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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [7 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1583 journals]
  • Hematopoietic Stem/Progenitor Cell Differentiation towards Myeloid Lineage
           is Modulated by LIGHT/LIGHT Receptor Signaling
    • Authors: Weikai Chen; Xin Lv, Changlong Liu, Ruoping Chen, Jianhe Liu, Haiyan Dai, Gang-Ming Zou
      Abstract: The cytokine LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a member of the tumor necrosis factor (TNF) superfamily. It is expressed primarily on activated T lymphocytes, and detectable on monocytes, granulocytes, and immune dendritic cells. It mainly plays a role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role as an inducer in embryonic stem cell differentiation, but its role in regulation of adult stem cell has not been defined. In the present study, we examined the expression of LIGHT receptor in Lin-c-kit+Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte-monocyte colony stimulating factor (GM-CSF). Further studies showed that LIGHT enhances both GM-CSF and GM-CSF receptor (GM-CSFR) expression in HSCs. LIGHT stimulation increases PU.1 expression in HSC/HPCs. In vivo administration of LIGHT increases the colony-forming unit-granulocyte/monocyte (CFU-GM) colony formation and plasma GM-CSF level. Altogether, the data suggest LIGHT promote myeloid differentiation of HSC/HPCs. This article is protected by copyright. All rights reserved
      PubDate: 2017-04-19T05:57:38.439815-05:
      DOI: 10.1002/jcp.25967
  • Isorhapontigenin induced cell growth inhibition and apoptosis by targeting
           EGFR-related pathways in prostate cancer
    • Authors: Cuicui Zhu; Qingyi Zhu, Zhaomeng Wu, Yingying Yin, Dan Kang, Shan Lu, Ping Liu
      Abstract: Isorhapontigenin (ISO), a naturally phytopolyphenol compound existing in Chinese herb, apples and various vegetables, has attracted extensive interest in recent years for its diverse pharmacological characteristics. Increasing evidences reveal that ISO can inhibit cancer cell growth by induced apoptosis, however the molecular mechanisms is not fully understood. In this study, we found for the first time that ISO apparently induced cell growth inhibition and apoptosis by targeting EGFR and its downstream signal pathways in prostate cancer (PCa) cells both in vitro and in vivo, whereas no obviously effect on normal prostate cells. From the results, we found that ISO competitively targeted EGFR with EGF and inhibited EGFR auto-phosphorylation, and then decreased the levels of p-Erk1/2, p-PI3K and p-AKT, and further induced down-regulation of p-FOXO1 and promoted FOXO1 nuclear translocation; and finally resulted in a significantly up-regulation of Bim/p21/27/Bax/cleaved Caspase-3/cleaved PARP-1 and a markedly down-regulation of Sp1/Bcl-2/XIAP/Cyclin D1. Moreover, our experimental data demonstrated that treatment of ISO decreased protein level of AR via both inhibiting the expression of AR gene and promoting the ubiquitination/degradation of AR proteins in proteasome. In vivo, we also found that ISO inhibited the growth of subcutaneous xenotransplanted tumor in nude mice by inducing PCa cell growth inhibition and apoptosis. Taken together, all findings here clearly implicated that EGFR-related signal pathways, including EGFR-PI3K-Akt and EGFR-Erk1/2 pathways, were involved in ISO-induced cell growth inhibition and apoptosis in PCa cells, providing a more solid theoretical basis for the application of ISO to treat patients with prostate cancer in clinic. This article is protected by copyright. All rights reserved
      PubDate: 2017-04-19T05:54:27.290005-05:
      DOI: 10.1002/jcp.25968
  • Statin Regulated ERK5 Stimulates Tight Junction Formation and Reduces
           Permeability in Human Cardiac Endothelial Cells
    • Abstract: The MEKK3/MEK5/ERK5 signalling axis is required for cardiovascular development in vivo. We analysed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilised human microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localisation with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provides the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity. This article is protected by copyright. All rights reserved
  • ESRRB plays a crucial role in the promotion of porcine cell reprogramming
    • Abstract: The estrogen-related receptor b (ESRRB) is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency. In mouse ES cells, overexpression of ESRRB can maintain LIF-independent self-renewal in the absence of Nanog. However, the fundamental features of porcine ESRRB remain elusive. In this study, we revealed the expression profiles of ESRRB in both porcine pluripotent stem cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor that enhanced porcine pluripotent gene activation. Addition of ESRRB into the cocktail of core pluripotent factors Oct4, Sox2, Klf4, and c-Myc (OSKM + E) could significantly enhance the reprogramming efficiency and the formation of alkaline phosphatase positive colonies. Conversely, knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes, minimized the alkaline phosphatase activity, and initiated the porcine induced pluripotent stem cell differentiation. Therefore, porcine ESRRB is a crucial transcription factor to improve the self-renewal of piPSCs. This article is protected by copyright. All rights reserved
  • 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 
  • High fat diet attenuates hyperglycemia, body composition changes, and bone
           loss in male streptozotocin-induced type 1 diabetic mice
    • Abstract: There is a growing and alarming prevalence of obesity and the metabolic syndrome in type I diabetic patients (T1DM), particularly in adolescence. In general, low bone mass, higher fracture risk and increased marrow adipose tissue (MAT) are features of diabetic osteopathy in insulin deficient subjects. On the other hand, type 2 diabetes (T2DM) is associated with normal or high bone mass, a greater risk of peripheral fractures and no change in MAT. Therefore, we sought to determine the effect of weight gain on bone turnover in insulin deficient mice. We evaluated the impact of a 6-week high-fat (HFD) rich in medium chain fatty acids or low-fat diet (LFD) on bone mass and MAT in a streptozotocin (STZ)-induced model using male C57BL/6J mice at 8 weeks of age. Dietary intervention was initiated after diabetes confirmation. At the endpoint, lower non-fasting glucose levels were observed in diabetic mice fed with high fat diet compared to diabetic mice fed the low fat diet (STZ-LFD). Compared to euglycemic controls, the STZ-LFD had marked polydipsia and polyphagia, as well as reduced lean mass, fat mass and bone parameters. Interestingly, STZ-HFD mice had higher bone mass, namely less cortical bone loss and more trabecular bone than STZ-LFD. Thus, we found that a HFD, rich in medium chain fatty acids, protects against bone loss in a T1DM mouse model. Whether this may also translate to T1DM patients who are overweight or obese in respect to maintenance of bone mass remains to be determined through longitudinal studies. This article is protected by copyright. All rights reserved
  • MicroRNA-mediated drug resistance in ovarian cancer
    • Abstract: The development of intrinsic or acquired resistance to chemotherapeutic agents used in the treatment of various human cancers is a major obstacle for the successful abolishment of cancer. The accumulated efforts in the understanding the exact mechanisms of development of multidrug resistance (MDR) have led to the introduction of several unique and common mechanisms. Recent studies demonstrate the regulatory role of small noncoding RNA or miRNA in the several parts of cancer biology. Practically all aspects of cell physiology under normal and disease conditions are reported to be controlled by miRNAs. In this review, we discuss how the miRNA profile is changed upon MDR development and the pivotal regulatory role played by miRNAs in overcoming resistance to chemotherapeutic agents. It is hoped that further studies will support the use of these differentially expressed miRNAs as prognostic and predictive markers, as well as novel therapeutic targets to overcome resistance in ovarian cancer. This article is protected by copyright. All rights reserved
  • Troxerutin with copper generates oxidative stress in cancer cells: its
           possible chemotherapeutic mechanism against hepatocellular carcinoma
    • Abstract: Troxerutin (TXER) a rutin derivative is known for its anticancer effect against hepatocellular carcinoma (HCC). As part of large study, recently we have shown TXER interact with genetic material and its anti-mutagenic property. In the present study we have explored its possible mode of action in HCC. Since TXER alone did not show significant anticancer effect on Huh-7 cells, in vitro biochemical assays were performed for determining anticancer efficacy of TXER + metal complex using transition metals such as Cu, Zn & Fe. The anticancer efficacy of TXER + Cu on Huh-7 cells were evaluated using MTT assay, DCFDA, JC-1 staining, comet assay, cell cycle analysis, immunocytochemistry and western blotting. Non-toxic nature of TXER was analysed on primary rat hepatocytes. The in vivo efficacy of TXER was tested in N-nitrosodiethylamine initiated and γ-benzene hexachloride & partial hepatectomy promoted rat liver cancer. Liver markers, transition metal levels, histopathological examination and expression levels of GST-P, 8-OHdG and Ki-67 were studied to assess the in vivo anticancer effect of TXER. We observed that TXER + Cu induced extensive cellular death on Huh-7 cells through generating free radicals and did not possess any toxic effect on normal hepatocytes. The in vivo studies revealed that TXER possess significant anti-cancer effect as assessed through improved liver markers and suppressed GST-P, 8-OHdG, and Ki-67 expression. TXER treatment reduced the hepatic Cu level in cancer bearing animals. Current study brings the putative mechanism involved in anti-cancer effect of TXER, further it will help to formulate phytoconstituents coupled anti-cancer drug for effective treatment of HCC. This article is protected by copyright. All rights reserved
    • Abstract: Low-intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm2 LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7d, 14d, 21d). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21d CD73 + /CD90 + : 6%; OCT-3/4 + /NANOG + /SOX2 + : 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation. This article is protected by copyright. All rights reserved
  • Microtubule actin crosslinking factor 1 promotes osteoblast
           differentiation by promoting β-catenin/TCF1/Runx2 signaling axis
    • Abstract: Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1 knockdown increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. This article is protected by copyright. All rights reserved
  • Poly r(C) binding protein 1-mediated regulation of microRNA expression
           underlies post-sevoflurane amelioration of acute lung injury in rats
    • Abstract: Background: Acute lung injury (ALI) presents a pervasive health burden due to the high morbidity and mortality associated with it. Volatile anesthetics like sevoflurane has been previously shown to have organ-protective effect, both in the context of normal physiological function in liver, and during LPS-induced ALI. Sevoflurane was shown to exert lung protective effect during LPS-induced ALI by modulating expression level of microRNAs (miRNAs), specifically miR-155. The objective of the current study was to define the underlying mechanism by which sevoflurane alters miRNA expression levels. Methods: Lung injury caused by LPS and its amelioration post sevoflurane administration was first confirmed. Expression levels of different miRNA and messenger RNAs (mRNAs) encoding inflammatory cytokines were measured in a rat model of lipopolysaccharide (LPS)-induced ALI, which were subsequently treated with either sevoflurane or vehicle control. Results: Host of miRNAs and messenger RNAs encoding pro-inflammatory cytokines are overexpressed during LPS-induced ALI, which are reversed following sevoflurane administration. Mass spectrometry analysis revealed that the RNA-binding protein, poly r(C) binding protein 1 (PCBP1) expression is induced in ALI and is repressed following sevoflurane treatment. RNA immunoprecipitation experiments revealed that PCBP1 expression dictates the altered miRNA expression and sevoflurane altered miRNA expression by suppressing PCBP1 expression. Conclusion: Our study thus elucidates a unique mechanism of lung protective effect of sevoflurane mediated by suppression of expression of a RNA-binding protein that potentiates expression of pro-inflammatory miRNAs. This article is protected by copyright. All rights reserved
  • Three-dimensional cell culture models for anticancer drug screening: worth
           the effort'
    • Abstract: High attrition of new oncology drug candidates in clinical trials is partially caused by the poor predictive capacity of artificial monolayer cell culture assays early in drug discovery. Monolayer assays do not take the natural three-dimensional (3D) microenvironment of cells into account. As a result, false positive compounds often enter clinical trials, leading to high dropout rates and a waste of time and money. Over the past two decades, tissue engineers and cell biologists have developed a broad range of 3D in vitro culturing tools that better represent in vivo cell biology. These tools preserve the 3D architecture of cells and can be used to predict toxicity of and resistance against antitumor agents. Recent progress in tissue engineering further improves 3D models by taking into account the tumor microenvironment, which is important for metastatic progression and vascularization. However, the widespread implementation of 3D cell cultures into cell-based research programs has been limited by various factors, including their cost and reproducibility. In addition, different 3D cell culture techniques often produce spheroids of different size and shape, which can strongly influence drug efficacy and toxicity. Hence, it is imperative to morphometrically characterize multicellular spheroids to avoid generalizations among different spheroid types. Standardized 3D culturing procedures could further reduce data variability and enhance biological relevance. Here, we critically evaluate the benefits and challenges inherent to growing cells in 3D, along with an overview of the techniques used to form spheroids. This is done with a specific focus on antitumor drug screening. This article is protected by copyright. All rights reserved
  • MicroRNA: A Novel Target of Curcumin in Cancer Therapy
    • Abstract: Curcumin is known as a natural dietary polyphenol which is extracted from Curcuma longa L. It has been shown that curcumin has a variety of pharmacological effects such as antioxidant, anti-cancer, anti-inflammatory, and anti-microbial activities. Anti-cancer effects of curcumin are due to targeting of a wide range of cellular and molecular pathways involved in cancer pathogenesis including NF-kB, MAPK, PTEN, P53, and microRNAs (miRNA) network. Multiple lines of evidence have indicated that curcumin exerts its therapeutic effects via regulating miRNA expression (e.g. miR-1, miR-7, miR-9, miR-34a, miR-181, miR-21 and miR-19) which could lead to the regulation of underlying cellular and molecular pathways involved in cancer pathogenesis. Exosomes are one of the important classes of biological vehicles which could be released from various types of cells such as cancer cells and stem cells and could change the behavior of recipient cells. It has been shown that treatment of cancer cells with different dose of curcumin leads to the release of exosomes containing curcumin. These exosomes could induce anti-cancer properties in recipient cells and reduce tumor growth. Hence, exosomes containing curcumin could be applied as powerful tools for cancer treatment. Here, we highlighted various miRNAs which could be affected by curcumin in various types of cancer. Moreover, we highlight exosomes containing curcumin as suitable therapeutic tools in cancer therapy. This article is protected by copyright. All rights reserved
  • Adenosine: an endogenous mediator in the pathogenesis of gynecological
    • 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
    • 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
  • Apigenin Induces ROS Dependent Apoptosis and ER Stress in Human
           Endometriosis Cells
    • Abstract: Apigenin is a plant-derived flavonoid having anti-proliferative, anti-inflammatory and anti-angiogenic properties in chronic and metabolic diseases, and cancers. However, the functional role of apigenin remains to be identified in human endometriosis that is a benign inflammatory disease causing infertility, dysmenorrhea, dyspareunia, and chronic abdominal or pelvic pain. In the present study, we determined the effects of apigenin on two well-established human endometriosis cell lines (VK2/E6E7 and End1/E6E7). Apigenin reduced proliferation and induced cell cycle arrest and apoptosis in the both endometriosis cell lines. In addition, it disrupted mitochondrial membrane potential (MMP) which was accompanied by an increase in concentration of calcium ions in the cytosol and in pro-apoptotic proteins including Bax and cytochrome c in the VK2/E6E7 and End1/E6E7 cells. Moreover, apigenin treated cells accumulated excessive reactive oxygen species (ROS), and experienced lipid peroxidation and endoplasmic reticulum (ER) stress with activation of the unfolded protein response (UPR) regulatory proteins. Furthermore, the apigenin-induced apoptosis in endometriosis cells was regulated via the ERK1/2, JNK and AKT cell signaling pathways. Taken together, apigenin is a potential novel therapeutic agent to overcome current limitations in the treatment to endometriosis. This article is protected by copyright. All rights reserved
  • A Perspective on Stem Cell Therapy for Ear Disorders
    • Abstract: The use of stem cells in cell-based therapy is an emerging concept for the treatment of ear disorders. Tympanic membrane perforation (TMP) and inner ear disorders are some of the most commonly presented otologic disorders that can benefit from advances in cell-based therapy. Studies have already demonstrated that stem cell-based therapy can potentially be an effective treatment modality for acute and chronic TMP. Recent studies have also shown promise in application of cell-based approach to treat inner ear dysfunction. In this perspective, we will discuss the recent advancements regarding the use of cell-based therapy for ear disorders. This article is protected by copyright. All rights reserved
  • Angiogenesis biomarkers and their targeting ligands as potential targets
           for tumor angiogenesis
    • Abstract: Angiogenesis is known as one of the hallmarks in cancer which could play a key role in providing oxygen and nutrients for tumor cells. It has been shown that tumor cannot grow without sufficient development of new blood vessels. Accordingly, targeting angiogenesis, especially endothelial cells, could be considered as a common therapeutic target in tumors and more investigation on already existing biomarkers and potentially new biomarkers of endothelial cells seems to be necessary in cancer therapy. Moreover, the use of effective targeting approaches such as proteins and peptides, aptamers, and small molecules is an important step for targeting biomarkers associated with endothelial cells and angiogenesis in cancer therapy. These agents are FDA approved, or are currently under investigation in pre-clinical and clinical studies. Among various biomarkers for angiogenesis microRNAs are suitable candidates for target therapy. These molecules play key roles in tumor angiogenesis which exert their effect via targeting a variety of cellular and molecular pathways involved in tumor angiogenesis. Here, we summarize a variety of biomarkers which their expressions or their functions could change the function of endothelial cells in tumor microenvironments. Moreover, we highlighted various therapeutic agents which could target these biomarkers. This article is protected by copyright. All rights reserved
  • Lipoprotein(a): A missing culprit in the management of
    • Abstract: Lipoprotein(a) Lp(a) is a cholesterol-rich, LDL-like particle that is independently associated with an increased risk for ischemic heart disease, atherosclerosis, thrombosis, and stroke. Genetic variation in the Lp(a) locus and some other genes related to Lp(a) synthesis and metabolism play a critical role in regulating plasma Lp(a) levels. The pathophysiological potential of Lp(a) is related to proatherogenic and prothrombotic effects on the vasculature. Different molecular mechanisms underlying the atherothrombotic potential of Lp(a), free apolipoprotein(a), and oxidized-Lp(a) have been proposed. However, plasma Lp(a) assay is complicated by problems associated with quantification and standardization owing to the polymorphic nature of this lipoprotein. This review has focused on the physicochemical properties of Lp(a), the genetic aspects of Lp(a), the need for accurate determination of Lp(a), the synthesis and recent findings on metabolism of Lp(a). Lastly, the patho-physiological mechanisms by which Lp(a) may increase athero-thrombosis and an overview on the therapeutic modalities to interfere with Lp(a) are summarized. This article is protected by copyright. All rights reserved
  • Combination of nanotechnology with vascular targeting agents for effective
           cancer therapy
    • Abstract: As a young science, nanotechnology promptly integrated into the current oncology practice. Accordingly, various nanostructure particles were developed to reduce drug toxicity and allow the targeted delivery of various diagnostic and therapeutic compounds to the cancer cells. New sophisticated nanosystems constantly emerge to improve the performance of current anticancer modalities. Targeting tumor vasculature is an attractive strategy to fight cancer. Though the idea was swiftly furthered from basic science to the clinic, targeting tumor vasculature had a limited potential in patients, where tumors relapse due to the development of multiple drug resistance and metastasis. The aim of this review is to discuss the advantages of nanosystem incorporation with various vascular targeting agents, including endogen anti-angiogenic agents; (ii) inhibitors of angiogenesis-related growth factors, (iii) inhibitors of tyrosine kinase receptors; (iv) inhibitors of angiogenesis-related signaling pathways, (v) inhibitors of tumor endothelial cell-associated markers, and (vi) tumor vascular disrupting agents. We also review the efficacy of nanostructures as natural vascular targeting agents. The efficacy of each approach in cancer therapy is further discussed. This article is protected by copyright. All rights reserved
    • Abstract: Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes. This article is protected by copyright. All rights reserved
  • Cholinergic and Dopaminergic Neuronal Differentiation of Human Adipose
           Tissue Derived Mesenchymal Stem Cells
    • Abstract: Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP-9, retinoic acid and heparin. Adipose-derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q-PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases. This article is protected by copyright. All rights reserved
  • Artesunate Inhibits RANKL-induced Osteoclastogenesis and Bone Resorption
           In Vitro and Prevents LPS-induced Bone Loss In Vivo
    • Abstract: Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side-effects caused by the currently available drugs, a continuous search for novel bone-protective therapies is essential. Artesunate (Art), the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, the mRNA expression of osteoclastic-specific genes, and resorption pit formation in a dose-dependent manner in primary bone marrow-derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL-induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF-κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)-induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL-induced osteoclastogenesis by suppressing the NF-κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases. This article is protected by copyright. All rights reserved
  • Cytoskeletal remodeling and regulation of cell fate in the hypertensive
           neonatal pulmonary artery in response to stress
    • Abstract: Neonatal pulmonary hypertension (PHN) is a lethal progressive disease that occurs in prenatal circulatory transition. Mechanical wall strain caused by cardiac pulsation integrates with hypoxia to generate rapidly progressive myocyte cytoskeleton disassembly and failure to exert force generation. The physiological responses to such an interaction have not been investigated. The persistent phenotype does not respond to traditional vasodilator therapy; hence, there is a need for new treatment strategies to improve the morbidity and mortality outcomes. We reviewed the current research methods, models, and markers of persistent PHN relevant to oxidative and nitrosative stress as well as cell fate commitment, with an emphasis on apoptosis and proliferation. We surveyed potential investigations into the role of senescence in neonatal PHN cell fate decision programming during vasodilator treatment and suggested putative drug targets to improve clinical outcomes. We identified important signaling intermediates of senescence and cell cycle entry regulation in hypertensive pulmonary arterial tissues.Identifying the concerted interplay between ROS/RNS generation, senescence, and inflammatory signaling will lead to therapeutic targets to combat vascular remodeling in PHN; the factor most limiting to treatment responses is irreversible fibrotic thickening. Prevention of the latter will help reduce mortality and morbidity in infants with PHN and prolong the window of opportunity for vasodilator therapies.
  • IFN-gamma priming of adipose-derived stromal cells at “physiological”
           hypoxia and under acute hypoxic stress
    • Abstract: Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodelling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2) were assessed as alterations of ASCs' CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2-adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in “wound closure”. Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodelling functions. This article is protected by copyright. All rights reserved
  • Humanized mouse models: Application to human diseases
    • Abstract: Humanized mice are superior to rodents for preclinical evaluation of the efficacy and safety of drug candidates using human cells or tissues. During the past decade, humanized mouse technology has been greatly advanced by the establishment of novel platforms of genetically modified immunodeficient mice. Several human diseases can be recapitulated using humanized mice due to the improved engraftment and differentiation capacity of human cells or tissues. In this review, we discuss current advanced humanized mouse models that recapitulate human diseases including cancer, allergy, and graft-versus-host disease. This article is protected by copyright. All rights reserved
  • Therapeutic Relevance of Ozone Therapy in degenerative diseases: Focus on
           Diabetes and Spinal Pain
    • Abstract: Ozone, one of the most important air pollutants, is a triatomic molecule containing three atoms of oxygen that results in an unstable form due to its mesomeric structure. It has been well-known that ozone has potent ability to oxidize organic compounds and can induce respiratory irritation. Although ozone has deleterious effects, many therapeutic effects have also been suggested. Since last decades, the therapeutic potential of ozone has gained much attention through its strong capacity to induce controlled and moderated oxidative stress when administered in precise therapeutic doses. A plethora of scientific evidence showed that the activation of hypoxia inducible factor-1α (HIF-1a), nuclear factor of activated T-cells (NFAT), nuclear factor-erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE), and activated protein-1 (AP-1) pathways are the main molecular mechanisms underlying the therapeutic effects of ozone therapy. Activation of these molecular pathways leads to up-regulation of endogenous antioxidant systems, activation of immune functions as well as suppression of inflammatory processes, which is important for correcting oxidative stress in diabetes and spinal pain. The present study intended to review critically the available scientific evidence concerning the beneficial properties of ozone therapy for treatment of diabetic complications and spinal pain. It finds benefit for integrating the therapy with ozone into pharmacological procedures, instead of a substitutive or additional option to therapy. This article is protected by copyright. All rights reserved
  • The Roles of Signaling Pathways in Bone Repair and Regeneration
    • Abstract: Regenerative medicine has sparked interest in potential strategies for bone repair. Bone defects are widespread and could be caused by trauma, congenital malformations, infections, and surgery. Although bone has a large self-healing capacity, some defects or fractures are too big to regenerate. To regenerate bone structures which can be used for treatment of patients, bone growth must be induced by a number of bioactive implantable materials, cell types and intracellular and extracellular molecular signaling pathways. Since mesenchymal stem cells (MSCs) and their differentiation during remodeling processes have important roles in bone regeneration, it is believed that understanding molecular signaling pathways involved is crucial to the development of bone implants, bone substitute materials, and cell-based scaffolds for bone regeneration. In this review, we briefly introduce concepts in fracture repair and regeneration following bone injuries, and then discuss the current clinical methods in bone regeneration. In the next section, we review the involvement of the various key signaling pathways in bone regeneration. This article is protected by copyright. All rights reserved
  • Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts
           from prolonged stress and contribute to survival and rejuvenation of human
           skin equivalents
    • Abstract: Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine. This article is protected by copyright. All rights reserved
  • Hypoxia-induced apoptosis of mouse spermatocytes is mediated by HIF-1α
           through a death receptor pathway and a mitochondrial pathway
    • Abstract: Hypoxia in vivo induces oligozoospermia, azoospermia and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia-induced apoptosis of mouse GC-2spd (GC-2) cells and the relationship between HIF-1α and apoptosis of GC-2 cells induced by hypoxia. GC-2 cells were subjected to 1% oxygen for 48 h. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase-3/8/9 in the absence and presence of HIF-1α siRNA. The protein levels of apoptosis-related markers were determined by western blot in the presence and absence of HIF-1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC-1 staining. Cell viability was assessed upon treatment of caspase-8 and caspase-9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 h induced apoptosis of GC-2 cells. A prolonged exposure of GC-2 cells to hypoxic conditions caused downregulation of c-FLIP, DcR2 and Bcl-2 and upregulation of DR5, TRAIL, Fas, p53 and Bax, with an overproduction of caspase-3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase-8/9 partially suppressed hypoxia-induced GC-2 cell apoptosis, and the anti-apoptotic effects of the caspase inhibitors were additive. Of note, HIF-1α knockdown attenuated hypoxia and induced apoptosis of GC-2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF-1α, contribute to hypoxia-induced GC-2 cell apoptosis. This article is protected by copyright. All rights reserved
  • 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
  • Activation of ClC-3 chloride channel by 17β-estradiol relies on the
           estrogen receptor α expression in breast cancer
    • Abstract: Although extensively studied, the mechanisms by which estrogen promotes breast cancer growth remain to be fully elucidated. Tamoxifen, an antiestrogen agent to treat ERα+ breast cancer, is also a high-affinity blocker of the chloride channels. In this study, we explored the involvement of the chloride channels in the action of estrogen in breast cancer. We found that 17β-estradiol (17β-E2) concentration-dependently activated the chloride currents in ERα+ breast cancer MCF-7 cells. Extracellular hypertonic challenge and chloride channel blockers, NPPB and DIDS inhibited the 17β-E2-activated chloride currents. Decreased the ClC-3 protein expression caused the depletion of the 17β-E2-activated chloride currents. 17β-E2-activated chloride currents which relied on the ERα expression were demonstrated by the following evidences. Firstly, 17β-E2-activated chloride currents could not be observed in ERα- breast cancer MDA-MB-231 cells. Secondly, ER antagonists, tamoxifen and ICI 182,780, and downregulation of ERα expression inhibited or abolished the 17β-E2-activated chloride currents. Thirdly, ERα expression was induced in MDA-MB-231 cells by ESR1 gene transfection, and then 17β-E2-activated chloride currents could be observed. In MCF-7 cells, ERα and ClC-3 mainly located in nucleus and translocated to cell plasma and membrane with respect to co-localization following treatment of 17β-E2. Downregulation of ERα expression could decrease the expression of ClC-3 protein. Conversely, downregulation of ClC-3 expression did not influence the ERα expression. Taken together, our findings demonstrated that ClC-3 is a potential target of 17β-E2 and is modulated by the ERα in breast cancer cell. Pharmacological modulation of ClC-3 may provide a deep understanding in antiestrogen treatment of breast cancer patients.
  • State of the art in microRNA as diagnostic and therapeutic biomarkers in
           chronic lymphocytic leukemia
    • Abstract: Early diagnostic is one of the most important steps in cancer therapy which helps to design and choose a better therapeutic approach. The finding of biomarkers in various levels including genomics, transcriptomics, and proteomics levels could provide better treatment for various cancers such as chronic lymphocytic leukemia (CLL). The CLL is the one of main lymphoid malignancies which is specified by aggregation of mature B lymphocytes. Among different biomarkers (e.g., CD38, chromosomes abnormalities, ZAP-70, TP53, and microRNA [miRNA]), miRNAs have appeared as new diagnostic and therapeutic biomarkers in patients with the CLL disease. Multiple lines of evidence indicated that deregulation of miRNAs could be associated with pathological events which are present in the CLL. These molecules have an effect on a variety of targets such as Bcl2, c-fos, c-Myc, TP53, TCL1, and STAT3 which play critical roles in the CLL pathogenesis. It has been shown that expression of miRNAs could lead to the activation of B cells and B cell antigen receptor (BCR). Moreover, exosomes containing miRNAs are one of the other molecules which could contribute to BCR stimulation and progression of CLL cells. Hence, miRNAs and exosomes released from CLL cells could be used as potential diagnostic and therapeutic biomarkers for CLL. This critical review focuses on a very important aspect of CLL based on biomarker discovery covers the pros and cons of using miRNAs as important diagnostics and therapeutics biomarkers for this deadly disease.
  • Anti-tumor effects of crocetin and related molecular targets
    • Abstract: Natural products have gained a wide popularity as chemopreventive and anti-cancer agents owing to their multi-mechanistic mode of action, availability and synergism with several conventional chemotherapeutic agents. Crocetin is a carotenoid compound isolated from the stigma of Crocus sativus L. (saffron). Crocetin has shown promising effects as an anti-tumor agent in animal models and cell culture systems. Crocetin retards the growth of cancer cells via inhibiting nucleic acid synthesis, enhancing anti-oxidative system, and inducing apoptosis and differentiation pathways. The present review outlines natural sources of crocetin, and its pharmacokinetic and pharmacological properties relevant to the prevention and treatment of cancer. Also, we discuss molecular targets underlying the putative anti-tumor effects of crocetin.The present review outlines natural sources of crocetin, and its pharmacokinetic and pharmacological properties relevant to the prevention and treatment of cancer. Also, we discuss molecular targets underlying the putative anti-tumor effects of crocetin.
  • 2-methoxyestradiol impacts on amino acids-mediated metabolic reprogramming
           in osteosarcoma cells by its interaction with NMDA receptor
    • Abstract: Deregulation of serine and glycine metabolism, have been identified to function as metabolic regulators in supporting tumor cell growth. The role of serine and glycine in regulation of cancer cell proliferation is complicated, dependent on concentrations of amino acids and tissue-specific. D-serine and glycine are coagonists of N-methyl-D-aspartate (NMDA) receptor subunit GRIN1. Importantly, NMDA receptors are widely expressed in cancer cells and play an important role in regulation of cell death, proliferation, and metabolism of numerous malignancies. The aim of the present work was to associate the metabolism of glycine and D-serine with the anticancer activity of 2-methoxyestradiol. 2-methoxyestradiol is a potent anticancer agent but also a physiological 17β- estradiol metabolite. In the study we have chosen two malignant cell lines expressing functional NMDA receptors, that is osteosarcoma 143B and breast cancer MCF7. We used MTS assay, migration assay, flow cytometric analyses, Western blotting and immunoprecipitation techniques as well as molecular modeling studies. We have demonstrated the extensive crosstalk between the deregulated metabolic network and cancer cell signaling. Herein, we observed an anticancer effect of high concentrations of glycine and D-serine in osteosarcoma cells. In contrast, the amino acids when used at low, physiological concentrations induced the proliferation and migration of osteosarcoma cells. Importantly, the pro-cancergogenic effects of both glycine and D-serine where abrogated by the usage of 2-methoxyestradiol at both physiological and pharmacological relevant concentrations. The obtained data confirmed that 2-methoxyestradiol may be a physiological anticancer molecule.Interaction of 2-methoxyestradiol with NMDA receptor. GluN1/GluN2A Ligand Binding Domain containing 2-metoxyestradiol and agonists-Glycine or D-Serine.
  • 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.
  • Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide,
           inducing apoptosis and membrane disruption
    • Abstract: Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 μM VRP treatment and at 350 μM, all kinds of sperm motility ceased within 20 s as assessed by the Sander–Cramer assay. The spermicidal effect was confirmed by eosin–nigrosin assay and HOS test. VRP treatment (100 μM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 μM VRP. MTT assay showed after treatments with 100 and 350 μM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality.VRP designed on the basis of heptad repeat of valine, inhibited progressive forward motility and had spermicidal activity in human sperm cells by inducing both extrinsic and intrinsic apoptotic pathways. It also morphologically disrupted the sperm cells and altered the mitrochondrial membrane permeability but the viability of vaginal microflora was not affected much. Therefore, VRP may be a promising microbicidal vaginal contraceptive molecule, worthy of further consideration for the future contraceptive drug development.
  • An improved method for primary culture of normal cervical epithelial cells
           and establishment of cell model in vitro with HPV-16 E6 gene by lentivirus
    • Abstract: Normal human cervical epitheliums infected with HPVs gene in vitro are underlying molecular models to investigate physiological mechanisms of cervical epithelia and cervical disease. The current study aimed to establish a modified culture method for cervical epithelium and explore the feasibility of transfection with HPV-16 E6 gene mediated by lentivirus in primary cervical cells. The cells were dissociated enzymatically using Dispase II combined with 0.25% Trypsin–0.01% ethylenediamine tetracetic acid (EDTA) or Collagenase I. The detached effectiveness of Dispase II at different times was compared. Isolated cells were cultured and subcultured in modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum (FBS) or K-SFM alone. Cytokeratin was used as the identification of cervical epitheliums. Proliferative capacity and growth curve of cervical epitheliums were evaluated by cell counting kit-8 (CCK-8). The cells at passages 3 were used to infect with HPV-16 E6 gene by lentivirus. The expression of green fluorescent protein (GFP) presented in the infected cells was observed via fluorescence microscopy and the levels of E6 mRNA were detected by quantitative real-time PCR (qRT-PCR). The results indicate that cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin–0.01% EDTA method for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS. The present study proposed a brief and high-yield protocol for isolation and culture of human cervical epitheliums. Moreover, an infected cell model with HPV-16 E6 gene mediated by lentivirus was established which can do duty for studies in vitro on the carcinogenic mechanism of HR-HPVs.Normal cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin–0.01% EDTA for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS.The establishment of the cell model with HPV-16 E6 gene mediated by lentivirus can provide a foundation for further study of the carcinogenic mechanism of HR-HPVs.
  • WNT4 acts downstream of BMP2 to mediate the regulation of ATRA signaling
           on RUNX1 expression: Implications for terminal differentiation of antler
    • Abstract: Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1.ATRA may activate the expression of RARα/RXRα and induce terminal differentiation of antler chondrocytes through BMP2-WNT4-RUNX1 pathway. CRABP2 can enhance the sensitivity of antler chondrocytes to ATRA, while CYP26A1 and CYP26B1 render chondrocytes hyposensitive to ATRA.
  • The skeletal cell-derived molecule sclerostin drives bone marrow
    • Abstract: The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.Sclerostin, a gene that encodes the SOST protein, is expressed by osteocytes within cortical and trabecular bone. We found that SOST protein can regulate bone marrow progenitor cell differentiation by not only inhibiting osteogenesis, but also inducing bone marrow adipogenesis. This novel role for sclerostin in development of bone marrow adipose suggests that sclerostin-targeting therapies may not only increase osteogenesis, but also decrease bone marrow adipogenesis, which could have wide clinical implications.
  • Osteogenic differentiation potential of mesenchymal stem cells cultured on
           nanofibrous scaffold improved in the presence of pulsed electromagnetic
    • Abstract: Nowadays, tissue engineering by using stem cells in combination with scaffolds and bioactive molecules has made significant contributions to the regeneration of damaged bone tissues. Since the usage of bioactive molecules including, growth factors to induce differentiation is safety limited in clinical applications, and it has also been previously observed that extremely low frequency pulsed electromagnetic fields (PEMF) can be effective in the enhancement of proliferation rate and osteogenic differentiation of stem cells, the aim of this study was investigating the osteoinductive potential of PEMF in combination with Poly(caprolactone) (PCL) nanofibrous scaffold. To achieve this aim, Adipose-derived mesenchymal stem cells (ADSCs) isolated and characterized and then osteogenic differentiation of them was investigated after culturing on the surface of PCL scaffold under treatments of PEMF, PEMF plus osteogenic medium (OM) and OM. Analysis of common osteogenic markers such as Alizarin red staining, ALP activity, calcium content and four important bone-related genes in days of 7, 14, and 21 confirmed that the effects of PEMF on the osteogenic differentiation of ADSCs are very similar to the effects of osteogenic medium. Thus, regarding the immunological concerns about the application of bioactive molecules for tissue engineering, PEMF could be a good alternative for osteogenic medium. Although, results were showed a synergetic effect for simultaneous application of PEMF and PCL scaffold in the osteogenesis process of ADSCs. Taking together, ADSCs-seeded PCL nanofibrous scaffold in combination with PEMF could be a great option for use in bone tissue engineering applications.Tissue engineering (stem cells in combination with scaffolds and bioactive molecules) has made significant contributions to the damaged bone regeneration. PEMF can be effective in the enhancement of proliferation rate and osteogenic differentiation of stem cells. ADSCs-seeded PCL nanofibers in combination with PEMF could be a great option for use in bone tissue engineering applications.
  • BacteRiophage EXclusion (BREX): A novel anti-phage mechanism in the
           arsenal of bacterial defense system
    • Abstract: The arm race between bacteria and phages leads to the development of many phage resistance mechanisms. Bacteria are perpetually changing their strategy to avert phage infection and killing. Recently, a study published that shed light on a novel bacterial defense system called bacteriophage exclusion (BREX). BREX system is a six-gene cassette in Bacillus cereus which provides complete phage resistance to a broad range of phages, including lytic and temperate ones. It is a novel defense strategy which involves DNA methylation of the host cell and block phage DNA replication.
  • Effects of electrical stimulation on cell proliferation and apoptosis
    • Abstract: The application of exogenous electrical stimulation (ES) to cells in order to manipulate cell apoptosis and proliferation has been widely investigated as a possible method of treatment in a number of diseases. Alteration of the transmembrane potential of cells via ES can affect various intracellular signaling pathways which are involved in the regulation of cellular function. Controversially, several types of ES have proved to be effective in both inhibiting or inducing apoptosis, as well as increasing proliferation. However, the mechanisms through which ES achieves this remain fairly unclear. The aim of this review was to comprehensively summarize current findings from in vitro and in vivo studies on the effects of different types of ES on cell apoptosis and proliferation, highlighting the possible mechanisms through which ES induced these effects and define the optimum parameters at which ES can be used. Through this we hope to provide a greater insight into how future studies can most effectively use ES at the clinical trial stage.Roles of electrical stimulation on cells to manipulate cell apoptosis and proliferation has been widely investigated. This review summarizes the reports of various types of electrical stimulation on these effects.
  • Platelet-derived microparticles regulates thrombin generation via
           phophatidylserine in abdominal sepsis
    • Abstract: Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis Thrombin generation triggered by PMPs is mediated via both intrinsic and extrinsic pathway of coagulation.
  • CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome
    • Abstract: CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for knockout of an individual gene as well as insertion of one gene and/or control of gene transcription. Studies have shown that CRISPR/Cas9 is a great tool to edit many genes in a variety of plant species, including the model plant species as well as agriculturally important crops, such as cotton, maize, wheat, and rice. CRISPR/Cas9-based genome editing can be used for plant functional studies and plant improvement to yield, quality, and tolerance to environmental stress.CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals, including the model plant species as well as agriculturally important crops, such as cotton, maize, and rice. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive.
  • Leptin accelerates the pathogenesis of heterotopic ossification in rat
           tendon tissues via mTORC1 signaling
    • Abstract: Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.Leptin promotes the osteogenic differentiation of tendon-derived stem cells (TDSCs) in heterotopic ossification (HO). mTORC1 signaling favors differentiation of TDSCs into osteoblasts and promotes expression of Runx2 and OSX in HO of Achilles tendon.
  • 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.
  • Complex osteoclastogenic inductive effects of nicotine over hydroxyapatite
    • Abstract: Cigarette smoke is associated to pathological weakening of bone tissue, being considered an important playmaker in conditions such as osteoporosis and periodontal bone loss. In addition, it is also associated with an increased risk of failure in bone regeneration strategies. The present work aimed to characterize the effects of nicotine on human osteoclastogenesis over a hydroxyapatite substrate. Osteoclast precursors were maintained in the absence or presence of the osteoclastogenesis enhancers M-CSF and RANKL, and were further treated with nicotine levels representative of the concentrations observed in the plasma and saliva of smokers. It was observed that nicotine at low concentrations elicit an increase in osteoclast differentiation, but only in the presence of M-CSF and RANKL it was also able to significantly increase the resorbing ability of osteoclasts. A slight downregulation of NFkB pathway and an increase in the production of TNF-α and, particularly PGE2, were involved in the observed effects of nicotine. At high concentrations, nicotine revealed cytotoxic effects, causing a decrease in cell density. In conclusion, nicotine at levels found in the plasma of the smokers, has the ability to act directly on osteoclast precursors, inducing its osteoclastogenic differentiation. The stimulatory behavior appears to be dependent on the stage of osteoclastic differentiation of the precursor cells, which means, in the absence of M-CSF and RANKL, it only favors the initial stages of osteoclast differentiation, while in the presence of the growth factors, a significant increase in their resorbing ability is also achieved.The effects of nicotine on human osteoclastogenesis was evaluated, in the presence of hydroxyapatite. At low doses, nicotine displayed a pro-osteoclastogenic behavior, while at high doses, it was lethal to the cells. The observed effects were accompanied by significant changes in important intracellular signaling pathways, as well as in the producation of TNF-alpha and PGE2.
  • 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.
  • HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR
           expression for hypertension-induced cardiomyocyte hypertrophy
    • Abstract: Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT1R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II-induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients.These results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy.
  • 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.
  • A highly efficient method for extracting next-generation sequencing
           quality RNA from adipose tissue of recalcitrant animal species
    • Abstract: The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Currently available approaches yield variable and unsatisfactory results with AT samples.A highly efficient novel protocol was developed for the extraction of NGS quality RNA from AT across a broad range of animal species.
  • HOTAIR role in melanoma progression and its identification in the blood of
           patients with advanced disease
    • Abstract: The molecular mechanisms responsible for the metastatic progression of melanoma have not been fully defined yet. We have recently shown that an important role in this process is certainly played by HOX genes, whose regulation is under control of particular non-coding RNAs, some of which are present within the HOX locus. HOTAIR is the most studied among them, whose aberrant expression is associated with the metastatic progression of many malignancies. The aim of this study was to verify the role played by HOTAIR in metastatic progression of melanoma and to evaluate the circulating levels of HOTAIR in the blood of patients with metastatic melanoma. A series of melanocytic lesions were selected to evaluate the potential changes in the expression of HOTAIR during the evolution of the disease through in situ and molecular approaches. None of the benign melanocytic lesions showed the presence of HOTAIR. The staining of HOTAIR resulted very weak in the primary pT1 lesions, while it was very strong in all pairs of primary tissues and corresponding metastases. Surprisingly, we found the presence of HOTAIR in some intratumoral lymphocytes, while this positivity decreased in lymphocyte component further away from the tumor. HOTAIR was also detected in the serum of selected metastatic patients. These data allowed us to speculate on the fundamental role played by HOTAIR in tumor evolution of melanoma. Its presence in intratumoral lymphocytes might suggest that its involvement in the modulation of tumor microenvironment and the detection in the serum could be used in the management of melanoma patients.The staining of HOTAIR was very weak in melanoma pT1a primary lesions while it showed a significant expression in all pairs of melanoma pT3–pT4 primary tissues and corresponding lymph node, cutaneous, and visceral metastases. HOTAIR staining was highlighted in intra-tumor lymphocytes, while this positivity tended to decline in the lymphocytic component more distant from tumor. HOTAIR was identified in the blood of melanoma metastatic patients, while patients with pT1a stage showed a baseline levels of expression.
  • Gingival spheroids possess multilineage differentiation potential
    • Abstract: Recently studies have demonstrated HGMSCs as ideal candidates for regenerative study. Interestingly we found that HGMSCs derived spheroids are more potent and maintain the properties of stemness convincingly compared to conventional culture methods. During the culture, GMSCs instinctively accumulated into spheroids and display multipotent STRO-1 and Vimentin-positive cells. Reduced phenotypic expression of CD73, CD105, and elevated expression STRO-1 and CD-34. Pluripotent nature of S-GMSCs putatively shown the expression of OCT4A, NANOG, SOX-2, SSEA4, TRA-1-60, and TRA-181. Also, levels of protein are much higher in spheroid than dissociated culture. On endothelial induction, spheroid differentiated and developed a vascular structure with positive expression of CD31 and on neuronal induction showed positivity for TUJ1 and E-Cadherin. Importantly, undifferentiated state of S-GMSCs exhibited significant upregulation of aforementioned pluripotent genes and lack of pro-inflammatory cytokines IL-6 and amplified ARF signal confirming that the spheroids are not teratoma formation. However, higher of CAP1, CP, TGFβ, OPN, PPARɣ, TUJ1, and NESTIN expression observed in spheroids, and minimal expression of the same markers were observed in adherent GMSCs respectively. Ahead of dissociated gingival culture, spheroid provides enhanced viable, pluripotent, and multilineage ability. This study suggested that S-GMSCs increased the chances of therapeutic efficacy in the regenerative applications.This study suggested that Spheroid-GMSCs increased the chances of therapeutic efficacy in the regenerative applications.
  • 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.
  • 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.
  • G protein-coupled receptor 84 controls osteoclastogenesis through
           inhibition of NF-κB and MAPK signaling pathways
    • Abstract: GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. This article is protected by copyright. All rights reserved
  • Stress-responsive microRNAs are involved in re-programming of metabolic
           functions in hibernators
    • Abstract: Mammalian hibernation includes re-programming of metabolic capacities, partially, encouraged by microRNAs (miRNAs). Albeit much is known about the functions of miRNAs, we need learning on low temperature miRNAs target determination. As hibernators can withstand low body temperatures (TB) for a long time without anguish tissue damage, understanding the means and mechanisms that empower them to do as such are of restorative intrigue.Nonetheless, these mechanisms by which miRNAs and the hibernators react to stressful conditions are not much clear. It is evident from recent data that the gene expression and the translation of mRNA to protein are controlled by miRNAs. The miRNAs also influence regulation of major cellular processes. As the significance of miRNAs in stress conditions adaptation are getting clearer, this audit article abridges the key alterations in miRNA expression and the mechanism that facilitates stress survival. This article is protected by copyright. All rights reserved
  • The significant role of the Golgi apparatus in cardiovascular diseases
    • Abstract: The Golgi apparatus is a ribbon-like system of stacks which consist of multiple closely apposed flattened cisternae and vesicles usually localized in the juxta-nuclear area. As for the biological functions, the Golgi apparatus plays a major role in protein biosynthesis, post-translational modification and sorting protein from ER to plasma membrane and other destinations. Structural changes and functional disorder of the Golgi apparatus is associated with various diseases. Moreover, increasing evidence revealed that swelling, poor development and other morphological alterations of the Golgi apparatus are linked to cardiovascular diseases such as heart failure, arrhythmia and dilated cardiomyopathy. Furthermore, dysfunction of the Golgi apparatus is also related to cardiovascular diseases since the Golgi apparatus is extremely responsible for transport, glycosylation, biosynthesis and subcellular distribution of cardiovascular proteins. This review gives a brief overview of the intricate relationship between the Golgi apparatus and cardiovascular diseases. In addition, we provide a further prospective that the Golgi apparatus may provide diagnosis reference for cardiovascular diseases, and changes in the ultrastructure and morphology of the Golgi apparatus such as swelling, poor development and fragmentation may serve as a reliable index for cardiovascular diseases. This article is protected by copyright. All rights reserved
  • Bone marrow adipocytes support haematopoietic stem cell survival
    • Abstract: In bone marrow (BM), haematopoietic elements are mingled with adipocytes (BM-A), which are the most abundant stromal component in the niche. BM-A progressively increase with ageing, eventually occupying up to 50% of BM cavities.In this work, the role played by BM-A was explored by studying primary human BM-A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human haematopoietic stem cells (HSC) by the long-term culture initiating cell (LTC-IC) assay.Findings demonstrated that BM-A are capable of supporting HSC survival in the LTC-IC assay, since after 5 weeks of co-culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C-X-C motif chemokine 12 (CXCL12), interleukin (IL)-8, colony-stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM-A and in primary human BM mesenchymal stromal cells (BM-MSC), whereas IL-3 was higher in BM-A.Interestingly, BM-A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT-A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes and white-to-brown differentiation pathways. Accordingly, analysis of the gene pathways involved in haematopoiesis regulation showed that BM-A are more closely related to BM-MSC than to AT-A.The present data suggest that BM-A play a supporting role in the haematopoietic niche and directly sustain HSC survival. This article is protected by copyright. All rights reserved
  • PCSK9 and infection: a potentially useful or dangerous association'
    • Abstract: Elevated plasma low-density lipoprotein-cholesterol (LDL-C) concentration is the most important risk factor for atherosclerotic cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a ubiquitously expressed serine proteinase which plays a key role in cholesterol metabolism, but has been found to be implicated in some other lipid-independent physiological processes. In this review, the role of PCSK9 was evaluated not only concerning lipid metabolism but also hepatitis C virus (HCV) infection, bacterial infections/sepsis and septic shock. Collected data from clinical trials revealed that treatment with PCSK9 inhibitors has beneficial effects in lowering LDL-C via inhibition of LDL-receptors (LDL-R), an antiviral effect on HCV infection via down-regulating the surface expression of LDL-R and CD81 on hepatic cells, and a positive association with increased inflammatory responses, as well as with septic shock by down-regulation of hepatocyte LDL-R. On the other hand, PCSK9 inhibition by therapeutic fully humanized antibodies has positive effects in reducing elevated LDL-C. However, their safety and tolerability is an important issue which has to be taken into consideration. This article is protected by copyright. All rights reserved
  • Targeting the tumor microenvironment as a potential therapeutic approach
           in colorectal cancer: rational and progress
    • Abstract: Colorectal cancer (CRC) is often diagnosed at a late stage when tumor metastasis may have already occurred. Current treatments are often ineffective in metastatic disease, and consequently late diagnosis is often associated with poor outcomes in CRC. Alternative strategies are therefore urgently required. An interaction between epithelial cancer cells and their tissue microenvironment is a contributor to metastasis, and therefore recent studies are beginning to focus on the properties of the tumor microenvironment and the mechanism by which the metastatic cells exploit the tumor microenvironment for survival, immune evasion, and growth. We have reviewed the development of the combined therapeutic approaches that have focused on targeting the microenvironment of colorectal cancer. This article is protected by copyright. All rights reserved
  • Roles of macrophage migration inhibitory factor in cartilage tissue
    • Abstract: To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif + / +) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif + /+ constructs deteriorated by 8 weeks. Since the Mif + /+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term. This article is protected by copyright. All rights reserved
  • The Fam50a positively regulates ameloblast differentiation via interacting
           with Runx2
    • Abstract: Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation. This article is protected by copyright. All rights reserved
  • 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.
  • Biological roles of glial fibrillary acidic protein as a biomarker in
           cartilage regenerative medicine
    • Abstract: Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology.We provide a rational that GFAP of the auricular chondrocytes can be used as a matrix production marker in culture auricular chondrocytes.
  • Hydroxychloroquine affects bone resorption both in vitro and in vivo
    • Abstract: We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC-sorted monocytes for 14 days and treated with different HCQ doses (control, 1 and 5 µg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker β-CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum β-CTx was significantly decreased after six months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker β-CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss. This article is protected by copyright. All rights reserved
  • Microtubules Regulate Brush Border Formation
    • Abstract: Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation towards the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity. This article is protected by copyright. All rights reserved
  • Co-culture systems-based strategies for articular cartilage tissue
    • 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
  • Nanoparticles as new tools for inhibition of cancer angiogenesis
    • Abstract: Angiogenesis is known as one of the hallmarks of cancer. Multiple lines evidence indicated that vascular endothelium growth factor (VEGF) is a key player in the progression of angiogenesis and exerts its functions via interaction with tyrosine kinase receptors (TKRs). These receptors could trigger a variety of cascades that lead to the supply of oxygen and nutrients to tumor cells and survival of these cells. With respect to pivotal role of angiogenesis in the tumor growth and survival, finding new therapeutic approaches via targeting angiogenesis could open a new horizon in cancer therapy. Among various types of therapeutic strategies, nanotechnology has emerged as new approach for the treatment of various cancers. Nanoparticles (NPs) could be used as effective tools for targeting a variety of therapeutic agents. According to in vitro and in vivo studies, NPs are efficient in depriving tumor cells from nutrients and oxygen by inhibiting angiogenesis. However, the utilization of NPs are associated with a variety of limitations. It seems that new approaches such as NPs conjugated with hydrogels could overcome to some limitations. In the present review, we summarize various mechanisms involved in angiogenesis, common anti-angiogenesis strategies and application of NPs for targeting angiogenesis in various cancers. This article is protected by copyright. All rights reserved
  • Integrated transcriptomic and metabolomic analysis reveals adaptive
           changes of hibernating retinas
    • Abstract: Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities. This article is protected by copyright. All rights reserved
  • Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to
           modulate endothelial cell number in angiogenic vessels
    • Abstract: During embryonic development, new arteries and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis. This article is protected by copyright. All rights reserved
  • The nucleus is irreversibly shaped by motion of cell boundaries in cancer
           and non-cancer cells
    • Abstract: Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume. This article is protected by copyright. All rights reserved
  • The effect of substrate stiffness on cancer cell volume homeostasis
    • Abstract: Existing studies on the mechanism of cell volume regulation are mainly relevant to ion channels and osmosis in extracellular fluid. Recently, accumulating evidence has shown that cellular mechanical microenvironment also influences the cell volume. Herein, we investigated the regulation of substrate stiffness on the cell volume homeostasis of MCF-7 cells and their following migration behaviors. We found that cell volume increases with increasing substrate stiffness, which could be affected by blocking the cell membrane anion permeability and dopamine receptor. In addition, the cell migration is significantly inhibited by decreasing the cell volume using tamoxifen and such inhibition effect on migration is enhanced by increasing substrate stiffness. The cell membrane anion permeability might be the linker between cellular mechanical microenvironment and cellular volume homeostasis regulation. This work revealed the regulation of substrate stiffness on cell volume homeostasis for the first time, which would provide a new perspective into the understanding of cancer metastasis and a promising anti-cancer therapy through regulation of cell volume homeostasis. This article is protected by copyright. All rights reserved
  • Estrogen receptor β regulates the tumoral suppressor PTEN to modulate
           pituitary cell growth
    • Abstract: In this study, we focused on ERβ regulation in the adenohypophysis under different estrogenic milieu, by analyzing whether ER modulates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression and its subcellular localization on anterior pituitary glands from Wistar rats and GH3 lactosomatotroph cells that over-expressed ERβ. ERβ was regulated in a cyclic manner, and underwent dynamic changes throughout the estrous cycle, with decreased ERβ+ cells in estrus and under E2 treatment, but increased in ovariectomized rats. In addition, the ERα/β ratio increased in estrus and under E2 stimulation, but decreased in ovariectomized rats. Double immunofluorescence revealed that lactotroph and somatotroph ERβ+ were significantly decreased in estrus. Also, variations in the PTEN expression was observed, which was diminished with high E2 conditions but augmented with low E2 milieu. The subcellular localization of this phosphatase was cell cycle–dependent, with remarkable changes in the immunostaining pattern: nuclear in arrested pituitary cells but cytoplasmic in stimulated cells, and responding differently to ER agonists, with only DPN being able to increase PTEN expression and retaining it in the nucleus. Finally, ERβ over-expression increased PTEN with a noticeable subcellular redistribution, and with a significant nuclear signal increase in correlation with an increase of cells in G0/G1 phase. These results showed that E2 is able to inhibit ERβ expression and suggests that the tumoral suppressor PTEN might be one of the signaling proteins by which E2, through ERβ, acts to modulate pituitary cell proliferation, thereby adapting endocrine populations in relation with hormonal necessities. This article is protected by copyright. All rights reserved
  • TNF-α has both stimulatory and inhibitory effects on mouse
           monocyte-derived osteoclastogenesis
    • Abstract: Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31hiLy-6C-), myeloid blasts (CD31+Ly-6C+) and monocytes (CD31-Ly-6Chi) were sorted from mouse bone marrow using flow cytometry and cultured with M-CSF and RANKL, with or without TNF-α. Surprisingly, TNF-α prevented the differentiation of TRAcP+ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL-1β or IL-6. When monocytes were pre-cultured with M-CSF and RANKL followed by exposure to TNF-α, a stimulatory effect was found. TNF-α also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF-α was added to monocytes cultured on plastic, RANK, NFATc1 and TRAcP were significantly down-regulated while TNF-αR1 and TNF-αR2 were up-regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF-α, indicating an altered ratio of bound-RANK/unbound-RANK. Our findings suggest a diverse role of TNF-α on monocytes' osteoclastogenesis: it affects the RANK-signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre-cultured with M-CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte-derived osteoclast formation away from the bone. This article is protected by copyright. All rights reserved
  • GLUT 1 receptor expression and circulating levels of fasting glucose in
           high grade serous ovarian cancer
    • Abstract: In recent years, the poorly remarkable goals achieved in terms of patients' important outcomes for ovarian cancer have fueled our interest towards the study of its metabolic roots. Within this research pipeline, we assessed the association between the expression of the glucose transporter GLUT1, as expressed at the tumor tissue level, and circulating pre-surgical levels of fasting glucose in a case series including data from 40 patients with high FIGO stage serous ovarian cancer. Patients who provided data to the current analysis were randomly selected from a larger cohort. To our purposes, the procedures related to serum and tissue collection, storage and biomarker assessment were highly standardized and centralized at the institutional laboratories. The GLUT1 antibody SPM498 SPRING (REF. E13810) was used at a 1:500 dilution in 2 µm slides. Staining for GLUT1 was observed at the cell membrane level in all the cases assessed, but strong staining was described in 29 (72.5) of them. The agreement between the two independent reviewers was 100%. Strong GLUT1 staining was inversely associated with the circulating levels of fasting glucose, with a particularly striking difference in the distribution for patients in the lowest fasting glucose tertile (p = 0.044). These results support the biological plausibility of the association of interest. If confirmed in larger studies, our findings may help clarify the potentials of biomarkers related to energy metabolism in terms of prognosis definition, treatment assignment and outcome interpretation for patients with high FIGO stage serous ovarian cancer. This article is protected by copyright. All rights reserved
    • Abstract: Model animals naturally differ from humans in various respects and results from the former are not directly translatable to the latter. One approach to address this issue is humanized mice that are defined as mice engrafted with functional human cells or tissues. In humanized mice, we can investigate the development and function of human cells or tissues (including their products encoded by human genes) in the in vivo context of a small animal. As such, humanized mouse models have played important roles that cannot be substituted by other animal models in various areas of biomedical research. Although there are obvious limitations in humanized mice and we may need some caution in interpreting the results obtained from them, it is reasonably expected that they will be utilized in increasingly diverse areas of biomedical research, as the technology for preparing humanized mice are rapidly improved. In this review, I will describe the methodology for generating humanized mice and overview their recent applications in various disciplines including immunology, infectious diseases, drug metabolism, and neuroscience. This article is protected by copyright. All rights reserved
  • The genetic factors contributing to the development of Wilm's tumor and
           their clinical utility in its diagnosis and prognosis
    • Abstract: Mutations in the Wilm's tumor 1 (WT1) gene are associated with a wide spectrum of renal manifestations, ultimately leading to end-stage kidney failure. There is an inadequate understanding of the molecular functions of WT1 in renal development, and this has limited the potential for therapeutic interventions in WT1-related diseases. In this review we discuss the existing data on the genetic and epigenetic abnormalities that have been described in WTs and their potential utility as biomarkers for risk stratification, prediction and prognosis in patients withWTs. This article is protected by copyright. All rights reserved
  • End stage renal disease-induced hypercalcemia may promote aortic valve
           calcification via Annexin VI enrichment of valve interstitial cell
           derived-matrix vesicles
    • Abstract: Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p 
  • Cover Image, Volume 232, Number 9, September 2017
    • Abstract: Cover: The cover image, by Alessandro Poli et al., is based on the Original Research Article Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression,
      DOI : 10.1002/jcp.25642.
  • Table of Contents, Editor's Choice, Highlights
  • TNT-Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of
           Pro-Phagocytic Signals From Apoptotic to Viable Cells
    • Abstract: The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat-me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV-treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS-exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro-phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti-phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS-membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV-treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed-PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro-phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals Inc.Tunneling nanotubes (TNTs) connected UV-treated apoptotic PC12 cells with neighboring untreated cells. These phosphatidylserine (PS)-positive TNTs facilitated the transfer of pro-phagocytic signals from apoptotic to viable cells. Notably, the phagocytosis of viable PC12 cells by macrophages increased when apoptotic cells were present, but only under conditions that allowed for the formation of TNTs. Our findings suggest that TNTs are a means of transferring “eat-me” signals from dying to living cells, and thus induce phagocytosis of previously healthy cells.
  • Cancer Cell Plasticity: Rapid Reversal of Chemosensitivity and Expression
           of Stemness Markers in Lung and Breast Cancer Tumorspheres
    • Abstract: In cancer cells, the reversible nature of the stemness status in terms of chemoresistance has been poorly characterized. In this study, we have simulated one cycle of environmental conditions to study such reversibility by first generating floating tumorspheres (FTs) from lung and breast cancer cells by culturing them in serum-free media without the addition of any external mitogenic stimulation, and subsequently (after 2 weeks) re-incubating them back in serum-containing media to simulate routine culture conditions (RCCs). We found that cancer cells are extremely plastic: cells grown under RCCs become multidrug-resistant when grown as FTs, but upon re-incubation under RCCs quickly re-attach and lose the acquired resistance. These phenotypic changes are accompanied by concomitant changes in the expression of key proteins associated with multiple pathways important for chemoresistance, survival, and stemness maintenance. Therefore, our strategy provides an excellent experimental model to study environmental factors that modulate the plasticity of cancer cells. J. Cell. Physiol. 232: 2280–2286, 2017. © 2016 Wiley Periodicals, Inc.In this report, we have developed a protocol to grow lung and breast cancer cells as floating tumorspheres (FTs) in the absence of any external mitogenic stimulation. By using routine culture conditions (RCCs), FTs, and revertant cells (FTs cell that reattach when incubated back under RCCs), we simulated one cycle of environmental conditions in order to study the plasticity of cancer cells. We found that cancer cells are extremely plastic: cells grown under RCCs become multidrug-resistant, when grown as FTs but upon re-incubation under RCCs quickly re-attach and lose the acquired resistance. These phenotypic changes are accompanied by concomitant changes in the expression of key proteins associated with multiple pathways important for chemoresistance, survival, and stemness maintenance.
  • Promoting Cancer Control in Africa With “Ubuntu”: A Report of the
           African Organization for Research and Training in Africa (AORTIC) 10th
           Conference, 2015 in Marrakech, Morocco
    • Abstract: The objectives of the African Organization for Research and Training in Cancer (AORTIC), includes bringing products of decades of advances in cancer research to African populations through local and international collaboration. The consistent and huge growth in participation in the conferences and the diversity of the nations is a witness to the success of the organization thus far. The theme for the Tenth AORTIC International Conference on Cancer in Africa in Morocco in 2015 was “Road map to Cancer Control in Africa” and topics of discussion of paramount importance for low- and middle-income African countries included childhood cancers such as BL, cancers of the cervix, breast, and prostate; cancers associated with HIV-infection such as cervical, vulvar, and anal; as well as cancer care challenges associated with palliative care. The role of environmental factors that underlie some epigenetic changes in some of the cancers was emphasized. Oral and poster presentations from various parts of the continent indicate the growth of basic and translational science of cancer in the region, with studies revealing regional diversity in the frequencies of the triple-negative breast cancer, cervical cancer, prostate cancer, HCC, and Burkitt's lymphoma. There was a sign that Africa is trying to keep pace with the paradigm shift and focusing on translational medicine. This was shown by suggestions for application of genome-wide association studies, new generation sequencing, as well as the evaluation of single nucleotide polymorphisms that may be responsible for variable susceptibility in some of the prevalent cancers in people of African descent. J. Cell. Physiol. 232: 2287–2295, 2017. © 2016 Wiley Periodicals, Inc.
  • New Roles of Lkb1 in Regulating Adipose Tissue Development and
    • Abstract: Adipose tissues regulate energy metabolism and reproduction. There are three types of adipocytes (brown, white, and beige adipocytes) in mammals. White adipocytes store energy and are closely associated with obesity and other metabolic diseases. The beige and brown adipocytes have numerous mitochondria and high levels of UCP1 that dissipates lipid to generate heat and defend against obesity. The global epidemic of obesity and its associated metabolic diseases urge an imperative need for understating the regulation of adipogenesis. Liver kinase B1 (Lkb1), also called STK11, is a master kinase of the AMPK subfamily and plays crucial roles in regulating glucose and energy homeostasis in various metabolic tissues. In this review, we focus on the regulatory roles of Lkb1 in regulating preadipocyte differentiation, adipose tissue development, and thermogenesis. J. Cell. Physiol. 232: 2296–2298, 2017. © 2016 Wiley Periodicals, Inc.(i) Lkb1 regulates adipocyte progenitors differentiation; (ii) Adipocyte-specific deletion of Lkb1 promotes brown adipose tissue development and browning of white adipose tissues; (iii) Lkb1 affects Ucp1 expression and thermogenesis.
  • Polydeoxyribonucleotides (PDRNs) From Skin to Musculoskeletal Tissue
           Regeneration via Adenosine A2A Receptor Involvement
    • Abstract: Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production, and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo, and clinical studies (1990–2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area. A total of 29 studies were found by PubMed and searches: 20 were on skin (six in vitro, six in vivo, one vitro/vivo, seven clinical studies), while the other nine regarded bone (one in vitro, two in vivo, one clinical studies), cartilage (one in vitro, one vitro/vivo, two clinical studies), or tendon (one clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity, and reduced pain and inflammation, through the activation of adenosine A2A receptor. PDRNs are currently used for bone, cartilage, and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of three to a maximum of five times and they act trough the activation of A2A receptor. Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. J. Cell. Physiol. 232: 2299–2307, 2017. © 2016 Wiley Periodicals, Inc.Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production, and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. A total of 29 studies were found by PubMed and searches: 20 were on skin (six in vitro, six in vivo, one vitro/vivo, seven clinical studies), while the other nine regarded bone (one in vitro, two in vivo, one clinical studies), cartilage (one in vitro, one vitro/vivo, two clinical studies), or tendon (one clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity, and reduced pain and inflammation, through the activation of adenosine A2A receptor.
  • SirT3 and p53 Deacetylation in Aging and Cancer
    • Abstract: The mammalian Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase. The mammalian Sirtuins family has identified seven different types of sirtuin. Sirtuins 3(SirT3) is localized to mitochondria, and is a multiple functional protein deacylase through deacetylating a variety of substrates. Cellular senescence represents a permanent withdraw from cell cycle in response to diverse stress; it is controlled by the p53 and retinoblastoma protein (RB) tumor suppressors, and constitutes a potent anticancer mechanisms. p53 can be deacetylated by a protein complex containing histone deacetylases (HDAC1). There is possibility that SirT3 may also deacetylate p53. We introduce the recent research on the SirT3-p53 interplay directly and indirectly, and discuss the significance of p53 deacetylation by SirT3 in the aging and cancer. J. Cell. Physiol. 232: 2308–2311, 2017. © 2016 Wiley Periodicals, Inc.Cellular senescence is controlled by the p53 and retinoblastoma protein (RB) tumor suppressors, and constitutes a potent anticancer mechanisms. p53 may be deacetylated by Sirtuins 3.
  • The Sabotaging Role of Myeloid Cells in Anti-Angiogenic Therapy:
           Coordination of Angiogenesis and Immune Suppression by Hypoxia
    • Abstract: Tumor angiogenesis has become a promising target for anti-tumor therapy. Unfortunately, the somewhat inevitable occurrence of resistance has limited the efficacy of anti-angiogenic therapy. In addition to their well-established role in immune suppression, bone marrow-derived myeloid cells actively contribute to tumor angiogenesis. More importantly, myeloid cells constitute one of the major mechanisms of resistance to angiogenesis inhibition. As the most pervasive feature in tumor microenvironment, hypoxia is able to initiate both pro-angiogenic and immunosuppressive capacities of myeloid cells. Tumor adapts to hypoxic stress primarily through signaling mediated by hypoxic inducible factors (HIFs) and consequently utilizes hypoxia to its own advantage. In this regard, hypoxia orchestrates both angiogenesis and immune evasion to support tumor growth. In this article, we will review available information on the sabotaging role of myeloid cells in anti-angiogenic therapy. We will also discuss how hypoxia coordinates the dual-role cellular and molecular participants in microenvironment to maximize the efficiency of angiogenesis and immunosuppression to promote tumor progression. J. Cell. Physiol. 232: 2312–2322, 2017. © 2016 Wiley Periodicals, Inc.The signaling mediated by hypoxia-inducible factors (HIFs) provides tumor cells and stromal cells with adaptation to the hypoxic microenvironment. In this regards, hypoxia and hypoxia-driven signaling orchestrate the cellular and molecular participants in angiogenesis and immune suppression, maximizing the efficiency of both programs.
  • Proinflammatory signaling functions of thrombin in cancer
    • Abstract: Thrombin-induced activation of protease-activated receptors (PARs) represents a link between inflammation and cancer. Proinflammatory signaling functions of thrombin are associated with several inflammatory diseases including neurodegenerative, cardiovascular, and of special interest in this review cancer. Thrombin-induced inflammatory responses up-regulates expression of cytokines, adhesion molecules, angiogenic factors, and matrix-degrading proteases that facilitate tumor cells proliferation, angiogenesis, invasion, and metastasis. This review summarizes the current knowledge about the mechanisms of thrombin-mediated proinflammatory responses in cancer pathology for a better understanding and hence a better management of this disease.Thrombin-induced inflammatory responses up-regulates expression of cytokines, adhesion molecules, angiogenic factors, and matrix-degrading proteases that facilitate tumor cells proliferation, angiogenesis, invasion, and metastasis. This review summarizes the current knowledge about the mechanisms of thrombin-mediated proinflammatory responses in cancer pathology for a better understanding and hence a better management of this disease.
  • PCSK9 at the crossroad of cholesterol metabolism and immune function
           during infections
    • Abstract: Sepsis, a complex and dynamic syndrome resulting from microbial invasion and immune system dysregulation, is associated with an increased mortality, reaching up to 35% worldwide. Cholesterol metabolism is often disturbed during sepsis, with low plasma cholesterol levels being associated with poor prognosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of the low-density lipoprotein receptor (LDLR), thus regulating intracellular and plasma cholesterol levels. PCSK9 is often upregulated during sepsis and might have a detrimental effect on immune host response and survival. Accordingly, PCSK9 reduces lipopolysaccharide uptake and clearance by human hepatocytes. Moreover, PCSK9 upregulation exacerbates organ dysfunction and tissue inflammation during sepsis, whereas a protective effect of PCSK9 deficiency has been documented in septic patients. Although a possible detrimental impact of PCSK9 on survival has been described, some beneficial effects of PCSK9 on immune response may be hypothesized. First, PCSK9 is associated with increased plasma cholesterol levels, which might be protective during sepsis. Second, PCSK9, by stimulating LDLR degradation and inhibiting reverse cholesterol transport (RCT), might promote preferential cholesterol accumulation in macrophages and other immune cells; these events might improve lipid raft composition and augment toll-like receptor function thus supporting inflammatory response. Hence, a more clear definition of the role of PCSK9 in septic states might provide additional insight in the understanding of the sepsis-associated immune dysregulation and enhance therapeutic outcomes.
  • Pin1, the Master Orchestrator of Bone Cell Differentiation
    • Abstract: Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT-P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1−/− mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt-Related Transcription Factor 2), SMAD1/5, and β-catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine-rich nucleic acid binding protein 1), C-FOS, and DC-STAMP. The phenotype of Pin1−/− mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. J. Cell. Physiol. 232: 2339–2347, 2017. © 2016 Wiley Periodicals, Inc.Pin1 is a peptidyl prolyl cis–trans isomerase that isomerizes only phosphorylated Ser/Thr-Pro motifs. Upon binding, Pin1 mediates structural modifications resulting in dramatic changes in substrate activity. In this review, we have summarized the known roles of Pin1 in molecular regulation of bone cell differentiation. Pin1 binds to Runx2, Smad1/5, and β-catenin, which act downstream of the FGF, BMP, and Wnt signaling pathways, respectively. Binding of Pin1 increases the stability and transcriptional activity of Runx2 and Smad1/5. Pin1 also inhibits the nuclear export of β-catenin and increases the level of transcriptionally active nuclear β-catenin.
  • The Role of Exercise and TFAM in Preventing Skeletal Muscle Atrophy
    • Abstract: Skeletal muscle atrophy is the consequence of protein degradation exceeding protein synthesis. This arises for a multitude of reasons including the unloading of muscle during microgravity, post-surgery bedrest, immobilization of a limb after injury, and overall disuse of the musculature. The development of therapies prior to skeletal muscle atrophy settings to diminish protein degradation is scarce. Mitochondrial dysfunction is associated with skeletal muscle atrophy and contributes to the induction of protein degradation and cell apoptosis through increased levels of ROS observed with the loss of organelle function. ROS binds mtDNA, leading to its degradation and decreasing functionality. Mitochondrial transcription factor A (TFAM) will bind and coat mtDNA, protecting it from ROS and degradation while increasing mitochondrial function. Exercise stimulates cell signaling pathways that converge on and increase PGC-1α, a well-known activator of the transcription of TFAM and mitochondrial biogenesis. Therefore, in the present review we are proposing, separately, exercise and TFAM treatments prior to atrophic settings (muscle unloading or disuse) alleviate skeletal muscle atrophy through enhanced mitochondrial adaptations and function. Additionally, we hypothesize the combination of exercise and TFAM leads to a synergistic effect in targeting mitochondrial function to prevent skeletal muscle atrophy. J. Cell. Physiol. 232: 2348–2358, 2017. © 2016 The
      Authors . Journal of Cellular Physiology Published by © 2016 Wiley Periodicals, Inc.Skeletal muscle atrophy via unloading and disuse of the musculature is the consequence of protein degradation exceeding protein synthesis. This state is correlated with increased markers of mitochondrial dysfunction. We hypothesize targeting increased mitochondrial function through exercise and mitochondrial transcription factor A treatments will diminish skeletal muscle atrophy after a period of muscle unloading and disuse.
  • Neurotransmitters: The Critical Modulators Regulating Gut–Brain Axis
    • Abstract: Neurotransmitters, including catecholamines and serotonin, play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the “fight or flight” response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in GI and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, GI innate immune system, and the microbiome. Furthermore, in pathological states, such as inflammatory bowel disease (IBD) and Parkinson's disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of GI symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state-of-the-art research and literature regarding the role of neurotransmitters in regulation of normal GI physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. J. Cell. Physiol. 232: 2359–2372, 2017. © 2016 Wiley Periodicals, Inc.Neurotransmitters affect microbiota in the gut: Neurotransmitters, including serotonin, alter the microbiota in the gut that can modulate the production of cytokines and bacterial by-products, leading to either healthy or diseased state.
  • Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6
           Myotubes Incubated With Palmitate
    • Abstract: The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160−/PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160−). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160− vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160−/PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373–2386, 2017. © 2016 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals Inc.AS160/TBC1D4 is a key regulator of GLUT4 translocation to the plasma membrane in myocytes, moreover, it role in the control of protein mediated fatty acids transport is also likely. The current study indicates that AS160/TBC1D4 may influence not only FA transporters content, and therefore their influx, but also their storage and oxidation. However, the effect differs depending on the events sequence, i.e., AS160 knock-down prior to or after palmitate incubation.
  • Super-resolution structure of DNA significantly differs in buccal cells of
           controls and Alzheimer's patients
    • Abstract: The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p 
  • Staphylococcal protein A promotes osteoclastogenesis through MAPK
           signaling during bone infection
    • Abstract: Bone infection is a common and serious complication in the orthopedics field, which often leads to excessive bone destruction and non-union. Osteoclast is the only type of cells which have the function of bone resorption. Its over activation is closely related to excessive bone loss. Staphylococcus aureus (S. aureus) is a major pathogen causing bone infection, which can produce a large number of strong pathogenic substances staphylococcal protein A (SPA). However, few studies were reported about the effects of SPA on osteoclastogenesis. In our study, we observed that S. aureus activated osteoclasts and promoted bone loss in bone infection specimens. Then, we investigated the effects of SPA on RANKL-induced osteoclastogenesis in vitro, the results revealed that SPA promoted osteoclastic differentiation and fusion, and enhanced osteoclastic bone resorption. In addition, we also showed that SPA upregulated the expression of NFATc1 and c-FOS through the activation of MAPK signaling to promote osteoclastogenesis. Our findings might help us better understand the pathogenic role of S. aureus in bone infection and develop new therapeutic strategies for infectious bone diseases.Staphylococcal protein A (SPA) is one of the most important pathogenic components of Staphylococcus aureus (S. aureus). Here we reported that S. aureus caused bone loss in bone infection specimens. In vitro study showed that SPA promoted osteoclastic differentiation, fusion and bone resorption activity by upregulating NFATc1 and c-FOS through MAPK signaling.
  • Reduced primary cilia length and altered Arl13b expression are associated
           with deregulated chondrocyte Hedgehog signaling in alkaptonuria
    • Abstract: Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.This study investigates whether the rare inherited disease alkaptonuria (AKU) is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling, which is known to mediate cartilage degradation in osteoarthritis. AKU results in alterations to primary cilia structure which are associated with ciliary trafficking defects with the potential to impact a range of fundamental signaling pathways. In particular, AKU chondrocytes demonstrated a complete inability to activate Hedgehog signaling in response to endogenous ligand; which we propose may be the result of the observed dysfunction of cilia structure and trafficking. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.
  • Aging Selectively Modulates Vitamin C Transporter Expression Patterns in
           the Kidney
    • Abstract: In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium-vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging-induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2-photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT-PCR, and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over-expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. J. Cell. Physiol. 232: 2418–2426, 2017. © 2016 Wiley Periodicals, Inc.Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging-induced changes in the expression of vitamin C transporters in renal tissue.
  • MAP Kinase-Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic
           Modification of Chromatin During Osteoblast Differentiation
    • Abstract: RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427–2435, 2017. © 2016 Wiley Periodicals, Inc.The RUNX2 transcription factor requires MAP kinase-dependent phosphorylation to become transcriptionally active. Here, we show that phosphorylation is required for transcription-related changes in histone acetylation and methylation and recruitment of cofactors such as P300/CBP and RNA polymerase II to promoter regions of bone-related genes.
  • Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume
           Regulation at the End of Oogenesis
    • Abstract: Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [3H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436–2446, 2017. © 2016 Wiley Periodicals, Inc.Independent cell volume regulation in the mouse oocyte normally becomes active after ovulation is triggered in a two-step process: the strong adhesion of the oocyte to the surrounding zona pellucida is released and glycine transport via the GLYT1 transporter is activated. Both mechanisms develop at the end of oocyte growth but remain suppressed within the ovarian follicle.
  • Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein
           Phosphorylation, Cell Shape, and Adhesion in Trabecular Meshwork Cells
    • Abstract: Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-β2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-β2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447–2460, 2017. © 2016 Wiley Periodicals, Inc.Our results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma.
  • Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light
           and Methods to Alleviate it
    • Abstract: In live cell imaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells and tested methods that have or could be used to alleviate photodamage. Unlabeled RPE1 cells were given single 0.5–2.5 min irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. Four hundred eighty-eight nanometer light produced a dose-dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell death for the highest dose. For 546 nm light we found a 10–15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell death for the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488 nm light irradiations of unlabeled cells, reducing the intensity 10-fold or spreading the exposures out as a series of 10 sec pulses at 1 min intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis. Thus, for long-term imaging there can be value to using RFP constructs and for GFP-tagged proteins reducing oxidative processes. J. Cell. Physiol. 232: 2461–2468, 2017. © 2016 Wiley Periodicals, Inc.We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells. Four hundred eighty-eight nanometer light produced a dose-dependent decrease in the percentage of cells that progressed to mitosis, and slowing of the cell cycle for some of those entering mitosis and for 546 nm light, we found a 10–15% reduction in the percentage of cells entering mitosis, but no strong dose dependency. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis.
  • Cardiotrophin-1 Regulates Adipokine Production in 3T3-L1 Adipocytes and
           Adipose Tissue From Obese Mice
    • Abstract: Cardiotrophin-1 (CT-1) belongs to the IL-6 family of cytokines. Previous studies of our group revealed that CT-1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT-1 on the production of several adipokines involved in body weight regulation, nutrient metabolism, and inflammation. For this purpose, 3T3-L1 adipocytes were incubated with recombinant protein CT-1 (rCT-1) (1–40 ng/ml) for 1 and 18 h. Moreover, the acute effects of rCT-1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high-fat fed obese mice. In 3T3-L1 adipocytes, rCT-1 treatment downregulated the expression and secretion of leptin, resistin, and visfatin. However, rCT-1 significantly stimulated apelin mRNA and secretion. rCT-1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2, and STAT3. Interestingly, pre-treatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT-1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT-1 on apelin production. In contrast, acute administration of rCT-1 (30 min and 3 h) to diet-induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT-1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT-1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. J. Cell. Physiol. 232: 2469–2477, 2017. © 2016 Wiley Periodicals, Inc.The current study demonstrates that CT-1 is capable of regulating the secretory pattern of adipokines by adipocytes, decreasing the production of pro-inflammatory adipokines, such as leptin, resistin, and visfatin, while stimulating the secretion of apelin. These suggest that the beneficial actions of CT-1 on glucose and lipid metabolism could be also related to its ability to control adipokine secretion and therefore the cross-talk between adipose tissue and other key metabolic organs.
  • Overexpression of Large-Conductance Calcium-Activated Potassium Channels
           in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration
    • Abstract: Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478–2488, 2017. © 2016 Wiley Periodicals, Inc.We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated in GBM stem-like cells expressing CD133, as well as other stem cell markers. In addition, transwell migration tests showed that GBM stem-like cell migration was very sensitive to BK channel block.
  • Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal
           Epithelial Cells
    • Abstract: Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-β), or pro-inflammatory mediators (IL-1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489–2496, 2017. © 2016 Wiley Periodicals, Inc.We have developed a method for culturing primary intestinal epithelial cells useful for the study of their interaction with microbes.
  • Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and
           Lung Tumorspheres
    • Abstract: Lung cancer is a leading cause of cancer-related death in the United States. Although several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides—Convallatoxin and Peruvoside on lung cancer cells. Although both drugs had significant anti-proliferative effects on H460 and Calu-3 lung cancer cells, Convallatoxin demonstrated twofold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs—p53-underexpressing cells were less sensitive as compared to p53-positive H460 cells. Further, assessment of p53-underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose-dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation, and EMT. Our data suggest, a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti-cancer potential of this class of drugs. J. Cell. Physiol. 232: 2497–2507, 2017. © 2016 Wiley Periodicals, Inc.The cardiac glycosides convallatoxin and peruvoside exert potent anti-cancer effects in lung cancer cells. A significant inhibition of cell migration, invasion, and lung tumorsphere formation was observed in response to both drugs.
  • The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on
           Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive
           Myocardial Hypertrophy
    • Abstract: Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 (ET-1) system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET-1 on cardiomyocytes. Indeed, ET-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET-1-dependent reduction in cell shortening and attenuated up-regulation of CaR. Down-regulation of RAMP-1 reduced CaR responsiveness. In conclusion, ET-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508–2518, 2017. © 2016 Wiley Periodicals, Inc.Current manuscript is the first report indicating the ET-1-dependent upregulation of CaR receptor in cardiomyocytes and it shows that this upregulation allows cardiac cells to maintain proper function. Second new finding indicates involvement of ETA and /or ETB1 receptors in adaptive hypertrophy in cardiomyocytes, whereas ETB2 receptors activation leads to mal-adaptive effect. Finally, chaperone activity of RAMP1 is required for the functional coupling of CaR in cardiomyocytes.
  • Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control
           Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem
    • Abstract: Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519–2527, 2017. © 2016 Wiley Periodicals, Inc.Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) are an attractive potential source of multipotent cells for tissue replacement therapies. Here, we study the ability of WJ-MSCs to engage the osteoblast differentiation by analyzing epigenetic signatures at the promoter regions of the two master regulators of this process, RUNX2/P57 and SP7. We find that the RUNX2/p57, but not SP7, gene is expressed in WJ-MSCs stimulated to differentiate to osteoblasts. This process is accompanied by the enrichment of activating histone marks at the RUNX2-P57 P1 promoter and maintenance of epigenetic silencing histone marks at the SP7 promoter.
  • Bone Shaft Revascularization After Marrow Ablation Is Dramatically
           Accelerated in BSP-/- Mice, Along With Faster Hematopoietic Recolonization
    • Abstract: The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin− (×3) as well as LSK (Lin− IL-7Rα− Sca-1hi c-Kithi, ×2) and hematopoietic stem cells (HSC: Flt3− LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin− was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528–2537, 2017. © 2016 Wiley Periodicals, Inc.In mice with a knockout of the SIBLING gene, Bone Sialoprotein (BSP-/-) vascular and hematopoietic recolonization are faster than in wild type, despite lower medullary bone formation. This goes with enhanced expression of osteopontin and VEGF, and downregulation of Dentin Matrix Protein 1, another SIBLING shown to inhibit angiogenesis. Lack of BSP thus affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, in part through the mediation of pro- and antiangiogenic factors.
  • Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates
           Osteoclast Differentiation via the Ca2+/NFATc1 Axis
    • Abstract: DC-STAMP is a multi-pass transmembrane protein essential for cell–cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP−/− mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC-STAMP−/− cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell–cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538–2549, 2017. © 2016 Wiley Periodicals, Inc.
  • Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is
           Involved in Cell Cycle Progression
    • Abstract: Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550–2557, 2017. © 2016 Wiley Periodicals, Inc.
  • Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate
           Immunity of Human Keratinocytes In Vitro
    • Abstract: Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558–2568, 2017. © 2016 Wiley Periodicals, Inc.Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from acacia or baobab seeds induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses.
  • Maintenance of Bone Homeostasis by DLL1-Mediated Notch Signaling
    • Abstract: Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals Inc.The proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-Notch signaling directly controls the physiological expansion and differentiation of osteoblasts and indirectly affects the maturation and function of osteoclasts, thereby ensuring the homeostasis of bone mass.
  • Let-7i-Induced Atg4B Suppression Is Essential for Autophagy of Placental
           Trophoblast in Preeclampsia
    • Abstract: Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let-7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let-7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let-7i mimics suppressed the autophagic activities in human HTR-8/SVneo trophoblast cell line (HTR-8) and human placental choriocarcinoma cell line JEG-3, whereas let-7i inhibitor enhanced the activities. As a potential target of let-7i, autophagy-related 4B cysteine peptidase (Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let-7i using dual luciferase reporter assay. Furthermore, these trophoblast-like cells transfected with the let-7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let-7i mimic-reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let-7i inhibitor-induced the level of LC3II/I expression. Taken together, these findings suggest that let-7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. J. Cell. Physiol. 232: 2581–2589, 2017. © 2016 Wiley Periodicals, Inc.This study provided insight into the role of let-7i in the pathophysiology of PE. Our study demonstrates for the first time that a novel let-7i-Atg4B-autophagy signaling pathway plays a key role in PE. This finding suggests that miRNAs are key molecules involved in autophagic activity, and might be regard as a new therapeutic target against PE.
  • 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
  • Intimate Connections: Inositol Pyrophosphates at the Interface of
           Metabolic Regulation and Cell-Signaling
    • Abstract: Inositol pyrophosphates are small, diffusible signaling molecules that possess the most concentrated three-dimensional array of phosphate groups in Nature; up to eight phosphates are crammed around a six-carbon inositol ring. This review discusses the physico-chemical properties of these unique molecules, and their mechanisms of action. Also provided is information on the enzymes that regulate the levels and hence the signaling properties of these molecules. This review pursues the idea that many of the biological effects of inositol pyrophosphates can be rationalized by their actions at the interface of cell signaling and metabolism that is essential to cellular and organismal homeostasis. This article is protected by copyright. All rights reserved
  • Effects of Mechanical Loading on Human Mesenchymal Stem Cells for
           Cartilage Tissue Engineering
    • Abstract: Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. This article is protected by copyright. All rights reserved
  • Low shear stress induces endothelial reactive oxygen species via the
           AT1R/eNOS/NO pathway
    • Abstract: Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS-mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyne/cm2) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time-dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time-dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L-arginine, SNP) and increased by the eNOS inhibitor L-NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO. This article is protected by copyright. All rights reserved
    • Abstract: The naturally-occurring sulfonium compound S-adenosyl-L-methionine (AdoMet) is an ubiquitous sulfur-nucleoside that represents the main methyl donor in numerous methylation reactions. In recent years, it has been shown that AdoMet possesses antiproliferative properties in various cancer cells, but the molecular mechanisms at the basis of the effect induced by AdoMet have been only in part investigated. In the present study, we found that AdoMet strongly inhibited the proliferation of breast cancer cells MCF-7 by inducing both autophagy and apoptosis. AdoMet consistently enhanced the levels of the autophagy markers beclin-1 and LC3B-II, and caused a significant increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by poly (ADP ribose) polymerase (PARP) and caspase 9, and 6 cleavage. Notably, AdoMet, already at low doses, raised the percentage of cells in G2/M phase of cell cycle by down-regulating the expression of cell cycle-regulatory proteins cyclin B and cyclin E with a remarkable increase of p53, p27 and p21. We also evaluated the combination of AdoMet and the autophagy inhibitor chloroquine (CLC) showing that autophagy block is synergistic in inducing both growth inhibition and apoptosis. These effects were paralleled by a strong inhibition of the activity of AKT and of the downstream effector mTOR and by an increased cleavage of caspase-6 and PARP.These data suggest, for the first time, that autophagy can act as an escape mechanism from the apoptotic activity of AdoMet, and that AdoMet could be used in combination with CLC or its analogs in the treatment of breast cancer. This article is protected by copyright. All rights reserved
  • NAD+: A big player in cardiac and skeletal muscle remodeling and aging
    • Abstract: In the past decade, NAD+ has gained importance for its beneficial effects as antioxidant and antiaging molecule. A paper in science by Zhang et al (2016) has described that NAD+ when replenished, ameliorates muscle dystrophy in mice by improving mitochondrial function. NAD+ was also demonstrated by the authors to improve the life span of mice. Cox et al (2002) demonstrated the cardiac effects of NAD+ which mitigated chronic heart failure via mitochondrial redox state mechanism. Cox et al (2002) also demonstrated that NAD+ is provided in the drinking water, it improves cardiac relaxation in volume overload model of heart failure. Although NAD+ has a profound anti-aging and anti-oxidant effects, its effect on humans and use as a dietary supplement needs more exploration. This article is protected by copyright. All rights reserved
  • PP2A deactivation is a common event in oral cancer and reactivation by
           FTY720 shows promising therapeutic potential
    • Abstract: Protein phosphatase 2A (PP2A) is a tumor suppressor gene, that has been frequently deactivated in many types of cancer. However its molecular and clinical relevance in oral squamous cell carcinoma (OSCC) remain unclear. Here we show that, PP2A deactivation is a common event in oral cancer cells and hyperphosphorylation in its tyrosine-307 (Y307) residue contributes to PP2A deactivation. PP2A restoration by FTY720 treatment reduced cell growth and decreased GSK-3β phosphorylation without significantly altering other PP2A targets. We further detected PP2A phosphorylation in 262 OSCC tissues. Increased expression of p-PP2A in the tumor tissues was significantly correlated with higher N2/N3- stage (aOR =2.1, 95% CI: 1.2-3.8). Patients with high p-PP2A expression had lower overall survival rates than those with low expression. Hazard ratio analysis showed that, high p-PP2A expression was significantly associated with mortality density (aOR= 2.2, 95% CI: 1.2-4.0) and lower 10-year overall survival (P = 0.027) in lymph node metastasis. However, no interaction was observed between p-PP2A expression and lymph node metastasis. All our results suggest that PP2A is frequently deactivated is oral cancer and determines poor outcome, restoring its expression by FTY720 can be an alternative therapeutic approach in OSCC. This article is protected by copyright. All rights reserved
  • Loss of TET1 facilitates DLD1 colon cancer cell migration via
           H3K27me3-mediated down-regulation of E-cadherin
    • Abstract: Epigenetic modifications such as histone modifications and cytosine hydroxymethylation are linked to tumorigenesis. Loss of 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation 1 (TET1) down-regulation facilitates tumor initiation and development. However, the mechanisms by which loss of TET1 knockdown promotes malignancy development remains unclear. Here, we report that TET1 knockdown induced epithelial-mesenchymal transition (EMT) and increased cancer cell growth, migration and invasion in DLD1 cells. Loss of TET1 increased EZH2 expression and reduced UTX-1 expression, thus increasing histone H3K27 tri-methylation causing repression of the target gene E-cadherin. Ectopic expression of the H3K27 demethylase UTX-1 or EZH2 depletion both impeded EZH2 binding caused a loss of H3K27 methylation at epithelial gene E-cadherin promoter, thereby suppressing EMT and tumor invasion in shTET1 cells. Conversely, UTX-1 depletion and ectopic expression of EZH2 enhanced EMT and tumor metastasis in DLD1 cells. These findings provide insight into the regulation of TET1 and E-cadherin and identify EZH2 as a critical mediator of E-cadherin repression and tumor progression.This article is protected by copyright. All rights reserved
  • The emerging role of NPNT in tissue injury repair and bone homeostasis
    • Abstract: Nephronectin (NPNT), a highly conserved extracellular matrix protein, plays an important role in regulating cell adhesion, differentiation, spreading and survival. NPNT protein belongs to epidermal growth factor (EGF)-like superfamily and exhibits several common structural determinants; including EGF-like repeat domains, MAM domain (Meprin, A5 Protein, and Receptor Protein-Tyrosine Phosphatase µ), RGD motif (Arg-Gly-Asp) and a coiled-coil domain. It regulates integrins-mediated signaling pathways via the interaction of its RGD motif with integrin α8β1. Recent studies revealed that NPNT is involved in kidney development, renal injury repair, atrioventricular canal differentiation, pulmonary function, and muscle cell niche maintenance. Moreover, NPNT regulates osteoblast differentiation and mineralization, as well as osteogenic angiogenesis. Altered expression of NPNT has been linked with the progression of certain types of cancers, such as spontaneous breast tumor metastasis and malignant melanoma. Interestingly, NPNT gene expression can be regulated by a range of external factors such as tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), oncostatin M (OSM), bone morphogenic protein 2 (BMP2), Wnt3a, Vitamin D3 and microRNA-378 (miR378). Further understanding the cellular and molecular mechanisms by which NPNT regulates tissue homeostasis in an organ-specific manner is critical in exploring NPNT as a therapeutic target for tissue regeneration and tissue engineering. This article is protected by copyright. All rights reserved
  • Histomorphology and Innate Immunity during the Progression of
           Osteoarthritis: Does Synovitis Affect Cartilage Degradation'
    • Abstract: Osteoarthritis (OA) is a common chronic degenerative disease that affects all joints. At present, the pathological processes and mechanisms of OA are still unclear. Innate immunity, a key player in damage to the structure of the joint and the mechanism by which the host attempts to repair OA, affects all pathological stages of the disease. In the present study, our aim was to assess changes in innate immunity during the pathological processes of OA in articular cartilage (AC) and the synovial membrane (SM), which are the major structures in joints, and to systematically examine the histological changes in AC and SM in mild, moderate and severe cases of OA, in order to further speculate about the manner in which the interactions of AC and SM are facilitated by innate immunity. Histological methods (including HE and Safranin O-fast green staining) were used to assess the morphological changes within AC and SM tissues in healthy and mild, moderate or severe OA rats. Mankin's and Wakitani scorings were conducted on the AC to assess the degradation of the cartilage and the extent of synovial inflammation. Localization and intensity of expression of the three key proteins: TLR4 (upstream), MyD88 (midstream) and NF-κB (downstream) of the inflammatory pathway believed to be involved in innate immune system–induced AC and SM structural degeneration, were examined using immunofluorescent staining. This technique allows a more stereoscopic visualization of the cellular structure by labeling the relative positions of the proteins of interest using fluorescent markers. In addition, significantly enhanced fluorescence signals could be detected by fluorescence microscopy in areas with more severe tissue damage. Combined with the terminal-deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the phenomenon of apoptosis in chondrocytes and synoviocytes was observed. We found that TLR4, MyD88 and NF-κB were significantly expressed in the AC and SM of the OA rats, while very little expression was detected in healthy rats. We speculate that innate immunity is programmed to play a role in mediating the repair of tissue damage. Considering the differences in the expression levels of these proteins in AC and SM during different stages of the disease, the SM might play a driving role in the process of creating lesions. Western blots (WBs) were combined to confirm the trend of expression of TLR4, MyD88 and NF-κB in SM tissues during different stages of OA. The expression of an important specificity protein TRAF6, in addition to IL-1β and TNF-α, representative downstream inflammatory mediators, were also measured in order to verify the key role of the signaling pathway in OA. In addition, we systematically describe the principal cell types in the SM which participate in innate immunity. Our results showed that the damage to AC and SM within the joints progressively worsened during the course of the disease, and that the innate immune system was closely involved in the AC and SM during each stage of OA. These findings also confirmed that SM may affect the pathological changes in AC through the innate immune system, and therefore affect the progress of OA. This article is protected by copyright. All rights reserved
  • Intranuclear and higher-order chromatin organization of the major histone
           gene cluster in breast cancer
    • Abstract: Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression. This article is protected by copyright. All rights reserved
  • MSCs Ameliorates DPN induced Cellular Pathology via [Ca2+]i Homeostasis
           and Scavenging the Pro-inflammatory cytokines
    • Abstract: The MSCs of various origins are known to ameliorate or modulate cell survival strategies. We investigated, whether UCB MSCs could improve the survival of the human neuronal cells and/or fibroblast assaulted with DPN sera. The results showed, the co-culture of UCB MSCs with human neuronal cells and/or fibroblasts could effectively scavenge the pro-inflammatory cytokines TNF-α, IL-1β, IFN-γ and IL − 12 and control the pro-apoptotic expression of p53/Bax. Further co-culture of UCB MSCs have shown to induce anti-inflammatory cytokines like IL-4, IL-10 and TGF-β and anti-apoptotic Bclxl/Bcl2 expression in the DPN sera stressed cells. Amelioration of elevated [Ca2+]i and cROS, the portent behind the NFκB/Caspase-3 mediated inflammation in DPN rescued the cells from apoptosis. The results of systemic administration of BM MSCs improved DPN pathology in rat as extrapolated from human cell model. The BM MSCs ameliorated prolonged distal motor latency (control: 0.70 ± 0.06, DPN: 1.29 ± 0.13 m/s DPN + BM MSCs: 0.89 ± 0.02 m/s, p < 0.05) and lowered high amplitude of compound muscle action potentials (CMAPs) (control: 12.36 ± 0.41, DPN: 7.52 ± 0.61 mV, DPN + MSCs: 8.79 ± 0.53 mV, p < 0.05), while slowly restoring the plasma glucose levels. Together, all these results showed that administration of BM or UCB MSCs improved the DPN via ameliorating pro-inflammatory cytokine signaling and [Ca2+]i homeostasis. This article is protected by copyright. All rights reserved
  • Biological function and histone recognition of family IV
           bromodomain-containing proteins
    • Abstract: Bromodomain proteins function as epigenetic readers that recognize acetylated histone tails to facilitate the transcription of target genes. There are approximately 60 known human bromodomains, which are divided into 8 sub-families based on structural conservation. The bromodomain-containing proteins in family IV include 7 members (BRPF1, BRPF2, BRPF3, BRD7, BRD9, ATAD2 and ATAD2b). The bromodomains of each of these proteins recognize and bind acetyllysine residues on histone tails protruding from the nucleosome. However, the histone marks recognized by each bromodomain protein can be very different. The BRPF1 subunit of the MOZ histone acetyltransferase (HAT) recognizes acetylated histones H2AK5ac, H4K12ac, H3K14ac, H4K8ac and H4K5ac. While the bromodomain of BRD7, a member of the SWI/SNF complex, was shown to preferentially recognize acetylated histones H3K9ac, H3K14ac, H4K8ac, H4K12ac and H4K16ac. The bromodomains of BRPF2 and BRPF3 have similar sequences, and function as part of the HBO1 HAT complex, but there is limited data on which histone ligands they bind. Similarly, there is little known about the histone targets of the BRD9 and ATAD2b bromodomain proteins. Interestingly, the ATAD2 bromodomain was recently shown to preferentially bind to the di-acetylated H4K5acK12ac mark found in newly synthesized histones following DNA replication. However, despite the physiological importance of the family IV bromodomains, little is known about how they function at the molecular or atomic level. In this review we summarize our understanding of how family IV bromodomains recognize and select for acetyllysine marks and discuss the importance of acetylated histone recognition for their biological functions. This article is protected by copyright. All rights reserved
  • High density lipoprotein (HDL) reverses palmitic acid induced energy
           metabolism imbalance by switching CD36 and GLUT4 signaling pathways in
    • Abstract: In our previous study palmitic acid (PA) induced lipotoxicity and switches energy metabolism from CD36 to GLUT4 in H9c2 cells. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. Therefore we in the present study investigated whether HDL can reverse PA induced lipotoxicity in H9c2 cardiomyoblast cells. In this study, we treated H9c2 cells with PA to create a hyperlipidemia model in vitro and analyzed for CD36 and GLUT4 metabolic pathway proteins. CD36 metabolic pathway proteins (phospho-AMPK, SIRT1, PGC1α, PPARα, CPT1β and CD36) were decreased by high PA (150 and 200 µg/µL) concentration. Interestingly, expression of GLUT4 metabolic pathway proteins (p-PI3K and pAKT) were increased at low concentration (50 µg/µL) and decreased at high PA concentration. Whereas, phospho-PKCζ, GLUT4 and PDH proteins expression was increased in a dose dependent manner. PA treated H9c2 cells were treated with HDL and analyzed for cell viability. Results showed that HDL treatment induced cell proliferation efficiency in PA treated cells. In addition, HDL reversed the metabolic effects of PA: CD36 translocation was increased and reduced GLUT4 translocation, but HDL treatment significantly increased CD36 metabolic pathway proteins and reduced GLUT4 pathway proteins. Rat neonatal cardiomyocytes showed similar results. In conclusion, HDL reversed palmatic acid-induced lipotoxicity and energy metabolism imbalance in H9c2 cardiomyoblast cells and in neonatal rat cardiomyocyte cells. This article is protected by copyright. All rights reserved
  • Differentiation of human olfactory bulb-derived neural stem cell towards
           oligodendrocyte development
    • Abstract: In the central nervous system, oligodendrocytes are the glial element in charge of myelin formation. Obtaining an overall presence of oligodendrocyte precursor cells/oligodendrocytes. (OPCs/OLs) in culture from different sources of NSCs is an important research area, because OPCs/OLs may provide a promising therapeutic strategy for diseases affecting myelination of axons. The present study was designed to differentiate human olfactory bulb NSCs (OBNSCs) into OPCs/OLs and using expression profiling (RT-qPCR) gene, immunocytochemistry and specific protein expression to highlight molecular mechanism(s) underlying differentiation of human OBNSCs into OPCs/OLs. The differentiation of OBNSCs was characterized by a simultaneous appearance of neurons and glial cells. The differentiation medium, containing cAMP, PDGFA, T3, and all-trans-retinoic acid (RA), promotes OBNSCs to generate mostly oligodendrocytes displaying morphological changes and appearance of long cytoplasmic processes. OBNSCs showed, after five days in oligodendrocytes differentiation medium, a considerable decrease in the number of nestin positive cells, which was associated with a concomitant increase of NG2 immunoreactive cells and few O4(+)-OPCs. In addition, a significant up regulation in gene and protein expression profile of stage specific cell markers for OPCs/OLs (CNPase, Galc, NG2, MOG, OLIG1, OLIG2, MBP), neurons and astrocytes (MAP2, β-TubulinIII, GFAP) and concomitant decrease of OBNSCs pluripotency markers (Oct4, Sox2, Nestin), was demonstrated following induction of OBNSCs differentiation.Taken together, the present study demonstrate the marked ability of a cocktail of factors containing PDGFA, T3, cAMP and ATRA, to induce OBNSCs differentiation into OPCs/OLs and shed light on the key genes and pathological pathways involved in this process. This article is protected by copyright. All rights reserved
  • The novel role of pyrvinium in cancer therapy
    • Abstract: Pyrvinium pamoate (PP) is a quinoline-derived cyanine dye which was officially approved by FDA for its anthelmintic properties and therapeutic function against animal-like protists such as Cryp-tosporidium parvum and Plasmodium falciparum in the 1950 s. In the last 10 years, several studies have shown the novel activity of pyrvinium in tumor therapy. Some investigations have indicated that pyrvinium could delay or inhibit tumor cell proliferation in cancer models including colon, breast, lung and prostate cancer and some hematological malignancies. In this review, we discuss multiple critical signaling pathways and mechanisms underlying the anticancer effects of PP. In details, pyrvinium acts through the following main mechanisms: i) energy and autophagy depletion, and ii) inhibition of Akt and Wnt-β-catenin-dependent pathways. Interestingly, pyrvinium has also shown potent anti-cancer stem cell activity. The overwhelming insights into the mechanism of anticancer properties of PP can help establishing novel and future anti-tumor treatment strategies. This article is protected by copyright. All rights reserved
  • E2F is Involved in Radioresistance of Carbon Ion Induced Apoptosis via
           Bax/caspase 3 Signal Pathway in Human Hepatoma Cell
    • Abstract: Deletion of p53, most common genetic alteration, is observed in human tumors and reported to lead to improve in cell radioresistance. Heavy-ion irradiation (IR) could induce p53-/- cancer cells apoptosis. However, little is known regarding the molecular mechanism in this type of cell apoptosis. The present studies have focused on mechanisms state of signaling pathways as an activator of the cell fate decisions induced by heavy ion IR without p53. Carbon ion IR could induce up-regulation of E2F1 expression in cancer cells. This phenomenon was not observed in X-ray IR group. Up-regulation of E2F1 could cause a higher reduction in clonogenic survival, low level of cellular activity, G2/M phase arrest, promotion of apoptosis rate, up-regulation of phosphor-Rb, Bax, and cleaved-caspase 3 proteins expressions without p53. Changes of E2F1 expressions could partly alter radioresistance in cancer cells. The results were suggested that heavy ion IR could induce p53-/- cancer cells apoptosis via E2F1 signal pathway. Our study provides a scientific rationale for the clinical use of heavy ion as radiotherapy in patients with p53-deficient tumors, which are often resistant to radiotherapy. This article is protected by copyright. All rights reserved
  • Application of a novel bioreactor for in vivo engineering of pancreas
    • Abstract: Type 1 diabetes is characterized by autoimmune destruction of pancreatic cells. Organ transplantation is an acceptable treatment for native organ failure. However, it is associated with several problems due to a number of reasons, such as the lack of appropriate donors and immunosuppression. In our present study, a novel model is presented for in vivo recellularization of acellular pancreas by implanting between the host pancreas and the adjacent omental flap. In this study, the pancreases were harvested and cannulated via the common bile duct and then, the scaffolds were acellularized by a detergent-based protocol. Afterthat, the abdomens of thirty-five rats were opened and the spleen was extracted with the adjacent omentum and placed outside the abdomen. The acellularized scaffold was stretched over the host pancreas and the omentum was wrapped around it to make a sandwich-like structure, which was then fixed with Chromic Sutures 6-0 and marked with Prolene 4-0 on four sides. All samples were biopsied at 14, 30, 60, 90, and 120 days post-transplantation. The result showed marked recellularization of acellularized pancreas with visible neovascularization and neoβ-cells with minimal inflammatory response. This study provides a new approach to produces a normal-like pancreas by allograft transplantation for pancreas tissue engineering. We observed that in vivo transplantation of acellularized pancreas can promote recellularization, proliferation, and differentiation by blood circulation. These findings support that in vivo studies can contribute to finding faster solutions for the treatment of diabetes. This article is protected by copyright. All rights reserved
  • Dysregulation of Mitochondrial Bioenergetics and Quality Control by HIV-1
           Tat in Cardiomyocytes
    • Abstract: Cardiovascular disease remains a leading cause of morbidity and mortality in HIV positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+]m) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis. This article is protected by copyright. All rights reserved
  • What role does the stress response have in congestive heart failure'
    • Abstract: This review is concerned with cardiac malfunction as a result of an imbalance in protein proteostasis, the homeostatic balance between protein removal and regeneration in a long remodeling process involving the endoplasmic reticulum (ER) and the unfolded protein response (UPR). The importance of this is of special significance with regard to cardiac function as a high energy requiring muscular organ that has a high oxygen requirement and is highly dependent on mitochondria. The importance of mitochondria is not only concerned with high energy dependence on mitochondrial electron transport, but it also has a role in the signaling between the mitochondria and the ER under stress. Proteins made in the ER are folded as a result of sulfhydryl groups (–SH) and attractive and repulsive reactions in the tertiary structure. We discuss how this matters with respect to an imbalance between muscle breakdown and repair in a stressful environment, especially as a result of oxidative and nitrosative byproducts of mitochondrial activity. The normal repair is a remodeling, but under this circumstance, the cell undergoes or even lysosomal “self eating” autophagy, or even necrosis instead of apoptosis. We shall discuss the relationship of the UPR pathway to chronic congestive heart failure (CHF). This article is protected by copyright. All rights reserved
  • Nociceptor plasticity: a closer look
    • Abstract: Nociceptors are receptors specifically involved in detecting a tissue damage and transducing it in an electrical signal. Nociceptor activation provoked by any kind of acute lesion is related to the release of several mediators of inflammation, within the framework of a process defined as “peripheral sensitization”. This results in an exaggerated response to the painful stimulus, clinically defined as “primary hyperalgesia”. The concept of “neuroplasticity” may explain the adaptive mechanisms carried out by the Nervous System in relation to a “harmful” damage; also, neuroplasticity mechanisms are also fundamental for rehabilitative intervention protocols. Here we review several studies that addressed the role of different receptors and ionic channels discovered on nociceptor surface and their role in pain perception. The changes in expression, distribution and functioning of receptors and ionic channels are thought to be a part of the neuroplasticity property, through which the Nervous System constantly adapts to external stimuli. Moreover, some of the reviewed mediators are also been associated to “central sensitization”, a process that results in pain chronicization when the painful stimulation is particularly prolonged or intense, and lastly leads to the memorization of the uncomfortable painful perception. This article is protected by copyright. All rights reserved
  • Involvement of the Warburg effect in non-tumor diseases processes
    • Abstract: Warburg effect, as an energy shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, is extensively found in various cancers. Interestingly, increasing researchers show that Warburg effect plays a crucial role in non-tumor diseases. For instance, inhibition of Warburg effect can alleviate pulmonary vascular remodeling in the process of pulmonary hypertension. Interference of Warburg effect improves mitochondrial function and cardiac function in the process of cardiac hypertrophy and heart failure. Additionally, the Warburg effect induces vascular smooth muscle cell proliferation and contributes to atherosclerosis. Warburg effect may also involve in axonal damage and neuronal death, which are related with multiple sclerosis. Furthermore, Warburg effect significantly promotes cell proliferation and cyst expansion in polycystic kidney disease. Besides, Warburg effect relieves amyloid β-mediated cell death in Alzheimer's disease. And Warburg effect also improves the mycobacterium tuberculosis infection. Finally, we also introduce some glycolytic agonists. This review focuses on the newest researches about the role of Warburg effect in non-tumor diseases, including pulmonary hypertension, tuberculosis, idiopathic pulmonary fibrosis, failing heart, cardiac hypertrophy, atherosclerosis, Alzheimer's diseases, multiple sclerosis and polycystic kidney disease. Obviously, Warburg effect may be a potential therapeutic target for those non-tumor diseases. This article is protected by copyright. All rights reserved
  • Hypomorphic conditional deletion of E11/Podoplanin reveals a role in
           osteocyte dendrite elongation
    • Abstract: The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (P 
  • Selective expression of long non-coding RNAs in a breast cancer cell
           progression model
    • Abstract: Long non-coding RNAs are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (pre-malignant), and MCF-10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF-7 (early stage, luminal), and MDA-MB-231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGFL2-AS1, of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2-AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression. This article is protected by copyright. All rights reserved
  • Antitumoral Potential, Antioxidant Activity and Carotenoid Content of Two
           Southern Italy Tomato Cultivars Extracts: San Marzano and Corbarino
    • Abstract: Gastric cancer represents a diffuse and aggressive neoplasm, whose mortality index is among the highest in the world. Predisposing factors are E-cadherin mutations, Helicobacter pylori infection and a diet rich in salted and smoked food, with a low intake of fresh fruits and vegetables. Here we analyzed the effect of total lipophilic extracts of two Southern Italy tomato varieties, San Marzano and Corbarino, on an in vitro model of gastric cancer, YCC-1, YCC-2 and YCC-3 cell lines, characterized by different aggressiveness. Our results showed a possible role of these two varieties of tomatoes against typical neoplastic features. The treatment with tomato extracts affected cancer cell ability to grow both in adherence and in semisolid medium, reducing also cell migration ability. No toxic effects were observed on non-tumoral cells. We found, on gastric cancer cell lines, effects on both cell cycle progression and apoptosis modulation. The extent of antineoplastic effects, however, did not seem to correlate with the carotenoid content and antioxidant activity of the two tomato varieties. Our data indicate that San Marzano and Corbarino intake might be further considered as nutritional support not only in cancer prevention, but also for cancer patient diet. This article is protected by copyright. All rights reserved
  • MicroRNA-224-5p regulates adipocyte apoptosis induced by TNFα via
           controlling NF-κB activation
    • Abstract: Tumor necrosis factor (TNF) α can induce cell apoptosis and activate nuclear transcription (NF)-κB in different cell types. Activated NF-κB further promotes or suppresses cellular apoptosis in different cases. The present study explored the effect of activated NF-κB on adipocyte apoptosis induced by TNFα and which microRNAs (miRNAs) were involved in the process. Our findings demonstrated that treatment of differentiated 3T3-L1 adipocytes with TNFα (20ng/mL) rapidly activated NF-κB and induced moderate apoptosis. Pyrrolidinedithiocarbamic acid (PDTC, 60µM), a specific NF-κB inhibitor, abated NF-κB activation that rendered the adipocytes vulnerable to TNFα-induced apoptosis. Dozens of miRNAs exhibited significant expression changes following TNFα treatment and the addition of PDTC. In which, miRNA-224-5p (miR-224) was up-regulated by TNFα exposure but down-regulated by PDTC addition. Furthermore, over-expression of miR-224 promoted NF-κB activation and prevented the adipocyte apoptosis induced by TNFα, while miR-224 deficiency showed the opposite effects. The TRAF-associated NF-κB activator (TANK) gene was identified as a direct target of miR-224 by computational and luciferase reporter assays. Additionally, silencing the TANK gene by the small interfering RNA similarly promoted NF-κB activation and attenuated the cellular apoptosis. In conclusion, these findings demonstrate that miR-224 plays an essential role in adipocyte apoptosis caused by TNFα through control of NF-κB activation via targeting the TANK gene. This article is protected by copyright. All rights reserved
  • Mild mitochondrial uncoupling induces HSL/ATGL-independent lipolysis
           relying on a form of autophagy in 3T3-L1 adipocytes
    • Abstract: Obesity is characterized by an excessive triacylglycerol accumulation in white adipocytes. Various mechanisms allowing the tight regulation of triacylglycerol storage and mobilization by lipid droplet-associated proteins as well as lipolytic enzymes have been identified. Increasing energy expenditure by inducing a mild uncoupling of mitochondria in adipocytes might represent a putative interesting anti-obesity strategy as it reduces the adipose tissue triacylglycerol content (limiting alterations caused by cell hypertrophy) by stimulating lipolysis through yet unknown mechanisms, limiting the adverse effects of adipocyte hypertrophy. Herein, the molecular mechanisms involved in lipolysis induced by a mild uncoupling of mitochondria in white 3T3-L1 adipocytes were characterized. Mitochondrial uncoupling-induced lipolysis was found to be independent from canonical pathways that involve lipolytic enzymes such as HSL and ATGL. Finally, enhanced lipolysis in response to mitochondrial uncoupling relies on a form of autophagy as lipid droplets are captured by endolysosomal vesicles. This new mechanism of triacylglycerol breakdown in adipocytes exposed to mild uncoupling provides new insights on the biology of adipocytes dealing with mitochondria forced to dissipate energy. This article is protected by copyright. All rights reserved
  • The influence of rAAV2-mediated SOX2 delivery into neonatal and adult
           human RPE cells; a comparative study
    • Abstract: Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed towards persuading for cells that can differentiate into retinal neurons. In this report we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprograming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into Adeno-Associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real time PCR and ICC for neural/ retinal progenitor cells' markers. Gene expression analysis showed 80 fold and 12 fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8 fold and 2.5 fold respectively. PAX6 expression was increased 3 fold and 2.5 fold in neonatal/adult treated cultures. Howbeit we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1 and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells. This article is protected by copyright. All rights reserved
  • Therapeutic Application of Multipotent Stem Cells
    • Abstract: Cell therapy is an emerging fields in the treatment of various diseases such as cardiovascular, pulmonary, hepatic and neoplastic diseases. Stem cells are an integral tool for cell therapy. Multipotent stem cells are an important class of stem cells which have the ability to self-renew through dividing and developing into multiple specific cell types in a specific tissue or organ. These cells are capable to activate or inhibit a sequence of cellular and molecular pathways leading to anti-inflammatory and anti-apoptotic effects which might contribute to the treatment of various diseases. It has been showed that multipotent stem cells exert their therapeutic effects via inhibition/activation of a sequence of cellular and molecular pathways.Although the advantages of multipotent stem cells are numerous, further investigation is still necessary to clarify the biology and safety of these cells before they could be considered as a potential treatment for different types of diseases. This review summarizes different features of multipotent stem cells including isolation, differentiation and therapeutic applications. This article is protected by copyright. All rights reserved
  • E2F1interactive with BRCA1 pathway induces HCC two different small
           molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to
    • Abstract: Breast cancer 1 (BRCA1) and E2F transcription factor 1 (E2F1) are related to metabolism and cell cycle regulation. However, the corresponding mechanism is not clear in HCC. High BRCA1 direct pathway was constructed with 11 molecules from E2F1 feedback-interactive network in HCC by GRNInfer based on 39 Pearson mutual positive corelation CC >0.25 molecules with E2F1. Integration of GRNInfer with GO, KEGG, BioCarta, GNF_U133A, UNIGENE_EST, Disease, GenMAPP databases by DAVID and MAS 3.0, E2F1 feedback-interactive BRCA1 indirect mitochondrion to cytosol pathway was identified as upstream LAPTM4B activation, feedback UNG, downstream BCAT1-HIST1H2AD-TK1 reflecting protein and DNA binding with enrichment of small molecule metabolism; The corresponding BRCA1 indirect membrane to cytosol pathway as upstream CCNB2-NUSAP1 activation, feedback TTK-HIST1H2BJ-CENPF, downstream MCM4-TK1 reflecting ATP and microtubule binding with enrichment of CD4+T-related cell cycle regulation in HCC. Therefore, we propose that E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol. Knowledge analysis demonstrates our E2F1 feedback-interactive BRCA1 pathway wide disease distribution and reflects a novel common one of tumor and cancer. This article is protected by copyright. All rights reserved
  • Role of follistatin in muscle and bone alterations induced by gravity
           change in mice
    • Abstract: Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin-related factors in the hypergravity-induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity-enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor-κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change. This article is protected by copyright. All rights reserved
  • Fasting inhibits hepatic stellate cells activation and potentiates
           anti-cancer activity of Sorafenib in hepatocellular cancer cells
    • Abstract: BACKGROUND: Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown.METHODS: 24 hour fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. 24 hours fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib.RESULTS: Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short-term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells.CONCLUSIONS: Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. This article is protected by copyright. All rights reserved
  • Folic Acid is Necessary for Proliferation and Differentiation of C2C12
    • Abstract: Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells..This article is protected by copyright. All rights reserved
  • Roles of platelet-derived growth factor in vascular calcification
    • Abstract: Vascular calcification (VC) is prevalent in aging, and patients with hypertension, chronic kidney disease (CKD) or diabetes. VC is regarded as an active and complex process that involves multiple mechanisms responsible for calcium deposition in vessel wall. In light of the complicated pathogenesis of VC, effective therapy for ameliorating VC is limited. Thus, it is urgent to explore the potential mechanisms and find new targets for the therapy of VC. Platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant, has been found to disturb the vascular homeostasis by inducing inflammation, oxidative stress and phenotype transition, all of which accelerate the process of VC. The aim of current review is to present a review about the roles of PDGF in affecting VC and to establish a potential target for treating VC. This article is protected by copyright. All rights reserved
  • Environmental Temperature affect Physiology and Survival of Nanosecond
           Pulsed Electric Field Treated Cells
    • Abstract: Nanosecond pulsed electric field (nsPEF) is a novel non-thermal tumor ablation technique. However, how nsPEF affect cell physiology at different environmental temperature is still kept unknown. But this issue is of critical clinical practice relevance. This work aim to investigate how nsPEF treated cancer cells react to different environmental temperatures (0°C, 4°C, 25°C and 37°C). Their cell viability, apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) were examined. Lower temperature resulted in higher apoptosis rate, decreased mitochondria membrane potential and increased ROS levels. Sucrose and N-acetylcysteine (NAC) pre-incubation inhibit ROS generation and increase cell survival, protecting nsPEF-treated cells from low temperature-caused cell death. This work provides an experimental basis for hypothermia and fluid transfusion during nsPEF ablation with anesthesia. This article is protected by copyright. All rights reserved
  • Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on
           Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells
    • Abstract: Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 [CV2, also known as BMP endothelial cell precursor derived regulator (BMPER)] is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model.Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells. This article is protected by copyright. All rights reserved
  • Overexpression of HOXA4 and HOXA9 genes promotes self-renewal and
           contributes to colon cancer stem cell overpopulation
    • Abstract: Because HOX genes encode master regulatory transcription factors that regulate stem cells (SCs) during development and aberrant expression of HOX genes occurs in various cancers, our goal was to determine if dysregulation of HOX genes is involved in the SC origin of colorectal cancer (CRC). We previously reported that HOXA4 and HOXD10 are expressed in the colonic SC niche and are overexpressed in CRC. HOX gene expression was studied in SCs from human colon tissue and CRC cells (CSCs) using qPCR and immunostaining. siRNA-mediated knockdown of HOX expression was used to evaluate the role of HOX genes in modulating CSC phenotype at the level of proliferation, SC marker expression, and sphere formation. All-trans-retinoic-acid (ATRA), a differentiation-inducing agent was evaluated for its effects on HOX expression and CSC growth. We found that HOXA4 and HOXA9 are up-regulated in CRC SCs. siRNA knockdown of HOXA4 and HOXA9 reduced: (i) proliferation and sphere-formation and (ii) gene expression of known SC markers (ALDH1, CD166, LGR5). These results indicate that proliferation and self-renewal ability of CRC SCs are reduced in HOXA4 and HOXA9 knockdown cells. ATRA decreased HOXA4, HOXA9 and HOXD10 expression in parallel with reduction in ALDH1 expression, self-renewal, and proliferation. Overall, our findings indicate that overexpression of HOXA4 and HOXA9 contributes to self-renewal and overpopulation of SCs in CRC. Strategies designed to modulate HOX expression may provide ways to target malignant SCs and to develop more effective therapies for CRC. This article is protected by copyright. All rights reserved
  • ABC of multifaceted Dystrophin glycoprotein complex (DGC)
    • Abstract: Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signalling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signalling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signalling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role and functioning of DGC and may lead to a deeper understanding of its role in several human diseases. This article is protected by copyright. All rights reserved
  • Surface Functionalized Magnetic Nanoparticles Shift Cell Behavior with
           On/Off Magnetic Fields
    • Abstract: Magnetic nanoparticles (MNPs) are used as contrast agents and targeted drug delivery systems (TDDS) due to their favorable size, surface charge, and magnetic properties. Unfortunately, the toxicity associated with MNPs limits their biological applications. Surface functionalization of MNPs with selective polymers alters the surface chemistry to impart better biocompatibility. We report the preparation of surface functionalized MNPs using iron oxide NPs (MNPs), poly (lactic-co-glycolic acid) (PLGA) and sodium alginate via co-precipitation, emulsification, and electro-spraying, respectively. The NPs are in the nanosize range and negatively charged. Morphological and structural analyses affirm the surface functionalized nanostructure of the NPs. The surface functionalized MNPs are biocompatible, and demonstrate enhanced intracellular delivery under an applied magnetic field (H), which evinces the targeting ability of MNPs. After NP treatment, the physico-mechanical properties of fibroblasts are decided by the selective MNP uptake under “on” or “off” magnetic field conditions. We envision potential use of biocompatible surface functionalized MNP for intracellular-, targeted-DDS, imaging, and for investigating cellular mechanics. This article is protected by copyright. All rights reserved
  • A simple answer to complex questions: C. elegans as an experimental model
           for examining the DNA damage response and disease genes
    • Abstract: The genetic information is constantly challenged by genotoxic attacks. DNA repair mechanisms evolved early in evolution and recognize and remove the various lesions. A complex network of DNA damage responses (DDR) orchestrates a variety of physiological adaptations to the presence of genome instability. Erroneous repair or malfunctioning of the DDR causes cancer development and the accumulation of DNA lesions drives the aging process. For understanding the complex DNA repair and DDR mechanisms it is pivotal to employ simple metazoan as model systems. The nematode Caenorhabditis elegans has become a well-established and popular experimental organism that allows dissecting genome stability mechanisms in dynamic and differentiated tissues and under physiological conditions. We provide an overview of the distinct advantages of the nematode system for studying DDR and provide a range of currently applied methodologies. This article is protected by copyright. All rights reserved
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