BioMed Research International: Cell Biology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. Shape and Boundary Similarity Features for Accurate HCC Image Recognition Tue, 07 Nov 2017 00:00:00 +0000 Nucleus morphology is of great importance in conventional cancer pathological diagnosis, which could provide information difference between normal and abnormal nuclei visually. Therefore, this paper proposes two novel kinds of features for normal and hepatocellular carcinoma (HCC) nucleus recognition, including shape and boundary similarity. First, each individual nucleus patch with the fixed size is obtained using center-proliferation segmentation (CPS) method. Then, nucleus shape library is constructed based on manual selection by pathologists, which is utilized to measure nucleus shape similarity via Dice, Jaccard, precision, and recall coefficients. Meanwhile, boundary similarity is evaluated through triangles composed of some boundary feature points for each nucleus. Finally, the conventional random forest (RF) is used to train and test the classification model for HCC nucleus recognition. Extensive cross-validation tests could facilitate the selection of the optimal feature set and the experiment comparison results demonstrate that our proposed morphological features are more beneficial for classification compared with other traditional characteristics. Xiaoyu Duan, Huiyan Jiang, and Siqi Li Copyright © 2017 Xiaoyu Duan et al. All rights reserved. Proanthocyanidins Attenuation of H2O2-Induced Oxidative Damage in Tendon-Derived Stem Cells via Upregulating Nrf-2 Signaling Pathway Sun, 22 Oct 2017 09:02:56 +0000 Proanthocyanidins (PCs) have shown inhibition of oxidative damage by improving Nrf-2 expression in many tissues. However, the cytoprotective effects of PCs on H2O2-induced tendon damage have not been verified. The current study was aimed at assessing the cytoprotection of PCs on the oxidative cellular toxicity of tendon-derived stem cells (TDSCs) induced by H2O2. The TDSCs were isolated from patellar tendons of Sprague Dawley (SD) rats, and the cells after third passage were used for subsequent experiments. The isolated cells were identified by flow cytometry assay and multidifferentiation potential assay. Cell Counting Kit-8 assay was performed to examine cell viability. Real-Time PCR and Western Blot were employed to, respectively, assess the mRNA and protein expressions of Nrf-2, GCLM, NQO-1, and HO-1. PCs significantly improved the cell viability of TDSCs. Furthermore, H2O2 upregulated Nrf-2, GCLM, NQO-1, and HO-1 without significant difference, while the proteins expressions were increased with significant difference in PCs group and PCs + H2O2 cotreated group. All the findings indicated that PCs could protect against the oxidative damage induced by H2O2 in TDSCs, and the cytoprotective effects might be due to the ability of PCs to activate the expressions of GCLM, HO-1, and NQO-1 via upregulating Nrf-2 signaling pathway. Wenshuang Sun, Jia Meng, Zhenheng Wang, Tao Yuan, Hong Qian, Wenxiang Chen, Jian Tong, Yu Xie, Ya Zhang, Jianning Zhao, and Nirong Bao Copyright © 2017 Wenshuang Sun et al. All rights reserved. Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators Thu, 19 Oct 2017 00:00:00 +0000 Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications. Xiaojing Zou, Mingyi Qu, Fang Fang, Zeng Fan, Lin Chen, Wen Yue, Xiaoyan Xie, and Xuetao Pei Copyright © 2017 Xiaojing Zou et al. All rights reserved. GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells Tue, 17 Oct 2017 00:00:00 +0000 It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), fatty acid binding protein 4 (FABP4), diacylglycerol acyltransferase 1 (DGAT1), perilipin 2 (PLIN2), perilipin 3 (PLIN3), and C/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPARγ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression of Bcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPARγ in mouse mammary glands. Gaoxiao Xu, Saixing Duan, Jianye Hou, Zhongxin Wei, and Guangwei Zhao Copyright © 2017 Gaoxiao Xu et al. All rights reserved. Corrigendum to “Expression of HIF-2α and VEGF in Cervical Squamous Cell Carcinoma and Its Clinical Significance” Mon, 16 Oct 2017 00:00:00 +0000 Lixia Zhang, Qiang Chen, Jing Hu, Yue Chen, Chenglong Liu, and Changshui Xu Copyright © 2017 Lixia Zhang et al. All rights reserved. Extracorporeal Shock Wave Rebuilt Subchondral Bone In Vivo and Activated Wnt5a/Ca2+ Signaling In Vitro Sun, 15 Oct 2017 00:00:00 +0000 Background. This study aimed to identify the optimal extracorporeal shock wave (ESW) intensity and to investigate its effect on subchondral bone rebuilt in vivo and Wnt5a/Ca2+ signaling in vitro using an osteoarthritis (OA) rat model and bone marrow mesenchymal stem cells (BMMSCs), respectively. Methods. OA rats treated with (OA + ESW group) or without (OA group) ESW (/group) were compared with healthy controls (control group, ). Gait patterns and subchondral trabecular bone changes were measured. Western blot and quantitative real-time polymerase chain reaction detected protein expression and gene transcription, respectively. Results. The gait disturbances of OA + ESW group were significantly improved compared with the OA group at 6th and 8th weeks. The micro-CT analysis indicated that the BMD, BSV/BV, BV/TV, Tr.S, and Tr.Th are significantly different between OA group and OA + ESW group. Expression of Wnt5a was increased rapidly after ESW treatment at 0.6 bar and peaked after 30 min. Conclusions. ESW were positive for bone remodeling in joint tibial condyle subchondral bone of OA rat. ESW prevented histological changes in OA and prevented gait disturbance associated with OA progression. Optimal intensity of ESW induced changes in BMMSCs via activation of the Wnt5a/Ca2+ signaling pathway. Lai Yu, Shuitao Liu, Zhe Zhao, Lin Xia, Haochong Zhang, Jing Lou, Jun Yang, Gengmei Xing, and Gengyan Xing Copyright © 2017 Lai Yu et al. All rights reserved. miR-31 Functions as an Oncomir Which Promotes Epithelial-Mesenchymal Transition via Regulating BAP1 in Cervical Cancer Wed, 11 Oct 2017 00:00:00 +0000 MicroRNA-31 (miR-31) functions as tumor suppressors or oncogenes that are involved in tumor behavior. However, the function of miR-31 in cervical carcinogenesis remains unclear. The aim of this study was to validate the potential role of miR-31 and BRCA1-associated protein-1 (BAP1) on regulating epithelial-mesenchymal transition (EMT) in cervical cancer. In the present study, qRT-PCR assay revealed that the expression of miR-31 was upregulated in human cervical cancer cells and clinical tissues. Results of wound healing and cell migration assay revealed that knockdown of miR-31 inhibited cell metastasis and migration. Bioinformatic and dual-luciferase reporter gene assay showed that BAP1 was the direct target of miR-31. Furthermore, the results revealed that miR-31 promoted proliferation and EMT in cervical cancer cells and accelerated the development of tumor growth in vivo xenograft experiment by inhibiting BAP1 expression. Overall, these results highlight an important role of miR-31 functioning as an oncomir which could promote EMT in cervical cancer via downregulating BAP1 expression. Thus, downregulation of miR-31 could be a novel approach for the molecular treatment of cervical cancers and other malignancies. Nan Wang, Yong Li, and Jianhong Zhou Copyright © 2017 Nan Wang et al. All rights reserved. Santamarine Inhibits NF-κB Activation and Induces Mitochondrial Apoptosis in A549 Lung Adenocarcinoma Cells via Oxidative Stress Mon, 02 Oct 2017 00:00:00 +0000 Santamarine (STM), a sesquiterpene lactone component of Magnolia grandiflora and Ambrosia confertiflora, has been shown to possess antimicrobial, antifungal, antibacterial, anti-inflammatory, and anticancer activities. However, no study has yet been conducted to investigate the molecular mechanism of STM-mediated anticancer activity. In the present study, we found that STM inhibits growth and induces apoptosis in A549 lung adenocarcinoma cells through induction of oxidative stress. STM induces oxidative stress by promoting reactive oxygen species (ROS) generation, depleting intracellular glutathione (GSH), and inhibiting thioredoxin reductase (TrxR) activity in a dose-dependent manner. Further mechanistic study demonstrated that STM induces apoptosis by modulation of Bax/Bcl-2 expressions, disruption of mitochondrial membrane potential, activation of caspase-3, and cleavage of PARP in a dose-dependent manner. Moreover, STM inhibited the constitutive and inducible translocation of NF-κBp65 into the nucleus. IKK-16 (I-κB kinase inhibitor) augmented the STM-induced apoptosis, indicating that STM induces apoptosis in A549 cells at least in part through NF-κB inhibition. Finally, STM-induced apoptosis and expressions of apoptosis regulators were effectively inhibited by thiol antioxidant N-acetyl-L-cysteine (NAC), indicating that STM exerts its anticancer effects mainly through oxidative stress. To the best of our knowledge, this is the first report providing evidence of anticancer activity and molecular mechanism of STM. Xuefeng Wu, Hua Zhu, Jingzhe Yan, Muhammad Khan, and Xiuyan Yu Copyright © 2017 Xuefeng Wu et al. All rights reserved. New Roles of the Primary Cilium in Autophagy Wed, 23 Aug 2017 00:00:00 +0000 The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies. Yenniffer Ávalos, Daniel Peña-Oyarzun, Mauricio Budini, Eugenia Morselli, and Alfredo Criollo Copyright © 2017 Yenniffer Ávalos et al. All rights reserved. MET18 Deficiency Increases the Sensitivity of Yeast to Oxidative Stress and Shortens Replicative Lifespan by Inhibiting Catalase Activity Sun, 30 Jul 2017 00:00:00 +0000 Yeast MET18, a subunit of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery which is responsible for the maturation of Fe/S proteins, has been reported to participate in the oxidative stress response. However, the underlying molecular mechanisms remain unclear. In this study, we constructed a MET18/met18Δ heterozygous mutant yeast strain and found that MET18 deficiency in yeast cells impaired oxidative stress resistance as evidenced by increased sensitivity to hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP). Mechanistically, the mRNA levels of catalase A (CTA1) and catalase T (CTT1) as well as the total catalase activity were significantly reduced in MET18-deficient cells. In contrast, overexpression of CTT1 or CTA1 in MET18-deficient cells significantly increased the intracellular catalase activity and enhanced the resistance ability against H2O2 and CHP. In addition, MET18 deficiency diminished the replicative capacity of yeast cells as evidenced by the shortened replicative lifespan, which can be restored by CTT1 overexpression, but not by CTA1, in the MET18-deficient cells. These results suggest that MET18, in a catalase-dependent manner, plays an essential role in enhancing the resistance of yeast cells to oxidative stress and increasing the replicative capacity of yeast cells. Ya-qin Chen, Xin-guang Liu, Wei Zhao, Hongjing Cui, Jie Ruan, Yuan Yuan, and Zhiguang Tu Copyright © 2017 Ya-qin Chen et al. All rights reserved. Tumor Proteins D52 and D54 Have Opposite Effects on the Terminal Differentiation of Chondrocytes Mon, 17 Jul 2017 00:00:00 +0000 The tumor protein D (TPD) family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase) activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification. Chihiro Ito, Yoshiki Mukudai, Masakatsu Itose, Kosuke Kato, Hiromi Motohashi, Toshikazu Shimane, Seiji Kondo, and Tatsuo Shirota Copyright © 2017 Chihiro Ito et al. All rights reserved. Comparative Analysis of the Cell Fates of Induced Schwann Cells from Subcutaneous Fat Tissue and Naïve Schwann Cells in the Sciatic Nerve Injury Model Tue, 20 Jun 2017 00:00:00 +0000 Purpose. The fate and function of the induced Schwann cells (iSCs) like cells from adipose tissue have not been critically evaluated in vivo after transplantation. The objective of this study is to compare the fate of iSCs with naïve SCs (nSCs) after transplantation into the lesion sites of sciatic nerve, respectively. Methods. Adipose-derived stem cells from eGFP-expressing transgenic rat’s subcutaneous fat were induced to iSCs in vitro. iSCs were injected to the sciatic nerve lesion area after crush injury and the cells fate was comparatively analyzed with that of nSCs from the same rat. Results. At 12 weeks after transplantation, nSCs were detected only in the restricted area of cell transplantation site but iSCs were widely distributed all over the sciatic nerve. Based on double fluorescence observations, both iSCs and naïve ones were colocalized with P0-expressing myelin sheath, outbound by laminin-expressing basal membrane, and terminated at contactin-associated protein-expressing doublets. However, some of iSCs were also differentiated to the fibrocyte/fibroblast-like cells. In the histological analysis of repaired sciatic nerves, axon density was higher in iSC-received group than in the nSCs group and normal sciatic nerve. Conclusion. iSCs induced from subcutaneous fat tissues have higher engraftment and migration capacity than nSCs. Mingzi Zhang, Mei Hua Jiang, Dae-Wook Kim, Woosung Ahn, Eunkyung Chung, Youngsook Son, and Guangfan Chi Copyright © 2017 Mingzi Zhang et al. All rights reserved. Effects of Cobalt Chloride, a Hypoxia-Mimetic Agent, on Autophagy and Atrophy in Skeletal C2C12 Myotubes Mon, 19 Jun 2017 07:22:38 +0000 Background. Hypoxia-induced autophagy and muscle wasting occur in several environmental and pathological conditions. However, the molecular mechanisms underlying the effects of the hypoxia-mimetic agent CoCl2 on autophagy and muscle atrophy are still unclear. Methods. C2C12 myotubes were exposed to increasing concentrations of CoCl2 for 24 hours. Quantitative RT-PCR, Western blotting, and transmission electron microscopy were performed to confirm autophagy occurs. Autophagy proteins were measured to understand the molecule mechanisms. We also inhibited hypoxic autophagy and examined the changes in myogenin expression, myotubes formation, and apoptosis. Results. Our results showed that CoCl2-mimicked hypoxia upregulated the expression of the autophagy-related proteins LC3, HIF-1α, BNIP3, p-AMPKα, and beclin-1, whereas p62 and p-mTOR were downregulated. In addition, the autophagosome could be observed after CoCl2 induction. The expression of the autophagy-related E3 ligase parkin and the muscle-specific ubiquitin ligase atrogin-1 was increased by CoCl2. Inhibition of autophagy by 3MA increased myogenin expression and promoted myotubes formation and the percentage of cell death was decreased. Conclusions. Our results confirmed that CoCl2-mimicked hypoxia induced autophagy via the HIF-1α/BNIP3/beclin-1 and AMPK/mTOR pathways. Our results also revealed an important link between autophagy and muscle atrophy under hypoxia, which may help to develop new therapeutic strategies for muscle diseases. Rui Chen, Ting Jiang, Yanling She, Jiehua Xu, Cheng Li, Shanyao Zhou, Huijuan Shen, Huacai Shi, and Shuang Liu Copyright © 2017 Rui Chen et al. All rights reserved. Dab1 Contributes to Angiotensin II-Induced Apoptosis via p38 Signaling Pathway in Podocytes Mon, 05 Jun 2017 00:00:00 +0000 Numerous studies have found that angiotensin II (Ang II) participates in podocyte apoptosis and exacerbates progression of end-stage kidney disease (ESKD). However, its underlying mechanism remains largely unexplored. As a homolog of Drosophila disabled (Dab) protein, Dab1 plays a vital role in cytoskeleton, neuronal migration, and proliferation. In the present study, our data revealed that Ang II-infused rats developed hypertension, proteinuria, and podocyte injury accompanied by Dab1 phosphorylation and increased reelin expression in kidney. Moreover, Ang II induced podocyte apoptosis in vitro. Dab1 phosphorylation and reelin expression in podocytes were increased after exposure to Ang II. Conversely, Dab1 small interfering RNA (siRNA) exerted protective effects on Ang II-induced podocyte apoptosis, resulting in decreased p38 phosphorylation and reelin expression. These results indicated that Dab1 mediated Ang II-induced podocyte apoptosis via p38 signaling pathway. Zhao Gao, Xinghua Chen, Kai Zhu, Ping Zeng, and Guohua Ding Copyright © 2017 Zhao Gao et al. All rights reserved. The Protective Effect of N-Acetylcysteine on Ionizing Radiation Induced Ovarian Failure and Loss of Ovarian Reserve in Female Mouse Sun, 21 May 2017 00:00:00 +0000 Ionizing radiation may cause irreversible ovarian failure, which, therefore, calls for an effective radioprotective reagent. The aim of the present study was to evaluate the potential radioprotective effect of N-acetylcysteine (NAC) on ionizing radiation induced ovarian failure and loss of ovarian reserve in mice. Kun-Ming mice were either exposed to X-irradiation (4 Gy), once, and/or treated with NAC (300 mg/kg), once daily for 7 days before X-irradiation. We examined the serum circulating hormone levels and the development of ovarian follicles as well as apoptosis, cell proliferation, and oxidative stress 24 hours after X-irradiation. In addition, morphological observations on the endometrial luminal epithelium and the fertility assessment were performed. We found that NAC successfully restored the ovarian and uterine function, enhanced the embryo implantation, improved the follicle development, and altered the abnormal hormone levels through reducing the oxidative stress and apoptosis level in granulosa cells while promoting the proliferation of granulosa cells. In conclusion, the radioprotective effect of NAC on mice ovary from X-irradiation was assessed, and our results suggested that NAC can be a potential radioprotector which is capable of preventing the ovarian failure occurrence and restoring the ovarian reserve. Wei Gao, Jin-Xiao Liang, Chi Ma, Jing-yin Dong, and Qiu Yan Copyright © 2017 Wei Gao et al. All rights reserved. Asymmetric Dimethylarginine Induced Apoptosis and Dysfunction of Endothelial Progenitor Cells: Role of Endoplasmic Reticulum Stress Pathway Sun, 14 May 2017 00:00:00 +0000 Asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, is a novel risk factor of cardiovascular disease. Endothelial progenitor cells (EPCs) bear typical endothelial characteristics and are thought to contribute to neovascularization by providing new endothelial cells (ECs) after arterial injury. Many studies have shown that ADMA can induce EPC apoptosis and dysfunction, but the underlying mechanism is not well understood. EPCs from umbilical cord blood were cultured in EGM-2 medium with particular growth factors and supplemented with 10% fetal bovine serum. The cells were treated with different concentrations of ADMA (5, 10, and 50 μmol/L). Endoplasmic reticulum (ER) stress marker levels were examined by western blot analysis. After 24-hour incubation, ADMA induced apoptosis of EPCs and significantly decreased the proliferation, migration, and vasculogenesis capacity of EPCs. We also found that ADMA treatment activated phosphorylated protein kinase RNA-activated-like ER kinase (PERK), a stress sensor protein in the endoplasmic reticulum (ER). The activated PERK induced 78 kDa glucose-regulated protein (GRP-78) and C/EBP homologous protein (CHOP) expression. Additionally, the inhibition of the ER stress pathway by Salubrinal (a specific ER stress inhibitor) can attenuate ADMA-induced apoptosis of EPCs. Overall, these observations indicate that ADMA may induce the apoptosis and dysfunction of EPCs through the ER stress pathway. Sheng Ye, Xi Zhou, Jiafeng Lin, and Peng Chen Copyright © 2017 Sheng Ye et al. All rights reserved. Tumor-Selective Cytotoxicity of Nitidine Results from Its Rapid Accumulation into Mitochondria Wed, 26 Apr 2017 00:00:00 +0000 We identified a nitidine- (NTD-) accumulating organelle and evaluated the net cytotoxicity of accumulated NTD. To evaluate tumor cell selectivity of the drug, we evaluated its selective cytotoxicity against 39 human cancer cell lines (JFCR39 panel), and the profile was compared with those of known anticancer drugs. Organelle specificity of NTD was visualized using organelle-targeted fluorescent proteins. Real-time analysis of cell growth, proliferation, and cytotoxicity was performed using the xCELLigence system. Selectivity of NTD in the JFCR39 panel was evaluated. Mitochondria-specific accumulation of NTD was observed. Real-time cytotoxicity analysis suggested that the mechanism of NTD-induced cell death is independent of the cell cycle. Short-term treatment indicated that this cytotoxicity only resulted from the accumulation of NTD into the mitochondria. The results from the JFCR39 panel indicated that NTD-mediated cytotoxicity resulted from unique mechanisms compared with those of other known anticancer drugs. These results suggested that the cytotoxicity of NTD is only induced by its accumulation in mitochondria. The drug triggered mitochondrial dysfunction in less than 2 h. Similarity analysis of the selectivity of NTD in 39 tumor cell lines strongly supported the unique tumor cell specificity of NTD. Thus, these features indicate that NTD may be a promising antitumor drug for new combination chemotherapies. Hironori Iwasaki, Masashi Inafuku, Naoyuki Taira, Seikoh Saito, and Hirosuke Oku Copyright © 2017 Hironori Iwasaki et al. All rights reserved. The Effect of L-Carnitine, Hypotaurine, and Taurine Supplementation on the Quality of Cryopreserved Chicken Semen Mon, 24 Apr 2017 08:46:45 +0000 The objective of this study was to investigate the effect of L-carnitine (LC), hypotaurine (HT), and taurine (T) on the quality of frozen-thawed chicken semen. Pooled semen samples were divided into seven aliquots (control, 1 mM LC, 5 mM LC, 1 mM HT, 10 mM HT, 1 mM T, and 10 mM T) and subjected to cryopreservation. Postthaw sperm motility was determined by IVOS system and sperm characteristics were assessed with fluorochromes and flow cytometry. The highest sperm motility and the highest percentage of viable sperm were in the HT1 group ( and ) following cryopreservation. After thawing, we observed a higher percentage of sperm without apoptosis and membrane reorganization changes in the LC1 and T1 group when compared to the control (). There was a higher percentage of live sperm without lipid peroxidation (LPO) in all treatments (; ), when compared to the control group. The percentage of sperm with high mitochondrial potential significantly increased with LC1, T1, and T10 (). Supplementation of the diluent with LC1, LC5, and T1 significantly () reduced DNA susceptibility to fragmentation, compared to the control and HT1 groups. These results indicate that the addition of examined antioxidants improves the quality of cryopreserved chicken semen. Agnieszka Partyka, Olga Rodak, Joanna Bajzert, Joanna Kochan, and Wojciech Niżański Copyright © 2017 Agnieszka Partyka et al. All rights reserved. High Glucose Promotes CD36 Expression by Upregulating Peroxisome Proliferator-Activated Receptor γ Levels to Exacerbate Lipid Deposition in Renal Tubular Cells Wed, 12 Apr 2017 00:00:00 +0000 Diabetic kidney disease (DKD) appears to be closely related to lipid deposition in kidney. The aim of this study was to determine whether high glucose (HG) exacerbated lipid deposition by increasing CD36 expression via AKT-PPARγ signaling pathway. Our results showed that HG activated AKT signaling pathway, followed by an increase in PPARγ that induced CD36 overexpression, ultimately causing lipid deposition in HK-2 cells. We also found that inhibition of AKT-PPARγ signaling pathway or knockdown of CD36 could reduce HG-induced lipid accumulation in HK-2 cells. These results indicated that AKT-PPARγ signaling pathway mediated HG-induced lipid deposition by upregulating CD36 expression in HK-2 cells and that inhibition of AKT-PPARγ signaling pathway had the potential beneficial effects of reducing lipid deposition in diabetic kidney. Lei Feng, Chengwu Gu, Yanxia Li, and Jiasui Huang Copyright © 2017 Lei Feng et al. All rights reserved. OCT4B1 Regulates the Cellular Stress Response of Human Dental Pulp Cells with Inflammation Tue, 04 Apr 2017 07:04:13 +0000 Introduction. Infection and apoptosis are combined triggers for inflammation in dental tissues. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel spliced variant of OCT4 family, could respond to the cellular stress and possess antiapoptotic property. However, its specific role in dental pulpitis remains unknown. Methods. To investigate the effect of OCT4B1 on inflammation of dental pulp cells (DPCs), its expression in inflamed dental pulp tissues and DPCs was examined by in situ hybridization, real-time PCR, and FISH assay. OCT4B1 overexpressed DPCs model was established, confirmed by western blot and immunofluorescence staining, and then stimulated with Lipopolysaccharide (LPS). Apoptotic rate was determined by Hoechst/PI staining and FACS. Cell survival rate was calculated by CCK8 assay. Results. In situ hybridization, real-time PCR, and FISH assay revealed that OCT4B1 was extensively expressed in inflamed dental pulp tissues and DPCs with LPS stimulation. Western blot and immunofluorescence staining showed the expression of OCT4B1 and OCT4B increased after OCT4B1 transfection. Hoechst/PI staining and FACS demonstrated that less red/blue fluorescence was detected and apoptotic percentage decreased (3.45%) after transfection. CCK8 demonstrated that the survival rate of pCDH-OCT4B1-flag cells increased. Conclusions. OCT4B1 plays an essential role in inflammation and apoptosis of DPCs. OCT4B might operate synergistically with OCT4B1 to reduce apoptosis. Lu Liu, Rong Huang, Ruiqi Yang, and Xi Wei Copyright © 2017 Lu Liu et al. All rights reserved. Platinum-Based Drugs Differentially Affect the Ultrastructure of Breast Cancer Cell Types Thu, 09 Mar 2017 07:34:03 +0000 Breast cancer (BC) is the most common cause of cancer-related death worldwide. Although platinum-based drugs (PBDs) are effective anticancer agents, responsive patients eventually become resistant. While resistance of some cancers to PBDs has been explored, the cellular responses of BC cells are not studied yet. Therefore, we aim to assess the differential effects of PBDs on BC ultrastructure. Three representative cells were treated with different concentrations and timing of Cisplatin, Carboplatin, and Oxaliplatin. Changes on cell surface and ultrastructure were detected by scanning (SEM) and transmission electron microscope (TEM). In SEM, control cells were semiflattened containing microvilli with extending lamellipodia while treated ones were round with irregular surface and several pores, indicating drug entry. Prolonged treatment resembled distinct apoptotic features such as shrinkage, membrane blebs, and narrowing of lamellipodia with blunt microvilli. TEM detected PBDs’ deposits that scattered among cellular organelles inducing structural distortion, lumen swelling, chromatin condensation, and nuclear fragmentation. Deposits were attracted to fat droplets, explained by drug hydrophobic properties, while later they were located close to cell membrane, suggesting drug efflux. Phagosomes with destructed organelles and deposits were detected as defending mechanism. Understanding BC cells response to PBDs might provide new insight for an effective treatment. Shadia Al-Bahlani, Buthaina Al-Dhahli, Kawther Al-Adawi, Abdurahman Al-Nabhani, and Mohamed Al-Kindi Copyright © 2017 Shadia Al-Bahlani et al. All rights reserved. Esculetin Neutralises Cytotoxicity of t-BHP but Not of H2O2 on Human Leukaemia NB4 Cells Tue, 07 Mar 2017 06:46:55 +0000 The coumarin esculetin shows antioxidant action on some cell types, both by scavenging ROS and by decreasing ROS production. We have previously demonstrated the induction of apoptosis by esculetin on NB4 human leukaemia cells by an ill-defined mechanism related to ROS levels. In this work, we analyze the effect of the simultaneous treatment with esculetin and two oxidants to observe the early events in the mechanism of esculetin-induced apoptosis. Our results show that, from the early time of 15 min, esculetin acts synergistically with H2O2 to decrease cell viability and metabolic activity and to increase apoptosis in NB4 cells. In contrast, the early oxidative effects of t-BHP are neutralised by esculetin, protecting human leukaemia NB4 cells from apoptosis. Esculetin seems to restrict the increase in peroxides caused by H2O2 or t-BHP in the time interval analyzed. These results contribute to a better understanding of the cytotoxic effect caused by esculetin on NB4 cells. At the same time, the early neutralisation of exogenous oxidants could be of interest to prevent diseases related to oxidative stress imbalance. Virginia Rubio, Ana I. García-Pérez, M. Cristina Tejedor, Angel Herráez, and José C. Diez Copyright © 2017 Virginia Rubio et al. All rights reserved. Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway Thu, 02 Mar 2017 00:00:00 +0000 Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway. Shuqing Chen, Yongqian Wang, Yubin Yang, Ting Xiang, Jiahui Liu, Houming Zhou, and Xinlin Wu Copyright © 2017 Shuqing Chen et al. All rights reserved. YCl3 Promotes Neuronal Cell Death by Inducing Apoptotic Pathways in Rats Thu, 23 Feb 2017 00:00:00 +0000 The pollutants rare earth elements (REEs) have posed great threats to human health. To investigate the cytotoxicity of yttrium (Y), a model that rats have free access to water containing YCl3 for 6 months is utilized. The results showed that YCl3 treatment promoted neuronal cell apoptosis by upregulating the proapoptotic factors Bax, caspase-3, Cyto c, and DAPK and by downregulating the antiapoptotic factors Bcl-2 and XIAP at both mRNA and protein levels. Conclusively, YCl3 exhibited cytotoxicity and promoted neuronal cell death by the induction of apoptotic pathways. Yechun Ding, Yuantong Tian, Zhaoyi Zeng, Ping Shuai, Haiying Lan, Xianshen Zhu, Yi Zhong, Longhuo Wu, and Xiaona Fan Copyright © 2017 Yechun Ding et al. All rights reserved. Effect of Wnt Signaling on the Differentiation of Islet β-Cells from Adipose-Derived Stem Cells Mon, 20 Feb 2017 00:00:00 +0000 The Wnt signaling is critical for pancreatic development and islet function; however, its precise effects on the development and function of the β-cells remain controversial. Here we examined mRNA and protein expression of components of the Wnt signaling throughout the differentiation of islet β-cells from adipose-derived stem cells (ADSCs). After induction, ADSCs expressed markers of β-cells, including the insulin, PDX1, and glucagon genes, and the PDX1, CK19, nestin, insulin, and C-peptide proteins, indicating their successful differentiation. Compared with pancreatic adult stem cells (PASCs), the quantities of insulin, GLUT2, and Irs2 mRNA decreased, whereas Gcg, Gck, and Irs1 mRNA increased. Over time, during differentiation, insulin mRNA and protein expression increased, Gcg and Gck mRNA expression increased, Irs1 mRNA expression decreased and then increased, and Irs2 mRNA increased and then decreased (all ). The expression of Dvl-2, LRP5, and GSK3β mRNA as well as the Dvl-2, GSK3β, and p-GSK3β proteins also increased (). Expression of TCF7L2 (6–10 d) and β-catenin mRNA as well as the β-catenin protein increased but not significantly (). Our results indicate that the Wnt signaling is activated during ADSC differentiation into islet β-cells, but there was no obvious enrichment of nonphosphorylated β-catenin protein. Hefei Wang, Yu Ren, Xiao Hu, Min Ma, Xiao Wang, Hao Liang, and Dongjun Liu Copyright © 2017 Hefei Wang et al. All rights reserved. Mechanical Stress Regulates Osteogenesis and Adipogenesis of Rat Mesenchymal Stem Cells through PI3K/Akt/GSK-3β/β-Catenin Signaling Pathway Tue, 14 Feb 2017 00:00:00 +0000 Osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) are regarded as being of great importance in the regulation of bone remodeling. In this study, rat BMSCs were exposed to different levels of cyclic mechanical stress generated by liquid drops and cultured in general medium or adipogenic medium. Markers of osteogenic (Runx2 and Collagen I) and adipogenic (C/EBPα, PPARγ, and lipid droplets) differentiation were detected using Western blot and histological staining. The protein levels of members of the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway were also examined. Results showed that small-magnitude stress significantly upregulated Runx2 and Collagen I and downregulated PPARγ and C/EBPα expression in BMSCs cultured in adipogenic medium, while large-magnitude stress reversed the effect when compared with unloading groups. The PI3K/Akt signaling pathway could be strongly activated by mechanical stimulation; however, large-magnitude stress led to decreased activation of the signaling pathway when compared with small-magnitude stress. Activation of β-catenin with LiCl led to increased expression of Runx2 and Collagen I and reduction of C/EBPα and PPARγ expression in BMSCs. Inhibition of PI3K/Akt signaling partially blocked the expression of β-catenin. Taken together, our results indicate that mechanical stress-regulated osteogenesis and adipogenesis of rat BMSCs are mediated, at least in part, by the PI3K/Akt/GSK-3β/β-catenin signaling pathway. Fanglong Song, Dawei Jiang, Tianchen Wang, Yi Wang, Yi Lou, Yinquan Zhang, Hui Ma, and Yifan Kang Copyright © 2017 Fanglong Song et al. All rights reserved. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site Mon, 16 Jan 2017 00:00:00 +0000 RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (). Patients with higher Dnd1 expression level had longer overall survival () by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3′UTR, the stability of Bim-5′UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3′UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3′UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development. Feng Cheng, Ying Pan, Yi-Min Lu, Lei Zhu, and Shuzheng Chen Copyright © 2017 Feng Cheng et al. All rights reserved. Proteome Changes of Human Bone Marrow Mesenchymal Stem Cells Induced by 1,4-Benzoquinone Thu, 29 Dec 2016 10:07:30 +0000 Benzene is metabolized to hydroquinone in liver and subsequently transported to bone marrow for further oxidization to 1,4-benzoquinone (1,4-BQ), which may be related to the leukemia and other blood disorders. In the present study, we investigated the proteome profiles of human primary bone marrow mesenchymal stem cells (hBM-MSCs) treated by 1,4-BQ. We identified 32 proteins that were differentially expressed. Two of them, HSP27 and Vimentin, were verified at both mRNA and protein levels and their cellular localization was examined by immunofluorescence. We also found increased mRNA level of RAP1GDS1, a critical factor of metabolism that has been identified as a fusion partner in various hematopoietic malignancies. Therefore, these differentially expressed proteins can play important roles in benzene-mediated hematoxicity. Wei Wu, Ling Zhang, Min Zhang, Li-Jin Zhu, Hai-Ling Xia, Jian-Lin Lou, Jia-Ren Liu, and Yun Xiao Copyright © 2016 Wei Wu et al. All rights reserved. The Expression of HMGB1 in Bone Marrow MSCs Is Upregulated by Hypoxia with Regulatory Effects on the Apoptosis and Adhesion Tue, 06 Dec 2016 11:15:20 +0000 Background and Aims. Hypoxia regulates the survival of mesenchymal stem cells (MSCs) but the mechanism is unclear. In hypoxia, the level of high mobility group box 1 (HMGB1) was increased in many cells which may be involved in the regulation of cell biology. The aim is to determine whether hypoxia affects the expression of HMGB1 in bone marrow MSCs (BM-MSCs) and to investigate the role of HMGB1 in the apoptosis and adhesion. Methods. BM-MSCs were exposed to hypoxia (1% O2) and normoxia (20% O2) and the expression of HMGB1 was measured by RT-PCR and western blotting. The apoptosis and adhesion of BM-MSCs were evaluated after interfered by different concentrations of HMGB1. Results. Expression of HMGB1 in BM-MSCs showed a significant upregulation in hypoxia when compared to those in normoxia. The adhesion of BM-MSCs was increased by HMGB1 in a concentration-dependent manner; the apoptosis effect of HMGB1 depended on its concentrations: HMGB1 at low concentration (50 ng/mL) promoted the apoptosis of BM-MSCs while HMGB1 at high concentration (≥100 ng/mL) reduced this apoptosis. Conclusions. Hypoxia enhanced the expression of HMGB1 in BM-MSCs with influences on apoptosis and adhesion and this could have a significant effect on the regenerative potential of MSC-based strategies. Mei-Yun Tan, Cai-Dong Zhang, Bo Xia, Jiang Guo, Zhong-Wei Fan, Tian-Hao Wu, Sen Wang, Shao-Feng Liu, Li Deng, Xing Guo, and Yong-Can Huang Copyright © 2016 Mei-Yun Tan et al. All rights reserved. Corrigendum to “The Effects of Cytokines in Adipose Stem Cell-Conditioned Medium on the Migration and Proliferation of Skin Fibroblasts In Vitro” Tue, 29 Nov 2016 08:41:42 +0000 Jiajia Zhao, Li Hu, Jiarong Liu, Niya Gong, and Lili Chen Copyright © 2016 Jiajia Zhao et al. All rights reserved. In Vitro Protective Effects of Lycium barbarum Berries Cultivated in Umbria (Italy) on Human Hepatocellular Carcinoma Cells Mon, 14 Nov 2016 13:32:41 +0000 Lycium barbarum is a famous plant in the traditional Chinese medicine. The plant is known to have health-promoting bioactive components. The properties of Lycium barbarum berries cultivated in Umbria (Italy) and their effect on human hepatocellular carcinoma cells (HepG2) have been investigated in this work. The obtained results demonstrated that the Lycium barbarum berries from Umbria region display high antioxidant properties evaluated by total phenolic content and ORAC method, on hydrophilic and lipophilic fractions. Moreover, on HepG2 cell line Lycium barbarum berries extract did not change cell viability analyzed by MTT and Trypan blue exclusion assay and did not induce genotoxic effect analyzed by comet assay. Furthermore, it was demonstrated, for the first time, that the berries extract showed a protective effect on DNA damage, expressed as antigenotoxic activity in vitro. Finally, Lycium barbarum berries extract was able to modulate the expression of genes involved in oxidative stress, proliferation, apoptosis, and cancer. In particular, downexpression of genes involved in tumor migration and invasion (CCL5), in increased risk of metastasis and antiapoptotic signal (DUSP1), and in carcinogenesis (GPx-3 and PTGS1), together with overexpression of tumor suppressor gene (MT3), suggested that Umbrian Lycium barbarum berries could play a protective role against hepatocellular carcinoma. M. R. Ceccarini, S. Vannini, S. Cataldi, M. Moretti, M. Villarini, B. Fioretti, E. Albi, T. Beccari, and M. Codini Copyright © 2016 M. R. Ceccarini et al. All rights reserved. Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice Sun, 13 Nov 2016 09:53:52 +0000 Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (). A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP) was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells’ tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo. Wei Xin Cai, Li Wu Zheng, Li Ma, Hong Zhang Huang, Ru Qing Yu, and Roger A. Zwahlen Copyright © 2016 Wei Xin Cai et al. All rights reserved. miR-20b Inhibits T Cell Proliferation and Activation via NFAT Signaling Pathway in Thymoma-Associated Myasthenia Gravis Wed, 19 Oct 2016 11:42:30 +0000 Purpose. We examined the role of miR-20b in development of thymoma-associated myasthenia gravis, especially in T cell proliferation and activation. Materials and Methods. Using qRT-PCR, we assessed expression levels of miR-20b and its target genes in cultured cells and patient samples and examined the proliferation of cultured cells, using MTT cell proliferation assays and flow cytometry based cell cycle analysis. Activation of T cells was determined by both flow cytometry and qRT-PCR of activation-specific marker genes. Results. Expression of miR-20b was downregulated in samples of thymoma tissues and serum from patients with thymoma-associated myasthenia gravis. In addition, T cell proliferation and activation were inhibited by ectopic overexpression of miR-20b, which led to increased T cell proliferation and activation. NFAT5 and CAMTA1 were identified as targets of miR-20b. Expression levels of NFAT5 and CAMTA1 were inhibited by miR-20b expression in cultured cells, and the expression levels of miR-20b and NFAT5/CAMTA1 were inversely correlated in patients with thymoma-associated myasthenia gravis. Conclusion. miR-20b acts as a tumor suppressor in the development of thymoma and thymoma-associated myasthenia gravis. The tumor suppressive function of miR-20b in thymoma could be due to its inhibition of NFAT signaling by repression of NFAT5 and CAMTA1 expression. Yanzhong Xin, Hongfei Cai, Tianyu Lu, Yan Zhang, Yue Yang, and Youbin Cui Copyright © 2016 Yanzhong Xin et al. All rights reserved. Effect of Phosphatase and Tensin Homologue on Chromosome 10 on Angiotensin II-Mediated Proliferation, Collagen Synthesis, and Akt/P27 Signaling in Neonatal Rat Cardiac Fibroblasts Sun, 25 Sep 2016 07:16:45 +0000 Cardiac fibroblasts (CFs) play a key role in cardiac fibrosis by regulating the balance between extracellular matrix synthesis and breakdown. Although phosphatase and tensin homologue on chromosome 10 (PTEN) has been found to play an important role in cardiovascular disease, it is not clear whether PTEN is involved in functional regulation of CFs. In the present study, PTEN was overexpressed in neonatal rat CFs via recombinant adenovirus-mediated gene transfer. The effects of PTEN overexpression on cell-cycle progression and angiotensin II- (Ang II-) mediated regulation of collagen metabolism, synthesis of matrix metalloproteinases, and Akt/P27 signaling were investigated. Compared with uninfected cells and cells infected with green fluorescent protein-expressing adenovirus (Ad-GFP), cells infected with PTEN-expressing adenovirus (Ad-PTEN) significantly increased PTEN protein and mRNA levels in CFs (). The proportion of CFs in the G1/S cell-cycle phase was significantly higher for PTEN-overexpressing cells. In addition, Ad-PTEN decreased mRNA expression and the protein synthesis rate of collagen types I and III and antagonized Ang II-induced collagen synthesis. Overexpression of PTEN also decreased Ang II-induced matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) production as well as gelatinase activity. Moreover, Ad-PTEN decreased Akt expression and increased P27 expression independent of Ang II stimulation. These results suggest that PTEN could regulate its functional effects in neonatal rat CFs partially via the Akt/P27 signaling pathway. Ling Nie, Jing-Hong Zhao, Jiang Wang, Rong Song, and Shan-Jun Zhu Copyright © 2016 Ling Nie et al. All rights reserved. Retracted: Leaf Rolling and Stem Fasciation in Grass Pea (Lathyrus sativus L.) Mutant Are Mediated through Glutathione-Dependent Cellular and Metabolic Changes and Associated with a Metabolic Diversion through Cysteine during Phenotypic Reversal Thu, 01 Sep 2016 08:37:14 +0000 BioMed Research International Copyright © 2016 BioMed Research International. All rights reserved. The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models Wed, 24 Aug 2016 17:58:56 +0000 Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by in hepatocyte-Kupffer cell, 2 : 1, and by in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice. Judit Pomothy, Gergely Szombath, Patrik Rokonál, Gábor Mátis, Zsuzsanna Neogrády, Torsten Steinmetzer, and Erzsébet Pászti-Gere Copyright © 2016 Judit Pomothy et al. All rights reserved. Preparation and In Vivo Pharmacokinetics of the Tongshu Suppository Tue, 16 Aug 2016 09:10:56 +0000 Astragalus polysaccharide (APS) (used for intestinal protection) was added to formulate the Tongshu suppository to improve the pharmacokinetics of Aceclofenac, which were assessed in New Zealand rabbits using an orthogonal experimental design. The single-agent Aceclofenac was taken as the control formulation. The concentration-time and drug release curves were drawn, and (min), (μg·mL−1), , and MRT were compared using a pharmacokinetic systems program. The formulated Tongshu suppository had moderate hardness, a smooth surface with uniform color, and theoretical drug-loading rate of 8%. Its release rate was in accordance with the drug preparation requirements. The concentration-time curves and drug release curves revealed that the maximum concentrations () were  μg·mL−1 and  μg·mL−1 for the Tongshu and Aceclofenac suppositories, respectively, showing statistically insignificant difference, while the peak times were  min and  min, respectively, also showing statistically insignificant difference. Compared with the Aceclofenac suppository, the relative bioavailability of the Tongshu suppository was 104.4%, and the difference between them was statistically insignificant. In this experiment, the Tongshu suppository was prepared using the hot-melt method. In vivo pharmacokinetic studies confirmed it had higher bioavailability than the Aceclofenac suppository. Guoqiang Liu, Leilei Dong, Kuan Lu, Sisi Liu, and Yingying Zheng Copyright © 2016 Guoqiang Liu et al. All rights reserved. HDAC Inhibitors as Epigenetic Regulators of the Immune System: Impacts on Cancer Therapy and Inflammatory Diseases Sun, 31 Jul 2016 10:05:49 +0000 Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments. Elizabeth E. Hull, McKale R. Montgomery, and Kathryn J. Leyva Copyright © 2016 Elizabeth E. Hull et al. All rights reserved. Corrigendum to “α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism” Tue, 19 Jul 2016 11:49:52 +0000 Jaeson Santos Calla-Choque, Elisa Elvira Figueroa-Angulo, Leticia Ávila-González, and Rossana Arroyo Copyright © 2016 Jaeson Santos Calla-Choque et al. All rights reserved. The Epithelial Sodium Channel and the Processes of Wound Healing Thu, 14 Jul 2016 09:29:40 +0000 The epithelial sodium channel (ENaC) mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation. Silvia Chifflet and Julio A. Hernandez Copyright © 2016 Silvia Chifflet and Julio A. Hernandez. All rights reserved. Molecular Imaging for Comparison of Different Growth Factors on Bone Marrow-Derived Mesenchymal Stromal Cells’ Survival and Proliferation In Vivo Tue, 21 Jun 2016 14:10:59 +0000 Introduction. Bone marrow-derived mesenchymal stromal cells (BMSCs) have emerged as promising cell candidates but with poor survival after transplantation. This study was designed to investigate the efficacy of VEGF, bFGF, and IGF-1 on BMSCs’ viability and proliferation both in vivo and in vitro using bioluminescence imaging (BLI). Methods. BMSCs were isolated from β-actin-Fluc+ transgenic FVB mice, which constitutively express firefly luciferase. Apoptosis was induced by hypoxia preconditioning for up to 24 h followed by flow cytometry and TUNEL assay. 106 BMSCs with/without growth factors were injected subcutaneously into wild type FVB mice’s backs. Survival of BMSCs was longitudinally monitored using bioluminescence imaging (BLI) for 5 weeks. Protein expression of Akt, p-Akt, PARP, and caspase-3 was detected by Western blot. Results. Hypoxia-induced apoptosis was significantly attenuated by bFGF and IGF-1 compared with VEGF and control group in vitro (). When combined with matrigel, IGF-1 showed the most beneficial effects in protecting BMSCs from apoptosis in vivo. The phosphorylation of Akt had a higher ratio in the cells from IGF-1 group. Conclusion. IGF-1 could protect BMSCs from hypoxia-induced apoptosis through activation of p-Akt/Akt pathway. Hongyu Qiao, Ran Zhang, Lina Gao, Yanjie Guo, Jinda Wang, Rongqing Zhang, Xiujuan Li, Congye Li, Yundai Chen, and Feng Cao Copyright © 2016 Hongyu Qiao et al. All rights reserved. Expression of HIF-2α and VEGF in Cervical Squamous Cell Carcinoma and Its Clinical Significance Mon, 20 Jun 2016 14:16:27 +0000 CSCC is a systemic disease involving polygenic alteration and multiple steps, and HIF and VEGF are closely associated with tumorigenesis. Specimens surgically resected from 64 cases of CSCC and 22 cases of normal cervical tissue were selected randomly to detect the expression of HIF-2α and VEGF in CSCC for exploring their clinical significance; information regarding the age, lymph node metastasis, and FIGO staging were collected as well; expression of HIF-2α and VEGF was detected by qPCR and immunohistochemistry. We found that the expression of HIF-2α and VEGF mRNA in CSCC was significantly higher than that of normal cervical tissues and showed a positive correlation between them. The positive rates of HIF-2α and VEGF protein expression in CSCC and normal cervical tissues were 93.8% and 18.2%, respectively, with correlation between them. The expression of both HIF-2α and VEGF mRNA did not relate closely to age but the FIGO staging and lymph node metastasis. Compared with the counterpart control group, CSCC tissues with high FIGO staging and lymph node metastasis had a higher level of HIF-2α and VEGF mRNA expression. So, HIF-2α and VEGF were overexpressed in CSCC, which has a great clinical significance for its diagnosis. Lixia Zhang, Qiang Chen, Jing Hu, Yue Chen, Chenglong Liu, and Changshui Xu Copyright © 2016 Lixia Zhang et al. All rights reserved. The Effect of Adipose-Derived Stem Cells on Full-Thickness Skin Grafts Mon, 20 Jun 2016 06:34:43 +0000 Background. The purpose of this study was to evaluate the effects of ASCs on full-thickness skin grafts. Specifically, we investigated the anti-inflammatory effects of ASCs that are mediated via regulation of the phenotypes of activated macrophages. Methods. ASCs were isolated, cultured, and injected under full-thickness skin grafts in 15 rats (ASC group). An additional 15 rats served as controls (PBS group). Skin graft survival assessment and vascularization detection were assessed with H&E staining and laser Doppler blood flowmetry (LDF). The effects of ASCs on angiogenesis, anti-inflammation, collagen accumulation-promoting, and antiscarring were assessed. Results. We found that the skin graft survival rate was significantly increased in the ASC group. The neovascularization, collagen deposition, collagen type I to type III ratio, and levels of VEGF and TGF-β3 in the ASC group were markedly higher than those in the PBS group at day 14. Additionally, in the ASC group, the levels of iNOS, IL-1β, and TNF-α were remarkably decreased, whereas the levels of IL-10 and Arg-1 were substantially increased. Conclusions. Our results confirm that ASCs transplantation can effectively improve full-thickness skin graft survival. Additionally, the anti-inflammatory role of ASCs may indirectly contribute to skin graft survival via its effect on macrophage polarization. Juan Wang, Haojie Hao, Hong Huang, Deyun Chen, Yan Han, and Weidong Han Copyright © 2016 Juan Wang et al. All rights reserved. The Preclinical Research Progress of Stem Cells Therapy in Parkinson’s Disease Thu, 09 Jun 2016 13:48:46 +0000 Parkinson’s disease (PD) is a type of degenerative disorder of the basal ganglia, causing tremor at rest, muscle rigidity hypokinesia, and dementia. The effectiveness of drug treatments gradually diminishes because the conversion to dopamine within the brain is increasingly disrupted by the progressive degeneration of the dopaminergic terminals. After long-term treatment, most patients with PD suffer from disability that cannot be satisfactorily controlled. To solve these issues, stem cells have recently been used for cell therapy of PD. In this review, the characteristics of different stem cells and their therapeutic effects on PD treatment will be discussed. Jun Zhang, Xianyun Wang, Jing Li, Rui Huang, Xuerui Yu, Ci Dong, Pujuan Liu, Fan Zhang, Jie Hu, Yixin Qi, Jing Zhang, Quanhai Li, and Baoyong Yan Copyright © 2016 Jun Zhang et al. All rights reserved. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation Thu, 02 Jun 2016 16:09:28 +0000 Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. Chandrama Shrestha, Yuanyuan Tang, Hong Fan, Lusha Li, Qin Zeng, Sally D. Pennypacker, Daniel D. Bikle, and Zhongjian Xie Copyright © 2016 Chandrama Shrestha et al. All rights reserved. Effects of Lycium barbarum Polysaccharides on Apoptosis, Cellular Adhesion, and Oxidative Damage in Bone Marrow Mononuclear Cells of Mice Exposed to Ionizing Radiation Injury Thu, 26 May 2016 13:10:37 +0000 Lycium barbarum has been used for more than 2500 years as a traditional herb and food in China. We investigated the effects of Lycium barbarum polysaccharides (LBP) on apoptosis, oxidative damage, and expression of adhesion molecules in bone marrow mononuclear cells (BMNC) of mice injured by ionizing radiation. Kunming mice were exposed to X-rays; then mice in the LBP groups were continuously injected with various concentrations of LBP intraperitoneally for 14 days. Mice in the control group were continuously injected with normal saline (NS) by the same route for 14 days. A normal group was set up. After 1, 7, and 14 days of treatment, mice were killed and BMNC were extracted. Cell cycle, apoptosis, and the expression of adhesion molecules CD44 and CD49d were detected by flow cytometry. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were identified by colorimetric analyses. LBP significantly decreased the percentage of G0/G1 phase, apoptosis, MDA level, and expression of CD44 and CD49d and distinctly increased the activity of SOD. LBP showed a protective effect on BMNC against ionizing radiation-induced apoptosis and oxidative damage and altered the expression of adhesion molecule. Jing Zhou, Hua Pang, Wenbo Li, Qiong Liu, Lu Xu, Qian Liu, and Ying Liu Copyright © 2016 Jing Zhou et al. All rights reserved. miR-145a-5p Promotes Myoblast Differentiation Thu, 28 Apr 2016 11:39:27 +0000 MicroRNAs are a class of 18–22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation. Jingjing Du, Qiang Li, Linyuan Shen, Huaigang Lei, Jia Luo, Yihui Liu, Peiwen Zhang, Qiang Pu, Yi Zhang, Surong Shuai, Xuewei Li, Shunhua Zhang, and Li Zhu Copyright © 2016 Jingjing Du et al. All rights reserved. Nicotinamide Mononucleotide Adenylyl Transferase 2: A Promising Diagnostic and Therapeutic Target for Colorectal Cancer Wed, 27 Apr 2016 06:30:45 +0000 Colorectal cancer (CRC) is one of the most common cancers all over the world. It is essential to search for more effective diagnostic and therapeutic methods for CRC. Abnormal nicotinamide adenine dinucleotide (NAD) metabolism has been considered as a characteristic of cancer cells. In this study, nicotinamide mononucleotide adenylyl transferases (NMNATs) as well as p53-mediated cancer signaling pathways were investigated in patients with colorectal cancer. The CRC tissues and adjacent normal tissues were obtained from 95 untreated colorectal cancer patients and were stained for expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and p53. The survival rate was analyzed by the Kaplan-Meier method and the log-rank test. The multivariate Cox proportional hazard regression analysis was conducted as well. Our data demonstrated that expression of NMNAT2 and p53 was significantly higher in CRC tissues, while NMNAT2 expression is in correlation with the invasive depth of tumors and TNM stage. Significant positive correlation was found between the expression of NMNAT2 and the expression of p53. However, NMNAT2 expression was not a statistically significant prognostic factor for overall survival. In conclusion, our results indicated that NMNAT2 might participate in tumorigenesis of CRC in a p53-dependent manner and NMNAT2 expression might be a potential therapeutic target for CRC. Chunhui Cui, Jia Qi, Quanwen Deng, Rihong Chen, Duanyang Zhai, and Jinlong Yu Copyright © 2016 Chunhui Cui et al. All rights reserved. Protective Effect of an Antibody against Specific Extracellular Domain of TLR2 on Agonists-Driven Inflammatory and Allergic Response Sun, 24 Apr 2016 08:30:58 +0000 Specific blocking strategies of TLR2-mediated inflammatory signaling and hypersensitivity reactions may offer novel therapeutic strategies to prevent a variety of diseases. In this study, we investigated the blocking effects of a new anti-TLR2 antibody anti-T20 against a 20 mer peptide T20 located in the extracellular specific domain of mouse TLR2. In addition, the effects of the anti-T20 in vitro, measuring the inhibition of the IL-6 and TNF-α production in response to PGN, LTA, and Pam3CSK4-stimulated RAW264.7 cells, were determined. In vivo, the effects of anti-T20 on a lethal anaphylaxis model using PGN-challenged OVA allergic mice, including the rectal temperature and mortality, and serum levels of TNF-α, IL-6, and LTC4 were assayed. The results showed that anti-T20 specifically bound to TLR2 and significantly inhibited PGN, LTA, and Pam3CSK4-driven TNF-α and IL-6 production by RAW264.7 cells. Also, anti-T20 protected OVA allergic mice from PGN-induced lethal anaphylaxis, and the serum levels of TNF-α, IL-6, and LTC4 of anti-T20 treated PGN-challenged OVA allergic mice were decreased as compared to isotype control of anti-T20 treated mice. In summary, this study produced a new antibody against the specific extracellular domain of TLR2 which has protective effect on TLR2 agonists-driven inflammatory and allergic response. Tianwu Guo, Jun Cai, Yanxia Peng, Lifang Zhang, Qiaofen Lan, Yanwen Chen, Huanjin Liao, Tong Xie, Ping Wu, and Qingjun Pan Copyright © 2016 Tianwu Guo et al. All rights reserved. The Possible Roles of Biological Bone Constructed with Peripheral Blood Derived EPCs and BMSCs in Osteogenesis and Angiogenesis Mon, 18 Apr 2016 16:32:06 +0000 This study aimed to determine the possible potential of partially deproteinized biologic bone (PDPBB) seeded with bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) in osteogenesis and angiogenesis. BMSCs and EPCs were isolated, identified, and cocultured in vitro, followed by seeding on the PDPBB. Expression of osteogenesis and vascularization markers was quantified by immunofluorescence (IF) staining, immunohistochemistry (IHC), and quantitive real-time polymerase chain reaction (qRT-PCR). Scanning electron microscope (SEM) was also employed to further evaluate the morphologic alterations of cocultured cells in the biologic bone. Results demonstrated that the coculture system combined with BMSCs and EPCs had significant advantages of (i) upregulating the mRNA expression of VEGF, Osteonectin, Osteopontin, and Collagen Type I and (ii) increasing ALP and OC staining compared to the BMSCs or EPCs only group. Moreover, IHC staining for CD105, CD34, and ZO-1 increased significantly in the implanted PDPBB seeded with coculture system, compared to that of BMSCs or EPCs only, respectively. Summarily, the present data provided evidence that PDPBB seeded with cocultured system possessed favorable cytocompatibility, provided suitable circumstances for different cell growth, and had the potential to provide reconstruction for cases with bone defection by promoting osteogenesis and angiogenesis. Li Wu, Xian Zhao, Bo He, Jie Jiang, Xiao-Jie Xie, and Liu Liu Copyright © 2016 Li Wu et al. All rights reserved. Apical External Root Resorption and Repair in Orthodontic Tooth Movement: Biological Events Tue, 29 Mar 2016 12:11:21 +0000 Some degree of external root resorption is a frequent, unpredictable, and unavoidable consequence of orthodontic tooth movement mediated by odontoclasts/cementoclasts originating from circulating precursor cells in the periodontal ligament. Its pathogenesis involves mechanical forces initiating complex interactions between signalling pathways activated by various biological agents. Resorption of cementum is regulated by mechanisms similar to those controlling osteoclastogenesis and bone resorption. Following root resorption there is repair by cellular cementum, but factors mediating the transition from resorption to repair are not clear. In this paper we review some of the biological events associated with orthodontically induced external root resorption. Liviu Feller, Razia A. G. Khammissa, George Thomadakis, Jeanine Fourie, and Johan Lemmer Copyright © 2016 Liviu Feller et al. All rights reserved. Hydrogen Peroxide and Sodium Transport in the Lung and Kidney Thu, 17 Mar 2016 09:55:02 +0000 Renal and lung epithelial cells are exposed to some significant concentrations of H2O2. In urine it may reach 100 μM, while in the epithelial lining fluid in the lung it is estimated to be in micromolar to tens-micromolar range. Hydrogen peroxide has a stimulatory action on the epithelial sodium channel (ENaC) single-channel activity. It also increases stability of the channel at the membrane and slows down the transcription of the ENaC subunits. The expression and the activity of the channel may be inhibited in some other, likely higher, oxidative states of the cell. This review discusses the role and the origin of H2O2 in the lung and kidney. Concentration-dependent effects of hydrogen peroxide on ENaC and the mechanisms of its action have been summarized. This review also describes outlooks for future investigations linking oxidative stress, epithelial sodium transport, and lung and kidney function. V. Shlyonsky, A. Boom, and F. Mies Copyright © 2016 V. Shlyonsky et al. All rights reserved. Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line Sun, 21 Feb 2016 16:46:05 +0000 The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-term in vitro studies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunit β (CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes. Yufei Zhang, Jing Shi, and Shuying Liu Copyright © 2016 Yufei Zhang et al. All rights reserved. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation Tue, 16 Feb 2016 14:06:55 +0000 Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. Chen-Shuang Li, Zhong Zheng, Xiao-Xia Su, Fei Wang, Michelle Ling, Min Zou, and Hong Zhou Copyright © 2016 Chen-Shuang Li et al. All rights reserved. Effects of Ginger Phenylpropanoids and Quercetin on Nrf2-ARE Pathway in Human BJ Fibroblasts and HaCaT Keratinocytes Thu, 28 Jan 2016 11:44:29 +0000 Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells. Ermin Schadich, Jan Hlaváč, Tereza Volná, Lakshman Varanasi, Marián Hajdúch, and Petr Džubák Copyright © 2016 Ermin Schadich et al. All rights reserved. Effect of miR-146a/bFGF/PEG-PEI Nanoparticles on Inflammation Response and Tissue Regeneration of Human Dental Pulp Cells Sun, 24 Jan 2016 14:19:16 +0000 Introduction. Inflammation in dental pulp cells (DPCs) initiated by Lipopolysaccharide (LPS) results in dental pulp necrosis. So far, whether there is a common system regulating inflammation response and tissue regeneration remains unknown. miR-146a is closely related to inflammation. Basic fibroblast growth factor (bFGF) is an important regulator for differentiation. Methods. To explore the effect of miR-146a/bFGF on inflammation and tissue regeneration, polyethylene glycol-polyethyleneimine (PEG-PEI) was synthesized, and physical characteristics were analyzed by dynamic light scattering and gel retardation analysis. Cell absorption, transfection efficiency, and cytotoxicity were assessed. Alginate gel was combined with miR-146a/PEG-PEI nanoparticles and bFGF. Drug release ratio was measured by ultraviolet spectrophotography. Proliferation and odontogenic differentiation of DPCs with 1 μg/mL LPS treatment were determined. Results. PEG-PEI prepared at N/P 2 showed complete gel retardation and smallest particle size and zeta potential. Transfection efficiency of PEG-PEI was higher than lipo2000. Cell viability decreased as N/P ratio increased. Drug release rate amounted to 70% at the first 12 h and then maintained slow release afterwards. Proliferation and differentiation decreased in DPCs with LPS treatment, whereas they increased in miR-146a/bFGF gel group. Conclusions. PEG-PEI is a promising vector for gene therapy. miR-146a and bFGF play critical roles in inflammation response and tissue regeneration of DPCs. Lu Liu, Shan Shu, Gary Shunpan Cheung, and Xi Wei Copyright © 2016 Lu Liu et al. All rights reserved. Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM Thu, 21 Jan 2016 07:20:53 +0000 Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2–6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy. Ibrahim Mustafa, Asma Al Marwani, Khuloud Mamdouh Nasr, Noora Abdulla Kano, and Tameem Hadwan Copyright © 2016 Ibrahim Mustafa et al. All rights reserved. After Myocardial Ischemia-Reperfusion, miR-29a, and Let7 Could Affect Apoptosis through Regulating IGF-1 Wed, 30 Dec 2015 11:42:38 +0000 Cardiovascular and cerebrovascular ischemic disease is a large class of diseases that is harmful to human health. The primary treatment for the ischemic disease is to recover the blood perfusion and relieve the tissue hypoxia and the shortage of the nutrients in the supply of nutrients. In recent years, investigations found that IGF-1 has a protective effect on cardiovascular disease, especially in myocardial ischemia-reperfusion injury. Investigation into molecular mechanism of ischemia-reperfusion injury may offer potential targets for the development of novel diagnostic strategies. In this study we defined IGF-1 was differentially expressed in the I/R model of the Mus musculus and IGF-1 was the target gene of miR-29a and Let7f. After ischemia-reperfusion, the expression of miR-29a and Let7f increased, while the expression of IGF-1 decreased significantly in the animal model assay. Further studies have found that IGF-1 could inhibit cell apoptosis signaling pathway, thus protecting the reperfusion injury. These results provide new understanding of ischemia-reperfusion injury, with the hope of offering theoretical support for future therapeutic studies. Lei Wang, Xuehong Niu, Jihua Hu, Haijian Xing, Min Sun, Juanli Wang, Qiang Jian, and Hua Yang Copyright © 2015 Lei Wang et al. All rights reserved. Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells Wed, 16 Dec 2015 06:38:31 +0000 Our previous study reported that muscle cell enhancement factor 2C (MEF2C) was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK) in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors. Zhenguo Dong, Wei Pan, Haiqing Wu, Dongjun Liu, and Ming Cang Copyright © 2015 Zhenguo Dong et al. All rights reserved. Transmission of Information in Neoplasia by Extracellular Vesicles Mon, 30 Nov 2015 07:23:49 +0000 Aurelio Lorico, Denis Corbeil, John M. Pawelek, and Riccardo Alessandro Copyright © 2015 Aurelio Lorico et al. All rights reserved. Antileukemic Effect of Tualang Honey on Acute and Chronic Leukemia Cell Lines Tue, 03 Nov 2015 06:38:20 +0000 Complementary medicine using natural product as antitumor is on the rise. Much research has been performed on Tualang Honey and it was shown to have therapeutic potential in wound healing, and antimicrobial activity and be antiproliferative against several cancer models such as human osteosarcoma (HOS), human breast (MCF-7 and MDA-MB-231), and cervical (HeLa) cancer cell lines. To date, there was limited study on antileukemic properties of Tualang (Koompassia excelsa) Honey. The aim of this study was to evaluate the antileukemic effect of Tualang Honey on acute and chronic leukemia cell lines. Leukemia cell lines (K562 and MV4-11) and human mononuclear cell isolated from peripheral blood were grown in RPM1 1640 culture medium. The cells were incubated with increasing concentrations of Tualang Honey. After incubation, the evaluation of viability and apoptosis was performed. The morphological changes of leukemia cells were the presence of cytoplasmic blebs followed by apoptotic bodies and round shape of cells. IC50 against K562 and MV4-11 was determined. Tualang Honey gave 53.9% and 50.6% apoptosis activity on K562 and MV4-11, respectively, while on human mononuclear cell it was 37.4%. Tualang Honey has the apoptosis-inducing ability for acute and chronic myeloid leukemia (K562 and MV4-11) cell lines. Nik Muhd Khuzaimi Nik Man, Rosline Hassan, Cheng Yong Ang, Abu Dzarr Abdullah, Muhammad Amiro Rasheeq Mohd Radzi, and Siti Amrah Sulaiman Copyright © 2015 Nik Muhd Khuzaimi Nik Man et al. All rights reserved. Breast Cancer-Derived Extracellular Vesicles: Characterization and Contribution to the Metastatic Phenotype Tue, 27 Oct 2015 14:04:43 +0000 The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers. Toni M. Green, Mary L. Alpaugh, Sanford H. Barsky, Germana Rappa, and Aurelio Lorico Copyright © 2015 Toni M. Green et al. All rights reserved. The Emerging Role of Extracellular Vesicle-Mediated Drug Resistance in Cancers: Implications in Advanced Prostate Cancer Mon, 26 Oct 2015 08:30:10 +0000 Emerging evidence has shown that the extracellular vesicles (EVs) regulate various biological processes and can control cell proliferation and survival, as well as being involved in normal cell development and diseases such as cancers. In cancer treatment, development of acquired drug resistance phenotype is a serious issue. Recently it has been shown that the presence of multidrug resistance proteins such as Pgp-1 and enrichment of the lipid ceramide in EVs could have a role in mediating drug resistance. EVs could also mediate multidrug resistance through uptake of drugs in vesicles and thus limit the bioavailability of drugs to treat cancer cells. In this review, we discussed the emerging evidence of the role EVs play in mediating drug resistance in cancers and in particular the role of EVs mediating drug resistance in advanced prostate cancer. The role of EV-associated multidrug resistance proteins, miRNA, mRNA, and lipid as well as the potential interaction(s) among these factors was probed. Lastly, we provide an overview of the current available treatments for advanced prostate cancer, considering where EVs may mediate the development of resistance against these drugs. Carolina Soekmadji and Colleen C. Nelson Copyright © 2015 Carolina Soekmadji and Colleen C. Nelson. All rights reserved. The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment Sun, 25 Oct 2015 11:49:18 +0000 Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer. Zhongrun Qian, Qi Shen, Xi Yang, Yongming Qiu, and Wenbin Zhang Copyright © 2015 Zhongrun Qian et al. All rights reserved. High LIN28A Expressing Ovarian Cancer Cells Secrete Exosomes That Induce Invasion and Migration in HEK293 Cells Sun, 25 Oct 2015 11:28:40 +0000 Epithelial ovarian cancer is the most aggressive and deadly form of ovarian cancer and is the most lethal gynecological malignancy worldwide; therefore, efforts to elucidate the molecular factors that lead to epithelial ovarian cancer are essential to better understand this disease. Recent studies reveal that tumor cells release cell-secreted vesicles called exosomes and these exosomes can transfer RNAs and miRNAs to distant sites, leading to cell transformation and tumor development. The RNA-binding protein LIN28 is a known marker of stem cells and when expressed in cancer, it is associated with poor tumor outcome. We hypothesized that high LIN28 expressing ovarian cancer cells secrete exosomes that can be taken up by nontumor cells and cause changes in gene expression and cell behavior associated with tumor development. IGROV1 cells were found to contain high LIN28A and secrete exosomes that were taken up by HEK293 cells. Moreover, exposure to these IGROV1 secreted exosomes led to significant increases in genes involved in Epithelial-to-Mesenchymal Transition (EMT), induced HEK293 cell invasion and migration. These changes were not observed with exosomes secreted by OV420 cells, which contain no detectable amounts of LIN28A or LIN28B. No evidence was found of LIN28A transfer from IGROV1 exosomes to HEK293 cells. Vanessa A. Enriquez, Ellane R. Cleys, Juliano C. Da Silveira, Monique A. Spillman, Quinton A. Winger, and Gerrit J. Bouma Copyright © 2015 Vanessa A. Enriquez et al. All rights reserved. Role of Extracellular Vesicles in Hematological Malignancies Sun, 25 Oct 2015 11:20:36 +0000 In recent years the role of tumor microenvironment in the progression of hematological malignancies has been widely recognized. Recent studies have focused on how cancer cells communicate within the microenvironment. Among several factors (cytokines, growth factors, and ECM molecules), a key role has been attributed to extracellular vesicles (EV), released from different cell types. EV (microvesicles and exosomes) may affect stroma remodeling, host cell functions, and tumor angiogenesis by inducing gene expression modulation in target cells, thus promoting cancer progression and metastasis. Microvesicles and exosomes can be recovered from the blood and other body fluids of cancer patients and contain and deliver genetic and proteomic contents that reflect the cell of origin, thus constituting a source of new predictive biomarkers involved in cancer development and serving as possible targets for therapies. Moreover, due to their specific cell-tropism and bioavailability, EV can be considered natural vehicles suitable for drug delivery. Here we will discuss the recent advances in the field of EV as actors in hematological cancer progression, pointing out the role of these vesicles in the tumor-host interplay and in their use as biomarkers for hematological malignancies. Stefania Raimondo, Chiara Corrado, Lavinia Raimondi, Giacomo De Leo, and Riccardo Alessandro Copyright © 2015 Stefania Raimondo et al. All rights reserved. Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions Sun, 25 Oct 2015 10:35:00 +0000 Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies. Gabriella Schiera, Carlo Maria Di Liegro, and Italia Di Liegro Copyright © 2015 Gabriella Schiera et al. All rights reserved. Role of Exosomal Noncoding RNAs in Lung Carcinogenesis Sun, 25 Oct 2015 09:37:19 +0000 Lung cancer is the major cause of cancer death worldwide. Novel, recently discovered classes of noncoding RNAs (ncRNAs) have diverse functional and regulatory activities and increasing evidence suggests crucial roles for deregulated ncRNAs in the onset and progression of cancer, including lung cancer. Exosomes are small extracellular membrane vesicles of endocytic origin that are released by many cells and are found in most body fluids. Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth and metastasis. MicroRNAs (miRNAs) are a subclass of ncRNAs that are present in exosomes. miRNAs are taken up by neighboring or distant cells and modulate various functions of recipient cells. Here, we review exosome-derived ncRNAs with a focus on miRNAs and their role in lung cancer biology. Tao Sun, Bill Kalionis, Guoying Lv, Shijin Xia, and Wen Gao Copyright © 2015 Tao Sun et al. All rights reserved. Comparative Analysis of the Hematopoietic Progenitor Cells from Placenta, Cord Blood, and Fetal Liver, Based on Their Immunophenotype Wed, 05 Aug 2015 06:18:39 +0000 We have investigated the characteristics of human hematopoietic progenitor cells (HPCs) with the CD34+CD45lowSSClow phenotype from full-term placental tissue (FTPT) as compared to cord blood (CB) and fetal liver (FL) cells. We demonstrated the presence of cell subpopulations at various stages of the differentiation with such immunophenotypes as CD34+/low, CD34++, CD34+++, CD45hi, and CD34++CD45hi in both first trimester placental tissue (FiTPT) and FTPT which implies their higher phenotypic heterogeneity compared to CB. HPCs of the FTPT origin expressed the CD90 antigen at a higher level compared to its expression by the CB HPCs and the CD133 antigen expression being at the same level in both cases. The HPCs compartment of FTPT versus CB contained higher number of myeloid and erythroid committed cells but lower number of myeloid and lymphoid ones compared to FL HPCs. HPCs of the FTPT and CB origin possess similar potentials for the multilineage differentiation in vitro and similar ratios of myeloid and erythroid progenitors among the committed cells. This observation suggests that the active hematopoiesis occurs in the FTPT. We obtained viable HPCs from cryopreserved placental tissue fragments allowing us to develop procedures for banking and testing of placenta-derived HPCs for clinical use. Maria D. Kuchma, Vitaliy M. Kyryk, Hanna M. Svitina, Yulia M. Shablii, Lubov L. Lukash, Galina S. Lobyntseva, and Volodymyr A. Shablii Copyright © 2015 Maria D. Kuchma et al. All rights reserved. Noncoding RNAs, Emerging Regulators of Skeletal Muscle Development and Diseases Tue, 14 Jul 2015 08:56:21 +0000 A healthy and independent life requires skeletal muscles to maintain optimal function throughout the lifespan, which is in turn dependent on efficient activation of processes that regulate muscle development, homeostasis, and metabolism. Thus, identifying mechanisms that modulate these processes is of crucial priority. Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), have emerged as a class of previously unrecognized transcripts whose importance in a wide range of biological processes and human disease is only starting to be appreciated. In this review, we summarize the roles of recently identified miRNAs and lncRNAs during skeletal muscle development and pathophysiology. We also discuss several molecular mechanisms of these noncoding RNAs. Undoubtedly, further systematic understanding of these noncoding RNAs’ functions and mechanisms will not only greatly expand our knowledge of basic skeletal muscle biology, but also significantly facilitate the development of therapies for various muscle diseases, such as muscular dystrophies, cachexia, and sarcopenia. Mao Nie, Zhong-Liang Deng, Jianming Liu, and Da-Zhi Wang Copyright © 2015 Mao Nie et al. All rights reserved. Noninflammatory Joint Contractures Arising from Immobility: Animal Models to Future Treatments Mon, 13 Jul 2015 11:29:06 +0000 Joint contractures, defined as the limitation in the passive range of motion of a mobile joint, can be classified as noninflammatory diseases of the musculoskeletal system. The pathophysiology is not well understood; limited information is available on causal factors, progression, the pathophysiology involved, and prediction of response to treatment. The clinical heterogeneity of joint contractures combined with the heterogeneous contribution of joint connective tissues to joint mobility presents challenges to the study of joint contractures. Furthermore, contractures are often a symptom of a wide variety of heterogeneous disorders that are in many cases multifactorial. Extended immobility has been identified as a causal factor and evidence is provided from both experimental and epidemiology studies. Of interest is the involvement of the joint capsule in the pathophysiology of joint contractures and lack of response to remobilization. While molecular pathways involved in the development of joint contractures are being investigated, current treatments focus on physiotherapy, which is ineffective on irreversible contractures. Future treatments may include early diagnosis and prevention. Kayleigh Wong, Guy Trudel, and Odette Laneuville Copyright © 2015 Kayleigh Wong et al. All rights reserved. Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells Mon, 13 Jul 2015 11:14:01 +0000 Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling. Rinaldo Florencio-Silva, Gisela Rodrigues da Silva Sasso, Estela Sasso-Cerri, Manuel Jesus Simões, and Paulo Sérgio Cerri Copyright © 2015 Rinaldo Florencio-Silva et al. All rights reserved. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System Thu, 11 Jun 2015 14:31:12 +0000 The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment. Takayuki Ueno, Jun Utsumi, Masakazu Toi, and Kazuharu Shimizu Copyright © 2015 Takayuki Ueno et al. All rights reserved. Angiogenesis and Vasculogenesis in Health and Disease Wed, 10 Jun 2015 12:08:29 +0000 Alessio D’Alessio, Francesco Moccia, Jie-Hui Li, Alessandra Micera, and Themis R. Kyriakides Copyright © 2015 Alessio D’Alessio et al. All rights reserved. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells Sun, 07 Jun 2015 08:58:18 +0000 Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. Bruno Lorusso, Angela Falco, Denise Madeddu, Caterina Frati, Stefano Cavalli, Gallia Graiani, Andrea Gervasi, Laura Rinaldi, Costanza Lagrasta, Davide Maselli, Letizia Gnetti, Enrico M. Silini, Eugenio Quaini, Luca Ampollini, Paolo Carbognani, and Federico Quaini Copyright © 2015 Bruno Lorusso et al. All rights reserved. Epoetin Alpha and Epoetin Zeta: A Comparative Study on Stimulation of Angiogenesis and Wound Repair in an Experimental Model of Burn Injury Thu, 04 Jun 2015 16:53:42 +0000 Deep second-degree burns are characterized by delayed formation of granulation tissue and impaired angiogenesis. Erythropoietin (EPO) is able to stimulate angiogenesis and mitosis, activating vascularization and cell cycle. The aim of our study was to investigate whether two biosimilar recombinant human erythropoietins, EPO-α and EPO-Z, may promote these processes in an experimental model of burn injury. A total of 84 mice were used and a scald burn was produced on the back after shaving, in 80°C water for 10 seconds. Mice were then randomized to receive EPO-α (400 units/kg/day/sc) or EPO-Z (400 units/kg/day/sc) or their vehicle (100 μL/day/sc 0.9% NaCl solution). After 12 days, both EPO-α and EPO-Z increased VEGF protein expression. EPO-α caused an increased cyclin D1/CDK6 and cyclin E/CDK2 expression compared with vehicle and EPO-Z (). Our study showed that EPO-α and EPO-Z accelerated wound closure and angiogenesis; however EPO-α resulted more effectively in achieving complete skin regeneration. Our data suggest that EPO-α and EPO-Z are not biosimilars for the wound healing effects. The higher efficacy of EPO-α might be likely due to its different conformational structure leading to a more efficient cell proliferation and skin remodelling. Natasha Irrera, Alessandra Bitto, Gabriele Pizzino, Mario Vaccaro, Francesco Squadrito, Mariarosaria Galeano, Francesco Stagno d’Alcontres, Ferdinando Stagno d’Alcontres, Michele Buemi, Letteria Minutoli, Michele Rosario Colonna, and Domenica Altavilla Copyright © 2015 Natasha Irrera et al. All rights reserved. Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies Thu, 04 Jun 2015 16:51:20 +0000 The cardiovascular system ensures the delivery of oxygen and nutrients to all cells, tissues, and organs. Under extended exposure to reduced oxygen levels, cells are able to survive through the transcriptional activation of a series of genes that participate in angiogenesis, glucose metabolism, and cell proliferation. The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions. The HIF-1 pathway was found to be a master regulator of angiogenesis. Whether the process is physiological or pathological, HIF-1 seems to participate in vasculature formation by synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor), PlGF (placental growth factor), or angiopoietins. Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis, it should be considered a promising target for treating ischaemic diseases or cancer. In this review, we discuss the roles of HIF-1 in both physiological/pathophysiological angiogenesis and potential strategies for clinical therapy. Agnieszka Zimna and Maciej Kurpisz Copyright © 2015 Agnieszka Zimna and Maciej Kurpisz. All rights reserved. OSM Enhances Angiogenesis and Improves Cardiac Function after Myocardial Infarction Thu, 04 Jun 2015 16:46:41 +0000 Oncostatin M (OSM) has been reported to stimulate angiogenesis by upregulating VEGF and bFGF, implying that it could be a therapeutic strategy in treating ischemic diseases. The present study was aimed at investigating whether OSM could improve cardiac function via prompting angiogenesis following myocardial infarction (MI). Wild type (WT) and Oβ knock-out (Oβ−/−) mice were, respectively, randomized into sham group, MI + vehicle group, and MI + OSM group. WT mice displayed significantly impaired cardiac function after MI. OSM treatment attenuated cardiac dysfunction in WT MI mice, while Oβ deletion abrogated the protective effects. Besides, OSM attenuated heart hypertrophy and pulmonary congestion evidenced by decreased heart weight/body weight and lung weight/body weight ratio. Further, reduction of apoptosis and fibrosis in infarct border zone was observed in OSM treated WT MI mice compared with vehicle. Moreover, in WT mice subjected to MI, OSM treatment significantly increased capillary density along with upregulation of p-Akt and angiogenic factors VEGF and bFGF in comparison with vehicle, and this phenomenon was not found in Oβ−/− mice. In conclusion, OSM treatment preserved cardiac function, inhibited apoptosis and fibrosis, and stimulated angiogenesis via upregulating VEGF and bFGF in infarct border zone of ischemic myocardium, indicating that OSM could be a novel therapeutic target for MI. Xiaotian Zhang, Di Zhu, Liping Wei, Zhijing Zhao, Xin Qi, Zongjin Li, and Dongdong Sun Copyright © 2015 Xiaotian Zhang et al. All rights reserved. Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis Thu, 04 Jun 2015 14:22:18 +0000 Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo. Wulf D. Ito, Natalie Lund, Ziyang Zhang, Friedrich Buck, Heinrich Lellek, Andrea Horst, Hans-Günther Machens, Heribert Schunkert, Wolfgang Schaper, and Thomas Meinertz Copyright © 2015 Wulf D. Ito et al. All rights reserved. The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis Thu, 04 Jun 2015 14:15:41 +0000 An association between mast cells and tumor angiogenesis is known to exist, but the exact role that mast cells play in this process is still unclear. It is thought that the mediators released by mast cells are important in neovascularization. However, it is not known how individual mediators are involved in this process. The major constituents of mast cell secretory granules are the mast cell specific proteases chymase, tryptase, and carboxypeptidase A3. Several previous studies aimed to understand the way in which specific mast cell granule constituents act to induce tumor angiogenesis. A body of evidence indicates that mast cell proteases are the pivotal players in inducing tumor angiogenesis. In this review, the likely mechanisms by which tryptase and chymase can act directly or indirectly to induce tumor angiogenesis are discussed. Finally, information presented here in this review indicates that mast cell proteases significantly influence angiogenesis thus affecting tumor growth and progression. This also suggests that these proteases could serve as novel therapeutic targets for the treatment of various types of cancer. Devandir Antonio de Souza Junior, Ana Carolina Santana, Elaine Zayas Marcelino da Silva, Constance Oliver, and Maria Celia Jamur Copyright © 2015 Devandir Antonio de Souza Junior et al. All rights reserved. Production of Human Endothelial Cells Free from Soluble Xenogeneic Antigens for Bioartificial Small Diameter Vascular Graft Endothelization Thu, 04 Jun 2015 14:14:07 +0000 Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease, but most lack adequate saphenous vein or other conduits for bypass procedures and would benefit from a bioartificial conduit. This study aimed to produce human endothelial cells (hECs) in large scale, free from xenogeneic antigens, to develop a small diameter, compatible vessel for potential use as a vascular graft. Human adipose-derived stromal cells (hASCs) were isolated, cultured, and differentiated in the presence of human serum and used for the reendothelization of a decellularized rat aorta. hASC derived ECs (hASC-ECs) expressed VEGFR2, vWf and CD31 endothelial cell markers, the latter in higher levels than hASCs and HUVECs, and were shown to be functional. Decellularization protocol yielded aortas devoid of cell nuclei, with preserved structure, including a preserved basement membrane. When seeded with hASC-ECs, the decellularized aorta was completely reendothelized, and the hASC-ECs maintained their phenotype in this new condition. hASCs can be differentiated into functional hECs without the use of animal supplements and are capable of reendothelizing a decellularized rat aorta while maintaining their phenotype. The preservation of the basement membrane following decellularization supported the complete reendothelization of the scaffold with no cell migration towards other layers. This approach is potentially useful for rapid obtention of compatible, xenogeneic-free conduit. Juliana Lott de Carvalho, Alessandra Zonari, Ana Cláudia Chagas de Paula, Thaís Maria da Mata Martins, Dawidson Assis Gomes, and Alfredo Miranda Goes Copyright © 2015 Juliana Lott de Carvalho et al. All rights reserved. Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways Thu, 04 Jun 2015 13:47:09 +0000 Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+ signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps. Irene Pafumi, Annarita Favia, Guido Gambara, Francesca Papacci, Elio Ziparo, Fioretta Palombi, and Antonio Filippini Copyright © 2015 Irene Pafumi et al. All rights reserved. Expression of Adiponectin Receptors on Peripheral Blood Leukocytes of Hypertensive Children Is Associated with the Severity of Hypertension Thu, 04 Jun 2015 13:46:59 +0000 The aim of the study was to find out whether peripheral blood leukocyte adiponectin receptors 1 and 2 (AdipoR1, AdipoR2) protein expression patterns (flow cytometry) differ between the primary hypertension children () and healthy controls () and if their expression levels are related to selected clinical parameters. The group of 26 patients [AdipoR(−)] showed lower and the group of 31 patients [AdipoR(+)] showed higher AdipoRs protein expression than the control and each other ( for neutrophils, for monocytes). The AdipoR(+) leukocytes expressed higher AdipoR1 mRNA levels (RT-PCR) than AdipoR(−) ones and controls ( and , resp.). Despite greater BMI, the AdipoR(−) patients had unchanged serum adiponectin levels. In contrast, AdipoR(+) patients had lower serum adiponectin concentrations than the AdipoR(−) ones and controls (). The AdipoR(+) patients had higher blood pressure () and greater carotid intima-media thickness () than the AdipoR(−) ones. The stage of hypertension was associated with increased neutrophil but not monocyte AdipoR1 density (AdipoR1 MFI) (). Severe ambulatory hypertension was presented more often in AdipoR(+) patients than in AdipoR(−) ones (51.6% versus 26.9%, resp.; ). In conclusion, neutrophil AdipoRs upregulation was associated with early stages of vascular injury, hypertension severity, and low serum levels of adiponectin. Lidia Gackowska, Mieczyslaw Litwin, Joanna Trojanek, Andrzej Eljaszewicz, Izabela Kubiszewska, Anna Niemirska, Aldona Wierzbicka, and Jacek Michalkiewicz Copyright © 2015 Lidia Gackowska et al. All rights reserved. Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts Thu, 21 May 2015 13:03:13 +0000 Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles. Lirija Alili, Swetlana Chapiro, Gernot U. Marten, Annette M. Schmidt, Klaus Zanger, and Peter Brenneisen Copyright © 2015 Lirija Alili et al. All rights reserved. Molecular Analysis of AFP and HSA Interactions with PTEN Protein Wed, 20 May 2015 11:24:17 +0000 Human cytoplasmic alpha-fetoprotein (AFP) has been classified as a member of the albuminoid gene family. The protein sequence of AFP has significant homology to that of human serum albumin (HSA), but its biological characteristics are vastly different from HSA. The AFP functions as a regulator in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, but HSA plays a key role as a transport protein. To probe their molecular mechanisms, we have applied colocalization, coimmunoprecipitation (co-IP), and molecular docking approaches to analyze the differences between AFP and HSA. The data from colocalization and co-IP displayed a strong interaction between AFP and PTEN (phosphatase and tensin homolog), demonstrating that AFP did bind to PTEN, but HSA did not. The molecular docking study further showed that the AFP domains I and III could contact with PTEN. In silicon substitutions of AFP binding site residues at position 490M/K and 105L/R corresponding to residues K490 and R105 in HSA resulted in steric clashes with PTEN residues R150 and K46, respectively. These steric clashes may explain the reason why HSA cannot bind to PTEN. Ultimately, the experimental results and the molecular modeling data from the interactions of AFP and HSA with PTEN will help us to identify targets for designing drugs and vaccines against human hepatocellular carcinoma. Mingyue Zhu, Bo Lin, Peng Zhou, and Mengsen Li Copyright © 2015 Mingyue Zhu et al. All rights reserved. Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro Mon, 18 May 2015 07:52:23 +0000 It has been proved that terminally differentiated mature adipocytes possess abilities to dedifferentiate into fibroblast-like progeny cells with self-renewal and multiple differentiation, termed dedifferentiated fat (DFAT) cells. However, the biological properties of DFAT cells during long-term culture in vitro have not been elucidated. Here, we obtained fibroblast-like morphology of porcine DFAT cells by ceiling culture. During the dedifferentiation process, round mature adipocytes with single large lipid droplets changed into spindle-shaped cells accompanied by the adipogenic markers PPARγ, aP2, LPL, and Adiponectin significant downregulation. Flow cytometric analysis showed that porcine DFAT cells displayed similar cell-surface antigen profile to mesenchymal stem cells (MSCs). Furthermore, different passages of porcine DFAT cells during long-term culture in vitro retained high levels of cell viabilities (>97%), efficient proliferative capacity including population doubling time ranged from 20 h to 22 h and population doubling reached by 58 days of culture. In addition, porcine DFAT cells maintained the multiple differentiation capabilities into adipocytes, osteoblasts, and skeletal myocytes and displayed normal chromosomal karyotypes for prolonged passaging. Therefore, porcine DFAT cells may be a novel model of stem cells for studying the functions of gene in the different biological events. Xuewu Peng, Tongxing Song, Xiaoming Hu, Yuanfei Zhou, Hongkui Wei, Jian Peng, and Siwen Jiang Copyright © 2015 Xuewu Peng et al. All rights reserved. Magnolia officinalis Extract Contains Potent Inhibitors against PTP1B and Attenuates Hyperglycemia in db/db Mice Thu, 07 May 2015 08:29:18 +0000 Protein tyrosine phosphatase 1B (PTP1B) is an established therapeutic target for type 2 diabetes mellitus (T2DM) and obesity. The aim of this study was to investigate the inhibitory activity of Magnolia officinalis extract (ME) on PTP1B and its anti-T2DM effects. Inhibition assays and inhibition kinetics of ME were performed in vitro. 3T3-L1 adipocytes and C2C12 myotubes were stimulated with ME to explore its bioavailability in cell level. The in vivo studies were performed on db/db mice to probe its anti-T2DM effects. In the present study, ME inhibited PTP1B in a reversible competitive manner and displayed good selectivity against PTPs in vitro. Furthermore, ME enhanced tyrosine phosphorylation levels of cellular proteins, especially the insulin-induced tyrosine phosphorylations of insulin receptor β-subunit (IRβ) and ERK1/2 in a dose-dependent manner in stimulated 3T3-L1 adipocytes and C2C12 myotubes. Meanwhile, ME enhanced insulin-stimulated GLUT4 translocation. More importantly, there was a significant decrease in fasting plasma glucose level of db/db diabetic mice treated orally with 0.5 g/kg ME for 4 weeks. These findings indicated that improvement of insulin sensitivity and hypoglycemic effects of ME may be attributed to the inhibition of PTP1B. Thereby, we pioneered the inhibitory potential of ME targeted on PTP1B as anti-T2DM drug discovery. Jing Sun, Yongsen Wang, Xueqi Fu, Yingli Chen, Deli Wang, Wannan Li, Shu Xing, and Guodong Li Copyright © 2015 Jing Sun et al. All rights reserved. Ca2+ Signaling in Cytoskeletal Reorganization, Cell Migration, and Cancer Metastasis Wed, 22 Apr 2015 12:32:59 +0000 Proper control of Ca2+ signaling is mandatory for effective cell migration, which is critical for embryonic development, wound healing, and cancer metastasis. However, how Ca2+ coordinates structural components and signaling molecules for proper cell motility had remained elusive. With the advance of fluorescent live-cell Ca2+ imaging in recent years, we gradually understand how Ca2+ is regulated spatially and temporally in migrating cells, driving polarization, protrusion, retraction, and adhesion at the right place and right time. Here we give an overview about how cells create local Ca2+ pulses near the leading edge, maintain cytosolic Ca2+ gradient from back to front, and restore Ca2+ depletion for persistent cell motility. Differential roles of Ca2+ in regulating various effectors and the interaction of roles of Ca2+ signaling with other pathways during migration are also discussed. Such information might suggest a new direction to control cancer metastasis by manipulating Ca2+ and its associating signaling molecules in a judicious manner. Feng-Chiao Tsai, Guan-Hung Kuo, Shu-Wei Chang, and Pei-Ju Tsai Copyright © 2015 Feng-Chiao Tsai et al. All rights reserved. TTBK2: A Tau Protein Kinase beyond Tau Phosphorylation Thu, 09 Apr 2015 12:39:45 +0000 Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions. Jung-Chi Liao, T. Tony Yang, Rueyhung Roc Weng, Ching-Te Kuo, and Chih-Wei Chang Copyright © 2015 Jung-Chi Liao et al. All rights reserved. Emerging Roles of Focal Adhesion Kinase in Cancer Tue, 31 Mar 2015 12:19:30 +0000 Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that enables activation by growth factor receptors or integrins in various types of human cancers. The kinase-dependent and kinase-independent scaffolding functions of FAK modulate the authentic signaling and fundamental functions not only in cancer cells but also in tumor microenvironment to facilitate cancer progression and metastasis. The overexpression and activation of FAK are usually investigated in primary or metastatic cancers and correlated with the poor clinical outcome, highlighting FAK as a potential prognostic marker and anticancer target. Small molecule inhibitors targeting FAK kinase activity or FAK-scaffolding functions impair cancer development in preclinical or clinical trials. In this review, we give an overview for FAK signaling in cancer cells as well as tumor microenvironment that provides new strategies for the invention of cancer development and malignancy. Yu-Ling Tai, Lih-Chyang Chen, and Tang-Long Shen Copyright © 2015 Yu-Ling Tai et al. All rights reserved. Functional Roles of Calreticulin in Cancer Biology Tue, 31 Mar 2015 11:36:28 +0000 Calreticulin is a highly conserved endoplasmic reticulum chaperone protein which participates in various cellular processes. It was first identified as a Ca2+-binding protein in 1974. Accumulated evidences indicate that calreticulin has great impacts for the development of different cancers and the effect of calreticulin on tumor formation and progression may depend on cell types and clinical stages. Cell surface calreticulin is considered as an “eat-me” signal and promotes phagocytic uptake of cancer cells by immune system. Moreover, several reports reveal that manipulation of calreticulin levels profoundly affects cancer cell proliferation and angiogenesis as well as differentiation. In addition to immunogenicity and tumorigenesis, interactions between calreticulin and integrins have been described during cell adhesion, which is an essential process for cancer metastasis. Integrins are heterodimeric transmembrane receptors which connect extracellular matrix and intracellular cytoskeleton and trigger inside-out or outside-in signaling transduction. More and more evidences reveal that proteins binding to integrins might affect integrin-cytoskeleton interaction and therefore influence ability of cell adhesion. Here, we reviewed the biological roles of calreticulin and summarized the potential mechanisms of calreticulin in regulating mRNA stability and therefore contributed to cancer metastasis. Yi-Chien Lu, Wen-Chin Weng, and Hsinyu Lee Copyright © 2015 Yi-Chien Lu et al. All rights reserved. Polymorphonuclear Leukocyte Apoptosis Is Accelerated by Sulfatides or Sulfatides-Treated Salmonella Typhimurium Bacteria Thu, 26 Mar 2015 13:00:23 +0000 Neutrophils die by apoptosis following activation and uptake of microbes or enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Here we report that sulfatides or sulfatides-treated Salmonella Typhimurium bacteria accelerated human neutrophil apoptosis. Neutrophil apoptosis was examined by flow cytometry. Sulfatides caused prominent increase in percentage of apoptotic cells after 2.5 hrs of incubation. Salmonella Typhimurium bacteria by themselves did not affect the basal level of apoptosis in neutrophil population. When neutrophils were added to S. Typhimurium “opsonized” by sulfatides, apoptotic index significantly increased, whereas the number of phagocyting cells was not influenced. Sulfatides’ proapoptotic effect was strongly dependent on the activity of β-galactosidase; inhibition of this enzyme impaired its potency to accelerate apoptosis. These data support the mechanism of neutrophil apoptosis triggering based on sulfatides’ ability to accumulate in intracellular compartments and mediate successive increase in ceramide content resulting from β-galactosidase activity. Zoryana V. Grishina, Galina M. Viryasova, Yulia M. Romanova, and Galina F. Sud’ina Copyright © 2015 Zoryana V. Grishina et al. All rights reserved. PVT1: A Rising Star among Oncogenic Long Noncoding RNAs Thu, 26 Mar 2015 07:26:50 +0000 It is becoming increasingly clear that short and long noncoding RNAs critically participate in the regulation of cell growth, differentiation, and (mis)function. However, while the functional characterization of short non-coding RNAs has been reaching maturity, there is still a paucity of well characterized long noncoding RNAs, even though large studies in recent years are rapidly increasing the number of annotated ones. The long noncoding RNA PVT1 is encoded by a gene that has been long known since it resides in the well-known cancer risk region 8q24. However, a couple of accidental concurrent conditions have slowed down the study of this gene, that is, a preconception on the primacy of the protein-coding over noncoding RNAs and the prevalent interest in its neighbor MYC oncogene. Recent studies have brought PVT1 under the spotlight suggesting interesting models of functioning, such as competing endogenous RNA activity and regulation of protein stability of important oncogenes, primarily of the MYC oncogene. Despite some advancements in modelling the PVT1 role in cancer, there are many questions that remain unanswered concerning the precise molecular mechanisms underlying its functioning. Teresa Colombo, Lorenzo Farina, Giuseppe Macino, and Paola Paci Copyright © 2015 Teresa Colombo et al. All rights reserved. MicroRNAs: Emerging Novel Targets of Cancer Therapies Thu, 19 Mar 2015 06:31:01 +0000 Chengfeng Yang, Yiguo Jiang, Ajay P. Singh, and Fumitaka Takeshita Copyright © 2015 Chengfeng Yang et al. All rights reserved. Modulation of MicroRNAs by Phytochemicals in Cancer: Underlying Mechanisms and Translational Significance Tue, 17 Mar 2015 06:22:31 +0000 MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that regulate a variety of biological processes such as differentiation, development, and survival. Recent studies suggest that miRNAs are dysregulated in cancer and play critical roles in cancer initiation, progression, and chemoresistance. Therefore, exploitation of miRNAs as targets for cancer prevention and therapy could be a promising approach. Extensive evidence suggests that many naturally occurring phytochemicals regulate the expression of numerous miRNAs involved in the pathobiology of cancer. Therefore, an understanding of the regulation of miRNAs by phytochemicals in cancer, their underlying molecular mechanisms, and functional consequences on tumor pathophysiology may be useful in formulating novel strategies to combat this devastating disease. These aspects are discussed in this review paper with an objective of highlighting the significance of these observations from the translational standpoint. Sanjeev K. Srivastava, Sumit Arora, Courey Averett, Seema Singh, and Ajay P. Singh Copyright © 2015 Sanjeev K. Srivastava et al. All rights reserved. Epigenetic Regulation in Hepatocellular Carcinoma Requires Long Noncoding RNAs Tue, 10 Mar 2015 13:53:13 +0000 Recent evidence has proven the relevance of epigenetic changes in the development of hepatocellular carcinoma (HCC), the major adult liver malignancy. Moreover, HCC onset and progression correlate with the deregulation of several long noncoding RNAs (lncRNAs), exhibiting great biological significance. As discussed in this review, many of these transcripts are able to specifically act as tumor suppressors or oncogenes by means of their role as molecular platforms. Indeed, these lncRNAs are able to bind and recruit epigenetic modifiers on specific genomic loci, ultimately resulting in regulation of the gene expression relevant in cancer development. The evidence presented in this review highlights that lncRNAs-mediated epigenetic regulation should be taken into account for potential targeted therapeutic approaches. Laura Amicone, Franca Citarella, and Carla Cicchini Copyright © 2015 Laura Amicone et al. All rights reserved. Elevated Serum Levels of Cysteine and Tyrosine: Early Biomarkers in Asymptomatic Adults at Increased Risk of Developing Metabolic Syndrome Wed, 04 Mar 2015 14:14:31 +0000 As there is effective intervention for delaying or preventing metabolic diseases, which are often present for years before becoming clinically apparent, novel biomarkers that would mark metabolic complications before the onset of metabolic disease should be identified. We investigated the role of fasting serum amino acids and their associations with inflammatory markers, adipokines, and metabolic syndrome (MetS) components in subjects prior to the onset of insulin resistance (IR). Anthropometric measurements, food records, adipokines, biochemical markers, and serum levels of amino acids were determined in 96 asymptomatic subjects aged 25–49 years divided into three groups according to the number of MetS components present. Cysteine and tyrosine were significantly higher already in group with one component of MetS present compared to subjects without MetS components. Serum amino acid levels correlated with markers of inflammation and adipokines. Alanine and glycine explained 10% of insulin resistance variability. The role of tyrosine and cysteine, that were higher already with 1 component of MetS present, should be further investigated as they might point to future insulin disturbances. Nina Mohorko, Ana Petelin, Mihaela Jurdana, Gianni Biolo, and Zala Jenko-Pražnikar Copyright © 2015 Nina Mohorko et al. All rights reserved. Ox-LDL Induces Dysfunction of Endothelial Progenitor Cells via Activation of NF-B Wed, 04 Mar 2015 07:53:19 +0000 Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity. Endothelial progenitor cells (EPCs) are thought to contribute to endothelial recovery after arterial injury. Oxidized low-density lipoprotein (ox-LDL) can induce EPC dysfunction, but the underlying mechanism is not well understood. Human EPCs were cultured in endothelial growth medium supplemented with VEGF (10 ng/mL) and bFGF (10 ng/mL). The cells were treated with ox-LDL (50 µg/mL). EPC proliferation was assayed by using CCK8 kits. Expression and translocation of nuclear factor-kabba B (NF-κB) were evaluated. The level of reactive oxygen species (ROS) in cells was measured using H2DCF-DA as a fluorescence probe. The activity of NADPH oxidase activity was determined by colorimetric assay. Ox-LDL significantly decreased the proliferation, migration, and adhesion capacity of EPCs, while significantly increased ROS production and NADPH oxidase expression. Ox-LDL induced NF-κB P65 mRNA expression and translocation in EPCs. Thus ox-LDL can induce EPC dysfunction at least by increasing expression and translocation of NF-κB P65 and NADPH oxidase activity, which represents a new mechanism of lipidemia-induced vascular injury. Kang-ting Ji, Lu Qian, Jin-liang Nan, Yang-jing Xue, Su-qin Zhang, Guo-qiang Wang, Ri-peng Yin, Yong-jin Zhu, Lu-ping Wang, Jun Ma, Lian-ming Liao, and Ji-fei Tang Copyright © 2015 Kang-ting Ji et al. All rights reserved. Macrophage Migration Inhibitory Factor Polymorphism Is Associated with Susceptibility to Inflammatory Coronary Heart Disease Wed, 04 Mar 2015 06:59:23 +0000 Background. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. This study explored the association of 173G/C polymorphism of the MIF gene with coronary heart disease (CHD). Methods. Sequencing was carried out after polymerase chain reaction with DNA specimens from 186 volunteers without CHD and 70 patients with CHD. Plasma MIF levels on admission were measured by ELISA. Patients were classified into either stable angina pectoris (SAP) or unstable angina pectoris (UAP). Genotype distribution between cases and controls and the association of patients’ genotypes with MIF level and plaque stability were statistically evaluated (ethical approval number: 2012-01). Results. The frequency of the C genotype was higher in CHD patients than in the control (). The frequency of the 173*CC genotype was higher in CHD patients than in the control (). The plasma MIF level was higher in MIF173*C carriers than in MIF173*G carriers (). CHD patients had higher plasma MIF levels than the control (). Patients with UAP had higher plasma MIF levels than patients with SAP (). Conclusions. These data suggest that MIF −173G/C polymorphism may be related to the development of CHD in a Chinese population. Plasma MIF level is a predictor of plaque stability. This trial is registered with NCT01750502 . Kangting Ji, Xiaoyan Wang, Ji Li, Qin Lu, Guoqiang Wang, Yangjing Xue, Suqin Zhang, Lu Qian, Wenwu Wu, Yongjin Zhu, Luping Wang, Lianming Liao, and Jifei Tang Copyright © 2015 Kangting Ji et al. All rights reserved. Effect of Luteolin and Apigenin on the Expression of Oct-4, Sox2, and c-Myc in Dental Pulp Cells with In Vitro Culture Thu, 26 Feb 2015 11:25:09 +0000 Introduction. Dental pulp cells (DPCs) are promising cell source for dental tissue regeneration. Recently, small molecules which optimize microenvironment or activate the reprogramming network provide a new way to enhance the pluripotency. Two promising bioflavonoids luteolin and apigenin were reported to enhance reprogramming efficiency in mouse embryonic fibroblast (MEF). However, their effect and underlying mechanism in cell fate determination of human DPCs remain unclear. Methods. To elucidate the effect of luteolin and apigenin on the cell fate determination of DPCs, we explored the cell proliferation, cell cycle, senescence, apoptosis, expression of pluripotency markers Oct-4, Sox2, and c-Myc, and multilineage differentiation capability of DPCs with luteolin or apigenin treatment. Results. We demonstrated that luteolin and apigenin inhibited cell proliferation, arrested DPCs in G2/M and S phase, and upregulated PI value and apoptosis. Moreover, luteolin and apigenin increased telomerase activity, maintained DPCs in a presenescent state, and activated the expression of Oct-4, Sox2, and c-Myc at a dose- and time-dependent pattern in DPCs even at late passages, albeit repressed lineage-specific differentiation. Conclusions. Addition of luteolin and apigenin in the culture medium might provide an effective way to maintain DPCs in an undifferentiated stage and inhibit lineage-specific differentiation. Lu Liu, Zhengjun Peng, Zezhen Xu, and Xi Wei Copyright © 2015 Lu Liu et al. All rights reserved. Serum/Plasma MicroRNAs as Biomarkers for HBV-Related Hepatocellular Carcinoma in China Thu, 22 Jan 2015 13:05:27 +0000 MicroRNAs (miRNAs) are a group of small RNAs with a fundamental role in the regulation of gene expression. These RNAs have been shown to participate in various cellular and physiological processes, including cellular development, apoptosis, proliferation, and differentiation. Aberrant expression of several miRNAs was found to be involved in a large variety of neoplasms, including hepatocellular carcinoma (HCC). Previous studies have shown the existence of a large amount of stable miRNAs in human serum/plasma, which laid the foundation for studying the role of serum/plasma miRNAs in the diagnosis and prognosis of HCC. Here, we review the recent progress in research on serum miRNAs as biomarkers for HCC in Chinese patients. Wen Yin, Yan Zhao, Yong-Jing Ji, Li-Ping Tong, Ya Liu, Shui-Xiang He, and Ai-Qin Wang Copyright © 2015 Wen Yin et al. All rights reserved. How Microgravity Affects the Biology of Living Systems Thu, 15 Jan 2015 12:39:21 +0000 Mariano Bizzarri, Monica Monici, and Jack J. W. A. van Loon Copyright © 2015 Mariano Bizzarri et al. All rights reserved. Nanoparticle Labeling of Bone Marrow-Derived Rat Mesenchymal Stem Cells: Their Use in Differentiation and Tracking Wed, 14 Jan 2015 14:05:49 +0000 Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies due to their ability to migrate to damaged tissue without inducing immune reaction. Many techniques have been developed to trace MSCs and their differentiation efficacy; however, all of these methods have limitations. Conjugated polymer based water-dispersible nanoparticles (CPN) represent a new class of probes because they offer high brightness, improved photostability, high fluorescent quantum yield, and noncytotoxicity comparing to conventional dyes and quantum dots. We aimed to use this tool for tracing MSCs’ fate in vitro and in vivo. MSC marker expression, survival, and differentiation capacity were assessed upon CPN treatment. Our results showed that after CPN labeling, MSC markers did not change and significant number of cells were found to be viable as revealed by MTT. Fluorescent signals were retained for 3 weeks after they were differentiated into osteocytes, adipocytes, and chondrocytes in vitro. We also showed that the labeled MSCs migrated to the site of injury and retained their labels in an in vivo liver regeneration model. The utilization of nanoparticle could be a promising tool for the tracking of MSCs in vivo and in vitro and therefore can be a useful tool to understand differentiation and homing mechanisms of MSCs. Ece Akhan, Donus Tuncel, and Kamil C. Akcali Copyright © 2015 Ece Akhan et al. All rights reserved. Simulated Microgravity: Critical Review on the Use of Random Positioning Machines for Mammalian Cell Culture Wed, 14 Jan 2015 12:55:07 +0000 Random Positioning Machines (RPMs) have been used since many years as a ground-based model to simulate microgravity. In this review we discuss several aspects of the RPM. Recent technological development has expanded the operative range of the RPM substantially. New possibilities of live cell imaging and partial gravity simulations, for example, are of particular interest. For obtaining valuable and reliable results from RPM experiments, the appropriate use of the RPM is of utmost importance. The simulation of microgravity requires that the RPM’s rotation is faster than the biological process under study, but not so fast that undesired side effects appear. It remains a legitimate question, however, whether the RPM can accurately and reliably simulate microgravity conditions comparable to real microgravity in space. We attempt to answer this question by mathematically analyzing the forces working on the samples while they are mounted on the operating RPM and by comparing data obtained under real microgravity in space and simulated microgravity on the RPM. In conclusion and after taking the mentioned constraints into consideration, we are convinced that simulated microgravity experiments on the RPM are a valid alternative for conducting examinations on the influence of the force of gravity in a fast and straightforward approach. Simon L. Wuest, Stéphane Richard, Sascha Kopp, Daniela Grimm, and Marcel Egli Copyright © 2015 Simon L. Wuest et al. All rights reserved. RhoGTPases as Key Players in Mammalian Cell Adaptation to Microgravity Tue, 13 Jan 2015 12:54:28 +0000 A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions. Fiona Louis, Christophe Deroanne, Betty Nusgens, Laurence Vico, and Alain Guignandon Copyright © 2015 Fiona Louis et al. All rights reserved. The Roads to Mitochondrial Dysfunction Tue, 13 Jan 2015 12:50:48 +0000 Marcos Roberto de Oliveira, Namasivayam Elangovan, Marko Ljubkovic, and Ancha Baranova Copyright © 2015 Marcos Roberto de Oliveira et al. All rights reserved. Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight Tue, 13 Jan 2015 12:49:21 +0000 Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness. Corey Nislow, Anna Y. Lee, Patricia L. Allen, Guri Giaever, Andrew Smith, Marinella Gebbia, Louis S. Stodieck, Jeffrey S. Hammond, Holly H. Birdsall, and Timothy G. Hammond Copyright © 2015 Corey Nislow et al. All rights reserved. Regulation of ICAM-1 in Cells of the Monocyte/Macrophage System in Microgravity Tue, 13 Jan 2015 12:41:39 +0000 Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells. Katrin Paulsen, Svantje Tauber, Claudia Dumrese, Gesine Bradacs, Dana M. Simmet, Nadine Gölz, Swantje Hauschild, Christiane Raig, Stephanie Engeli, Annett Gutewort, Eva Hürlimann, Josefine Biskup, Felix Unverdorben, Gabriela Rieder, Daniel Hofmänner, Lisa Mutschler, Sonja Krammer, Isabell Buttron, Claudia Philpot, Andreas Huge, Hartwin Lier, Ines Barz, Frank Engelmann, Liliana E. Layer, Cora S. Thiel, and Oliver Ullrich Copyright © 2015 Katrin Paulsen et al. All rights reserved. A Tissue Retrieval and Postharvest Processing Regimen for Rodent Reproductive Tissues Compatible with Long-Term Storage on the International Space Station and Postflight Biospecimen Sharing Program Tue, 13 Jan 2015 12:35:34 +0000 Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at −80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA’s Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities. Vijayalaxmi Gupta, Lesya Holets-Bondar, Katherine F. Roby, George Enders, and Joseph S. Tash Copyright © 2015 Vijayalaxmi Gupta et al. All rights reserved. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity Tue, 13 Jan 2015 11:57:06 +0000 Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. Cora S. Thiel, Swantje Hauschild, Svantje Tauber, Katrin Paulsen, Christiane Raig, Arnold Raem, Josefine Biskup, Annett Gutewort, Eva Hürlimann, Felix Unverdorben, Isabell Buttron, Beatrice Lauber, Claudia Philpot, Hartwin Lier, Frank Engelmann, Liliana E. Layer, and Oliver Ullrich Copyright © 2015 Cora S. Thiel et al. All rights reserved. A Whole-Genome Microarray Study of Arabidopsis thaliana Semisolid Callus Cultures Exposed to Microgravity and Nonmicrogravity Related Spaceflight Conditions for 5 Days on Board of Shenzhou 8 Tue, 13 Jan 2015 11:53:06 +0000 The Simbox mission was the first joint space project between Germany and China in November 2011. Eleven-day-old Arabidopsis thaliana wild type semisolid callus cultures were integrated into fully automated plant cultivation containers and exposed to spaceflight conditions within the Simbox hardware on board of the spacecraft Shenzhou 8. The related ground experiment was conducted under similar conditions. The use of an in-flight centrifuge provided a 1 g gravitational field in space. The cells were metabolically quenched after 5 days via RNAlater injection. The impact on the Arabidopsis transcriptome was investigated by means of whole-genome gene expression analysis. The results show a major impact of nonmicrogravity related spaceflight conditions. Genes that were significantly altered in transcript abundance are mainly involved in protein phosphorylation and MAPK cascade-related signaling processes, as well as in the cellular defense and stress responses. In contrast to short-term effects of microgravity (seconds, minutes), this mission identified only minor changes after 5 days of microgravity. These concerned genes coding for proteins involved in the plastid-associated translation machinery, mitochondrial electron transport, and energy production. Svenja Fengler, Ina Spirer, Maren Neef, Margret Ecke, Kay Nieselt, and Rüdiger Hampp Copyright © 2015 Svenja Fengler et al. All rights reserved. Space Flight Effects on Antioxidant Molecules in Dry Tardigrades: The TARDIKISS Experiment Tue, 13 Jan 2015 11:53:02 +0000 The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload, a set of multidisciplinary experiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in 2011. This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravity environment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space Station. TARDIKISS was composed of three sample sets: one flight sample and two ground control samples. These samples provided the biological material used to test as space stressors, including microgravity, affected animal survivability, life cycle, DNA integrity, and pathways of molecules working as antioxidants. In this paper we compared the molecular pathways of some antioxidant molecules, thiobarbituric acid reactive substances, and fatty acid composition between flight and control samples in two tardigrade species, namely, Paramacrobiotus richtersi and Ramazzottius oberhaeuseri. In both species, the activities of ROS scavenging enzymes, the total content of glutathione, and the fatty acids composition between flight and control samples showed few significant differences. TARDIKISS experiment, together with a previous space experiment (TARSE), further confirms that both desiccated and hydrated tardigrades represent useful animal tool for space research. Angela Maria Rizzo, Tiziana Altiero, Paola Antonia Corsetto, Gigliola Montorfano, Roberto Guidetti, and Lorena Rebecchi Copyright © 2015 Angela Maria Rizzo et al. All rights reserved. RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures Tue, 13 Jan 2015 11:32:00 +0000 We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. Caterina Morabito, Nathalie Steimberg, Giovanna Mazzoleni, Simone Guarnieri, Giorgio Fanò-Illic, and Maria A. Mariggiò Copyright © 2015 Caterina Morabito et al. All rights reserved. Large Artery Remodeling and Dynamics following Simulated Microgravity by Prolonged Head-Down Tilt Bed Rest in Humans Tue, 13 Jan 2015 11:16:38 +0000 The effects of simulated microgravity on the static and dynamic properties of large arteries are still mostly unknown. The present study evaluated, using an integrated vascular approach, changes in structure and function of the common carotid and femoral arteries (CCA and CFA) after prolonged head-down tilt bed rest (HDTBR). Ten healthy men were enrolled in a 5-week HDTBR study endorsed by the Italian Space Agency (ASI). Arterial geometry, flow, stiffness, and shear rate were evaluated by ultrasound. Local carotid pulse pressure and wave reflection were studied by applanation tonometry. After five weeks of HDTBR, CFA showed a decrease in lumen diameter without significant changes in wall thickness (IMT), resulting in an inward remodeling. Local carotid pulse pressure decreased and carotid-to-brachial pressure amplification increased. The ratio of systolic-to-diastolic volumetric flow in CFA decreased, whereas in CCA it tended to increase. Indices of arterial stiffness and shear rate did not change during HDTBR, either in CCA or CFA. In summary, prolonged HDTBR has a different impact on CCA and CFA structure and flow, probably depending on the characteristics of the vascular bed perfused. Carlo Palombo, Carmela Morizzo, Martino Baluci, Daniela Lucini, Stefano Ricci, Gianni Biolo, Piero Tortoli, and Michaela Kozakova Copyright © 2015 Carlo Palombo et al. All rights reserved. The Impact of Microgravity and Hypergravity on Endothelial Cells Tue, 13 Jan 2015 10:41:10 +0000 The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature. Jeanette A. M. Maier, Francesca Cialdai, Monica Monici, and Lucia Morbidelli Copyright © 2015 Jeanette A. M. Maier et al. All rights reserved. The Roads to Mitochondrial Dysfunction in a Rat Model of Posttraumatic Syringomyelia Tue, 13 Jan 2015 09:27:58 +0000 The pathophysiology of posttraumatic syringomyelia is incompletely understood. We examined whether local ischemia occurs after spinal cord injury. If so, whether it causes neuronal mitochondrial dysfunction and depletion, and subsequent energy metabolism impairment results in cell starvation of energy and even cell death, contributing to the enlargement of the cavity. Local blood flow was measured in a rat model of posttraumatic syringomyelia that had received injections of quisqualic acid and kaolin. We found an % reduction of local blood flow at C8 where a cyst formed at 6 weeks after syrinx induction procedure , and no difference in blood flow rate between the laminectomy and intact controls. Electron microscopy confirmed irreversible neuronal mitochondrion depletion surrounding the cyst, but recoverable mitochondrial loses in laminectomy rats. Profound energy loss quantified in the spinal cord of syrinx animals, and less ATP and ADP decline observed in laminectomy rats. Our findings demonstrate that an excitotoxic injury induces local ischemia in the spinal cord and results in neuronal mitochondrial depletion, and profound ATP loss, contributing to syrinx enlargement. Ischemia did not occur following laminectomy induced trauma in which mitochondrial loss and decline in ATP were reversible. This confirms excitotoxic injury contributing to the pathology of posttraumatic syringomyelia. Zhiqiang Hu and Jian Tu Copyright © 2015 Zhiqiang Hu and Jian Tu. All rights reserved. Dose Response of Endotoxin on Hepatocyte and Muscle Mitochondrial Respiration In Vitro Mon, 12 Jan 2015 11:39:22 +0000 Introduction. Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. Methods. Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1–100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. Results. In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). Conclusion. LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner. Victor Jeger, Sebastian Brandt, Francesca Porta, Stephan M. Jakob, Jukka Takala, and Siamak Djafarzadeh Copyright © 2015 Victor Jeger et al. All rights reserved. Molecular and Cellular Basis of Autosomal Recessive Primary Microcephaly Mon, 08 Dec 2014 00:10:43 +0000 Autosomal recessive primary microcephaly (MCPH) is a rare hereditary neurodevelopmental disorder characterized by a marked reduction in brain size and intellectual disability. MCPH is genetically heterogeneous and can exhibit additional clinical features that overlap with related disorders including Seckel syndrome, Meier-Gorlin syndrome, and microcephalic osteodysplastic dwarfism. In this review, we discuss the key proteins mutated in MCPH. To date, MCPH-causing mutations have been identified in twelve different genes, many of which encode proteins that are involved in cell cycle regulation or are present at the centrosome, an organelle crucial for mitotic spindle assembly and cell division. We highlight recent findings on MCPH proteins with regard to their role in cell cycle progression, centrosome function, and early brain development. Marine Barbelanne and William Y. Tsang Copyright © 2014 Marine Barbelanne and William Y. Tsang. All rights reserved. Deregulation of Serum MicroRNA Expression Is Associated with Cigarette Smoking and Lung Cancer Mon, 20 Oct 2014 09:28:51 +0000 Lung cancer is the leading cause of cancer-related death and cigarette smoking is the main risk factor for lung cancer. Circulating microRNAs (miRNAs) are considered potential biomarkers of various cancers, including lung cancer. However, it is unclear whether changes in circulating miRNAs are associated with smoking and smoking-related lung cancer. In this study, we determined the serum miRNA profiles of 10 nonsmokers, 10 smokers, and 10 lung-cancer patients with miRCURY LNA microRNA arrays. The differentially expressed miRNAs were then confirmed in a larger sample. We found that let-7i-3p and miR-154-5p were significantly downregulated in the sera of smokers and lung-cancer patients, so the serum levels of let-7i-3p and miR-154-5p are associated with smoking and smoking-related lung cancer. The areas under receiver operating characteristic curves for let-7i-3p and miR-154-5p were approximately 0.892 and 0.957, respectively. In conclusion, our results indicate that changes in serum miRNAs are associated with cigarette smoking and lung cancer and that let-7i-3p and miR-154-5p are potential biomarkers of smoking-related lung cancer. Jinkun Huang, Jianjun Wu, Yuanqi Li, Xun Li, Ti Yang, Qiaoyuan Yang, and Yiguo Jiang Copyright © 2014 Jinkun Huang et al. All rights reserved. Planarians Sense Simulated Microgravity and Hypergravity Wed, 17 Sep 2014 06:44:19 +0000 Planarians are flatworms, which belong to the phylum Platyhelminthes. They have been a classical subject of study due to their amazing regenerative ability, which relies on the existence of adult totipotent stem cells. Nowadays they are an emerging model system in the field of developmental, regenerative, and stem cell biology. In this study we analyze the effect of a simulated microgravity and a hypergravity environment during the process of planarian regeneration and embryogenesis. We demonstrate that simulated microgravity by means of the random positioning machine (RPM) set at a speed of 60 °/s but not at 10 °/s produces the dead of planarians. Under hypergravity of 3 g and 4 g in a large diameter centrifuge (LDC) planarians can regenerate missing tissues, although a decrease in the proliferation rate is observed. Under 8 g hypergravity small planarian fragments are not able to regenerate. Moreover, we found an effect of gravity alterations in the rate of planarian scission, which is its asexual mode of reproduction. No apparent effects of altered gravity were found during the embryonic development. Teresa Adell, Emili Saló, Jack J. W. A. van Loon, and Gennaro Auletta Copyright © 2014 Teresa Adell et al. All rights reserved. A Functional Interplay between 5-Lipoxygenase and μ-Calpain Affects Survival and Cytokine Profile of Human Jurkat T Lymphocyte Exposed to Simulated Microgravity Tue, 16 Sep 2014 07:08:26 +0000 A growing body of evidence strongly indicates that both simulated and authentic weightlessness exert a broad range of effects on mammalian tissues and cells, including impairment of immune cell function and increased apoptotic death. We previously reported that microgravity-dependent activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of apoptosis in human T lymphocytes, suggesting that the upregulation of this enzyme might be (at least in part) responsible for immunodepression observed in astronauts during space flights. Herein, we supplement novel information about the molecular mechanisms underlying microgravity-triggered apoptotic cell death and immune system deregulation, demonstrating that under simulated microgravity human Jurkat T cells increase the content of cytosolic DNA fragments and cytochrome c (typical hallmarks of apoptosis) and have an upregulated expression and activity of µ-calpain. These events were paralleled by the unbalance of interleukin- (IL-) 2 and interferon- (INF-) γ, anti- and proapoptotic cytokines, respectively, that seemed to be dependent on the functional interplay between 5-LOX and µ-calpain. Indeed, we report unprecedented evidence that 5-LOX inhibition reduced apoptotic death, restored the initial IL-2/INF-γ ratio, and more importantly reverted µ-calpain activation induced by simulated microgravity. Valeria Gasperi, Cinzia Rapino, Natalia Battista, Monica Bari, Nicolina Mastrangelo, Silvia Angeletti, Enrico Dainese, and Mauro Maccarrone Copyright © 2014 Valeria Gasperi et al. All rights reserved. Genetic Networks Lead and Follow Tumor Development: MicroRNA Regulation of Cell Cycle and Apoptosis in the p53 Pathways Thu, 11 Sep 2014 06:37:07 +0000 During the past ten years, microRNAs (miRNAs) have been shown to play a more significant role in the formation and progression of cancer diseases than previously thought. With an increase in reports about the dysregulation of miRNAs in diverse tumor types, it becomes more obvious that classic tumor-suppressive molecules enter deep into the world of miRNAs. Recently, it has been demonstrated that a typical tumor suppressor p53, known as the guardian of the genome, regulates some kinds of miRNAs to contribute to tumor suppression by the induction of cell-cycle arrest and apoptosis. Meanwhile, miRNAs directly/indirectly control the expression level and activity of p53 to fine-tune its functions or to render p53 inactive, indicating that the interplay between p53 and miRNA is overly complicated. The findings, along with current studies, will underline the continuing importance of understanding this interlocking control system for future therapeutic strategies in cancer treatment and prevention. Kurataka Otsuka and Takahiro Ochiya Copyright © 2014 Kurataka Otsuka and Takahiro Ochiya. All rights reserved. How Microgravity Changes Galectin-3 in Thyroid Follicles Thu, 11 Sep 2014 06:26:35 +0000 After long-term exposure to real microgravity thyroid gland in vivo undergoes specific changes, follicles are made up of larger thyrocytes that produce more cAMP and express more thyrotropin-receptor, caveolin-1, and sphingomyelinase and sphingomyelin-synthase; parafollicular spaces lose C cells with consequent reduction of calcitonin production. Here we studied four immunohistochemical tumor markers (HBME-1, MIB-1, CK19, and Galectin-3) in thyroid of mice housed in the Mouse Drawer System and maintained for 90 days in the International Space Station. Results showed that MIB-1 proliferative index and CK19 are negative whereas HBME-1 and Galectin-3 are overexpressed. The positivity of Galectin-3 deserves attention not only for its expression but also and especially for its localization. Our results highlighted that, in microgravity conditions, Galectin-3 leaves thyrocytes and diffuses in colloid. It is possible that the gravity force contributes to the maintenance of the distribution of the molecules in both basal membrane side and apical membrane side and that the microgravity facilitates slippage of Galectin-3 in colloid probably due to membrane remodelling-microgravity induced. Elisabetta Albi, Francesco Curcio, Andrea Lazzarini, Alessandro Floridi, Samuela Cataldi, Remo Lazzarini, Elisabetta Loreti, Ivana Ferri, and Francesco Saverio Ambesi-Impiombato Copyright © 2014 Elisabetta Albi et al. All rights reserved. The Impact of Extracellular Vesicle-Encapsulated Circulating MicroRNAs in Lung Cancer Research Thu, 11 Sep 2014 00:00:00 +0000 Lung cancer is the leading cause of cancer-related deaths. Biomarkers for lung cancer have raised great expectations in their clinical applications for early diagnosis, survival, and therapeutic responses. MicroRNAs (miRNAs), a family of short endogenous noncoding RNAs, play critical roles in cell growth, differentiation, and the development of various types of cancers. Current studies have shown that miRNAs are present in the extracellular spaces, packaged into various membrane-bound vesicles. Tumor-specific circulating miRNAs have been developed as early diagnostic biomarkers for lung cancer. Remarkably, some studies have succeeded in discovering circulating miRNAs with prognostic or predictive significance. Extracellular vesicles (EVs), such as exosomes and microvesicles, are recognized as novel tools for cell-cell communication and as biomarkers for various diseases. Their vesicle composition and miRNA content have the ability to transfer biological information to recipient cells and play an important role in cancer metastasis and prognosis. This review provides an in-depth summary of current findings on circulating miRNAs in lung cancer patients used as diagnostic biomarkers. We also discuss the role of EV miRNAs in cell-cell communication and explore the effectiveness of these contents as predictive biomarkers for cancer malignancy. Yu Fujita, Kazuyoshi Kuwano, Takahiro Ochiya, and Fumitaka Takeshita Copyright © 2014 Yu Fujita et al. All rights reserved. Lung Cancer Stem Cell Lose Their Stemness Default State after Exposure to Microgravity Sun, 07 Sep 2014 12:10:33 +0000 Microgravity influences cell differentiation by modifying the morphogenetic field in which stem cells are embedded. Preliminary data showed indeed that stem cells are committed to selective differentiation when exposed to real or simulated microgravity. Our study provides evidence that a similar event occurs when cancer stem cells (CSCs) are cultured in microgravity. In the same time, a significant increase in apoptosis was recorded: those data point out that microgravity rescues CSCs from their relative quiescent state, inducing CSCs to lose their stemness features, as documented by the decrease in ALDH and the downregulation of both Nanog and Oct-4 genes. Those traits were stably acquired and preserved by CSCs when cells were placed again on a 1 g field. Studies conducted in microgravity on CSCs may improve our understanding of the fundamental role exerted by biophysical forces in cancer cell growth and function. Maria Elena Pisanu, Alessia Noto, Claudia De Vitis, Maria Grazia Masiello, Pierpaolo Coluccia, Sara Proietti, Maria Rosaria Giovagnoli, Alberto Ricci, Enrico Giarnieri, Alessandra Cucina, Gennaro Ciliberto, Mariano Bizzarri, and Rita Mancini Copyright © 2014 Maria Elena Pisanu et al. All rights reserved. Silencing of Plasma Membrane Ca2+-ATPase Isoforms 2 and 3 Impairs Energy Metabolism in Differentiating PC12 Cells Sun, 07 Sep 2014 11:33:38 +0000 A close link between Ca2+, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca2+ may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+ in cytosol. In differentiation process plasma membrane Ca2+ ATPase (PMCA) is considered as one of the major players for Ca2+ homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation. Tomasz Boczek, Malwina Lisek, Bozena Ferenc, Antoni Kowalski, Magdalena Wiktorska, and Ludmila Zylinska Copyright © 2014 Tomasz Boczek et al. All rights reserved. Regulation of MicroRNAs by Natural Agents: New Strategies in Cancer Therapies Mon, 01 Sep 2014 05:44:54 +0000 MicroRNAs (miRNAs) are short noncoding RNA which regulate gene expression by messenger RNA (mRNA) degradation or translation repression. The plethora of published reports in recent years demonstrated that they play fundamental roles in many biological processes, such as carcinogenesis, angiogenesis, programmed cell death, cell proliferation, invasion, migration, and differentiation by acting as tumour suppressor or oncogene, and aberrations in their expressions have been linked to onset and progression of various cancers. Furthermore, each miRNA is capable of regulating the expression of many genes, allowing them to simultaneously regulate multiple cellular signalling pathways. Hence, miRNAs have the potential to be used as biomarkers for cancer diagnosis and prognosis as well as therapeutic targets. Recent studies have shown that natural agents such as curcumin, resveratrol, genistein, epigallocatechin-3-gallate, indole-3-carbinol, and 3,3′-diindolylmethane exert their antiproliferative and/or proapoptotic effects through the regulation of one or more miRNAs. Therefore, this review will look at the regulation of miRNAs by natural agents as a means to potentially enhance the efficacy of conventional chemotherapy through combinatorial therapies. It is hoped that this would provide new strategies in cancer therapies to improve overall response and survival outcome in cancer patients. Neoh Hun Phuah and Noor Hasima Nagoor Copyright © 2014 Neoh Hun Phuah and Noor Hasima Nagoor. All rights reserved. Autophagy in Development, Cell Differentiation, and Homeodynamics: From Molecular Mechanisms to Diseases and Pathophysiology Thu, 28 Aug 2014 12:39:18 +0000 Ioannis P. Nezis, Maria I. Vaccaro, Rodney J. Devenish, and Gábor Juhász Copyright © 2014 Ioannis P. Nezis et al. All rights reserved. The Influence of Simulated Microgravity on Purinergic Signaling Is Different between Individual Culture and Endothelial and Smooth Muscle Cell Coculture Thu, 28 Aug 2014 00:00:00 +0000 Exposure to microgravity conditions causes cardiovascular deconditioning in astronauts during spaceflight. Until now, no specific drugs are available for countermeasure, since the underlying mechanism is largely unknown. Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in various vascular functions, many of which are regulated by purinergic 2 (P2) receptors. However, their function in ECs and SMCs under microgravity conditions is still unclear. In this study, primary ECs and SMCs were isolated from bovine aorta and verified with specific markers. We show for the first time that the P2 receptor expression pattern is altered in ECs and SMCs after 24 h exposure to simulated microgravity using a clinostat. However, conditioned medium compensates this change in specific P2 receptors, for example, P2X7. Notably, P2 receptors such as P2X7 might be the important players during the paracrine interaction. Additionally, ECs and SMCs secreted different cytokines under simulated microgravity, leading into a pathogenic proliferation and migration. In conclusion, our data indicate P2 receptors might be important players responding to gravity changes in ECs and SMCs. Since some artificial P2 receptor ligands are applied as drugs, it is reasonable to assume that they might be promising candidates against cardiovascular deconditioning in the future. Yu Zhang, Patrick Lau, Andreas Pansky, Matthias Kassack, Ruth Hemmersbach, and Edda Tobiasch Copyright © 2014 Yu Zhang et al. All rights reserved. Human Locomotion under Reduced Gravity Conditions: Biomechanical and Neurophysiological Considerations Thu, 28 Aug 2014 00:00:00 +0000 Reduced gravity offers unique opportunities to study motor behavior. This paper aims at providing a review on current issues of the known tools and techniques used for hypogravity simulation and their effects on human locomotion. Walking and running rely on the limb oscillatory mechanics, and one way to change its dynamic properties is to modify the level of gravity. Gravity has a strong effect on the optimal rate of limb oscillations, optimal walking speed, and muscle activity patterns, and gait transitions occur smoothly and at slower speeds at lower gravity levels. Altered center of mass movements and interplay between stance and swing leg dynamics may challenge new forms of locomotion in a heterogravity environment. Furthermore, observations in the lack of gravity effects help to reveal the intrinsic properties of locomotor pattern generators and make evident facilitation of nonvoluntary limb stepping. In view of that, space neurosciences research has participated in the development of new technologies that can be used as an effective tool for gait rehabilitation. Francesca Sylos-Labini, Francesco Lacquaniti, and Yuri P. Ivanenko Copyright © 2014 Francesca Sylos-Labini et al. All rights reserved. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity Tue, 19 Aug 2014 07:21:27 +0000 Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. Alessandra Cazzaniga, Sara Castiglioni, and Jeanette A. M. Maier Copyright © 2014 Alessandra Cazzaniga et al. All rights reserved. Phenotypic Switch Induced by Simulated Microgravity on MDA-MB-231 Breast Cancer Cells Mon, 18 Aug 2014 08:24:31 +0000 Microgravity exerts dramatic effects on cell morphology and functions, by disrupting cytoskeleton and adhesion structures, as well as by interfering with biochemical pathways and gene expression. Impairment of cells behavior has both practical and theoretical significance, given that investigations of mechanisms involved in microgravity-mediated effects may shed light on how biophysical constraints cooperate in shaping complex living systems. By exposing breast cancer MDA-MB-231 cells to simulated microgravity (~0.001 g), we observed the emergence of two morphological phenotypes, characterized by distinct membrane fractal values, surface area, and roundness. Moreover, the two phenotypes display different aggregation profiles and adherent behavior on the substrate. These morphological differences are mirrored by the concomitant dramatic functional changes in cell processes (proliferation and apoptosis) and signaling pathways (ERK, AKT, and Survivin). Furthermore, cytoskeleton undergoes a dramatic reorganization, eventually leading to a very different configuration between the two populations. These findings could be considered adaptive and reversible features, given that, by culturing microgravity-exposed cells into a normal gravity field, cells are enabled to recover their original phenotype. Overall these data outline the fundamental role gravity plays in shaping form and function in living systems. Maria Grazia Masiello, Alessandra Cucina, Sara Proietti, Alessandro Palombo, Pierpaolo Coluccia, Fabrizio D’Anselmi, Simona Dinicola, Alessia Pasqualato, Veronica Morini, and Mariano Bizzarri Copyright © 2014 Maria Grazia Masiello et al. All rights reserved. Oxidative Stress and NO Signalling in the Root Apex as an Early Response to Changes in Gravity Conditions Sun, 17 Aug 2014 11:15:12 +0000 Oxygen influx showed an asymmetry in the transition zone of the root apex when roots were placed horizontally on ground. The influx increased only in the upper side, while no changes were detected in the division and in the elongation zone. Nitric oxide (NO) was also monitored after gravistimulation, revealing a sudden burst only in the transition zone. In order to confirm these results in real microgravity conditions, experiments have been set up by using parabolic flights and drop tower. The production of reactive oxygen species (ROS) was also monitored. Oxygen, NO, and ROS were continuously monitored during normal and hyper- and microgravity conditions in roots of maize seedlings. A distinct signal in oxygen and NO fluxes was clearly detected only in the apex zone during microgravity, with no significant changes in normal and in hypergravity conditions. The same results were obtained by ROS measurement. The detrimental effect of D’orenone, disrupting the polarised auxin transport, on the onset of the oxygen peaks during the microgravity period was also evaluated. Results indicates an active role of NO and ROS as messengers during the gravitropic response, with probable implications in the auxin redistribution. Sergio Mugnai, Camilla Pandolfi, Elisa Masi, Elisa Azzarello, Emanuela Monetti, Diego Comparini, Boris Voigt, Dieter Volkmann, and Stefano Mancuso Copyright © 2014 Sergio Mugnai et al. All rights reserved. Bcl-2 Family Proteins Are Involved in the Signal Crosstalk between Endoplasmic Reticulum Stress and Mitochondrial Dysfunction in Tumor Chemotherapy Resistance Sun, 10 Aug 2014 12:22:49 +0000 Tumor cells overexpress antiapoptotic proteins of the Bcl-2 (B-cell leukemia/lymphoma-2) family, which can lead to both escape from cell death and resistance to chemotherapeutic drugs. Recent studies suggest that the endoplasmic reticulum (ER) can produce proapoptotic signals, amplifying the apoptotic signaling cascade. The crosstalk between mitochondria and ER plays a decisive role in many cellular events but especially in cell death. Bcl-2 family proteins located in the ER and mitochondria can influence not only the function of the two organelles but also the interaction between them. Therefore, the Bcl-2 family of proteins may also be involved in the mechanism of tumor chemotherapy resistance by influencing crosstalk between the ER and mitochondria. In this review we will briefly discuss evidence to support this concept. Jing Su, Lei Zhou, Mei-hui Xia, Ye Xu, Xi-yan Xiang, and Lian-kun Sun Copyright © 2014 Jing Su et al. All rights reserved. Inflammation in Muscle Repair, Aging, and Myopathies Mon, 04 Aug 2014 08:38:06 +0000 Marina Bouché, Pura Muñoz-Cánoves, Fabio Rossi, and Dario Coletti Copyright © 2014 Marina Bouché et al. All rights reserved. Upregulation of Pluripotency Markers in Adipose Tissue-Derived Stem Cells by miR-302 and Leukemia Inhibitory Factor Wed, 23 Jul 2014 11:40:48 +0000 The expression pattern of pluripotency markers in adipose tissue-derived stem cells (ADSCs) is a subject of controversy. Moreover, there is no data about the signaling molecules that regulate these markers in ADSCs. In the present study, we studied the roles of leukemia inhibitory factor (LIF) and miR-302 in this regard. Freshly isolated mouse ADSCs expressed hematopoietic, mesenchymal, and pluripotency markers. One day after plating, ADSCs expressed OCT4 and Sox2 proteins. After three passages, the expression of hematopoietic and pluripotency markers decreased, while the expression of mesenchymal stem cell markers exhibited a striking rise. Both supplementation of culture media with LIF and transfection of the ADSCs with miR-302 family upregulated the expression levels of OCT4, Nanog, and Sox2 mRNAs. These findings showed that mouse adipose tissue contains a population of cells with molecular resemblance to embryonic stem cells, and LIF and miR-302 family positively affect the expression of pluripotency markers. Masoumeh Fakhr Taha, Arash Javeri, Sara Rohban, and Seyed Javad Mowla Copyright © 2014 Masoumeh Fakhr Taha et al. All rights reserved. Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells Sun, 20 Jul 2014 10:19:51 +0000 High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer. Zheng Hu, Lan Yu, Da Zhu, Wencheng Ding, Xiaoli Wang, Changlin Zhang, Liming Wang, Xiaohui Jiang, Hui Shen, Dan He, Kezhen Li, Ling Xi, Ding Ma, and Hui Wang Copyright © 2014 Zheng Hu et al. All rights reserved. Cytoskeleton Modifications and Autophagy Induction in TCam-2 Seminoma Cells Exposed to Simulated Microgravity Thu, 17 Jul 2014 08:43:41 +0000 The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one. Francesca Ferranti, Maria Caruso, Marcella Cammarota, Maria Grazia Masiello, Katia Corano Scheri, Cinzia Fabrizi, Lorenzo Fumagalli, Chiara Schiraldi, Alessandra Cucina, Angela Catizone, and Giulia Ricci Copyright © 2014 Francesca Ferranti et al. All rights reserved. A Molecular View of Autophagy in Lepidoptera Wed, 16 Jul 2014 07:24:05 +0000 Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm, Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications. Davide Romanelli, Barbara Casati, Eleonora Franzetti, and Gianluca Tettamanti Copyright © 2014 Davide Romanelli et al. All rights reserved. Gravity Affects the Closure of the Traps in Dionaea muscipula Tue, 15 Jul 2014 07:26:53 +0000 Venus flytrap (Dionaea muscipula Ellis) is a carnivorous plant known for its ability to capture insects thanks to the fast snapping of its traps. This fast movement has been long studied and it is triggered by the mechanical stimulation of hairs, located in the middle of the leaves. Here we present detailed experiments on the effect of microgravity on trap closure recorded for the first time during a parabolic flight campaign. Our results suggest that gravity has an impact on trap responsiveness and on the kinetics of trap closure. The possible role of the alterations of membrane permeability induced by microgravity on trap movement is discussed. Finally we show how the Venus flytrap could be an easy and effective model plant to perform studies on ion channels and aquaporin activities, as well as on electrical activity in vivo on board of parabolic flights and large diameter centrifuges. Camilla Pandolfi, Elisa Masi, Boris Voigt, Sergio Mugnai, Dieter Volkmann, and Stefano Mancuso Copyright © 2014 Camilla Pandolfi et al. All rights reserved. The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells Thu, 10 Jul 2014 07:27:40 +0000 How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-) is currently a hot topic in biomedicine. In r- and s-, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s- devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of on the aforementioned cells and an outlook for future perspectives in tissue engineering. Claudia Ulbrich, Markus Wehland, Jessica Pietsch, Ganna Aleshcheva, Petra Wise, Jack van Loon, Nils Magnusson, Manfred Infanger, Jirka Grosse, Christoph Eilles, Alamelu Sundaresan, and Daniela Grimm Copyright © 2014 Claudia Ulbrich et al. All rights reserved. Store-Operated Ca2+ Entry Does Not Control Proliferation in Primary Cultures of Human Metastatic Renal Cellular Carcinoma Wed, 09 Jul 2014 11:53:59 +0000 Store-operated Ca2+ entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca2+ levels within the endoplasmic reticulum (ER) Ca2+ reservoir, and a number of a Ca2+-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca2+ store, but not by InsP3-dependent Ca2+ release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd3+ and Pyr6, while it was inhibited by 100 µM Gd3+, 2-APB, and carboxyamidotriazole (CAI). Neither Gd3+ nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca2+ signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors. Silvia Dragoni, Ilaria Turin, Umberto Laforenza, Duilio Michele Potenza, Cinzia Bottino, Toma N. Glasnov, Martina Prestia, Federica Ferulli, Anna Saitta, Alessandra Mosca, Germano Guerra, Vittorio Rosti, Ombretta Luinetti, Carlo Ganini, Camillo Porta, Paolo Pedrazzoli, Franco Tanzi, Daniela Montagna, and Francesco Moccia Copyright © 2014 Silvia Dragoni et al. All rights reserved. A Simple Method to Estimate the Number of Autophagic Elements by Electron Microscopic Morphometry in Real Cellular Dimensions Thu, 03 Jul 2014 07:33:27 +0000 Autophagic elements typically appear as spherical bodies. During their life they undergo a series of changes (e.g., fusion, degradation of content, and swelling) which influence their size in a way that may be characteristic for cell type, stage of maturation, or various experimentally manipulated parameters. A simple and time efficient method is suggested here to use exactly calculated specific surface values and estimate average diameter and number of autophagic elements in real cellular dimensions. The method is based on the easiest morphometric determination of relative surface (surface density) and volume (volume density) data by electron microscopy. A series of data from real experimental samples of liver and exocrine pancreatic cells are offered to illustrate the potential of these measurements and calculations. Attila L. Kovács Copyright © 2014 Attila L. Kovács. All rights reserved. Investigations of Curcumin and Resveratrol on Neurite Outgrowth: Perspectives on Spinal Muscular Atrophy Wed, 02 Jul 2014 14:11:50 +0000 Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease with progressive muscle weakness and atrophy. SMA is caused by low levels of the Survival of Motor Neuron (SMN) protein, which also leads to neurite outgrowth defects in neuronal cells. Rescue of the outgrowth defect is thought to be a strategy for SMA treatment. Polyphenolic histone deacetylase (HDAC) inhibitors might be good candidates due to their neuritogenic properties. In the present study, it was investigated whether neurite outgrowth defects could be rescued by curcumin and resveratrol, which are SMN-inducing polyphenols, having HDAC inhibition activity. According to our results, although curcumin and resveratrol failed to restore the neurite outgrowth defects, the SMN protein was found to be necessary for the neurite-promoting activity of curcumin in neuron-like PC12 cells. Gamze Bora-Tatar and Hayat Erdem-Yurter Copyright © 2014 Gamze Bora-Tatar and Hayat Erdem-Yurter. All rights reserved. MicroRNAs: Novel Players in Cancer Diagnosis and Therapies Wed, 02 Jul 2014 06:49:22 +0000 First discovered in 1993, microRNAs (miRNAs) have been one of the hottest research areas over the past two decades. Oftentimes, miRNAs levels are found to be dysregulated in cancer patients. The potential use of miRNAs in cancer therapies is an emerging and promising field, with research finding miRNAs to play a role in cancer initiation, tumor growth, and metastasis. Therefore, miRNAs could become an integral part from cancer diagnosis to treatment in future. This review aims to examine current novel research work on the potential roles of miRNAs in cancer therapies, while also discussing several current challenges and needed future research. Aaron L. Oom, Brock A. Humphries, and Chengfeng Yang Copyright © 2014 Aaron L. Oom et al. All rights reserved. Multisensory Integration and Internal Models for Sensing Gravity Effects in Primates Tue, 01 Jul 2014 10:43:06 +0000 Gravity is crucial for spatial perception, postural equilibrium, and movement generation. The vestibular apparatus is the main sensory system involved in monitoring gravity. Hair cells in the vestibular maculae respond to gravitoinertial forces, but they cannot distinguish between linear accelerations and changes of head orientation relative to gravity. The brain deals with this sensory ambiguity (which can cause some lethal airplane accidents) by combining several cues with the otolith signals: angular velocity signals provided by the semicircular canals, proprioceptive signals from muscles and tendons, visceral signals related to gravity, and visual signals. In particular, vision provides both static and dynamic signals about body orientation relative to the vertical, but it poorly discriminates arbitrary accelerations of moving objects. However, we are able to visually detect the specific acceleration of gravity since early infancy. This ability depends on the fact that gravity effects are stored in brain regions which integrate visual, vestibular, and neck proprioceptive signals and combine this information with an internal model of gravity effects. Francesco Lacquaniti, Gianfranco Bosco, Silvio Gravano, Iole Indovina, Barbara La Scaleia, Vincenzo Maffei, and Myrka Zago Copyright © 2014 Francesco Lacquaniti et al. All rights reserved. Stem Cell Transplantation for Muscular Dystrophy: The Challenge of Immune Response Thu, 26 Jun 2014 06:52:58 +0000 Treating muscle disorders poses several challenges to the rapidly evolving field of regenerative medicine. Considerable progress has been made in isolating, characterizing, and expanding myogenic stem cells and, although we are now envisaging strategies to generate very large numbers of transplantable cells (e.g., by differentiating induced pluripotent stem cells), limitations directly linked to the interaction between transplanted cells and the host will continue to hamper a successful outcome. Among these limitations, host inflammatory and immune responses challenge the critical phases after cell delivery, including engraftment, migration, and differentiation. Therefore, it is key to study the mechanisms and dynamics that impair the efficacy of cell transplants in order to develop strategies that can ultimately improve the outcome of allogeneic and autologous stem cell therapies, in particular for severe disease such as muscular dystrophies. In this review we provide an overview of the main players and issues involved in this process and discuss potential approaches that might be beneficial for future regenerative therapies of skeletal muscle. Sara Martina Maffioletti, Maddalena Noviello, Karen English, and Francesco Saverio Tedesco Copyright © 2014 Sara Martina Maffioletti et al. All rights reserved. Integration Analysis of MicroRNA and mRNA Expression Profiles in Human Peripheral Blood Lymphocytes Cultured in Modeled Microgravity Mon, 23 Jun 2014 05:37:37 +0000 We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition, simulated by a ground-based rotating wall vessel (RWV) bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1 g incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs, we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death, and regulation of cell proliferation. We identified the correlation of miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p expression with that of genes involved in immune/inflammatory response (e.g., IFNG and IL17F), apoptosis (e.g., PDCD4 and PTEN), and cell proliferation (e.g., NKX3-1 and GADD45A). Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation. C. Girardi, C. De Pittà, S. Casara, E. Calura, C. Romualdi, L. Celotti, and M. Mognato Copyright © 2014 C. Girardi et al. All rights reserved. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics Sun, 22 Jun 2014 12:52:24 +0000 Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Macugen” is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions. Baby Santosh and Pramod K. Yadava Copyright © 2014 Baby Santosh and Pramod K. Yadava. All rights reserved. Multiple Effects of Berberine Derivatives on Colon Cancer Cells Wed, 18 Jun 2014 06:19:10 +0000 The pharmacological use of the plant alkaloid berberine is based on its antibacterial and anti-inflammatory properties; recently, anticancer activity has been attributed to this compound. To exploit this interesting feature, we synthesized three berberine derivatives, namely, NAX012, NAX014, and NAX018, and we tested their effects on two human colon carcinoma cell lines, that is, HCT116 and SW613-B3, which are characterized by wt and mutated p53, respectively. We observed that cell proliferation is more affected by cell treatment with the derivatives than with the lead compound; moreover, the derivatives proved to induce cell cycle arrest and cell death through apoptosis, thus suggesting that they could be promising anticancer drugs. Finally, we detected typical signs of autophagy in cells treated with berberine derivatives. Luis Miguel Guamán Ortiz, Micol Tillhon, Michael Parks, Ilaria Dutto, Ennio Prosperi, Monica Savio, Andrea G. Arcamone, Franco Buzzetti, Paolo Lombardi, and Anna Ivana Scovassi Copyright © 2014 Luis Miguel Guamán Ortiz et al. All rights reserved. Histological, Histochemical, and Protein Changes after Induced Malocclusion by Occlusion Alteration of Wistar Rats Tue, 17 Jun 2014 06:17:25 +0000 Although disorders of the stomatognathic system are common, the mechanisms involved are unknown. Our objective was to study the changes in the masseter muscles after unilateral exodontia. Molar extraction was performed on Wistar rats (left side), and the animals were sacrificed after either 14 or 26 days. The masseter muscle was processed for histological analysis, conventional and in situ zymography, and immunohistochemistry. The morphological analysis showed unique and specific characteristics for the experimental group. By conventional zymography no significant values of 72 kDa MMP-2 () were found in both of the sides of masseter muscle after 14 and 26 days of unilateral extraction. The in situ zymography showed gelatinolytic activity on all deep masseter muscles, with significant increase on the contralateral side after 14 and 26 days (). The immunohistochemistry demonstrated greater expression of MMP-2 than MMP-9 and MMP-14 in all masseter muscles and there were few differences in the staining of 4 TIMPs. This knowledge about morphology and molecular masticatory muscle remodeling following environmental interventions can be used to develop clinically successful treatments. Carolina de Souza Guerra, Yamba Carla Lara Pereira, João Paulo Mardegan Issa, Kelly Galisteu Luiz, Elaine A. Del Bel Guimarães, Raquel Fernanda Gerlach, and Mamie Mizusaki Iyomasa Copyright © 2014 Carolina de Souza Guerra et al. All rights reserved. From Innate to Adaptive Immune Response in Muscular Dystrophies and Skeletal Muscle Regeneration: The Role of Lymphocytes Mon, 16 Jun 2014 13:10:01 +0000 Skeletal muscle is able to restore contractile functionality after injury thanks to its ability to regenerate. Following muscle necrosis, debris is removed by macrophages, and muscle satellite cells (MuSCs), the muscle stem cells, are activated and subsequently proliferate, migrate, and form muscle fibers restoring muscle functionality. In most muscle dystrophies (MDs), MuSCs fail to properly proliferate, differentiate, or replenish the stem cell compartment, leading to fibrotic deposition. However, besides MuSCs, interstitial nonmyogenic cells and inflammatory cells also play a key role in orchestrating muscle repair. A complete understanding of the complexity of these mechanisms should allow the design of interventions to attenuate MDs pathology without disrupting regenerative processes. In this review we will focus on the contribution of immune cells in the onset and progression of MDs, with particular emphasis on Duchenne muscular dystrophy (DMD). We will briefly summarize the current knowledge and recent advances made in our understanding of the involvement of different innate immune cells in MDs and will move on to critically evaluate the possible role of cell populations within the acquired immune response. Revisiting previous observations in the light of recent evidence will likely change our current view of the onset and progression of the disease. Luca Madaro and Marina Bouché Copyright © 2014 Luca Madaro and Marina Bouché. All rights reserved. Mechanisms of T-Cell Immunosuppression by Mesenchymal Stromal Cells: What Do We Know So Far? Mon, 16 Jun 2014 00:00:00 +0000 Mesenchymal stromal cells (MSCs) are multipotent cells, which can give rise to several cell types including osteoblasts, adipocytes, and chondroblasts. These cells can be found in a variety of adult and fetal tissues, such as bone marrow, adipose tissue, cord blood, and placenta. In recent years, the biological properties of MSCs have attracted the attention of researchers worldwide due to their potential application for treating a series of clinical situations. Among these properties, special attention should be given to the immunoregulatory potential of those cells. MSCs are able to act on all cells of the immune system, which includes the capacity to inhibit the proliferation and function of T-cells. This feature renders them natural candidates to treat several diseases in which cellular immune response is exacerbated. In this review, we outline the main mechanisms by which MSCs immunosuppress T-cell response, focusing on cell-cell contact, secretion of soluble factors, and regulatory T-cell generation. The influence of surface markers in the immunosuppression process and features of MSCs isolated from different sources are also discussed. Finally, the influences of toll-like receptors and cytokines on the inflammatory microenvironment are highlighted regarding the activation of MSCs to exert their immunoregulatory function. Rodrigo Haddad and Felipe Saldanha-Araujo Copyright © 2014 Rodrigo Haddad and Felipe Saldanha-Araujo. All rights reserved. The Role of the Selective Adaptor p62 and Ubiquitin-Like Proteins in Autophagy Thu, 12 Jun 2014 07:23:32 +0000 The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family. Mónika Lippai and Péter Lőw Copyright © 2014 Mónika Lippai and Péter Lőw. All rights reserved. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro Mon, 09 Jun 2014 09:00:22 +0000 Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. Xingfu Bao, Xianjun Liu, Yi Zhang, Yue Cui, Jindan Yao, and Min Hu Copyright © 2014 Xingfu Bao et al. All rights reserved. Cell Biology of Pathogenic Protozoa and Their Interaction with Host Cells Thu, 05 Jun 2014 11:59:02 +0000 Marlene Benchimol, Juan C. Engel, Kevin S. W. Tan, and Wanderley de Souza Copyright © 2014 Marlene Benchimol et al. All rights reserved. Cardioprotective Effects of Osteopontin-1 during Development of Murine Ischemic Cardiomyopathy Thu, 29 May 2014 15:35:13 +0000 Repetitive brief ischemia and reperfusion (I/R) is associated with ventricular dysfunction in pathogenesis of murine ischemic cardiomyopathy and human hibernating myocardium. We investigated the role of matricellular protein osteopontin-1 (OPN) in murine model of repetitive I/R. One 15-min LAD-occlusion followed by reperfusion was performed daily over 3, 5, and 7 consecutive days in C57/Bl6 wildtype- (WT-) and OPN−/−-mice (/group). After echocardiography hearts were processed for histological and mRNA-studies. Cardiac fibroblasts were isolated, cultured, and stimulated with TGF-β1. WT-mice showed an early, strong, and cardiomyocyte-specific osteopontin-expression leading to interstitial macrophage infiltration and consecutive fibrosis after 7 days I/R in absence of myocardial infarction. In contrast, OPN−/−-mice showed small, nontransmural infarctions after 3 days I/R associated with significantly worse ventricular dysfunction. OPN−/−-mice had different expression of myocardial contractile elements and antioxidative mediators and a lower expression of chemokines during I/R. OPN−/−-mice showed predominant collagen deposition in macrophage-rich small infarctions. We found lower induction of tenascin-C, MMP-9, MMP-12, and TIMP-1, whereas MMP-13-expression was higher in OPN−/−-mice. Cultured OPN−/−-myofibroblasts confirmed these findings. In conclusion, osteopontin seems to modulate expression of contractile elements, antioxidative mediators, and inflammatory response and subsequently remodel in order to protect cardiomyocytes in murine ischemic cardiomyopathy. Georg D. Duerr, Bettina Mesenholl, Jan C. Heinemann, Martin Zoerlein, Peter Huebener, Prisca Schneider, Andreas Feisst, Alexander Ghanem, Klaus Tiemann, Daniela Dewald, Armin Welz, and Oliver Dewald Copyright © 2014 Georg D. Duerr et al. All rights reserved. Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Chylomicron and VLDL Synthesis and Secretion in Caco-2 Cells Wed, 28 May 2014 09:02:42 +0000 The present research was undertaken to determine the effects of EPA (20 : 5 n-3) and DHA (22 : 6 n-3) on chylomicron and VLDL synthesis and secretion by Caco-2 cells. Cells were incubated for 12 to 36 h with 400 μM OA, EPA, and DHA; then 36 h was chosen for further study because EPA and DHA decreased de novo triglycerides synthesis in a longer incubation compared with OA  (). Neither the uptake nor oxidation was different in response to the respective fatty acids (). Compared with OA, intercellular and secreted nascent apolipoprotein B48 and B100 were decreased by EPA and DHA (). Both DHA and EPA resulted in a lower secretion of chylomicron and VLDL (). In contrast to OA, EPA and DHA were preferentially incorporated into phospholipids instead of triacylglycerols (). These discoveries demonstrated that exposure of DHA and EPA reduced the secretion of chylomicron and VLDL partly by regulating the synthesis of TG and apoB. Yue Wang, Qiaowei Lin, Peipei Zheng, Lulu Li, Zhengxi Bao, and Feiruo Huang Copyright © 2014 Yue Wang et al. All rights reserved. Leaf Rolling and Stem Fasciation in Grass Pea (Lathyrus sativus L.) Mutant Are Mediated through Glutathione-Dependent Cellular and Metabolic Changes and Associated with a Metabolic Diversion through Cysteine during Phenotypic Reversal Wed, 28 May 2014 08:29:22 +0000 A Lathyrus sativus L. mutant isolated in ethylmethane sulfonate-treated M2 progeny of mother variety BioL-212 and designated as rlfL-1 was characterized by inwardly rolled-leaf and stem and bud fasciations. The mutant exhibited karyomorphological peculiarities in both mitosis and meiosis with origin of aneuploidy. The mitosis was vigorous with high frequency of divisional cells and their quick turnover presumably steered cell proliferations. Significant transcriptional upregulations of cysteine and glutathione synthesis and concomitant stimulations of glutathione-mediated antioxidant defense helped rlfL-1 mutant to maintain balanced reactive oxygen species (ROS) metabolisms, as deduced by ROS-imaging study. Glutathione synthesis was shut down in buthionine sulfoximine- (BSO-) treated mother plant and mutant, and leaf-rolling and stems/buds fasciations in the mutant were reversed, accompanied by normalization of mitotic cell division process. Antioxidant defense was downregulated under low glutathione-redox but cysteine-desulfurations and photorespiratory glycolate oxidase transcripts were markedly overexpressed, preventing cysteine overaccumulation but resulted in excess H2O2 in BSO-treated mutant. This led to oxidative damage in proliferating cells, manifested by severe necrosis in rolled-leaf and fasciated stems. Results indicated vital role of glutathione in maintaining abnormal proliferations in plant organs, and its deficiency triggered phenotypic reversal through metabolic diversions of cysteine and concomitant cellular and metabolic modulations. Dibyendu Talukdar and Tulika Talukdar Copyright © 2014 Dibyendu Talukdar and Tulika Talukdar. All rights reserved. The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy Sun, 25 May 2014 07:10:39 +0000 Corticosteroids cause muscle atrophy by acting on proteasomal and lysosomal systems and by affecting pathways related to muscular trophysm, such as the IGF-1/PI-3k/Akt/mTOR. Omega-3 fatty acid (n-3) has been used beneficially to attenuate muscle atrophy linked to sepsis and cachexia; however, its effect on dexamethasone-induced muscle atrophy has not been evaluated. Objectives. We evaluated whether n-3 supplementation could mitigate the development of dexamethasone-induced muscle atrophy. Methods. Two groups of Wistar rats were orally supplemented with n-3 or vehicle solution for 40 days. In the last 10 days, dexamethasone, or saline solution, was administrated establishing four groups: control, dexamethasone, n-3, and dexamethasone + n-3. The cross-sectional areas of muscle fibers, gene expression (MyoD, Myogenin, MuRF-1, and Atrogin-1), and protein expression (Akt, GSK3, FOXO3a, and mTOR) were assessed. Results. Dexamethasone induced a significant loss in body and muscle weight, atrophy in type 2B fibers, and decreased expression of P-Akt, P-GSK3, and P-FOXO3a. N-3 supplementation did not attenuate the negative effects of dexamethasone on skeletal muscle; instead, it caused atrophy in type 1, 2A, reduced the expression of Myogenin, and increased the expression of Atrogin-1. Conclusion. Food supplements containing n-3 are usually healthful, but they may potentiate some of the side effects of glucocorticoids. Alan Fappi, Tiago S. Godoy, Jessica R. Maximino, Vanessa R. Rizzato, Juliana de C. Neves, Gerson Chadi, and Edmar Zanoteli Copyright © 2014 Alan Fappi et al. All rights reserved. IL-6 Impairs Myogenic Differentiation by Downmodulation of p90RSK/eEF2 and mTOR/p70S6K Axes, without Affecting AKT Activity Wed, 21 May 2014 11:14:14 +0000 IL-6 is a multifaceted pleiotropic cytokine, which is produced by a variety of cell types and targets different cells and tissues. In physiological conditions, IL-6 can be locally and transiently produced by skeletal muscle and plays an important role in muscle homeostasis. Circulating IL-6 levels are normally very low or undetectable but are dramatically increased in several pathologic conditions. In this study, we aimed to define the potential molecular mechanisms underlying the effects of IL-6 on myogenic program. We explored the molecular mechanisms through which exogenous IL-6, or the conditioned medium from the murine C-26 adenocarcinoma cells (a cellular model that secretes high levels of IL-6 and induces cancer cachexia in mice), interferes with the myogenic program. Our study revealed that IL-6 induces the activation of the Stat3 signaling and promotes the downmodulation of the p90RSK/eEF2 and mTOR/p70S6K axes, while it does not affect the activation of AKT. We thus identified potential molecular mediators of the inhibitory effects of IL-6 on myogenic program. Michele Pelosi, Manuela De Rossi, Laura Barberi, and Antonio Musarò Copyright © 2014 Michele Pelosi et al. All rights reserved. Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development Wed, 21 May 2014 09:34:26 +0000 Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation. Péter Lőrincz, Zsolt Lakatos, Tamás Maruzs, Zsuzsanna Szatmári, Viktor Kis, and Miklós Sass Copyright © 2014 Péter Lőrincz et al. All rights reserved. Local Overexpression of V1a-Vasopressin Receptor Enhances Regeneration in Tumor Necrosis Factor-Induced Muscle Atrophy Tue, 20 May 2014 07:24:39 +0000 Skeletal muscle atrophy occurs during disuse and aging, or as a consequence of chronic diseases such as cancer and diabetes. It is characterized by progressive loss of muscle tissue due to hypotrophic changes, degeneration, and an inability of the regeneration machinery to replace damaged myofibers. Tumor necrosis factor (TNF) is a proinflammatory cytokine known to mediate muscle atrophy in many chronic diseases and to inhibit skeletal muscle regeneration. In this study, we investigated the role of Arg-vasopressin-(AVP-)dependent pathways in muscles in which atrophy was induced by local overexpression of TNF. AVP is a potent myogenesis-promoting factor and is able to enhance skeletal muscle regeneration by stimulating Ca2+/calmodulin-dependent kinase and calcineurin signaling. We performed morphological and molecular analyses and demonstrated that local over-expression of the AVP receptor V1a enhances regeneration of atrophic muscle. By upregulating the regeneration/differentiation markers, modulating the inflammatory response, and attenuating fibrogenesis, the stimulation of AVP-dependent pathways creates a favourable environment for efficient and sustained muscle regeneration and repair even in the presence of elevated levels of TNF. This study highlights a novel in vivo role for AVP-dependent pathways, which may represent an interesting strategy to counteract muscle decline in aging or in muscular pathologies. Alessandra Costa, Angelica Toschi, Ivana Murfuni, Laura Pelosi, Gigliola Sica, Sergio Adamo, and Bianca Maria Scicchitano Copyright © 2014 Alessandra Costa et al. All rights reserved. Influence of Immune Responses in Gene/Stem Cell Therapies for Muscular Dystrophies Mon, 19 May 2014 00:00:00 +0000 Muscular dystrophies (MDs) are a heterogeneous group of diseases, caused by mutations in different components of sarcolemma, extracellular matrix, or enzymes. Inflammation and innate or adaptive immune response activation are prominent features of MDs. Various therapies under development are directed toward rescuing the dystrophic muscle damage using gene transfer or cell therapy. Here we discussed current knowledge about involvement of immune system responses to experimental therapies in MDs. Andrea Farini, Clementina Sitzia, Silvia Erratico, Mirella Meregalli, and Yvan Torrente Copyright © 2014 Andrea Farini et al. All rights reserved. Autophagy in Drosophila: From Historical Studies to Current Knowledge Sun, 18 May 2014 12:41:23 +0000 The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy. Nitha C. Mulakkal, Peter Nagy, Szabolcs Takats, Radu Tusco, Gábor Juhász, and Ioannis P. Nezis Copyright © 2014 Nitha C. Mulakkal et al. All rights reserved. Antitumor Activity of Ethanolic Extract of Dendrobium formosum in T-Cell Lymphoma: An In Vitro and In Vivo Study Sun, 18 May 2014 09:07:11 +0000 Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton’s lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton’s lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton’s lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton’s lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved. Ritika Prasad and Biplob Koch Copyright © 2014 Ritika Prasad and Biplob Koch. All rights reserved. Biological Characterization and Pluripotent Identification of Sheep Dermis-Derived Mesenchymal Stem/Progenitor Cells Sun, 18 May 2014 07:14:24 +0000 Dermis-derived mesenchymal stem/progenitor cells (DMS/PCs) were a multipotential stem cell population, which has potential applications in the tissue damage repair and skin transplant. Although a large number of studies deal with the human DMS/PCs self-renewal and regulation, however, the study of livestock-derived DMS/PCs has rarely been reported. Here, sheep DMS/PCs were isolated from one-month-old sheep embryos and studied at the cellular and molecular level. And then the DMS/PCs biological characteristics were analysed by RT-PCR and immunofluorescence. Experimental results showed that DMS/PCs could be expanded for 48 passages and the cells viability and hereditary character were steady. In addition, the DMS/PCs maker β-integrin, CD71, CD44, and CD73 were expressed positively through RT-PCR and immunofluorescence. Passage 3 DMS/PCs were successfully induced to differentiate into adipocytes, osteoblasts, chondrocytes, and neurocytes, respectively. The above results suggest that DMS/PCs not only have strong self-renewal capacity but also have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, and neurocytes. The study provides theoretical basis and experimental evidence for potential clinical application. Peng Cui, Xiaohong He, Yabin Pu, Wenxiu Zhang, Ping Zhang, Changli Li, Weijun Guan, Xiangchen Li, and Yuehui Ma Copyright © 2014 Peng Cui et al. All rights reserved. The Putative HORMA Domain Protein Atg101 Dimerizes and Is Required for Starvation-Induced and Selective Autophagy in Drosophila Thu, 08 May 2014 14:45:17 +0000 The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes. Krisztina Hegedűs, Péter Nagy, Zoltán Gáspári, and Gábor Juhász Copyright © 2014 Krisztina Hegedűs et al. All rights reserved. Three Consecutive Days of Interval Runs to Exhaustion Affects Lymphocyte Subset Apoptosis and Migration Thu, 08 May 2014 00:00:00 +0000 This investigation assessed the lymphocyte subset response to three days of intermittent run exercise to exhaustion. Twelve healthy college-aged males () and females () (age = 26 ± 4 years; height = 170.2 ± 10 cm; body mass = 75 ± 18 kg) completed an exertion test (maximal running speed and VO2max) and later performed three consecutive days of an intermittent run protocol to exhaustion (30 sec at maximal running speed and 30 sec at half of the maximal running speed). Blood was collected before exercise (PRE) and immediately following the treadmill bout (POST) each day. When the absolute change from baseline was evaluated (i. e., Δ baseline), a significant change in CD4+ and CD8+ for CX3CR1 cells was observed by completion of the third day. Significant changes in both apoptosis and migration were observed following two consecutive days in CD19+ lymphocytes, and the influence of apoptosis persisted following the third day. Given these lymphocyte responses, it is recommended that a rest day be incorporated following two consecutive days of a high-intensity intermittent run program to minimize immune cell modulations and reduce potential susceptibility. James W. Navalta, Ramires Alsamir Tibana, Elizabeth A. Fedor, Amilton Vieira, and Jonato Prestes Copyright © 2014 James W. Navalta et al. All rights reserved. Functional Interactions between 17β-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures Wed, 07 May 2014 15:02:28 +0000 Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2) and progesterone (P4). Mammary epithelial cells (MECs) cultured on reconstituted basement membrane (rBM) form three-dimensional (3D) acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR) involved in autophagy induction. Katarzyna Zielniok, Tomasz Motyl, and Malgorzata Gajewska Copyright © 2014 Katarzyna Zielniok et al. All rights reserved. Vitamin D Receptor Agonists: Suitable Candidates as Novel Therapeutic Options in Autoimmune Inflammatory Myopathy Wed, 07 May 2014 06:38:10 +0000 The primary aim in the treatment of autoimmune inflammatory myopathies (IMs) is to recover muscle function. The presence of immune/inflammatory cell infiltrates within muscle tissues represents the common feature of different IM subtypes, albeit a correlation between muscular damage extent and inflammation degree is often lacking. Treatments for IMs are based on life-long immunosuppressive therapy, with the well known adverse effects; recovery is incomplete for many patients. More effective therapies, with reduced side-effects, are highly desirable. Vitamin D receptor (VDR) agonists emerge to retain pleiotropic anti-inflammatory properties, since they regulate innate and adaptive immunity by switching the immune response from proinflammatory T helper 1 (Th1) type to tolerogenic T helper 2 (Th2) type dominance. In skeletal muscle cells less hypercalcemic VDR ligands target powerful mediators of inflammation, such as TNFα and TNFα driven paths, without affecting immune or muscle cells viability, retaining the potentiality to counteract Th1 driven overreactivity established by the self-enhancing inflammatory loop between immune and skeletal muscle cells. This review summarizes those features of VDR agonists as candidates in future treatment of IM. Clara Crescioli Copyright © 2014 Clara Crescioli. All rights reserved. 7-Tesla Magnetic Resonance Imaging Precisely and Noninvasively Reflects Inflammation and Remodeling of the Skeletal Muscle in a Mouse Model of Antisynthetase Syndrome Mon, 05 May 2014 00:00:00 +0000 Inflammatory myopathies comprise heterogeneous disorders. Their etiopathogenesis is poorly understood, because of the paucity of informative experimental models and of approaches for the noninvasive study of inflamed tissues. Magnetic resonance imaging (MRI) provides information about the state of the skeletal muscle that reflects various facets of inflammation and remodeling. This technique has been scarcely used in experimental models of inflammatory myopathies. We characterized the performance of MRI in a well-established mouse model of myositis and the antisynthetase syndrome, based on the immunization of wild-type mice with the amino-terminal fragment of histidyl-tRNA synthetase (HisRS). Over an eight-week period following myositis induction, MRI enabled precise identification of pathological events taking place in muscle tissue. Areas of edema and of active inflammation identified by histopathology paralleled muscle modifications detected noninvasively by MRI. Muscles changes were chronologically associated with the establishment of autoimmunity, as reflected by the development of anti-HisRS antibodies in the blood of immunized mice. MR imaging easily appreciated muscle damage and remodeling even if actual disruption of myofiber integrity (as assessed by serum concentrations of creatinine phosphokinase) was limited. Thus, MR imaging represents an informative and noninvasive analytical tool for studying in vivo immune-mediated muscle involvement. Clara Sciorati, Antonio Esposito, Lara Campana, Tamara Canu, Antonella Monno, Anna Palmisano, Francesco De Cobelli, Alessandro Del Maschio, Dana P. Ascheman, Angelo A. Manfredi, and Patrizia Rovere-Querini Copyright © 2014 Clara Sciorati et al. All rights reserved. Fatty Acids in Energy Metabolism of the Central Nervous System Sun, 04 May 2014 08:03:53 +0000 In this review, we analyze the current hypotheses regarding energy metabolism in the neurons and astroglia. Recently, it was shown that up to 20% of the total brain’s energy is provided by mitochondrial oxidation of fatty acids. However, the existing hypotheses consider glucose, or its derivative lactate, as the only main energy substrate for the brain. Astroglia metabolically supports the neurons by providing lactate as a substrate for neuronal mitochondria. In addition, a significant amount of neuromediators, glutamate and GABA, is transported into neurons and also serves as substrates for mitochondria. Thus, neuronal mitochondria may simultaneously oxidize several substrates. Astrocytes have to replenish the pool of neuromediators by synthesis de novo, which requires large amounts of energy. In this review, we made an attempt to reconcile -oxidation of fatty acids by astrocytic mitochondria with the existing hypothesis on regulation of aerobic glycolysis. We suggest that, under condition of neuronal excitation, both metabolic pathways may exist simultaneously. We provide experimental evidence that isolated neuronal mitochondria may oxidize palmitoyl carnitine in the presence of other mitochondrial substrates. We also suggest that variations in the brain mitochondrial metabolic phenotype may be associated with different mtDNA haplogroups. Alexander Panov, Zulfiya Orynbayeva, Valentin Vavilin, and Vyacheslav Lyakhovich Copyright © 2014 Alexander Panov et al. All rights reserved. Impairment of Electron Transfer Chain Induced by Acute Carnosine Administration in Skeletal Muscle of Young Rats Sun, 04 May 2014 00:00:00 +0000 Serum carnosinase deficiency is an inherited disorder that leads to an accumulation of carnosine in the brain tissue, cerebrospinal fluid, skeletal muscle, and other tissues of affected patients. Considering that high levels of carnosine are associated with neurological dysfunction and that the pathophysiological mechanisms involved in serum carnosinase deficiency remain poorly understood, we investigated the in vivo effects of carnosine on bioenergetics parameters, namely, respiratory chain complexes (I–III, II, and II-III), malate dehydrogenase, succinate dehydrogenase, and creatine kinase activities and the expression of mitochondrial-specific transcription factors (NRF-1, PGC-1α, and TFAM) in skeletal muscle of young Wistar rats. We observed a significant decrease of complexes I–III and II activities in animals receiving carnosine acutely, as compared to control group. However, no significant alterations in respiratory chain complexes, citric acid cycle enzymes, and creatine kinase activities were found between rats receiving carnosine chronically and control group animals. As compared to control group, mRNA levels of NRF-1, PGC-1α, and TFAM were unchanged. The present findings indicate that electron transfer through the respiratory chain is impaired in skeletal muscle of rats receiving carnosine acutely. In case these findings are confirmed by further studies and ATP depletion is also observed, impairment of bioenergetics could be considered a putative mechanism responsible for the muscle damage observed in serum carnosinase-deficient patients. José Roberto Macarini, Soliany Grassi Maravai, José Henrique Cararo, Nádia Webber Dimer, Cinara Ludvig Gonçalves, Luiza Wilges Kist, Mauricio Reis Bogo, Patrícia Fernanda Schuck, Emilio Luiz Streck, and Gustavo Costa Ferreira Copyright © 2014 José Roberto Macarini et al. All rights reserved. Inflammation Based Regulation of Cancer Cachexia Sun, 04 May 2014 00:00:00 +0000 Cancer cachexia, consisting of significant skeletal muscle wasting independent of nutritional intake, is a major concern for patients with solid tumors that affects surgical, therapeutic, and quality of life outcomes. This review summarizes the clinical implications, background of inflammatory cytokines, and the origin and sources of procachectic factors including TNF-, IL-6, IL-1, INF-, and PIF. Molecular mechanisms and pathways are described to elucidate the link between the immune response caused by the presence of the tumor and the final result of skeletal muscle wasting. Jill K. Onesti and Denis C. Guttridge Copyright © 2014 Jill K. Onesti and Denis C. Guttridge. All rights reserved. Understanding the Process of Fibrosis in Duchenne Muscular Dystrophy Sun, 04 May 2014 00:00:00 +0000 Fibrosis is the aberrant deposition of extracellular matrix (ECM) components during tissue healing leading to loss of its architecture and function. Fibrotic diseases are often associated with chronic pathologies and occur in a large variety of vital organs and tissues, including skeletal muscle. In human muscle, fibrosis is most readily associated with the severe muscle wasting disorder Duchenne muscular dystrophy (DMD), caused by loss of dystrophin gene function. In DMD, skeletal muscle degenerates and is infiltrated by inflammatory cells and the functions of the muscle stem cells (satellite cells) become impeded and fibrogenic cells hyperproliferate and are overactivated, leading to the substitution of skeletal muscle with nonfunctional fibrotic tissue. Here, we review new developments in our understanding of the mechanisms leading to fibrosis in DMD and several recent advances towards reverting it, as potential treatments to attenuate disease progression. Yacine Kharraz, Joana Guerra, Patrizia Pessina, Antonio L. Serrano, and Pura Muñoz-Cánoves Copyright © 2014 Yacine Kharraz et al. All rights reserved. Retracted: Downregulation of ADAM10 Expression Inhibits Metastasis and Invasiveness of Human Hepatocellular Carcinoma HepG2 Cells Tue, 29 Apr 2014 07:36:31 +0000 BioMed Research International Copyright © 2014 BioMed Research International. All rights reserved. Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum Sun, 27 Apr 2014 12:13:33 +0000 Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1. Sabna Cheemadan, Ramya Ramadoss, and Zbynek Bozdech Copyright © 2014 Sabna Cheemadan et al. All rights reserved. An Historical Perspective on How Advances in Microscopic Imaging Contributed to Understanding the Leishmania Spp. and Trypanosoma cruzi Host-Parasite Relationship Sun, 27 Apr 2014 00:00:00 +0000 The literature has identified complex aspects of intracellular host-parasite relationships, which require systematic, nonreductionist approaches and spatial/temporal information. Increasing and integrating temporal and spatial dimensions in host cell imaging have contributed to elucidating several conceptual gaps in the biology of intracellular parasites. To access and investigate complex and emergent dynamic events, it is mandatory to follow them in the context of living cells and organs, constructing scientific images with integrated high quality spatiotemporal data. This review discusses examples of how advances in microscopy have challenged established conceptual models of the intracellular life cycles of Leishmania spp. and Trypanosoma cruzi protozoan parasites. P. T. V. Florentino, F. Real, A. Bonfim-Melo, C. M. Orikaza, E. R. Ferreira, C. C. Pessoa, B. R. Lima, G. R. S. Sasso, and R. A. Mortara Copyright © 2014 P. T. V. Florentino et al. All rights reserved. Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla Wed, 23 Apr 2014 08:58:19 +0000 Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs. Junjun Li, Ming Yan, Zilu Wang, Shuanglin Jing, Yao Li, Genxia Liu, Jinhua Yu, and Zhipeng Fan Copyright © 2014 Junjun Li et al. All rights reserved. Stability Analysis of a High Fibre Yield and Low Lignin Content “Thick Stem” Mutant in Tossa Jute (Corchorus olitorius L.) Tue, 22 Apr 2014 13:08:17 +0000 A “thick stem” mutant of Corchorus olitorius L. was induced at M2 (0.50%, 4 h, EMS) and the true breeding mutant is assessed across generations (M5 to M7) considering morphometric traits as well as SEM analysis of pollen grains and raw jute fibres, stem anatomy, cytogenetical attributes, and lignin content in relation to control. Furthermore, single fibre diameter and tensile strength are also analysed. The objective is to assess the stability of mutant for its effective exploration for raising a new plant type in tossa jute for commercial exploitation and efficient breeding. The mutant trait is monogenic recessive to normal. Results indicate that “thick stem” mutant is stable across generations () with distinctive high seed and fibre yield and significantly low lignin content. Stem anatomy of the mutant shows significant enhancement in fibre zone, number of fibre pyramids and fibre bundles per pyramid, and diameter of fibre cell in relation to control. Moreover, tensile strength of mutant fibre is significantly higher than control fibre and the trait is inversely related to fibre diameter. However the mutant is associated with low germination frequency, poor seed viability, and high pollen sterility, which may be eliminated through mutational approach followed by rigorous selection and efficient breeding. Aninda Mandal and Animesh K. Datta Copyright © 2014 Aninda Mandal and Animesh K. Datta. All rights reserved. How to Take Autophagy and Endocytosis Up a Notch Tue, 22 Apr 2014 11:52:39 +0000 The interconnection of the endocytic and autophagosomal trafficking routes has been recognized more than two decades ago with both pathways using a set of identical effector proteins and sharing the same ultimate lysosomal destination. More recent data sheds light onto how other pathways are intertwined into this network, and how degradation via the endosomal/autophagosomal system may affect signaling pathways in multicellular organisms. Here, we briefly review the common features of autophagy and endocytosis and discuss how other players enter this mix with particular respect to the Notch signaling pathway. Julia M. I. Barth and Katja Köhler Copyright © 2014 Julia M. I. Barth and Katja Köhler. All rights reserved. Cell Communication in a Coculture System Consisting of Outgrowth Endothelial Cells and Primary Osteoblasts Tue, 22 Apr 2014 09:03:52 +0000 Bone tissue is a highly vascularized and dynamic system with a complex construction. In order to develop a construct for implant purposes in bone tissue engineering, a proper understanding of the complex dependencies between different cells and cell types would provide further insight into the highly regulated processes during bone repair, namely, angiogenesis and osteogenesis, and might result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task. This study is based on an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used in different studies of bone repair processes with special regard to angiogenesis and osteogenesis. Coculture systems of OECs and pOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures. Although many studies have focused on cell communication, there are still numerous aspects which remain poorly understood. Therefore, the aim of this study is to investigate certain growth factors and cell communication molecules that are important during bone repair processes. Selected growth factors like VEGF, angiopoietins, BMPs, and IGFs were investigated during angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks of cultivation. In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed. Another important focus of this study was to investigate the role of “gap junctions,” small protein pores which connect adjacent cells. With these bridges cells are able to exchange signal molecules, growth factors, and other important mediators. It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane. In addition, areas in which two cells formed gap junctions were found. David Paul Eric Herzog, Eva Dohle, Iris Bischoff, and Charles James Kirkpatrick Copyright © 2014 David Paul Eric Herzog et al. All rights reserved. Entamoeba histolytica and E. dispar Calreticulin: Inhibition of Classical Complement Pathway and Differences in the Level of Expression in Amoebic Liver Abscess Tue, 22 Apr 2014 00:00:00 +0000 The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship. Cecilia Ximénez, Enrique González, Miriam E. Nieves, Angélica Silva-Olivares, Mineko Shibayama, Silvia Galindo-Gómez, Jaime Escobar-Herrera, Ma del Carmen García de León, Patricia Morán, Alicia Valadez, Liliana Rojas, Eric G. Hernández, Oswaldo Partida, and René Cerritos Copyright © 2014 Cecilia Ximénez et al. All rights reserved. Macrophage Plasticity in Skeletal Muscle Repair Thu, 17 Apr 2014 14:04:02 +0000 Macrophages are one of the first barriers of host defence against pathogens. Beyond their role in innate immunity, macrophages play increasingly defined roles in orchestrating the healing of various injured tissues. Perturbations of macrophage function and/or activation may result in impaired regeneration and fibrosis deposition as described in several chronic pathological diseases. Heterogeneity and plasticity have been demonstrated to be hallmarks of macrophages. In response to environmental cues they display a proinflammatory (M1) or an alternative anti-inflammatory (M2) phenotype. A lot of evidence demonstrated that after acute injury M1 macrophages infiltrate early to promote the clearance of necrotic debris, whereas M2 macrophages appear later to sustain tissue healing. Whether the sequential presence of two different macrophage populations results from a dynamic shift in macrophage polarization or from the recruitment of new circulating monocytes is a subject of ongoing debate. In this paper, we discuss the current available information about the role that different phenotypes of macrophages plays after injury and during the remodelling phase in different tissue types, with particular attention to the skeletal muscle. Elena Rigamonti, Paola Zordan, Clara Sciorati, Patrizia Rovere-Querini, and Silvia Brunelli Copyright © 2014 Elena Rigamonti et al. All rights reserved. Peroxynitrite and Peroxiredoxin in the Pathogenesis of Experimental Amebic Liver Abscess Tue, 15 Apr 2014 00:00:00 +0000 The molecular mechanisms by which Entamoeba histolytica causes amebic liver abscess (ALA) are still not fully understood. Amebic mechanisms of adherence and cytotoxic activity are pivotal for amebic survival but apparently do not directly cause liver abscess. Abundant evidence indicates that chronic inflammation (resulting from an inadequate immune response) is probably the main cause of ALA. Reports referring to inflammatory mechanisms of liver damage mention a repertoire of toxic molecules by the immune response (especially nitric oxide and reactive oxygen intermediates) and cytotoxic substances released by neutrophils and macrophages after being lysed by amoebas (e.g., defensins, complement, and proteases). Nevertheless, recent evidence downplays these mechanisms in abscess formation and emphasizes the importance of peroxynitrite (ONOO−). It seems that the defense mechanism of amoebas against ONOO−, namely, the amebic thioredoxin system (including peroxiredoxin), is superior to that of mammals. The aim of the present text is to define the importance of ONOO− as the main agent of liver abscess formation during amebic invasion, and to explain the superior capacity of amoebas to defend themselves against this toxic agent through the peroxiredoxin and thioredoxin system. Judith Pacheco-Yepez, Rosa Adriana Jarillo-Luna, Manuel Gutierrez-Meza, Edgar Abarca-Rojano, Bruce Allan Larsen, and Rafael Campos-Rodriguez Copyright © 2014 Judith Pacheco-Yepez et al. All rights reserved. Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner Mon, 14 Apr 2014 07:50:15 +0000 Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine. Zhaona Wu, Haris Mirza, Joshua D. W. Teo, and Kevin S. W. Tan Copyright © 2014 Zhaona Wu et al. All rights reserved. Quantitative Assessment of Heteroplasmy of Mitochondrial Genome: Perspectives in Diagnostics and Methodological Pitfalls Thu, 10 Apr 2014 09:40:43 +0000 The role of alterations of mitochondrial DNA (mtDNA) in the development of human pathologies is not understood well. Most of mitochondrial mutations are characterized by the phenomenon of heteroplasmy which is defined as the presence of a mixture of more than one type of an organellar genome within a cell or tissue. The level of heteroplasmy varies in wide range, and the expression of disease is dependent on the percent of alleles bearing mutations, thus allowing consumption that an upper threshold level may exist beyond which the mitochondrial function collapses. Recent findings have demonstrated that some mtDNA heteroplasmic mutations are associated with widely spread chronic diseases, including atherosclerosis and cancer. Actually, each etiological mtDNA mutation has its own heteroplasmy threshold that needs to be measured. Therefore, quantitative evaluation of a mutant allele of mitochondrial genome is an obvious methodological challenge, since it may be a keystone for diagnostics of individual genetic predisposition to the disease. This review provides a comprehensive comparison of methods applicable to the measurement of heteroplasmy level of mitochondrial mutations associated with the development of pathology, in particular, in atherosclerosis and its clinical manifestations. Igor A. Sobenin, Konstantin Y. Mitrofanov, Andrey V. Zhelankin, Margarita A. Sazonova, Anton Y. Postnov, Victor V. Revin, Yuri V. Bobryshev, and Alexander N. Orekhov Copyright © 2014 Igor A. Sobenin et al. All rights reserved. Apoptotic and Inhibitory Effects on Cell Proliferation of Hepatocellular Carcinoma HepG2 Cells by Methanol Leaf Extract of Costus speciosus Thu, 10 Apr 2014 09:16:35 +0000 Costus speciosus is a medicinal plant commonly known as wild ginger distributed in South and Southeast Asian countries. Leaves of this plant are used for ayurvedic treatment regimes in malignancies and mental illness. Rhizome extract from the plant is used to treat malignancies, pneumonia, urinary disorders, jaundice, rheumatism, and diabetes. The goal of this study was to investigate the effects of methanol extract of leaves of C. speciosus on the growth of human hepatocellular carcinoma (HepG2) cells and understand possible mechanisms of its action. Viability of HepG2 cells were measured by MTS assay after 24 h and 48 h treatment with extracts of 1, 10, 50, 100, and 200 μg/mL concentrations. Cell cycle analysis and apoptosis were evaluated by flow cytometry and caspase-3 induction. HepG2 cells treated with 100 μg/mL methanol leaf extract for 24 h displayed a significant reduction in cell viability . The methanol extract perturbed cell cycle progression, modulated cell cycle and regulated, signal molecules were involved in induction of apoptosis in HepG2 cells. Our findings indicate that phytochemicals of leaves of C. speciosus shows potential for natural therapeutic product development for hepatocellular carcinoma. This is the first report to demonstrate in vitro anticancer activity of leaf extract of C. speciosus in relation to liver cancer. Sandhya V. G. Nair, Menik Hettihewa, and H. P. Vasantha Rupasinghe Copyright © 2014 Sandhya V. G. Nair et al. All rights reserved. Mitochondrial Aging and Age-Related Dysfunction of Mitochondria Thu, 10 Apr 2014 08:37:06 +0000 Age-related changes in mitochondria are associated with decline in mitochondrial function. With advanced age, mitochondrial DNA volume, integrity and functionality decrease due to accumulation of mutations and oxidative damage induced by reactive oxygen species (ROS). In aged subjects, mitochondria are characterized by impaired function such as lowered oxidative capacity, reduced oxidative phosphorylation, decreased ATP production, significant increase in ROS generation, and diminished antioxidant defense. Mitochondrial biogenesis declines with age due to alterations in mitochondrial dynamics and inhibition of mitophagy, an autophagy process that removes dysfunctional mitochondria. Age-dependent abnormalities in mitochondrial quality control further weaken and impair mitochondrial function. In aged tissues, enhanced mitochondria-mediated apoptosis contributes to an increase in the percentage of apoptotic cells. However, implementation of strategies such as caloric restriction and regular physical training may delay mitochondrial aging and attenuate the age-related phenotype in humans. Dimitry A. Chistiakov, Igor A. Sobenin, Victor V. Revin, Alexander N. Orekhov, and Yuri V. Bobryshev Copyright © 2014 Dimitry A. Chistiakov et al. All rights reserved. Gene Expression Changes Induced by Trypanosoma cruzi Shed Microvesicles in Mammalian Host Cells: Relevance of tRNA-Derived Halves Wed, 09 Apr 2014 11:21:45 +0000 At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay. Maria R. Garcia-Silva, Florencia Cabrera-Cabrera, Roberta Ferreira Cura das Neves, Thaís Souto-Padrón, Wanderley de Souza, and Alfonso Cayota Copyright © 2014 Maria R. Garcia-Silva et al. All rights reserved. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells Wed, 09 Apr 2014 09:24:09 +0000 Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response), a great number of transcription factors (including the majority of NFκB family members), and host metabolism (cholesterol, fatty acids, and phospholipids). These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination. María Laura Chiribao, Gabriela Libisch, Adriana Parodi-Talice, and Carlos Robello Copyright © 2014 María Laura Chiribao et al. All rights reserved. FOXO Transcription Factors: Their Clinical Significance and Regulation Thu, 03 Apr 2014 14:09:34 +0000 Members of the class O of forkhead box transcription factors (FOXO) have important roles in metabolism, cellular proliferation, stress resistance, and apoptosis. The activity of FOXOs is tightly regulated by posttranslational modification, including phosphorylation, acetylation, and ubiquitylation. Activation of cell survival pathways such as phosphoinositide-3-kinase/AKT/IKK or RAS/mitogen-activated protein kinase phosphorylates FOXOs at different sites which regulate FOXOs nuclear localization or degradation. FOXO transcription factors are upregulated in a number of cell types including hepatocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, pericytes, and cardiac myocytes. They are involved in a number of pathologic and physiologic processes that include proliferation, apoptosis, autophagy, metabolism, inflammation, cytokine expression, immunity, differentiation, and resistance to oxidative stress. These processes impact a number of clinical conditions such as carcinogenesis, diabetes, diabetic complications, cardiovascular disease, host response, and wound healing. In this paper, we focus on the potential role of FOXOs in different disease models and the regulation of FOXOs by various stimuli. Yu Wang, Yanmin Zhou, and Dana T. Graves Copyright © 2014 Yu Wang et al. All rights reserved. The Double-Edged Sword in Pathogenic Trypanosomatids: The Pivotal Role of Mitochondria in Oxidative Stress and Bioenergetics Mon, 31 Mar 2014 13:45:58 +0000 The pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids’ life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes. Rubem Figueiredo Sadok Menna-Barreto and Solange Lisboa de Castro Copyright © 2014 Rubem Figueiredo Sadok Menna-Barreto and Solange Lisboa de Castro. All rights reserved. Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma brucei with Inducible YFP-α-Tubulin Sun, 30 Mar 2014 13:56:40 +0000 The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division. Omar Sheriff, Li-Fern Lim, and Cynthia Y. He Copyright © 2014 Omar Sheriff et al. All rights reserved. Comparison of Long Noncoding RNA and mRNA Expression Profiles in Mesenchymal Stem Cells Derived from Human Periodontal Ligament and Bone Marrow Thu, 27 Mar 2014 09:57:39 +0000 Mesenchymal stem cells (MSCs) in different anatomic locations possess diverse biological activities. Maintaining the pluripotent state and differentiation depend on the expression and regulation of thousands of genes, but it remains unclear which molecular mechanisms underlie MSC diversity. Thus, potential MSC applications are restricted. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry of cellular processes. We investigated differences in lncRNA and mRNA expression profiles between bone marrow stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs) with lncRNA microarray assays and bioinformatics analysis. In PDLSCs, numerous lncRNAs were significantly upregulated () or downregulated () compared to BMSCs. Furthermore, 1,578 mRNAs were differentially expressed. These genes implicated cellular pathways that may be associated with MSC characteristics, including apoptosis, MAPK, cell cycle, and Wnt signaling pathway. Signal-net analysis indicated that phospholipase C beta 4, filamin B beta, calcium/calmodulin-dependent protein kinase II gamma, and the ionotropic glutamate receptor, AMPA 1, had the highest betweenness centrality among significant genes in the differential gene profile network. A comparison between the coding-noncoding gene coexpression networks of PDLSCs and BMSCs identified chemokine (C-X-C motif) ligand 12 as a core regulatory factor in MSC biology. These results provided insight into the mechanisms underlying MSC biology. Rui Dong, Juan Du, Liping Wang, Jinsong Wang, Gang Ding, Songlin Wang, and Zhipeng Fan Copyright © 2014 Rui Dong et al. All rights reserved. Cellular Players in Skeletal Muscle Regeneration Sun, 23 Mar 2014 09:41:55 +0000 Skeletal muscle, a tissue endowed with remarkable endogenous regeneration potential, is still under focused experimental investigation mainly due to treatment potential for muscle trauma and muscular dystrophies. Resident satellite cells with stem cell features were enthusiastically described quite a long time ago, but activation of these cells is not yet controlled by any medical interventions. However, after thorough reports of their existence, survival, activation, and differentiation there are still many questions to be answered regarding the intimate mechanism of tissue regeneration. This review delivers an up-to-date inventory of the main known key players in skeletal muscle repair, revealed by various models of tissue injuries in mechanical trauma, toxic lesions, and muscular dystrophy. A better understanding of the spatial and temporal relationships between various cell populations, with different physical or paracrine interactions and phenotype changes induced by local or systemic signalling, might lead to a more efficient approach for future therapies. Laura Cristina Ceafalan, Bogdan Ovidiu Popescu, and Mihail Eugen Hinescu Copyright © 2014 Laura Cristina Ceafalan et al. All rights reserved. Detection and Antibiotic Treatment of Mycoplasma arginini Contamination in a Mouse Epithelial Cell Line Restore Normal Cell Physiology Thu, 20 Mar 2014 07:26:47 +0000 Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material. Brianna Boslett, Subhra Nag, and Andrew Resnick Copyright © 2014 Brianna Boslett et al. All rights reserved. Intracerebroventricular Administration of Nerve Growth Factor Induces Gliogenesis in Sensory Ganglia, Dorsal Root, and within the Dorsal Root Entry Zone Sun, 16 Mar 2014 00:00:00 +0000 Previous studies indicated that intracerebroventricular administration of nerve growth factor (NGF) leads to massive Schwann cell hyperplasia surrounding the medulla oblongata and spinal cord. This study was designed to characterize the proliferation of peripheral glial cells, that is, Schwann and satellite cells, in the trigeminal ganglia and dorsal root ganglia (DRG) of adult rats during two weeks of NGF infusion using bromodeoxyuridine (BrdU) to label dividing cells. The trigeminal ganglia as well as the cervical and lumbar DRG were analyzed. Along the entire neuraxis a small number of dividing cells were observed within these regions under physiological condition. NGF infusion has dramatically increased the generation of new cells in the neuronal soma and axonal compartments of sensory ganglia and along the dorsal root and the dorsal root entry zone. Quantification of BrdU positive cells within sensory ganglia revealed a 2.3- to 3-fold increase in glial cells compared to controls with a similar response to NGF for the different peripheral ganglia examined. Immunofluorescent labeling with S100β revealed that Schwann and satellite cells underwent mitosis after NGF administration. These data indicate that intracerebroventricular NGF infusion significantly induces gliogenesis in trigeminal ganglia and the spinal sensory ganglia and along the dorsal root entry zone as well as the dorsal root. Johannes C. M. Schlachetzki, Donald P. Pizzo, Debbi A. Morrissette, and Jürgen Winkler Copyright © 2014 Johannes C. M. Schlachetzki et al. All rights reserved. Association of CHMP4B and Autophagy with Micronuclei: Implications for Cataract Formation Tue, 11 Mar 2014 12:49:17 +0000 Autophagy is a mechanism of cellular self-degradation that is very important for cellular homeostasis and differentiation. Components of the endosomal sorting complex required for transport (ESCRT) machinery are required for endosomal sorting and also for autophagy and the completion of cytokinesis. Here we show that the ESCRT-III subunit CHMP4B not only localizes to normal cytokinetic bridges but also to chromosome bridges and micronuclei, the latter surrounded by lysosomes and autophagosomes. Moreover, CHMP4B can be co-immunoprecipitated with chromatin. Interestingly, a CHMP4B mutation associated with autosomal dominant posterior polar cataract abolishes the ability of CHMP4B to localize to micronuclei. We propose that CHMP4B, through its association with chromatin, may participate in the autophagolysosomal degradation of micronuclei and other extranuclear chromatin. This may have implications for DNA degradation during lens cell differentiation, thus potentially protecting lens cells from cataract development. Antonia P. Sagona, Ioannis P. Nezis, and Harald Stenmark Copyright © 2014 Antonia P. Sagona et al. All rights reserved.