calcification (PFBC) is an autosomal dominant rare disorder characterized by bilateral and symmetric brain calcifications and neuropsychiatric manifestations. Four genes have been linked to PFBC: SLC20A2, PDGFRB, PDGFB and XPR1. In this study, we report molecular and clinical data of a PFBC patient carrying a novel SLC20A2 mutation and we investigate the impact of the mutation on PiT-2 expression and function. Sanger sequencing of SLC20A2, PDGFRB, PDGFB, XPR1 led to the identification of a novel duplication of twelve nucleotides (c.1876_1887dup/ p.Trp626_Thr629dup) in SLC20A2 gene. SLC20A2 encodes for a cell membrane transporter (PiT-2) involved in maintenance of inorganic phosphate homeostasis. We performed an analysis of expression and functionality of PiT-2 protein in patient primary cultured fibroblasts. In patient fibroblasts, the mutation does not affect PiT-2 expression but alter sub-cellular localization. The Pi-uptake assay revealed a less Pi depletion in patient than in control fibroblasts, suggesting that SLC20A2 duplication may impair Pi internalization. This is the first study reporting sub-cellular expression analysis of mutant PiT-2 in primary cultured fibroblasts from a PFBC patient, showing that p.Trp626_Thr629dup in SLC20A2 alters PiT-2 sub-cellular localization and reduces Pi-uptake, leading to onset of PFBC in our patient. This article is protected by copyright. All rights reserved
  • 12-O-Tetradecanoylphorbol-13-acetate and EZH2 inhibition: a novel approach
           for promoting myogenic differentiation in embryonal rhabdomyosarcoma cells
    • Abstract: Rhabdomyosarcoma (RMS) is a soft tissue sarcoma that arises from muscle precursors affecting predominately children and young adults. It can be divided into two main classes: embryonal (eRMS) and alveolar rhabodomyosarcomas (aRMS). Despite the expression of early muscle specific genes, RMS cells fail to complete myogenesis even in differentiation conditions.We previously demonstrated that Enhancer Zeste of Homolog 2 (EZH2), the catalytic subunits of PRC2 complex, contributes to inhibit muscle differentiation in eRMS and its down-regulation causes a partial recovery of myogenesis.12-O-Tetradecanoylphorbol-13-acetate (TPA) is a molecule able to induce differentiation in eRMS with a mechanism that involves the protein kinase C (PKC). In this paper we report that treatment with TPA reduces the expression of EZH2 without affecting levels of H3K27me3. The combination of TPA with GSK126, an inhibitor of the catalytic activity of EZH2, has a synergic effect on the induction of muscle differentiation in RD rhabdomyosarcoma cells, suggesting a new therapeutic combinatory approach for RMS treatment. This article is protected by copyright. All rights reserved
  • The Ubiquitin Ligase SCFFBXW7α Promotes ATA3 Degradation
    • Abstract: GATA3 is a key transcription factor in cell fate determination and its dysregulation has been implicated in various types of malignancies. However, how the abundance and function of GATA3 are regulated remains unclear. Here, we report that GATA3 is physically associated with FBXW7α, and FBXW7α destabilizes GATA3 through assembly of a SKP1-CUL1-F-box E3 ligase complex. Importantly, we showed that FBXW7α promotes GATA3 ubiquitination and degradation in a GSK3 dependent manner. Furthermore, we demonstrated that FBXW7α inhibits breast cancer cells survival through destabilizing GATA3, and the expression level of FBXW7αis negatively correlated with that of GATA3 in breast cancer samples. This study indicated that FBXW7α is a critical negative regulator of GATA3 and revealed a pathway for the maintenance of GATA3 abundance in breast cancer cells. This article is protected by copyright. All rights reserved
  • Structure-based release analysis of the JC virus agnoprotein regions: A
           role for the hydrophilic surface of the major alpha helix domain in
    • Abstract: Agnoprotein (Agno) is an important regulatory protein of JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) and these viruses are unable to replicate efficiently in the absence of this protein. Recent 3D-NMR structural data revealed that Agno contains two alpha-helices (a minor and a major) while the rest of the protein adopts an unstructured conformation (Coric et al., 2017, J Cell Biochem). Previously, release of the JCV Agno from the Agno-positive cells was reported. Here, we have further mapped the regions of Agno responsible for its release by a structure-based systematic mutagenesis approach. Results revealed that amino acid residues (Lys22, Lys 23, Phe31, Glu34 and Asp38) located either on or adjacent to the hydrophilic surface of the major alpha-helix domain of Agno play critical roles in release. Additionally, Agno was shown to strongly interact with unidentified components of the cell surface when cells are treated with Agno, suggesting additional novel roles for Agno during the viral infection cycle. This article is protected by copyright. All rights reserved
  • Cyanidin Suppresses Autophagic Activity Regulating Chondrocyte
           Hypertrophic Differentiation
    • Abstract: Cartilage is a kind of special connective tissue which does not contain neither blood vessels nor lymphatics and nerves. Therefore, the damage in cartilage is difficult to be repaired spontaneously. Constructing tissue engineered cartilage provides a new technique for cartilage repairing. Mesenchymal stem cells (MSCs) possess a unique capability of self-renew and can differentiate into pre-chondrocytes which are frequently applied as seed cells in tissue engineering. However, in regenerated cartilage the chondrocytes derived from MSCs can hardly maintain homeostasis and preferentially present hypertrophic like phenotype. We investigated the effects of cyanidin, a natural organic compound, on chondrogenic and subsequent hypertrophic differentiation of MSCs in order to seek approaches to inhibit chondrocyte hypertrophy. We evaluated the effects of cyanidin on expression of chondrogenic and hypertrophic marker genes through RT-PCR, western blot, alcian blue staining and immunocytochemistry. The results showed that both chondrogenic related genes Sox9, Col2a1 and hypertrophic marker genes Runx2, Col10a1 were inhibited by cyanidin. In addition, we found that cyanidin promoted Nrf2 and p62 expression and suppressed LC3B expression during chondrogenic stage of MSCs. Meanwhile phosphorylation of IκBα and autophagosome related protein LC3B were inactivated by cyanidin during chondrocyte hypertrophic stage. Furthermore, rapamycin, an autophagy activator, abrogated the inhibitory effect of cyanidin on chondrogenic and hypertrophic differentiation of MSCs. In conclusion, one potential mechanism of cyanidin, by which the chondrogenic and hypertrophic differentiation of MSCs were inhibited, was due to decreased autophagy activity. Our results indicated that cyanidin was a potential therapeutic agent for keeping mature chondrocyte functions. This article is protected by copyright. All rights reserved
  • Neoadjuvant Chemotherapy In Triple-Negative Breast Cancer: A Multicentric
           Retrospective Observational Study In Real-Life Setting
    • Abstract: We aimed to assess the efficacy of neoadjuvant chemotherapy (NACT) in a cohort of 213 triple-negative breast cancer (TNBC) patients treated in real-world practice at 8 Italian cancer centres. We computed descriptive statistics for all the variable of interest. Factors testing significant in univariate analysis were included in multivariate models. Survival data were compared by Kaplan-Meier curves and log-rank test.The median follow-up was 45 months. We observed 60 (28.2%) pathological complete response (pCR). The sequential anthracyclines-taxanes-based regimens produced the highest rate of pCR (42.6%), followed by concomitant anthracycline-taxane (24.2%), and other regimens (15.6%) (p = 0.008). When analyzing the role of baseline Ki-67, a 50% cut-off was the optimal threshold value for pCR prediction (p = 0.0005). The 5-year disease-free survival (DFS) was 57.3% and the 5-year overall survival (OS) was 70.8%. In patients not achieving pCR, the optimal Ki-67 variation between biopsy and surgical specimen with prognostic relevance on long-term outcomes was 13% (p = 0.04). Patients with a Ki-67 reduction (rKi-67) 6 cycles compared with their counterparts, p = 0.02). In multivariate analysis, node status, grading and bio-pathological treatment response (including pCR and rKi-67) impacted DFS and OS. Our results confirmed the advantage conferred by more than 6 cycles of a sequential antracycline-taxane-based NACT. Higher baseline Ki-67 values shows greater predictive significance on pathogical response, while the rKi-67 plays a prognostic role on long-term outcomes. This article is protected by copyright. All rights reserved
  • Differential protein modulation by ketoprofen and ibuprofen underlines
           different cellular response by gastric epithelium
    • Abstract: Ketoprofen L-lysine salt (KLS), is widely used due to its analgesic efficacy and tolerability, and L-lysine was reported to increase the solubility and the gastric tolerance of ketoprofen. In a recent report, L-lysine salification has been shown to exert a gastroprotective effect due to its specific ability to counteract the NSAIDs-induced oxidative stress and up-regulate gastroprotective proteins. In order to derive further insights into the safety and efficacy profile of KLS, in this study we additionally compared the effect of lysine and arginine, another amino acid counterion commonly used for NSAIDs salification, in control and in ethanol challenged human gastric mucosa model. KLS is widely used for the control of post-surgical pain and for the management of pain and fever in inflammatory conditions in children and adults. It is generally well tolerated in pediatric patients, and data from three studies in >900 children indicate that oral administration is well tolerated when administered for up to 3 weeks after surgery. Since only few studies have so far investigated the effect of ketoprofen on gastric mucosa maintenance and adaptive mechanisms, in the second part of the study we applied the cMap approach to compare ketoprofen-induced and ibuprofen-induced gene expression profiles in order to explore compound-specific targeted biological pathways. Among the several genes exclusively modulated by ketoprofen, our attention was particularly focused on genes involved in the maintenance of gastric mucosa barrier integrity (cell junctions, morphology and viability). The hypothesis was further validated by Real-time PCR. This article is protected by copyright. All rights reserved
  • The depletion of MARVELD1 leads to murine placenta accreta via integrin
           β4-dependent trophoblast cell invasion
    • Abstract: The placenta is a remarkable organ, it serves as the interface between the mother and the fetus. Proper invasion of trophoblast cells is required for a successful pregnancy. Previous studies have found that the adhesion molecule integrin β4 plays important roles during trophoblast cell invasion. Here, we found that the overall birth rate of the MARVELD1 knockout mouse is much lower than that of the wild-type mouse (P
  • Aquaporin1 and 3 modification as a result of chondrogenic differentiation
           of human mesenchymal stem cell
    • Abstract: Chondrocytes are cells of articular cartilage particularly sensitive to water transport and ionic and osmotic changes from extracellular environment and responsible for the production of the synovial fluid. Aquaporins (AQPs) are a family of water and small solute transport channel proteins identified in several tissues, involved in physiological pathways and in manifold human diseases. In a recent period, AQP1 and 3 seem to have a role in metabolic water regulation in articular cartilage of load bearing joints. The aim of this study was to examine the levels of AQP1 and 3 during the chondrogenic differentiation of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT). For the determination of chondrogenic markers and AQPs levels, glycosaminoglycans (GAGs) quantification, immunocytochemistry, RT-PCR and Western blot were used after 0, 7, 14, 21 and 28 days from the start of differentiation. At 21 days, chondrocytes derived from AT-MSCs were able to produce augmented content of GAGs and significant quantity of SOX-9, lubricin, aggrecan and collagen type II, suggesting hyaline cartilage formation, in combination with an increase of AQP3 and AQP1. However, while AQP1 level decreased after 21 days; AQP3 reached higher values at 28 days. The expression of AQP1 and 3 is a manifestation of physiological adaptation of functionally mature chondrocytes able to respond to the change of their internal environment influenced by extracellular matrix. The alteration or loss of expression of AQP1 and AQP3 could contribute to destruction of chondrocytes and to development of cartilage damage. This article is protected by copyright. All rights reserved
  • FHOD1 regulates cytoplasmic actin-based spindle migration for mouse oocyte
           asymmetric cell division
    • Abstract: FHOD1 is a member of Diaphanous related formins (DRFs) which belongs to the Formin family. Previous studies have shown that the DFRs might affect several cellular functions such as morphogenesis, cytokinesis, cell polarity and embryonic differentiation. However, there is no evidence showing the functions of FHOD1 during oocyte meiosis. This study is aimed at exploring the roles of FHOD1 during the mammalian oocyte maturation. Immunofluorescent staining showed that FHOD1 was restricted to the nucleus in germinal vesicle (GV) stage of the oocytes, after the GV breakdown FHOD1 was primarily located at two poles of the spindle at both metaphase I and metaphase II stages. Knockdown of FHOD1 by siRNA injection did not affect polar body extrusion but generated the large polar bodies. In addition, we observed the spindle migration failure in metaphase I oocytes, with a large number of meiotic spindles anchoring in the center of cytoplasm. The expression level of cytoplasmic actin but not cortex actin was significantly reduced, indicating that FHOD1 regulates cytoplasmic actin distribution for the spindle movement. Furthermore, we found that the disruption of ROCK (the Rho-dependent protein kinase) with inhibitor Y-27632 caused the decreased FHOD1 protein expression. Therefore, our data indicate that FHOD1 is regulated by ROCK for cytoplasm actin assembly and spindle migration during mouse oocyte meiosis. This article is protected by copyright. All rights reserved
  • miR-199a-3p is involved in estrogen-mediated autophagy through the
           IGF-1/mTOR pathway in osteocyte-like MLO-Y4 cells
    • Abstract: To date, evidence indicates that estrogen partially modulates cellular processes through microRNAs. Autophagy is a catabolic process that is regulated by multiple factors and is associated with skeletal diseases. However, whether estrogen regulates osteocyte autophagy via microRNAs is largely unknown. In this study, we observed the up-regulation of microRNA-199a-3p, a post-transcriptional regulatory factor, in osteocytic areas in ovariectomized (OVX) mice. The mature forms of miR-199a-3p and pri-miR-199a were produced in response to estrogen signaling in osteocyte-like MLO-Y4 cells. Western blotting, autophagic flux detection, mRFP-GFP-LC3 fluorescence and electron microscopy confirmed that miR-199a-3p induced autophagy in MLO-Y4 cells, although cellular apoptosis was not affected. Additionally, we documented the ability of estrogen to mediate osteocyte autophagy. Based on our in vivo data, estrogen deficiency induced autophagy in osteocytes. Treatment of starved MLO-Y4 cells with 17β-estradiol suppressed the excess autophagy induced by starvation via activation of mammalian target of rapamycin (mTOR)-related signaling cascades, while administration of rapamycin reversed the effects of 17β-estradiol. Meanwhile, miR-199a-3p overexpression reversed 17β-estradiol-mediated regulation of autophagy in MLO-Y4 cells. According to mechanistic studies, miR-199a-3p inhibited the mTOR pathway by directly binding to the 3'-untranslated regions of insulin growth factor-1 (IGF-1) and mTOR. However, overexpression of miR-199a-3p inhibited IGF-1 phosphorylation and mTOR-related pathways. Knockdown of mTOR and IGF-1 abolished estrogen signaling and restored LC3-II expression through mTOR re-activation, respectively. Thus, miR-199a-3p appears to be involved in the estrogen regulatory networks that mediate bone cell autophagy, potentially by targeting IGF-1 and mTOR. This article is protected by copyright. All rights reserved
  • miRNAs and ovarian cancer: An overview
    • Abstract: Ovarian cancer (OC) is the sixth most common cancer in women globally. However, even with the advances in detection andtherapeutics it still represents the most dangerous gynecologic malignancy in women of the industrialized countries. The discovery of micro- RNAs (miRNA), a small noncoding RNA molecule targeting multiple mRNAs and regulation of gene expression by triggering translation repression and/or RNA degradation, has revealed the existence of a new array for regulation of genes involved in cancer. This review summarizes the current knowledge regarding the role of miRNAs expression in OC. It also provides information about potential clinical relevance of circulating miRNAs for OC diagnosis, prognosis, and therapeutics. The identification of functional targets for miRNAs represents a major obstacle in our understanding of microRNA function in OC, but significant progress is being made. The better understanding of the role of microRNA expression in ovarian cancer may provide new array for the detection, diagnosis, and therapy of the OC. This article is protected by copyright. All rights reserved
  • Maresin 1 inhibits TNF-alpha-induced lipolysis and autophagy in 3T3-L1
    • Abstract: Obesity is associated with high pro-inflammatory cytokine levels like tumor necrosis factor-alpha (TNF-α), which promotes inflammation in adipose tissue. The omega-3 PUFAs, and their derived lipid mediators, such as Maresin 1 (MaR1) have anti-inflammatory effects on adipose tissue. This study aimed to analyze if MaR1 may counteract alterations induced by TNF-α on lipolysis and autophagy in mature 3T3-L1 adipocytes. Our data revealed that MaR1 (1-100 nM) inhibited the TNF-α-induced glycerol release after 48 h, which may be related to MaR1 ability of preventing the decrease of lipid droplet-coating protein perilipin and G0/G1 Switch 2 protein expression. MaR1 also reversed the decrease on total hormone sensitive lipase (total HSL), and the ratio of phosphoHSL at Ser-565/total HSL, while preventing the increased ratio of phosphoHSL at Ser-660/total HSL and phosphorylation of extracellular signal-regulated kinase 1/2 induced by TNF-α. Moreover, MaR1 counteracted the cytokine-induced decrease of p62 protein, a key autophagy indicator, and also prevented the induction of LC3II/LC3I, an important autophagosome formation marker. Current data suggest that MaR1 may ameliorate TNF-α-induced alterations on lipolysis and autophagy in adipocytes. This might also contribute to the beneficial actions of MaR1 on adipose tissue and insulin sensitivity in obesity. This article is protected by copyright. All rights reserved
  • The Xenoestrogens Biphenol-A and Nonylphenol differentially regulate
           metalloprotease-mediated shedding of EGFR ligands
    • Abstract: The xenoestrogens Bisphenol-A (BPA) and Nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF- α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF- α. In cells lacking ADAM17 (Adam17-/- mEFs) BPA-stimulated release of HB-EGF, but not TGF- α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF- α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17-/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF- α was comparable to wild type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2-/- mEFs. The defect in shedding of HB-EGF in iRhom2-/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17-/-, iRhom2-/- or iRhom1/2-/-, but not in Adam10/17-/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens. This article is protected by copyright. All rights reserved
  • Occludin as a functional marker of vascular endothelial cells on
           tube-forming activity
    • Abstract: Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G2/M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis. This article is protected by copyright. All rights reserved
  • Wnt5a Suppresses Osteoblastic Differentiation of Human Periodontal
           Ligament Stem Cell-like Cells Via Ror2/JNK signaling
    • Abstract: Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue. This article is protected by copyright. All rights reserved
  • Src nuclear localization and its prognostic relevance in human
    • Abstract: Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low- versus high-risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well-characterised membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so-far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high-stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan-Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N-myristoyltransferase enzymes expression and activity and the Src nuclear content.In conclusion, these results described a so-far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N-myristoyltransferase enzymes and the Src subcellular localization was described as well. This article is protected by copyright. All rights reserved
  • Transcription-Dependent Association of HDAC2 with Active Chromatin
    • Abstract: Histone deacetylase 2 (HDAC2) catalyzes deacetylation of histones at the promoter and coding regions of transcribed genes and regulates chromatin structure and transcription. To explore the role of HDAC2 and phosphorylated HDAC2 in gene regulation, we studied the location along transcribed genes, the mode of recruitment and the associated proteins with HDAC2 and HDAC2S394ph in chicken polychromatic erythrocytes. We show that HDAC2 and HDAC2S394ph are associated with transcriptionally active chromatin and located in the interchromatin channels. HDAC2S394ph was present primarly at the upstream promoter region of the transcribed CA2 and GAS41 genes, while total HDAC2 was also found within the coding region of the CA2 gene. Recruitment of HDAC2 to these genes was partially dependent upon on-going transcription. Unmodified HDAC2 was associated with RNA binding proteins and interacted with RNA bound to the initiating and elongating forms of RNA polymerase II. HDAC2S394ph was not associated with RNA polymerase II. These results highlight the differential properties of unmodified and phosphorylated HDAC2 and the organization of acetylated transcriptionally active chromatin in the chicken polychromatic erythrocyte. This article is protected by copyright. All rights reserved
  • Myeloid-derived Suppressor Cells: important contributors to tumor
           progression and metastasis
    • Abstract: Myeloid-derived suppressor cells (MDSCs) are traditionally considered among the major components of the immunosuppressive tumor microenvironment. However, there is currently increasing evidence indicating that MDSCs in addition to suppression of immune surveillance is also involved in an array of non-immunological functions like augmenting metastatic potential of tumor cells. Indeed, MDSCs can promote metastasis in animal models and cancer patients through promoting premetastatic niche formation, tumor angiogenesis and invasion. Moreover, MDSC frequency and function have been associated with progressive disease and correlated with clinical outcome. This review will summarize and discusses the data demonstrating the role for MDSCs in tumor metastasis. This article is protected by copyright. All rights reserved
  • Therapeutic potentials of adenosine receptors agonists and antagonists in
           colitis; current status and perspectives
    • Abstract: Increasing evidence suggests that adenosine is dysregulated in ulcerative colitis (UC), potentially affecting UC pathogenesis, diagnosis, and therapy. Dysregulation of the activity of adenosine generating enzymes including adenosine deaminase in serum of patients with acute colitis supports the role of this omnipresent metabolite in the pathogenesis of colitis. Adenosine regulates inflammatory responses including epithelial barrier hyper-permeability, myeloperoxidase activity, and neuromuscular motility in colitis, supporting the therapeutic potency of adenosine receptors agonists and antagonists in this disease. Depending upon the adenosine receptor subtype, activation or suppression of the receptor with pharmacological agonists or antagonists attenuates colitis pathological symptoms in colitis model. This review summarizes the role of adenosine receptors agonists and antagonists in the pathogenesis of colitis for a better understanding and hence a better management of this disease. This article is protected by copyright. All rights reserved
  • Establishment of in vitro model of corneal scar pathophysiology
    • Abstract: Corneal scarring is the major source of permanent blindness worldwide. The complex pathophysiology of corneal scarring is not comprehensibly understood as it involves the interaction of a constellation of pro-fibrotic cytokines influencing several signaling pathways involved in corneal scar development. In the present study, an attempt has been made to generate a relatively simple in vitro corneal scar model using primary corneal keratocytes by exogenously providing an optimized dose of combination of cytokines (TGF-β1, IL-6 and IL-8) involved in scar formation in situ. Data obtained from gene and protein expression analysis depicted enhanced ECM production with discrete expression of myofibroblast specific markers. The protein-protein interactions associated these proteins to various pathways involved in wound healing, cellular migration and cytoskeletal remodeling justifying high relevance to in vivo scar formation. Hence the developed model can be used to acquire understanding about corneal scar pathophysiology and thus might be useful for designing the treatment modalities and efficacies for controlling scar formation. This article is protected by copyright. All rights reserved
  • Ptn functions downstream of C/EBPβ to mediate the effects of cAMP on
           uterine stromal cell differentiation through targeting Hand2 in response
           to progesterone
    • Abstract: Ptn is a pleiotropic growth factor involving in the regulation of cellular proliferation and differentiation, but its biological function in uterine decidualization remains unknown. Here we showed that Ptn was highly expressed in the decidual cells, and could induce the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1 which were two well-established differentiation markers for decidualization, suggesting an important role of Ptn in decidualization. In the uterine stromal cells, progesterone stimulated the expression of Ptn accompanied with an accumulation of intracellular cAMP level. Silencing of Ptn impeded the induction of progesterone and cAMP on the differentiation of uterine stromal cells. Administration of PKA inhibitor H89 resulted in a blockage of progesterone on Ptn expression. Further analysis evidenced that regulation of progesterone and cAMP on Ptn was mediated by C/EBPβ. During in vitro decidualization, knockdown of Ptn could weaken the up-regulation of Prl8a2 and Prl3c1 elicited by C/EBPβ overexpression, while constitutive activation of Ptn reversed the repressive effects of C/EBPβ siRNA on the expression of Prl8a2 and Prl3c1. Meanwhile, Ptn might mediate the regulation of C/EBPβ on Hand2 which was a downstream target of Ptn in the differentiation of uterine stromal cells. Attenuation of Ptn or C/EBPβ by specific siRNA blocked the stimulation of Hand2 by progesterone and cAMP. Collectively, Ptn may play a vital role in the progesterone-induced decidualization pathway. This article is protected by copyright. All rights reserved
  • Silibinin Stimluates Apoptosis by Inducing Generation of ROS and ER Stress
           in Human Choriocarcinoma Cells
    • Abstract: Silibinin is a flavonolignan extracted from seeds of milk thistles. Traditionally, it has been used as a therapeutic agent for liver disorders, and now it is well-known for its anti-cancer effects. However, studies on anti-cancer effects of silibinin on choriocarcinoma are very limited. Therefore, we performed proliferation and apoptosis assays to determine effects of silibinin on the viability of human choriocarcinoma (JAR and JEG3) cells. Our results showed that silibinin significantly inhibited proliferation and induced apoptosis in both JAR and JEG3 cells, and significantly increased reactive oxygen species (ROS) and lipid peroxidation. Moreover, silibinin disrupted mitochondrial function by inducing permeabilization of mitochondrial membrane potential and calcium ion efflux in JAR and JEG3 cells. Furthermore, silibinin-induced apoptosis in choriocarcinoma cells via AKT, mitogen-activated protein kinases (MAPK) and unfolded protein response (UPR) signal transduction. Collectively, our results suggest that silibinin is a novel therapeutic agent or dietary supplement for management of human placental choriocarcinomas. This article is protected by copyright. All rights reserved
    • Abstract: One of the methods employed to improve healing of damaged tissues is the use of cellular based therapies. A number of regenerative medicine based strategies, from in vitro expanded mesenchymal stem cells (MSCs) to “one-step” procedures using bone marrow (BM) in toto (BM aspirate; BMA) or BM concentrate (BMC), have been developed. Recently, orthopedic researchers focused their attention on the clinical therapeutic potential of BMC and BMA for musculoskeletal regeneration. BMA is reported as an excellent source of cells and growth factors. However, the quality of BM harvest and aspirate is extremely technique-dependent and, due to the presence of megakaryocytes and platelets, BMA is prone to clot. BMA clot formation is usually considered a complication hampering the procedures on both BMC preparation and MSC expansion. Therefore, different protocols have been developed to avoid and/or degrade clots. However, from a biological point of view there is a strong rationale for the use of BMA clot for tissue engineering strategies. This descriptive systematic literature review summarizes preclinical and clinical studies dealing the use of BMA clot for orthopedic procedures and provided some evidence supporting its use as a cell based therapy for cartilage and bone regeneration. Despite these results, there are still few preclinical and clinical studies that carefully evaluate the safety and efficacy of BMA clot in orthopedic procedures. Thus, implementing biological knowledge and both preclinical and clinical studies could help researchers and clinicians to understand if BMA clots can really be considered a possible therapeutic tool. This article is protected by copyright. All rights reserved
  • Visualization of Stimulus-specific Heterogeneous Activation of Individual
           Vascular Smooth Muscle Cells in Aortic Tissues
    • Abstract: Intercellular communication among autonomic nerves, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus-specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately 3 fold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO-induced relaxation is quite high and approximately 10 fold higher than that of its counterpart, the Wister-Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure. This article is protected by copyright. All rights reserved
  • MicroRNA: Relevance to stroke diagnosis, prognosis, and therapy
    • Abstract: Stroke is a life-threatening disease that accounts for a considerable burden of mortality in both developing and developed world. Identification of specific biomarkers for stroke and its outcomes can greatly contribute to improved care of patients. MicroRNAs (miRNAs) are known as novel biomarkers that could be used as diagnostic, prognostic, and therapeutic biomarkers. Various studies have shown that miRNAs have key roles in the pathogenesis of stroke, and its complications and outcomes. In addition, there is evidence showing that mesenchaymal stromal cell-derived exosomes containing miRNAs can be used for monitoring and treatment of various diseases such as stroke. Here, we summarized various aspects of miRNA applications in different stages of stroke.
  • Statin Regulated ERK5 Stimulates Tight Junction Formation and Reduces
           Permeability in Human Cardiac Endothelial Cells
    • Abstract: The MEKK3/MEK5/ERK5 signalling axis is required for cardiovascular development in vivo. We analysed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilised human microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localisation with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provides the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity. This article is protected by copyright. All rights reserved
  • ESRRB plays a crucial role in the promotion of porcine cell reprogramming
    • Abstract: The estrogen-related receptor b (ESRRB) is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency. In mouse ES cells, overexpression of ESRRB can maintain LIF-independent self-renewal in the absence of Nanog. However, the fundamental features of porcine ESRRB remain elusive. In this study, we revealed the expression profiles of ESRRB in both porcine pluripotent stem cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor that enhanced porcine pluripotent gene activation. Addition of ESRRB into the cocktail of core pluripotent factors Oct4, Sox2, Klf4, and c-Myc (OSKM + E) could significantly enhance the reprogramming efficiency and the formation of alkaline phosphatase positive colonies. Conversely, knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes, minimized the alkaline phosphatase activity, and initiated the porcine induced pluripotent stem cell differentiation. Therefore, porcine ESRRB is a crucial transcription factor to improve the self-renewal of piPSCs. This article is protected by copyright. All rights reserved
  • High fat diet attenuates hyperglycemia, body composition changes, and bone
           loss in male streptozotocin-induced type 1 diabetic mice
    • Abstract: There is a growing and alarming prevalence of obesity and the metabolic syndrome in type I diabetic patients (T1DM), particularly in adolescence. In general, low bone mass, higher fracture risk and increased marrow adipose tissue (MAT) are features of diabetic osteopathy in insulin deficient subjects. On the other hand, type 2 diabetes (T2DM) is associated with normal or high bone mass, a greater risk of peripheral fractures and no change in MAT. Therefore, we sought to determine the effect of weight gain on bone turnover in insulin deficient mice. We evaluated the impact of a 6-week high-fat (HFD) rich in medium chain fatty acids or low-fat diet (LFD) on bone mass and MAT in a streptozotocin (STZ)-induced model using male C57BL/6J mice at 8 weeks of age. Dietary intervention was initiated after diabetes confirmation. At the endpoint, lower non-fasting glucose levels were observed in diabetic mice fed with high fat diet compared to diabetic mice fed the low fat diet (STZ-LFD). Compared to euglycemic controls, the STZ-LFD had marked polydipsia and polyphagia, as well as reduced lean mass, fat mass and bone parameters. Interestingly, STZ-HFD mice had higher bone mass, namely less cortical bone loss and more trabecular bone than STZ-LFD. Thus, we found that a HFD, rich in medium chain fatty acids, protects against bone loss in a T1DM mouse model. Whether this may also translate to T1DM patients who are overweight or obese in respect to maintenance of bone mass remains to be determined through longitudinal studies. This article is protected by copyright. All rights reserved
  • MicroRNA-mediated drug resistance in ovarian cancer
    • Abstract: The development of intrinsic or acquired resistance to chemotherapeutic agents used in the treatment of various human cancers is a major obstacle for the successful abolishment of cancer. The accumulated efforts in the understanding the exact mechanisms of development of multidrug resistance (MDR) have led to the introduction of several unique and common mechanisms. Recent studies demonstrate the regulatory role of small noncoding RNA or miRNA in the several parts of cancer biology. Practically all aspects of cell physiology under normal and disease conditions are reported to be controlled by miRNAs. In this review, we discuss how the miRNA profile is changed upon MDR development and the pivotal regulatory role played by miRNAs in overcoming resistance to chemotherapeutic agents. It is hoped that further studies will support the use of these differentially expressed miRNAs as prognostic and predictive markers, as well as novel therapeutic targets to overcome resistance in ovarian cancer. This article is protected by copyright. All rights reserved
  • Troxerutin with copper generates oxidative stress in cancer cells: its
           possible chemotherapeutic mechanism against hepatocellular carcinoma
    • Abstract: Troxerutin (TXER) a rutin derivative is known for its anticancer effect against hepatocellular carcinoma (HCC). As part of large study, recently we have shown TXER interact with genetic material and its anti-mutagenic property. In the present study we have explored its possible mode of action in HCC. Since TXER alone did not show significant anticancer effect on Huh-7 cells, in vitro biochemical assays were performed for determining anticancer efficacy of TXER + metal complex using transition metals such as Cu, Zn & Fe. The anticancer efficacy of TXER + Cu on Huh-7 cells were evaluated using MTT assay, DCFDA, JC-1 staining, comet assay, cell cycle analysis, immunocytochemistry and western blotting. Non-toxic nature of TXER was analysed on primary rat hepatocytes. The in vivo efficacy of TXER was tested in N-nitrosodiethylamine initiated and γ-benzene hexachloride & partial hepatectomy promoted rat liver cancer. Liver markers, transition metal levels, histopathological examination and expression levels of GST-P, 8-OHdG and Ki-67 were studied to assess the in vivo anticancer effect of TXER. We observed that TXER + Cu induced extensive cellular death on Huh-7 cells through generating free radicals and did not possess any toxic effect on normal hepatocytes. The in vivo studies revealed that TXER possess significant anti-cancer effect as assessed through improved liver markers and suppressed GST-P, 8-OHdG, and Ki-67 expression. TXER treatment reduced the hepatic Cu level in cancer bearing animals. Current study brings the putative mechanism involved in anti-cancer effect of TXER, further it will help to formulate phytoconstituents coupled anti-cancer drug for effective treatment of HCC. This article is protected by copyright. All rights reserved
    • Abstract: Low-intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm2 LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7d, 14d, 21d). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21d CD73 + /CD90 + : 6%; OCT-3/4 + /NANOG + /SOX2 + : 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation. This article is protected by copyright. All rights reserved
  • Microtubule actin crosslinking factor 1 promotes osteoblast
           differentiation by promoting β-catenin/TCF1/Runx2 signaling axis
    • Abstract: Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1 knockdown increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. This article is protected by copyright. All rights reserved
  • Poly r(C) binding protein 1-mediated regulation of microRNA expression
           underlies post-sevoflurane amelioration of acute lung injury in rats
    • Abstract: Background: Acute lung injury (ALI) presents a pervasive health burden due to the high morbidity and mortality associated with it. Volatile anesthetics like sevoflurane has been previously shown to have organ-protective effect, both in the context of normal physiological function in liver, and during LPS-induced ALI. Sevoflurane was shown to exert lung protective effect during LPS-induced ALI by modulating expression level of microRNAs (miRNAs), specifically miR-155. The objective of the current study was to define the underlying mechanism by which sevoflurane alters miRNA expression levels. Methods: Lung injury caused by LPS and its amelioration post sevoflurane administration was first confirmed. Expression levels of different miRNA and messenger RNAs (mRNAs) encoding inflammatory cytokines were measured in a rat model of lipopolysaccharide (LPS)-induced ALI, which were subsequently treated with either sevoflurane or vehicle control. Results: Host of miRNAs and messenger RNAs encoding pro-inflammatory cytokines are overexpressed during LPS-induced ALI, which are reversed following sevoflurane administration. Mass spectrometry analysis revealed that the RNA-binding protein, poly r(C) binding protein 1 (PCBP1) expression is induced in ALI and is repressed following sevoflurane treatment. RNA immunoprecipitation experiments revealed that PCBP1 expression dictates the altered miRNA expression and sevoflurane altered miRNA expression by suppressing PCBP1 expression. Conclusion: Our study thus elucidates a unique mechanism of lung protective effect of sevoflurane mediated by suppression of expression of a RNA-binding protein that potentiates expression of pro-inflammatory miRNAs. This article is protected by copyright. All rights reserved
  • Three-dimensional cell culture models for anticancer drug screening: worth
           the effort'
    • Abstract: High attrition of new oncology drug candidates in clinical trials is partially caused by the poor predictive capacity of artificial monolayer cell culture assays early in drug discovery. Monolayer assays do not take the natural three-dimensional (3D) microenvironment of cells into account. As a result, false positive compounds often enter clinical trials, leading to high dropout rates and a waste of time and money. Over the past two decades, tissue engineers and cell biologists have developed a broad range of 3D in vitro culturing tools that better represent in vivo cell biology. These tools preserve the 3D architecture of cells and can be used to predict toxicity of and resistance against antitumor agents. Recent progress in tissue engineering further improves 3D models by taking into account the tumor microenvironment, which is important for metastatic progression and vascularization. However, the widespread implementation of 3D cell cultures into cell-based research programs has been limited by various factors, including their cost and reproducibility. In addition, different 3D cell culture techniques often produce spheroids of different size and shape, which can strongly influence drug efficacy and toxicity. Hence, it is imperative to morphometrically characterize multicellular spheroids to avoid generalizations among different spheroid types. Standardized 3D culturing procedures could further reduce data variability and enhance biological relevance. Here, we critically evaluate the benefits and challenges inherent to growing cells in 3D, along with an overview of the techniques used to form spheroids. This is done with a specific focus on antitumor drug screening. This article is protected by copyright. All rights reserved
  • MicroRNA: A Novel Target of Curcumin in Cancer Therapy
    • Abstract: Curcumin is known as a natural dietary polyphenol which is extracted from Curcuma longa L. It has been shown that curcumin has a variety of pharmacological effects such as antioxidant, anti-cancer, anti-inflammatory, and anti-microbial activities. Anti-cancer effects of curcumin are due to targeting of a wide range of cellular and molecular pathways involved in cancer pathogenesis including NF-kB, MAPK, PTEN, P53, and microRNAs (miRNA) network. Multiple lines of evidence have indicated that curcumin exerts its therapeutic effects via regulating miRNA expression (e.g. miR-1, miR-7, miR-9, miR-34a, miR-181, miR-21 and miR-19) which could lead to the regulation of underlying cellular and molecular pathways involved in cancer pathogenesis. Exosomes are one of the important classes of biological vehicles which could be released from various types of cells such as cancer cells and stem cells and could change the behavior of recipient cells. It has been shown that treatment of cancer cells with different dose of curcumin leads to the release of exosomes containing curcumin. These exosomes could induce anti-cancer properties in recipient cells and reduce tumor growth. Hence, exosomes containing curcumin could be applied as powerful tools for cancer treatment. Here, we highlighted various miRNAs which could be affected by curcumin in various types of cancer. Moreover, we highlight exosomes containing curcumin as suitable therapeutic tools in cancer therapy. This article is protected by copyright. All rights reserved
  • Apigenin Induces ROS Dependent Apoptosis and ER Stress in Human
           Endometriosis Cells
    • Abstract: Apigenin is a plant-derived flavonoid having anti-proliferative, anti-inflammatory and anti-angiogenic properties in chronic and metabolic diseases, and cancers. However, the functional role of apigenin remains to be identified in human endometriosis that is a benign inflammatory disease causing infertility, dysmenorrhea, dyspareunia, and chronic abdominal or pelvic pain. In the present study, we determined the effects of apigenin on two well-established human endometriosis cell lines (VK2/E6E7 and End1/E6E7). Apigenin reduced proliferation and induced cell cycle arrest and apoptosis in the both endometriosis cell lines. In addition, it disrupted mitochondrial membrane potential (MMP) which was accompanied by an increase in concentration of calcium ions in the cytosol and in pro-apoptotic proteins including Bax and cytochrome c in the VK2/E6E7 and End1/E6E7 cells. Moreover, apigenin treated cells accumulated excessive reactive oxygen species (ROS), and experienced lipid peroxidation and endoplasmic reticulum (ER) stress with activation of the unfolded protein response (UPR) regulatory proteins. Furthermore, the apigenin-induced apoptosis in endometriosis cells was regulated via the ERK1/2, JNK and AKT cell signaling pathways. Taken together, apigenin is a potential novel therapeutic agent to overcome current limitations in the treatment to endometriosis. This article is protected by copyright. All rights reserved
  • A Perspective on Stem Cell Therapy for Ear Disorders
    • Abstract: The use of stem cells in cell-based therapy is an emerging concept for the treatment of ear disorders. Tympanic membrane perforation (TMP) and inner ear disorders are some of the most commonly presented otologic disorders that can benefit from advances in cell-based therapy. Studies have already demonstrated that stem cell-based therapy can potentially be an effective treatment modality for acute and chronic TMP. Recent studies have also shown promise in application of cell-based approach to treat inner ear dysfunction. In this perspective, we will discuss the recent advancements regarding the use of cell-based therapy for ear disorders. This article is protected by copyright. All rights reserved
  • Angiogenesis biomarkers and their targeting ligands as potential targets
           for tumor angiogenesis
    • Abstract: Angiogenesis is known as one of the hallmarks in cancer which could play a key role in providing oxygen and nutrients for tumor cells. It has been shown that tumor cannot grow without sufficient development of new blood vessels. Accordingly, targeting angiogenesis, especially endothelial cells, could be considered as a common therapeutic target in tumors and more investigation on already existing biomarkers and potentially new biomarkers of endothelial cells seems to be necessary in cancer therapy. Moreover, the use of effective targeting approaches such as proteins and peptides, aptamers, and small molecules is an important step for targeting biomarkers associated with endothelial cells and angiogenesis in cancer therapy. These agents are FDA approved, or are currently under investigation in pre-clinical and clinical studies. Among various biomarkers for angiogenesis microRNAs are suitable candidates for target therapy. These molecules play key roles in tumor angiogenesis which exert their effect via targeting a variety of cellular and molecular pathways involved in tumor angiogenesis. Here, we summarize a variety of biomarkers which their expressions or their functions could change the function of endothelial cells in tumor microenvironments. Moreover, we highlighted various therapeutic agents which could target these biomarkers. This article is protected by copyright. All rights reserved
  • Lipoprotein(a): A missing culprit in the management of
    • Abstract: Lipoprotein(a) Lp(a) is a cholesterol-rich, LDL-like particle that is independently associated with an increased risk for ischemic heart disease, atherosclerosis, thrombosis, and stroke. Genetic variation in the Lp(a) locus and some other genes related to Lp(a) synthesis and metabolism play a critical role in regulating plasma Lp(a) levels. The pathophysiological potential of Lp(a) is related to proatherogenic and prothrombotic effects on the vasculature. Different molecular mechanisms underlying the atherothrombotic potential of Lp(a), free apolipoprotein(a), and oxidized-Lp(a) have been proposed. However, plasma Lp(a) assay is complicated by problems associated with quantification and standardization owing to the polymorphic nature of this lipoprotein. This review has focused on the physicochemical properties of Lp(a), the genetic aspects of Lp(a), the need for accurate determination of Lp(a), the synthesis and recent findings on metabolism of Lp(a). Lastly, the patho-physiological mechanisms by which Lp(a) may increase athero-thrombosis and an overview on the therapeutic modalities to interfere with Lp(a) are summarized. This article is protected by copyright. All rights reserved
    • Abstract: Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes. This article is protected by copyright. All rights reserved
  • Humanized mouse models: Application to human diseases
    • Abstract: Humanized mice are superior to rodents for preclinical evaluation of the efficacy and safety of drug candidates using human cells or tissues. During the past decade, humanized mouse technology has been greatly advanced by the establishment of novel platforms of genetically modified immunodeficient mice. Several human diseases can be recapitulated using humanized mice due to the improved engraftment and differentiation capacity of human cells or tissues. In this review, we discuss current advanced humanized mouse models that recapitulate human diseases including cancer, allergy, and graft-versus-host disease. This article is protected by copyright. All rights reserved
  • Therapeutic Relevance of Ozone Therapy in degenerative diseases: Focus on
           Diabetes and Spinal Pain
    • Abstract: Ozone, one of the most important air pollutants, is a triatomic molecule containing three atoms of oxygen that results in an unstable form due to its mesomeric structure. It has been well-known that ozone has potent ability to oxidize organic compounds and can induce respiratory irritation. Although ozone has deleterious effects, many therapeutic effects have also been suggested. Since last decades, the therapeutic potential of ozone has gained much attention through its strong capacity to induce controlled and moderated oxidative stress when administered in precise therapeutic doses. A plethora of scientific evidence showed that the activation of hypoxia inducible factor-1α (HIF-1a), nuclear factor of activated T-cells (NFAT), nuclear factor-erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE), and activated protein-1 (AP-1) pathways are the main molecular mechanisms underlying the therapeutic effects of ozone therapy. Activation of these molecular pathways leads to up-regulation of endogenous antioxidant systems, activation of immune functions as well as suppression of inflammatory processes, which is important for correcting oxidative stress in diabetes and spinal pain. The present study intended to review critically the available scientific evidence concerning the beneficial properties of ozone therapy for treatment of diabetic complications and spinal pain. It finds benefit for integrating the therapy with ozone into pharmacological procedures, instead of a substitutive or additional option to therapy. This article is protected by copyright. All rights reserved
  • The Roles of Signaling Pathways in Bone Repair and Regeneration
    • Abstract: Regenerative medicine has sparked interest in potential strategies for bone repair. Bone defects are widespread and could be caused by trauma, congenital malformations, infections, and surgery. Although bone has a large self-healing capacity, some defects or fractures are too big to regenerate. To regenerate bone structures which can be used for treatment of patients, bone growth must be induced by a number of bioactive implantable materials, cell types and intracellular and extracellular molecular signaling pathways. Since mesenchymal stem cells (MSCs) and their differentiation during remodeling processes have important roles in bone regeneration, it is believed that understanding molecular signaling pathways involved is crucial to the development of bone implants, bone substitute materials, and cell-based scaffolds for bone regeneration. In this review, we briefly introduce concepts in fracture repair and regeneration following bone injuries, and then discuss the current clinical methods in bone regeneration. In the next section, we review the involvement of the various key signaling pathways in bone regeneration. This article is protected by copyright. All rights reserved
  • Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts
           from prolonged stress and contribute to survival and rejuvenation of human
           skin equivalents
    • Abstract: Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine. This article is protected by copyright. All rights reserved
  • Hypoxia-induced apoptosis of mouse spermatocytes is mediated by HIF-1α
           through a death receptor pathway and a mitochondrial pathway
    • Abstract: Hypoxia in vivo induces oligozoospermia, azoospermia and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia-induced apoptosis of mouse GC-2spd (GC-2) cells and the relationship between HIF-1α and apoptosis of GC-2 cells induced by hypoxia. GC-2 cells were subjected to 1% oxygen for 48 h. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase-3/8/9 in the absence and presence of HIF-1α siRNA. The protein levels of apoptosis-related markers were determined by western blot in the presence and absence of HIF-1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC-1 staining. Cell viability was assessed upon treatment of caspase-8 and caspase-9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 h induced apoptosis of GC-2 cells. A prolonged exposure of GC-2 cells to hypoxic conditions caused downregulation of c-FLIP, DcR2 and Bcl-2 and upregulation of DR5, TRAIL, Fas, p53 and Bax, with an overproduction of caspase-3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase-8/9 partially suppressed hypoxia-induced GC-2 cell apoptosis, and the anti-apoptotic effects of the caspase inhibitors were additive. Of note, HIF-1α knockdown attenuated hypoxia and induced apoptosis of GC-2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF-1α, contribute to hypoxia-induced GC-2 cell apoptosis. This article is protected by copyright. All rights reserved
  • G protein-coupled receptor 84 controls osteoclastogenesis through
           inhibition of NF-κB and MAPK signaling pathways
    • Abstract: GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. This article is protected by copyright. All rights reserved
  • Stress-responsive microRNAs are involved in re-programming of metabolic
           functions in hibernators
    • Abstract: Mammalian hibernation includes re-programming of metabolic capacities, partially, encouraged by microRNAs (miRNAs). Albeit much is known about the functions of miRNAs, we need learning on low temperature miRNAs target determination. As hibernators can withstand low body temperatures (TB) for a long time without anguish tissue damage, understanding the means and mechanisms that empower them to do as such are of restorative intrigue.Nonetheless, these mechanisms by which miRNAs and the hibernators react to stressful conditions are not much clear. It is evident from recent data that the gene expression and the translation of mRNA to protein are controlled by miRNAs. The miRNAs also influence regulation of major cellular processes. As the significance of miRNAs in stress conditions adaptation are getting clearer, this audit article abridges the key alterations in miRNA expression and the mechanism that facilitates stress survival. This article is protected by copyright. All rights reserved
  • The significant role of the Golgi apparatus in cardiovascular diseases
    • Abstract: The Golgi apparatus is a ribbon-like system of stacks which consist of multiple closely apposed flattened cisternae and vesicles usually localized in the juxta-nuclear area. As for the biological functions, the Golgi apparatus plays a major role in protein biosynthesis, post-translational modification and sorting protein from ER to plasma membrane and other destinations. Structural changes and functional disorder of the Golgi apparatus is associated with various diseases. Moreover, increasing evidence revealed that swelling, poor development and other morphological alterations of the Golgi apparatus are linked to cardiovascular diseases such as heart failure, arrhythmia and dilated cardiomyopathy. Furthermore, dysfunction of the Golgi apparatus is also related to cardiovascular diseases since the Golgi apparatus is extremely responsible for transport, glycosylation, biosynthesis and subcellular distribution of cardiovascular proteins. This review gives a brief overview of the intricate relationship between the Golgi apparatus and cardiovascular diseases. In addition, we provide a further prospective that the Golgi apparatus may provide diagnosis reference for cardiovascular diseases, and changes in the ultrastructure and morphology of the Golgi apparatus such as swelling, poor development and fragmentation may serve as a reliable index for cardiovascular diseases. This article is protected by copyright. All rights reserved
  • Bone marrow adipocytes support haematopoietic stem cell survival
    • Abstract: In bone marrow (BM), haematopoietic elements are mingled with adipocytes (BM-A), which are the most abundant stromal component in the niche. BM-A progressively increase with ageing, eventually occupying up to 50% of BM cavities.In this work, the role played by BM-A was explored by studying primary human BM-A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human haematopoietic stem cells (HSC) by the long-term culture initiating cell (LTC-IC) assay.Findings demonstrated that BM-A are capable of supporting HSC survival in the LTC-IC assay, since after 5 weeks of co-culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C-X-C motif chemokine 12 (CXCL12), interleukin (IL)-8, colony-stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM-A and in primary human BM mesenchymal stromal cells (BM-MSC), whereas IL-3 was higher in BM-A.Interestingly, BM-A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT-A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes and white-to-brown differentiation pathways. Accordingly, analysis of the gene pathways involved in haematopoiesis regulation showed that BM-A are more closely related to BM-MSC than to AT-A.The present data suggest that BM-A play a supporting role in the haematopoietic niche and directly sustain HSC survival. This article is protected by copyright. All rights reserved
  • PCSK9 and infection: a potentially useful or dangerous association'
    • Abstract: Elevated plasma low-density lipoprotein-cholesterol (LDL-C) concentration is the most important risk factor for atherosclerotic cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a ubiquitously expressed serine proteinase which plays a key role in cholesterol metabolism, but has been found to be implicated in some other lipid-independent physiological processes. In this review, the role of PCSK9 was evaluated not only concerning lipid metabolism but also hepatitis C virus (HCV) infection, bacterial infections/sepsis and septic shock. Collected data from clinical trials revealed that treatment with PCSK9 inhibitors has beneficial effects in lowering LDL-C via inhibition of LDL-receptors (LDL-R), an antiviral effect on HCV infection via down-regulating the surface expression of LDL-R and CD81 on hepatic cells, and a positive association with increased inflammatory responses, as well as with septic shock by down-regulation of hepatocyte LDL-R. On the other hand, PCSK9 inhibition by therapeutic fully humanized antibodies has positive effects in reducing elevated LDL-C. However, their safety and tolerability is an important issue which has to be taken into consideration. This article is protected by copyright. All rights reserved
  • Targeting the tumor microenvironment as a potential therapeutic approach
           in colorectal cancer: rational and progress
    • Abstract: Colorectal cancer (CRC) is often diagnosed at a late stage when tumor metastasis may have already occurred. Current treatments are often ineffective in metastatic disease, and consequently late diagnosis is often associated with poor outcomes in CRC. Alternative strategies are therefore urgently required. An interaction between epithelial cancer cells and their tissue microenvironment is a contributor to metastasis, and therefore recent studies are beginning to focus on the properties of the tumor microenvironment and the mechanism by which the metastatic cells exploit the tumor microenvironment for survival, immune evasion, and growth. We have reviewed the development of the combined therapeutic approaches that have focused on targeting the microenvironment of colorectal cancer. This article is protected by copyright. All rights reserved
  • Roles of macrophage migration inhibitory factor in cartilage tissue
    • Abstract: To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif + / +) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif + /+ constructs deteriorated by 8 weeks. Since the Mif + /+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term. This article is protected by copyright. All rights reserved
  • The Fam50a positively regulates ameloblast differentiation via interacting
           with Runx2
    • Abstract: Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation. This article is protected by copyright. All rights reserved
  • Hydroxychloroquine affects bone resorption both in vitro and in vivo
    • Abstract: We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC-sorted monocytes for 14 days and treated with different HCQ doses (control, 1 and 5 µg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker β-CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum β-CTx was significantly decreased after six months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker β-CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss. This article is protected by copyright. All rights reserved
  • Integrated transcriptomic and metabolomic analysis reveals adaptive
           changes of hibernating retinas
    • Abstract: Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities. This article is protected by copyright. All rights reserved
  • Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to
           modulate endothelial cell number in angiogenic vessels
    • Abstract: During embryonic development, new arteries and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis. This article is protected by copyright. All rights reserved
  • TNF-α has both stimulatory and inhibitory effects on mouse
           monocyte-derived osteoclastogenesis
    • Abstract: Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31hiLy-6C-), myeloid blasts (CD31+Ly-6C+) and monocytes (CD31-Ly-6Chi) were sorted from mouse bone marrow using flow cytometry and cultured with M-CSF and RANKL, with or without TNF-α. Surprisingly, TNF-α prevented the differentiation of TRAcP+ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL-1β or IL-6. When monocytes were pre-cultured with M-CSF and RANKL followed by exposure to TNF-α, a stimulatory effect was found. TNF-α also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF-α was added to monocytes cultured on plastic, RANK, NFATc1 and TRAcP were significantly down-regulated while TNF-αR1 and TNF-αR2 were up-regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF-α, indicating an altered ratio of bound-RANK/unbound-RANK. Our findings suggest a diverse role of TNF-α on monocytes' osteoclastogenesis: it affects the RANK-signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre-cultured with M-CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte-derived osteoclast formation away from the bone. This article is protected by copyright. All rights reserved
  • GLUT 1 receptor expression and circulating levels of fasting glucose in
           high grade serous ovarian cancer
    • Abstract: In recent years, the poorly remarkable goals achieved in terms of patients' important outcomes for ovarian cancer have fueled our interest towards the study of its metabolic roots. Within this research pipeline, we assessed the association between the expression of the glucose transporter GLUT1, as expressed at the tumor tissue level, and circulating pre-surgical levels of fasting glucose in a case series including data from 40 patients with high FIGO stage serous ovarian cancer. Patients who provided data to the current analysis were randomly selected from a larger cohort. To our purposes, the procedures related to serum and tissue collection, storage and biomarker assessment were highly standardized and centralized at the institutional laboratories. The GLUT1 antibody SPM498 SPRING (REF. E13810) was used at a 1:500 dilution in 2 µm slides. Staining for GLUT1 was observed at the cell membrane level in all the cases assessed, but strong staining was described in 29 (72.5) of them. The agreement between the two independent reviewers was 100%. Strong GLUT1 staining was inversely associated with the circulating levels of fasting glucose, with a particularly striking difference in the distribution for patients in the lowest fasting glucose tertile (p = 0.044). These results support the biological plausibility of the association of interest. If confirmed in larger studies, our findings may help clarify the potentials of biomarkers related to energy metabolism in terms of prognosis definition, treatment assignment and outcome interpretation for patients with high FIGO stage serous ovarian cancer. This article is protected by copyright. All rights reserved
  • The genetic factors contributing to the development of Wilm's tumor and
           their clinical utility in its diagnosis and prognosis
    • Abstract: Mutations in the Wilm's tumor 1 (WT1) gene are associated with a wide spectrum of renal manifestations, ultimately leading to end-stage kidney failure. There is an inadequate understanding of the molecular functions of WT1 in renal development, and this has limited the potential for therapeutic interventions in WT1-related diseases. In this review we discuss the existing data on the genetic and epigenetic abnormalities that have been described in WTs and their potential utility as biomarkers for risk stratification, prediction and prognosis in patients withWTs. This article is protected by copyright. All rights reserved
  • NAD+: A big player in cardiac and skeletal muscle remodeling and aging
    • Abstract: In the past decade, NAD+ has gained importance for its beneficial effects as antioxidant and antiaging molecule. A paper in science by Zhang et al (2016) has described that NAD+ when replenished, ameliorates muscle dystrophy in mice by improving mitochondrial function. NAD+ was also demonstrated by the authors to improve the life span of mice. Cox et al (2002) demonstrated the cardiac effects of NAD+ which mitigated chronic heart failure via mitochondrial redox state mechanism. Cox et al (2002) also demonstrated that NAD+ is provided in the drinking water, it improves cardiac relaxation in volume overload model of heart failure. Although NAD+ has a profound anti-aging and anti-oxidant effects, its effect on humans and use as a dietary supplement needs more exploration. This article is protected by copyright. All rights reserved
  • High density lipoprotein (HDL) reverses palmitic acid induced energy
           metabolism imbalance by switching CD36 and GLUT4 signaling pathways in
    • Abstract: In our previous study palmitic acid (PA) induced lipotoxicity and switches energy metabolism from CD36 to GLUT4 in H9c2 cells. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. Therefore we in the present study investigated whether HDL can reverse PA induced lipotoxicity in H9c2 cardiomyoblast cells. In this study, we treated H9c2 cells with PA to create a hyperlipidemia model in vitro and analyzed for CD36 and GLUT4 metabolic pathway proteins. CD36 metabolic pathway proteins (phospho-AMPK, SIRT1, PGC1α, PPARα, CPT1β and CD36) were decreased by high PA (150 and 200 µg/µL) concentration. Interestingly, expression of GLUT4 metabolic pathway proteins (p-PI3K and pAKT) were increased at low concentration (50 µg/µL) and decreased at high PA concentration. Whereas, phospho-PKCζ, GLUT4 and PDH proteins expression was increased in a dose dependent manner. PA treated H9c2 cells were treated with HDL and analyzed for cell viability. Results showed that HDL treatment induced cell proliferation efficiency in PA treated cells. In addition, HDL reversed the metabolic effects of PA: CD36 translocation was increased and reduced GLUT4 translocation, but HDL treatment significantly increased CD36 metabolic pathway proteins and reduced GLUT4 pathway proteins. Rat neonatal cardiomyocytes showed similar results. In conclusion, HDL reversed palmatic acid-induced lipotoxicity and energy metabolism imbalance in H9c2 cardiomyoblast cells and in neonatal rat cardiomyocyte cells. This article is protected by copyright. All rights reserved
  • What role does the stress response have in congestive heart failure'
    • Abstract: This review is concerned with cardiac malfunction as a result of an imbalance in protein proteostasis, the homeostatic balance between protein removal and regeneration in a long remodeling process involving the endoplasmic reticulum (ER) and the unfolded protein response (UPR). The importance of this is of special significance with regard to cardiac function as a high energy requiring muscular organ that has a high oxygen requirement and is highly dependent on mitochondria. The importance of mitochondria is not only concerned with high energy dependence on mitochondrial electron transport, but it also has a role in the signaling between the mitochondria and the ER under stress. Proteins made in the ER are folded as a result of sulfhydryl groups (–SH) and attractive and repulsive reactions in the tertiary structure. We discuss how this matters with respect to an imbalance between muscle breakdown and repair in a stressful environment, especially as a result of oxidative and nitrosative byproducts of mitochondrial activity. The normal repair is a remodeling, but under this circumstance, the cell undergoes or even lysosomal “self eating” autophagy, or even necrosis instead of apoptosis. We shall discuss the relationship of the UPR pathway to chronic congestive heart failure (CHF). This article is protected by copyright. All rights reserved
  • Hypomorphic conditional deletion of E11/Podoplanin reveals a role in
           osteocyte dendrite elongation
    • Abstract: The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (P 
  • Fasting inhibits hepatic stellate cells activation and potentiates
           anti-cancer activity of Sorafenib in hepatocellular cancer cells
    • Abstract: BACKGROUND: Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown.METHODS: 24 hour fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. 24 hours fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib.RESULTS: Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short-term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells.CONCLUSIONS: Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. This article is protected by copyright. All rights reserved
  • Overexpression of HOXA4 and HOXA9 genes promotes self-renewal and
           contributes to colon cancer stem cell overpopulation
    • Abstract: Because HOX genes encode master regulatory transcription factors that regulate stem cells (SCs) during development and aberrant expression of HOX genes occurs in various cancers, our goal was to determine if dysregulation of HOX genes is involved in the SC origin of colorectal cancer (CRC). We previously reported that HOXA4 and HOXD10 are expressed in the colonic SC niche and are overexpressed in CRC. HOX gene expression was studied in SCs from human colon tissue and CRC cells (CSCs) using qPCR and immunostaining. siRNA-mediated knockdown of HOX expression was used to evaluate the role of HOX genes in modulating CSC phenotype at the level of proliferation, SC marker expression, and sphere formation. All-trans-retinoic-acid (ATRA), a differentiation-inducing agent was evaluated for its effects on HOX expression and CSC growth. We found that HOXA4 and HOXA9 are up-regulated in CRC SCs. siRNA knockdown of HOXA4 and HOXA9 reduced: (i) proliferation and sphere-formation and (ii) gene expression of known SC markers (ALDH1, CD166, LGR5). These results indicate that proliferation and self-renewal ability of CRC SCs are reduced in HOXA4 and HOXA9 knockdown cells. ATRA decreased HOXA4, HOXA9 and HOXD10 expression in parallel with reduction in ALDH1 expression, self-renewal, and proliferation. Overall, our findings indicate that overexpression of HOXA4 and HOXA9 contributes to self-renewal and overpopulation of SCs in CRC. Strategies designed to modulate HOX expression may provide ways to target malignant SCs and to develop more effective therapies for CRC. This article is protected by copyright. All rights reserved
  • ABC of multifaceted Dystrophin glycoprotein complex (DGC)
    • Abstract: Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signalling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signalling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signalling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role and functioning of DGC and may lead to a deeper understanding of its role in several human diseases. This article is protected by copyright. All rights reserved
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