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Journal Cover Journal of Cellular Physiology
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   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  [1579 journals]
  • A New Parameter of Growth Inhibition for Cell Proliferation Assays
    • Abstract: Cell proliferation assays are performed by four decades to test the anti‐proliferative activity of natural products and synthetic compounds in cell cultures. In cancer research, they are widely employed to evaluate drug efficacy in in vitro tumor models, such as established cell lines, primary cultures and recently developed three‐dimensional tumor organoids. In this manuscript, we demonstrated that current employed parameters used by researchers to quantify in vitro growth inhibition, IC50 and GI50, lead to a misinterpretation of results based on the exponential, and not linear, proliferation of the cells in culture. Therefore, we introduce a new parameter for the analysis of growth inhibition in cell proliferation assays, termed relative population doubling capacity, that can be employed to properly quantify the anti‐proliferative activity of tested compounds and to compare drug efficacy between distinct cell models. This article is protected by copyright. All rights reserved
       
  • C‐C motif chemokine ligand 23 abolishes ER stress‐ and LPS‐induced
           reduction in proliferation of bovine endometrial epithelial cells
    • Abstract: To reduce embryonic losses in domestic animals for economic production of livestock meat and milk, chemokines and their receptors are required for proper implantation and placentation during early pregnancy. In this study, we investigated the effects of C‐C‐motif chemokine ligand 23 (CCL23) on the proliferation of bovine endometrial (BEND) epithelial cells. CCL23 treatment improved BEND cell proliferation by enhancing PCNA and cyclin D1 expression via activation of the PI3K/AKT and MAPK signaling pathways. In addition, a combination of CCL23 and tunicamycin reversed the ER stress‐induced reduction in cell proliferation and the decreased expression of UPR‐mediated signaling proteins, including IRE1α, PERK, and ATF6α. Moreover, it regulated the lipopolysaccharide‐induced inflammation in BEND cells by inhibiting the expression of pro‐inflammatory cytokines (IL‐6 and IL‐8), and by restoring intracellular Ca2+ levels. These findings demonstrate that CCL23 improves endometrial development and uterine receptivity required for implantation and placentation during early pregnancy. This article is protected by copyright. All rights reserved
       
  • Melatonin application in targeting oxidative‐induced liver injuries:
           a review
    • Abstract: It is believed that oxidative stress is a key causing factor of liver damage induced by a variety of agents, and it is a major contributing factor in almost all conditions compromising liver function, including ischemia–reperfusion injury (IRI), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). Liver is the organ that high concentration of melatonin (N‐acetyl‐5‐methoxytryptamine) accumulates, and it is the sole organ where circulating melatonin is metabolized. Melatonin is one of the best antioxidants that protects liver, and its metabolites also have antioxidative function. Melatonin exerts its antioxidative function directly through its radical scavenging ability and indirectly through stimulation of antioxidant enzymes. The antioxidative response from melatonin in liver affects from various factors, including its dosage, route, time and duration of administration, the type of oxidative‐induced agent and species aging. This indoleamine is also an effective and promising antioxidative choice for targeting liver IRI, NAFLD, NASH, fibrosis, cirrhosis and HCC. This article is protected by copyright. All rights reserved
       
  • Inhibition of arterial medial calcification and bone mineralization by
           extracellular nucleotides: The same functional effect mediated by
           different cellular mechanisms
    • Abstract: Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast‐like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non‐calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100‐fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto‐nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi. In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.The extracellular nucleotides, ATP and UTP, inhibit bone mineralization and arterial medial calcification. However, the cellular mechanisms mediating these effects are different.
       
  • Downregulation of P2Y12 in the superior cervical ganglia alleviates
           abnormal sympathetic activity after myocardial ischemia
    • Abstract: Background/Aims: Superior cervical ganglia (SCG) innervate the myocardium and participate in sympathoexcitatory transmission. P2Y12 receptor is expressed in satellite glial cells (SGCs). This study seeks to clarify whether the P2Y12 receptor is involved in the sympathoexcitation reflex after myocardial ischemia (MI). Methods: MI model was induced by occlusion of the left coronary artery. P2Y12 were assayed by real time PCR and Western blotting. Results: Our results showed that expression levels of P2Y12 mRNA and protein were significantly higher in the MI group than in the sham group. Administration of P2Y12 short hairpin RNA (shRNA) caused downregulation of the P2Y12 receptor in the SCG. In MI rats plus P2Y12 shRNA treatment group, the abnormal changes in diastolic blood pressure (DBP), systolic blood pressure (SBP), heart rate (HR), electrocardiograms (ECGs), and cardiac tissue structures were alleviated. When the treatment of P2Y12 shRNA in MI rats, upregulated co‐expression values of P2Y12 and glial fibrillary acidic protein (GFAP), the upregulation of tumor necrosis factor α (TNF‐α) and phosphorylated P38 mitogen activated protein kinase (p‐P38 MAPK) in the SCG were decreased. Conclusion: Downregulation of the P2Y12 receptor in the SCG after MI may improve cardiac function by alleviating the sympathoexcitatory reflex. This article is protected by copyright. All rights reserved
       
  • Suppressing Angiogenesis Regulates the Irradiation‐induced Stimulation
           on Osteoclastogenesis in vitro
    • Abstract: Ionizing radiation‐induced bone loss is a potential health concern in radiotherapy, occupational exposure and astronauts. Although impaired bone vasculature and reduced proliferation of bone‐forming osteoblasts has been implicated in this process, it has not been clearly characterized that whether radiation affects the growth of bone‐resorbing osteoclasts. The molecular crosstalk between different cell populations in the skeletal system has not yet been elucidated in detail, especially between the increased bone resorption at early stage of post‐irradiation and bone marrow‐derived endothelial progenitor cells (BM‐EPCs). In order to further understand the mechanisms involved in radiation‐induced bone loss at the cellular level, we assessed the effects of irradiation on angiogenesis of BM‐EPCs and osteoclastogenesis of receptor activator for nuclear factor‐κB ligand (RANKL)‐stimulated RAW 264.7 cells and crosstalk between these cell populations. We herein found significantly dysfunction of BM‐EPCs in response to irradiation at a dose of 2 Gy, including inhibited proliferation, migration, tube‐forming abilities and downregulated expression of pro‐angiogenesis vascular endothelial growth factors A (VEGF A). Meanwhile, we observed that irradiation promoted osteoclastogenesis of RANKL‐stimulated RAW 264.7 cells directly or indirectly. These results provide quantitative evidences of irradiation induced osteoclastogenesis at a cellular level, and strongly suggest the involvement of osteoclastogenesis, angiogenesis and crosstalk between bone marrow cells in the radiation‐induced bone loss. This study may provide new insights for the early diagnosis and intervention of bone loss post‐irradiation.This article is protected by copyright. All rights reserved
       
  • Targeted therapy of human glioblastoma via delivery of a toxin through a
           peptide directed to cell surface nucleolin
    • Abstract: Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell‐surface nucleolin known as N6L, previously reported to decrease both tumour growth and tumour angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells.The pseudopeptide N6L cross‐linked to saporin‐S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID50 four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary “in vivo” study demonstrated efficiency in reducing the tumour mass in an orthotopic mouse model of glioblastoma This article is protected by copyright. All rights reserved
       
  • Substrate stiffness regulated migration and angiogenesis potential of A549
           cells and HUVECs
    • Abstract: Tumor tissue tends to stiffen during solid tumor progression. Substrate stiffness is known to alter cell behaviors, such as proliferation and migration, during which angiogenesis is requisite. Mono‐culture and co‐culture systems of lung cancer cell line A549 and human umbilical vein endothelial cells (HUVECs), on polydimethylsiloxane substrates (PDMS) with varying stiffness, were used for investigating the effects of substrate stiffness on the migration and angiogenesis of lung cancer. The expressions of matrix metalloproteinases (MMPs) and angiogenesis‐related growth factors were up‐regulated with the increase of substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase (TIMPs) were down‐regulated with increasing substrate stiffness. Our data not only suggested that stiff substrate may promote the migration and angiogenesis capacities of lung cancer, but also suggested that therapeutically targeting lung tumor stiffness or response of ECs to lung tumor stiffness may help reduce migration and angiogenesis of lung tumor. This article is protected by copyright. All rights reserved
       
  • Adenosine, adenosine receptors and their role in glucose homeostasis and
           lipid metabolism
    • Abstract: Adenosine is an endogenous metabolite that is released from all tissues and cells including liver, pancreas, muscle and fat, particularly under stress, intense exercise, or during cell damage. The role of adenosine in glucose homeostasis has been attributed to its ability to regulate, through its membrane receptors, processes such as insulin secretion, glucose release and clearance, glycogenolysis, and glycogenesis. Additionally, adenosine and its multiple receptors have been connected to lipid metabolism by augmenting insulin‐mediated inhibition of lipolysis, and the subsequent increase in free fatty acids and glycerol levels. Furthermore, adenosine was reported to control liver cholesterol synthesis, consequently affecting plasma levels of cholesterol and triglycerides, and the amount of fat tissue. Alterations in the balance of glucose and lipid homeostasis have implications in both cardiovascular disease and diabetes. The ability of different adenosine receptors to activate and inhibit the same signaling cascades has made it challenging to study the influence of adenosine, adenosine analogs and their receptors in health and disease. This review focuses on the role and significance of different adenosine receptors in mediating the effect of adenosine on glucose and lipid homeostasis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
       
  • Cover Image, Volume 233, Number 1, January 2018
    • Abstract: Cover: The cover image, by Rachelle Frenette‐Cotton et al., is based on the Original Research Article Phosphoregulation of K+‐Cl‐ cotransporters during cell swelling: Novel insights,
      DOI : 10.1002/jcp.25899.
       
  • Table of Contents, Editor's Choice, Highlights
    •  
  • Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric
           oxide signaling in regulating murine sperm functional activity
    • Abstract: Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca2+‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca2+ efflux pump, PMCA4. This is the major Ca2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca2+] in capacitated and Ca2+ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca2+] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p 
       
  • Effects of reduced natural background radiation on Drosophila melanogaster
           growth and development as revealed by the FLYINGLOW program
    • Abstract: Natural background radiation of Earth and cosmic rays played a relevant role during the evolution of living organisms. However, how chronic low doses of radiation can affect biological processes is still unclear. Previous data have indicated that cells grown at the Gran Sasso Underground Laboratory (LNGS, L'Aquila) of National Institute of Nuclear Physics (INFN) of Italy, where the dose rate of cosmic rays and neutrons is significantly reduced with respect to the external environment, elicited an impaired response against endogenous damage as compared to cells grown outside LNGS. This suggests that environmental radiation contributes to the development of defense mechanisms at cellular level. To further understand how environmental radiation affects metabolism of living organisms, we have recently launched the FLYINGLOW program that aims at exploiting Drosophila melanogaster as a model for evaluating the effects of low doses/dose rates of radiation at the organismal level. Here, we will present a comparative data set on lifespan, motility and fertility from different Drosophila strains grown in parallel at LNGS and in a reference laboratory at the University of L'Aquila. Our data suggest the reduced radiation environment can influence Drosophila development and, depending on the genetic background, may affect viability for several generations even when flies are moved back to normal background radiation. As flies are considered a valuable model for human biology, our results might shed some light on understanding the effect of low dose radiation also in humans.Reduced natural background radiation at the Gran Sasso laboratories affects life span and fertility of Drosophila melanogaster flies. It also favors selection of flies bearing mutations in the ATM encoding gene.
       
  • Fasting up‐regulates ferroportin 1 expression via a
           Ghrelin/GHSR/MAPK signaling pathway
    • Abstract: The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft‐L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O‐acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft‐L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft‐L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre‐treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway.Fasting increases the expression of ghrelin, GHSR1α, GOAT as well as Ft‐L and Fpn1 proteins in the spleens of mice in vivo. Ghrelin increases the expression of Ft‐L and Fpn1 proteins as well as phosphorylation of ERK in macrophages in vitro. Fasting upregulates Fpn1 expression probably via a ghrelin/GHSR/MAPK signaling pathway.
       
  • The endoplasmic reticulum‐associated protein, OS‐9, behaves as a
           lectin in targeting the immature calcium‐sensing receptor
    • Abstract: The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.We identified osteosarcoma‐9 (OS‐9) protein as a binding partner of the calcium‐sensing receptor (CaSR) in a yeast two‐hybrid screen. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway via its mannose‐6‐phosphate receptor homology (MRH) domain. We investigated the role of OS‐9 in CaSR processing and show that the CaSR and OS‐9 co‐localize in the ER and that when the two proteins are coexpressed in COS‐1 cells, OS‐9 specifically binds the immature ER‐resident form of the CaSR with binding dependent on the MRH domain, indicating that OS‐9 acts as a lectin in targeting misfolded CaSR to ERAD.
       
  • Anti‐CRISPR proteins: Counterattack of phages on bacterial defense
           (CRISPR/Cas) system
    • Abstract: Since the dawn of life there is a never ending strife between bacteria and phages. Both are perpetually changing their strategies to take over each other. CRISPR/Cas is the most widespread defense system used by bacteria against mobile genetic elements (MGEs) such as phages, cojugative palsmids, transoposons, and pathogenicity islands. This system utilizes small guide RNA molecules to protect against phages infection and invasion by MGEs. Phages circumvent to these antiviral barriers by point mutation in PAM (protospacer‐adjacent motif) sequence, genome rearrangements and by using anti‐CRISPR proteins.Acr proteins have the potential to become powerful weapon for regulate gene expression and could be used them as “off‐switch” for Cas9‐based activity. These proteins provide an opportunity to researchers to halt any unpredicted consequences within cells or tissues, would ameliorate accuracy and safety of CRISPR applications.
       
  • 4PBA strongly attenuates endoplasmic reticulum stress, fibrosis, and
           mitochondrial apoptosis markers in cyclosporine treated human gingival
           fibroblasts
    • Abstract: Cyclosporine induces overgrowth of human gingiva. Previously we have shown (i) cyclosporine—inducing ER stress in human gingival fibroblasts (HGF), (ii) increased matrix protein expression, and (iii) interference with mitochondrial pro‐ and anti—apoptotic factors. This study was undertaken to assess the effects of melatonin (an antioxidant), 4PBA (an ER stress inhibitor), and simvastatin on the expression of ER Stress markers as well as on matrix and mitochondrial markers. HGF incubated with cyclosporine, or without melatonin/4PBA/statin. After 24 hr of incubation, mRNA expression of ER stress markers (GRP78, CHOP, XBP1, and XBPs) and matrix protein markers (like α‐SMA, VEGF, TGF‐β, CTGF), and mitochondrial apoptosis markers estimated and compared with housekeeping gene GAPDH. Compared to the control cyclosporine significantly augmented ER Stress and matrix proteins, which decreased significantly with the use of melatonin, 4PBA, and simvastatin. The mitochondrial proapoptotic molecule cyclophilin D, as well as Bcl2 expression also decreased after PBA treatment, paralleling an increase in cytochrome c expression. The effect of 4PBA was much more pronounced than the influence of other two. In conclusion, 4PBA could be a viable therapeutic option for drug‐induced gingival overgrowth.
       
  • Emerging roles of ER stress and unfolded protein response pathways in
           skeletal muscle health and disease
    • Abstract: Skeletal muscle is the most abundant tissue in the human body and can adapt its mass as a consequence of physical activity, metabolism, growth factors, and disease conditions. Skeletal muscle contains an extensive network of endoplasmic reticulum (ER), called sarcoplasmic reticulum, which plays an important role in the regulation of proteostasis and calcium homeostasis. In many cell types, environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen that ultimately leads to ER stress. To alleviate the stress and restore homeostasis, the ER activates a signaling network called the unfolded protein response (UPR). The UPR has three arms, which regulate protein synthesis and expression of many ER chaperone and regulatory proteins. However, the role of individual UPR pathways in skeletal muscle has just begun to be investigated. Recent studies suggest that UPR pathways play pivotal roles in muscle stem cell homeostasis, myogenic differentiation, and regeneration of injured skeletal muscle. Moreover, markers of ER stress and the UPR are activated in skeletal muscle in diverse conditions such as exercise, denervation, starvation, high fat diet, cancer cachexia, and aging. Accumulating evidence also suggests that ER stress may have important roles in the pathogenesis of inflammatory myopathies and genetic muscle disorders. The purpose of this review article is to discuss the role and potential mechanisms by which ER stress and the individual arms of the UPR regulate skeletal muscle formation, plasticity, and function in various physiological and pathophysiological conditions.ER stress and unfolded protein response (UPR) pathways play important roles in the regulation of tissue homeostasis. In this article, we have highlighted the role of ER stress and UPR pathways in skeletal muscle formation, regeneration, atrophy, aging, metabolic function, and muscle diseases. We also provided suggestions for future research that would elucidate the role of individual arms of the UPR in the regulation of skeletal muscle physiology and diseases.
       
  • New advances of TMEM88 in cancer initiation and progression, with special
           emphasis on Wnt signaling pathway
    • Abstract: Transmembrane protein 88 (TMEM88), a newly discovered protein localized on the cell membrane. Recent studies showed that TMEM88 was involved in the regulation of several types of cancer. TMEM88 was expressed at significantly higher levels in breast cancer (BC) cell line than in normal breast cell line with co‐localized with Dishevelled (DVL) in the cytoplasm of BC cell line. TMEM88 silencing in the ovarian cancer cell line CP70 resulted in significant upregulation of Wnt downstream genes (c‐Myc, cyclin‐D1) and other Wnt target genes including JUN, PTIX2, CTNNB1 (β‐catenin), further supporting that TMEM88 inhibits canonical Wnt signaling pathway. Wnt signaling pathway has been known to play important roles in many diseases, especially in cancer. For instance, hepatocellular carcinoma (HCC) has become one of the most common tumors harboring mutations in the Wnt signaling pathway. As the inhibitor of Wnt signaling, TMEM88 has been considered to act as an oncogene or a tumor suppressor. Up‐regulated TMEM88 or gene therapy approaches could be an effective therapeutic approach against tumor as TMEM88 inhibits Wnt signaling through direct interaction with DVL. Here, we review the current knowledge on the functional role and potential clinical application of TMEM88 in the control of various cancers.HighlightsWnt signaling displays an important role in several pathogenesis of cancer.Wnt signaling pathway is activated during cancer development.TMEM88 has an impact on cancer by inhibiting canonical Wnt signaling.We discuss the importance and new applications of TMEM88 in cancer therapy.As the inhibitor of Wnt signaling, TMEM88 has been considered to act as an oncogene or a tumor suppressor. Up‐regulated TMEM88 or gene therapy approaches could be an effective therapeutic approach against tumor as TMEM88 inhibits Wnt signaling through direct interaction with DVL.
       
  • Adipocytes properties and crosstalk with immune system in
           obesity‐related inflammation
    • Abstract: Obesity is a condition likely associated with several dysmetabolic conditions or worsening of cardiovascular and other chronic disturbances. A key role in this mechanism seem to be played by the onset of low‐grade systemic inflammation, highlighting the importance of the interplay between adipocytes and immune system cells. Adipocytes express a complex and highly adaptive biological profile being capable to selectively activate different metabolic pathways in order to respond to environmental stimuli. It has been demonstrated how adipocytes, under appropriate stimulation, can easily differentiate and de‐differentiate thereby converting themselves into different phenotypes according to metabolic necessities. Although underlying mechanisms are not fully understood, growing in adipocyte size and the inability of storing triglycerides under overfeeding conditions seem to be crucial for the switching to a dysfunctional metabolic profile, which is characterized by inflammatory and apoptotic pathways activation, and by the shifting to pro‐inflammatory adipokines secretion. In obesity, changes in adipokines secretion along with adipocyte deregulation and fatty acids release into circulation contribute to maintain immune cells activation as well as their infiltration into regulatory organs. Over the well‐established role of macrophages, recent findings suggest the involvement of new classes of immune cells such as T regulatory lymphocytes and neutrophils in the development inflammation and multi systemic worsening. Deeply understanding the pathways of adipocyte regulation and the de‐differentiation process could be extremely useful for developing novel strategies aimed at curbing obesity‐related inflammation and related metabolic disorders.
       
  • TGFβ signaling and the control of myofibroblast differentiation:
           Implications for chronic inflammatory disorders
    • Abstract: The myofibroblast is a highly specialized cell type that plays a critical role during normal tissue wound healing, but also contributes pathologically to chronic inflammatory conditions such as fibrosis and cancer. As fibrotic conditions continue to be a major burden to the public health system, novel therapies that target the function of myofibroblasts may show promise in the clinic. The cytokine transforming growth factor β (TGFβ) is the most potent known inducer of myofibroblast differentiation and thus represents a powerful target to modify myofibroblast function during disease. This review focuses on our current understanding of the key signaling pathways activated by TGFβ during myofibroblast differentiation.The myofibroblast is a highly specialized cell type that plays a critical role during normal tissue wound healing, but also contributes pathologically to chronic inflammatory conditions such as fibrosis and cancer. TGFβ is the most potent known inducer of myofibroblast differentiation and thus represents a powerful target to modify myofibroblast function during disease. This review focuses on our current understanding of the key signaling pathways activated by TGFβ during myofibroblast differentiation.
       
  • Nuclear phosphoinositide regulation of chromatin
    • Abstract: Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear‐driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology.Nuclear phosphoinositide signaling has recently gained prominence with the discovery of several novel and specifically nuclear lipid signaling mechanisms. This review attempts to highlight those discoveries, and put them into a broader historical context.
       
  • Biological and pharmacological evaluation of dimethoxycurcumin: A
           metabolically stable curcumin analogue with a promising therapeutic
           potential
    • Abstract: Dimethoxycurcumin (DiMC) is a synthetic analog of curcumin with superior inter‐related pro‐oxidant and anti‐cancer activity, and metabolic stability. Numerous studies have shown that DiMC reserves the biologically beneficial features, including anti‐inflammatory, anti‐carcinogenic, and cytoprotective properties, almost to the same extent as curcumin exhibits. DiMC lacks the phenolic‐OH groups as opposed to curcumin, dimethoxycurcumin, and bis‐demethoxycurcumin that all vary in the number of methoxy groups per molecule, and has drawn the attentions of researchers who attempted to discover the structure‐activity relationship (SAR) of curcumin. In this regard, tetrahydrocurcumin (THC), the reduced and biologically inert metabolite of curcumin, denotes the significance of the conjugated α,β diketone moiety for the curcumin activity. DiMC exerts unique molecular activities compared to curcumin, including induction of androgen receptor (AR) degradation and suppression of the transcription factor activator protein‐1 (AP‐1). The enhanced AR degradation on DiMC treatment suggests it as a novel anticancer agent against resistant tumors with androgenic etiology. Further, DiMC might be a potential treatment for acne vulgaris. DiMC induces epigenetic alteration more effectively than curcumin, although both showed no direct DNA hypomethylating activity. Given the metabolic stability, nanoparticulation of DiMC is more promising for in vivo effectiveness. However, studies in this regard are still in its infancy. In the current review, we portray the various molecular and biological functions of DiMC reported so far. Whenever possible, the efficiency is compared with curcumin and the reasons for DiMC being more metabolically stable are elaborated. We also provide future perspective investigations with respect to varying DiMC‐nanoparticles.This review, being the first of its kind, is a comprehensive evaluation of the molecular, cellular, and clinical properties of dimethoxycurcumin as a promising analog of curcumin. We have discussed various molecular and biological functions of DiMC reported so far. Whenever possible, the efficiency compared with curcumin and the reasons for DiMC being more metabolically stable than curcumin has been explained. We have also provided future perspective investigations with respect to varying DiMC‐nanoparticles.
       
  • Curcumin as a potential candidate for treating hyperlipidemia: A review of
           cellular and metabolic mechanisms
    • Abstract: Curcumin is an herbal polyphenol extensively investigated for antioxidant, anti‐inflammatory, and hypolipidaemic properties. In the present review, the efficacy of curcumin for improving a plasma lipid profile has been evaluated and compared with statins, a well‐known class of medicines for treating hypercholesterolemia and hyperlipidaemia. Curcumin is presumably most effective in reducing triglyceride (TG), while statins are most efficient in lowering low‐density lipoproteins‐cholesterol (LDL‐C). Additionally, various molecular and metabolic mediators of cholesterol and plasma lipid homeostasis are discussed in relation to how they are modulated by curcumin or statins. Overall, curcumin influences the same mediators of plasma lipid alteration as statins do. Almost all the pathways through which cholesterol trafficking takes place are affected by these agents. These include gastrointestinal absorption of dietary cholesterol, hepatocellular removal of plasma cholesterol, the mediators of reverse cholesterol transport, and removal of cholesterol from peripheral tissues. Moreover, the reactive oxygen species (ROS) scavenging potential of curcumin limits the risk of lipid peroxidation that triggers inflammatory responses causing cardiovascular diseases (CVD) and atherosclerosis. Taken together, curcumin could be used as a safe and well‐tolerated adjunct to statins to control hyperlipidaemia more effectively than statins alone.In the present review, the efficacy of curcumin for improving a plasma lipid profile has been evaluated and compared with statins, a well‐known class of medicines for treating hypercholesterolemia and hyperlipidaemia.
       
  • The role of various peroxisome proliferator‐activated receptors and
           their ligands in clinical practice
    • Abstract: Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors involved in several physiological processes including modulation of cellular differentiation, development, metabolism of carbohydrates, lipids, proteins, and tumorigenesis. The aim of this review is to examine how different PPAR ligands act, and discuss their use in clinical practice. PPAR ligands have a lot of effects and applications in clinical practice. Some PPAR ligands such as fibrates (PPAR‐α ligands) are currently used for the treatment of dyslipidemia, while pioglitazone and rosiglitazone (PPAR‐γ ligands) are anti‐diabetic and insulin‐sensitizing agents. Regarding new generation drugs, acting on both α/γ, β/δ, or α/δ receptors simultaneously, preliminary data on PPAR‐α/γ dual agonists revealed a positive effect on lipid profile, blood pressure, atherosclerosis, inflammation, and anti‐coagulant effects, while the overexpression of PPAR‐β/δ seems to prevent obesity and to decrease lipid storage in cardiac cells. Finally, PPAR‐α/δ dual agonist induces resolution of nonalcoholic steatohepatitis without fibrosis worsening.Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors involved in several physiological processes. PPAR ligands have a lot of effects and applications in clinical practice. The aim of this review is to examine how different PPAR ligands act, and discuss their use in clinical practice.
       
  • A systems approach to physiologic evolution: From micelles to
           consciousness
    • Abstract: A systems approach to evolutionary biology offers the promise of an improved understanding of the fundamental principles of life through the effective integration of many biologic disciplines. It is presented that any critical integrative approach to evolutionary development involves a paradigmatic shift in perspective, more than just the engagement of a large number of disciplines. Critical to this differing viewpoint is the recognition that all biological processes originate from the unicellular state and remain permanently anchored to that phase throughout evolutionary development despite their macroscopic appearances. Multicellular eukaryotic development can, therefore, be viewed as a series of connected responses to epiphenomena that proceeds from that base in continuous iterative maintenance of collective cellular homeostatic equipoise juxtaposed against an ever‐changing and challenging environment. By following this trajectory of multicellular eukaryotic evolution from within unicellular First Principles of Physiology forward, the mechanistic nature of complex physiology can be identified through a step‐wise analysis of a continuous arc of vertebrate evolution based upon serial exaptations.There is no systematic way of understanding physiology at the present time. By reducing the process of physiologic evolution to an integrated series of cellular‐molecular steps from the unicellular state to complex traits, this goal has now been accomplished.
       
  • Understanding the role of structural integrity and differential expression
           of integrin profiling to identify potential therapeutic targets in breast
           cancer
    • Abstract: Breast cancer is found to be the most prevalent neoplasm in women worldwide. Despite the function of physically tethering cells to the matrix, transmembrane protein integrins are crucially involved in diverse cellular functions such as cell differentiation, proliferation, invasion, migration, and metastasis. Dysregulation of integrins and their interactions with the cells and their microenvironment can trigger several signaling cues that determine the cell fate decision. In this review, we spotlight all pre‐existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumors and their microenvironment to conventional therapy and overall suppress their metastatic phenotype.In this review, we spotlight all pre‐existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumors and their microenvironment to conventional therapy and overall suppress their metastatic phenotype.
       
  • Statin regulated ERK5 stimulates tight junction formation and reduces
           permeability in human cardiac endothelial cells
    • Abstract: The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed 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 utilized human cardiac 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‐localization 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 provide 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.Extracellular signal regulated kinase 5 (ERK5) is required for cardiovascular development in vivo. We have utilized human cardiac endothelial cells to show that ERK5 regulates tight junction formation and permeability in these cells. Statins stimulate ERK5 activity resulting in decreased permeability, which can potentially protect against the adverse effects of cardiotoxic drugs.
       
  • Transforming growth factor β induces bone marrow mesenchymal stem cell
           migration via noncanonical signals and N‐cadherin
    • Abstract: Transforming growth factor‐beta (TGF‐β) induces the migration and mobilization of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF‐β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM‐MSCs mediated by TGF‐β are unclear. In this study, we showed that TGF‐β activated noncanonical signaling molecules, such as Akt, extracellular signal‐regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF‐β type I receptor in human BM‐MSCs and murine BM‐MSC‐like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF‐β‐mediated migration. These results suggested that the Smad‐independent, noncanonical signals activated by TGF‐β were necessary for migration. We also showed that N‐cadherin‐dependent intercellular interactions were required for TGF‐β‐mediated migration using functional inhibition of N‐cadherin with EDTA treatment and a neutralizing antibody (GC‐4 antibody) or siRNA‐mediated knockdown of N‐cadherin. However, N‐cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF‐β. Therefore, these results suggested that the migration of BM‐MSCs in response to TGF‐β was mediated through N‐cadherin and noncanonical TGF‐β signals.N‐cadherin‐dependent intercellular interaction is required for TGF‐β‐mediated migration of both BM‐MSCs and the BM‐MSC like cell line, ST2. It is likely that in addition to N‐cadherin, non‐canonical TGF‐β signals including Akt, ERKs, p38, FAK, and Rac1 are also required for the cell migration.
       
  • Mitochondria‐targeted esculetin inhibits PAI‐1 levels by modulating
           STAT3 activation and miR‐19b via SIRT3: Role in acute coronary artery
           syndrome
    • Abstract: In this study we explored the microRNAs responsible for the regulation of PAI‐1 during LPS‐stimulated inflammation in human aortic endothelial cells and subsequently studied the effect of a newly synthesized mitochondria‐targeted esculetin (Mito‐Esc) that was shown for its anti‐atherosclerotic potential, in modulating PAI‐1 levels and its targeted miRs during angiotensin‐II‐induced atherosclerosis in ApoE−/− mice. LPS‐stimulated PAI‐1 was accompanied with an upregulation of miR‐19b and down‐regulation of miR‐30c. These effects of LPS on PAI‐1 were reversed in the presence of both parent esculetin and Mito‐Esc. However, the effect of Mito‐Esc was more pronounced in the regulation of PAI‐1. In addition, LPS‐stimulated PAI‐1 expression was significantly decreased in cells treated with Anti‐miR‐19b, thereby suggesting that miR‐19b co‐expression plays a key role in PAI‐1 regulation. The results also show that incubation of cells with Stattic, an inhibitor of STAT‐3, inhibited LPS‐stimulated PAI‐1 expression. Interestingly, knockdown of SIRT3, a mitochondrial biogenetic marker, enhanced PAI‐1 levels via modulation of miR‐19b and ‐30c. Mito‐Esc treatment significantly inhibited Ang‐II‐induced PAI‐1, possibly via altering miR‐19b and 30c in ApoE−/− mice. The association between PAI‐1, miR‐19b and ‐30c were further confirmed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees. Taken together, LPS‐induced PAI‐1 involves co‐expression of miR‐19b and down regulation of miR‐30c, and Mito‐Esc treatment by modulating miR‐19b and miR‐30c through SIRT3 activation, inhibits PAI‐1 levels that, in part, contribute to its anti‐atherosclerotic effects. Moreover, there exists a strong positive correlation between miR‐19b and PAI‐1 in patients suffering from ST‐elevated myocardial infarction.LPS‐induced PAI‐1 involves co‐expression of miR‐19b and down regulation of miR‐30c. Mito‐Esc treatment by modulating miR‐19b and miR‐30c through SIRT3 activation, inhibits PAI‐1 levels. An association between PAI‐1, miR‐19b, and ‐30c was observed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees.
       
  • MAPK3/1 participates in the activation of primordial follicles through
           mTORC1‐KITL signaling
    • Abstract: The majority of ovarian primordial follicles are preserved in a dormant state to maintain the female reproductive lifespan, and only a few primordial follicles are activated to enter the growing follicle pool in each wave. Recent studies have shown that primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)‐KIT ligand (KITL) signaling in pre‐granulosa cells and its receptor (KIT)‐phosphoinositol 3 kinase (PI3K) signaling in oocytes. However, the upstream regulator of mTORC1 signaling is unclear. The results of the present study showed that the phosphorylated mitogen‐activated protein kinase3/1 (MAPK3/1) protein is expressed in some primordial follicles and all growing follicles. Culture of 3 days post‐parturition (dpp) ovaries with the MAPK3/1 signaling inhibitor U0126 significantly reduced the number of activated follicles and was accompanied by dramatically reduced granulosa cell proliferation and increased oocyte apoptosis. Western blot and immunofluorescence analyses showed that U0126 significantly decreased the phosphorylation levels of Tsc2, S6K1, and rpS6 and the expression of KITL, indicating that U0126 inhibits mTORC1‐KITL signaling. Furthermore, U0126 decreased the phosphorylation levels of Akt, resulting in a decreased number of oocytes with Foxo3 nuclear export. To further investigate MAPK3/1 signaling in primordial follicle activation, we used phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor bpV(HOpic) to promote primordial follicle activation. In this model, U0126 also inhibited the activation of primordial follicles and mTORC1 signaling. Thus, these results suggest that MAPK3/1 participates in primordial follicle activation through mTORC1‐KITL signaling.Primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)‐KIT ligand (KITL) signaling in pre‐granulosa cells and its receptor (KIT)‐phosphoinositol 3 kinase (PI3K) signaling in oocytes. Here, we have shown that MAPK3/1 signaling participates in the activation of primordial follicles through mTORC1 signaling and the expression of KITL in pre‐granulosa cells to activate the PI3K‐Akt pathway.
       
  • MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin‐1
           dependent mechanism
    • Abstract: Cellular migration is important during many physiological as well as pathological conditions and is regulated very tightly by an intricate network of signaling and effector molecules. One of the important players during cellular migration are matrix metalloproteases and their levels have been reported to be important in determining the cellular migratory properties during metastasis. MMPs and regulators of MMPs therefore, present themselves as potent candidates for manipulation, to control conditions where they get dysregulated. Micro RNAs are a group of micro regulators that can modulate expression of a gene through transcriptional and post transcriptional regulations. Owing to the fact that many microRNAs have already been reported to regulate MMPs and that miR106a, a member of oncomir17 family has been implicated in metastatic conditions, the present study intended to analyze if miR106a can regulate levels of MMP9, an important inducible matrix metalloproteinase. The results of the in vitro experiments demonstrated that under conditions of migration cells showed elevated levels of miR106a, which could regulate the expression of major MMP9 regulator, SIRT‐1. Decreased levels of SIRT1thus resulted in an increase in the expression and activity of MMP9. Over expression and mRNA stability studies carried out also suggested regulatory role of miR106a. The overall results thus suggested that the levels of miR106a gets modulated during cellular migration, causing a change in the levels of SIRT‐1 mRNA by affecting its stability and the levels of SIRT‐1 in turn can regulate the levels of MMP9.During cellular migration, the level of miRNA 106a is upregulated, which in turn regulates the levels of SIRT‐1 and further, SIRT‐1 modulates the levels of MMP‐9.
       
  • Soft tissue engineering with micronized‐gingival connective tissues
    • Abstract: The free gingival graft (FGG) and connective tissue graft (CTG) are currently considered to be the gold standards for keratinized gingival tissue reconstruction and augmentation. However, these procedures have some disadvantages in harvesting large grafts, such as donor‐site morbidity as well as insufficient gingival width and thickness at the recipient site post‐treatment. To solve these problems, we focused on an alternative strategy using micronized tissue transplantation (micro‐graft). In this study, we first investigated whether transplantation of micronized gingival connective tissues (MGCTs) promotes skin wound healing. MGCTs (≤100 µm) were obtained by mincing a small piece (8 mm3) of porcine keratinized gingiva using the RIGENERA system. The MGCTs were then transplanted to a full skin defect (5 mm in diameter) on the dorsal surface of immunodeficient mice after seeding to an atelocollagen matrix. Transplantations of atelocollagen matrixes with and without micronized dermis were employed as experimental controls. The results indicated that MGCTs markedly promote the vascularization and epithelialization of the defect area 14 days after transplantation compared to the experimental controls. After 21 days, complete wound closure with low contraction was obtained only in the MGCT grafts. Tracking analysis of transplanted MGCTs revealed that some mesenchymal cells derived from MGCTs can survive during healing and may function to assist in wound healing. We propose here that micro‐grafting with MGCTs represents an alternative strategy for keratinized tissue reconstruction that is characterized by low morbidity and ready availability.Micronized‐gingival connective tissues promote the skin wound healing.
       
  • Profilin1 is expressed in osteocytes and regulates cell shape and
           migration
    • Abstract: Osteocytes are the most abundant cells in bone and regulate bone metabolism in coordination with osteoblasts and osteoclasts. However, the molecules that control osteocytes are still incompletely understood. Profilin1 is an actin‐binding protein that is involved in actin polymerization. Osteocytes possess characteristic dendritic process formed based on actin cytoskeleton. Here, we examined the expression of profilin1 and its function in osteocytes. Profilin1 mRNA was expressed in osteocytic MLO‐Y4 cells and its levels were gradually increased along with the time in culture. With regard to functional aspect, knockdown of profilin1 by siRNA enhanced BMP‐induced increase in alkaline phosphatase expression levels in MLO‐Y4 cells. Profilin1 knockdown suppressed the levels of dendritic processes and migration of MLO‐Y4 cells. Since aging causes an increase in ROS in the body, we further examined the effects of hydrogen peroxide on the expression of profilin1. Hydrogen peroxide treatment increased the levels of profilin1 mRNA in MLO‐Y4 cells in contrast to the decline in alkaline phosphatase. Profilin1 was expressed not only in MLO‐Y4cells but also in the primary cultures of osteocytes. Importantly, profilin1 mRNA levels in primary cultures of osteocytes were higher than those in primary cultures of osteoblasts. To examine in vivo role of profilin1 in osteocytes, profilin1 was conditionally knocked out by using DMP1‐cre and profilin1 floxed mice. This conditional deletion of profilin1 specifically in osteocytes resulted in reduction in the levels of bone volume and bone mineral density. These data indicate that profilin1 is expressed in osteocytes and regulates cell shape, migration and bone mass.Profilin1 knockdown reduces dendritic process formation in osteocytic MLO‐Y4 cells.
       
  • T11TS repress gliomagenic apoptosis of bone marrow hematopoietic stem
           cells
    • Abstract: Combating gliomagenic global immunosuppression is one of the emerging key for improving prognosis in malignant glioma. Apoptosis plays a pivotal role within the adult hematopoietic system particularly in regulating the cells of immune system. Gliomagenic regulation of apoptotic mediators within bone marrow milieu has not been elucidated. We previously demonstrated that administration of membrane glycopeptides T11 target structure (T11TS) not only rejuvenate bone marrow hematopoietic stem cells (BMHSCs) from glioma mediated hibernation by inhibiting gliomagenic overexpression of Ang‐1/Tie‐2 but also stimulate glioma mediated diminution of expression CD34, c‐kit, and Sca‐1 markers. In the present study, we investigated the impact of glioma on apoptotic signaling cascades of BMHSCs and consequences following T11TS therapy. Bone marrow smear and Annexin V staining confirm gliomagenic acceleration of apoptotic fate of BMHSCs whereas T11TS treatment in glioma‐bearing rats disrupted apoptosis of BMHSCs. Flowcytometry, immunoblotting, and immunofluorescence imagining results revealed multi potent T11TS not only significantly downregulates gliomagenic overexpression of Fas, Fas L, Bid, and caspase‐8, the pro‐apoptotic extrinsic mediators but also strongly inhibits cytosolic release of cytochrome‐c, Apf‐1, and Bax to deactivate gliomagenic caspase‐9, 3 the key intrinsic apoptotic mediators followed by up modulation of anti‐apoptotic Bcl‐2 in glioma associated HSCs. T11TS is also able to diminish the perforin‐granzyme B mediated apoptotic verdict of BMHSCs during gliomagenesis. The anti‐apoptotic action of T11TS on glioma associated BMHSCs provide a crucial insight into how T11TS exerts its immunomodulatory action against glioma mediated immune devastation.T11TS inhibits gliomagenic acceleration of premature apoptosis of bone marrow hematopoietic stem cells by repressing extrinsic, intrinsic, and granzyme B mediated apoptogenic cascades. Flowcytometry, immunoblotting experiments and immunofluorescence imagining results revealed multi potent T11TS significantly downregulates gliomaogenic overexpression of the key apoptotic mediators of all the above three pathways.
       
  • An advanced tri‐culture model to evaluate the dynamic interplay among
           osteoblasts, osteoclasts, and endothelial cells
    • Abstract: The dynamic metabolism and the numerous roles of bone tissue necessitate a suitable in vitro model to represent them. In order to investigate the interaction among the several cell types composing bone microenvironment, we studied a tri‐culture model including human osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (HUVEC). While OBs are essential for bone deposition and OCs for bone resorption, the vasculature is necessary to provide growth factors, nutrients, and oxygen in the mature tissue. The results of this study showed a strong mutual influence between OBs, OCs, and HUVEC in term of proliferation, viability, and activity (release of ALP, Coll I, OPG, RANKL, VEGF, CTSK, TGFβ, and IL‐6). The behavior of the single cultures demonstrated to be different compared to the bi‐ or tri‐cultures and depending on the cell types involved: the coexistence of OBs and OCs stimulated the synthetic activity of both cell types, while the presence of HUVEC induced a stimulating role for OBs but mainly an inhibitory effect for OC. In addition, evidence of the effects of OBs and OCs on HUVEC is highlighted by their morphology: regular and able to “sketch” little vessels in presence of OBs, more disorganized and heterogeneous in presence of OCs. Taken together, these observations well characterize an advanced cellular model to be used as starting point for mimicking bone microenvironment in vivo, thus reducing the use of animals in the preclinical phase and offering a more reliable tool to test new and innovative biomaterials.In vitro model of direct co‐colture showing the mutual influence among osteoblast for bone deposition, osteoclast for bone resorption and endothelial cells for angiogenesis, as starting point in mimicking in vivo microenvironment.
       
  • Melatonin‐induced increase of lipid droplets accumulation and in vitro
           maturation in porcine oocytes is mediated by mitochondrial quiescence
    • Abstract: Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA‐encoded genes was significantly down‐regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin‐treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up‐regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin‐induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence.
       
  • GM13133 is a negative regulator in mouse white adipocytes differentiation
           and drives the characteristics of brown adipocytes
    • Abstract: Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non‐shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown‐like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, β3‐adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre‐adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown‐like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white‐to‐brown adipocytes conversion. Our results identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.Our results provide identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and further indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.
       
  • Identification of murine phosphodiesterase 5A isoforms and their
           functional characterization in HL‐1 cardiac cell line
    • Abstract: Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). Overexpression of mPDE5As induced cell cycle progression of HL‐1 cells. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms.
       
  • Steady state peripheral blood provides cells with functional and metabolic
           characteristics of real hematopoietic stem cells
    • Abstract: Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non‐SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non‐SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia‐induced factors HIF‐1α and HIF‐2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non‐SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.We provided evidence that Side Population (SP) cells from Steady State Peripheral Blood (SSPB) displayed properties of very primitive cells and HSC based on in vitro and in vivo functional assays. Moreover these SP cells are better maintained during culture at low O2 concentrations, and they displayed a reduced mitochondrial mass and mitochondrial activity compared to non‐SP cells, while exhibiting a similar level of glucose incorporation. These cells are thus an interesting model for research and cell therapy.
       
  • Transcription factor EGR1 promotes differentiation of bovine skeletal
           muscle satellite cells by regulating MyoG gene expression
    • Abstract: The transcription factor, early growth response 1 (EGR1), has important roles in various cell types in response to different stimuli. EGR1 is thought to be involved in differentiation of bovine skeletal muscle‐derived satellite cells (MDSCs); however, the precise effects of EGR1 on differentiation of MDSCs and its mechanism of action remain unknown. In the present study, a time course of EGR1 expression and the effects of EGR1 on MDSC differentiation were determined. The results demonstrated that the expression of EGR1 mRNA and protein increased significantly in differentiating MDSCs relative to that in proliferating cells. Over‐expression of the EGR1 gene in MDSCs promoted their differentiation and inhibited proliferation. Conversely, knock‐down of EGR1 inhibited differentiation of MDSCs and promoted their proliferation, indicating that EGR1 promotes MDSC differentiation. Moreover, over‐expression of EGR1 in MDSCs increased the expression of MyoG mRNA and protein, whereas its knock‐down had the opposite effect. Furthermore, ChIP‐PCR analyses demonstrated that EGR1 could bind directly to its putative binding site within the promoter region of MyoG, and determination of ERG1 subcellular localization in MDSCs demonstrated that it could relocate to the nucleus, indicating MyoG is likely an EGR1 target gene whose expression is positively regulated by this transcription factor. In conclusion, EGR1 can promote MDSC differentiation through positive regulation of MyoG gene expression.The transcription factor, EGR1, can promote differentiation of bovine skeletal muscle‐derived satellite cells. In addition, EGR1 can relocate to the nucleus and bind directly to the MyoG gene promoter, hence positively regulating MyoG gene expression. These data prove that EGR1 promotes MDSC differentiation by regulation of MyoG gene expression.
       
  • Swelling‐induced chloride current in glioblastoma proliferation,
           migration, and invasion
    • Abstract: Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current ICl,swell. In this study, we investigated the effects of ICl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of ICl,swell, DCPIB, potently reduced the ICl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the ICl,swell may be a potential drug target for GBM.Illustration of the cellular pathways involved in the effects of ICl,swell inhibition on GBM proliferation, migration and invasion.
       
  • Bone loss in C57BL/6J‐OlaHsd mice, a substrain of C57BL/6J carrying
           mutated alpha‐synuclein and multimerin‐1 genes
    • Abstract: The inbred mouse strain C57BL/6 is commonly used for the generation of transgenic mouse and is a well established strain in bone research. Different vendors supply different substrains of C57BL/6J as wild‐type animals when genetic drift did not incur any noticeable phenotype. However, we sporadically observed drastic differences in the bone phenotype of “WT” C57BL/6J mice originating from different labs and speculated that these variations are attributable, at least in part, to the variation between C57BL/6J substrains, which is often overlooked. C57BL/6J‐OlaHsd is a commonly used substrain that despite a well defined deletion in the alpha‐synuclein (Snca) and multimerin‐1 (Mmrn1) genes, was reported to display no obvious phenotype and is used as WT control. Here, we compared the bone phenotype of C57BL/6J‐OlaHsd (6J‐OLA) to C57BL/6J‐RccHsd (6J‐RCC) and to the original C57BL/6J (6J‐JAX). Using μCT analysis, we found that 6J‐OLA mice display a significantly lower trabecular bone mass compared to 6J‐RCC and 6J‐JAX. PCR analysis revealed that both the Snca and Mmrn1 genes are expressed in bone tissue of 6J‐RCC animals but not of 6J‐OLA mutants, suggesting either one or both genes play a role in bone metabolism. In vitro analysis demonstrated increase in osteoclasts number and decreased osteoblast mineralization in cells derived from 6J‐OLA compared with 6J‐RCC. Our data may shed light on unexplained differences in basal bone measurements between different research centers and reiterate the importance of specifying the exact substrain type. In addition, our findings describe the physiological role for Mmrn1 and/or Snca in bone remodeling.The C57Bl/6J‐OlaHsd substrain commonly considered as wild‐type mice, display a low bone mass phenotype compared to the original 6J substrain. These differences are attributable to the physiological role of alpha‐synuclein and multimerin in osteoblast and osteoclast functions. These findings also reiterate the critical importance of using littermates as controls for transgenic mice.
       
  • The prognostic value of MGMT promoter methylation in glioblastoma: A
           meta‐analysis of clinical trials
    • Abstract: The DNA repair protein O6‐Methylguanine‐DNA methyltransferase (MGMT) is suggested to be associated with resistance to alkylating agents such as Temozolomide which is being used in treatment of patients with glioblastoma (GBM). Therefore, we evaluated the associations between MGMT promoter methylation and prognosis of patients with glioblastoma (GBM). Data were extracted from publications in Embase, PubMed, and the Cochrane Library. Data on overall survival (OS), progression‐free survival (PFS), and MGMT methylation status were obtained and 4,097 subjects were enrolled. Data from 34 studies showed that MGMT methylated patients had better OS, compared to GBM unmethylated patients (pooled HRs, 0.494; 95%CI 0.412–0.591; p = 0.001). Meta‐analysis of 10 eligible studies reporting on PFS, demonstrated that MGMT promoter methylation was not significantly associated with better PFS (pooled HRs, 0.653; 95%CI 0.414–1.030; p = 0.067). GBM patients with MGMT methylation were associated with longer overall survival, although this effect was not detected for PFS. Moreover, we performed further analysis in patients underwent a comprehensive imaging evaluation. This data showed a significant association with better OS and PFS, although further studies are warranted to assess the value of emerging marker in prospective setting in patients with glioblastoma as a risk stratification biomarker in clinical management of the patients.
       
  • Comparative proteomic profiling of human osteoblast‐derived
           extracellular matrices identifies proteins involved in mesenchymal stromal
           cell osteogenic differentiation and mineralization
    • Abstract: The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.In vitro cell‐derived extracellular matrices (ECM) mimic the ECM role in tissue architecture and in actively modulating cell behavior, and they have been proposed for tissue engineering applications. We created three models of human osteoblast‐derived ECM with extremely different mineralization phenotypes, to identify regulatory proteins to modulate mesenchymal stromal cell (MSC) differentiation. Comparative proteomic profiles of these ECMs reveal that the protein composition is important to regulate MSC behavior, and could be used to modulate MSC properties and bone quality for bone tissue engineering applications.
       
  • Phosphoregulation of K+‐Cl− cotransporters during cell
           swelling: Novel insights
    • Abstract: The K+‐Cl− cotransporters (KCCs) belong to the cation‐Cl− cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl− ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl−] (Cli), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K+‐Cl− cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.We have found that both cell swelling and a T926A substitution in the C‐terminus of full‐lenght KCC4 lead to higher levels of heterologous K‐Cl cotranport by increasing overall carrier phosphorylation. These results imply that the response to cell swelling could involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.
       
  • Osteoactivin regulates head and neck squamous cell carcinoma invasion by
           modulating matrix metalloproteases
    • Abstract: Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP‐10 expression in UMSCC14a (p = 0.0431) and SCC15 (p 
       
  • Fibroblast dynamics as an in vitro screening platform for anti‐fibrotic
           drugs in primary myelofibrosis
    • Abstract: Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA‐approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications—cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA‐approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications—cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.
       
  • 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 threefold 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.Impairment of intercellular communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is linked to development of vascular diseases such as hypertension, cerebral, and coronary vasospasms, aortic aneurism, and erectile dysfunction. 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.
       
  • Lipopolysaccharide can modify differentiation and immunomodulatory
           potential of periodontal ligament stem cells via ERK1,2 signaling
    • Abstract: Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4+ and the ratio of CD4+CD25high/CD4+CD25low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.In this study, we examined functional properties of human periodontal ligament stem cells (PDLSCs) in presence of LPS (E. coli). Our results indicated that LPS modified PDLSCs’ mesodermal lineage commitment, favoring myofibroblastic‐like phenotype, without affecting their growth and immunophenotype. LPS‐treated PDLSCs strongly inhibited transendothelial migration (TEM) of human peripheral blood mononuclear cells (MNCs). LPS‐treated PDLSCs decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity, predominantly the CFU‐GM number, of MNCs. Our results indicate that changes in differentiation and immunomodulatory properties of PDLSCs may be in partly governed by LPS‐activated ERK1,2 signaling cascade.
       
  • Mitochondrial ROS‐induced ERK1/2 activation and HSF2‐mediated AT1R
           upregulation are required for doxorubicin‐induced cardiotoxicity
    • Abstract: Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT1R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT1R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT1R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT1R upregulation causing DOX‐damaged heart failure in vitro and in vivo.DOX induces mitochondrial ROS accumulation to activate ERK activation for HSF2‐mediated AT1R upregulation.
       
  • 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.
       
  • Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is
           associated with altered pancreatic islets redox status
    • Abstract: In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H2O2), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H2O2 and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H2O2 production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H2O2 (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.Our data suggest that short‐term early life protein malnourishment induces reduction in redox GIIS signaling only at extreme oxidant challenges, elicited here by high H2O2 pre‐incubation followed by elevated glucose concentration incubation, probably due to lower antioxidant scavenging capacity.
       
  • Autophagy mediates cytotoxicity of human colorectal cancer cells treated
           with garcinielliptone FC
    • Abstract: The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT‐29 cells. In the present study, we observed that many autophagy‐related genes in GFC‐treated HT‐29 cells were up‐ and down‐regulated using a cDNA microarray containing oncogenes and kinase genes. GFC‐induced autophagy of HT‐29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double‐membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5‐Atg12 and PI3K/Beclin‐1 complexes were observed using Western blot. Administration of autophagy inhibitor (3‐methyladenine and shRNA Atg5) and apoptosis inhibitor Z‐VAD showed that the GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC‐induced anticancer mechanisms of human colorectal cancer.The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy.
       
  • Knockout of ATG5 leads to malignant cell transformation and resistance to
           Src family kinase inhibitor PP2
    • Abstract: Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.ATG5 knockout cells show morphological transformation and anchorage‐independent growth‐ ATG5 KO cells have a lower proportion of the apoptotic cell population as a way to escape cell death in response to PP2.
       
  • Rapamycin attenuates BAFF‐extended proliferation and survival via
           disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid
           cells
    • Abstract: B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.Rapamycin, a macrocyclic lactone, prevented B cells from hsBAFF‐promoted proliferation/survival, not only by targeting mTORC1‐mediated S6K1/4E‐BP1 pathways, but also via targeting mTORC2‐mediated Akt pathway. This study highlights that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.
       
  • HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic
           neurogenesis and in schizophrenia‐like animals
    • Abstract: Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1‐deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro‐differentiation was almost suppressed. Neuro‐differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia‐like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia‐like brains that were treated with the cannabinoid receptor‐1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co‐regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype.We showed that co‐regulation of H3K9 acetylation by histone deacetylases HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype. This knowledge contributes to a basic understanding of epigenetics of the brain.
       
  • Identification and characterization of site‐specific N‐glycosylation
           in the potassium channel Kv3.1b
    • Abstract: The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex3HexNAc4Fuc1 glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man8GlcNAc2 glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.Site‐specific N‐glycosylation and N‐glycan structure regulate cell surface trafficking and expression of the Kv3.1b potassium channel. Mass spectrometry analysis identified Man8GlcNAc2 (high‐mannose type) and Hex3HexNAc4Fuc1 (complex type) as the glycan moieties on N220 and N229.
       
  • The role of Runx2 in facilitating autophagy in metastatic breast cancer
           cells
    • Abstract: Breast cancer metastases cause significant patient mortality. During metastases, cancer cells use autophagy, a catabolic process to recycle nutrients via lysosomal degradation, to overcome nutritional stress for their survival. The Runt‐related transcription factor, Runx2, promotes cell survival under metabolic stress, and regulates breast cancer progression and bone metastases. Here, we identify that Runx2 enhances autophagy in metastatic breast cancer cells. We defined Runx2 function in cellular autophagy by monitoring microtubule‐associated protein light chain (LC3B‐II) levels, an autophagy‐specific marker. The electron and confocal microscopic analyses were utilized to identify alterations in autophagic vesicles. The Runx2 knockdown cells accumulate LC3B‐II protein and autophagic vesicles due to reduced turnover. Interestingly, Runx2 promotes autophagy by enhancing trafficking of LC3B vesicles. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α‐tubulin sub‐units of microtubules. Inhibiting autophagy decreased cell adhesion and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide molecular insights into the role of autophagy in metastatic cancer cells.We demonstrate a novel function of Runx2 in facilitating autophagy by altering cytoskeletal elements during cellular stress conditions such as nutrient deprivation. Our results in bone metastatic MDA‐MB‐231 breast cancer cells show that Runx2 promotes autophagy via α‐tubulin acetylation and autophagosome trafficking. Since autophagy promotes tumor cell adhesion, the Runx2‐mediated autophagy supporting cell adhesion highlights its function in metastatic breast cancer cell survival.
       
  • Gax suppresses chemerin/CMKLR1‐induced preadipocyte biofunctions through
           the inhibition of Akt/mTOR and ERK signaling pathways
    • Abstract: Adipose tissue is closely associated with angiogenesis and vascular remodeling. Chemerin is involved in inflammatory reaction and vascular dysfunction. However, the mechanisms of chemerin participating in vascular remodeling and whether Growth arrest‐specific homeobox (Gax) can effectively intervene it remain obscured. Here, 3T3‐F442A preadipocytes were cultured, injected into athymic mice to model fat pads, and treated respectively with Ad‐chemerin, Ad‐Gax, or specific inhibitors in vitro and in vivo. MTT, flow cytometry, Western blotting, and imunohisto(cyto)‐chemistry analyses showed that chemerin enhanced the expression of FABP4 and VEGF, activated Akt/mTOR and ERK pathways, increased the cell percent of S phase, decreased the percent of G0‐G1 phase and apoptotic cells, and augmented neovascular density in fat pads. Inversely, Gax suppressed the expression of these adipogenic and vasifactive markers and these signaling proteins, decreased the percent of S phase cells, and increased those of G0‐G1 phase and apoptotic cells, and reduced the neovascular density. Our results indicate that chemerin‐CMKLR1 activates Akt/mTOR and ERK pathways and facilitates preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on preadipocyte biofunctions.Chemerin/CMKLR1 activates Akt/mTOR/p70SK1/4EBP1 and ERK1/2 pathways and enhances preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on the preadipocyte biofunctions.
       
  • Transplantation of mesenchymal stem cells overexpressing IL10 attenuates
           cardiac impairments in rats with myocardial infarction
    • Abstract: Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin‐10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow‐derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen‐glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10‐MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10‐MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10‐MSC treatment. IL10 overexpression and MSC may exert a synergistic anti‐inflammatory effect to alleviate cardiac injury after MI.IL10 overexpression in mesenchymal stem cells enhances cell viability and decreases apoptosis. Implantation of IL10‐overexpressed mesenchymal stem cells into myocardial infarct animals results in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis.
       
  • TGF‐β inhibits osteogenesis by upregulating the expression of ubiquitin
           ligase SMURF1 via MAPK‐ERK signaling
    • Abstract: High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK‐ERK signaling in the TGF‐β‐mediated osteoblastic differentiation. Our results indicated that TGF‐β activated the MAPK‐ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK‐ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF‐β, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF‐β induced protein expression of E3 ubiquitin‐protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK‐ERK inhibitor U0126 in TGF‐β‐treated differentiating preosteoblasts, suggesting that MAPK‐ERK regulated the transcription of osteogenesis‐related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)‐2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF‐β inhibited osteoblastic differentiation by inducing the MAPK‐ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP‐2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.TGF‐β activates MAPK‐ERK signaling to inhibit osteogenesis through Smurf‐1.MAPK‐ERK inhibitor U0126 and BMP‐2 have a synergistic positive effect on osteoblast differentiation and bone formation.
       
  • All‐trans retinoic acid ameliorates hepatic stellate cell activation via
           suppression of thioredoxin interacting protein expression
    • Abstract: Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome‐wide screening of RA‐responsive genes by in silico analysis of RA‐response elements, and identified 26 RA‐responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all‐trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP‐mediated HSC activation was suppressed by antioxidant N‐acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.
       
  • Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates
           osteoclast fusion in a cell heterotypic manner
    • Abstract: Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.This study investigate the molecular mechanisms regulating osteoclast fusion. Osteoclast fusion involve a fusion initiation cell termed fusion founder which fuse to cells lacking fusion initiation abilities termed fusion followers. This study show for the fist time that the protein Dyrk2 inhibit fusion by limiting the distribution of fusion follower cells.
       
  • Involvement of testicular DAAM1 expression in zinc protection against
           cadmium‐induced male rat reproductive toxicity
    • Abstract: In order to verify the effects of exposure to Cd and Zn on testicular DAAM1 gene and protein expression and also to ascertain their involvement in the protective role of Zn in prevent the testicular toxicity Cd‐induced in male offspring rats at adult age after gestational and lactational exposure, male offspring rats, from mothers receiving either tap water, Cd, Zn, or Cd + Zn during gestation and lactation periods, were scarified on postnatal days (PND) 70. The reproductive organ (testis, epididymis, and vesicle seminal) were collected, weighed, and analyzed. The results showed that exposure to Cd in utero and through lactation decreased the relative reproductive organ weight, altered the testicular histology at the interstitial and tubular levels, and causing a significant reduction in the daily sperm production (DSP) per testis and per gram of testis, and other then altering the epididymal sperm quality. Furthermore, both mRNA and protein expression of rat testicular DAAM1 were also inhibited in Cd‐treated group. Zn supply has completely corrected the most of these toxic effects. Our results imply that Zn could prevent Cd‐induced testicular toxicity and sperm quality alteration in adult male rat after gestational and lactational exposure, probably via the restoration of the testicular DAAM1 expression inhibited by Cd.The effects of exposure to Cd and Zn on testicular DAAM1 gene and protein expression and heir involvement in the protective role of Zn in prevent the testicular toxicity Cd‐induced in male offspring rats have been studied. The reproductive organ were collected, weighed and analyzed. The results showed that Cd exposure decreased the relative reproductive organ weight, altered the testicular histology, and causing a significant reduction in the daily sperm production other then altering the epididymal sperm quality. In addition, both mRNA and protein expression of rat testicular DAAM1 were also inhibited. Zn supply has completely corrected the most of these toxic effects. Our results imply that Zn could prevent Cd‐induced testicular toxicity probably via the restoration of the testicular DAAM1 expression inhibited by Cd.
       
  • Retinoic acid receptor‐related orphan receptor RORα regulates
           differentiation and survival of keratinocytes during hypoxia
    • Abstract: Low O2 pressures present in the microenvironment of epidermis control keratinocyte differentiation and epidermal barrier function through hypoxia inducible factors (HIFs) dependent gene expression. This study focuses on investigating relations of the retinoic acid receptor‐related orphan receptor alpha (RORα) to HIF‐1α in keratinocytes under hypoxic conditions. The expression level of RORα is significantly elevated under hypoxia in both human and murine keratinocytes. Gene silencing of RORA attenuates hypoxia‐stimulated expression of genes related to late differentiation and epidermal barrier function, and leads to an enhanced apoptotic response. While the hypoxic induction of RORα is dependent on HIF‐1α, RORα is in turn critical for nuclear accumulation of HIF‐1α and activation of HIF transcriptional activity. These results collectively suggest that RORα functions as an important mediator of HIF‐1α activities in regulating keratinocyte differentiation/survival and epidermal barrier function during the oxygen sensing stage.We have identified RORα as a regulator imperative to HIF‐1α activities in promoting terminal differentiation, epidermal barrier function, and survival of keratinocytes under low O2 tension. Given highly diversified functions of HIF targets in maintaining skin homeostasis, manipulations of HIF activities through RORα ligands can represent a novel strategy for therapeutic treatment of pathophysiological conditions such as cutaneous diseases related to defective epidermal barrier functions and wound healing.
       
  • Mechanism of prolactin inhibition of miR‐135b via methylation in
           goat mammary epithelial cells
    • Abstract: Prolactin is an important endocrine activator of lactogenesis. This study investigated the function and mechanism of miR‐135b in the enhancement of lactation by prolactin in goat mammary epithelial tissue. We utilized S‐Poly (T) sequencing to evaluate changes in gene regulation in the goat mammary gland after incubation with 2.5 μg/ml prolactin and 2.5 μg/ml IGF‐1 by examining highly expressed miRNAs during early lactation and late‐lactation. The results illustrated that miR‐135b is highly expressed in the goat mammary gland during early lactation and late‐lactation, and also after treatment with 2.5 μg/ml prolactin and 2.5 μg/ml IGF‐1. We used Q‐RT PCR, Western Blot, immunofluorescence, and luciferase reporter assay analysis, and found that PRL was significantly down‐regulated in response to the expression of miR‐135b in a manner that was functionally related to TAG synthesis via the large tumor suppressor 2 gene (Lats2), an important regulator of adipocyte proliferation via Hippo Signaling. Furthermore, using bisulfite‐sequencing PCR (BSP), Q‐PCR, and Western Blot we discovered an increase in expression of DNMT I (DNA methyl transferase I) in goat mammary epithelial cells with the 2.5 μg/ml PRL incubation, which led to DNA methylation of the CpG island upstream of miR‐135b and inhibited the transcription and expression of miR‐135b.This study clarified the mechanism by which PRL induces milk formation and identified a role of miR‐135b in this process. With PRL stimulation, the expression of DNMT I in mammary epithelial cells increased, resulting in DNA methylation of the CpG island upstream of miR‐135b's, thus inhibiting transcription of miR‐135b. At the same time, down‐regulation of miR‐135b resulted in increased expression of the target gene LATS2 thereby causing proliferation and metabolic changes in the mammary gland, eventually leading to lactation.
       
  • Primary cilium alterations and expression changes of Patched1 proteins in
           niemann‐pick type C disease
    • Abstract: Niemann‐Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease‐causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3‐ß‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one (U18666A), a cholesterol transport‐inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A‐treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A‐treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A‐treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients. We demonstrated altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia in fibroblasts from NPC1 patients.
       
  • miR‐124 and miR‐9 mediated downregulation of HDAC5 promotes neurite
           development through activating MEF2C‐GPM6A pathway
    • Abstract: The class IIa histone deacetylases (HDACs) play important roles in the central nervous system during diverse biological processes such as synaptic plasticity, axon regeneration, cell apoptosis, and neural differentiation. Although it is known that HDAC5 regulates neuronal differentiation, neither the physiological function nor the regulation of HDAC5 in neuronal differentiation is clear. Here, we identify HDAC5 as an inhibitor of neurite elongation and show that HDAC5 is regulated by the brain enriched microRNA miR‐124 and miR‐9. We discover that HDAC5 inhibits neurite extension both in differentiated P19 cells and primary neurons. We also show that the neuronal membrane glycoprotein GPM6A (M6a) is a direct target gene of HDAC5 regulated transcriptional factor MEF2C. HDAC5 inhibits neurite elongation, acting at least partially via a MEF2C/M6a signaling pathway. We also confirmed the miR‐124/miR‐9 regulated HDAC5‐MEF2C‐M6a pathway regulates neurite development in primary neurons. Thus, HDAC5 emerges as a cellular conductor of MEF2C and M6a activity and is regulated by miR‐124 and miR‐9 to control neurite development.We provide evidence that through the direct repression of HDAC5, miR‐124, and miR‐9 intrinsically regulate MEF2C transcription activity and M6a expression, and this ensures proper neurite elongation. Our findings further suggest that by acting in neurons, brain‐enriched miRNAs can play key roles in coordinating vertebrate central nervous system development.
       
  • Autophagy maintains the integrity of endothelial barrier in
           LPS‐induced lung injury
    • Abstract: Understanding the role and underlying regulation mechanism of autophagy in lipopolysaccharide‐induced lung injury (LPS‐LI) may provide potentially new pharmacological targets for treatment of acute lung injury. The aim of this study was to investigate the functional significance of autophagy in LPS‐LI. The autophagy of human pulmonary microvascular endothelial cells (HPMVECs) and mice was inhibited before they were challenged with LPS. In vitro, permeability, vitality, and the LDH release rate of the cells were detected, the zonula occluden‐1 (ZO‐1) expression and the stress fiber formation were determined. In vivo, the lung injury was assessed. We found LPS caused high permeability and increased lactate dehydrogenase (LDH) release rate, lowered viability of the cells, inhibited the ZO‐1 expression and induced stress fiber formation, these effects were further aggravated by prohibiting the level of autophagy. Consistently, in in vivo experiments, LPS‐induced serious lung injury, which was reflected as edema, leukocyte infiltration and hemorrhage in lung tissue, and the high concentration of pro‐inflammation cytokines tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β in bronchoalveolar lavage fluid (BALF). Inhibiting autophagy further exacerbated LPS‐LI. It appears that autophagy played a protective role in LPS‐LI in part through restricting the injury of lung microvascular barrier.
       
  • Global chondrocyte gene expression after a single anabolic loading period:
           Time evolution and re‐inducibility of mechano‐responses
    • Abstract: Aim of this study was a genome‐wide identification of mechano‐regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode and characterization of time evolution and re‐inducibility of the response. Osteochondral constructs consisting of a chondrocyte‐seeded collagen‐scaffold connected to β‐tricalcium‐phosphate were pre‐cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9 × 10 min over 3 hr) before microarray‐profiling was performed. Proteoglycan synthesis was determined by 35S‐sulfate‐incorporation over 24 hr. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45‐fold, p = 0.016). Among 115 significantly regulated genes, 114 were up‐regulated, 48 of them ≥ twofold. AP‐1‐relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2‐phosphorylation and rising MAP3K4 expression. Expression of proteoglycan‐synthesizing enzymes CHSY1 and GALNT4 was load‐responsive as were factors associated with the MAPK‐, TGF‐β‐, calcium‐, retinoic‐acid‐, Wnt‐, and Notch‐signaling pathway which were significantly upregulated SOX9, and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9‐signaling showed no consistent regulation. This study associates novel genes with mechanoregulation in chondrocytes, raising SOX9 protein levels with anabolic loading and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback‐regulators. Upregulation of mechanosensitive indicators extending differentially into the resting time provides crucial knowledge to maximize cartilage matrix deposition for the generation of high‐level cartilage replacement tissue.We here identified mechano‐regulation of SOX9 in chondrocytes in the center of a complex signaling network of an anabolic loading context and describe its robust re‐inducibility in daily intervals without desensitization. We suggest to use SOX9 protein levels and a promising selection of critical mechanosensitive genes as diagnostic markers to optimize loading regimes for future refinement of anabolic loading protocols to enhance cartilage tissue functionality in vitro and of tissue formation after surgical therapy in patients in vivo.
       
  • Horizontal transfer of miR‐23a from hypoxic tumor cell colonies can
           induce angiogenesis
    • Abstract: Neo vessel formation by angiogenesis is an important event during many pathological conditions including cancer, where it is indispensable for tumor growth and survival. Although, various pro‐angiogenic cytokines and soluble factors, secreted by tumor cells, have been reported to promote angiogenesis, recent studies have shown regulatory role of exosomes, secreted by tumor cells in the process of angiogenesis. These exosomes are capable of carrying nucleic acids, proteins etc. as their cargo. Under the light of these facts and considering the presence of miRNAs, the non‐coding RNAs capable of regulating target gene expression, as one of the major cargos in the exosomes, we investigated, whether exosomes derived from normoxic and hypoxic tumor cell colonies exhibit difference in levels of miR‐23∼27∼24 cluster members and if so, to check the significance of their horizontall transfer on the process of angiogenesis. Results of our study showed that exosomes secreted by hypoxic tumor cell colonies possess significantly higher levels of miR23a and can induce angiogenesis.. Further we have shown that exosomes secreted by cells that ectopically over express miR23a is capable of inducing angiogenesis in different angiogenic model systems such as CAM, in ovo Xenograft and HUVEC models systems. Further, mechanistic analysis revealed that miR23a driven regulation of angiogenesis is brought about by down regulation of SIRT1 in the recipient cells. Collectively, the results presented here suggest that exosomal transfer of miR23a from tumor cell colonies can induce the process of angiogenesis by targeting SIRT1 in the recipient endothelial cells. This article is protected by copyright. All rights reserved
       
  • Long non‐coding RNA LINC00968 acts as oncogene in NSCLC by activating
           the Wnt signaling pathway
    • Abstract: Long non‐coding RNAs (lncRNAs) have played critical roles in a variety of cancers, including non‐small cell lung cancer (NSCLC). In our study, we focused on the biological function and clinical significance of lncRNA LINC00968 in NSCLC. It was indicated that LINC00968 was significantly increased in LUAD tissues, LUSC tissues and NSCLC cells compared to their corresponding controls. Inhibition of LINC00968 was able to repress NSCLC growth, migration and invasion in vitro while upregulation of LINC00968 reversed this process. Additionally, downregulation of LINC00968 induced apoptosis capacity of A549 cell. Apoptosis‐related proteins BCL‐2 were decreased and BAX was increased by knockdown of LINC00968 respectively. Meanwhile we observed that Wnt signaling pathway was involved in the LINC00968‐induced NSCLC progression. Finally, in vivo tumor xenografts were established using A549 cells to detect the function of LINC00968 in NSCLC tumorigenesis. Silencing LINC00968 greatly inhibited NSCLC tumor progression, which was consistent with the in vitro tests. In conclusion, we have uncovered that LINC00968 could be regarded as a novel prognostic biomarker and therapeutic target in NSCLC diagnosis and treatment. This article is protected by copyright. All rights reserved
       
  • Long non‐coding RNA MALAT1 promotes oral squamous cell carcinoma
           development via microRNA‐125b/STAT3 axis
    • Abstract: Oral squamous cell carcinoma (OSCC), as the most common type of oral cancer, is responsible for almost 3% of all malignant tumors worldwide. Non‐coding RNAs such as lncRNAs and microRNAs have been involved in many cancers including OSCC. Recently, lncRNA metastasis‐associated lung adenocarcinoma transcript‐1 (MALAT1) has been reported to play an oncogenic role in OSCC metastasis. However, the underlying mechanism of MALAT1 in regulating OSCC progression remains unclear. The aim of this study was to investigate the specific role of MALAT1 in OSCC development. It was observed that MALAT1 was upregulated in OSCC cell lines. Inhibition of MALAT1 can prevent OSCC proliferation while overexpressing MALAT1 promoted OSCC progression. In addition, bioinformatics search was used to identify that miR‐125b was a direct target of MALAT1, which indicated a negative correlation between MALAT1 and miR‐125b. Besides these, STAT3 was predicted as a binding target of miR‐125b in OSCC. Overexpression of MALAT1 was able to suppress the tumor inhibitory effect of miR‐125b mimics via upregulating STAT3. Moreover, the function of MALAT1 in OSCC development was further investigated by using in vivo assays. The established nude mice models revealed that downregulated MALAT1 greatly inhibited OSCC tumor growth and reversely upregualated MALAT1 promoted OSCC development via miR‐125b/STAT3 axis respectively. In conclusion, MALAT1 can function as a competing endogenous RNA (ceRNA) to modulate STAT3 expression by absorbing miR‐125b in OSCC and could be used as a novel therapeutic target in OSCC diagnosis and treatment. This article is protected by copyright. All rights reserved
       
  • Functional Role of Mesenchymal Stem Cells in the Treatment of Chronic
           Neurodegenerative Diseases
    • Abstract: Mesenchymal stem cells (MSCs) can differentiate into not only cells of mesodermal lineages, but also into endodermal and ectodermal derived elements, including neurons and glial cells. For this reason, MSCs have been extensively investigated to develop cell‐based therapeutic strategies, especially in pathologies whose pharmacological treatments give poor results, if any. As in the case of irreversible neurological disorders characterized by progressive neuronal death, in which behavioral and cognitive functions of patients inexorably decline as the disease progresses. In this review, we focus on the possible functional role exerted by MSCs in the treatment of some disabling neurodegenerative disorders such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease and Parkinson's Disease. Investigations have been mainly performed in vitro and in animal models by using MSCs generally originated from umbilical cord, bone marrow or adipose tissue. Positive results obtained have prompted several clinical trials, the number of which is progressively increasing worldwide. To date, many of them have been primarily addressed to verify the safety of the procedures but some improvements have already been reported, fortunately. Although the exact mechanisms of MSC‐induced beneficial activities are not entirely defined, they include neurogenesis and angiogenesis stimulation, antiapoptotic, immunomodulatory and anti‐inflammatory actions. Most effects would be exerted through their paracrine expression of neurotrophic factors and cytokines, mainly delivered at damaged regions, given the innate propensity of MSCs to home to injured sites. Hopefully, in the near future more efficacious cell‐replacement therapies will be developed to substantially restore disease‐disrupted brain circuitry. This article is protected by copyright. All rights reserved
       
  • Pulmonary Hypertension: Molecular Aspects of Current Therapeutic
           Intervention and Future Direction
    • Abstract: Pulmonary hypertension (PH) is a life‐threatening lung disorder with towering prevalence and risk for future has been gradually rising worldwide. Even, no specific medications are available for pulmonary hypertension; various classes of treatment based upon the origin and magnitude of hypertension are still used for the treatment of PH. Consideration of molecular or signaling modulation is the imperative approach that can offer a new notion for prevalent pharmacotherapeutic agents. Instead of concurrent targets, including endothelin receptor antagonists (ETA/ETB), phosphodiesterase 5 inhibitor (PDF‐5), calcium channel blockers, anticoagulants, diuretics and long acting prostacyclin analogue, recent scientific reports revealed the numerous potential alternative therapeutic approaches that can significantly target the pathological signaling alteration associated with PH. Understanding precise molecular cascade involved in PH can be useful for designing preclinical animal experiments and human clinical trials to evaluate target specific novel therapeutic interventions for the treatment of PH. In this review, we discussed the possible molecular signaling involved in the pathogenesis of PH and detailed account of the current status of medications employed for the treatment of PH. Moreover, the newly identified potential target sites and alternative approaches for treating the PH have been discussed. This article is protected by copyright. All rights reserved
       
  • Efficacy of different chemotherapy regimens in treatment of advanced or
           metastatic pancreatic cancer: a network meta‐analysis
    • Abstract: We performed a network meta‐analysis (NMA) to compare the short‐ and long‐term efficacy of Gemcitabine, Gemcitabine + S‐1 (tegafur), Gemcitabine + nab‐paclitaxel, Gemcitabine + Capecitabine, Gemcitabine + Cisplatin, FOLFIRINOX (oxaliplatin + irinotecan + fluorouracil + leucovorin), Gemcitabine + oxaliplatin, Gemcitabine + irinotecan, Gemcitabine + Exatecan, Gemcitabine + pemetrexed, Gemcitabine + 5‐FU and S‐1 in treating advanced or metastatic pancreatic cancer (PC). The odds radios (OR) or weighted mean difference (WMD) and surface under the cumulative ranking curves (SUCRA) were evaluated by a combination of direct evidence and indirect evidence. In total twenty studies were included in this paper. For short‐term efficacy, the overall response rate (ORR) was lower for patients treated with Gemcitabine compared with Gemcitabine + S‐1, Gemcitabine + Cisplatin, Gemcitabine + irinotecan and S‐1. The ORR for FOLFIRINOX was higher compared with Gemcitabine, Gemcitabine + Capecitabine and Gemcitabine + Cisplatin. The disease control rate (DCR) for Gemcitabine was lower compared with Gemcitabine + S‐1, Gemcitabine + Cisplatin and FOLFIRINOX. For long‐term efficacy, the 12‐month overall survival (OS) rate for FOLFIRINOX was higher compared with Gemcitabine, Gemcitabine + Capecitabine, Gemcitabine + Cisplatin, Gemcitabine + irinotecan, Gemcitabine + Exatecan and Gemcitabine + pemetrexed. The SUCRA revealed that FOLFIRINOX was relatively better in both short‐term and long‐term efficacy, while Gemcitabine was relatively poorer. In both short‐term and long‐term efficacy, FOLFIRINOX had the best short term and long term efficacy among the 12 chemotherapy regimens while efficacy of Gemcitabine was relatively poorer in the treatment of advanced or metastatic PC. This article is protected by copyright. All rights reserved
       
  • Key‐genes regulating the liposecretion process of mature adipocytes
    • Abstract: White mature adipocytes (MAs) are plastic cells able to reversibly transdifferentiate toward fibroblast‐like cells maintaining stem cell gene signatures. The main morphologic aspect of this transdifferentiation process, called liposecretion, is the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion.There is a considerable interest in the adipocyte plastic properties involving liposecretion process, but the molecular details are incompletely explored.This review analyzes the gene expression of MAs isolated from human subcutaneous fat tissue with respect to bone marrow (BM)‐derived mesenchymal stem cells (MSC) focusing on gene regulatory pathways involved into cellular morphology changes, cellular proliferation and transports of molecules through the membrane, suggesting potential ways to guide liposecretion.In particular, Wnt, MAPK/ERK and AKT pathways were accurately described, studying up‐ and down‐stream molecules involved. Moreover, adipogenic extra‐ and intra‐cellular interactions were analyzed studying the role of CDH2, CDH11, ITGA5, E‐Syt1, PAI‐1, IGF1 and INHBB genes. Additionally, PLIN1 and PLIN2 could be key‐genes of liposecretion process regulating molecules transport through the membrane.All together data demonstrated that liposecretion is regulated through a complex molecular networks that are able to respond to microenvironment signals, cytokines and growth factors. Autocrine as well as external signaling molecules might activate liposecretion affecting adipocytes physiology. This article is protected by copyright. All rights reserved
       
  • Curcumin regulates proliferation, autophagy and apoptosis in gastric
           cancer cells by affecting PI3K and P53 signaling
    • Abstract: In this study, we aimed to investigate the effects of curcumin on cell activities of gastric cancer (GC), and the connection between curcumin and P53 as well as PI3K signaling. This study was conducted with two cell lines SGC‐7901 and BGC‐823, both were exposed to curcumin at the concentrations of 0 µM, 10 µM, 20 µM and 40 µM. MTT assay, flow cytometry (FCM) assay, transmission electron microscopy (TEM) were used to study the underlying mechanisms of curcumin in respective of proliferation, apoptosis and autophagy. Western blot assay was also employed to detect impacts of curcumin on tophosphatidylinositol‐3 kinase (PI3K) and P53 signaling pathways‐related proteins. MTT assay displayed that curcumin inhibited GC cell proliferation. FCM results indicated that curcumin induced the apoptosis of GC cells. TEM revealed that curcumin induced autophagy in GC cells. Western blot results showed that curcumin activated P53 signaling pathway and inhibited PI3K signaling pathway. Curcumin may inhibit proliferation and induce the autophagy and apoptosis in GC cells. Additionally, curcumin activated the P53 signaling pathway by up‐regulating P53 and P21, which also inhibited PI3K pathway through down‐regulating PI3K, p‐Akt and p‐mTOR. This article is protected by copyright. All rights reserved
       
  • Anti‐osteoporosis activity of Sanguinarine in preosteoblast MC3T3‐E1
           cells and an ovariectomized rat model
    • Abstract: Sanguinarine, a benzophenanthridine alkaloid, has been previously demonstrated to exert antimicrobial, anti‐inflammatory and anti‐tumor activities. A previous study has identified Sanguinarine as a potential drug candidate for osteoporosis treatment by computational bioinformatics analysis. This study further evaluated the effects of Sanguinarine on the differentiation of murine preosteoblast MC3T3‐E1 cells and its anti‐osteoporosis activity in an ovarietomized rat model. Sanguinarine treatment (0.25, 0.5, 1 and 2 µM) of MC3T3‐E1 cells significantly increased alkaline phosphatase (ALP) activity and the phoshporalyation of AMP‐activated protein kinase α subunit (AMPKα), but did not affect cell proliferation. The induction effects of Sanguinarine treatment (2 µM) on ALP activity, AMPKα phosphorylation, Smad1 phosphorylation and the expression of three osteoblast differentiation‐regulators (bone morphogenetic protein 2 [BMP2], osterix [OSX] and osteoprotegerin [OPG]) were partially reversed by Compound C treatment. More importantly, Sanguinarine treatment promoted bone tissue growth in an ovariectomized (OVX) osteoporosis rat model as evaluated by histological examination, micro‐CT analysis and serum parameter detection. In conclusion, these results indicate that Sanguinarine induces the differentiation of MC3T3‐E1 cells through the activation of the AMPK/Smad1 signaling pathway. Sanguinarine can stimulate bone growth in vivo and may be an effective drug for osteoporosis treatment. This article is protected by copyright. All rights reserved
       
  • PACAP and PAC1R are differentially expressed in motor cortex of
           amyotrophic lateral sclerosis patients and support survival of
           iPSC‐derived motor neurons
    • Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal and disabling neurodegenerative disease characterized by upper and lower motor neurons depletion. In our previous work, comprehensive genomic profiling of 41 motor cortex samples enabled to discriminate controls from sporadic ALS patients, and segregated these latter into two distinct subgroups (SALS1 and SALS2), each associated with different deregulated genes. In the present study, we focused our attention on two of them, Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) and its type 1 receptor (PAC1R), and validated the results of the transcriptome experiments by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), immunohistochemistry and western blot analysis. To assess the functional role of PACAP and PAC1R in ALS, we developed an in vitro model of human induced Pluripotent Stem Cells (iPSC)‐derived motor neurons and examined the trophic effects of exogenous PACAP following neurodegenerative stimuli. Treatment with 100 nM PACAP was able to effectively rescue iPSC‐derived motor neurons from apoptosis, as shown by cell viability assay and protein dosage of the apoptotic marker (BAX). All together, these data suggest that perturbations in the PACAP‐PAC1R pathway may be involved in ALS pathology and represent a potential drug target to enhance motor neuron viability. This article is protected by copyright. All rights reserved
       
  • Regulating Osteogenesis and Adipogenesis in Adipose‐Derived Stem Cells
           by Controlling Underlying Substrate Stiffness
    • Abstract: Cells reside in a complex microenvironment (niche) in which the biochemical and biophysical properties of the extracellular matrix profoundly affect cell behavior. Extracellular stiffness, one important bio‐mechanical characteristic of the cell niche, is important in regulating cell proliferation, migration, and lineage specification. However, the mechanism by which mechanical signals guide osteogenic and adipogenic commitment of stem cells remains difficult to dissect. To explore this question, we generated a range of polydimethylsiloxane‐based matrices with differing degrees of stiffness that mimicked the stiffness seen in natural tissues and examined adipose stem cell morphology, spreading, vinculin expression and differentiation along the osteogenic and adipogenic pathways. Rigid matrices allowed broader cell spreading, faster growth rate and stronger expression of vinculin in adipose‐derived stem cells. In the presence of inductive culture media, stiffness‐dependent osteogenesis and adipogenesis of the adipose stem cells indicated that there was a combinatorial effect of biophysical and biochemical cues; no such lineage specification was observed in normal media. Osteogenic differentiation behavior showed a correlation with matrix rigidity, as well as with elevated expression of RhoA, ROCK‐1/‐2 and related proteins in the Wnt/β‐catenin pathway. The result provides a comprehensive understanding of how stem cells respond to the surrounding microenvironment and points to the fact that matrix stiffness is a critical element in biomaterial design and this will be an important advance in stem cell‐based tissue engineering. This article is protected by copyright. All rights reserved
       
  • Oroxyloside inhibits angiogenesis through suppressing internalization of
           VEGFR2/Flk‐1 in endothelial cells
    • Abstract: Increasing flavonoids have been reported to possess anti‐angiogenic effects. Inhibition of angiogenesis plays a critical role in the treatment of cancer, especially in advanced metastatic cancer. In this study, we assessed the effect of Oroxylin A‐7‐glucuronide (Oroxyloside), a main metabolite of Oroxylin A, on angiogenesis in human endothelial cell‐like EA.hy926 cells. Oroxyloside suppressed the migration and tube formation of EA.hy926 cells. Meanwhile, microvessels sprouting from aortic rings and new blood vessels on the chicken chorioallantoic membrane (CAM) were also inhibited. Mechanism studies showed that Oroxyloside reduced the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2/Flk‐1) while it up‐regulated the expression of R‐Ras and VE‐cadherin. In consequence, Oroxyloside inhibited the downstream Akt/MAPK/NF‐κB pathways and then decreased the nuclear translocation and DNA binding ability of NF‐κB. Furthermore, in‐vivo study showed that Oroxyloside exhibited a potential anti‐angiogenic effect in Matrigel plug assay and inhibited growth of xenografted tumors with low systemic toxicity, which could be ascribed to the inhibition of VEGFR2 internalization. Taken together, these results suggested that Oroxyloside could inhibit angiogenesis in vitro and in vivo via suppressing the internalization of VEGFR2 and might serve as a potential antitumor agent. This article is protected by copyright. All rights reserved
       
  • LOSS OF RUNX1 IS ASSOCIATED WITH AGGRESSIVE LUNG ADENOCARCINOMAS
    • Abstract: The mammalian runt‐related factor 1 (RUNX1) is a master transcription factor that regulates lineage specification of hematopoietic stem cells. RUNX1 translocations result in the development of myeloid leukemias. Recently, RUNX1 has been implicated as a tumor suppressor in other cancers. We postulated RUNX1 expression may be associated with lung adenocarcinoma etiology and/or progression. We evaluated the association of RUNX1 mRNA expression with overall survival data from The Cancer Genome Atlas (TCGA), a publically available database. Compared to high expression levels, Low RUNX1 levels from lung adenocarcinomas were associated with a worse overall survival (Hazard Ratio = 2.014 (1.042 to 3.730 95% confidence interval), log‐rank P = 0.035) compared to those that expressed high RUNX1 levels. Further immunohistochemical examination of 85 surgical specimens resected at the University of Vermont Medical Center identified that low RUNX1 protein expression was associated with larger tumors (P = 0.038). Gene expression network analysis was performed on the same subset of TCGA cases that demonstrated differential survival by RUNX1 expression. This analysis, which reveals regulatory relationships, showed that reduced RUNX1 levels were closely linked to upregulation of the transcription factor E2F1. To interrogate this relationship, RUNX1 was depleted in a lung cancer cell line that expresses high levels of RUNX1. Loss of RUNX1 resulted in enhanced proliferation, migration, and invasion. RUNX1 depletion also resulted in increased mRNA expression of E2F1 and multiple E2F1 target genes. Our data implicate loss of RUNX1 as driver of lung adenocarcinoma aggression, potentially through deregulation of the E2F1 pathway. This article is protected by copyright. All rights reserved
       
  • FAM20C regulates osteoblast behaviors and intracellular signaling pathways
           in a cell‐autonomous manner
    • Abstract: Recent studies indicate that Family with sequence similarity 20 member C (FAM20C) catalyzes the phosphorylation of secreted proteins, and participates in a variety of biological processes, including cell proliferation, migration, mineralization and phosphate homeostasis. To explore the local influences of FAM20C on osteoblast, Fam20c‐deficient osteoblasts were generated by treating the immortalized Fam20cf/f osteoblasts with CMV‐Cre‐IRES‐EGFP lentivirus. Compared with the normal Fam20cf/f osteoblasts, the expression of Bone sialoprotein (Bsp), Osteocalcin (Ocn), Fibroblast growth factor 23 (Fgf23) and transcription factors that promote osteoblast maturation were up‐regulated in the Fam20c‐deficient osteoblasts. In contrast, the expression of Dental matrix protein 1 (Dmp1), Dentin sialophosphoprotein (Dspp), Osteopontin (Opn), type I Collagen a 1 (Col1a1) and Alkine phosphatase (Alp) were down‐regulated in the Fam20c‐deficient cells. These alterations disclosed the primary regulation of Fam20c on gene expression. The Fam20c‐deficient osteoblasts showed a remarkable reduction in the ability of forming mineralized nodules. However, supplements of extracellular matrix proteins extracted from the normal bone failed to rescue the reduced mineralization, suggesting that FAM20C may affect the biomineralization by the means more than local phosphorylation of extracellular matrix proteins and systemic phosphorus homeostasis. Moreover, although Fam20c deficiency had little impact on cell proliferation, it significantly reduced cell migration and lowered the levels of p‐Smad1/5/8, p‐Erk and p‐p38, suggesting that the kinase activity of FAM20C might be essential to cell mobility and the activity of BMP ligands. In summary, these findings provide evidences that FAM20C may regulate osteoblast maturation, migration, mineralization and BMP signaling pathways in a cell‐autonomous manner. This article is protected by copyright. All rights reserved
       
  • Distinct mechanisms of regulation of the ITGA6 and ITGB4 genes by RUNX1 in
           myeloid cells
    • Abstract: Integrins are transmembrane adhesion receptors that play an important role in hematopoiesis by facilitating interactions between hematopoietic cells and extracellular matrix components of the bone marrow and hematopoietic tissues. These interactions are important in regulating the function, proliferation and differentiation of hematopoietic cells, as well as their homing and mobilization in the bone marrow. Not surprisingly altered expression and function of integrins plays a key role in the development and progression of cancer including leukemias. However, the regulation of integrin gene expression is not well characterized and the mechanisms by which integrin genes are disrupted in cancer remain unclear. Here we demonstrate for the first time that a key regulator of hematopoiesis, RUNX1, binds to and regulates the promoters of both the ITGA6 and ITGB4 genes in myeloid cells. The ITGA6 and ITGB4 integrin genes form the α6β4 integrin receptor. However our data indicates that RUNX1 functions differently at these two promoters. RUNX1 regulates ITGA6 through a consensus RUNX1 binding motif in its promoter. In contrast, although the ITGB4 promoter is also activated by RUNX1, it does so in the absence of a recognized consensus RUNX1 binding motif. Further, our data suggest that regulation of ITGB4 may involve interactions between the promoter and upstream regulatory elements. This article is protected by copyright. All rights reserved
       
  • FENOFIBRATE REVERSES CHANGES INDUCED BY HIGH‐FAT DIET ON METABOLISM IN
           MICE MUSCLE AND VISCERAL ADIPOCYTES
    • Abstract: The effect of fenofibrate on the metabolism of skeletal muscle and visceral white adipose tissue of diet‐induced obese (DIO) mice was investigated. C57BL/6J male mice were fed either a control or high‐fat diet for eight weeks. Fenofibrate (50 mg/Kg b.w., daily) was administered by oral gavage during the last two weeks of the experimental period. Insulin‐stimulated glucose metabolism in soleus muscles, glucose tolerance test, insulin tolerance test, indirect calorimetry, lipolysis of visceral white adipose tissue, expression of miR‐103‐3p in adipose tissue and miR‐1a, miR‐133a/b, miR‐206, let7b‐5p, miR‐23b‐3p, miR‐29‐3p, miR‐143‐3p in soleus muscle, genes related to glucose and fatty acid metabolism in adipose tissue and soleus muscle, and proteins (phospho‐AMPKα2, Pgc1α, Cpt1b), intramuscular lipid staining, and activities of fatty acid oxidation enzymes in skeletal muscle were investigated. In DIO mice, fenofibrate prevented weight gain induced by HFD feeding by increasing energy expenditure; improved whole body glucose homeostasis, and in skeletal muscle, increased insulin dependent glucose uptake, miR‐1a levels, reduced intramuscular lipid accumulation, and phospho‐AMPKα2 levels. In visceral adipose tissue of obese mice, fenofibrate decreased basal lipolysis rate and visceral adipocytes hypertrophy, and induced the expression of Glut‐4, Irs1 and Cav‐1 mRNA and miR‐103‐3p suggesting a higher insulin sensitivity of the adipocytes. The evidence is presented herein that beneficial effects of fenofibrate on body weight, glucose homeostasis and muscle metabolism might be related to its action in adipose tissue. Moreover, fenofibrate regulates miR‐1a‐3p in soleus and miR‐103‐3p in adipose tissue, suggesting these microRNAs might contribute to fenofibrate beneficial effects on metabolism. This article is protected by copyright. All rights reserved
       
  • Role of Viruses in Gastrointestinal cancer
    • Abstract: Gastrointestinal cancers are a global public health problem, which represent a vast majority of all cancer‐caused deaths in both men and women. On the other hand, viral pathogens have been long implicated as etiological factors in the onset of certain human cancers, including gastrointestinal tumors. In this regard, Human Papilloma Virus (HPV), Epstein ‐ Barr Virus (EBV) and John Cunningham Virus (JCV) have been more strongly suggested to be involved in gastrointestinal carcinogenesis; so that, the association of HPV with oropharyngeal and anal cancers and also the association of EBV with gastric cancer have been etiologically confirmed by epidemiological and experimental investigations. Although, the association of other viruses is less evident, but may rely on co‐factors for their oncogenic roles. Therefore, to improve the prevention and treatment of these classes of cancer, their association with viral agents as potential risk factors should be investigated with care. In this respect, the present review has focused on the existing literature on the subject of viral involvement in gastrointestinal tumorgenesis, by covering and discussing various gastrointestinal cancers, corresponding viral agents and their oncogenic aspects and then summarizing evidences either supporting or rejecting a causal role of these pathogens in gastrointestinal malignancies. This article is protected by copyright. All rights reserved
       
  • Bone morphogenetic protein‐7 inhibits endothelial‐mesenchymal
           transition in pulmonary artery endothelial cell under hypoxia
    • Abstract: Pulmonary artery hypertension (PAH) is characterized by structural changes in pulmonary arteries. Increased numbers of cells expressing α‐smooth muscle actin (α‐SMA) is a nearly universal finding in the remodeled artery. It has been confirmed endothelial‐to‐mesenchymal transition (EndoMT) may be a source of those α‐SMA–expressing cells. In addition, the EndoMT is reversible. Here, we show that under hypoxia, the expression of bone morphogenetic protein 7 (BMP‐7) was decreased both in vivo and in vitro. We also found that under normoxia, BMP‐7 deficiency induced spontaneous EndoMT and cell migration. The hypoxia‐induced EndoMT and cell migration were markedly attenuated after pretreatment with rh‐BMP‐7. Moreover, m‐TOR phosphorylation was involved in EndoMT and BMP‐7 suppressed hypoxia‐induced m‐TORC1 phosphorylation in pulmonary artery endothelial cells. Our results demonstrate that BMP‐7 attenuates the hypoxia‐induced EndoMT and cell migration by suppressing the m‐TORC1 signaling pathway. Our study revealed a novel mechanism underlying the hypoxia‐induced EndoMT in pulmonary artery endothelial cells and suggested a new therapeutic strategy targeting EndoMT for the treatment of pulmonary arterial hypertension. This article is protected by copyright. All rights reserved
       
  • Glucose stimulates intestinal epithelial crypt proliferation by modulating
           cellular energy metabolism
    • Abstract: The intestinal epithelium plays an essential role in nutrient absorption, hormone release and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make‐up of the tissue. The mechanism underlying the nutrient‐driven changes in proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study 1) the effect of glucose on crypt proliferation, and 2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real‐time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis, and the glycolytic pathway inhibitor 2‐Deoxy‐D‐glucose (2‐DG) attenuated glucose‐induced crypt proliferation. Glucose did not enhance glucose oxidation, but did increase the maximum mitochondrial respiratory capacity, which may contribute to glucose‐induced increases in proliferation. Glucose activated Akt/HIF‐1α signaling pathway, which might be at least in part responsible for glucose‐induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation. This article is protected by copyright. All rights reserved
       
  • Gap Junctional Intercellular Communication in Adipose‐derived
           Stromal/Stem Cells is Cell Density‐dependent and Positively Impacts
           Adipogenic Differentiation
    • Abstract: Adipose‐derived stromal/stem cells (ASCs) represent a widely used cell source with multi‐lineage differentiation capacity in approaches for tissue engineering and regenerative medicine. Despite the multitude of literature on their differentiation capacity, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction‐forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in human ASCs, and its significance for adipogenic differentiation of these cells.Cx43 expression in ASCs was demonstrated histologically and on the gene and protein expression level, and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of ASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α‐glycyrrhetinic acid (AGA) significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process.Altogether, this study demonstrates the impact of direct cell‐cell communication via gap junctions on the adipogenic differentiation process of ASCs, and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches. This article is protected by copyright. All rights reserved
       
  • Involvement of nutrients and nutritional mediators in mitochondrial
           3‐hydroxy‐3‐methylglutaryl‐CoA synthase gene expression
    • Abstract: Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase (HMGCS2) catalyses the first step of ketogenesis and is critical in various metabolic conditions. Several nutrient molecules were able to differentially modulate HMGCS2 expression levels. Docosahexaenoic acid (DHA, C22:6, n‐3), eicosapentaenoic acid (EPA, C20:5, n‐3), arachidonic acid (AA, C20:4, n‐6) and glucose increased HMGCS2 mRNA and protein levels in HepG2 hepatoma cells, while fructose decreased them. The effect of n‐6 AA resulted significantly higher than that of n‐3 PUFA, but when combined all these molecules were far less efficient. Insulin reduced HMGCS2 mRNA and protein levels in HepG2 cells, even when treated with PUFA and monosaccharides. Several nuclear receptors and transcription factors are involved in HMGCS2 expression regulation. While peroxysome proliferator activated receptor α (PPAR‐α) agonist WY14643 increased HMGCS2 expression, this treatment was unable to affect PUFA‐mediated regulation of HMGCS2 expression. Forkhead box O1 (FoxO1) inhibitor AS1842856 reduced HMGCS2 expression and suppressed induction promoted by fatty acids. Cells treatment with liver X receptor alpha (LXRα) agonist T0901317 reduced HMGCS2 mRNA, indicating a role for this transcription factor as suppressor of HMGCS2 gene. Previous observations already indicated HMGCS2 expression as possible nutrition status reference: our results show that several nutrients as well as specific nutritional related hormonal conditions are able to affect significantly HMGCS2 gene expression, indicating a relevant role for PUFA, which are mostly derived from nutritional intake. These insights into mechanisms of its regulation, specifically through nutrients commonly associated with disease risk, indicate HMGCS2 expression as possible reference marker of metabolic and nutritional status. This article is protected by copyright. All rights reserved
       
  • Blunting of estrogen modulation of cardiac cellular chymase/RAS activity
           and function in SHR
    • Abstract: The relatively low efficacy of ACE‐inhibitors in the treatment of heart failure in women after estrogen loss may be due to their inability to reach the intracellular sites at which angiotensin (Ang) II is generated and/or the existence of cell‐specific mechanisms in which ACE is not the essential processing pathway for Ang II formation. We compared the metabolic pathway for Ang II formation in freshly isolated myocytes (CMs) and non‐myocytes (NCMs) in cardiac membranes extracted from hearts of gonadal‐intact and ovariectomized (OVX) adult WKY and SHR rats. Plasma Ang II levels were higher in WKY vs. SHR (strain effect: WKY: 62 ± 6 pg/mL vs. SHR: 42 ± 9 pg/mL; P 
       
  • Comparison of genome‐wide analysis techniques to DNA methylation
           analysis in human cancer
    • Abstract: DNA methylation was the first epigenetic modification to be detected in human cancers with specific relation to aberrant gene expression. Herein, DNA methylation analysis explains how epigenetic patterns affect gene expression level. Hypermethylation at tumor suppressor gene loci leads to increased tumorigenesis due to tumor suppressor gene silencing, whereas global hypomethylation of CpG islands (CGIs) is followed by genomic instability and aberrant activation of multiple oncogenes. Therefore, characterization of the genes which silenced or activated epigenetically in human tumor cells can improve our understanding of cancer biology. Different genome‐wide methodologies are applied to evaluate methylation status. Various commonly conducted techniques for this evaluation are reviewed in this paper. We provided comparative description of the procedures, advantages, and drawbacks of genome‐wide DNA methylation analysis methods and biological applications, to give information on selecting the appropriate method for different methylation studies. This article is protected by copyright. All rights reserved
       
  • Gut microbiota‐derived endotoxin enhanced the incidence of cardia
           bifida during cardiogenesis
    • Abstract: BackgroundCytotoxicity and inflammation‐associated toxic responses could be induced by bacterial lipopolysaccharides (LPS) in vitro and in vivo respectively. However, the mechanism involved in LPS‐induced cardiac malformation in prenatal fetus is still unknown. Methods and resultsIn this study, we demonstrated that LPS was induced in gut microbiota imbalance mice, and next, LPS exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Gene transfection and tissue transplantation trajectory indicated that LPS exposure restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. In vitro explant allograft of GFP‐labeled anterior primitive streak demonstrated that LPS treatments could inhibit cell migration. A similar observation was also obtained from the cell migration assay of scratch wounds using primary culture of cardiomyocytes or H9c2 cells. In the embryos exposed to LPS, expressions of Nkx2.5 and GATA5 were disturbed. These genes are associated with cardiomyocyte differentiation when heart tube fusion occurs. Furthermore, pHIS3, C‐caspase3 immunohistological staining indicated that cell proliferation decreased, cell apoptosis increased in the heart tube of chick embryo. Meanwhile, in vivo, pHIS3 immunohistological staining and Hochest/PI staining also draw the similar conclusions. The LPS exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. At last, we showed that LPS‐induced cardia bifida could be partially rescued through the addition of antioxidants. ConclusionsTogether, these results reveal that excess ROS generation is involved in the LPS‐induced defects in heart tube during chick embryo development. This article is protected by copyright. All rights reserved
       
  • Systematic‐analysis of mRNA Expression Profiles in Skeletal Muscle of
           Patients with Type II Diabetes: the Glucocorticoid was Central in
           Pathogenesis
    • Abstract: Since the past thirty years, the prevalence of diabetes has more than doubled, making it an urgent challenge globally. We carried out systematic analysis with the public data of mRNA expression profiles in skeletal muscle to study the pathogenesis, since insulin resistance in the skeletal muscle is an early feature. We utilized three GEO datasets, containing total 60 cases and 63 normal samples. After the background removal, R package QC was utilized to finish the preprocessing of datasets. We obtained a dataset containing 2481 genes and 123 samples after the pre‐processing. Quantitative quality control measures were calculated to represent the quality of these datasets. MetaDE package provides functions for conducting different systematic analysis methods for differential expression analysis. The GO term enrichment was carried out using PANTHER. Protein‐protein interactions, drug‐gene interactions and genetic association of the identified differentially expressed genes were analyzed using STRING v10.0 online tool, DGIdb and the Genetic Association Database, respectively. The datasets had good performances on IQC and EQC, which suggested that the datasets had good internal and external quality. Totally 96 differentially expressed genes were detected using 0.01 as cutoff of AW. The enriched GO terms were mainly associated with the response to glucocorticoid. There were seven genes involving in the gluconeogenesis were differentially expressed, which might be the potential treatment target for this disease. The closely connected networks and potential targets of existed drugs suggested that some of the drugs might be applied to the treatment of diabetes as well. This article is protected by copyright. All rights reserved
       
  • Fibroblast growth factor 2 induces proliferation and distribution of G2/M
           phase of bovine endometrial cells involving activation of PI3K/AKT and
           MAPK cell signaling and prevention of effects of ER stress
    • Abstract: Fibroblast growth factor 2 (FGF2) is abundantly expressed in conceptuses and endometria during pregnancy in diverse animal models including domestic animals. However, its intracellular mechanism of action has not been reported for bovine endometrial cells. Therefore, the aim of this study was to identify functional roles of FGF2 in bovine endometrial (BEND) cell line which has served as a good model system for investigating regulation of signal transduction following treatment with interferon‐tau (IFNT) in vitro. Results of present study demonstrated that administration of FGF2 to BEND cells increased their proliferation and regulated the cell cycle through DNA replication by an increase of PCNA and Cyclin D1. FGF2 also increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, and P38 in BEND cells in a dose‐dependent manner, and expression of each of those transcription factors was inhibited by their respective pharmacological inhibitor including Wormannin, U0126 and SP600125. In addition, the increase in proliferation of BEND cells and activation of the protein kinases in response to FGF2 was suppressed by BGJ398, a FGFR inhibitor. Furthermore, proliferation of BEND cells was inhibited by tunicamycin, but treatment of BEND cells with FGF2 restored proliferation of BEND cells. Consistent with this result, the stimulated unfolded protein response (UPR) regulatory proteins induced by tunicamycin were down‐regulated by FGF2. Results of this study suggest that FGF2 promotes proliferation of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating cell signaling via the PI3K and MAPK pathways and by restoring ER stress through the FGFR. This article is protected by copyright. All rights reserved
       
  • Global analysis of gene expression profiles in the submandibular salivary
           gland of klotho knockout mice
    • Abstract: Salivary dysfunction commonly occurs in many older adults and is considered a physiological phenomenon. However, the genetic changes in salivary glands during aging have not been characterized. The present study analyzed the gene expression profile in salivary glands from accelerated aging klotho deficient mice (klotho ‐/‐, 4 weeks old). Microarray analysis showed that 195 genes were differentially expressed (z‐score > 2 in two independent arrays) in klotho null mice compared to wild‐type mice. Importantly, alpha2‐Na+/K+‐ATPase (Atp1a2), Ca2+‐ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to be regulators of submandibular salivary gland function, were significantly decreased. When a network was constructed from the differentially expressed genes, proliferator‐activated receptor‐γ (PPAR γ), which regulates energy homeostasis and insulin sensitivity, was located at the core of the network. In addition, the expression of genes proposed to regulate various PPAR γ‐related cellular pathways, such as Klk1b26, Egfbp2, Cox8b, Gpx3, Fabp3, EGF, and NGFβ, was altered in the submandibular salivary glands of klotho ‐/‐ mice. Our results may provide clues for the identification of novel genes involved in salivary gland dysfunction. Further characterization of these differentially expressed genes will be useful in elucidating the genetic basis of aging‐related changes in the submandibular salivary gland. This article is protected by copyright. All rights reserved
       
  • Overexpression of miR‐216b: prognostic and predictive value in acute
           myeloid leukemia
    • Abstract: Accumulating studies have shown that miR‐216b acted as a tumor suppressor and was down‐regulated in solid tumors. However, little studies revealed the role or clinical implication of miR‐216b in blood cancers. Herein, we reported miR‐216b expression and its clinical significance in patients with acute myeloid leukemia (AML). In the current study, we analyzed bone marrow (BM) miR‐216b expression in 115 de novo AML patients examined by real‐time quantitative PCR. Notably, BM miR‐216b expression was significantly up‐regulated in AML patients, and could serve as a potential biomarker distinguishing AML from controls. No significant correlations of BM miR‐216 expression were found with sex, age, white blood cells, hemoglobin, platelets, BM blasts, French‐American‐British classifications, and karyotypes. Significantly, patients with high miR‐216b expression tended to have a lower frequency of FLT3‐ITD mutation and higher incidence of U2AF1 and IDH1/2 mutations. Moreover, complete remission (CR) rate and overall survival were negatively affected by BM miR‐216b overexpression among cytogenetically normal AML (CN‐AML). Cox regression analyses showed that high BM miR‐216b expression may act as an independent risk factor in CN‐AML patients. Among the follow‐up patients, BM miR‐216b level in CR phase was markedly lower than in diagnosis time, and was returned in relapse phase. Collectively, our findings indicated that miR‐216b overexpression was a frequent event in de novo AML, and independently conferred a poor prognosis in CN‐AML. Moreover, miR‐216b expression was a valuable biomarker correlated with disease recurrence in AML. This article is protected by copyright. All rights reserved
       
  • RNA binding protein Musashi‐2 regulates PIWIL1 and TBX1 in mouse
           spermatogenesis
    • Abstract: RNA‐binding proteins (RBP) are important facilitators of post‐transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi‐2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalisation, identified differential expression and localisation of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis‐expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post‐transcriptional regulation during male gamete development. This article is protected by copyright. All rights reserved
       
  • Neutrophil extracellular traps in acrolein promoted hepatic ischemia
           reperfusion injury: therapeutic potential of NOX2 and p38MAPK inhibitors
    • Abstract: Neutrophil is a significant contributor to ischemia reperfusion (IR) induced liver tissue damage. However, the underlying mechanisms of IR induced innate immune activation and liver damage is not quite clear. Our study sheds light on the role of chronic oxidative stress end products in worsening the IR inflammatory process by neutrophil recruitment and activation following liver surgery. We employed specific inhibitors for molecular targets – NOX2 (NADPH oxidase 2) and P38 MAPK (Mitogen activated protein kinase) signal to counteract neutrophil activation and neutrophil extracellular trap (NET) release induced liver damage in IR injury. We found that acrolein initiated neutrophil chemotaxis and induced NET release both in vitro and in vivo. Acrolein exposure caused NET induced nuclear and mitochondrial damage in HepG2 cells as well as aggravated the IR injury in rat liver. Pretreatment with F‐apocynin and naringin, efficiently suppressed acrolein induced NET release in vitro. Notably, it suppressed the expression of inflammatory cytokines, P38MAPK‐ERK activation and apoptotic signals in rat liver exposed to acrolein and subjected to IR. Moreover, this combination effectively attenuated acrolein induced NET release and hepatic IR injury. Conclusion: In the current study we have shown that acrolein accumulation in liver due to chronic stress, is responsible for neutrophil recruitment and its activation leading to NET induced liver damage during surgery. Our study shows that therapeutic targeting of NOX2 and p38MAPK signaling in patients with chronic hepatic disorders would improve post operative hepatic function and survival. This article is protected by copyright. All rights reserved
       
  • The inhibitory effect in Fraxinellone on oxidative stress‐induced
           senescence correlates with AMP‐activated protein kinase‐dependent
           autophagy restoration
    • Abstract: As a natural metabolite of limonoids from Dictamnus dasycarpus, fraxinellone has been reported to be neuroprotective and anti‐inflammatory. However, its influence on cellular metabolism remains largely unknown. In the present study, we investigated the effect of fraxinellone on cellular senescence‐induced by oxidative stress and the potential mechanism. We found that fraxinellone administration caused growth arrest and certainly repressed the activity of senescence associated β‐galactosidase as well as the expression of senescence‐associated‐genes. Interestingly, this effect of fraxinellone is closely correlated with the restoration of impaired autophagy and the activation of AMPK. Notably, fraxinellone reacts in an AMPK‐dependent but mTORC1‐independent manner. Together, our study demonstrates for the first time that fraxinellone has the effect on senescence inhibition and AMPK activation, and supports the notion that autophagic mechanism is important for aging prevention. These findings expanded the list of natural compounds and will be potentially utilized for aging decay and/or AMPK activation. This article is protected by copyright. All rights reserved
       
  • Adult‐onset brain tumors and neurodegeneration: Are polyphenols
           protective'
    • Abstract: Aging is a primary risk factor for both neurodegenerative disorders (NDs) and tumors such as adult‐onset brain tumors. Since NDs and tumors are severe, disabling, progressive and often incurable conditions, they represent a pressing problem in terms of human suffering and economic costs to the healthcare systems. The current challenge for physicians and researchers is to develop new therapeutic strategies in both areas to improve the patients' quality of life. In addition to genetics and environmental stressors, the increase in cellular oxidative stress as one of the potential common etiologies has been reported for both disorders. Recently, the scientific community has focused on the beneficial effects of dietary antioxidant classes, known as nutraceuticals, such as carotenoids, vitamins and polyphenols. Among these compounds, polyphenols are considered to be one of the most bioactive agents in neurodegeneration and tumor prevention. Despite the beneficial activity of polyphenols, their poor bioavailability and inefficient delivery systems are the main factors limiting their use in medicine and functional food. The development of polymeric nanoparticle‐based delivery systems able to encapsulate and preserve polyphenolic compounds may represent a promising tool to enhance their stability, solubility and cell membrane permeation.In the present review we provide an overview of the main polyphenolic compounds used for ND and brain tumor prevention and treatment that explores their mechanisms of action, recent clinical findings and principal factors limiting their application in medicine. This article is protected by copyright. All rights reserved
       
  • Loss of Hdac3 in osteoprogenitors increases bone expression of
           osteoprotegerin, improving systemic insulin sensitivity
    • Abstract: Type 2 diabetes is an emerging global health epidemic. Foundations for new therapies are arising from understanding interactions between body systems. Bone-derived factors that reduce RANKL (receptor activator of NF-kappa B ligand) signaling in the liver may prevent insulin resistance and the onset of type 2 diabetes. Here we demonstrate that deletion of the epigenetic regulator, Hdac3, in Osx1-expressing osteoprogenitors prevents insulin resistance induced by high fat diet by increasing serum and skeletal gene expression levels of osteoprotegerin (Opg), a natural inhibitor of RANKL signaling. Removal of one Opg allele in mice lacking Hdac3 in Osx1+ osteoprogenitors increases the insulin resistance of the Hdac3-deficient mice on a high fat diet. Thus, Hdac3-depletion in osteoblasts increases expression of Opg, subsequently preserving insulin sensitivity. The Hdac inhibitor vorinostat also increased Opg transcription and histone acetylation of the Opg locus. These results define a new mechanism by which bone regulates systemic insulin sensitivity. This article is protected by copyright. All rights reserved
       
  • CCL18-dependent translocation of AMAP1 is critical for epithelial to
           mesenchymal transition in breast cancer
    • Abstract: AMAP1 was a GTPase-activating protein that regulates cytoskeletal structures in focal adhesions, circular dorsal ruffles, and promote cell differentiation in tumor cells. But the activation and function of AMAP1 in breast cancer remain largely unexplored. Here we show that AMAP1 was phosphorylated and translocated to plasma membrane and formed a stable complex with Pyk2 in response to CCL18. Moreover, CCL18-dependent AMAP1 translocation interfered the AMAP1-IKK-β interaction, resulting in nuclear factor-kappaB (NF-Κb)activation. Depletion of AMAP1 expression by RNAi efficiently reversed the CCL18-induced epithelial to mesenchymal transition (EMT) of breast cancer cells and as well as CCL18-induced adhesion, migration and invasion. Strikingly, AMAP1 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognosis in breast cancers. Given that AMAP1 mediated CCL18-induce activation of NF-κB and promoted breast cancer metastasis, AMAP1 may represent a therapeutic target for the eradication of breast cancer metastasis. This article is protected by copyright. All rights reserved
       
  • Nestin expression is dynamically regulated in cardiomyocytes during
           embryogenesis
    • Abstract: The transcriptional factors implicated in the expression of the intermediate filament protein nestin in cardiomyocytes during embryogenesis remain undefined. In the heart of 9,5-10,5 day embryonic mice, nestin staining was detected in atrial and ventricular cardiomyocytes and a subpopulation co-expressed Tbx5. At later stages of development, nestin immunoreactivity in cardiomyocytes gradually diminished and was absent in the heart of 17,5 day embryonic mice. In the heart of wild type 11,5 day embryonic mice, 54 ± 7% of the trabeculae expressed nestin and the percentage was significantly increased in the hearts of Tbx5+/− and Gata4+/− embryos. The cell cycle protein Ki67 and transcriptional coactivator Yap-1 were still prevalent in the nucleus of nestin(+)-cardiomyocytes identified in the heart of Tbx5+/− and Gata4+/− embryonic mice. Phorbol 12,13-dibutyrate treatment of neonatal rat ventricular cardiomyocytes increased Yap-1 phosphorylation and co-administration of the p38 MAPK inhibitor SB203580 led to significant dephosphorylation. Antagonism of dephosphorylated Yap-1 signalling with verteporfin inhibited phorbol 12,13-dibutyrate/SB203580-mediated nestin expression and BrdU incorporation of neonatal cardiomyocytes. Nestin depletion with an AAV9 containing a shRNA directed against the intermediate filament protein significantly reduced the number of neonatal cardiomyocytes that re-entered the cell cycle. These findings demonstrate that Tbx5- and Gata4-dependent events negatively regulate nestin expression in cardiomyocytes during embryogenesis. By contrast, dephosphorylated Yap-1 acting via upregulation of the intermediate filament protein nestin plays a seminal role in the cell cycle re-entry of cardiomyocytes. Based on these data, an analogous role of Yap-1 may be prevalent in the heart of Tbx5+/− and Gata4+/− mice. This article is protected by copyright. All rights reserved
       
  • Claudin7b is required for the formation and function of inner ear in
           zebrafish
    • Abstract: Zebrafish has become an excellent model for studying the development and function of inner ear. We report here a zebrafish line in which claudin 7b (cldn7b) locus is interrupted by a Tol2 transposon at its first intron. The homozygous mutants have enlarged otocysts, smaller or no otoliths, slowly formed semicircular canals, and insensitiveness to sound stimulation. These abnormal phenotypes and hearing loss of inner ear could be mostly rescued by injection of cldn7b-mRNA into one-cell stage homozygous mutant embryos. Mechanistically, cldn7b-deficiency interrupted the formation of apical junction complexes (AJCs) in otic epithelial cells of inner ear and the ion-homeostasis of endolymph, which then led to the loss of proper contact between otoliths and normally developed hair cells in utricle and saccule or aberrant mechanosensory transduction. Thus, Cldn7b is essential for the formation and proper function of inner ear through its unique role in keeping an initial integrity of otic epithelia during zebrafish embryogenesis. This article is protected by copyright. All rights reserved
       
  • Osteocalcin-dependent regulation of glucose metabolism and fertility:
           Skeletal implications for the development of insulin resistance
    • Abstract: The skeleton has recently emerged as a critical insulin target tissue that regulates whole body glucose metabolism and male reproductive function. While our understanding of these new regulatory axes remains in its infancy, the bone-specific protein, osteocalcin, has been shown to be centrally involved. Undercarboxylated osteocalcin acts as a secretagogue in a feed-forward loop to stimulate pancreatic β-cell proliferation and insulin secretion, improve insulin sensitivity and promote testosterone production. Importantly, dysregulation of insulin signaling in bone causes a reduction in serum osteocalcin levels that is associated with elevated blood glucose and reduced serum insulin levels, suggesting that the skeleton may play a significant role in the development of diet-induced insulin resistance. Insulin signaling is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) which becomes hyper-activated in response to nutrient overload. Loss- and gain-of function models suggest that mTORC1 function in bone is essential for normal skeletal development; however, the role of this complex in the regulation of glucose metabolism remains to be determined. This review highlights our current understanding of the role played by osteocalcin in the skeletal regulation of glucose metabolism and fertility. In particular, it examines data emerging from transgenic mouse models which have revealed a pancreas-bone-testis regulatory axis and discusses recent human studies which seek to corroborate findings from mouse models with clinical observations. Moreover, we review recent studies which suggest dysregulation of insulin signaling in bone leads to the development of insulin resistance and discuss the potential role of mTORC1 signaling in this process This article is protected by copyright. All rights reserved
       
  • Butyrate stimulates the growth of human intestinal smooth muscle cells by
           activation of Yes-Associated Protein
    • Abstract: Intestinal smooth muscle cells play a critical role in the remodeling of intestinal structure and functional adaptation after bowel resection. Recent studies have shown that supplementation of butyrate (Bu) contributes to the compensatory expansion of a muscular layer of the residual intestine in a rodent model of short-bowel syndrome (SBS). However, the underlying mechanism remains elusive. In this study, we found that the growth of human intestinal smooth muscle cells (HISMCs) was significantly stimulated by Bu via activation of Yes-Associated Protein (YAP). Incubation with 0.5 mM Bu induced a distinct proliferative effect on HISMCs, as indicated by the promotion of cell cycle progression and increased DNA replication. Notably, YAP silencing by RNA interference or its specific inhibitor significantly abolished the proliferative effect of Bu on HISMCs. Furthermore, Bu induced YAP expression and enhanced the translocation of YAP from the cytoplasm to the nucleus, which led to changes in the expression of mitogenesis genes, including TEAD1, TEAD4, CTGF and Cyr61. These results provide evidence that Bu stimulates the growth of human intestinal muscle cells by activation of YAP, which may be a potential treatment for improving intestinal adaptation. This article is protected by copyright. All rights reserved
       
  • Mammalian Target of Rapamycin As a Therapeutic Target in Osteoporosis
    • Abstract: The mechanistic target of rapamycin (mTOR) plays a key role in sensing and integrating large amounts of environmental cues to regulate organismal growth, homeostasis, and many major cellular processes. Recently, mounting evidences highlight its roles in regulating bone homeostasis, which sheds light on the pathogenesis of osteoporosis. The activation/inhibition of mTOR signaling is reported to positively/negatively regulate bone marrow mesenchymal stem cells (BMSCs)/osteoblasts-mediated bone formation, adipogenic differentiation, osteocytes homeostasis, and osteoclasts-mediated bone resorption, which result in the changes of bone homeostasis, thereby resulting in or protect against osteoporosis. Given the likely importance of mTOR signaling in the pathogenesis of osteoporosis, here we discuss the detailed mechanisms in mTOR machinery and its association with osteoporosis therapy. This article is protected by copyright. All rights reserved
       
  • Triptolide prevents extracellular matrix accumulation in experimental
           diabetic kidney disease by targeting microRNA-137/Notch1 pathway
    • Abstract: MicroRNAs (miRNAs) are involved in multiple biological functions via suppressing target genes. Triptolide is a monomeric compound isolated from a traditional Chinese herb, which exerts protective roles in many kinds of glomerular diseases. However, our understanding of the triptolide effect on miRNAome is still limited. In this study, we found that triptolide significantly decreased albuminuria and improved glomerulosclerosis in rats with diabetic kidney disease (DKD). And triptolide also inhibited extracellular matrix (ECM) protein accumulation and the notch1 pathway activation under diabetic conditions. MiR-137 was significantly decreased in the HG (high glucose)-treated HRMCs and in the kidney tissues of the diabetic rats, but was upregulated by triptolide. In addition, overexpression of miR-137 exerted similar effects to those of triptolide, while miR-137 inhibition aggravated ECM protein accumulation. Luciferase reporter assay results demonstrated that miR-137 directly targets Notch1. Furthermore, the miR-137-dependent effects were due to Notch1 suppression that in turn inhibited ECM protein expression, key mediators of glomerulosclerosis. Finally, downregulation of miR-137 reversed the ECM inhibition role of triptolide in HG cultured HRMCs. Taken together, these findings indicate that triptolide is a potential therapeutic option for DKD and that miR-137/Notch1 pathway play roles in the anti-glomerulosclerosis mechanism of triptolide. This article is protected by copyright. All rights reserved
       
  • Yes-associated protein 1 promotes the differentiation and mineralization
           of cementoblast
    • Abstract: Yes-associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1-knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad-dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP-stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad-dependent BMP and Erk1/2 signaling pathways. This article is protected by copyright. All rights reserved
       
  • Targeting P-glycoprotein and SORCIN: Dihydromyricetin strengthens
           anti-proliferative efficiency of adriamycin via MAPK/ERK and Ca2+-mediated
           apoptosis pathways in MCF-7/ADR and K562/ADR
    • Abstract: Recently, a new target Ca2+-binding protein SORCIN was reported to participate in multidrug resistance (MDR) in cancer. Here we aim to investigate whether dihydromyricetin (DMY), a dihydroflavonol compound with anti-inflamatory, anti-oxidant, anti-bacterial and anti-tumor actions, reverses MDR in MCF-7/ADR and K562/ADR and to elucidate its potential molecular mechanism. DMY enhanced cytotoxicity of adriamycin (ADR) by downregulating MDR1 mRNA and P-gp expression through MAPK/ERK pathway and also inhibiting the function of P-gp significantly. Meanwhile, DMY decreased mRNA and protein expression of SORCIN, which resulted in elevating intracellular free Ca2+. Finally, we investigated co-administration ADR with DMY remarkably increased ADR-induced apoptosis. Further study showed DMY elevated ROS levels and caspase-12 protein expression, which signal apoptosis in endoplasmic reticulum. At the same time, proteins related to mitochondrial apoptosis were also changed such as Bcl-2, Bax, caspase-3, caspase-9 and PARP. Finally, nude mice model also demonstrated that DMY strengthened anti-tumor activity of ADR in vivo. In conclusion, DMY reverses MDR by downregulating P-gp, SORCIN expression and increasing free Ca2+, as well as, inducing apoptosis in MCF-7/ADR and K562/ADR. These fundamental findings provide evidence for further clinical research in application of DMY as an assistant agent in the treatment of cancer. This article is protected by copyright. All rights reserved
       
  • LncRNA-PVT1 promotes pancreatic cancer cells proliferation and migration
           through acting as a molecular sponge to regulate miR-448
    • Abstract: The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological process has currently developed rapidly. LncRNA-PVT1, located adjacent to the MYC locus on chromosomal region 8q24, has been reported to be associated with many biological processes. However, the function and mechanism of PVT1 in pancreatic carcinoma (PC) is poorly understood. In this present study, we first measured the level of PVT1 in the PC cell lines and tissues by quantitative real-time PCR (qRT-PCR), and then employed loss-of-function and gain-of-function approaches to explore the association between PVT1 expression levels and PC cell proliferation/migration ability. Furthermore, bioinformatics analysis was utilized to show that PVT1 contains binding site for miR-448 and an inverse correlation between PVT1 and miR-448 was obtained in PC specimens. Additionally, dual luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) and applied biotin-avidin pulldown system were applied to further confirm that PVT1 directly bind with microRNA binding site harboring in the PVT1 sequence. Then, SERBP1 was identified as a target of miR-448 according to the gene expression array analysis of PC clinical samples. Together, we revealed that PVT1 functions as an endogenous ‘sponge’ by competing for miR-448 binding to regulate the miRNA target SERBP1 and therefore promotes the proliferation and migration of PC cells. This article is protected by copyright. All rights reserved
       
  • Relationship of Human Herpes Virus 6 and Multiple Sclerosis: A Systematic
           Review and Meta-analysis
    • Abstract: BackgroundInfection with human herpes viruses has been suggested to contribute to multiple sclerosis (MS), while interaction between human herpes 6 (HHV6) and MS remain unclear yet. Here, we conducted a meta-analysis on the relationship of HHV6 infection and MS.Materials and MethodsAll related studies were collected from major databases. The analyses were performed by STATA 14 and Comprehensive Meta-Analysis V2.0 softwares. Pooled odds ratios (ORs) and 95% CIs were calculated from the raw data of the including studies by the random effects models when I2 >50% and fix model when I2 6 score that used serum/blood sample with OR of [6.7 (95% CI 4.8-8.6), P 
       
  • Adenosine: an endogenous mediator in the pathogenesis of gynecological
           cancer
    • Abstract: Extracellular concentration of adenosine increases in the hypoxic tumor microenvironment. Adenosine signaling regulates apoptosis, angiogenesis, metastasis and immune suppression in cancer cells. Adenosine-induced cell responses depend upon different subtypes of adenosine receptors activation and type of cancer. Suppression of adenosine signaling via inhibition of adenosine receptors or adenosine generating enzymes including CD39 and CD73 on ovarian or cervical cancer cells is a potentially novel therapeutic approach for gynecological cancer patients. This review summarizes the role of adenosine in the pathogenesis of gynecological cancer for a better understanding and hence a better management of this disease. This article is protected by copyright. All rights reserved
       
  • Conditionally Targeted Deletion of PSEN1 Leads to Diastolic Heart
           Dysfunction
    • Abstract: Recently, PSEN1 has been reported to have mutations in dilated cardiomyopathy pedigrees. However, the function and mechanism of PSEN1 in cardiomyopathy remains unresolved. Here, we established 4 types of genetically modified mice to determine the function of PSEN1 in cardiac development and pathology. PSEN1 null mutation resulted in perinatal death, retardation of heart growth, ventricular dilatation, septum defects, and valvular thickening. PSEN1 knockout in adults led to decreased muscle fibers, widened sarcomere Z lines and reduced lengths of sarcomeres in cardiomyocytes. Cardiovascular loss of function of PSEN1 induced by Sm22a-Cre or Myh6-Cre/ER/tamoxifen also resulted in severe ultrastructural abnormalities, such as relaxed gap junctions between neighboring cardiomyocytes. Functionally, cardiovascular deletion of PSEN1 caused spontaneous mortality from birth to adulthood and led to diastolic heart dysfunction, including decreased volume of the left ventricle at the end-systolic and end-diastolic stages. Additionally, in a myocardial ischemia model, deletion of PSEN1 in the cardiovascular system first protected mice by inducing adaptive hypertrophy but ultimately resulted in severe heart failure. Furthermore, a collection of genes was abnormally expressed in the hearts of cardiac-specific PSEN1 knockout mice. They were enriched in cell proliferation, calcium regulation, and so on. Taken together, dynamic regulation and abnormal function of PSEN1 underlie the pathogenesis of cardiovascular diseases due to ultrastructural abnormality of cardiomyocytes. This article is protected by copyright. All rights reserved
       
  • Co-culture systems-based strategies for articular cartilage tissue
           engineering
    • Abstract: Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. This article is protected by copyright. All rights reserved
       
  • Genetic susceptibility in cervical cancer: from bench to bedside
    • Abstract: Cervical cancer (CC) is the third most common malignancy in women globally, and persistent infection with the oncogenic human papillomaviruses (HPV) is recognized as the major risk factor. The pathogenesis of CC relies on the interplay between the tumorigenic properties of the HPV and host factors. Host-related genetic factors, including the presence of susceptibility loci for cervix tumor is substantial importance. Preclinical and genome-wide association studies (GWAS) have reported the associations of genetic variations in several susceptibility loci for the development of cervical cancer. However, many of these reports are inconsistent. In this review, we discuss the findings to date of candidate gene association studies, and GWAS in cervical cancer. The associations between these genetic variations with response to chemotherapy are also discussed. This article is protected by copyright. All rights reserved
       
  • Nap Counteracts Hyperglycemia/Hypoxia Induced Retinal Pigment Epithelial
           Barrier Breakdown Through Modulation Of HIFs and VEGF Expression
    • Abstract: Diabetic macular edema (DME) is a common complication leading to a central vision loss in patients with diabetes. In this eye pathology, the hyperglycaemic/hypoxic microenvironment of pigmented epithelium is responsible for outer blood retinal barrier integrity changes.More recently, we have shown that a small peptide derived from the activity-dependent neuroprotective protein (ADNP), known as NAP, counteracts damages occurring during progression of diabetic retinopathy by modulating HIFs/VEGF pathway. Here, we have investigated for the first time the role of this peptide on outer blood retinal barrier (BRB) integrity exposed to hyperglycaemic/hypoxic insult mimicking a model in vitro of DME. To characterize NAP role on disease's pathogenesis, we have analysed its effect on HIFs /VEGF system in human retinal pigmented epithelial cells, ARPE-19, grown in high glucose and low oxygen tension. The results have shown that NAP prevents outer BRB breakdown by reducing HIF1α /HIF2α, VEGF/VEGFRs and increasing HIF3 α expression, moreover it is able to reduce the percentage of apoptotic cells by modulating the expression of two death related genes, BAX and Bcl2. Further investigations are needed to determine the possible use of NAP in DME treatment. This article is protected by copyright. All rights reserved
       
  • Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for
           Cardiac Tissue Engineering
    • Abstract: Electrospinning is a well-established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non-toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200-800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days.Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved
       
  • Protein Kinase C Is Involved in the Induction of ATP-Binding Cassette
           Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor Agonist
           in Human Macrophages
    • Abstract: The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved
       
  • Genetic variation in the DNA repair pathway as a potential determinant of
           response to platinum-based chemotherapy in breast cancer
    • Abstract: Platinum-based chemotherapy is often used as a first-line treatment for patient with breast cancer. Platinum agents bind to DNA, forming adducts that contain intra and inter-strand crosslinks. It is possible that genetic variations of the DNA repair pathways may affect the activity, or efficacy of platinum, and hence resistance to platinum chemotherapy may be related to these genetic variants. We have summarized the known variants in the DNA repair pathway that have been reported to predict the response to platinum-based therapy in breast cancer.
       
  • Endogenous bioelectric currents promote differentiation of the mammalian
           lens
    • Abstract: The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and β1 subunits of Na+/K+-ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem. Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of β-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.Mature, denucleated lens fibers expressed high levels of the α1 and β1 subunits of Na+/K+-ATPase In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1. An applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. The applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2.
       
  • 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
           commitment
    • 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
           osteoclastogenesis
    • 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
       
  • DHQZ-17, A POTENT INHIBITOR OF THE TRANSCRIPTION FACTOR HNF4A, SUPPRESSES
           TUMORIGENICITY OF HEAD & NECK SQUAMOUS CELL CARCINOMA IN VIVO
    • 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
       
  • 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
       
  • PHOSPHATIDYLINOSITOL 3-KINASE INHIBITION POTENTIATES GLUCOCORTICOID
           RESPONSE IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
    • 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
       
  • Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling
           pathway
    • 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
       
  • 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
       
  • 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
       
  • 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
       
  • 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
       
  • 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.
       
  • 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
       
  • 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
       
  • 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
       
  • 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
       
 
 
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