BioMed Research International: Cell Biology The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . 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. 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. 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. 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. 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. 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. 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. Effects of Quercetin on CYP450 and Cytokines in Aroclor 1254 Injured Endometrial Cells of the Pregnant Rats Mon, 10 Mar 2014 08:51:48 +0000 Polychlorinated biphenyls (PCBs) are widespread persistent residual environmental pollutants, which affect seriously the growth and reproductive alterations in humans and animals. Aroclor 1254 is a commercial mixture of PCBs. Quercetin is a flavonoid, which acts on estrogen receptors and causes the development of estrogen-related diseases. In this paper, the primary cultured endometrial cells in the pregnant rats were isolated and Aroclor 1254 was used to induce the injured endometrial cells model. The cells were treated with gradient quercetin, the viability of the endometrial cells, the expressions of CYP450, the contents of TNF-α, IL-6, estradiol (E2), and progesterone (P4) were measured. It showed that the viability of the cultured endometrial cells, the expression of CYP1A1 and CYP2B1, and the contents of TNF-α, E2, and IL-6 in the injured endometrial cells increased with the treatment of quercetin. It shows that quercetin has protective effect on the injured endometrial cells in the pregnant rats, this provide a basis on herbal medicine protection for animal reproductive diseases caused by environmental endocrine disruptors. Lina Xu, Liyun Sun, Liqin Lu, Xiuhui Zhong, Yuzhong Ma, and Jianhua Qin Copyright © 2014 Lina Xu et al. All rights reserved. Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells Sun, 09 Mar 2014 11:39:42 +0000 The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells. Karla Dias Cruz, Thayana Araújo Cruz, Gabriela Veras de Moraes, Tatiana Christina Paredes-Santos, Marcia Attias, and Wanderley de Souza Copyright © 2014 Karla Dias Cruz et al. All rights reserved. α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism Sun, 02 Mar 2014 00:00:00 +0000 Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system. Jaeson Santos Calla-Choque, Elisa Elvira Figueroa-Angulo, Leticia Ávila-González, and Rossana Arroyo Copyright © 2014 Jaeson Santos Calla-Choque et al. All rights reserved. Telomere Length Reprogramming in Embryos and Stem Cells Thu, 27 Feb 2014 09:33:19 +0000 Telomeres protect and cap linear chromosome ends, yet these genomic buffers erode over an organism’s lifespan. Short telomeres have been associated with many age-related conditions in humans, and genetic mutations resulting in short telomeres in humans manifest as syndromes of precocious aging. In women, telomere length limits a fertilized egg’s capacity to develop into a healthy embryo. Thus, telomere length must be reset with each subsequent generation. Although telomerase is purportedly responsible for restoring telomere DNA, recent studies have elucidated the role of alternative telomeres lengthening mechanisms in the reprogramming of early embryos and stem cells, which we review here. Keri Kalmbach, LeRoy G. Robinson Jr., Fang Wang, Lin Liu, and David Keefe Copyright © 2014 Keri Kalmbach et al. All rights reserved. Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirre Cells Tue, 25 Feb 2014 14:10:13 +0000 Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+ cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-β with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38− cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs. Muti ur Rehman Khan, Ijaz Ali, Wei Jiao, Yun Wang, Saima Masood, Muhammad Zubair Yousaf, Aqeel Javaid, Shafique Ahmad, and Meifu Feng Copyright © 2014 Muti ur Rehman Khan et al. All rights reserved. Dual Effects of Cigarette Smoke Extract on Proliferation of Endothelial Progenitor Cells and the Protective Effect of 5-aza-2′-deoxycytidine on EPCs against the Damage Caused by CSE Tue, 18 Feb 2014 16:26:12 +0000 Cigarette smoke is a major public health problem associated with multitude of diseases, including pulmonary and vascular diseases. Endothelial progenitor cells (EPCs) contribute to neovascularization and play an important role in the development of these diseases. The effect of CSE on EPCs is seldom studied. The aim of the current study is to observe the effect of CSE on biological behavior of EPCs and, further, to search for potential candidate agent in protection of proliferation of EPCs against the damage caused by CSE exposure in vitro. Methods. The proliferations of EPCs isolated from bone marrow of C57BL/6J mice were assessed by MTT after incubating the EPCs with a series of concentrations of CSE (1.0%, 2.5%, 5.0%, and 10.0%) for different times (3, 6, and 24 hours) as well as with 1.0% CSE in presence of 5-AZA-CdR for 24 hours. Results. The proliferations of EPCs were significantly enhanced after 3 hours of exposure to concentrations of 1.0% and 2.5% CSE but depressed when exposed to concentrations of 5.0% and 10.0% CSE. Furthermore, the 5-AZA-CdR in concentrations of 2.0 μmol/L and 5.0 μmol/L partly protected against the depression of proliferation of EPCs caused by CSE exposure. Conclusions. The CSE showed dual effects on proliferation of EPCs isolated from mice. The 5-AZA-CdR partly protected the proliferation of EPCs against the damage caused by CSE exposure in vitro, suggesting that DNA methylation may be involved in the dysfunction of EPCs induced by CSE. Zhi-Hui He, Ping Chen, Yan Chen, Ying-Qun Zhu, Sheng-Dong He, Ji-Ru Ye, Da Liu, and Yue Yang Copyright © 2014 Zhi-Hui He et al. All rights reserved. Nodal Promotes the Self-Renewal of Human Colon Cancer Stem Cells via an Autocrine Manner through Smad2/3 Signaling Pathway Mon, 17 Feb 2014 08:00:10 +0000 Colorectal cancer is one of the most common and fatal tumors. However, molecular mechanisms underlying carcinogenesis of colorectal cancer remain largely undefined. Here, we explored the expression and function of Nodal in colon cancer stem cells (CCSCs). Nodal and its receptors were present in numerous human colorectal cancer cell lines. NODAL and ALK-4 were coexpressed in human colon cancerous tissues, and NODAL, CD24, and CD44, markers for CCSCs, were expressed at higher levels in human colon cancerous tissues than adjacent noncancerous colon tissues. Human CCSCs were isolated by magnetic activated cell sorting using anti-CD24 and anti-CD44. Nodal transcript and protein were hardly detectable in CD44- or CD24-negative human colorectal cancer cell lines, whereas Nodal and its receptors were present in CCSCs. Notably, Nodal facilitated spheroid formation of human CCSCs, and phosphorylation of Smad2 and Smad3 was activated by Nodal in cells of spheres derived from human CCSCs. Collectively, these results suggest that Nodal promotes the self-renewal of human CCSCs and mediate carcinogenesis of human colorectal cancer via an autocrine manner through Smad2/3 pathway. This study provides a novel insight into molecular mechanisms controlling fate of human CCSCs and offers new targets for gene therapy of human colorectal cancer. Yuehua Gong, Ying Guo, Yanan Hai, Hao Yang, Yang Liu, Shi Yang, Zhenzhen Zhang, Meng Ma, Linhong Liu, Zheng Li, and Zuping He Copyright © 2014 Yuehua Gong et al. All rights reserved. Towards Scarless Wound Healing: A Comparison of Protein Expression between Human, Adult and Foetal Fibroblasts Thu, 30 Jan 2014 13:53:24 +0000 Proteins from human adult and foetal fibroblast cell lines were compared, focusing on those involved in wound healing. Proteins were separated through two-dimensional gel electrophoresis (2DE). Differences in protein spot intensity between the lineages were quantified through 3D gel scanning densitometry. Selected protein spots were excised, subjected to tryptic digests, prior to separation using HPLC with a linear ion trap mass spectrometer, and identified. Protein maps representing the proteomes from adult and foetal fibroblasts showed similar distributions but revealed differences in expression levels. Heat shock cognate 71 kDA protein, Tubulin alpha-1A chain, actin cytoplasmic-1, and neuron cytoplasmic protein were all expressed in significantly higher concentrations by foetal fibroblasts, nearly double those observed for their adult counterparts. Fructose bisphosphate aldolase A, Cofilin-1, Peroxiredoxin-1, Lactotransferrin Galectin-1, Profilin-1, and Calreticulin were expressed at comparatively higher concentrations by the adult fibroblasts. Significant differences in the expression levels of some proteins in human adult and foetal fibroblasts correlated with known differences in wound healing behaviour. This data may assist in the development of technologies to promote scarless wound healing and better functional tissue repair and regeneration. Sonia Ho, Helder Marçal, and Leslie John Ray Foster Copyright © 2014 Sonia Ho et al. All rights reserved. mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes of Trypanosoma cruzi Epimastigotes Thu, 23 Jan 2014 13:24:04 +0000 Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway of T. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinant T. cruzi cruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes of T. cruzi. Cassiano Martin Batista, Lia Carolina Soares Medeiros, Iriane Eger, and Maurilio José Soares Copyright © 2014 Cassiano Martin Batista et al. All rights reserved. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin Thu, 23 Jan 2014 07:24:01 +0000 Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases. Sebastian Gehmert, Carina Wenzel, Markus Loibl, Gero Brockhoff, Michaela Huber, Werner Krutsch, Michael Nerlich, Martin Gosau, Silvan Klein, Stephan Schreml, Lukas Prantl, and Sanga Gehmert Copyright © 2014 Sebastian Gehmert et al. All rights reserved. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro Mon, 20 Jan 2014 13:28:53 +0000 Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs) and endothelial progenitor cells (EPCs). We identified stabilising pericytes (PCs) as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts. Markus Loibl, Andreas Binder, Marietta Herrmann, Fabian Duttenhoefer, R. Geoff Richards, Michael Nerlich, Mauro Alini, and Sophie Verrier Copyright © 2014 Markus Loibl et al. All rights reserved. Biological Activity of Blackcurrant Extracts (Ribes nigrum L.) in Relation to Erythrocyte Membranes Thu, 16 Jan 2014 13:39:22 +0000 Compounds contained in fruits and leaves of blackcurrant (Ribes nigrum L.) are known as agents acting preventively and therapeutically on the organism. The HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and flavonoids in leaves, that have antioxidant activity and are beneficial for health. The aim of the research was to determine the effect of blackcurrant fruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant properties. The effect of the extracts on osmotic resistance, shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined with spectrophotometric methods. The FTIR investigation showed that extracts modify the erythrocyte membrane and protect it against free radicals induced by UV radiation. The results show that the extracts do not induce hemolysis and even protect erythrocytes against the harmful action of UVC radiation, while slightly strengthening the membrane and inducing echinocytes. The compounds contained in the extracts do not penetrate into the hydrophobic region, but bind to the membrane surface inducing small changes in the packing arrangement of the polar head groups of membrane lipids. The extracts have a high antioxidant activity. Their presence on the surface of the erythrocyte membrane entails protection against free radicals. Dorota Bonarska-Kujawa, Sylwia Cyboran, Romuald Żyłka, Jan Oszmiański, and Halina Kleszczyńska Copyright © 2014 Dorota Bonarska-Kujawa et al. All rights reserved. Cell Surface Proteomics Analysis Indicates a Neural Lineage Bias of Rat Bone Marrow Mesenchymal Stromal Cells Thu, 16 Jan 2014 08:32:02 +0000 Mesenchymal stromal cells (MSCs) are one of the most intensively studied stem cell types with application aims. However, the molecular characterisation and the relationship between the molecular characterisation and functional properties of MSCs are largely unknown. In this study, we purified the surface proteins from rat bone marrow MSCs (rBMMSCs) and characterised their surface proteome by LC-MS/MS. Moreover, we comparatively analysed the data from this study with the surface proteomics data of mouse and human embryonic stem (ES) cells and human mesenchymal stromal cells (hMSCs). The data showed that, in contrast to ES cells and human mesenchymal stromal cells, rBMMSCs possessed a surface proteomics pattern biased to neural and neural-endocrine lineages, indicating a neural/neural crest bias, and suggested a neural differentiation tendency of these cells. The different surface proteomics pattern between rBMMSCs and hMSCs also suggested that MSCs of different origin might possess a different lineage bias. Guiyun Zhao, Huijiao Ji, Shihao Wang, Bin Gu, Xiuli Song, Jiarong Zhang, Yukan Liu, Liangbiao Chen, and Ming Zhang Copyright © 2014 Guiyun Zhao et al. All rights reserved. Individual Osmotic Fragility Distribution: A New Parameter for Determination of the Osmotic Properties of Human Red Blood Cells Thu, 02 Jan 2014 16:25:46 +0000 The aim of our experiments was to characterise and to validate the osmotic fragility test when applied to human blood samples with no significant alterations of osmotic fragility but with a differentiating shape of the haemolysis curve. All experiments were carried out on human erythrocytes taken from the Regional Centre of Blood Donation and Blood Therapy in Wrocław. The washed erythrocytes were exposed to near-infrared radiation (NIR) or ozonated, and the osmotic fragility test was applied. The osmotic fragility, calculated from the experimental haemolysis curve for the control and cells irradiated for 15 min, is the same within the empirical error. Calculation of the first derivative of the haemolysis curve allowed us to visualise the changes in osmotic fragility distribution after exposure to NIR. By contrast, significant changes both to the osmotic fragility value and the distribution of osmotic properties were observed after an erythrocytes ozonation procedure. Description of cell osmotic properties requires at least two parameters—the value of osmotic fragility and the slope of the haemolysis curve in the region where absorbance sharply increases due to cell haemolysis. Tomasz Walski, Ludmiła Chludzińska, Małgorzata Komorowska, and Wojciech Witkiewicz Copyright © 2014 Tomasz Walski et al. All rights reserved. Protective Effect of Thunbergia laurifolia (Linn.) on Lead Induced Acetylcholinesterase Dysfunction and Cognitive Impairment in Mice Sat, 21 Dec 2013 10:32:12 +0000 Thunbergia laurifolia (linn., TL), a natural phenolic compound, has been reported to have many benefits and medicinal properties. The current study ascertains the total phenolic content present in TL aqueous leaf extract and also examines the antioxidant ability of the extract in preserving acetylcholinesterase (AChE) activity of mice exposed to lead in vivo and in vitro model. Mice were given lead acetate (Pb) in drinking water (1 g/L) together with TL 100 and 200 mg/kg/day. The result showed that Pb induced AChE dysfunction in both in vitro and in vivo studies. TL significantly prevented Pb induced neurotoxicity in a dose-dependent manner which was indicated by comparatively better performance of TL treated mice in Morris Water Maze Swimming Test and increased AChE activity in the tissue sample collected from the brains of these mice. TL also exhibited the greatest amount of phenolic content, which has a significant positive correlation with its antioxidant capacity (). Taken together, these data suggested that the total phenolic compounds in TL could exhibit antioxidant and in part neuroprotective properties. It may play a potential treatment strategy for Pb contamination. Moe Pwint Phyu and Jitbanjong Tangpong Copyright © 2013 Moe Pwint Phyu and Jitbanjong Tangpong. All rights reserved. The Effects of Cytokines in Adipose Stem Cell-Conditioned Medium on the Migration and Proliferation of Skin Fibroblasts In Vitro Sun, 15 Dec 2013 16:04:36 +0000 Although adipose stem cell-conditioned medium (ASC-CM) has demonstrated the effect of promoting the cutaneous wound healing, the mechanism for this response on the effector cells (e.g., dermal fibroblasts) during the process remains to be determined. In this study, we aim to investigate the types and contents of cytokines in ASC-CM and the effects of some kinds of common cytokines in ASC-CM, such as EGF, PDGF-AA, VEGF, and bFGF, on dermal fibroblasts proliferation and migration in wound healing process. Results showed that these four cytokines had high concentrations in ASC-CM. The migration of skin fibroblasts could be significantly stimulated by VEGF, bFGF, and PDGF-AA, and the proliferation could be significantly stimulated by bFGF and EGF in ASC-CM. Additionally, ASC-CM had more obvious promoting effect on fibroblasts proliferation and migration than single cytokine. These observations suggested that ASC-CM played an important role in the cutaneous injury partly by the synergistic actions of several cytokines in promoting dermal fibroblasts proliferation and migration, and ASC-CM was more adaptive than each single cytokine to be applied in promoting the wound healing. Jiajia Zhao, Li Hu, Jiarong Liu, Niya Gong, and Lili Chen Copyright © 2013 Jiajia Zhao et al. All rights reserved. The Cell Surface GRP78 Facilitates the Invasion of Hepatocellular Carcinoma Cells Sun, 08 Dec 2013 09:17:04 +0000 Invasion is a major characteristic of hepatocellular carcinoma and one of the main causes of refractory to treatment. We have previously reported that GRP78 promotes the invasion of hepatocellular carcinoma although the mechanism underlying this change remains uncertain. In this paper, we explored the role of the cell surface GRP78 in the regulation of cancer cell invasion in hepatocellular carcinoma cells. We found that neutralization of the endogenous cell surface GRP78 with the anti-GRP78 antibody inhibited the adhesion and invasion in hepatocellular carcinoma cell lines Mahlavu and SMMC7721. However, forced expression of the cell surface GRP78 facilitated the adhesion and invasion in SMMC7721. We further demonstrated that inhibition of the endogenous cell surface GRP78 specifically inhibited the secretion and activity of MMP-2 but did not affect the secretion and activity of MMP-9. We also found that inhibition of the cell surface GRP78 increased E-Cadherin expression and decreased N-Cadherin level. On the contrary, forced expression of the cell surface GRP78 increased N-Cadherin expression and decreased E-Cadherin level, suggesting that the cell surface GRP78 plays critical role in the regulation of EMT process. These findings suggest that the cell surface GRP78 plays a stimulatory role in the invasion process and may be a potential anti-invasion target for the treatment of hepatocellular carcinoma. Xiu-Xiu Zhang, Hong-Dan Li, Song Zhao, Liang Zhao, Hui-Juan Song, Guan Wang, Qing-Jun Guo, Zhi-Dong Luan, and Rong-Jian Su Copyright © 2013 Xiu-Xiu Zhang et al. All rights reserved. Neuroprotective Function of 14-3-3 Proteins in Neurodegeneration Mon, 02 Dec 2013 14:21:44 +0000 14-3-3 proteins are abundantly expressed adaptor proteins that interact with a vast number of binding partners to regulate their cellular localization and function. They regulate substrate function in a number of ways including protection from dephosphorylation, regulation of enzyme activity, formation of ternary complexes and sequestration. The diversity of 14-3-3 interacting partners thus enables 14-3-3 proteins to impact a wide variety of cellular and physiological processes. 14-3-3 proteins are broadly expressed in the brain, and clinical and experimental studies have implicated 14-3-3 proteins in neurodegenerative disease. A recurring theme is that 14-3-3 proteins play important roles in pathogenesis through regulating the subcellular localization of target proteins. Here, we review the evidence that 14-3-3 proteins regulate aspects of neurodegenerative disease with a focus on their protective roles against neurodegeneration. Tadayuki Shimada, Alyson E. Fournier, and Kanato Yamagata Copyright © 2013 Tadayuki Shimada et al. All rights reserved. Growth Arrest on Inhibition of Nonsense-Mediated Decay Is Mediated by Noncoding RNA GAS5 Mon, 11 Nov 2013 09:42:02 +0000 Nonsense-mediated decay is a key RNA surveillance mechanism responsible for the rapid degradation of mRNAs containing premature termination codons and hence prevents the synthesis of truncated proteins. More recently, it has been shown that nonsense-mediated decay also has broader significance in controlling the expression of a significant proportion of the transcriptome. The importance of this mechanism to the mammalian cell is demonstrated by the observation that its inhibition causes growth arrest. The noncoding RNA growth arrest specific transcript 5 (GAS5) has recently been shown to play a key role in growth arrest induced by several mechanisms, including serum withdrawal and treatment with the mTOR inhibitor rapamycin. Here we show that inhibition of nonsense-mediated decay in several human lymphocyte cell lines causes growth arrest, and siRNA-mediated downregulation of GAS5 in these cells significantly alleviates the inhibitory effects observed. These observations hold true for inhibition of nonsense-mediated decay both through RNA interference and through pharmacological inhibition by aminoglycoside antibiotics gentamycin and G418. These studies have important implications for ototoxicity and nephrotoxicity caused by gentamycin and for the proposed use of NMD inhibition in treating genetic disease. This report further demonstrates the critical role played by GAS5 in the growth arrest of mammalian cells. Mirna Mourtada-Maarabouni and Gwyn T. Williams Copyright © 2013 Mirna Mourtada-Maarabouni and Gwyn T. Williams. All rights reserved. Cellular Microenvironment in Human Pathologies Thu, 07 Nov 2013 10:35:43 +0000 Davide Vigetti, Martin Götte, Mauro S. G. Pavão, and Achilleas D. Theocharis Copyright © 2013 Davide Vigetti et al. All rights reserved. Salamander-Derived, Human-Optimized nAG Protein Suppresses Collagen Synthesis and Increases Collagen Degradation in Primary Human Fibroblasts Thu, 31 Oct 2013 09:36:14 +0000 Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF-β1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects. Mohammad M. Al-Qattan, Medhat K. Shier, Mervat M. Abd-AlWahed, Ola H. Mawlana, Mohammed S. El-Wetidy, Reginald S. Bagayawa, Hebatallah H. Ali, May S. Al-Nbaheen, and Abdullah M. Aldahmash Copyright © 2013 Mohammad M. Al-Qattan et al. All rights reserved. Role of Gap Junctions and Hemichannels in Parasitic Infections Wed, 23 Oct 2013 14:03:11 +0000 In vertebrates, connexins (Cxs) and pannexins (Panxs) are proteins that form gap junction channels and/or hemichannels located at cell-cell interfaces and cell surface, respectively. Similar channel types are formed by innexins in invertebrate cells. These channels serve as pathways for cellular communication that coordinate diverse physiologic processes. However, it is known that many acquired and inherited diseases deregulate Cx and/or Panx channels, condition that frequently worsens the pathological state of vertebrates. Recent evidences suggest that Cx and/or Panx hemichannels play a relevant role in bacterial and viral infections. Nonetheless, little is known about the role of Cx- and Panx-based channels in parasitic infections of vertebrates. In this review, available data on changes in Cx and gap junction channel changes induced by parasitic infections are summarized. Additionally, we describe recent findings that suggest possible roles of hemichannels in parasitic infections. Finally, the possibility of new therapeutic designs based on hemichannel blokers is presented. José Luis Vega, Mario Subiabre, Felipe Figueroa, Kurt Alex Schalper, Luis Osorio, Jorge González, and Juan Carlos Sáez Copyright © 2013 José Luis Vega et al. All rights reserved. The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo Wed, 25 Sep 2013 12:02:33 +0000 Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform. Jeoffrey Schageman, Emily Zeringer, Mu Li, Tim Barta, Kristi Lea, Jian Gu, Susan Magdaleno, Robert Setterquist, and Alexander V. Vlassov Copyright © 2013 Jeoffrey Schageman et al. All rights reserved. Proregenerative Properties of ECM Molecules Mon, 09 Sep 2013 17:55:08 +0000 After traumatic injuries to the nervous system, regrowing axons encounter a complex microenvironment where mechanisms that promote regeneration compete with inhibitory processes. Sprouting and axonal regrowth are key components of functional recovery but are often counteracted by inhibitory molecules. This review covers extracellular matrix molecules that support neuron axonal outgrowth. Stefan Plantman Copyright © 2013 Stefan Plantman. All rights reserved. MicroRNAs in Kidney Fibrosis and Diabetic Nephropathy: Roles on EMT and EndMT Sun, 08 Sep 2013 08:58:17 +0000 MicroRNAs (miRNAs) are a family of small, noncoding RNAs that regulate gene expression in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. As a result, miRNAs emerged as major area of biomedical research with relevance to kidney fibrosis. Fibrosis is characterized by the excess deposition of extracellular matrix (ECM) components, which is the end result of an imbalance of metabolism of the ECM molecule. Recent evidence suggests that miRNAs participate in the fibrotic process in a number of organs including the heart, kidney, liver, and lung. Epithelial mesenchymal transition (EMT) and endothelial mesenchymal transition (EndMT) programs play vital roles in the development of fibrosis in the kidney. A growing number of the extracellular and intracellular molecules that control EMT and EndMT have been identified and could be exploited in developing therapeutics for fibrosis. This review highlights recent advances on the role of miRNAs in the kidney diseases; diabetic nephropathy especially focused on EMT and EndMT program responsible for the development of kidney fibrosis. These miRNAs can be utilized as a potential novel drug target for the studying of underlying mechanism and treatment of kidney fibrosis. Swayam Prakash Srivastava, Daisuke Koya, and Keizo Kanasaki Copyright © 2013 Swayam Prakash Srivastava et al. All rights reserved. The Roles of Hyaluronan/RHAMM/CD44 and Their Respective Interactions along the Insidious Pathways of Fibrosarcoma Progression Thu, 05 Sep 2013 19:15:10 +0000 Fibrosarcomas are rare malignant mesenchymal tumors originating from fibroblasts. Importantly, fibrosarcoma cells were shown to have a high content and turnover of extracellular matrix (ECM) components including hyaluronan (HA), proteoglycans, collagens, fibronectin, and laminin. ECMs are complicated structures that surround and support cells within tissues. During cancer progression, significant changes can be observed in the structural and mechanical properties of the ECM components. Importantly, hyaluronan deposition is usually higher in malignant tumors as compared to benign tissues, predicting tumor progression in some tumor types. Furthermore, activated stromal cells are able to produce tissue structure rich in hyaluronan in order to promote tumor growth. Key biological roles of HA result from its interactions with its specific CD44 and RHAMM (receptor for HA-mediated motility) cell-surface receptors. HA-receptor downstream signaling pathways regulate in turn cellular processes implicated in tumorigenesis. Growth factors, including PDGF-BB, TGFβ2, and FGF-2, enhanced hyaluronan deposition to ECM and modulated HA-receptor expression in fibrosarcoma cells. Indeed, FGF-2 through upregulation of specific HAS isoforms and hyaluronan synthesis regulated secretion and net hyaluronan deposition to the fibrosarcoma pericellular matrix modulating these cells’ migration capability. In this paper we discuss the involvement of hyaluronan/RHAMM/CD44 mediated signaling in the insidious pathways of fibrosarcoma progression. Dragana Nikitovic, Katerina Kouvidi, Nikos K. Karamanos, and George N. Tzanakakis Copyright © 2013 Dragana Nikitovic et al. All rights reserved. The Role of Changes in Extracellular Matrix of Cartilage in the Presence of Inflammation on the Pathology of Osteoarthritis Wed, 28 Aug 2013 10:47:08 +0000 Osteoarthritis (OA) is a degenerative disease that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments. No therapy is currently available to completely prevent the initiation or progression of the disease partly due to poor understanding of the mechanisms of the disease pathology. Cartilage is the main tissue afflicted by OA, and chondrocytes, the sole cellular component in the tissue, actively participate in the degeneration process. Multiple factors affect the development and progression of OA including inflammation that is sustained during the progression of the disease and alteration in biomechanical conditions due to wear and tear or trauma in cartilage. During the progression of OA, extracellular matrix (ECM) of cartilage is actively remodeled by chondrocytes under inflammatory conditions. This alteration of ECM, in turn, changes the biomechanical environment of chondrocytes, which further drives the progression of the disease in the presence of inflammation. The changes in ECM composition and structure also prevent participation of mesenchymal stem cells in the repair process by inhibiting their chondrogenic differentiation. This review focuses on how inflammation-induced ECM remodeling disturbs cellular activities to prevent self-regeneration of cartilage in the pathology of OA. Maricela Maldonado and Jin Nam Copyright © 2013 Maricela Maldonado and Jin Nam. All rights reserved. Development, Disease, and Regeneration of Tissues in the Dental-Craniofacial Complex Tue, 27 Aug 2013 12:48:04 +0000 Brian L. Foster, Yong-Hee P. Chun, Erica L. Scheller, Zhao Lin, Chad M. Novince, and Avina Paranjpe Copyright © 2013 Brian L. Foster et al. All rights reserved. Combined Treatment with Low Concentrations of Decitabine and SAHA Causes Cell Death in Leukemic Cell Lines but Not in Normal Peripheral Blood Lymphocytes Tue, 13 Aug 2013 15:05:20 +0000 Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat) on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line. Barbora Brodská, Aleš Holoubek, Petra Otevřelová, and Kateřina Kuželová Copyright © 2013 Barbora Brodská et al. All rights reserved. The Therapeutic Potential of Umbilical Cord Mesenchymal Stem Cells in Mice Premature Ovarian Failure Tue, 13 Aug 2013 08:09:24 +0000 Mesenchymal stem cells, which are poorly immunogenic and have potent immunosuppressive activities, have emerged as promising cellular therapeutics for the treatment of several diseases. Mesenchymal-like cells derived from Wharton’s Jelly, called umbilical cord matrix stem cells (UCMSCs), reportedly secrete a variety of cytokines and growth factors, acting as trophic suppliers. Here, we used UCMSCs to treat premature ovarian failure (POF). Ovarian function was evaluated by ovulation and the number of follicles. Apoptosis of the granulosa cells (GC) was analyzed by TUNEL staining. We found that after transplantation of the UCMSCs, apoptosis of cumulus cells in the ovarian damage model was reduced and the function of the ovary had been recovered. The sex hormone level was significantly elevated in mice treated with UCMSCs. The number of follicles in the treated group was higher than in the control group. Our results demonstrate that UCMSCs can effectively restore ovary functionality and reduce apoptosis of granulosa cells. We compared the RNA expression of the UCMSCs treated group with the POF model and wild-type control group and found that the UCMSC group is most similar to the wild-type group. Our experiments provide new information regarding the treatment of ovarian function failure. Shufang Wang, Ling Yu, Min Sun, Sha Mu, Changyong Wang, Deqing Wang, and Yuanqing Yao Copyright © 2013 Shufang Wang et al. All rights reserved. The Role of Epidermal Growth Factor Receptor in Cancer Metastasis and Microenvironment Wed, 07 Aug 2013 08:06:17 +0000 Despite significant improvements in diagnosis, surgical techniques, and advancements in general patient care, the majority of deaths from cancer are caused by the metastases. There is an urgent need for an improved understanding of the cellular and molecular factors that promote cancer metastasis. The process of cancer metastasis depends on multiple interactions between cancer cells and host cells. Studies investigating the TGFα-EGFR signaling pathways that promote the growth and spread of cancer cells. Moreover, the signaling activates not only tumor cells, but also tumor-associated endothelial cells. TGFα-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFα-positive cancers. In this review, we describe the recent advances in our understanding of the molecular basis of cancer metastasis. Takamitsu Sasaki, Kuniyasu Hiroki, and Yuichi Yamashita Copyright © 2013 Takamitsu Sasaki et al. All rights reserved. Heparan Sulfate and Heparanase as Modulators of Breast Cancer Progression Wed, 31 Jul 2013 12:50:05 +0000 Breast cancer is defined as a cancer originating in tissues of the breast, frequently in ducts and lobules. During the last 30 years, studies to understand the biology and to treat breast tumor improved patients’ survival rates. These studies have focused on genetic components involved in tumor progression and on tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are involved in cell signaling, adhesion, extracellular matrix assembly, and growth factors storage. As a central molecule, HSPG regulates cell behavior and tumor progression. HS accompanied by its glycosaminoglycan counterparts regulates tissue homeostasis and cancer development. These molecules present opposite effects according to tumor type or cancer model. Studies in this area may contribute to unveil glycosaminoglycan activities on cell dynamics during breast cancer exploring these polysaccharides as antitumor agents. Heparanase is a potent tumor modulator due to its protumorigenic, proangiogenic, and prometastatic activities. Several lines of evidence indicate that heparanase is upregulated in all human sarcomas and carcinomas. Heparanase seems to be related to several aspects regulating the potential of breast cancer metastasis. Due to its multiple roles, heparanase is seen as a target in cancer treatment. We will describe recent findings on the function of HSPGs and heparanase in breast cancer behavior and progression. Angélica M. Gomes, Mariana P. Stelling, and Mauro S. G. Pavão Copyright © 2013 Angélica M. Gomes et al. All rights reserved. Membrane Localization of Membrane Type 1 Matrix Metalloproteinase by CD44 Regulates the Activation of Pro-Matrix Metalloproteinase 9 in Osteoclasts Sun, 28 Jul 2013 09:47:58 +0000 CD44, MT1-MMP, and MMP9 are implicated in the migration of osteoclast and bone resorption. This study was designed to determine the functional relationship between CD44 and MT1-MMP in the activation of pro-MMP9. We used osteoclasts isolated from wild-type and CD44-null mice. Results showed that MT1-MMP is present in multiple forms with a molecular mass ~63, 55, and 45 kDa in the membrane of wild-type osteoclasts. CD44-null osteoclasts demonstrated a 55 kDa active MT1-MMP form in the membrane and conditioned medium. It failed to activate pro-MMP9 because TIMP2 binds and inhibits this MT1-MMP (~55 kDa) in CD44-null osteoclasts. The role of MT1-MMP in the activation of pro-MMP9, CD44 expression, and migration was confirmed by knockdown of MT1-MMP in wild-type osteoclasts. Although knockdown of MMP9 suppressed osteoclast migration, it had no effects on MT1-MMP activity or CD44 expression. These results suggest that CD44 and MT1-MMP are directly or indirectly involved in the regulation of pro-MMP9 activation. Surface expression of CD44, membrane localization of MT1-MMP, and activation of pro-MMP9 are the necessary sequence of events in osteoclast migration. Meenakshi A. Chellaiah and Tao Ma Copyright © 2013 Meenakshi A. Chellaiah and Tao Ma. All rights reserved. Interaction between a Regenerative Matrix and Wound Bed in Nonhealing Ulcers: Results with 16 Cases Thu, 18 Jul 2013 09:15:10 +0000 A chronic wound is a wound that is delayed in one of the wound-healing stages and cannot progress any further. A chronic wound leaves the patient at risk of infection and hospitalization. In these case series, 16 patients affected by venous ulcers underwent Hyalomatrix PA grafting for reconstructive surgery. Hyalomatrix PA is a bilayered, sterile, flexible, and conformable three-dimensional matrix made of fibers of HYAFF, a benzyl ester of hyaluronic acid, and a semipermeable silicone membrane. Hyalomatrix PA acts as a substitutive and regenerative permanent matrix able to replace the dermis providing a three-dimensional matrix for cellular invasion and capillary growth. The silicon layer controls water vapor loss avoiding an excessive loss of fluids and acts as a semipermeable barrier to the external agents. In the presented cases, the average area grafted per procedure was 153 cm2. The length of followup ranged from 0.5 to 1 year. The final results were considered to be good in 12 cases, fair in 3 cases, and poor in one case. This study suggests that the combination of wound bed preparation with application of the hyaluronic regenerative matrix can be a valid approach for treatment of partial thickness ulcers. Alberico Motolese, Francesca Vignati, Roberto Brambilla, Michele Cerati, and Alberto Passi Copyright © 2013 Alberico Motolese et al. All rights reserved. Downregulation of ADAM10 Expression Inhibits Metastasis and Invasiveness of Human Hepatocellular Carcinoma HepG2 Cells Sun, 14 Jul 2013 10:51:36 +0000 Objective. This study aims to investigate the effects of ADAM10 expression on metastasis and invasiveness of human hepatocellular carcinoma HepG2 cells. Methods. The HepG2 cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10, respectively. Cell migration assay and Transwell invasiveness assay were performed to detect the effects of ADAM10 knockdown on migration and invasiveness of HepG2 cells. Western blotting and real-time RT PCR were performed to investigate the effects of knock-down of ADAM10 on protein and mRNA levels of E-cadherin gene. Results. Cell migration and invasiveness of HepG2 cells transfected with ADAM10 siRNA were significantly decreased, when compared with the cells transfected with the control siRNA, suggesting that the downregulation of ADAM10 expression inhibits cell migration and invasiveness. The Western blotting results suggest that the down-regulation of ADAM10 expression increases E-cadherin protein levels. The real-time RT-PCR results indicated that the mRNA level of E-cadherin is not detectably affected by the knock-down of ADAM10 gene. Conclusions. Expression of ADAM10 may be related to cell migration and invasiveness of human hepatocellular carcinoma HepG2 cells via a mechanism related to E-cadherin. Yuan Yue, Yuan Shao, Qing Luo, Lei Shi, and Zuoren Wang Copyright © 2013 Yuan Yue et al. All rights reserved. The Possible Role of Mena Protein and Its Splicing-Derived Variants in Embryogenesis, Carcinogenesis, and Tumor Invasion: A Systematic Review of the Literature Sun, 14 Jul 2013 09:49:35 +0000 The Ena/VASP (enabled/vasodilator stimulated phosphoprotein) family includes the binding actin proteins such as mammalian Ena (Mena), VASP, and Ena-VASP-like. It is known that the perturbation of actin cycle could determine alteration in the mobility of cells and in consequence of organogenesis. Few recent studies have revealed that Mena protein could play a role in breast or pancreatic carcinogenesis. Based on our researches, we observed that the intensity of Mena expression increased from premalignant to malignant lesions in some organs such as large bowel, stomach, cervix, and salivary glands. These findings prove that Mena could be a marker of premalignant epithelial lesions. In premalignant lesions, it could be helpful to define more accurately the risk for malignant transformation. In malignant tumors, correlation of expression of its splice variants could indicate metastatic behavior. In conclusion, we consider that it is necessary to analyze the expression of Mena splice variants in a higher number of cases, in different epithelial lesions, and also in experimental studies to define its exact role in carcinogenesis and also its possible prognostic and predictive values. Simona Gurzu, Diana Ciortea, Istvan Ember, and Ioan Jung Copyright © 2013 Simona Gurzu et al. All rights reserved. LRP-1: A Checkpoint for the Extracellular Matrix Proteolysis Wed, 10 Jul 2013 08:49:05 +0000 Low-density lipoprotein receptor-related protein-(LRP-1) is a large endocytic receptor that binds more than 35 ligands and exhibits signaling properties. Proteinases capable of degrading extracellular matrix (ECM), called matrix proteinases in this paper, are mainly serine proteinases: the activators of plasminogen into plasmin, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, and the members of the matrix metalloproteinase (MMP) family. LRP-1 is responsible for clearing matrix proteinases, complexed or not with inhibitors. This paper attempts to summarize some aspects on the cellular and molecular bases of endocytic and signaling functions of LRP-1 that modulate extra- and pericellular levels of matrix proteinases. Nicolas Etique, Laurie Verzeaux, Stéphane Dedieu, and Hervé Emonard Copyright © 2013 Nicolas Etique et al. All rights reserved. Prostate Stem Cells in the Development of Benign Prostate Hyperplasia and Prostate Cancer: Emerging Role and Concepts Mon, 08 Jul 2013 08:33:29 +0000 Benign Prostate hyperplasia (BPH) and prostate cancer (PCa) are the most common prostatic disorders affecting elderly men. Multiple factors including hormonal imbalance, disruption of cell proliferation, apoptosis, chronic inflammation, and aging are thought to be responsible for the pathophysiology of these diseases. Both BPH and PCa are considered to be arisen from aberrant proliferation of prostate stem cells. Recent studies on BPH and PCa have provided significant evidence for the origin of these diseases from stem cells that share characteristics with normal prostate stem cells. Aberrant changes in prostate stem cell regulatory factors may contribute to the development of BPH or PCa. Understanding these regulatory factors may provide insight into the mechanisms that convert quiescent adult prostate cells into proliferating compartments and lead to BPH or carcinoma. Ultimately, the knowledge of the unique prostate stem or stem-like cells in the pathogenesis and development of hyperplasia will facilitate the development of new therapeutic targets for BPH and PCa. In this review, we address recent progress towards understanding the putative role and complexities of stem cells in the development of BPH and PCa. Akhilesh Prajapati, Sharad Gupta, Bhavesh Mistry, and Sarita Gupta Copyright © 2013 Akhilesh Prajapati et al. All rights reserved. The Exposure of Breast Cancer Cells to Fulvestrant and Tamoxifen Modulates Cell Migration Differently Tue, 02 Jul 2013 10:23:38 +0000 There is no doubt that there are increased benefits of hormonal therapy to breast cancer patients; however, current evidence suggests that estrogen receptor (ER) blockage using antiestrogens is associated with a small induction of invasiveness in vitro. The mechanism by which epithelial tumor cells escape from the primary tumor and colonize to a distant site is not entirely understood. This study investigates the effect of two selective antagonists of the ER, Fulvestrant (Fulv) and Tamoxifen (Tam), on the invasive ability of breast cancer cells. We found that 17β-estradiol (E2) demonstrated a protective role regarding cell migration and invasion. Fulv did not alter this effect while Tam stimulated active cell migration according to an increase in Snail and a decrease in E-cadherin protein expression. Furthermore, both tested agents increased expression of matrix metalloproteinases (MMPs) and enhanced invasive potential of breast cancer cells. These changes were in line with focal adhesion kinase (FAK) rearrangement. Our data indicate that the anti-estrogens counteracted the protective role of E2 concerning migration and invasion since their effect was not limited to antiproliferative events. Although Fulv caused a less aggressive result compared to Tam, the benefits of hormonal therapy concerning invasion and metastasis yet remain to be investigated. Dionysia Lymperatou, Efstathia Giannopoulou, Angelos K. Koutras, and Haralabos P. Kalofonos Copyright © 2013 Dionysia Lymperatou et al. All rights reserved. The Adaptive Nature of the Bone-Periodontal Ligament-Cementum Complex in a Ligature-Induced Periodontitis Rat Model Tue, 02 Jul 2013 09:35:21 +0000 The novel aspect of this study involves illustrating significant adaptation of a functionally loaded bone-PDL-cementum complex in a ligature-induced periodontitis rat model. Following 4, 8, and 15 days of ligation, proinflammatory cytokines (TNF-α and RANKL), a mineral resorption indicator (TRAP), and a cell migration and adhesion molecule for tissue regeneration (fibronectin) within the complex were localized and correlated with changes in PDL-space (functional space). At 4 days of ligation, the functional space of the distal complex was widened compared to controls and was positively correlated with an increased expression of TNF-α. At 8 and 15 days, the number of RANKL(+) cells decreased near the mesial alveolar bone crest (ABC) but increased at the distal ABC. TRAP(+) cells on both sides of the complex significantly increased at 8 days. A gradual change in fibronectin expression from the distal PDL-secondary cementum interfaces through precementum layers was observed when compared to increased and abrupt changes at the mesial PDL-cementum and PDL-bone interfaces in ligated and control groups. Based on our results, we hypothesize that compromised strain fields can be created in a diseased periodontium, which in response to prolonged function can significantly alter the original bone and apical cementum formations. Ji-Hyun Lee, Jeremy D. Lin, Justine I. Fong, Mark I. Ryder, and Sunita P. Ho Copyright © 2013 Ji-Hyun Lee et al. All rights reserved. Extracellular Matrix Degradation and Tissue Remodeling in Periprosthetic Loosening and Osteolysis: Focus on Matrix Metalloproteinases, Their Endogenous Tissue Inhibitors, and the Proteasome Wed, 19 Jun 2013 12:58:56 +0000 The leading complication of total joint replacement is periprosthetic osteolysis, which often results in aseptic loosening of the implant, leading to revision surgery. Extracellular matrix degradation and connective tissue remodeling around implants have been considered as major biological events in the periprosthetic loosening. Critical mediators of wear particle-induced inflammatory osteolysis released by periprosthetic synovial cells (mainly macrophages) are inflammatory cytokines, chemokines, and proteolytic enzymes, mainly matrix metalloproteinases (MMPs). Numerous studies reveal a strong interdependence of MMP expression and activity with the molecular mechanisms that control the composition and turnover of periprosthetic matrices. MMPs can either actively modulate or be modulated by the molecular mechanisms that determine the debris-induced remodeling of the periprosthetic microenvironment. In the present study, the molecular mechanisms that control the composition, turnover, and activity of matrix macromolecules within the periprosthetic microenvironment exposed to wear debris are summarized and presented. Special emphasis is given to MMPs and their endogenous tissue inhibitors (TIMPs), as well as to the proteasome pathway, which appears to be an elegant molecular regulator of specific matrix macromolecules (including specific MMPs and TIMPs). Furthermore, strong rationale for potential clinical applications of the described molecular mechanisms to the treatment of periprosthetic loosening and osteolysis is provided. Spyros A. Syggelos, Alexios J. Aletras, Ioanna Smirlaki, and Spyros S. Skandalis Copyright © 2013 Spyros A. Syggelos et al. All rights reserved. Identification, Selection, and Enrichment of Cardiomyocyte Precursors Tue, 18 Jun 2013 13:50:58 +0000 The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. The main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5′-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP+. These GFP+ cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs), which can then be differentiated terminally for cell therapy and tissue engineering. Bianca Ferrarini Zanetti, Walter José Gomes, and Sang Won Han Copyright © 2013 Bianca Ferrarini Zanetti et al. All rights reserved. Regulatory Systems in Bone Marrow for Hematopoietic Stem/Progenitor Cells Mobilization and Homing Mon, 17 Jun 2013 17:12:48 +0000 Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM) and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs) cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a) the role of different factors, such as stromal cell derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and vascular cell adhesion molecule-1 (VCAM-1), among other ligands; (b) the stem cell count in peripheral blood and BM and influential factors; (c) the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d) the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases. P. Alvarez, E. Carrillo, C. Vélez, F. Hita-Contreras, A. Martínez-Amat, F. Rodríguez-Serrano, H. Boulaiz, R. Ortiz, C. Melguizo, J. Prados, and A. Aránega Copyright © 2013 P. Alvarez et al. All rights reserved. Expression of Syndecan-4 and Correlation with Metastatic Potential in Testicular Germ Cell Tumours Sat, 15 Jun 2013 16:04:20 +0000 Although syndecan-4 is implicated in cancer progression, there is no information for its role in testicular germ cell tumours (TGCTs). Thus, we examined the expression of syndecan-4 in patients with TGCTs and its correlation with the clinicopathological findings. Immunohistochemical staining in 71 tissue specimens and mRNA analysis revealed significant overexpression of syndecan-4 in TGCTs. In seminomas, high percentage of tumour cells exhibited membranous and/or cytoplasmic staining for syndecan-4 in all cases. Stromal staining for syndecan-4 was found in seminomas and it was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Reduced tumour cell associated staining for syndecan-4 was observed in nonseminomatous germ cell tumours (NSGCTs) compared to seminomas. This loss of syndecan-4 was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Stromal staining for syndecan-4 in NSGCTs did not correlate with any of the clinicopathological variables. The stromal expression of syndecan-4 in TGCTs was correlated with microvessel density (). Our results indicate that syndecan-4 is differentially expressed in seminomas and NSGCTs and might be a useful marker. Stromal staining in seminomas and reduced levels of syndecan-4 in tumour cells in NSGCTs are related to metastatic potential, whereas stromal staining in TGCTs is associated with neovascularization. Vassiliki T. Labropoulou, Spyros S. Skandalis, Panagiota Ravazoula, Petros Perimenis, Nikos K. Karamanos, Haralabos P. Kalofonos, and Achilleas D. Theocharis Copyright © 2013 Vassiliki T. Labropoulou et al. All rights reserved. Urine Bikunin as a Marker of Renal Impairment in Fabry's Disease Wed, 12 Jun 2013 09:32:52 +0000 Fabry’s disease is a rare lysosomal storage disorder caused by the deficiency of α-galactosidase A that leads to the accumulation of neutral glycosphingolipids in many organs including kidney, heart, and brain. Since end-stage renal disease represents a major complication of this pathology, the aim of the present work was to evaluate if urinary proteoglycan/glycosaminoglycan excretion could represent a useful marker for monitoring kidney function in these patients at high risk. Quali-quantitative and structural analyses were conducted on plasma and urine from 24 Fabry’s patients and 43 control subjects. Patients were sorted for presence and degree of renal impairment (proteinuria/renal damage). Results showed that levels of urine bikunin, also known as urinary trypsin inhibitor (UTI), are significantly higher in patients with renal impairment than in controls. In this respect, no differences were evidenced in plasma chondroitin sulfate isomers level/structure indicating a likely direct kidney involvement. Noteworthy, urine bikunin levels are higher in patients since early symptoms of renal impairment occur (proteinuria). Overall, our findings suggest that urine bikunin level, as well as proteinuria, could represent a useful parameter for monitoring renal function in those patients that do not present any symptoms of renal insufficiency. Antonio Junior Lepedda, Laura Fancellu, Elisabetta Zinellu, Pierina De Muro, Gabriele Nieddu, Giovanni Andrea Deiana, Piera Canu, Daniela Concolino, Simona Sestito, Marilena Formato, and Gianpietro Sechi Copyright © 2013 Antonio Junior Lepedda et al. All rights reserved. An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-1 in Salivary Gland Epithelial and Mesenchymal Differentiation Sun, 09 Jun 2013 09:38:37 +0000 Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-1 on salivary gland cell differentiation. TGF-1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-R1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF- signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. Kajohnkiart Janebodin, Worakanya Buranaphatthana, Nicholas Ieronimakis, Aislinn L. Hays, and Morayma Reyes Copyright © 2013 Kajohnkiart Janebodin et al. All rights reserved. Expression and Function of NUMB in Odontogenesis Thu, 06 Jun 2013 16:48:23 +0000 NUMB is a multifunctional protein implicated to function in self-renewal and differentiation of progenitors in several tissues. To characterize the transcripts and to analyze the expression pattern of NUMB in odontogenesis, we isolated 2 full-length clones for NUMB from mouse dental pulp mRNA. One novel sequence contained 200 bp insertion in the phosphotyrosine binding domain (PTB). Confocal microscopy analysis showed strong NUMB expression in human dental pulp stem cells (hDPSC) and preameloblasts. Western blot analysis indicated that NUMB isoforms were differentially expressed in various dental tissues. Immunohistochemical analysis showed that in postnatal mouse tooth germs, NUMB was differentially expressed in the preameloblasts, odontoblasts, cervical loop region, and in the dental pulp stem cells during development. Interestingly, overexpression of NUMB in HAT-7, a preameloblast cell line, had dramatic antagonizing effects on the protein expression level of activated Notch 1. Further analysis of the Notch signaling pathway showed that NUMB significantly downregulates sonic hedgehog (Shh) expression in preameloblasts. Therefore, we propose that NUMB maintains ameloblast progenitor phenotype at the cervical loop by downregulating the activated Notch1 protein and thereby inhibiting the mRNA expression of Shh. Haitao Li, Amsaveni Ramachandran, Qi Gao, Sriram Ravindran, Yiqiang Song, Carla Evans, and Anne George Copyright © 2013 Haitao Li et al. All rights reserved. Mineral and Matrix Changes in Brtl/+ Teeth Provide Insights into Mineralization Mechanisms Wed, 29 May 2013 16:28:31 +0000 The Brtl/+ mouse is a knock-in model for osteogenesis imperfecta type IV in which a Gly349Cys substitution was introduced into one COL1A1 allele. To gain insight into the changes in dentin structure and mineral composition in these transgenic mice, the objective of this study was to use microcomputed tomography (micro-CT), scanning electron microscopy (SEM), and Fourier transform infrared imaging (FTIRI) to analyze these structures at 2 and 6 months of age. Results, consistent with the dental phenotype in humans with type IV OI, showed decreased molar volume and reduced mineralized tissue volume in the teeth without changes in enamel properties. Increased acid phosphate content was noted at 2 and 6 months by FTIRI, and a trend towards altered collagen structure was noted at 2 but not 6 months in the Brtl/+ teeth. The increase in acid phosphate content suggests a delay in the mineralization process, most likely associated with the defect in the collagen structure. It appears that in the Brtl/+ teeth slow maturation of the mineralized structures allows correction of altered mineral content and acid phosphate distribution. Adele L. Boskey, Kostas Verdelis, Lyudmila Spevak, Lyudmila Lukashova, Elia Beniash, Xu Yang, Wayne A. Cabral, and Joan C. Marini Copyright © 2013 Adele L. Boskey et al. All rights reserved. New Therapeutics in Promoting and Modulating Mandibular Growth in Cases with Mandibular Hypoplasia Wed, 29 May 2013 15:17:54 +0000 Children with mandibular growth deficiency may develop airway obstruction. The standard treatment of severe airway obstruction involves invasive procedures such as tracheostomy. Mandibular distraction osteogenesis has been proposed in neonates with mandibular deficiency as a treatment option to avoid tracheostomy procedure later in life. Both tracheostomy and distraction osteogenesis procedures suffer from substantial shortcomings including scarring, unpredictability, and surgical complications. Forward jaw positioning appliances have been also used to enhance mandible growth. However, the effectiveness of these appliances is limited and lacks predictability. Current and future approaches to enhance mandibular growth, both experimental and clinical trials, and their effectiveness are presented and discussed. Tarek El-Bialy and Adel Alhadlaq Copyright © 2013 Tarek El-Bialy and Adel Alhadlaq. All rights reserved. Regulation of Tissue Fibrosis by the Biomechanical Environment Tue, 28 May 2013 17:45:47 +0000 The biomechanical environment plays a fundamental role in embryonic development, tissue maintenance, and pathogenesis. Mechanical forces play particularly important roles in the regulation of connective tissues including not only bone and cartilage but also the interstitial tissues of most organs. In vivo studies have correlated changes in mechanical load to modulation of the extracellular matrix and have indicated that increased mechanical force contributes to the enhanced expression and deposition of extracellular matrix components or fibrosis. Pathological fibrosis contributes to dysfunction of many organ systems. A variety of in vitro models have been utilized to evaluate the effects of mechanical force on extracellular matrix-producing cells. In general, application of mechanical stretch, fluid flow, and compression results in increased expression of extracellular matrix components. More recent studies have indicated that tissue rigidity also provides profibrotic signals to cells. The mechanisms whereby cells detect mechanical signals and transduce them into biochemical responses have received considerable attention. Cell surface receptors for extracellular matrix components and intracellular signaling pathways are instrumental in the mechanotransduction process. Understanding how mechanical signals are transmitted from the microenvironment will identify novel therapeutic targets for fibrosis and other pathological conditions. Wayne Carver and Edie C. Goldsmith Copyright © 2013 Wayne Carver and Edie C. Goldsmith. All rights reserved. An Immunohistochemistry Study of Sox9, Runx2, and Osterix Expression in the Mandibular Cartilages of Newborn Mouse Thu, 16 May 2013 15:42:12 +0000 The purpose of this study is to investigate the spacial expression pattern and functional significance of three key transcription factors related to bone and cartilage formation, namely, Sox9, Runx2, and Osterix in cartilages during the late development of mouse mandible. Immunohistochemical examinations of Sox9, Runx2, and Osterix were conducted in the mandibular cartilages of the 15 neonatal C57BL/6N mice. In secondary cartilages, both Sox9 and Runx2 were weakly expressed in the polymorphic cell zone, strongly expressed in the flattened cell zone and throughout the entire hypertrophic cell zone. Similarly, both transcriptional factors were weakly expressed in the uncalcified Meckel’s cartilage while strongly expressed in the rostral cartilage. Meanwhile, Osterix was at an extremely low level in cells of the flattened cell zone and the upper hypertrophic cell zone in secondary cartilages. Surprisingly, Osterix was intensely expressed in hypertrophic chondrocytes in the center of the uncalcified Meckel’s cartilage while moderately expressed in part of hypertrophic chondrocytes in the rostral process. Consequently, it is suggested that Sox9 is a main and unique positive regulator in the hypertrophic differentiation process of mandibular secondary cartilages, in addition to Runx2. Furthermore, Osterix is likely responsible for phenotypic conversion of Meckel’s chondrocytes during its degeneration. Hong Zhang, Xiaopeng Zhao, Zhiguang Zhang, Weiwei Chen, and Xinli Zhang Copyright © 2013 Hong Zhang et al. All rights reserved. Craniosynostosis-Associated Fgfr2C342Y Mutant Bone Marrow Stromal Cells Exhibit Cell Autonomous Abnormalities in Osteoblast Differentiation and Bone Formation Thu, 09 May 2013 11:43:26 +0000 We recently reported that cranial bones of craniosynostotic mice are diminished in density when compared to those of wild type mice, and that cranial bone cells isolated from the mutant mice exhibit inhibited late stage osteoblast differentiation. To provide further support for the idea that craniosynostosis-associated Fgfr mutations lead to cell autonomous defects in osteoblast differentiation and mineralized tissue formation, here we tested bone marrow stromal cells isolated from mice for their ability to differentiate into osteoblasts. Additionally, to determine if the low bone mass phenotype of Crouzon syndrome includes the appendicular skeleton, long bones were assessed by micro CT. cells showed increased osteoblastic gene expression during early osteoblastic differentiation but decreased expression of alkaline phosphatase mRNA and enzyme activity, and decreased mineralization during later stages of differentiation, when cultured under 2D in vitro conditions. Cells isolated from mice also formed less bone when allowed to differentiate in a 3D matrix in vivo. Cortical bone parameters were diminished in long bones of mice. These results demonstrate that marrow stromal cells of mice have an autonomous defect in osteoblast differentiation and bone mineralization, and that the mutation influences both the axial and appendicular skeletons. J. Liu, T.-G. Kwon, H. K. Nam, and N. E. Hatch Copyright © 2013 J. Liu et al. All rights reserved. Cardiac Fibroblast-Derived Extracellular Matrix (Biomatrix) as a Model for the Studies of Cardiac Primitive Cell Biological Properties in Normal and Pathological Adult Human Heart Thu, 02 May 2013 11:53:49 +0000 Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration. Clotilde Castaldo, Franca Di Meglio, Rita Miraglia, Anna Maria Sacco, Veronica Romano, Ciro Bancone, Alessandro Della Corte, Stefania Montagnani, and Daria Nurzynska Copyright © 2013 Clotilde Castaldo et al. All rights reserved. Stem Cells in Reproductive Tissues: From the Basics to Clinics Sun, 07 Apr 2013 14:23:32 +0000 Irma Virant-Klun, Thomas Skutella, Deepa Bhartiya, and Xuan Jin Copyright © 2013 Irma Virant-Klun et al. All rights reserved. Isolation, Characterization, and Transduction of Endometrial Decidual Tissue Multipotent Mesenchymal Stromal/Stem Cells from Menstrual Blood Sun, 31 Mar 2013 10:11:06 +0000 Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expanded in vitro as fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications. Filippo Rossignoli, Anna Caselli, Giulia Grisendi, Serena Piccinno, Jorge S. Burns, Alba Murgia, Elena Veronesi, Pietro Loschi, Cristina Masini, Pierfranco Conte, Paolo Paolucci, Edwin M. Horwiz, and Massimo Dominici Copyright © 2013 Filippo Rossignoli et al. All rights reserved. Platelet-Rich Fibrin Promotes Periodontal Regeneration and Enhances Alveolar Bone Augmentation Tue, 26 Mar 2013 16:42:13 +0000 In the present study we have determined the suitability of platelet-rich fibrin (PRF) as a complex scaffold for periodontal tissue regeneration. Replacing PRF with its major component fibrin increased mineralization in alveolar bone progenitors when compared to periodontal progenitors, suggesting that fibrin played a substantial role in PRF-induced osteogenic lineage differentiation. Moreover, there was a 3.6-fold increase in the early osteoblast transcription factor RUNX2 and a 3.1-fold reduction of the mineralization inhibitor MGP as a result of PRF application in alveolar bone progenitors, a trend not observed in periodontal progenitors. Subcutaneous implantation studies revealed that PRF readily integrated with surrounding tissues and was partially replaced with collagen fibers 2 weeks after implantation. Finally, clinical pilot studies in human patients documented an approximately 5 mm elevation of alveolar bone height in tandem with oral mucosal wound healing. Together, these studies suggest that PRF enhances osteogenic lineage differentiation of alveolar bone progenitors more than of periodontal progenitors by augmenting osteoblast differentiation, RUNX2 expression, and mineralized nodule formation via its principal component fibrin. They also document that PRF functions as a complex regenerative scaffold promoting both tissue-specific alveolar bone augmentation and surrounding periodontal soft tissue regeneration via progenitor-specific mechanisms. Qi Li, Shuang Pan, Smit J. Dangaria, Gokul Gopinathan, Antonia Kolokythas, Shunli Chu, Yajun Geng, Yanmin Zhou, and Xianghong Luan Copyright © 2013 Qi Li et al. All rights reserved. HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors Tue, 19 Mar 2013 15:27:59 +0000 Introduction. JEG3 is a choriocarcinoma—and HTR8/SVneo a transformed extravillous trophoblast—cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo’s self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of “stemness-” associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum. Maja Weber, Ilka Knoefler, Ekkehard Schleussner, Udo R. Markert, and Justine S. Fitzgerald Copyright © 2013 Maja Weber et al. All rights reserved. Expression of Pluripotency and Oocyte-Related Genes in Single Putative Stem Cells from Human Adult Ovarian Surface Epithelium Cultured In Vitro in the Presence of Follicular Fluid Thu, 28 Feb 2013 10:57:46 +0000 The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The majority of primitive oocyte-like cells was mononuclear and expressed several genes related to pluripotency and oocytes, including genes related to meiosis, although they did not express some important oocyte-specific genes. Our work reveals the presence of putative stem cells in the ovarian surface epithelium of women with premature ovarian failure. Irma Virant-Klun, Thomas Skutella, Mikael Kubista, Andrej Vogler, Jasna Sinkovec, and Helena Meden-Vrtovec Copyright © 2013 Irma Virant-Klun et al. All rights reserved. Expression of N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis Wed, 20 Feb 2013 15:20:34 +0000 Chondroitin sulfate (CS) containing E-disaccharide units, glucuronic acid-N-acetylgalactosamine(4, 6-O-disulfate), at surfaces of tumor cells plays a key role in tumor metastasis. However, the molecular mechanism of the metastasis involving the CS chain-containing E-units is not fully understood. In this study, to clarify the role of E-units in the metastasis and to search for potential molecular targets for anticancer drugs, the isolation and characterization of Lewis lung carcinoma (LLC) cells stably downregulated by the knockdown for the gene encoding N-acetylgalactosamine 4-O-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which is responsible for the formation of E-units in CS chains, were performed. Knockdown of GalNAc4S-6ST in LLC cells resulted in a reduction in the proportion of E-units, in adhesiveness to extracellular matrix adhesion molecules and in proliferation in vitro. Furthermore, the stable downregulation of GalNAc4S-6ST expression in LLC cells markedly inhibited the colonization of the lungs by inoculated LLC cells and invasive capacity of LLC cells. These results provide clear evidence that CS chain-containing E-units and/or GalNAc4S-6ST play a crucial role in pulmonary metastasis at least through the increased adhesion and the invasive capacity of LLC cells and also provides insights into future drug targets for anticancer treatment. Shuji Mizumoto, Moto Watanabe, Shuhei Yamada, and Kazuyuki Sugahara Copyright © 2013 Shuji Mizumoto et al. All rights reserved. Gene Expression Profiling of Human Oocytes Developed and Matured In Vivo or In Vitro Wed, 20 Feb 2013 07:58:25 +0000 The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in the in vitro fertilization programme is very limited. The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in the in vitro fertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocyte in vitro maturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developed in vitro from human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future. Irma Virant-Klun, Katja Knez, Tomaz Tomazevic, and Thomas Skutella Copyright © 2013 Irma Virant-Klun et al. All rights reserved. Isolation of Small SSEA-4-Positive Putative Stem Cells from the Ovarian Surface Epithelium of Adult Human Ovaries by Two Different Methods Mon, 18 Feb 2013 15:26:50 +0000 The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched. Irma Virant-Klun, Thomas Skutella, Matjaz Hren, Kristina Gruden, Branko Cvjeticanin, Andrej Vogler, and Jasna Sinkovec Copyright © 2013 Irma Virant-Klun et al. All rights reserved. Recovery of Fertility in Azoospermia Rats after Injection of Adipose-Tissue-Derived Mesenchymal Stem Cells: The Sperm Generation Mon, 18 Feb 2013 14:28:46 +0000 The recent reports on the treatment of azoospermia patients, in which spermatozoa could not be traced in their testes, are focused more on the potential use of adult stem cells, like mesenchymal stem cells (MSCs). The aim of this study was to demonstrate the potential use of MSCs derived from adipose tissue in the treatment of azoospermia using rat disease models. After busulfan application, the rats () were injected with the GFP+ MSCs into left rete testes. After 12 weeks, the testes with cell injection (right testes) were compared to control (left testes) after dimensional and immunohistochemical analyses. Testes treated with MSCs appeared morphologically normal, but they were atrophic in rats without stem cell treatment, in which the seminiferous tubules were empty. Spermatogenesis was detected, not in every but in some tubules of cell-treated testes. GFP+/VASA+ and GFP+/SCP1+ cells in testes indicated the transdifferentiation of MSCs into spermatogenetic cells in the appropriate microenvironment. Rats with cell treatment were mated to show the full recovery of spermatogenesis, and continuous generations were obtained. The expression of GFP was detected in the mesenchymal stem cells derived from adipose tissue and bone marrow and also in the sperms of offspring. In conclusion, MSCs might be studied for the same purpose in humans in future. Cihangir Cakici, Bugra Buyrukcu, Gokhan Duruksu, Ahmet Hakan Haliloglu, Ayca Aksoy, Ayca Isık, Orhan Uludag, Huseyin Ustun, Cansu Subası, and Erdal Karaoz Copyright © 2013 Cihangir Cakici et al. All rights reserved. Very Small Embryonic-Like Stem Cells: Implications in Reproductive Biology Wed, 13 Feb 2013 11:09:27 +0000 The most primitive germ cells in adult mammalian testis are the spermatogonial stem cells (SSCs) whereas primordial follicles (PFs) are considered the fundamental functional unit in ovary. However, this central dogma has recently been modified with the identification of a novel population of very small embryonic-like stem cells (VSELs) in the adult mammalian gonads. These stem cells are more primitive to SSCs and are also implicated during postnatal ovarian neo-oogenesis and primordial follicle assembly. VSELs are pluripotent in nature and characterized by nuclear Oct-4A, cell surface SSEA-4, and other pluripotent markers like Nanog, Sox2, and TERT. VSELs are considered to be the descendants of epiblast stem cells and possibly the primordial germ cells that persist into adulthood and undergo asymmetric cell division to replenish the gonadal germ cells throughout life. Elucidation of their role during infertility, endometrial repair, superovulation, and pathogenesis of various reproductive diseases like PCOS, endometriosis, cancer, and so on needs to be addressed. Hence, a detailed review of current understanding of VSEL biology is pertinent, which will hopefully open up new avenues for research to better understand various reproductive processes and cancers. It will also be relevant for future regenerative medicine, translational research, and clinical applications in human reproduction. Deepa Bhartiya, Sreepoorna Unni, Seema Parte, and Sandhya Anand Copyright © 2013 Deepa Bhartiya et al. All rights reserved. Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos Tue, 12 Feb 2013 11:18:12 +0000 Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage. Jared M. Campbell, Michelle Lane, Ivan Vassiliev, and Mark B. Nottle Copyright © 2013 Jared M. Campbell et al. All rights reserved. Heparanase Localization during Palatogenesis in Mice Tue, 12 Feb 2013 08:46:39 +0000 Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES. Azumi Hirata, Kentaro Katayama, Takehito Tsuji, Nagato Natsume, Toshio Sugahara, Yuichi Koga, Kazufumi Takano, Yoshinori Otsuki, and Hiroaki Nakamura Copyright © 2013 Azumi Hirata et al. All rights reserved. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells Tue, 05 Feb 2013 16:26:03 +0000 Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. Claudia González, Marisol Espinosa, María Trinidad Sánchez, Karla Droguett, Mariana Ríos, Ximena Fonseca, and Manuel Villalón Copyright © 2012 Claudia González et al. All rights reserved. Ovarian Cancer Stem Cells: A New Target for Cancer Therapy Wed, 30 Jan 2013 15:19:30 +0000 Ovarian cancer is a highly lethal disease among all gynecologic malignancies and is the fifth leading cause of cancer-related death in women. Although the standard combination of surgery and chemotherapy was initially effective in patients with ovarian cancer, disease relapse commonly occurred due to the generation of chemoresistance. It has been reported that cancer stem cells (CSCs) are involved in drug resistance and cancer recurrence. Over the past decades, increasing studies have been done to identify CSCs from human ovarian cancer cells. The present paper will summarize different investigations on ovarian CSCs, including isolation, mechanisms of chemoresistance, and therapeutic approaches. Although there are still numerous challenges to translate basic research to clinical applications, understanding the molecular details of CSCs is essential for developing effective strategies to prevent ovarian cancer and its recurrence. Qinglei Zhan, Chunmei Wang, and Saiming Ngai Copyright © 2013 Qinglei Zhan et al. All rights reserved. In Vitro Large Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells Wed, 30 Jan 2013 08:54:43 +0000 In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive β-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion. Jiafei Xi, Yanhua Li, Ruoyong Wang, Yunfang Wang, Xue Nan, Lijuan He, Peng Zhang, Lin Chen, Wen Yue, and Xuetao Pei Copyright © 2013 Jiafei Xi et al. All rights reserved. Establishment of a Lentiviral Vector Encoding Human HGF and the Infection of Human ADSCs Sun, 20 Jan 2013 09:43:38 +0000 The delivery of adipose-derived stem cells (ADSCs) for promoting tissue repair has become a potential new therapy, while hepatocyte growth factor (HGF) is an important growth factor with angiogenic, anti-fibrotic, and anti-inflammatory benefits. In this paper, hADSCs were separated, cultured and identified based on the expression of cell surface antigens and multiple differentiation potential. We successfully generated a lentiviral vector encoding human HGF, infected hADSCs with this vector and examined the protein expression pattern. Finally we found that the hHGF lentiviral vector was successfully generated, and the lentiviral vector was able to safely infect hADSCs with high infection efficiency, thereby producing cells that overexpressed hHGF, which may provide a new strategy for the treatment of ischemic heart disease (IHD) and other ischemic diseases. Xiaoyu Zhu, Lei Xu, Xin Liu, Jingsheng Wu, Weibo Zhu, Xiaoyan Cai, and Zimin Sun Copyright © 2012 Xiaoyu Zhu et al. All rights reserved. Cell Transplantation for Spinal Cord Injury: A Systematic Review Tue, 15 Jan 2013 14:06:57 +0000 Cell transplantation, as a therapeutic intervention for spinal cord injury (SCI), has been extensively studied by researchers in recent years. A number of different kinds of stem cells, neural progenitors, and glial cells have been tested in basic research, and most have been excluded from clinical studies because of a variety of reasons, including safety and efficacy. The signaling pathways, protein interactions, cellular behavior, and the differentiated fates of experimental cells have been studied in vitro in detail. Furthermore, the survival, proliferation, differentiation, and effects on promoting functional recovery of transplanted cells have also been examined in different animal SCI models. However, despite significant progress, a “bench to bedside” gap still exists. In this paper, we comprehensively cover publications in the field from the last years. The most commonly utilized cell lineages were covered in this paper and specific areas covered include survival of grafted cells, axonal regeneration and remyelination, sensory and motor functional recovery, and electrophysiological improvements. Finally we also review the literature on the in vivo tracking techniques for transplanted cells. Jun Li and Guilherme Lepski Copyright © 2013 Jun Li and Guilherme Lepski. All rights reserved. Developing a Clinical-Grade Cryopreservation Protocol for Human Testicular Tissue and Cells Mon, 14 Jan 2013 08:05:30 +0000 Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients. Jason Pacchiarotti, Thomas Ramos, Kyle Howerton, Scott Greilach, Karina Zaragoza, Marnie Olmstead, and Fariborz Izadyar Copyright © 2013 Jason Pacchiarotti et al. All rights reserved. Restoring Fertility in Sterile Childhood Cancer Survivors by Autotransplanting Spermatogonial Stem Cells: Are We There Yet? Thu, 03 Jan 2013 11:35:50 +0000 Current cancer treatment regimens do not only target tumor cells, but can also have devastating effects on the spermatogonial stem cell pool, resulting in a lack of functional gametes and hence sterility. In adult men, fertility can be preserved prior to cancer treatment by cryopreservation of ejaculated or surgically retrieved spermatozoa, but this is not an option for prepubertal boys since spermatogenesis does not commence until puberty. Cryopreservation of a testicular biopsy taken before initiation of cancer treatment, followed by in vitro propagation of spermatogonial stem cells and subsequent autotransplantation of these stem cells after cancer treatment, has been suggested as a way to preserve and restore fertility in childhood cancer survivors. This strategy, known as spermatogonial stem cell transplantation, has been successful in mice and other model systems, but has not yet been applied in humans. Although recent progress has brought clinical application of spermatogonial stem cell autotransplantation in closer range, there are still a number of important issues to address. In this paper, we describe the state of the art of spermatogonial stem cell transplantation and outline the hurdles that need to be overcome before clinical implementation. Robert B. Struijk, Callista L. Mulder, Fulco van der Veen, Ans M. M. van Pelt, and Sjoerd Repping Copyright © 2013 Robert B. Struijk et al. All rights reserved. Propagation of Adult SSCs: From Mouse to Human Tue, 01 Jan 2013 07:42:17 +0000 Adult spermatogonial stem cells (SSCs) represent a distinctive source of stem cells in mammals for several reasons. First, by giving rise to spermatogenesis, SSCs are responsible for the propagation of a father’s genetic material. As such, autologous SSCs have been considered for treatment of infertility and other purposes, including correction of inherited disorders. Second, adult spermatogonia can spontaneously produce embryonic-like stem cells in vitro, which could be used as an alternative for therapeutic, diagnostic, or drug discovery strategies for humans. Therefore, an increasing urgency is driving efforts to understand the biology of SSCs and improve techniques to manipulate them in vitro as a prerequisite to achieve the aforementioned goals. The characterization of adult SSCs also requires reproducible methods to isolate and maintain them in long-term culture. Herein, we describe recent major advances and challenges in propagation of adult SSCs from mice and humans during the past few years, including the use of unique cell surface markers and defined cultured conditions. Laura A. Martin and Marco Seandel Copyright © 2013 Laura A. Martin and Marco Seandel. All rights reserved. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes Sun, 30 Dec 2012 16:02:45 +0000 The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. Fernando Navarro-Garcia, Antonio Serapio-Palacios, Paul Ugalde-Silva, Gabriela Tapia-Pastrana, and Lucia Chavez-Dueñas Copyright © 2013 Fernando Navarro-Garcia et al. All rights reserved. Endothelial Progenitor Cell Dysfunction in Cardiovascular Diseases: Role of Reactive Oxygen Species and Inflammation Sun, 30 Dec 2012 14:36:17 +0000 Endothelial progenitor cells (EPCs) move towards injured endothelium or inflamed tissues and incorporate into foci of neovascularisation, thereby improving blood flow and tissue repair. Patients with cardiovascular diseases have been shown to exhibit reduced EPC number and function. It has become increasingly apparent that these changes may be effected in response to enhanced oxidative stress, possibly as a result of systemic and localised inflammatory responses. The interplay between inflammation and oxidative stress affects the initiation, progression, and complications of cardiovascular diseases. Recent studies suggest that inflammation and oxidative stress modulate EPC bioactivity. Clinical medications with anti-inflammatory and antioxidant properties, such as statins, thiazolidinediones, angiotensin II receptor 1 blockers, and angiotensin-converting enzyme inhibitors, are currently administered to patients with cardiovascular diseases. These medications appear to exert beneficial effects on EPC biology. This review focuses on EPC biology and explores the links between oxidative stress, inflammation, and development of cardiovascular diseases. Chih-Pei Lin, Feng-Yen Lin, Po-Hsun Huang, Yuh-Lien Chen, Wen-Chi Chen, Huey-Yi Chen, Yu-Chuen Huang, Wen-Ling Liao, Huey-Chun Huang, Po-Len Liu, and Yung-Hsiang Chen Copyright © 2013 Chih-Pei Lin et al. All rights reserved. Differentiation of Induced Pluripotent Stem Cells into Male Germ Cells In Vitro through Embryoid Body Formation and Retinoic Acid or Testosterone Induction Sun, 30 Dec 2012 14:34:39 +0000 Generation of germ cells from pluripotent stem cells in vitro could have great application for treating infertility and provides an excellent model for uncovering molecular mechanisms controlling gametogenesis. In this study, we explored the differentiation potential of mouse induced pluripotent stem (iPS) cells towards male germ cells. Embryoid body formation and retinoic acid/testosterone induction were applied to promote differentiation of mouse iPS cells into male germ cells in vitro. Quantitative RT-PCR and immunoflourescence were performed to characterize the iPS cell differentiation process, and notably there were different temporal expression profiles of male germ cell-associated genes. The expression of proteins, including MVH, CDH1, and SCP3, was remarkably increased. mRNA expression of Stra8, Odf2, Act, and Prm1 was upregulated in iPS cells by retinoic acid or testosterone induction, whereas Oct-4 transcription was reduced in these cells compared to the controls. Hormones were also measured in the EB medium. DNA content analysis by flow cytometry revealed that iPS cells could differentiate into haploid cells through retinoic acid or testosterone treatment. Collectively, our results suggest that mouse iPS cells possess the potency to differentiate into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction. Peng Li, Hongliang Hu, Shi Yang, Ruhui Tian, Zhenzhen Zhang, Wei Zhang, Meng Ma, Yong Zhu, Xizhi Guo, Yiran Huang, Zuping He, and Zheng Li Copyright © 2013 Peng Li et al. All rights reserved. Direct Effect of Chenodeoxycholic Acid on Differentiation of Mouse Embryonic Stem Cells Cultured under Feeder-Free Culture Conditions Sat, 29 Dec 2012 13:44:28 +0000 Chenodeoxycholic acid (CDCA), a farnesoid X receptor (FXR) ligand, is a member of the nuclear receptor family and is probably involved in regulating the cellular activities of embryonic stem (ES) cells. Recently, although it was reported that the FXR ligand can mediate differentiation, apoptosis, and/or growth arrest in several cell types, it is still not well known how CDCA mediates effects in ES cells. Therefore, we investigated the direct effect of CDCA on mES cells. Feeder-free mES cells were treated in a dose-dependent manner with CDCA (50, 100, and 200 μM) for 72 h, and then a 100 μM CDCA treatment was performed for an additional 72 h. We analyzed the morphology, cell growth, cell characteristics, immunocytochemistry, and RT-PCR. In CDCA-treated cells, we observed the disappearance of pluripotent stem cell markers including alkaline phosphatase, Oct4, and Nanog and a time- and dose-dependent increase in expression of nestin, PAX6, and α-smooth muscle actin, but not α-fetoprotein. The 100 μM CDCA-treated cells in their second passage continued this differentiation pattern similar to those in the controls. In conclusion, these results suggest that CDCA can guide mES cells by an FXR-independent pathway to differentiate into ectoderm and/or mesoderm, but not endoderm. Soon-Jung Park, Seul-Bi Lee, Dong-Sup Lee, Young-Joon Ryu, Gene Lee, and Jaejin Cho Copyright © 2013 Soon-Jung Park et al. All rights reserved. A DSPP Mutation Causing Dentinogenesis Imperfecta and Characterization of the Mutational Effect Thu, 27 Dec 2012 18:16:31 +0000 Mutations in the DSPP gene have been identified in nonsyndromic hereditary dentin defects, but the genotype-phenotype correlations are not fully understood. Recently, it has been demonstrated that the mutations of DSPP affecting the IPV leader sequence result in mutant DSPP retention in rough endoplasmic reticulum (ER). In this study, we identified a Korean family with dentinogenesis imperfecta type III. To identify the disease causing mutation in this family, we performed mutational analysis based on candidate gene sequencing. Exons and exon-intron boundaries of DSPP gene were sequenced, and the effects of the identified mutation on the pre-mRNA splicing and protein secretion were investigated. Candidate gene sequencing revealed a mutation (c.50C > T, p.P17L) in exon 2 of the DSPP gene. The splicing assay showed that the mutation did not influence pre-mRNA splicing. However, the mutation interfered with protein secretion and resulted in the mutant protein remaining largely in the ER. These results suggest that the mutation affects ER-to-Golgi apparatus export and results in the reduction of secreted DSPP and ER overload. This may induce cell stress and damage processing and/or transport of dentin matrix proteins or other critical proteins. Sook-Kyung Lee, Kyung-Eun Lee, Su Jeong Song, Hong-Keun Hyun, Sang-Hoon Lee, and Jung-Wook Kim Copyright © 2013 Sook-Kyung Lee et al. All rights reserved. Cells with Stem Cell Characteristics in Somatic Compartments of the Ovary Thu, 27 Dec 2012 14:18:58 +0000 Antral follicular growth in the ovary is characterized by rapid expansion of granulosa cells accompanied by a rising complexity of their functionality. Within two weeks the number of human granulosa cells increases from less than 500,000 to more than 50 millions cells per follicle and differentiates into groups of cells with a variety of specialized functions involved in steroidogenesis, nursing the oocyte, and forming a functional syncitium. Both the rapid proliferation and different specialized functions of the granulosa cells can only be explained through the involvement of stem cells. However, luteinizing granulosa cells were believed to be terminally differentiated cells. Only recently, stem and progenitor cells with FSH-receptor activity were identified in populations of luteinizing granulosa cells obtained during oocyte collected for assisted reproduction. In the presence of the leukaemia-inhibiting factor (LIF), it was possible to culture a subpopulation of the luteinizing granulosa cells over prolonged time periods. Furthermore, when embedded in a matrix consisting of collagen type I, these cells continued to express the FSH receptor over prolonged time periods, developed globular formations that surrogated as follicle-like structures, providing a promising tool for reproductive biology. Katarzyna Kossowska-Tomaszczuk and Christian De Geyter Copyright © 2013 Katarzyna Kossowska-Tomaszczuk and Christian De Geyter. All rights reserved. Glutamine and Alanyl-Glutamine Increase RhoA Expression and Reduce Clostridium difficile Toxin-A-Induced Intestinal Epithelial Cell Damage Thu, 27 Dec 2012 12:01:35 +0000 Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression. Ana A. Q. A. Santos, Manuel B. Braga-Neto, Marcelo R. Oliveira, Rosemeire S. Freire, Eduardo B. Barros, Thiago M. Santiago, Luciana M. Rebelo, Claudia Mermelstein, Cirle A. Warren, Richard L. Guerrant, and Gerly A. C. Brito Copyright © 2013 Ana A. Q. A. Santos et al. All rights reserved. Patients' Attitudes towards the Surplus Frozen Embryos in China Wed, 26 Dec 2012 18:15:07 +0000 Background. Assisted reproductive techniques have been used in China for more than 20 years. This study investigates the attitudes of surplus embryo holders towards embryos storage and donation for medical research. Methods. A total of 363 couples who had completed in vitro fertilization (IVF) treatment and had already had biological children but who still had frozen embryos in storage were invited to participate. Interviews were conducted by clinics in a narrative style. Results. Family size was the major reason for participants’ (dis)continuation of embryo storage; moreover, the moral status of embryos was an important factor for couples choosing embryo storage, while the storage fee was an important factor for couples choosing embryo disposal. Most couples discontinued the storage of their embryos once their children were older than 3 years. In our study, 58.8% of the couples preferred to dispose of surplus embryos rather than donate them to research, citing a lack of information and distrust in science as significant reasons for their decision. Conclusions. Interviews regarding frozen embryos, including patients’ expectations for embryo storage and information to assist them with decisions regarding embryo disposal, are beneficial for policies addressing embryo disposition and embryo donation in China. Xuan Jin, GongXian Wang, SiSun Liu, Ming Liu, Jing Zhang, and YuFa Shi Copyright © 2013 Xuan Jin et al. All rights reserved. Bodyweight Assessment of Enamelin Null Mice Wed, 26 Dec 2012 15:17:38 +0000 The Enam null mice appear to be smaller than wild-type mice, which prompted the hypothesis that enamel defects negatively influence nutritional intake and bodyweight gain (BWG). We compared the BWG of Enam−/− and wild-type mice from birth (D0) to Day 42 (D42). Wild-type (WT) and Enam−/− (N) mice were given either hard chow (HC) or soft chow (SC). Four experimental groups were studied: WTHC, WTSC, NHC, and NSC. The mother’s bodyweight (DBW) and the average litter bodyweight (ALBW) were obtained from D0 to D21. After D21, the pups were separated from the mother and provided the same type of food. Litter bodyweights were measured until D42. ALBW was compared at 7-day intervals using one-way ANOVA, while the influence of DBW on ALBW was analyzed by mixed-model analyses. The ALBW of Enam−/− mice maintained on hard chow (NHC) was significantly lower than the two WT groups at D21 and the differences persisted into young adulthood. The ALBW of Enam−/− mice maintained on soft chow (NSC) trended lower, but was not significantly different than that of the WT groups. We conclude that genotype, which affects enamel integrity, and food hardness influence bodyweight gain in postnatal and young adult mice. Albert H.-L. Chan, Rangsiyakorn Lertlam, James P. Simmer, Chia-Ning Wang, and Jan C. C. Hu Copyright © 2013 Albert H.-L. Chan et al. All rights reserved. Estradiol Synthesis and Release in Cultured Female Rat Bone Marrow Stem Cells Wed, 26 Dec 2012 11:58:24 +0000 Bone marrow stem cells (BMSCs) have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17β (E2) under high glucose culture conditions with or without retinoic acid (RA). The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency. Dalei Zhang, Bei Yang, Weiying Zou, Xiaying Lu, Mingdi Xiong, Lei Wu, Jinglei Wang, Junhong Gao, Sifan Xu, and Ting Zou Copyright © 2013 Dalei Zhang et al. All rights reserved. In Vitro Culture-Induced Pluripotency of Human Spermatogonial Stem Cells Mon, 24 Dec 2012 14:46:48 +0000 Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotency in vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs in in vitro cultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2–4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs during in vitro culture. Jung Jin Lim, Hyung Joon Kim, Kye-Seong Kim, Jae Yup Hong, and Dong Ryul Lee Copyright © 2013 Jung Jin Lim et al. All rights reserved. Role of the Microenvironment in Ovarian Cancer Stem Cell Maintenance Mon, 24 Dec 2012 14:22:50 +0000 Despite recent progresses in cancer therapy and increased knowledge in cancer biology, ovarian cancer remains a challenging condition. Among the latest concepts developed in cancer biology, cancer stem cells and the role of microenvironment in tumor progression seem to be related. Indeed, cancer stem cells have been described in several solid tumors including ovarian cancers. These particular cells have the ability to self-renew and reconstitute a heterogeneous tumor. They are characterized by specific surface markers and display resistance to therapeutic regimens. During development, specific molecular cues from the tumor microenvironment can play a role in maintaining and expanding stemness of cancer cells. The tumor stroma contains several compartments: cellular component, cytokine network, and extracellular matrix. These different compartments interact to form a permissive niche for the cancer stem cells. Understanding the molecular cues underlying this crosstalk will allow the design of new therapeutic regimens targeting the niche. In this paper, we will discuss the mechanisms implicated in the interaction between ovarian cancer stem cells and their microenvironment. Jennifer Pasquier and Arash Rafii Copyright © 2013 Jennifer Pasquier and Arash Rafii. All rights reserved. Role of TGF-β1/Smad3 Signaling Pathway in Secretion of Type I and III Collagen by Vascular Smooth Muscle Cells of Rats Undergoing Balloon Injury Tue, 02 Oct 2012 13:41:33 +0000 Antisense Smad3 adenoviral vectors were used to transfect vascular smooth muscle cells (VSMCs) from rats with balloon injury or infused into the rat balloon-catheter injured carotid arteries, and the role of TGF-β1/Smad3 signaling pathway in the secretion of type I and III collagen by VSMCs following balloon injury was investigated. Antisense Smad3 adenoviral vectors were used to transfect these VSMCs (antisense Smad3 group). A total 90 rats were randomly assigned into blank control group, experiment group, negative control group. In the in vitro study, the expression of type I and III collagen was markedly reduced in the antisense Smad3 group when compared with the control groups (𝑃<0.05). In the in vivo study, the expression of type I and III collagen was significantly lower than that in the negative control group at 3 days, 1 week and 2 weeks after injury (𝑃<0.05). At 2 weeks and 3 months after injury, the lumen area in the antisense Smad3 group was markedly increased but the intimal area dramatically reduced when compared with the negative control (𝑃<0.05). We conclude that transfection of VSMCs with antisense Smad3 can reduce the secretion of type I and III collagen which then inhibit intimal hyperplasia. Ping Lu, Songhao Wang, Wenwei Cai, and Jing Sheng Copyright © 2012 Ping Lu et al. All rights reserved. A New Candidate Substrate for Cell-Matrix Adhesion Study: The Acellular Human Amniotic Matrix Tue, 02 Oct 2012 13:34:36 +0000 In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Natural three-dimensional (3D) matrices, such as self-assembling matrices and Matrigel, have limitations in terms of their biomechanical properties. Here, we present a simple method to produce an acellular human amniotic matrix (AHAM) with preserved biomechanical properties and a favorable adhesion potential. On the stromal side of the AHAM, human foreskin fibroblasts (HFFs) attached and extended with bipolar spindle-shaped morphology proliferated to multilayer networks, invaded into the AHAM, and migrated in a straight line. Moreover, αV integrin, paxillin, and fibronectin were observed to colocalize after 24 h of HFF culture on the stromal side of the AHAM. Our results indicate that the AHAM may be an ideal candidate as a cell-matrix adhesion substrate to study cell adhesion and invasion as well as other functions in vitro under a tensile force that mimics the in vivo environment. Qianchen Guo, Xuya Lu, Yuan Xue, Hong Zheng, Xiaotao Zhao, and Huajian Zhao Copyright © 2012 Qianchen Guo et al. All rights reserved. Roles of p53 in Various Biological Aspects of Hematopoietic Stem Cells Wed, 20 Jun 2012 09:29:46 +0000 Hematopoietic stem cells (HSCs) have the capacity to self-renew as well as to differentiate into all blood cell types, and they can reconstitute hematopoiesis in recipients with bone marrow ablation. In addition, transplantation therapy using HSCs is widely performed for the treatment of various incurable diseases such as hematopoietic malignancies and congenital immunodeficiency disorders. For the safe and successful transplantation of HSCs, their genetic and epigenetic integrities need to be maintained properly. Therefore, understanding the molecular mechanisms that respond to various cellular stresses in HSCs is important. The tumor suppressor protein, p53, has been shown to play critical roles in maintenance of “cell integrity” under stress conditions by controlling its target genes that regulate cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In this paper, we summarize recent reports that describe various biological functions of HSCs and discuss the roles of p53 associated with them. Takenobu Nii, Tomotoshi Marumoto, and Kenzaburo Tani Copyright © 2012 Takenobu Nii et al. All rights reserved. Ischemia-Induced Apoptosis of Intestinal Epithelial Cells Correlates with Altered Integrin Distribution and Disassembly of F-Actin Triggered by Calcium Overload Wed, 30 May 2012 10:58:50 +0000 The present study examined intestinal epithelial cell (IEC) integrin distribution and disassembly of actin cytoskeleton in response to ischemia-anoxia. Protective effects of calcium channel blocker(CCB) were further examined to explore underlying mechanisms of cellular injury. Materials and Methods. Primary cultures of rat IECs and an in vitro model of ischemia/anoxia were established. IECs were exposed to ischemia/anoxia in the presence and absence of verapamil. The extent of exfoliation was determined using light microscopy while apoptosis rate was measured using flow cytometry. Changes in intracellular calcium, the distribution of integrins and the morphology of F-actin were assessed by confocal microscopy. Results. Detachment and apoptosis of IECs increased following ischemia/anoxia-induced injury. Treatment with verapamil inhibited the detachment and apoptosis. Under control conditions, the strongest fluorescent staining for integrins appeared on the basal surface of IECs while this re-distributed to the apical membrane in response to ischemic injury. Depolymerization of F-actin was also observed in the injured cells. Verapamil attenuated both changes of integrins and F-actin. Conclusions. Redistribution of integrins and disruption of F-actin under ischemia/anoxia injury is associated with IEC detachment and increased apoptosis. These events appeared to be triggered by an increase in Ca𝑖2+ suggesting a potential use for CCB in prevention and treatment of intestinal injury. Zhenyi Jia, Qian Chen, and Huanlong Qin Copyright © 2012 Zhenyi Jia et al. All rights reserved.