ALBERT

Description: O -GlcNAcylation is an inducible, highly dynamic and reversible post-translational modification, mediated by a unique enzyme named O -linked N -acetyl- d -glucosamine ( O -GlcNAc) transferase (OGT). In response to nutrients, O -GlcNAc levels are differentially regulated on many cellular proteins involved in gene expression, translation, immune reactions, protein degradation, protein–protein interaction, apoptosis and signal transduction. In contrast to eukaryotic cells, little is known about the role of O -GlcNAcylation in the viral life cycle. Here, we show that the overexpression of the OGT reduces the replication efficiency of Kaposi's sarcoma-associated herpesvirus (KSHV) in a dose-dependent manner. In order to investigate the global impact of O -GlcNAcylation in the KSHV life cycle, we systematically analyzed the 85 annotated KSHV-encoded open reading frames for O -GlcNAc modification. For this purpose, an immunoprecipitation (IP) strategy with three different approaches was carried out and the O -GlcNAc signal of the identified proteins was properly controlled for specificity. Out of the 85 KSHV-encoded proteins, 18 proteins were found to be direct targets for O -GlcNAcylation. Selected proteins were further confirmed by mass spectrometry for O -GlcNAc modification. Correlation of the functional annotation and the O -GlcNAc status of KSHV proteins showed that the predominant targets were proteins involved in viral DNA synthesis and replication. These results indicate that O -GlcNAcylation plays a major role in the regulation of KSHV propagation.

Description: Galectins are potent adhesion/growth-regulatory effectors with characteristic expression profiles. Understanding the molecular basis of gene regulation in each case requires detailed information on copy number of genes and sequence(s) of their promoter(s). Our report reveals plasticity in this respect between galectins and species. We here describe occurrence of a two-gene constellation for human galectin (Gal)-7 and define current extent of promoter-sequence divergence. Interestingly, cross-species genome analyses also detected single-copy display. Because the regulatory potential will then be different, extrapolations of expression profiles are precluded between respective species pairs. Gal-4 coding in chromosomal vicinity was found to be confined to one gene, whereas copy-number variation also applied to Gal-9. The example of rat Gal-9 teaches the lesson that the presence of multiple bands in Southern blotting despite a single-copy gene constellation is attributable to two pseudogenes. The documented copy-number variability should thus be taken into consideration when studying regulation of galectin genes, in a species and in comparison between species.

Description: In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous -〉 alpha-1 (AI-1) -〉 alpha-2 (AI-2) -〉 gamma (GI) -〉 delta (DI) -〉 zeta (ZI) -〉 epsilon (EI) -〉 omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1 H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

Description: The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N -linked glycans but up to now no one has addressed engineering the O -linked glycosylation pathway. Typically, O -linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O -linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O -linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein- O -linked-mannose β-1,2- N -acetylglucosaminyltransferase 1, resulted in the capping of the single O -linked mannose residues with N -acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O -linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N -linked glycosylated biotherapeutics to include molecules possessing O -linked glycans.

Description: Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Description: With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito- Pst I. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Description: Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the “vicious cycle” of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue it is difficult to study this system in vivo . In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; i.e. there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Aberrant glycosylation by N -acetylgalactosaminyl transferases (GALNTs) is a well-described pathological alteration that is widespread in hereditary diseases, prominently including human cancers, familial tumoral calcinosis and hyperostosis-hyperphosphatemia. In this study, we integrated different computational tools to perform the in silico analysis of clinically significant mutations (nsSNPs/ single amino acid change) at both functional and structural levels, found in human GALNT3, GALNT8, GALNT12 and GALNT13 genes. From function and structure based insights, mutations encoding R162Q, T359K, C574G, G359D, R297W, Y396C & D313N substitutions were concordantly predicted highly deleterious for relevant GALNTs proteins. From intriguing findings, T359K- GALNT3 was simulated with high contribution for disease susceptibility (tumor calcinosis) as compared to its partner variant T272K [Ichikawa et al., 2006]. Similarly, the prediction of high damaging behavior, evolutionary conservation and structural destabilization for C574G were proposed as major contributing factors to regulate metabolic disorder underlying tumor calcinosis and hyperostosis-hyperphosphatemia syndrome. In case of R297W- GALNT12 , prediction of highly deleterious effect and disruption in ionic interactions were anticipated with reduction in enzymatic activity, associated with bilateral breast cancer and primary colorectal cancers. The second GALNT12 mutation (D303N)-known splice variant- was predicted with disease severity as a result of decrease in charge density and buried behavior neighboring the catalytic B domain. In the lack of adequate in silico data about systematic characterization of clinically significant mutations in GALNTs genes, current study can be used as a significant tool to interpret the role of GALNTs reaction chemistry in disease-association risks in body. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Objective To investigate whether crosstalk between RUNX2 and miRNAs is involved in tooth eruption regulated by dental follicle cells(DFCs) and the possible molecular mechanism. Methods Blood samples and embedded dental follicles were collected from patients with cleidocranial dysplasia (CCD), and RUNX2 gene mutations were analyzed, then RUNX2 +/m DFCs were isolated and identified. The characteristics of RUNX2 +/m DFCs were analyzed. The differential expression of miRNAs was detected between the RUNX2 +/m DFCs and RUNX2 +/+ DFCs by microarray, and target genes were predicted by miRGen. miR-146a was chosen for further investigation, and its effects in DFCs were analyzed by transfecting its mimics and inhibitors, and expression of genes involved in tooth eruption were detected. Results A novel insertion mutation (c.309_310insTG) of RUNX2 gene was identified which had an effect on the characteristics of DFCs. Compared with the RUNX2 +/+ DFCs, there were 69 microRNAs more than 2-fold up-regulated and 54 microRNAs more than 2-fold down-regulated in the RUNX2 +/m DFCs. Among these, miR-146a decreased significantly in RUNX 2 +/m DFCs, and expression of RUNX2, CSF-1,EGFR and OPG was significantly altered when miR-146a was over-expressed or inhibited. Conclusion RUNX2 gene mutation contributes to the characteristic change of dental follicle cells, and the crosstalk between RUNX2 gene and miRNAs may be one of the key regulatory mechanisms of differentiation of dental follicle cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate -mitochondrial specific substrates- leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSCs energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Areca chewing is an important environmental risk factor for development of oral premalignant lesions and cancer. Epidemiological evidence indicates that areca chewing is tightly linked to oral carcinogenesis. However, the pathogenetic impacts of areca nut extract (ANE) on normal human oral keratinocytes (HOKs) are unclear and possibly involve oxidative stress via redox imbalance. Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that play an important role in regulating cellular reactive oxygen species (ROS) production. Recent studies have confirmed that ANE and other areca ingredients can induce ROS. In this study, we examined the role of SIRT3 in the regulation of ANE-induced ROS in HOK cells. We examined HOK cell viability following treatment with various ANE concentrations. ANE-induced cytotoxicity increased in a dose-dependent manner and was approximately 48% at a concentration of 50 μg/ml after 24 h. SIRT3 expression and enzyme activity were up-regulated in HOK cells by ANE-induced oxidative stress. Additionally, we identified that SIRT3 controls the enzymatic activity of mitochondrial proteins, such as forkhead box O3a (Foxo3a) transcription factor and antioxidant-encoding gene superoxide dismutase 2 (SOD2), by deacetylation in HOK cells. Moreover, SIRT3-mediated deacetylation and activation of Foxo3a promotes nuclear localization in vivo . These findings suggest that SIRT3 is an endogenous negative regulator in response to ANE-induced oxidative stress and demonstrate an essential role for redox balance in HOK cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Collagen is the most abundant structural protein in mammals and is expressed in various tissues. In recent years, sphingosine 1-phosphate receptors (S1PRs) have been proven to play an important role in the regulation of collagen expression. Our previous studies reported that S1PRs are involved in TGF-β1-induced collagen expression via up-regulating S1PR1/3 in mouse bone marrow-derived mesenchymal stem cells (BMSCs), and result in experimental mouse liver fibrogenesis. But it remains unclear whether this process happens in human bone marrow-derived mesenchymal stem cells (hMSCs). In this study, we provide evidences that S1PR1/3, but not S1PR2, negatively regulate the expression of collagen in hMSCs using cellular and molecular approaches in vitro . We find that treatment of hMSCs with TGF-β1 up-regulated collagen expression in a dose- and time-dependent manner. Meanwhile, TGF-β1 inhibited the expression of S1PR1/3, but not S1PR2, in hMSCs in a time-dependent manner. Furthermore, either selective knock-down of S1PR1 or silencing S1PR3 induced collagen α1(I) and collagen α1(III) expression in hMSCs. In contrast, inhibition of S1PR2 by siRNA had no effects on the expression of collagen. Altogether, all these findings demonstrated that collagen expression was negatively regulated by S1PR1 and S1PR3 in hMSCs. This study highlights the differences between hMSCs and mouse BMSCs, provides a new regulation mechanism for collagen expression, and points out the risk of utilizing hMSCs in clinical applications. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Cervical carcinoma represents the paradigm of virus-induced cancers, where virtually all cervical cancers come from previous “high-risk” HPV infection. The persistent expression of the HPV viral oncoproteins E6 and E7 is responsible for the reprogramming of fundamental cellular functions in the host cell, thus generating a noticeable, yet only partially explored, imbalance in protein molecular networks and cell signaling pathways. Eighty-eight cellular factors, identified as HPV direct or surrogate targets, were chosen and monitored in a retrospective analysis for their mRNA expression in HPV-induced cervical lesions, from dysplasia to cancer. Real-time quantitative PCR (qPCR) was performed by using formalin-fixed, paraffin embedded archival samples. Gene expression analysis identified 40 genes significantly modulated in LSIL, HSIL and squamous cervical carcinoma. Interestingly, among these, the expression level of a panel of four genes, TOP2A, CTNNB1, PFKM and GSN, was able to distinguish between normal tissues and cervical carcinomas. Immunohistochemistry was also done to assess protein expression of two genes among those up-regulated during the transition between dysplasia and carcinoma, namely E2F1 and CDC25A, and their correlation with clinical parameters. Besides the possibility of significantly enhancing the use of some of these factors in diagnostic or prognostic procedures, these data clearly outline specific pathways, and thus key biological processes, altered in cervical dysplasia and carcinoma. Deeper insight on how these molecular mechanisms work may help widen the spectrum of novel innovative approaches to these virus-induced cell pathologies. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Cellular pathways are numerous and are highly integrated in function in the control of cellular systems. They collectively regulate cell division, proliferation, survival and apoptosis of cells and mutagenesis of key genes that control these pathways can initiate neoplastic transformations. Understanding these pathways is crucial to future therapeutic and preventive strategies of the disease. Ovarian cancers are of three major types; epithelial, germ-cell and stromal. However, ovarian cancers of epithelial origin, arising from the mesothelium, are the predominant form. Of the subtypes of ovarian cancer, the high-grade serous tumors are fatal, with low survival rate due to late detection and poor response to treatments. Close examination of preserved ovarian tissues and in vitro studies have provided insights into the mechanistic changes occurring in cells mediated by a few key genes. This review will focus on pathways and key genes of the pathways that are mutated or have aberrant functions in the pathology of ovarian cancer. Non-genetic mechanisms that are gaining prominence in the pathology of ovarian cancer, miRNAs and epigenetics, will also be discussed in the review. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ET A R) and possibly type B (ET B R) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ET B R. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ET A R and ET B R. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12) and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ET A R and ET B R were examined using RT-PCR, Western blots, immunohistochemistry and immunofluorescence. Phenylephrine (Phe, 10 −5  M), KCl (51 mM) and ET-1 (10 −6  M) caused VSMC contraction that was in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with ET B R antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with the ET A R antagonist BQ123, ET-1 caused a small contraction; and the ET B R agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg 〈 mid-Preg and virgin rats. RT-PCR revealed similar ET A R, but greater ET B R mRNA expression in pregnant vs. virgin rats. Western blots revealed similar ET A R, and greater protein amount of ET B R in endothelium-intact vessels, but reduced ET B R in endothelium-denuded vessels of pregnant vs. virgin rats. Immunohistochemistry revealed prominent ET B R staining in the intima, but reduced ET A R and ET B R in the aortic media of pregnant rats. Immunofluorescence signal for ET A R and ET B R was less in VSMCs of pregnant vs. virgin rats. The pregnancy-associated decrease in ET A R- and ET B R-mediated VSMC contraction appears to involve downregulation of ET A R and ET B R expression/activity in VSM, and may play a role in the adaptive vasodilation during pregnancy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Neuroregenerative medicine is an ever-growing field in which regeneration of lost cells/tissues due to a neurodegenerative disease is the ultimate goal. With the scarcity of available replacement alternatives, stem cells provide an attractive source for regenerating neural tissue. While many stem cell sources exist, including: mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), the limited cellular potency, technical difficulties, and ethical considerations associated with these make finding alternate sources a desirable goal. Periodontal ligament stem cells (PDLSCs) derived from the neural crest were induced into neural-like cells using a combination of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Morphological changes were evident in our treated group, seen under both light microscopy and scanning electron microscopy (SEM). A statistically significant increase in the expression of neuron-specific β-tubulin III and the neural stem/progenitor cell marker nestin, along with positive immunohistochemical staining for glial fibrillary acidic protein (GFAP), demonstrated the success of our treatment in inducing both neuronal and glial phenotypes. Positive staining for synaptophysin demonstrated neural connections and electrophysiological recordings indicated that when subjected to whole cell patch clamping, our treated cells displayed inward currents conducted through voltage-gated sodium (Na + ) channels. Taken together, our results indicate the success of our treatment in inducing PDLSCs to neural-like cells. The ease of sourcing and expansion, their embryologic neural crest origin, and the lack of ethical implications in their use make PDLSCs an attractive source for use in neuroregenerative medicine. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Urotensin II (UII), a vasoactive peptide modulates renal hemodynamics. However, the physiological functions of UII in glomerular cells are unclear. In particular, whether UII alters mesangial tone remains largely unknown. The present study investigates the physiological effects of UII on intracellular Ca 2+ ([Ca 2+ ] i ) and contraction in glomerular mesangial cells (GMCs). This study also tested the hypothesis that the regulator of G-protein signaling (RGS) controls UII receptor (UTR) activity in GMCs. RT-PCR, Western immunoblotting, and immunofluorescence revealed UTR expression and localization in cultured murine GMCs. Mouse UII (mUII) stimulated [Ca 2+ ] i elevation in GMCs in the absence and presence of extracellular Ca 2+ . mUII also caused a reduction in planar GMC surface area. mUII-induced [Ca 2+ ] i elevation and contraction in GMCs were attenuated by SB 657510, a UTR antagonist, araguspongin B, an inositol 1,4,5-trisphosphate receptor antagonist, thapsigargin, a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor, and La 3+ , a store-operated Ca 2+ channel blocker, but not nimodipine, an L-type Ca 2+ channel blocker. In situ proximity ligation assay indicated molecular proximity between endogenous RGS2 and UTR in the cells. Treatment of GMCs with mUII increased plasma membrane association of RGS2 by ∼ 2-fold. mUII also increased the interaction between RGS2 and UTR in the cells. siRNA-mediated knockdown of RGS2 in murine GMCs increased mUII-induced [Ca 2+ ] i elevation and contraction by ∼ 35 and 31%, respectively. These findings indicate that mUII induces [Ca 2+ ] i elevation and contraction in murine GMCs. Data also suggest that UTR activation stimulates RGS2 recruitment to GMC plasma membrane as a negative feedback mechanism to regulate UTR signaling. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galβ1,4-GlcNAcβ1,3) n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosminyltransferase (β3GnT). Because β4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of 8 known β3GnT genes in mice. In the olfactory epithelium (OE), β3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here we show for the first time that the β1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that coexpress β3GnT2 and PLN. This highly specific coexpression suggests that GCNT2 and β3GnT2 function cooperatively in PLN synthesis. In support of this, β3GnT2 and GCNT2 cotransfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with β3GnT2 to determine PLN levels in olfactory neurons by regulating β1,6-branches that promote PLN extension. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Objective The aim of this study was to evaluate the effect of artemisinin on the proliferation and apoptosis of rat vascular smooth muscle cells (VSMCs). Method Primary rat VSMCs were treated with various doses of artemisinin. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the messenger RNA and protein expressions of proliferating cell nuclear antigen were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. Apoptosis was measured using annexin V and propidium iodide double staining evaluated by flow cytometry. Protein expression of Bax, Bcl2, and cyclin-dependent kinase 4 was determined by Western blot. Results After 72 h of treatment, artemisinin significantly inhibited VSMC proliferation in a dose-dependent manner. Treatment with 1 mM artemisinin for 72 h significantly reduced the expression of proliferating cell nuclear antigen messenger RNA. On the other hand, the same treatment increased the apoptosis of VSMCs, the activation of caspase-3, the Bax protein expression, and the Bax/Bcl2 ratio. Conclusion The results suggest that artemisinin can effectively inhibit VSMC proliferation and induce VSMC apoptosis. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Motivation: Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (~1%) in an infected host with high sensitivity and specificity. Results: We developed a new approach, referred to as Indel and Carryforward Correction (ICC), to cluster sequences without substitutions and locally correct only indel and carryforward sequencing errors within clusters to ensure that no rare variants are lost. ICC performs sequence clustering in the order of (i) homopolymer indel patterns only, (ii) indel patterns only and (iii) carryforward errors only, without the requirement of a distance cutoff value. Overall, ICC removed 93–95% of sequencing errors found in control datasets. On pyrosequencing data from a PCR fragment derived from 15 HIV-1 plasmid clones mixed at various frequencies as low as 0.1%, ICC achieved the highest sensitivity and similar specificity compared with other commonly used error correction and variant calling algorithms. Availability and implementation: Source code is freely available for download at http://indra.mullins.microbiol.washington.edu/ICC . It is implemented in Perl and supported on Linux, Mac OS X and MS Windows. Contact: jmullins@uw.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Tiki proteins appear to antagonize Wnt signalling pathway by acting as Wnt proteases, thereby affecting Wnt solubility by its amino-terminal cleavage. Tiki1 protease activity was shown to be metal ion-dependent and was inhibited by chelating agents and thus was tentatively proposed to be a metalloprotease. Nevertheless, Tiki proteins exhibit no detectable sequence similarity to previously described metalloproteases, but instead have been reported as being homologues of TraB proteins (Pfam ID: PF01963), a widely distributed family of unknown function and structure. Here, we show that Tiki proteins are members of a new superfamily of domains contained not just in TraB proteins, but also in erythromycin esterase (Pfam ID: PF05139), DUF399 (domain of unknown function 399; Pfam ID: PF04187) and MARTX toxins that contribute to host invasion and pathogenesis by bacteria. We establish the core fold of this enzymatic domain and its catalytic residues. Contact: luis.sanchezpulido@dpag.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The research area metabolomics achieved tremendous popularity and development in the last couple of years. Owing to its unique interdisciplinarity, it requires to combine knowledge from various scientific disciplines. Advances in the high-throughput technology and the consequently growing quality and quantity of data put new demands on applied analytical and computational methods. Exploration of finally generated and analyzed datasets furthermore relies on powerful tools for data mining and visualization. Results: To cover and keep up with these requirements, we have created MeltDB 2.0, a next-generation web application addressing storage, sharing, standardization, integration and analysis of metabolomics experiments. New features improve both efficiency and effectivity of the entire processing pipeline of chromatographic raw data from pre-processing to the derivation of new biological knowledge. First, the generation of high-quality metabolic datasets has been vastly simplified. Second, the new statistics tool box allows to investigate these datasets according to a wide spectrum of scientific and explorative questions. Availability: The system is publicly available at https://meltdb.cebitec.uni-bielefeld.de . A login is required but freely available. Contact: nkessler@cebitec.uni-bielefeld.de

Description: Motivation: High - throughput next - generation sequencing technologies enable increasingly fast and affordable sequencing of genomes and transcriptomes, with a broad range of applications. The quality of the sequencing data is crucial for all applications. A significant portion of the data produced contains errors, and ever more efficient error correction programs are needed. Results: We propose RACER (Rapid and Accurate Correction of Errors in Reads), a new software program for correcting errors in sequencing data. RACER has better error-correcting performance than existing programs, is faster and requires less memory. To support our claims, we performed extensive comparison with the existing leading programs on a variety of real datasets. Availability: RACER is freely available for non-commercial use at www.csd.uwo.ca/~ilie/RACER/ . Contact: ilie@csd.uwo.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Interactions between various types of molecules that regulate crucial cellular processes are extensively investigated by high-throughput experiments and require dedicated computational methods for the analysis of the resulting data. In many cases, these data can be represented as a bipartite graph because it describes interactions between elements of two different types such as the influence of different experimental conditions on cellular variables or the direct interaction between receptors and their activators/inhibitors. One of the major challenges in the analysis of such noisy datasets is the statistical evaluation of the relationship between any two elements of the same type. Here, we present SICOP (significant co-interaction patterns), an implementation of a method that provides such an evaluation based on the number of their common interaction partners, their so-called co-interaction. This general network analytic method, proved successful in diverse fields, provides a framework for assessing the significance of this relationship by comparison with the expected co-interaction in a suitable null model of the same bipartite graph. SICOP takes into consideration up to two distinct types of interactions such as up- or downregulation. The tool is written in Java and accepts several common input formats and supports different output formats, facilitating further analysis and visualization. Its key features include a user-friendly interface, easy installation and platform independence. Availability: The software is open source and available at cna.cs.uni-kl.de/SICOP under the terms of the GNU General Public Licence (version 3 or later). Contact: agnes.horvat@iwr.uni-heidelberg.de or zweig@cs.uni-kl.de

Description: : Molecular recognition features (MoRFs) are small, intrinsically disordered regions in proteins that undergo a disorder-to-order transition on binding to their partners. MoRFs are involved in protein–protein interactions and may function as the initial step in molecular recognition. The aim of this work was to collect, organize and store all membrane proteins that contain MoRFs. Membrane proteins constitute ~30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. MoRFs were classified according to their secondary structure, after interacting with their partners. We identified MoRFs in transmembrane and peripheral membrane proteins. The position of transmembrane protein MoRFs was determined in relation to a protein’s topology. All information was stored in a publicly available mySQL database with a user-friendly web interface. A Jmol applet is integrated for visualization of the structures. mpMoRFsDB provides valuable information related to disorder-based protein–protein interactions in membrane proteins. Availability: http://bioinformatics.biol.uoa.gr/mpMoRFsDB Contact: shamodr@biol.uoa.gr

Description: : The signaling Petri net (SPN) simulator, designed to provide insights into the trends of molecules’ activity levels in response to an external stimulus, contributes to the systems biology necessity of analyzing the dynamics of large-scale cellular networks. Implemented into the freely available software, BioLayout Express 3D , the simulator is publicly available and easy to use, provided the input files are prepared in the GraphML format, typically using the network editing software, yEd, and standards specific to the software. However, analysis of complex networks represented using other systems biology formatting languages (on which popular software, such as CellDesigner and Cytoscape, are based) requires manual manipulation, a step that is prone to error and limits the use of the SPN simulator in BioLayout Express 3D . To overcome this, we present a Cytoscape plug-in that enables users to automatically convert networks for analysis with the SPN simulator from the standard systems biology markup language. The automation of this step opens the SPN simulator to a far larger user group than has previously been possible. Availability and implementation: Distributed under the GNU General Public License Version 3 at http://apps.cytoscape.org/apps/spnconverter . Contact: christine@picb.ac.cn

Description: Motivation: Conformational diversity is a key concept in the understanding of different issues related with protein function such as the study of catalytic processes in enzymes, protein-protein recognition, protein evolution and the origins of new biological functions. Here, we present a database of proteins with different degrees of conformational diversity. Conformational Diversity of Native State (CoDNaS) is a redundant collection of three-dimensional structures for the same protein derived from protein data bank. Structures for the same protein obtained under different crystallographic conditions have been associated with snapshots of protein dynamism and consequently could characterize protein conformers. CoDNaS allows the user to explore global and local structural differences among conformers as a function of different parameters such as presence of ligand, post-translational modifications, changes in oligomeric states and differences in pH and temperature. Additionally, CoDNaS contains information about protein taxonomy and function, disorder level and structural classification offering useful information to explore the underlying mechanism of conformational diversity and its close relationship with protein function. Currently, CoDNaS has 122 122 structures integrating 12 684 entries, with an average of 9.63 conformers per protein. Availability: The database is freely available at http://www.codnas.com.ar/ . Contact: gusparisi@gmail.com

Description: Motivation: Recent experimental advancements allow determining positions of nucleosomes for complete genomes. However, the resulting nucleosome occupancy maps are averages of heterogeneous cell populations. Accordingly, they represent a snapshot of a dynamic ensemble at a single time point with an overlay of many configurations from different cells. To study the organization of nucleosomes along the genome and to understand the mechanisms of nucleosome translocation, it is necessary to retrieve features of specific conformations from the population average. Results: Here, we present a method for identifying non-overlapping nucleosome configurations that combines binary-variable analysis and a Monte Carlo approach with a simulated annealing scheme. In this manner, we obtain specific nucleosome configurations and optimized solutions for the complex positioning patterns from experimental data. We apply the method to compare nucleosome positioning at transcription factor binding sites in different mouse cell types. Our method can model nucleosome translocations at regulatory genomic elements and generate configurations for simulations of the spatial folding of the nucleosome chain. Availability: Source code, precompiled binaries, test data and a web-based test installation are freely available at http://bioinformatics.fh-stralsund.de/nucpos/ Contact: gero.wedemann@fh-stralsund.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (−2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Lymph nodes are often the first target of metastatic cancer which can then remetastasize to distant organs. The progression of lymph node metastasis is dependent on sufficient blood supply provided by angiogenesis. In the present study, we have developed a color-coded imaging model to visualize angiogenesis of lymph nodes metastasis using green fluorescent protein (GFP) and red fluorescent protein (RFP). Transgenic mice carrying GFP under the control of the nestin second-intron enhancer (ND-GFP mice) were used as hosts. Nascent blood vessels express GFP in these mice. B16F10-RFP melanoma cells were injected into the efferent lymph vessel of the inguinal lymph node of the ND-GFP nude mice, whereby the melanoma cells trafficked to the axillary lymph node. Three days after melanoma implantation, ND-GFP-expressing nascent blood vessels were imaged in the axillary lymph nodes. Seven days after implantation, ND-GFP-expressing nascent blood vessels formed a network in the lymph nodes. ND-GFP-positive blood vessels surrounded the tumor mass by 14 days after implantation. However, by 28 days after implantation, ND-GFP expression was diminished as the blood vessels matured. Treatment with doxorubicin significantly decreased the mean nascent blood vessel length per tumor volume. These results show that the dual-color ND-GFP blood vessels/RFP-tumor model is a powerful tool to visualize and quantitate angiogenesis of metastatic lymph nodes as well as for evaluation of its inhibition. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Spermatogenesis is a special process by which spermatogonial stem cells (SSCs) divide and differentiate to male gametes called mature spermatozoa. SSCs are the unique cells because they are adult stem cells that transmit genetic information to subsequent generations. Accumulating evidence has demonstrated that SSCs can be reprogrammed to acquire pluripotency to become embryonic stem-like cells that differentiate into all cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Recent studies from peers and us have made great achievements on the characterization, isolation and culture of mouse and human SSCs, which could lead to better understanding the biology of SSCs and the applications of SSCs in both reproductive and regenerative medicine. In this review, we first compared the cell identity and biochemical phenotypes between mouse SSCs and human SSCs. Notably, the cell types of mouse and human SSCs are distinct, and human SSCs share some but not all phenotypes with mouse SSCs. The approaches for isolating SSCs as well as short- and long- term culture of mouse SSCs and short-period culture of human SSCs were also discussed. We further addressed the new advances on the self-renewal of SSCs with an aim to establish the long-term culture of human SSCs which has not yet been achieved. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Long non-coding RNAs (lncRNAs) have recently gained increasing attention because of their crucial roles in gene regulatory processes. Functional studies using mammalian skin as a model system have revealed their role in controlling normal tissue homeostasis as well as the transition to a diseased state. Here, we describe how lncRNAs regulate differentiation to preserve an undifferentiated epidermal progenitor compartment, and to maintain a functional skin permeability barrier. Furthermore, we will reflect on recent work analyzing the impact of lncRNAs on the progression from normal epithelium to the development of skin disorders and cancer. Long non-coding RNAs (lncRNAs) have recently been shown to control a wide variety of gene regulatory processes. In mammalian skin, lncRNAs appear to regulate the intricate balance between progenitor cells undergoing continual regeneration in the basal layer and highly differentiated cells forming the epidermal permeability barrier.

Description: Motivation: Residue–residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize. Results: We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score 〉0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity 〉30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins. Availability: www.csbio.sjtu.edu.cn/bioinf/MemBrain/ Contact: hbshen@sjtu.edu.cn or zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Identification of protein–ligand binding sites is critical to protein function annotation and drug discovery. However, there is no method that could generate optimal binding site prediction for different protein types. Combination of complementary predictions is probably the most reliable solution to the problem. Results: We develop two new methods, one based on binding-specific substructure comparison (TM-SITE) and another on sequence profile alignment (S-SITE), for complementary binding site predictions. The methods are tested on a set of 500 non-redundant proteins harboring 814 natural, drug-like and metal ion molecules. Starting from low-resolution protein structure predictions, the methods successfully recognize 〉51% of binding residues with average Matthews correlation coefficient (MCC) significantly higher (with P -value 〈10 –9 in student t -test) than other state-of-the-art methods, including COFACTOR, FINDSITE and ConCavity. When combining TM-SITE and S-SITE with other structure-based programs, a consensus approach (COACH) can increase MCC by 15% over the best individual predictions. COACH was examined in the recent community-wide COMEO experiment and consistently ranked as the best method in last 22 individual datasets with the Area Under the Curve score 22.5% higher than the second best method. These data demonstrate a new robust approach to protein–ligand binding site recognition, which is ready for genome-wide structure-based function annotations. Availability: http://zhanglab.ccmb.med.umich.edu/COACH/ Contact: zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The nucleosome is the basic repeating unit of chromatin. It contains two copies each of the four core histones H2A, H2B, H3 and H4 and about 147 bp of DNA. The residues of the histone proteins are subject to numerous post-translational modifications, such as methylation or acetylation. Chromatin immunoprecipitiation followed by sequencing (ChIP-seq) is a technique that provides genome-wide occupancy data of these modified histone proteins, and it requires appropriate computational methods. Results: We present NucHunter, an algorithm that uses the data from ChIP-seq experiments directed against many histone modifications to infer positioned nucleosomes. NucHunter annotates each of these nucleosomes with the intensities of the histone modifications. We demonstrate that these annotations can be used to infer nucleosomal states with distinct correlations to underlying genomic features and chromatin-related processes, such as transcriptional start sites, enhancers, elongation by RNA polymerase II and chromatin-mediated repression. Thus, NucHunter is a versatile tool that can be used to predict positioned nucleosomes from a panel of histone modification ChIP-seq experiments and infer distinct histone modification patterns associated to different chromatin states. Availability: The software is available at http://epigen.molgen.mpg.de/nuchunter/ . Contact: chung@molgen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: In biomedical research a growing number of platforms and technologies are used to measure diverse but related information, and the task of clustering a set of objects based on multiple sources of data arises in several applications. Most current approaches to multisource clustering either independently determine a separate clustering for each data source or determine a single ‘joint’ clustering for all data sources. There is a need for more flexible approaches that simultaneously model the dependence and the heterogeneity of the data sources. Results: We propose an integrative statistical model that permits a separate clustering of the objects for each data source. These separate clusterings adhere loosely to an overall consensus clustering, and hence they are not independent. We describe a computationally scalable Bayesian framework for simultaneous estimation of both the consensus clustering and the source-specific clusterings. We demonstrate that this flexible approach is more robust than joint clustering of all data sources, and is more powerful than clustering each data source independently. We present an application to subtype identification of breast cancer tumor samples using publicly available data from The Cancer Genome Atlas. Availability: R code with instructions and examples is available at http://people.duke.edu/%7Eel113/software.html . Contact: Eric.Lock@duke.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: More and more evidences have indicated that long–non-coding RNAs (lncRNAs) play critical roles in many important biological processes. Therefore, mutations and dysregulations of these lncRNAs would contribute to the development of various complex diseases. Developing powerful computational models for potential disease-related lncRNAs identification would benefit biomarker identification and drug discovery for human disease diagnosis, treatment, prognosis and prevention. Results : In this article, we proposed the assumption that similar diseases tend to be associated with functionally similar lncRNAs. Then, we further developed the method of Laplacian Regularized Least Squares for LncRNA–Disease Association (LRLSLDA) in the semisupervised learning framework. Although known disease–lncRNA associations in the database are rare, LRLSLDA still obtained an AUC of 0.7760 in the leave-one-out cross validation, significantly improving the performance of previous methods. We also illustrated the performance of LRLSLDA is not sensitive (even robust) to the parameters selection and it can obtain a reliable performance in all the test classes. Plenty of potential disease–lncRNA associations were publicly released and some of them have been confirmed by recent results in biological experiments. It is anticipated that LRLSLDA could be an effective and important biological tool for biomedical research. Availability: The code of LRLSLDA is freely available at http://asdcd.amss.ac.cn/Software/Details/2 . Contact: xingchen@amss.ac.cn or yangy@amt.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Genomic repositories are rapidly growing, as witnessed by the 1000 Genomes or the UK10K projects. Hence, compression of multiple genomes of the same species has become an active research area in the past years. The well-known large redundancy in human sequences is not easy to exploit because of huge memory requirements from traditional compression algorithms. Results: We show how to obtain several times higher compression ratio than of the best reported results, on two large genome collections (1092 human and 775 plant genomes). Our inputs are variant call format files restricted to their essential fields. More precisely, our novel Ziv-Lempel-style compression algorithm squeezes a single human genome to ~400 KB. The key to high compression is to look for similarities across the whole collection, not just against one reference sequence, what is typical for existing solutions. Availability: http://sun.aei.polsl.pl/tgc (also as Supplementary Material) under a free license. Supplementary data: Supplementary data are available at Bioinformatics online. Contact: sebastian.deorowicz@polsl.pl

Description: Motivation:  Biological systems are understood through iterations of modeling and experimentation. Not all experiments, however, are equally valuable for predictive modeling. This study introduces an efficient method for experimental design aimed at selecting dynamical models from data. Motivated by biological applications, the method enables the design of crucial experiments: it determines a highly informative selection of measurement readouts and time points. Results:  We demonstrate formal guarantees of design efficiency on the basis of previous results. By reducing our task to the setting of graphical models, we prove that the method finds a near-optimal design selection with a polynomial number of evaluations. Moreover, the method exhibits the best polynomial-complexity constant approximation factor, unless P = NP. We measure the performance of the method in comparison with established alternatives, such as ensemble non-centrality, on example models of different complexity. Efficient design accelerates the loop between modeling and experimentation: it enables the inference of complex mechanisms, such as those controlling central metabolic operation. Availability:  Toolbox ‘NearOED’ available with source code under GPL on the Machine Learning Open Source Software Web site (mloss.org). Contact:   busettoa@inf.ethz.ch Supplementary information:   Supplementary data are available at Bioinformatics online.

Description: : Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA–mRNA interaction databases. Availability and Implementation: The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/ . Contact: rotter@genxpro.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: : We present PARSEC (PAtteRn Search and Contextualization), a new open source platform for guided discovery, allowing localization and biological characterization of short genomic sites in entire eukaryotic genomes. PARSEC can search for a sequence or a degenerated pattern. The retrieved set of genomic sites can be characterized in terms of (i) conservation in model organisms, (ii) genomic context (proximity to genes) and (iii) function of neighboring genes. These modules allow the user to explore, visualize, filter and extract biological knowledge from a set of short genomic regions such as transcription factor binding sites. Availability: Web site implemented in Java, JavaScript and C++, with all major browsers supported. Freely available at lbgi.fr/parsec. Source code is freely available at sourceforge.net/projects/genomicparsec. Contact: odile.lecompte@unistra.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : At the rate that prokaryotic genomes can now be generated, comparative genomics studies require a flexible method for quickly and accurately predicting orthologs among the rapidly changing set of genomes available. SPOCS implements a graph-based ortholog prediction method to generate a simple tab-delimited table of orthologs and in addition, html files that provide a visualization of the predicted ortholog/paralog relationships to which gene/protein expression metadata may be overlaid. Availability and Implementation: A SPOCS web application is freely available at http://cbb.pnnl.gov/portal/tools/spocs.html . Source code for Linux systems is also freely available under an open source license at http://cbb.pnnl.gov/portal/software/spocs.html ; the Boost C++ libraries and BLAST are required. Contact: leeann.mccue@pnnl.gov

Description: : Automated image processing has allowed cell migration research to evolve to a high-throughput research field. As a consequence, there is now an unmet need for data management in this domain. The absence of a generic management system for the quantitative data generated in cell migration assays results in each dataset being treated in isolation, making data comparison across experiments difficult. Moreover, by integrating quality control and analysis capabilities into such a data management system, the common practice of having to manually transfer data across different downstream analysis tools will be markedly sped up and made more robust. In addition, access to a data management solution creates gateways for data standardization, meta-analysis and structured public data dissemination. We here present CellMissy, a cross-platform data management system for cell migration data with a focus on wound healing data. CellMissy simplifies and automates data management, storage and analysis from the initial experimental set-up to data exploration. Availability and implementation: CellMissy is a cross-platform open-source software developed in Java. Source code and cross-platform binaries are freely available under the Apache2 open source license at http://cellmissy.googlecode.com . Contact: lennart.martens@ugent.be Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Motivation: With the expansion of high-throughput technologies, understanding different kinds of genome-level data is a common task. MicroRNA (miRNA) is increasingly profiled using high-throughput technologies (microarrays or next-generation sequencing). The downstream analysis of miRNA targets can be difficult. Although there are many databases and algorithms to predict miRNA targets, there are few tools to integrate miRNA–gene interaction data into high-throughput genomic analyses. Results: We present targetHub, a CouchDB database of miRNA–gene interactions. TargetHub provides a programmer-friendly interface to access miRNA targets. The Web site provides RESTful access to miRNA–gene interactions with an assortment of gene and miRNA identifiers. It can be a useful tool to integrate miRNA target interaction data directly into high-throughput bioinformatics analyses. Availability: TargetHub is available on the web at http://app1.bioinformatics.mdanderson.org/tarhub/_design/basic/index.html . Contact: coombes.3@osu.edu

Description: Angiogenin (ANG) undergoes nuclear translocation and promotes ribosomal RNA (rRNA) transcription thereby enhancing cell growth and proliferation. However, the mode of action of ANG in stimulating rRNA transcription is unclear. Here, we show that ANG enhances the formation of RNA polymerase I (Pol I) pre-initiation complex at the ribosomal DNA (rDNA) promoter. ANG binds at the upstream control element (UCE) of the promoter and enhances promoter occupancy of RNA Pol I as well as the selectivity factor SL1 components TAF I 48 and TAF I 110. We also show that ANG increases the number of actively transcribing rDNA by epigenetic activation through promoter methylation and histone modification. ANG binds to histone H3, inhibits H3K9 methylation, and activates H3K4 methylation as well as H4 acetylation at the rDNA promoter. These data suggest that one of the mechanisms by which ANG stimulates rRNA transcription is through an epigenetic activation of rDNA promoter. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: No abstract is available for this article.

Description: Pancreatic cancer (PC) has a high rate of mortality and a poorly understood mechanism of progression. Investigation of the molecular mechanism of PC and exploration of the specific markers for early diagnosis and specific targets of therapy are key points to prevent and treat PC effectively and to improve their prognosis. In our study, expression profiles experiment of para-carcinoma, carcinoma and relapse human PC was performed using Agilent human whole genomic oligonucleotide microarrays with 45 000 probes. Differentially expressed genes related with PC were screened and analysed further by Gene Ontology term analysis and Kyoto encyclopaedia of genes and genomes pathway analysis. Our results showed that there were 3853 differentially expressed genes associated with pancreatic carcinogenesis and relapse. In addition, our study found that PC was related to the Jak–STAT signalling pathway, PPAR signalling pathway and Calcium signalling pathway, indicating their potential roles in pancreatic carcinogenesis and progress. Copyright © 2013 John Wiley & Sons, Ltd.

Description: ABSTRACT Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1 -H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cell in endocrine tissues where prior in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1 -H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis. © 2013 Wiley Periodicals, Inc.

Description: The cytoplasmic signaling protein tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5), which was identified as a signal transducer for members of the TNF receptor super-family, has been implicated in several biological functions in T/B lymphocytes and the innate immune response against viral infection. However, the role of TRAF5 in cardiac hypertrophy has not been reported. In the present study, we investigated the effect of TRAF5 on the development of pathological cardiac hypertrophy induced by transthoracic aorta constriction (TAC) and further explored the underlying molecular mechanisms. Cardiac hypertrophy and function were evaluated with echocardiography, hemodynamic measurements, pathological and molecular analyses. For the first time, we found that TRAF5 deficiency substantially aggravated cardiac hypertrophy, cardiac dysfunction and fibrosis in response to pressure overload after 4 weeks of TAC compared to wild-type (WT) mice. Moreover, the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway was more activated in TRAF5-deficient mice than WT mice. In conclusion, our results suggest that as an intrinsic cardioprotective factor, TRAF5 plays a crucial role in the development of cardiac hypertrophy through the negative regulation of the MEK-ERK1/2 pathway. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules/centrosomes maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase-dependent apoptosis in HEp-2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp-2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome-dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t-Bid) but did not alter other Bcl-2 family members. The knock-down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t-Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer. Copyright © 2013 John Wiley & Sons, Ltd.

Description: General mechanism for exaggerated sexuallyselected traits. Many animals wield sexually-selected exaggerated traits. ‘Classic’ examples include the peacock's train, and the antlers observed in male deer, as well as fiddler crabs with enlarged claws, and the enlarged head-horns of rhinoceros beetles ( Trypoxylus dichotomus , cover). These traits are used for mate choice, or to deter rival males, because they act as unusually reliable signals of the condition of individual males: only the best-quality animals produce full-sized signal structures. But what keeps their expression honest? How can signal traits evolve that are resistant to invasion by cheaters who fake attractive signals? The answer may lie in the ancient insulin-like signalling pathway, which is found in all animals and directly links individual condition to growth in a dose dependent manner. Warren et al. discuss recent evidence suggesting that exaggerated sexually-selected signal traits arise when specific structures become extra-sensitive to physiological signals like insulins or insulin-like growth factors (pages 889–899 ).

Description: mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for functional genomics the use of nascent transcription rates should be restricted to the evaluation of the transcriptional process itself, whereas mature mRNA synthesis rates should be employed to address the transcriptional input to mRNA concentration balance leading to variation of gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. It is important to distinguish when to use each one.

Description: CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research. Sweetness, umami, and bitterness are transmitted to the nervous system via ion channel-mediated ATP release from taste cells. A recent study demonstrated that CALHM1 is essential for taste cell ATP release and perception of sweetness, umami, and bitterness. We discuss the new findings and unresolved issues in peripheral taste signaling.

Description: The precise orchestration of two opposing protein complexes – one in the cytoplasm (β-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) – is critical for controlling levels of the transcriptional co-factor β-catenin, and subsequent activation of the Wnt/β-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area. The mechanism by which the Wnt pathway stabilizes β-catenin, a key transcriptional co-factor, remains controversial. Recent studies have revealed that the phosphorylation state of an essential regulator of the pathway, Axin, controls its conformation and, consequently, its availability to scaffold two opposing Wnt pathway protein complexes that regulate β-catenin stability.

Description: Motivation: Human miRNAs have recently been found to have important roles in viral replication. Understanding the patterns and details of human miRNA interactions during virus–host interactions may help uncover novel antiviral therapies. Based on the abundance of knowledge available regarding protein–protein interactions (PPI), virus–host protein interactions, experimentally validated human miRNA-target pairs and transcriptional regulation of human miRNAs, it is possible to explore the complex regulatory network that exists between viral proteins and human miRNAs at the system level. Results: By integrating current data regarding the virus–human interactome and human miRNA-target pairs, the overlap between targets of viral proteins and human miRNAs was identified and found to represent topologically important proteins (e.g. hubs or bottlenecks) at the global center of the human PPI network. Viral proteins and human miRNAs were also found to significantly target human PPI pairs. Furthermore, an overlap analysis of virus targets and transcription factors (TFs) of human miRNAs revealed that viral proteins preferentially target human miRNA TFs, representing a new pattern of virus–host interactions. Potential feedback loops formed by viruses, human miRNAs and miRNA TFs were also identified, and these may be exploited by viruses resulting in greater virulence and more effective replication strategies. Contact: boxc@bmi.ac.cn or ni.ming@163.com or sqwang@bmi.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from http://www.well.ox.ac.uk/~rivas/mamba Contact: donnelly@well.ox.ac.uk

Description: Motivation: Structural information of macromolecular complexes provides key insights into the way they carry out their biological functions. Achieving high-resolution structural details with electron microscopy requires the identification of a large number (up to hundreds of thousands) of single particles from electron micrographs, which is a laborious task if it has to be manually done and constitutes a hurdle towards high-throughput. Automatic particle selection in micrographs is far from being settled and new and more robust algorithms are required to reduce the number of false positives and false negatives. Results: In this article, we introduce an automatic particle picker that learns from the user the kind of particles he is interested in. Particle candidates are quickly and robustly classified as particles or non-particles. A number of new discriminative shape-related features as well as some statistical description of the image grey intensities are used to train two support vector machine classifiers. Experimental results demonstrate that the proposed method: (i) has a considerably low computational complexity and (ii) provides results better or comparable with previously reported methods at a fraction of their computing time. Availability: The algorithm is fully implemented in the open-source Xmipp package and downloadable from http://xmipp.cnb.csic.es . Contact: vabrishami@cnb.csic.es or coss@cnb.csic.es Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: : Recent major cancer genome sequencing studies have used whole-genome sequencing to detect various types of genomic variation. However, a number of these studies have continued to rely on SNP array information to provide additional results for copy number and loss-of-heterozygosity estimation and assessing tumour purity. OncoSNP-SEQ is a statistical model-based approach for inferring copy number profiles directly from high-coverage whole genome sequencing data that is able to account for unknown tumour purity and ploidy. Availability: MATLAB code is available at the following URL: https://sites.google.com/site/oncosnpseq/ . Contact : c.yau@imperial.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Multi-Image Genome (MIG) viewer is a web-based application for visualizing, querying and filtering many thousands of genome browser regions as well as for exporting the data in a variety of formats. This methodology has been used successfully to analyze ChIP-Seq data and RNA-Seq data and to detect somatic mutations in genome resequencing projects. Availability: MIG is available at https://mig.molbiol.ox.ac.uk/mig/ Contact: simon.mcgowan@imm.ox.ac.uk

Description: : Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA–protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses. Availability: ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf . Contact: alper.kucukural@umassmed.edu or ccenik@stanford.edu

Description: Motivation: Kinases of the eukaryotic protein kinase superfamily are key regulators of most aspects eukaryotic cellular behavior and have provided several drug targets including kinases dysregulated in cancers. The rapid increase in the number of genomic sequences has created an acute need to identify and classify members of this important class of enzymes efficiently and accurately. Results: Kinannote produces a draft kinome and comparative analyses for a predicted proteome using a single line command, and it is currently the only tool that automatically classifies protein kinases using the controlled vocabulary of Hanks and Hunter [Hanks and Hunter (1995)]. A hidden Markov model in combination with a position-specific scoring matrix is used by Kinannote to identify kinases, which are subsequently classified using a BLAST comparison with a local version of KinBase, the curated protein kinase dataset from www.kinase.com . Kinannote was tested on the predicted proteomes from four divergent species. The average sensitivity and precision for kinome retrieval from the test species are 94.4 and 96.8%. The ability of Kinannote to classify identified kinases was also evaluated, and the average sensitivity and precision for full classification of conserved kinases are 71.5 and 82.5%, respectively. Kinannote has had a significant impact on eukaryotic genome annotation, providing protein kinase annotations for 36 genomes made public by the Broad Institute in the period spanning 2009 to the present. Availability: Kinannote is freely available at http://sourceforge.net/projects/kinannote . Contact: jmgold@broadinstitute.org Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Quantification of lipids is a primary goal in lipidomics. In direct infusion/injection (or shotgun) lipidomics, accurate downstream identification and quantitation requires accurate summarization of repetitive peak measurements. Imprecise peak summarization multiplies downstream error by propagating into species identification and intensity estimation. To our knowledge, this is the first analysis of direct infusion peak summarization in the literature. Results: We present two novel peak summarization algorithms for direct infusion samples and compare them with an off-machine ad hoc summarization algorithm as well as with the propriety Xcalibur algorithm. Our statistical agglomeration algorithm reduces peakwise error by 38% mass/charge (m/z) and 44% (intensity) compared with the ad hoc method over three datasets. Pointwise error is reduced by 23% (m/z). Compared with Xcalibur, our statistical agglomeration algorithm produces 68% less m/z error and 51% less intensity error on average on two comparable datasets. Availability: The source code for Statistical Agglomeration and the datasets used are freely available for non-commercial purposes at https://github.com/optimusmoose/statistical_agglomeration . Modified Bin Aggolmeration is freely available in MSpire, an open source mass spectrometry package at https://github.com/princelab/mspire/ . Contact: 2robsmith@gmail.com or jtprince@chem.byu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Liquid chromatography coupled to mass spectrometry (LC-MS) is the dominant technological platform for proteomics. An LC-MS analysis of a complex biological sample can be visualized as a ‘map’ of which the positional coordinates are the mass-to-charge ratio (m/z) and chromatographic retention time (RT) of the chemical species profiled. Label-free quantitative proteomics requires the alignment and comparison of multiple LC-MS maps to ascertain the reproducibility of experiments or reveal proteome changes under different conditions. The main challenge in this task lies in correcting inevitable RT shifts. Similar, but not identical, LC instruments and settings can cause peptides to elute at very different times and sometimes in a different order, violating the assumptions of many state-of-the-art alignment tools. To meet this challenge, we developed LWBMatch, a new algorithm based on weighted bipartite matching. Unlike existing tools, which search for accurate warping functions to correct RT shifts, we directly seek a peak-to-peak mapping by maximizing a global similarity function between two LC-MS maps. For alignment tasks with large RT shifts (〉500 s), an approximate warping function is determined by locally weighted scatterplot smoothing of potential matched features, detected using a novel voting scheme based on co-elution. For validation, we defined the ground truth for alignment success based on tandem mass spectrometry identifications from sequence searching. We showed that our method outperforms several existing tools in terms of precision and recall, and is capable of aligning maps from different instruments and settings. Availability: Available at https://sourceforge.net/projects/rt-alignment/ . Contact: kehlam@ust.hk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : We propose SW#, a new CUDA graphical processor unit-enabled and memory-efficient implementation of dynamic programming algorithm, for local alignment. It can be used as either a stand-alone application or a library. Although there are other graphical processor unit implementations of the Smith–Waterman algorithm, SW# is the only one publicly available that can produce sequence alignments on genome-wide scale. For long sequences, it is at least a few hundred times faster than a CPU version of the same algorithm. Availability: Source code and installation instructions freely available for download at http://complex.zesoi.fer.hr/SW.html . Contact: mile.sikic@fer.hr Supplementary information: Supplementary results are available at Bioinformatics online.

Description: Background Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte-specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. Materials and Methods After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC-transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Results Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside-induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e-overexpressing podocytes in the transwell migration assay. Internalization of FITC-transferrin also increased in Myo1e-overexpressing podocytes relative to control cells. Conclusions Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 ( SMN1 ) gene, retention of the survival motor neuron 2 ( SMN2 ) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy.

Description: Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA Thr 15927G〉A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G〉A mutation affects structure and function of tRNA Thr . Northern analysis revealed 80% decrease in the steady-state level of tRNA Thr in the mutant cell lines carrying the 15927G〉A mutation. The 15927G〉A mutation changed the conformation of tRNA Thr , as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ~39% reduction in aminoacylated efficiency of tRNA Thr was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ~53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N '-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G〉A mutation. These results provide the direct evidence that the tRNA Thr 15927G〉A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.

Description: Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.

Description: Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple ( brdp ) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b ( Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.

Description: CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet–Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo , at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Description: Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2 , to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2 , drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/– SMA mice), and positively impacts neuromuscular pathologies. In 2B/– SMA mice, RG3039 provided a 〉600% survival benefit (median 18 days to 〉112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo . These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/– SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Description: Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motoneurons. The primary triggers for motoneuron degeneration are still unknown, but inflammation is considered an important contributing factor. P2X7 receptor is a key player in microglia response to toxic insults and was previously shown to increase pro-inflammatory actions of SOD1-G93A ALS microglia. We therefore hypothesized that lack of P2X7 receptor could modify disease features in the SOD1-G93A mice. Hetero- and homozygous P2X7 receptor knock-out SOD1-G93A mice were thus generated and analysed for body weight, disease onset and progression (by behavioural scores, grip and rotarod tests) and survival. Although the lifespan of P2X7 +/– and P2X7 –/– /SOD1-G93A female mice was extended by 6–7% with respect to SOD1-G93A mice, to our surprise the clinical onset was significantly anticipated and the disease progression worsened in both male and female P2X7 –/– /SOD1-G93A mice. Consistently, we found increased astrogliosis, microgliosis, motoneuron loss, induction of the pro-inflammatory markers NOX2 and iNOS and activation of the MAPKs pathway in the lumbar spinal cord of end-stage P2X7 –/– /SOD1-G93A mice. These results show that the constitutive deletion of P2X7 receptor aggravates the ALS pathogenesis, suggesting that the receptor might have beneficial effects in at least definite stages of the disease. This study unravels a complex dual role of P2X7 receptor in ALS and strengthens the importance of a successful time window of therapeutic intervention in contrasting the pathology.

Description: There are certain de novo germline mutations associated with genetic disorders whose mutation rates per generation are orders of magnitude higher than the genome average. Moreover, these mutations occur exclusively in the male germ line and older men have a higher probability of having an affected child than younger ones, known as the paternal age effect (PAE). The classic example of a genetic disorder exhibiting a PAE is achondroplasia, caused predominantly by a single-nucleotide substitution (c.1138G〉A) in FGFR3 . To elucidate what mechanisms might be driving the high frequency of this mutation in the male germline, we examined the spatial distribution of the c.1138G〉A substitution in a testis from an 80-year-old unaffected man. Using a technology based on bead-emulsion amplification, we were able to measure mutation frequencies in 192 individual pieces of the dissected testis with a false-positive rate lower than 2.7 x 10 –6 . We observed that most mutations are clustered in a few pieces with 95% of all mutations occurring in 27% of the total testis. Using computational simulations, we rejected the model proposing an elevated mutation rate per cell division at this nucleotide site. Instead, we determined that the observed mutation distribution fits a germline selection model, where mutant spermatogonial stem cells have a proliferative advantage over unmutated cells. Combined with data on several other PAE mutations, our results support the idea that the PAE, associated with a number of Mendelian disorders, may be explained primarily by a selective mechanism.

Description: microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1) G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1 G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS.

Description: Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). It is characterized by the infiltration of autoreactive immune cells into the CNS, which target the myelin sheath, leading to the loss of neuronal function. Although it is accepted that MS is a multifactorial disorder with both genetic and environmental factors influencing its development and course, the molecular pathogenesis of MS has not yet been fully elucidated. Here, we studied the longitudinal gene expression profiles of whole-blood RNA from a cohort of 195 MS patients and 66 healthy controls. We analyzed these transcriptomes at both the individual transcript and the biological pathway level. We found 62 transcripts to be significantly up-regulated in MS patients; the expression of 11 of these genes was counter-regulated by interferon treatment, suggesting partial restoration of a ‘healthy’ gene expression profile. Global pathway analyses linked the proteasome and Wnt signaling to MS disease processes. Since genotypes from a subset of individuals were available, we were able to identify expression quantitative trait loci (eQTL), a number of which involved two genes of the MS gene signature. However, all these eQTL were also present in healthy controls. This study highlights the challenge posed by analyzing transcripts from whole blood and how these can be mitigated by using large, well-characterized cohorts of patients with longitudinal follow-up and multi-modality measurements.

Description: Cornelia de Lange syndrome (CdLS) is a developmental disorder caused by mutations in NIPBL, a protein which has functionally been associated with the cohesin complex. Mutations in core cohesin complex components have also been reported in individuals with CdLS-like phenotypes. In addition to its role in sister chromatid cohesion, cohesin is thought to play a role in regulating gene expression during development. The mechanism of this gene regulation remains unclear, but NIPBL and cohesin have been reported to affect long-range chromosomal interactions, both independently and through interactions with CTCF. We used fluorescence in situ hybridization to investigate whether the disruption of NIPBL affects chromosome architecture. We show that cells from CdLS patients exhibit visible chromatin decompaction, that is most pronounced across gene-rich regions of the genome. Cells carrying mutations predicted to have a more severe effect on NIPBL function show more extensive chromatin decompaction than those carrying milder mutations. This cellular phenotype was reproduced in normal cells depleted for NIPBL with siRNA, but was not seen following the knockdown of either the cohesin component SMC3, or CTCF. We conclude that NIPBL has a function in modulating chromatin architecture, particularly for gene-rich areas of the chromosome, that is not dependent on SMC3/cohesin or CTCF, raising the possibility that the aetiology of disorders associated with the mutation of core cohesin components is distinct from that associated with the disruption of NIPBL itself in classical CdLS.

Description: Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique -hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gaucher's disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg f/fK14CreERT2 mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg -mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPARβ/), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.

Description: A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies—MW1, 1C2 and 3B5H10—have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

Description: Charcot–Marie–Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.

Description: Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Description: Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin ( UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line Umod C93F , leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line Umod A227T . In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and Cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

Description: Hepatitis B virus (HBV) infection is the predominant risk factor for chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Recently, several genome-wide association studies (GWASs) of CHB identified human leukocyte antigen (HLA) loci, including HLA-DP and HLA-DQ in Asian populations, as being associated with the risk of CHB. To confirm and identify the host genetic factors related to CHB infection, we performed another GWAS using a higher-density chip in Korean CHB carriers. We analyzed 1400 samples from Korean population (400 CHB cases and 1000 population controls) using a higher-density GWAS chip [1 140 419 single nucleotide polymorphisms (SNPs)]. In subsequent replication analysis, we further analyzed in an independent study of a Korean CHB cohort consisting of 2909 Korean samples (971 cases and 1938 controls). Logistic regression methods were used for statistical analysis adjusting for age and sex as covariates. This study identified two new risk-associated loci for CHB on the HLA region of chromosome 6, e.g. rs652888 on euchromatic histone-lysine-methyltransferase 2 (EHMT2, P = 7.07 x 10 –13 ) and rs1419881 on transcription factor 19 (TCF19, P = 1.26 x 10 –18 ). Conditional analysis with nearby HLA CHB loci that were previously known, confirmed the independent genetic effects of these two loci on CHB. Conclusion : The GWAS and the subsequent validation study identified new variants associated with the risk of CHB. These findings may advance the understanding of genetic susceptibility to CHB.

Description: Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array ( n = 6) compared with fetuses with a non-pathogenic D4Z4 array ( n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with 〈11 D4Z4 . The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

Description: Indoleamine 2,3-dioxygenase-1 (IDO1) catabolizes the essential amino acid tryptophan, acting as a modifier of inflammation and immune tolerance. Recent work has implicated IDO1 in many human diseases, including in cancer, chronic infection, autoimmune disorders and neurodegenerative disease, stimulating a major surge in preclinical and clinical studies of its pathogenic functions. In the mouse, IDO1 is expressed widely but in situ detection of the enzyme in murine tissues has been unreliable due to the lack of specific antibodies that do not also react with tissues from animals that are genetically deficient in IDO1. Such probes are crucial to establish cellular mechanisms since IDO1 appears to act in different cell types depending on disease context, but reliable probes have been elusive in the field. In this report, we address this issue with the development of IDO1 monoclonal antibody 4B7 which specifically recognizes the murine enzyme in tissue sections, offering a reliable tool for immunohistology in preclinical disease models. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: There is a rapidly growing body of literature on the effects of topography and critically, nanotopography on cell adhesion, apoptosis and differentiation. Understanding the effects of nanotopography on cell adhesion and morphology and the consequences of cell shape changes in the nucleus, and consequently, gene expression offers new approaches to the elucidation and potential control of stem cell differentiation. In the current study we have used molecular approaches in combination with immunohistology and transcript analysis to understand the role of nanotopography on mesenchymal stem cell morphology and phenotype. Results demonstrate large changes in cell adhesion, nucleus and lamin morphologies in response to the different nanotopographies. Furthermore, these changes relate to alterations in packing of chromosome territories within the interphase nucleus. This, in turn, leads to changes in transcription factor activity and functional (phenotypical) signalling including cell metabolism. Nanotopography provides a useful, non-invasive tool for studying cellular mechanotransduction, gene and protein expression patterns, through effects on cell morphology. The different nanotopographies examined, result in different morphological changes in the cyto- and nucleo-skeleton. We propose that both indirect (biochemical) and direct (mechanical) signalling are important in these early stages of regulating stem cell fate as a consequence of altered metabolic changes and altered phenotype. The current studies provide new insight on cell–surface interactions and enhance our understanding of the modulation of stem cell differentiation with significant potential application in regenerative medicine. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Constitutive androstane receptor (CAR) was originally identified as xenobiotic sensor that regulates the expression of cytochrome P450 genes. However, recent studies suggest that this nuclear receptor is also involved in the regulation of energy metabolism including glucose and lipid homeostasis. This study investigated the role of CAR in the regulation of bone mass in vivo using CAR -/- mice. Endogenous mRNA expression of CAR was observed in both primary osteoblasts and osteoclast precursors. CAR -/- mice have exhibited significant increase in whole body bone mineral density (BMD) by 9.5% ( p  〈 0.01) and 5.5% (p 〈 0.05) at 10 and 15 weeks of age, respectively, compared with WT mice in males. Microcomputed tomography analysis of proximal tibia demonstrated a significant increase in trabecular bone volume (62.7%), trabecular number (54.1%) in male CAR -/- mice compared with WT mice. However, primary culture of calvarial cells exhibited no significant changes in osteogenic differentiation potential between CAR -/- and WT. In addition, the number of tartrate-resistant acid-phosphatase positive osteoclasts in the femur and serum level of CTx was not different between CAR -/- and WT mice. The higher BMD and microstructural parameters were not observed in female mice. Interestingly, serum level of testosterone in male CAR -/- mice was 2.5-fold higher compared with WT mice and the mRNA expressions of Cyp2b9 and 2b10 in the liver, which regulate testosterone metabolism, were significantly down-regulated in male CAR -/- mice. Furthermore, the difference in BMD between CAR -/- and WT mice disappeared at 8 weeks after performing orchiectomy. CAR -/- mice also exhibited significant increase in serum1,25(OH) 2 D 3 levels but Cyp 27B1 which converts 25(OH)D 3 to 1,25(OH) 2 D 3 was significantly down-regulated compared to WT mice. These results suggest that in vivo deletion of CAR resulted in higher bone mass, which appears to be a result from reduced metabolism of testosterone due to down-regulation of Cyp2b. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.