ALBERT

Description: by Ping-Ping Yao, Lei Qian, Yong Xia, Fang Xu, Zhang-Nv Yang, Rong-Hui Xie, Xiao Li, Wei-Feng Liang, Xiao-Xiao Huang, Zhi-Yong Zhu, Han-Ping Zhu A reliable disease model mimicking Enterovirus 71 (EV71) infection in humans is essential for understanding pathogenesis and for developing a safe and effective vaccine. Commonly used rodent models including mouse or rat models are not suitable for vaccine evaluation because the rodents are resistant to EV71 infection after they reach the age of 6 days. In this study, 21-day-old gerbils inoculated intraperitoneally (IP) with a non mouse-adapted EV71 strain developed neurological lesion-related signs including hind limb paralysis, slowness, ataxia and lethargy similar to those of central nervous system (CNS) infection of EV71 in humans. The infected gerbils eventually died of the neurological lesions and EV71 could be isolated from lung, liver, spleen, kidney, heart, spinal cord, brain cortex, brainstem and skeletal muscle. Significantly high virus replication was detected in spinal cord, brainstem and skeletal muscle by cellular analysis, real-time quantitative PCR (RT-PCR) and immunohistochemical staining. Histopathologic changes such as neuronal degeneration, neuronal loss and neuronophagia were observed in spinal cord, brain cortex, brainstem, and skeletal muscle along with necrotizing myositis and splenic atrophy. Gerbils that received two doses of inactive whole-virus vaccine showed no EV71-specific symptoms after challenged with EV71. In contrast, gerbils that received mock vaccination died of EV71-induced neuropathology after challenged with EV71. The result indicates that gerbils can serve as a reliable disease model for evaluating safety and efficacy of EV71 vaccine.

Description: by Anna Ligasová, Dmytro Strunin, Radek Liboska, Ivan Rosenberg, Karel Koberna A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2′-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.

Description: by Francesca Collu, Matteo Ceccarelli, Paolo Ruggerone Ligand-receptor interactions are at the basis of the mediation of our physiological responses to a large variety of ligands, such as hormones, neurotransmitters and environmental stimulants, and their tuning represents the goal of a large variety of therapies. Several molecular details of these interactions are still largely unknown. In an effort to shed some light on this important issue, we performed a computational study on the interaction of two related compounds differing by a single methyl group (clozapine and desmethylclozapine) with a -opioid receptor. According to experiments, desmethylclozapine is more active than clozapine, providing a system well suited for a comparative study. We investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by molecular dynamics simulations. Our results point out that the action of the compounds might be related to the spatial and temporal distribution of the affinity sites they visit during their permanency. Moreover, no particularly pronounced structural perturbations of the receptor were detected during the simulations, reinforcing the idea of a strong dynamical character of the interaction process, with an important role played by the solvent in addition.

Description: by Qiang Feng, Xia Zou, Ling Lu, Yun Li, Yunzhang Liu, Jianfeng Zhou, Cunming Duan REDD1/redd1 is a stress-response gene that is induced under various stressful conditions such as hypoxia, DNA damage, and energy stress. The increased REDD1 inhibits mTOR signaling and cell growth. Here we report an unexpected role of Redd1 in regulating dorsoventral patterning in zebrafish embryos and the underlying mechanisms. Zebrafish redd1 mRNA is maternally deposited. Although it is ubiquitously detected in many adult tissues, its expression is highly tissue-specific and dynamic during early development. Hypoxia and heat shock strongly induce redd1 expression in zebrafish embryos. Knockdown of Redd1 using two independent morpholinos results in dorsalized embryos and this effect can be rescued by injecting redd1 mRNA. Forced expression of Redd1 ventralizes embryos. Co-expression of Redd1 with Wnt3a or a constitutively active form of β-catenin suggests that Redd1 alters dorsoventral patterning by antagonizing the Wnt/β-catenin signaling pathway. These findings have unraveled a novel role of Redd1 in early development by antagonizing Wnt/β-catenin signaling.

Description: by Claudia Figueroa-Romero, Junguk Hur, Diane E. Bender, Colin E. Delaney, Michael D. Cataldo, Andrea L. Smith, Raymond Yung, Douglas M. Ruden, Brian C. Callaghan, Eva L. Feldman Amyotrophic lateral sclerosis (ALS) is a terminal disease involving the progressive degeneration of motor neurons within the motor cortex, brainstem and spinal cord. Most cases are sporadic (sALS) with unknown causes suggesting that the etiology of sALS may not be limited to the genotype of patients, but may be influenced by exposure to environmental factors. Alterations in epigenetic modifications are likely to play a role in disease onset and progression in ALS, as aberrant epigenetic patterns may be acquired throughout life. The aim of this study was to identify epigenetic marks associated with sALS. We hypothesize that epigenetic modifications may alter the expression of pathogenesis-related genes leading to the onset and progression of sALS. Using ELISA assays, we observed alterations in global methylation (5 mC) and hydroxymethylation (5 HmC) in postmortem sALS spinal cord but not in whole blood. Loci-specific differentially methylated and expressed genes in sALS spinal cord were identified by genome-wide 5mC and expression profiling using high-throughput microarrays. Concordant direction, hyper- or hypo-5mC with parallel changes in gene expression (under- or over-expression), was observed in 112 genes highly associated with biological functions related to immune and inflammation response. Furthermore, literature-based analysis identified potential associations among the epigenes. Integration of methylomics and transcriptomics data successfully revealed methylation changes in sALS spinal cord. This study represents an initial identification of epigenetic regulatory mechanisms in sALS which may improve our understanding of sALS pathogenesis for the identification of biomarkers and new therapeutic targets.

Description: by Wen-Yang Tsai, Szu-Chia Hsieh, Chih-Yun Lai, Hong-En Lin, Vivek R. Nerurkar, Wei-Kung Wang Background The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. Methodology/Principal Findings In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. Conclusions/Significance A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.

Description: by Quancheng Zhou, Guihua Sheng The thermal decomposition of Perilla frutescens polysaccharide was examined by thermogravimetry, differential thermogravimetry, and differential thermal analysis. The results showed that the mass loss of the substance proceeded in three steps. The first stage can be attributed to the expulsion of the water from ambient temperature to 182°C. The second stage corresponded to devolatilization from 182°C to 439°C. The residue slowly degraded in the third stage. The weight loss in air is faster than that in nitrogen, because the oxygen in air accelerated the pyrolytic reaction speed reaction. The heating rate significantly affected the pyrolysis of the sample. Similar activation energies of the degradation process (210–211 kJ mol −1 ) were obtained by the FWO, KAS, and Popescu techniques. According to Popescu mechanism functions, the possible kinetic model was estimated to be Avrami–Erofeev 20 g ( α ) = [−ln(1– α )] 4 .

Description: by Linda Irvine, Donald W. Falconer, Claire Jones, Ian W. Ricketts, Brian Williams, Iain K. Crombie Background Process evaluation is essential in developing, piloting and evaluating complex interventions. This often involves observation of intervention delivery and interviews with study participants. Mobile telephone interventions involve no face to face contact, making conventional process evaluation difficult. This study assesses the utility of novel techniques for process evaluation involving no face to face contact. Methods Text messages were delivered to 34 disadvantaged men as part of a feasibility study of a brief alcohol intervention. Process evaluation focused on delivery of the text messages and responses received from study participants. The computerized delivery system captured data on receipt of the messages. The text messages, delivered over 28 days, included nine which asked questions. Responses to these questions served as one technique for process evaluation by ascertaining the nature of engagement with the study and with steps on the causal chain to behavior change. Results A total of 646 SMS text messages were sent to participants. Of these, 613 messages (95%) were recorded as delivered to participants’ telephones. 88% of participants responded to messages that asked questions. There was little attenuation in responses to the questions across the intervention period. Content analysis of the responses revealed that participants engaged with text messages, thought deeply about their content and provided carefully considered personal responses to the questions. Conclusions Socially disadvantaged men, a hard to reach population, engaged in a meaningful way over a sustained period with an interactive intervention delivered by text message. The novel process measures used in the study are unobtrusive, low cost and collect real-time data on all participants. They assessed the fidelity of delivery of the intervention and monitored retention in the study. They measured levels of engagement and identified participants’ reactions to components of the intervention. These methods provide a valuable addition to conventional process evaluation techniques.

Description: by Golnar Karimian, Manon Buist-Homan, Bojana Mikus, Robert H. Henning, Klaas Nico Faber, Han Moshage Conclusion Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.

Description: by Nuria Kotecki, Nicolas Penel, Antoine Adenis, Charles Ferte, Stéphanie Clisant Background The diagnosis of tumour progression or progressive disease (PD) is a key element for designing and interpreting contemporary phase II trials. In some cases, PD is stated by the physician and is not formally confirmed by imaging. Purpose In this study, we intend to analyze the value of the PD based on clinical judgment and the risk of overestimating the occurrence of PD by clinical judgment. Methods We have conducted a single-centre retrospective study to analyse the diagnostic accuracy of this clinical judgment compared to planned imaging including all patients enrolled in our institution in phase II trials investigating systemic treatments for advanced solid tumours between January 2008 and November 2010. Results The positive predictive value (PPV) and the specificity of clinical judgment of PD was very high (〉90%). Conclusions According to this study, the clinical judgment of PD is highly predictive of radiological PD as assessed, for example, by RECIST. Physicians do not overestimate PD occurence. Clinical judgment of PD could be taken into account in the definition of PD.

Description: by Aziza El Harchi, Dario Melgari, Yi Hong Zhang, Henggui Zhang, Jules C. Hancox Background The familial Short QT Syndrome (SQTS) is associated with an increased risk of cardiac arrhythmia and sudden death. Gain-of-function mutations in the hERG K + channel protein have been linked to variant 1 of the SQTS. A hERG channel pore (T618I) mutation has recently been identified in families with heritable SQTS. This study aimed to determine effects of the T618I-hERG mutation on (i) hERG current (I hERG ) elicited by ventricular action potentials; (ii) the sensitivity of I hERG to inhibition by four clinically used antiarrhythmic drugs. Methods Electrophysiological recordings of I hERG were made at 37°C from HEK 293 cells expressing wild-type (WT) or T618I hERG. Whole-cell patch clamp recording was performed using both conventional voltage clamp and ventricular action potential (AP) clamp methods. Results Under conventional voltage-clamp, WT I hERG peaked at 0-+10 mV, whilst for T618I I hERG maximal current was right-ward shifted to ∼ +40 mV. Voltage-dependent activation and inactivation of T618I I hERG were positively shifted (respectively by +15 and ∼ +25 mV) compared to WT I hERG . The I hERG ‘window’ was increased for T618I compared to WT hERG. Under ventricular AP clamp, maximal repolarising WT I hERG occurred at ∼ -30 mV, whilst for T618I hERG peak I hERG occurred earlier during AP repolarisation, at ∼ +5 mV. Under conventional voltage clamp, half-maximal inhibitory concentrations (IC 50 ) for inhibition of I hERG tails by quinidine, disopyramide, D-sotalol and flecainide for T618I hERG ranged between 1.4 and 3.2 fold that for WT hERG. Under action potential voltage clamp, T618I IC 50 s ranged from 1.2 to 2.0 fold the corresponding IC 50 values for WT hERG. Conclusions The T618I mutation produces a more modest effect on repolarising I hERG than reported previously for the N588K-hERG variant 1 SQTS mutation. All drugs studied here appear substantially to retain their ability to inhibit I hERG in the setting of the SQTS-linked T618I mutation.

Description: by Adelene Ai-Lian Song, Janna Ong Abdullah, Mohd. Puad Abdullah, Norazizah Shafee, Roohaida Othman, Ee-Fun Tan, Normah Mohd. Noor, Abdul Rahim Raha Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus ). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis . The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

Description: by Larisa R. G. DeSantis, Blaine W. Schubert, Jessica R. Scott, Peter S. Ungar The saber-toothed cat, Smilodon fatalis , and American lion, Panthera atrox , were among the largest terrestrial carnivores that lived during the Pleistocene, going extinct along with other megafauna ∼12,000 years ago. Previous work suggests that times were difficult at La Brea (California) during the late Pleistocene, as nearly all carnivores have greater incidences of tooth breakage (used to infer greater carcass utilization) compared to today. As Dental Microwear Texture Analysis (DMTA) can differentiate between levels of bone consumption in extant carnivores, we use DMTA to clarify the dietary niches of extinct carnivorans from La Brea. Specifically, we test the hypothesis that times were tough at La Brea with carnivorous taxa utilizing more of the carcasses. Our results show no evidence of bone crushing by P. atrox , with DMTA attributes most similar to the extant cheetah, Acinonyx jubatus , which actively avoids bone. In contrast, S. fatalis has DMTA attributes most similar to the African lion Panthera leo , implying that S. fatalis did not avoid bone to the extent previously suggested by SEM microwear data. DMTA characters most indicative of bone consumption (i.e., complexity and textural fill volume) suggest that carcass utilization by the extinct carnivorans was not necessarily more complete during the Pleistocene at La Brea; thus, times may not have been “tougher” than the present. Additionally, minor to no significant differences in DMTA attributes from older (∼30–35 Ka) to younger (∼11.5 Ka) deposits offer little evidence that declining prey resources were a primary cause of extinction for these large cats.

Description: by Feng Qu, Cai-Sheng Wu, Jin-Feng Hou, Ying Jin, Jin-Lan Zhang Background Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism. Methods High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation. Results As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p

Description: by Thomas D. Pfister, Melinda Hollingshead, Robert J. Kinders, Yiping Zhang, Yvonne A. Evrard, Jiuping Ji, Sonny A. Khin, Suzanne Borgel, Howard Stotler, John Carter, Raymond Divelbiss, Shivaani Kummar, Yves Pommier, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow Background Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. Methodology/Principal Findings We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. Conclusions/Significance Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.

Description: by Lindsay Riley, Hua Zhou, Kenneth Lange, Janet S. Sinsheimer, Mary E Sehl Introduction Trastuzumab dramatically improves survival in breast cancer patients whose tumor overexpresses HER2. A subpopulation of cells in human breast tumors has been identified with characteristics of cancer stem cells. These breast cancer stem-like cells (BCSCs) rely on HER2 signaling for self-renewal, suggesting that HER2-targeted therapy targets BCSCs even when the bulk of the tumor does not overexpress HER2. In order to guide clinical trials examining HER2-targeted therapy in the adjuvant setting, we propose a mathematical model to examine BCSC population dynamics and predict optimal duration of therapy. Methods Varying the susceptibility of BCSCs to HER2-targeted therapy, we quantify the average time to extinction of BCSCs. We expand our model using stochastic simulation to include the partially differentiated tumor cells (TCs) that represent bulk tumor population and examine effects of plasticity on required duration of therapy. Results Lower susceptibility of BCSCs and increased rates of dedifferentiation entail longer extinction times, indicating a need for prolonged administration of HER2-targeted therapy. We predict that even when therapy does not appreciably reduce tumor size in the advanced cancer setting, it will eventually eradicate the tumor in the adjuvant setting as long as there is at least a modest effect on BCSCs. Conclusions We anticipate that our results will inform clinical trials of targeted therapies in planning the duration of therapy needed to eradicate BCSCs. Our predictions also address safety, as longer duration of therapy entails a greater potential impact on normal stem cells that may also be susceptible to stem cell-targeted therapies.

Description: by Mahir Karakas, Jens Baumert, Marcus E. Kleber, Barbara Thorand, Dhayana Dallmeier, Günther Silbernagel, Tanja B. Grammer, Wolfgang Rottbauer, Christa Meisinger, Thomas Illig, Winfried März, Wolfgang Koenig Background Elevated soluble (s) E-selectin levels have been associated with various cardiovascular diseases. Recently, genetic variants in the ABO blood group have been related to E-selectin levels in a small cohort of patients with type 1 diabetes. We evaluated whether this association is reproducible in two large samples of Caucasians. Methodology/ Principal Findings Data of the present study was drawn from the population-based MONICA/KORA Augsburg study (n = 1,482) and the patients-based LURIC study (n = 1,546). A high-density genotyping array (50K IBC Chip) containing single-nucleotide polymorphisms (SNPs) from E-selectin candidate genes selected on known biology of E-selectin metabolism, mouse genetic studies, and human genetic association studies, was used for genotyping. Linear regression analyses with adjustment for age and sex (and survey in KORA) were applied to assess associations between gene variants and sE-selectin concentrations. A number of 12 SNPs (in KORA) and 13 SNPs (in LURIC), all from the ABO blood group gene, were significantly associated with the log-transformed concentration of E-selectin. The strongest association was observed for rs651007 with a change of log-transformed sE-selectin per one copy of the minor allele of −0.37 ng/ml (p = 1.87×10 −103 ) in KORA and −0.35 ng/ml (p = 5.11×10 −84 ) in LURIC. Inclusion of rs651007 increased the explained sE-selectin variance by 0.256 in KORA and 0.213 in LURIC. All SNPs had minor allele frequencies above 20% showing a substantial gene variation. Conclusions/ Significance Our findings in two independent samples indicate that the genetic variants at the ABO locus affect sE-selectin levels. Since distinct genome-wide association studies linked the ABO gene with myocardial infarction (MI) in the presence of coronary atherosclerosis and with coronary artery disease, these findings may not only enhance our understanding of adhesion molecule biology, but may also provide a focus for several novel research avenues.

Description: by Hon-Yi Shi, Hao-Hsien Lee, Jinn-Tsong Tsai, Wen-Hsien Ho, Chieh-Fan Chen, King-Teh Lee, Chong-Chi Chiu Background Few studies of laparoscopic cholecystectomy (LC) outcome have used longitudinal data for more than two years. Moreover, no studies have considered group differences in factors other than outcome such as age and nonsurgical treatment. Additionally, almost all published articles agree that the essential issue of the internal validity (reproducibility) of the artificial neural network (ANN), support vector machine (SVM), Gaussian process regression (GPR) and multiple linear regression (MLR) models has not been adequately addressed. This study proposed to validate the use of these models for predicting quality of life (QOL) after LC and to compare the predictive capability of ANNs with that of SVM, GPR and MLR. Methodology/Principal Findings A total of 400 LC patients completed the SF-36 and the Gastrointestinal Quality of Life Index at baseline and at 2 years postoperatively. The criteria for evaluating the accuracy of the system models were mean square error (MSE) and mean absolute percentage error (MAPE). A global sensitivity analysis was also performed to assess the relative significance of input parameters in the system model and to rank the variables in order of importance. Compared to SVM, GPR and MLR models, the ANN model generally had smaller MSE and MAPE values in the training data set and test data set. Most ANN models had MAPE values ranging from 4.20% to 8.60%, and most had high prediction accuracy. The global sensitivity analysis also showed that preoperative functional status was the best parameter for predicting QOL after LC. Conclusions/Significance Compared with SVM, GPR and MLR models, the ANN model in this study was more accurate in predicting patient-reported QOL and had higher overall performance indices. Further studies of this model may consider the effect of a more detailed database that includes complications and clinical examination findings as well as more detailed outcome data.

Description: by Stacie L. Lambert, Chin-Fen Yang, Zheng Liu, Rosemary Sweetwood, Jackie Zhao, Lily Cheng, Hong Jin, Jennifer Woo Background Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood. Methodology and Principal Findings We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for T H 1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood. Conclusions/Significance While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

Description: by Lianne J. Woodward, Caron A. C. Clark, Samudragupta Bora, Terrie E. Inder Background Cerebral white matter abnormalities on term MRI are a strong predictor of motor disability in children born very preterm. However, their contribution to cognitive impairment is less certain. Objective Examine relationships between the presence and severity of cerebral white matter abnormalities on neonatal MRI and a range of neurocognitive outcomes assessed at ages 4 and 6 years. Design/Methods The study sample consisted of a regionally representative cohort of 104 very preterm (≤32 weeks gestation) infants born from 1998–2000 and a comparison group of 107 full-term infants. At term equivalent, all preterm infants underwent a structural MRI scan that was analyzed qualitatively for the presence and severity of cerebral white matter abnormalities, including cysts, signal abnormalities, loss of white matter volume, ventriculomegaly, and corpus callosal thinning/myelination. At corrected ages 4 and 6 years, all children underwent a comprehensive neurodevelopmental assessment that included measures of general intellectual ability, language development, and executive functioning. Results At 4 and 6 years, very preterm children without cerebral white matter abnormalities showed no apparent neurocognitive impairments relative to their full-term peers on any of the domain specific measures of intelligence, language, and executive functioning. In contrast, children born very preterm with mild and moderate-to-severe white matter abnormalities were characterized by performance impairments across all measures and time points, with more severe cerebral abnormalities being associated with increased risks of cognitive impairment. These associations persisted after adjustment for gender, neonatal medical risk factors, and family social risk. Conclusions Findings highlight the importance of cerebral white matter connectivity for later intact cognitive functioning amongst children born very preterm. Preterm born children without cerebral white matter abnormalities on their term MRI appear to be spared many of the cognitive impairments commonly associated with preterm birth. Further follow-up will be important to assess whether this finding persists into the school years.

Description: by Lynnae M. Smith, Mya C. Schiess, Mary P. Coffey, Andrea C. Klaver, David A. Loeffler α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n = 14), atypical Parkinson syndromes (n = 11), idiopathic rapid eye movement sleep behavior disorder (n = 10), and healthy controls (n = 9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho = 0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.

Description: by Jan Churan, Daniel Guitton, Christopher C. Pack Visual neurons have spatial receptive fields that encode the positions of objects relative to the fovea. Because foveate animals execute frequent saccadic eye movements, this position information is constantly changing, even though the visual world is generally stationary. Interestingly, visual receptive fields in many brain regions have been found to exhibit changes in strength, size, or position around the time of each saccade, and these changes have often been suggested to be involved in the maintenance of perceptual stability. Crucial to the circuitry underlying perisaccadic changes in visual receptive fields is the superior colliculus (SC), a brainstem structure responsible for integrating visual and oculomotor signals. In this work we have studied the time-course of receptive field changes in the SC. We find that the distribution of the latencies of SC responses to stimuli placed outside the fixation receptive field is bimodal: The first mode is comprised of early responses that are temporally locked to the onset of the visual probe stimulus and stronger for probes placed closer to the classical receptive field. We suggest that such responses are therefore consistent with a perisaccadic rescaling, or enhancement, of weak visual responses within a fixed spatial receptive field. The second mode is more similar to the remapping that has been reported in the cortex, as responses are time-locked to saccade onset and stronger for stimuli placed in the postsaccadic receptive field location. We suggest that these two temporal phases of spatial updating may represent different sources of input to the SC.

Description: by Chengchao Zhou, Jie Chu, Jinan Liu, Ruoyan Gai Tobe, Hong Gen, Xingzhou Wang, Wengui Zheng, Lingzhong Xu Adherence to TB treatment is the most important requirement for efficient TB control. Migrant TB patients’ “migratory” nature affects the adherence negatively, which presents an important barrier for National TB Control Program in China. Therefore, TB control among migrants is of high importance.The aim of this study is to describe adherence to TB treatment among migrant TB patients and to identify factors associated with adherence. A total of 12 counties/districts of Shandong Province, China were selected as study sites. 314 confirmed smear positive TB patients were enrolled between August 2 nd 2008 and October 17 th 2008, 16% of whom were non-adherent to TB therapy. Risk factors for non-adherence were: the divorced or bereft of spouse, patients not receiving TB-related health education before chemotherapy, weak incentives for treatment adherence, and self supervision on treatment. Based on the risk factors identified, measures are recommended such as implementing health education for all migrant patients before chemotherapy and encouraging primary care workers to supervise patients.

Description: by Hong-Lim Kim, Ji Hyun Jeon, Tae-Hyung Koo, U-Young Lee, Eojin Jeong, Myung-Hoon Chun, Jung-Il Moon, Stephen C. Massey, In-Beom Kim In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a , thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a . The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.

Description: by Bryan Bedrosian, Derek Craighead, Ross Crandall Studies suggest hunter discarded viscera of big game animals (i.e., offal) is a source of lead available to scavengers. We investigated the incidence of lead exposure in bald eagles in Wyoming during the big game hunting season, the influx of eagles into our study area during the hunt, the geographic origins of eagles exposed to lead, and the efficacy of using non-lead rifle ammunition to reduce lead in eagles. We tested 81 blood samples from bald eagles before, during and after the big game hunting seasons in 2005–2010, excluding 2008, and found eagles had significantly higher lead levels during the hunt. We found 24% of eagles tested had levels indicating at least clinical exposure (〉60 ug/dL) during the hunt while no birds did during the non-hunting seasons. We performed driving surveys from 2009–2010 to measure eagle abundance and found evidence to suggest that eagles are attracted to the study area during the hunt. We fitted 10 eagles with satellite transmitters captured during the hunt and all migrated south after the cessation of the hunt. One returned to our study area while the remaining nine traveled north to summer/breed in Canada. The following fall, 80% returned to our study area for the hunting season, indicating that offal provides a seasonal attractant for eagles. We fitted three local breeding eagles with satellite transmitters and none left their breeding territories to feed on offal during the hunt, indicating that lead ingestion may be affecting migrants to a greater degree. During the 2009 and 2010 hunting seasons we provided non-lead rifle ammunition to local hunters and recorded that 24% and 31% of successful hunters used non-lead ammunition, respectively. We found the use of non-lead ammunition significantly reduced lead exposure in eagles, suggesting this is a viable solution to reduce lead exposure in eagles.

Description: by Chandrashekhar T. Sreeramareddy, T. N. Sathyanarayana, H. N. Harsha Kumar Background Information about utilization of health services and associated factors are useful for improving service delivery to achieve universal health coverage. Methods Data on a sample of ever-married women from India Demographic and Health survey 2005–06 was used. Mothers of children aged 0–59 months were asked about child’s illnesses and type of health facilities where treatment was given during 15 days prior to the survey date. Type of health facilities were grouped as informal provider, public provider and private provider. Factors associated with utilization of health services for diarrhea and fever/cough was assessed according to Andersen’s health behavior model. Multinomial logistic regression analyses were done considering sampling weights for complex sampling design. Results A total of 48,679 of ever-married women reported that 9.1% 14.8% and 17.67% of their children had diarrhea, fever and cough respectively. Nearly one-third of the children with diarrhea and fever/cough did not receive any treatment. Two-thirds of children who received treatment were from private health care providers (HCPs). Among predisposing factors, children aged 1–2 years and those born at health facility (public/private) were more likely to be taken to any type of HCP during illness. Among enabling factors, as compared to poorer household, wealthier households were 2.5 times more likely to choose private HCPs for any illness. Children in rural areas were likely to be taken to any type of HCP for diarrhea but rural children were less likely to utilize private HCP for fever/cough. ‘Need’ factors i.e. children having severe symptoms were 2–3 times more likely to be taken to any type of HCP. Conclusion Private HCPs were preferred for treatment of childhood illnesses. Involvement of private HCPs may be considered while planning child health programs. Health insurance scheme for childhood illnesses may to protect economically weaker sections from out-of-pocket health expenditure during child illness.

Description: by Michael T. Cheeseman, Kate Vowell, Tertius A. Hough, Lynn Jones, Paras Pathak, Hayley E. Tyrer, Michelle Kelly, Roger Cox, Madhuri V. Warren, Jo Peters GNAS/Gnas encodes G s α that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G s α. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas . We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml . These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G s α. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G s α deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G s α and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G s α signalling pathways play a vital role in repressing ectopic bone formation.

Description: by Xavier Bosch, Cristina Hernández, Juan M. Pericas, Pamela Doti, Ana Marušić Background It is not clear which research misconduct policies are adopted by biomedical journals. This study assessed the prevalence and content policies of the most influential biomedical journals on misconduct and procedures for handling and responding to allegations of misconduct. Methods We conducted a cross-sectional study of misconduct policies of 399 high-impact biomedical journals in 27 biomedical categories of the Journal Citation Reports in December 2011. Journal websites were reviewed for information relevant to misconduct policies. Results Of 399 journals, 140 (35.1%) provided explicit definitions of misconduct. Falsification was explicitly mentioned by 113 (28.3%) journals, fabrication by 104 (26.1%), plagiarism by 224 (56.1%), duplication by 242 (60.7%) and image manipulation by 154 (38.6%). Procedures for responding to misconduct were described in 179 (44.9%) websites, including retraction, (30.8%) and expression of concern (16.3%). Plagiarism-checking services were used by 112 (28.1%) journals. The prevalences of all types of misconduct policies were higher in journals that endorsed any policy from editors’ associations, Office of Research Integrity or professional societies compared to those that did not state adherence to these policy-producing bodies. Elsevier and Wiley-Blackwell had the most journals included (22.6% and 14.8%, respectively), with Wiley journals having greater a prevalence of misconduct definition and policies on falsification, fabrication and expression of concern and Elsevier of plagiarism-checking services. Conclusions Only a third of top-ranking peer-reviewed journals had publicly-available definitions of misconduct and less than a half described procedures for handling allegations of misconduct. As endorsement of international policies from policy-producing bodies was positively associated with implementation of policies and procedures, journals and their publishers should standardize their policies globally in order to increase public trust in the integrity of the published record in biomedicine.

Description: by Ru Li, Ehsan Khafipour, Denis O. Krause, Martin H. Entz, Teresa R. de Kievit, W. G. Dilantha Fernando It has been debated how different farming systems influence the composition of soil bacterial communities, which are crucial for maintaining soil health. In this research, we applied high-throughput pyrosequencing of V1 to V3 regions of bacterial 16S rRNA genes to gain further insight into how organic and conventional farming systems and crop rotation influence bulk soil bacterial communities. A 2×2 factorial experiment consisted of two agriculture management systems (organic versus conventional) and two crop rotations (flax-oat-fababean-wheat versus flax-alfalfa-alfalfa-wheat) was conducted at the Glenlea Long-Term Crop Rotation and Management Station, which is Canada’s oldest organic-conventional management study field. Results revealed that there is a significant difference in the composition of bacterial genera between organic and conventional management systems but crop rotation was not a discriminator factor. Organic farming was associated with higher relative abundance of Proteobacteria , while Actinobacteria and Chloroflexi were more abundant in conventional farming. The dominant genera including Blastococcus, Microlunatus, Pseudonocardia, Solirubrobacter, Brevundimonas, Pseudomonas , and Stenotrophomonas exhibited significant variation between the organic and conventional farming systems. The relative abundance of bacterial communities at the phylum and class level was correlated to soil pH rather than other edaphic properties. In addition, it was found that Proteobacteria and Actinobacteria were more sensitive to pH variation.

Description: by Rohan Steel, Ryan S. Cross, Sarah L. Ellis, Robin L. Anderson Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

Description: by Jens Mani, Jasmina Makarević, Eva Juengel, Hanns Ackermann, Karen Nelson, Axel Haferkamp, Roman A. Blaheta The pathophysiologic mechanisms behind urologic disease are increasingly being elucidated. The object of this investigation was to evaluate the publication policies of urologic journals during a period of progressively better understanding and management of urologic disease. Based on the ISI Web of Knowledge Journal Citation Reports and the PubMed database, the number and percentage of original experimental, original clinical, review or commentarial articles published between 2002–2010 in six leading urologic journals were analyzed. “British Journal of Urology International”, “European Urology”, “Urologic Oncology-Seminars and Original Investigations” (“Urologic Oncology”), “Urology”, “The Journal of Urology”, and “World Journal of Urology” were chosen, because these journals publish articles in all four categories. The publication policies of the six journals were very heterogeneous during the time period from 2002 to 2010. The percentage of original experimental and original clinical articles, related to all categories, remained the same in “British Journal of Urology International”, “Urologic Oncology”, “Urology” and “The Journal of Urology”. The percentage of experimental reports in “World Journal of Urology” between 2002–2010 significantly increased from 10 to 20%. A distinct elevation in the percentage of commentarial articles accompanied by a reduction of clinical articles became evident in “European Urology” which significantly correlated with a large increase in the journal’s impact factor. No clearly superior policy could be identified with regard to a general increase in the impact factors from all the journals. The publication policy of urologic journals does not expressly reflect the increase in scientific knowledge, which has occurred over the period 2002–2010. One way of increasing the exposure of urologists to research and expand the interface between experimental and clinical research, would be to enlarge the percentage of experimental articles published. There is no indication that such policy would be detrimental to a journal’s impact factor.

Description: by Naresh C. Laddha, Mitesh Dwivedi, Rasheedunnisa Begum Abstract Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of TNF-α are involved in the regulation of its expression. The present study explores TNF -α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF -α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF -α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF -α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo.

Description: by Subhadeep Chakrabarti, Sandra T. Davidge Estrogen, the female sex hormone, is known to exert anti-inflammatory and anti-atherogenic effects. Traditionally, estrogen effects were believed to be largely mediated through the classical estrogen receptors (ERs). However, there is increasing evidence that G-protein coupled receptor 30 (GPR30), a novel estrogen receptor, can mediate many estrogenic effects on the vasculature. Despite this, the localization and functional significance of GPR30 in the human vascular endothelium remains poorly understood. Given this background, we examined the subcellular location and potential anti-inflammatory roles of GPR30 using human umbilical vein endothelial cells as a model system. Inflammatory changes were induced by treatment with tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in atherogenesis and many other inflammatory conditions. We found that GPR30 was located predominantly in the endothelial cell nuclei. Treatment with the selective GPR30 agonist G-1 partially attenuated the TNF induced upregulation of pro-inflammatory proteins such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was completely abolished by the selective GPR30 antagonist G-15, suggesting that it was indeed mediated in a GPR30 dependent manner. Interestingly, estrogen alone had no effects on TNF-treated endothelium. Concomitant activation of the classical ERs blocked the anti-inflammatory effects of G-1, indicating opposing effects of GPR30 and the classical ERs. Our findings demonstrate that endothelial GPR30 is a novel regulator of the inflammatory response which could be a potential therapeutic target against atherosclerosis and other inflammatory diseases.

Description: by Rosalinde K. E. Poortvliet, Ian Ford, Suzanne M. Lloyd, Naveed Sattar, Simon P. Mooijaart, Anton J. M. de Craen, Rudi G. J. Westendorp, J. Wouter Jukema, Christopher J. Packard, Jacobijn Gussekloo, Wouter de Ruijter, David J. Stott Variability in blood pressure predicts cardiovascular disease in young- and middle-aged subjects, but relevant data for older individuals are sparse. We analysed data from the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) study of 5804 participants aged 70–82 years with a history of, or risk factors for cardiovascular disease. Visit-to-visit variability in blood pressure (standard deviation) was determined using a minimum of five measurements over 1 year; an inception cohort of 4819 subjects had subsequent in-trial 3 years follow-up; longer-term follow-up (mean 7.1 years) was available for 1808 subjects. Higher systolic blood pressure variability independently predicted long-term follow-up vascular and total mortality (hazard ratio per 5 mmHg increase in standard deviation of systolic blood pressure = 1.2, 95% confidence interval 1.1–1.4; hazard ratio 1.1, 95% confidence interval 1.1–1.2, respectively). Variability in diastolic blood pressure associated with increased risk for coronary events (hazard ratio 1.5, 95% confidence interval 1.2–1.8 for each 5 mmHg increase), heart failure hospitalisation (hazard ratio 1.4, 95% confidence interval 1.1–1.8) and vascular (hazard ratio 1.4, 95% confidence interval 1.1–1.7) and total mortality (hazard ratio 1.3, 95% confidence interval 1.1–1.5), all in long-term follow-up. Pulse pressure variability was associated with increased stroke risk (hazard ratio 1.2, 95% confidence interval 1.0–1.4 for each 5 mmHg increase), vascular mortality (hazard ratio 1.2, 95% confidence interval 1.0–1.3) and total mortality (hazard ratio 1.1, 95% confidence interval 1.0–1.2), all in long-term follow-up. All associations were independent of respective mean blood pressure levels, age, gender, in-trial treatment group (pravastatin or placebo) and prior vascular disease and cardiovascular disease risk factors. Our observations suggest variability in diastolic blood pressure is more strongly associated with vascular or total mortality than is systolic pressure variability in older high-risk subjects.

Description: by Jonathan A. Hare, Mark J. Wuenschel, Matthew E. Kimball We couple a species range limit hypothesis with the output of an ensemble of general circulation models to project the poleward range limit of gray snapper. Using laboratory-derived thermal limits and statistical downscaling from IPCC AR4 general circulation models, we project that gray snapper will shift northwards; the magnitude of this shift is dependent on the magnitude of climate change. We also evaluate the uncertainty in our projection and find that statistical uncertainty associated with the experimentally-derived thermal limits is the largest contributor (∼ 65%) to overall quantified uncertainty. This finding argues for more experimental work aimed at understanding and parameterizing the effects of climate change and variability on marine species.

Description: by Tal Raz, Reut Avni, Yoseph Addadi, Yoni Cohen, Ariel J. Jaffa, Brian Hemmings, Joel R. Garbow, Michal Neeman In mammalian pregnancy, maternal cardiovascular adaptations must match the requirements of the growing fetus(es), and respond to physiologic and pathologic conditions. Such adaptations are particularly demanding for mammals bearing large-litter pregnancies, with their inherent conflict between the interests of each individual fetus and the welfare of the entire progeny. The mouse is the most common animal model used to study development and genetics, as well as pregnancy-related diseases. Previous studies suggested that in mice, maternal blood flow to the placentas occurs via a single arterial uterine loop generated by arterial-arterial anastomosis of the uterine artery to the uterine branch of the ovarian artery, resulting in counter bi-directional blood flow. However, we provide here experimental evidence that each placenta is actually supplied by two distinct arterial inputs stemming from the uterine artery and from the uterine branch of the ovarian artery, with position-dependent contribution of flow from each source. Moreover, we report significant positional- and inter-fetal dependent alteration of placental perfusion, which were detected by in vivo MRI and fluorescence imaging. Maternal blood flow to the placentas was dependent on litter size and was attenuated for placentas located centrally along the uterine horn. Distinctive apposing, inter-fetal hemodynamic effects of either reduced or elevated maternal blood flow, were measured for placenta of normal fetuses that are positioned adjacent to either pathological, or to hypovascular Akt1 -deficient placentas, respectively. The results reported here underscore the critical importance of confounding local and systemic in utero effects on phenotype presentation, in general and in the setting of genetically modified mice. The unique robustness and plasticity of the uterine vasculature architecture, as reported in this study, can explain the ability to accommodate varying litter sizes, sustain large-litter pregnancies and overcome pathologic challenges. Remarkably, the dual arterial supply is evolutionary conserved in mammals bearing a single offspring, including primates.

Description: by Zhipeng Qu, David L. Adelson ncRNAs (non-coding RNAs), in particular long ncRNAs, represent a significant proportion of the vertebrate transcriptome and probably regulate many biological processes. We used publically available ESTs (Expressed Sequence Tags) from human, mouse and zebrafish and a previously published analysis pipeline to annotate and analyze the vertebrate non-protein-coding transcriptome. Comparative analysis confirmed some previously described features of intergenic ncRNAs, such as a positionally biased distribution with respect to regulatory or development related protein-coding genes, and weak but clear sequence conservation across species. Significantly, comparative analysis of developmental and regulatory genes proximate to long ncRNAs indicated that the only conserved relationship of these genes to neighbor long ncRNAs was with respect to genes expressed in human brain, suggesting a conserved, ncRNA cis-regulatory network in vertebrate nervous system development. Most of the relationships between long ncRNAs and proximate coding genes were not conserved, providing evidence for the rapid evolution of species-specific gene associated long ncRNAs. We have reconstructed and annotated over 130,000 long ncRNAs in these three species, providing a significantly expanded number of candidates for functional testing by the research community.

Description: by David J. Leibly, Trang Nhu Nguyen, Louis T. Kao, Stephen N. Hewitt, Lynn K. Barrett, Wesley C. Van Voorhis Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli ( E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo . We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl 2 , proline, xylitol, NDSB 201, CTAB and K 2 PO 4 ) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

Description: by Orphal Colleye, Eric Parmentier Background Clownfishes (Pomacentridae) are brightly colored coral reef fishes well known for their mutualistic symbiosis with tropical sea anemones. These fishes live in social groups in which there is a size-based dominance hierarchy. In this structure where sex is socially controlled, agonistic interactions are numerous and serve to maintain size differences between individuals adjacent in rank. Clownfishes are also prolific callers whose sounds seem to play an important role in the social hierarchy. Here, we aim to review and to synthesize the diversity of sounds produced by clownfishes in order to emphasize the importance of acoustic signals in their way of life. Methodology/Principal Findings Recording the different acoustic behaviors indicated that sounds are divided into two main categories: aggressive sounds produced in conjunction with threat postures (charge and chase), and submissive sounds always emitted when fish exhibited head shaking movements (i.e. a submissive posture). Both types of sounds showed size-related intraspecific variation in dominant frequency and pulse duration: smaller individuals produce higher frequency and shorter duration pulses than larger ones, and inversely. Consequently, these sonic features might be useful cues for individual recognition within the group. This observation is of significant importance due to the size-based hierarchy in clownfish group. On the other hand, no acoustic signal was associated with the different reproductive activities. Conclusions/Significance Unlike other pomacentrids, sounds are not produced for mate attraction in clownfishes but to reach and to defend the competition for breeding status, which explains why constraints are not important enough for promoting call diversification in this group.

Description: by Staša Milojević References are an essential component of research articles and therefore of scientific communication. In this study we investigate referencing (citing) behavior in five diverse fields (astronomy, mathematics, robotics, ecology and economics) based on 213,756 core journal articles. At the macro level we find: (a) a steady increase in the number of references per article over the period studied (50 years), which in some fields is due to a higher rate of usage, while in others reflects longer articles and (b) an increase in all fields in the fraction of older, foundational references since the 1980s, with no obvious change in citing patterns associated with the introduction of the Internet. At the meso level we explore current (2006–2010) referencing behavior of different categories of authors (21,562 total) within each field, based on their academic age, productivity and collaborative practices. Contrary to some previous findings and expectations we find that senior researchers use references at the same rate as their junior colleagues, with similar rates of re-citation (use of same references in multiple papers). High Modified Price Index (MPI, which measures the speed of the research front more accurately than the traditional Price Index) of senior authors indicates that their research has the similar cutting-edge aspect as that of their younger colleagues. In all fields both the productive researchers and especially those who collaborate more use a significantly lower fraction of foundational references and have much higher MPI and lower re-citation rates, i.e., they are the ones pushing the research front regardless of researcher age. This paper introduces improved bibliometric methods to measure the speed of the research front, disambiguate lead authors in co-authored papers and decouple measures of productivity and collaboration.

Description: by James R. Petrie, Pushkar Shrestha, Xue-Rong Zhou, Maged P. Mansour, Qing Liu, Srinivas Belide, Peter D. Nichols, Surinder P. Singh Background Omega-3 long-chain (≥C 20 ) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C 20 EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high. Methodology/Principal Findings The transgenic seed production of the particularly important C 22 DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C 22 fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C 18 ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway. Conclusions/Significance The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.

Description: by Hyunju Kim, Jung-A Choi, Geun-Soo Park, Jae-Hong Kim Background The elevated production of interleukin (IL)–8 is critically associated with invasiveness and metastatic potential in breast cancer cells. However, the intracellular signaling pathway responsible for up-regulation of IL-8 production in breast cancer cells has remained unclear. Methodology/Principal Findings In this study, we report that the expression of BLT2 is markedly up-regulated in the highly aggressive human breast cancer cell lines MDA-MB-231 and MDA-MB-435 compared with MCF-10A immortalized human mammary epithelial cells, as determined by RT-PCR, real-time PCR and FACS analysis. Blockade of BLT2 with BLT2 siRNA knockdown or BLT2 inhibitor treatment downregulated IL-8 production and thereby diminished the invasiveness of aggressive breast cancer cells, analyzed by Matrigel invasion chamber assays. We further characterized the downstream signaling mechanism by which BLT2 stimulates IL-8 production and identified critical mediatory roles for the generation of reactive oxygen species (ROS) and the consequent activation of the transcription factor NF-κB. Moreover, blockade of BLT2 suppressed the formation of metastatic lung nodules by MDA-MB-231 cells in both experimental and orthotopic metastasis models. Conclusions/Significance Taken together, our study demonstrates that a BLT2–ROS–NF-κB pathway up-regulates IL-8 production in MDA-MB-231 and MDA-MB-435 cells, thereby contributing to the invasiveness of these aggressive breast cancer cells. Our findings provide insight into the molecular mechanism of invasiveness in breast cancer.

Description: by Farideh Mirzayans, Rotem Lavy, Jonathan Penner-Chea, Fred B. Berry Hierarchal transcriptional regulatory networks function to control the correct spatiotemporal patterning of the mammalian skeletal system. One such factor, the forkhead box transcription factor FOXC1 is necessary for the correct formation of the axial and craniofacial skeleton. Previous studies have demonstrated that the frontal and parietal bones of the skull fail to develop in mice deficient for Foxc1. Furthermore expression of the Msx2 homeobox gene, an essential regulator of calvarial bone development is absent in the skull mesenchymal progenitors of Foxc1 mutant mice. Thus we sought to determine whether Msx2 was a direct target of FOXC1 transcriptional regulation. Here, we demonstrate that elevated expression of FOXC1 can increase endogenous Msx2 mRNA levels. Chromatin immunoprecipitation experiments reveal that FOXC1 occupies a conserved element in the MSX2 promoter. Using a luciferase reporter assay, we demonstrate that FOXC1 can stimulate the activity of the both human and mouse MSX2 promoters. We also report that reducing FOXC1 levels by RNA interference leads to a decrease in MSX2 expression. Finally, we demonstrate that heterologous expression of Foxc1 in C2C12 cells results in elevated alkaline phosphatase activity and increased expression of Runx2 and Msx2. These data indicate that Foxc1 expression leads to a similar enhanced osteogenic differentiation phenotype as observed with Msx2 overexpression. Together these findings suggest that a Foxc1 -〉 Msx2 regulatory network functions in the initial stages of osteoblast differentiation.

Description: by Rocco Rossano, Marilena Larocca, Teresa Polito, Anna Maria Perna, Maria Carmela Padula, Giuseppe Martelli, Paolo Riccio Honey is a sweet and healthy food produced by honeybees ( Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

Description: by Aines Castro-Prieto, Bettina Wachter, Joerg Melzheimer, Susanne Thalwitzer, Heribert Hofer, Simone Sommer Background Genes under selection provide ecologically important information useful for conservation issues. Major histocompatibility complex (MHC) class I and II genes are essential for the immune defence against pathogens from intracellular (e.g. viruses) and extracellular (e.g. helminths) origins, respectively. Serosurvey studies in Namibian cheetahs ( Acinonyx juabuts ) revealed higher exposure to viral pathogens in individuals from north-central than east-central regions. Here we examined whether the observed differences in exposure to viruses influence the patterns of genetic variation and differentiation at MHC loci in 88 free-ranging Namibian cheetahs. Methodology/Principal Findings Genetic variation at MHC I and II loci was assessed through single-stranded conformation polymorphism (SSCP) analysis and sequencing. While the overall allelic diversity did not differ, we observed a high genetic differentiation at MHC class I loci between cheetahs from north-central and east-central Namibia. No such differentiation in MHC class II and neutral markers were found. Conclusions/Significance Our results suggest that MHC class I variation mirrors the variation in selection pressure imposed by viruses in free-ranging cheetahs across Namibian farmland. This is of high significance for future management and conservation programs of this species.

Description: by Nobuaki Shindoh, Akinori Yoda, Yuka Yoda, Timothy J. Sullivan, Oliver Weigert, Andrew A. Lane, Nadja Kopp, Liat Bird, Scott J. Rodig, Edward A. Fox, David M. Weinstock There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo .

Description: by Wei Zhao, Merina Varghese, Prashant Vempati, Anastasiya Dzhun, Alice Cheng, Jun Wang, Dale Lange, Amanda Bilski, Irene Faravelli, Giulio Maria Pasinetti Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and finally death. ALS patients suffer from asthenia and their progressive weakness negatively impacts quality of life, limiting their daily activities. They have impaired energy balance linked to lower activity of mitochondrial electron transport chain enzymes in ALS spinal cord, suggesting that improving mitochondrial function may present a therapeutic approach for ALS. When fed a ketogenic diet, the G93A ALS mouse shows a significant increase in serum ketones as well as a significantly slower progression of weakness and lower mortality rate. In this study, we treated SOD1-G93A mice with caprylic triglyceride, a medium chain triglyceride that is metabolized into ketone bodies and can serve as an alternate energy substrate for neuronal metabolism. Treatment with caprylic triglyceride attenuated progression of weakness and protected spinal cord motor neuron loss in SOD1-G93A transgenic animals, significantly improving their performance even though there was no significant benefit regarding the survival of the ALS transgenic animals. We found that caprylic triglyceride significantly promoted the mitochondrial oxygen consumption rate in vivo . Our results demonstrated that caprylic triglyceride alleviates ALS-type motor impairment through restoration of energy metabolism in SOD1-G93A ALS mice, especially during the overt stage of the disease. These data indicate the feasibility of using caprylic acid as an easily administered treatment with a high impact on the quality of life of ALS patients.

Description: by Gordon M. Cann, Zulfiqar G. Gulzar, Samantha Cooper, Robin Li, Shujun Luo, Mai Tat, Sarah Stuart, Gary Schroth, Sandhya Srinivas, Mostafa Ronaghi, James D. Brooks, AmirAli H. Talasaz Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Description: by Zong-xiu Wang, Rui-ping Deng, He-Wei Jiang, Shu-Juan Guo, Huang-ying Le, Xiao-dong Zhao, Chien-Sheng Chen, Ji-bin Zhang, Sheng-ce Tao Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli , our study greatly extends our understanding of protein glycosylation in prokaryotes.

Description: by Luke Mander, Cassandra J. Wesseln, Jennifer C. McElwain, Surangi W. Punyasena The interpretation of biotic changes in the geological past relies on the assumption that samples from different time intervals represent an equivalent suite of natural sampling conditions. As a result, detailed investigations of taphonomic regimes during intervals of major biotic upheaval, such as mass extinctions, are crucial. In this paper, we have used variations in the frequency of chemical and mechanical sporomorph (pollen and spore) damage as a guide to taphonomic regimes across the Triassic–Jurassic mass extinction (Tr-J; ∼201.3 Ma) at a boundary section at Astartekløft, East Greenland. We find that the frequency of sporomorph damage is extremely variable in samples from this locality. This likely reflects a combination of taxon-specific susceptibility to damage and the mixing of sporomorphs from a mosaic of environments and taphonomic regimes. The stratigraphic interval containing evidence of plant extinction and compositional change in the source vegetation at Astartekløft is not marked by a consistent rise or fall in the frequency of sporomorph damage. This indicates that natural taphonomic regimes did not shift radically during this critical interval. We find no evidence of a consistent relationship between the taxonomic richness of sporomorph assemblages and the frequency of damage among sporomorphs at Astartekløft. This indicates that previously reported patterns of sporomorph richness across the Tr-J at this locality are likely to be robust. Taken together, our results suggest that the patterns of vegetation change at Astartekløft represent a real biological response to environmental change at the Tr-J.

Description: by Janice A. Sabin, Maddalena Marini, Brian A. Nosek Overweight patients report weight discrimination in health care settings and subsequent avoidance of routine preventive health care. The purpose of this study was to examine implicit and explicit attitudes about weight among a large group of medical doctors (MDs) to determine the pervasiveness of negative attitudes about weight among MDs. Test-takers voluntarily accessed a public Web site, known as Project Implicit®, and opted to complete the Weight Implicit Association Test (IAT) (N = 359,261). A sub-sample identified their highest level of education as MD (N = 2,284). Among the MDs, 55% were female, 78% reported their race as white, and 62% had a normal range BMI. This large sample of test-takers showed strong implicit anti-fat bias (Cohen’s d =  1.0). MDs, on average, also showed strong implicit anti-fat bias (Cohen’s d =  0.93). All test-takers and the MD sub-sample reported a strong preference for thin people rather than fat people or a strong explicit anti-fat bias. We conclude that strong implicit and explicit anti-fat bias is as pervasive among MDs as it is among the general public. An important area for future research is to investigate the association between providers’ implicit and explicit attitudes about weight, patient reports of weight discrimination in health care, and quality of care delivered to overweight patients.

Description: by Shuo Liang, Jingsong Hu, Weijun Cao, Sanjun Cai Background CYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of colorectal cancer (CRC). In the past decade, the relationship between CYP2C9 common polymorphisms (R144C and I359L) and CRC has been reported in various ethnic groups; however, these studies have yielded contradictory results. To investigate this inconsistency, we performed this meta-analysis. Methods Databases including Pubmed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. Results A total of 13 articles involving 9,463 cases and 11,416 controls were included. Overall, the summary odds ratio of CRC was 0.98 (95% CI: 0.89−1.06) and 0.99 (95% CI: 0.87−1.14) for CYP2C9 144C and 359L alleles, respectively. No significant results were observed using dominant or recessive genetic model for these polymorphisms. In the stratified analyses according to ethnicity and sex, no evidence of any gene-disease association was obtained. Conclusions This meta-analysis suggests that the CYP2C9 may not be associated with colorectal cancer development.

Description: by Serge A. Wich, David Gaveau, Nicola Abram, Marc Ancrenaz, Alessandro Baccini, Stephen Brend, Lisa Curran, Roberto A. Delgado, Andi Erman, Gabriella M. Fredriksson, Benoit Goossens, Simon J. Husson, Isabelle Lackman, Andrew J. Marshall, Anita Naomi, Elis Molidena, Nardiyono, Anton Nurcahyo, Kisar Odom, Adventus Panda, Purnomo, Andjar Rafiastanto, Dessy Ratnasari, Adi H. Santana, Imam Sapari, Carel P. van Schaik, Jamartin Sihite, Stephanie Spehar, Eddy Santoso, Amat Suyoko, Albertus Tiju, Graham Usher, Sri Suci Utami Atmoko, Erik P. Willems, Erik Meijaard The geographic distribution of Bornean orang-utans and its overlap with existing land-use categories (protected areas, logging and plantation concessions) is a necessary foundation to prioritize conservation planning. Based on an extensive orang-utan survey dataset and a number of environmental variables, we modelled an orang-utan distribution map. The modelled orang-utan distribution map covers 155,106 km 2 (21% of Borneo's landmass) and reveals four distinct distribution areas. The most important environmental predictors are annual rainfall and land cover. The overlap of the orang-utan distribution with land-use categories reveals that only 22% of the distribution lies in protected areas, but that 29% lies in natural forest concessions. A further 19% and 6% occurs in largely undeveloped oil palm and tree plantation concessions, respectively. The remaining 24% of the orang-utan distribution range occurs outside of protected areas and outside of concessions. An estimated 49% of the orang-utan distribution will be lost if all forest outside of protected areas and logging concessions is lost. To avoid this potential decline plantation development in orang-utan habitats must be halted because it infringes on national laws of species protection. Further growth of the plantation sector should be achieved through increasing yields in existing plantations and expansion of new plantations into areas that have already been deforested. To reach this goal a large scale island-wide land-use masterplan is needed that clarifies which possible land uses and managements are allowed in the landscape and provides new standardized strategic conservation policies. Such a process should make much better use of non-market values of ecosystem services of forests such as water provision, flood control, carbon sequestration, and sources of livelihood for rural communities. Presently land use planning is more driven by vested interests and direct and immediate economic gains, rather than by approaches that take into consideration social equity and environmental sustainability.

Description: by Hideyuki Arata, Hiroshi Komatsu, Kazuo Hosokawa, Mizuo Maeda Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.

Description: by Alessia Garufi, Giuseppa Pistritto, Claudia Ceci, Livia Di Renzo, Roberta Santarelli, Alberto Faggioni, Mara Cirone, Gabriella D’Orazi Background Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo . HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE 2 ). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE 2 production after HIPK2 depletion and how to modulate it. Methodology/Principal findings We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE 2 generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE 2 /VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation. Conclusion/Significance These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE 2 /VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.

Description: by Rosaria Scozzari, Andrea Massaia, Eugenia D’Atanasio, Natalie M. Myres, Ugo A. Perego, Beniamino Trombetta, Fulvio Cruciani One hundred and forty-six previously detected mutations were more precisely positioned in the human Y chromosome phylogeny by the analysis of 51 representative Y chromosome haplogroups and the use of 59 mutations from literature. Twenty-two new mutations were also described and incorporated in the revised phylogeny. This analysis made it possible to identify new haplogroups and to resolve a deep trifurcation within haplogroup B2. Our data provide a highly resolved branching in the African-specific portion of the Y tree and support the hypothesis of an origin in the north-western quadrant of the African continent for the human MSY diversity.

Description: by Charlene S. Dezzutti, Elizabeth R. Brown, Bernard Moncla, Julie Russo, Marilyn Cost, Lin Wang, Kevin Uranker, Ratiya P. Kunjara Na Ayudhya, Kara Pryke, Jim Pickett, Marc-André LeBlanc, Lisa C. Rohan Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. Therefore, safety and anti-HIV-1 activity of over-the-counter (OTC) aqueous- (n = 10), lipid- (n = 2), and silicone-based (n = 2) products were tested. The rheological properties of the lipid-based lubricants precluded testing with the exception of explant safety testing. Six aqueous-based gels were hyperosmolar, two were nearly iso-osmolar, and two were hypo-osmolar. Evaluation of the panel of products showed Gynol II (a spermicidal gel containing 2% nonoxynol-9), KY Jelly, and Replens were toxic to Lactobacillus . Two nearly iso-osmolar aqueous- and both silicone-based gels were not toxic toward epithelial cell lines or ectocervical or colorectal explant tissues. Hyperosmolar lubricants demonstrated reduction of tissue viability and epithelial fracture/sloughing while the nearly iso-osmolar and silicon-based lubricants showed no significant changes in tissue viability or epithelial modifications. While most of the lubricants had no measurable anti-HIV-1 activity, three lubricants which retained cell viability did demonstrate modest anti-HIV-1 activity in vitro . To determine if this would result in protection of mucosal tissue or conversely determine if the epithelial damage associated with the hyperosmolar lubricants increased HIV-1 infection ex vivo , ectocervical tissue was exposed to selected lubricants and then challenged with HIV-1. None of the lubricants that had a moderate to high therapeutic index protected the mucosal tissue. These results show hyperosmolar lubricant gels were associated with cellular toxicity and epithelial damage while showing no anti-viral activity. The two iso-osmolar lubricants, Good Clean Love and PRÉ, and both silicone-based lubricants, Female Condom 2 lubricant and Wet Platinum, were the safest in our testing algorithm.

Description: by Marion de Toledo, Christelle Anguille, Laureline Roger, Pierre Roux, Gilles Gadea Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.

Description: by Jamal Stie, Deborah Fox The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans . Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis.

Description: by Lukasz Japtok, Katrin Schaper, Wolfgang Bäumer, Heinfried H. Radeke, Se Kyoo Jeong, Burkhard Kleuser Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P 2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P 2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.

Description: by Claudia Röser, Nadine Jordan, Sabine Balfanz, Arnd Baumann, Bernd Walz, Otto Baumann, Wolfgang Blenau Secretion in blowfly ( Calliphora vicina ) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP 3 )/Ca 2+ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs ( Cv5-ht2α , Cv5-ht7 ) that share high similarity with mammalian 5-HT 2 and 5-HT 7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α -transfected mammalian cells with 5-HT elevates cytosolic [Ca 2+ ] in a dose-dependent manner (EC 50  = 24 nM). In Cv5-ht7 -transfected cells, 5-HT produces a dose-dependent increase in [cAMP] i (EC 50  = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT 2α or Cv5-HT 7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT 2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT 7 receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv 5-HT 7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca 2+ - and cAMP-signalling cascades.

Description: by Ben J. Mans, Daniel de Klerk, Ronel Pienaar, Minique H. de Castro, Abdalla A. Latif Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae ( Nuttalliella namaqua ) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.

Description: by Hee-Jeong Jung, Hyo-Jin Jung, Nasar Uddin Ahmed, Jong-In Park, Kwon-Kyoo Kang, Yoonkang Hur, Yong-Pyo Lim, Ill-Sup Nou The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the S -locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same S -haplotype. Recognition of S -haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase ( SRK ) and S -locus protein 11 or S locus Cysteine-rich ( SP11/SCR ) protein. Here, we examined the function of S60, one SP11/SCR allele of B. rapa cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in B. rapa .

Description: by The PLOS ONE Editors

Description: by Na Wei, Lei Zhang, Huang Huang, Yue Chen, Jie Zheng, Xiao Zhou, Fan Yi, Quan Du, Zicai Liang It has been noted that target sites located in the coding region or the 3′-untranslated region (3′-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3′-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3′-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3′-UTR, while the silencing of nonnucleolytic AGO proteins (AGO1, AGO3 and AGO4) did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site.

Description: by Seiichi Tada, Takashi Andou, Takehiro Suzuki, Naoshi Dohmae, Eiry Kobatake, Yoshihiro Ito Polyethylene glycol (PEG) was genetically incorporated into a polypeptide. Stop-anticodon-containing tRNAs were acylated with PEG-containing amino acids and were then translated into polypeptides corresponding to DNA sequences containing the stop codons. The molecular weights of the PEG used were 170, 500, 700, 1000, and 2000 Da, and the translation was confirmed by mass spectrometry. The PEG incorporation ratio decreased as the molecular weight of PEG increased, and PEG with a molecular weight of 1000 Da was only slightly incorporated. Although improvement is required to increase the efficiency of the process, this study demonstrates the possibility of genetic PEGylation.

Description: by Jason M. Schmidt, Peter Sebastian, Shawn M. Wilder, Ann L. Rypstra While foraging theory predicts that predatory responses should be determined by the energy content and size of prey, it is becoming increasingly clear that carnivores regulate their intake of specific nutrients. We tested the hypothesis that prey nutrient composition and predator nutritional history affects foraging intensity, consumption, and prey selection by the wolf spider, Pardosa milvina . By altering the rearing environment for fruit flies, Drosophila melanogaster , we produced high quality flies containing more nitrogen and protein and less lipid than low quality fruit flies. In one experiment, we quantified the proportion of flies taken and consumption across a range of densities of either high or low quality flies and, in a second experiment, we determined the prey capture and consumption of spiders that had been maintained on contrasting diets prior to testing. In both cases, the proportion of prey captured declined with increasing prey density, which characterizes the Type II functional response that is typical of wolf spiders. Spiders with similar nutritional histories killed similar numbers of each prey type but consumed more of the low quality prey. Spiders provided high quality prey in the weeks prior to testing killed more prey than those on the low quality diet but there was no effect of prior diet on consumption. In the third experiment, spiders were maintained on contrasting diets for three weeks and then allowed to select from a mixture of high and low quality prey. Interestingly, feeding history affected prey preferences: spiders that had been on a low quality diet showed no preference but those on the high quality diet selected high quality flies from the mixture. Our results suggest that, even when prey size and species identity are controlled, the nutritional experience of the predator as well as the specific content of the prey shapes predator-prey interactions.

Description: by Kanyarat Thueng-in, Jeeraphong Thanongsaksrikul, Potjanee Srimanote, Kunan Bangphoomi, Ornnuthchar Poungpair, Santi Maneewatch, Kiattawee Choowongkomon, Wanpen Chaicumpa NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V H H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V H H from a humanized-camel VH/V H H display library. VH/V H H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V H H hallmark and were designated V H H6 and V H H24; other clones were conventional VH, designated VH9 and VH13. All VH/V H H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V H H6 and V H H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V H H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

Description: by Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Nora Schweitzer, Arndt Vogel, Hans Kreipe, Ulrich Lehmann Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the DLK1-MEG3 locus is frequently deregulated in HCC. Deregulation of DLK1 and MEG3 expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the DLK1-MEG3 locus correlates linearly with global loss of DNA methylation in HCC (r 2  = 0.63, p

Description: by Sophie Jasinski, Alain Lécureuil, Martine Miquel, Olivier Loudet, Sylvain Raffaele, Marine Froissard, Philippe Guerche Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana . Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2 , accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the β-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.

Description: by Rafi Shaik, Wusirika Ramakrishna Drought stress response is a complex trait regulated at multiple levels. Changes in the epigenetic and miRNA regulatory landscape can dramatically alter the outcome of a stress response. However, little is known about the scope and extent of these regulatory factors on drought related cellular processes and functions. To this end, we selected a list of 5468 drought responsive genes (DRGs) of rice identified in multiple microarray studies and mapped the DNA methylation regions found in a genome wide methylcytosine immunoprecipitation and sequencing (mCIP-Seq) study to their genic and promoter regions, identified the chromatin remodeling genes and the genes that are targets of miRNAs. We found statistically significant enrichment of DNA methylation reads and miRNA target sequences in DRGs compared to a random set of genes. About 75% of the DRGs annotated to be involved in chromatin remodeling were downregulated. We found one-third of the DRGs are targeted by two-thirds of all known/predicted miRNAs in rice which include many transcription factors targeted by more than five miRNAs. Clustering analysis of the DRGs with epigenetic and miRNA features revealed, upregulated cluster was enriched in drought tolerance mechanisms while the downregulated cluster was enriched in drought resistance mechanisms evident by their unique gene ontologies (GOs), protein-protein interactions (PPIs), specific transcription factors, protein domains and metabolic pathways. Further, we analyzed the proteome of two weeks old young rice plants treated with a global demethylating agent, 5-azacytidine (5-azaC), subjected to drought stress and identified 56 protein spots that are differentially expressed. Out of the 56 spots, 35 were differently expressed in the sample with both demethylation and drought stress treatments and 28 (50%) were part of DRGs considered in the bioinformatic analysis.

Description: by Siacia Broos, Laurent Malisoux, Daniel Theisen, Marc Francaux, Louise Deldicque, Martine A. Thomis A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3−/− (KO) mouse suggest a shift towards ‘slow’ metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an ‘equal environment condition’ what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier’s biopsy was the only one that presented type I fibers with a complete lack of type II x fibers. Properties of hybrid type II a /II x fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II a /II x fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm 2 ) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects ( P  = 0.75). Unloaded shortening velocity was highest in R-allele carriers ( P

Description: by Tal Varsano, Vanessa Taupin, Lixia Guo, Oscar Y. Baterina, Marilyn G. Farquhar Lysophosphatidic acid (LPA) mediates diverse cellular responses through the activation of at least six LPA receptors – LPA 1–6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA 1 contains a PDZ binding motif (SVV) identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA 1 but not that of other LPA receptors. LPA 1 colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA 1 to EEA1 early endosomes and promoted LPA 1 mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA 1 and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.

Description: by Ana Oliveira, Sandeep Singh, Axel Bidon-Chanal, Flavio Forti, Marcelo A. Martí, Leonardo Boechi, Dario A. Estrin, Kanak L. Dikshit, F. Javier Luque The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O 2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O 2 /CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Description: by Camilo Gouet, Belen Aburto, Cecilia Vergara, Magdalena Sanhueza Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca 2+ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.

Description: by Jocelyn Widagdo, Kylie M. Taylor, Peter W. Gunning, Edna C. Hardeman, Stephen J. Palmer GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxβ, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxβ. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxβ itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell.

Description: by Stephan Waldert, Laura Tüshaus, Christoph P. Kaller, Ad Aertsen, Carsten Mehring Functional near-infrared spectroscopy (fNIRS) has become an established tool to investigate brain function and is, due to its portability and resistance to electromagnetic noise, an interesting modality for brain-machine interfaces (BMIs). BMIs have been successfully realized using the decoding of movement kinematics from intra-cortical recordings in monkey and human. Recently, it has been shown that hemodynamic brain responses as measured by fMRI are modulated by the direction of hand movements. However, quantitative data on the decoding of movement direction from hemodynamic responses is still lacking and it remains unclear whether this can be achieved with fNIRS, which records signals at a lower spatial resolution but with the advantage of being portable. Here, we recorded brain activity with fNIRS above different cortical areas while subjects performed hand movements in two different directions. We found that hemodynamic signals in contralateral sensorimotor areas vary with the direction of movements, though only weakly. Using these signals, movement direction could be inferred on a single-trial basis with an accuracy of ∼65% on average across subjects. The temporal evolution of decoding accuracy resembled that of typical hemodynamic responses observed in motor experiments. Simultaneous recordings with a head tracking system showed that head movements, at least up to some extent, do not influence the decoding of fNIRS signals. Due to the low accuracy, fNIRS is not a viable alternative for BMIs utilizing decoding of movement direction. However, due to its relative resistance to head movements, it is promising for studies investigating brain activity during motor experiments.

Description: by Emilie Borgström, Peter Andersen, Fredrik Atterfelt, Inger Julander, Gunilla Källenius, Markus Maeurer, Ida Rosenkrands, Maria Widfeldt, Judith Bruchfeld, Hans Gaines Background There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood ( FASCIA ), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection. Methods and Findings Consecutive patients with suspected TB (n = 85), with microbiologically verified active pulmonary TB (n = 33), extra pulmonary TB (n = 21), clinical TB (n = 11), presumed latent TB infection (LTBI) (n = 23), patients negative for TB (n = 8) and 21 healthy controls were studied. Blood samples were analyzed with FASCIA and multiplex technology to determine and correlate proliferative responses and the value of 14 cytokines for diagnosis of Mtb infection: IFN- γ, IL-2, TNF-α, IP-10, IL-12, IL-6, IL-4, IL-5, IL-13, IL-17, MIP-1β, GM-CSF, IFN-α2 and IL-10. Cytokine levels for IFN-γ, IP-10, MIP-1β, IL-2, TNF-α, IL-6, IL-10, IL-13 and GM-CSF were significantly higher after stimulation with the Mtb specific antigens ESAT-6 and CFP-10 in patients with active TB compared to healthy controls (p

Description: by Lisanne J. Balk, Willemien A. E. J. de Vries–Knoppert, Axel Petzold Purpose Optical coherence tomography (OCT) allows quantification of the thickness of the retinal nerve fibre layer (RNFL) thickness, a potential biomarker for neurodegeneration. The estimated annual RNFL loss in multiple sclerosis amounts to 2 μ m using time domain OCT. The recognition of measurement artifacts exceeding this limit is relevant for the successful use of OCT as a secondary outcome measure in clinical trials. Methods Prospective study design. An exploratory pilot study (ring and volume scans) followed by a cohort study (1,980 OCT ring scans). The OCT measurement beam was placed off–axis to the left, right, top and bottom of the subjects pupil and RNFL thickness of these scans were compared to the centrally placed reference scans. Results Off–axis placement of the OCT measurement beam resulted in significant artifacts in RNFL thickness measurements (95%CI 9 μ m, maximal size of error 42 μ m). Off–axis placement gave characteristic patterns of the OCT live images which are not necessarily saved for review. Off–axis placement also causes regional inhomogeneity of reflectivity in the outer nuclear (ONL) and outer plexiform layers (OPL) which remains visible on scans saved for review. Conclusion Off–axis beam placement introduces measurement artifacts at a magnitude which may mask recognition of RNFL loss due to neurodegeneration in multiple sclerosis. The resulting pattern in the OCT live image can only be recognised by the technician capturing the scans. Once the averaged scans have been aligned this pattern is lost. Retrospective identification of this artifact is however possible by presence of regional inhomogeneity of ONL/OPL reflectivity. This simple and robust sign may be considered for quality control criteria in the setting of multicentre OCT studies. The practical advice of this study is to keep the OCT image in the acquisition window horizontally aligned whenever possible.

Description: by Baoxing Song, Xiaoquan Su, Jian Xu, Kang Ning The NGS (next generation sequencing)-based metagenomic data analysis is becoming the mainstream for the study of microbial communities. Faced with a large amount of data in metagenomic research, effective data visualization is important for scientists to effectively explore, interpret and manipulate such rich information. The visualization of the metagenomic data, especially multi-sample data, is one of the most critical challenges. The different data sample sources, sequencing approaches and heterogeneous data formats make robust and seamless data visualization difficult. Moreover, researchers have different focuses on metagenomic studies: taxonomical or functional, sample-centric or genome-centric, single sample or multiple samples, etc. However, current efforts in metagenomic data visualization cannot fulfill all of these needs, and it is extremely hard to organize all of these visualization effects in a systematic manner. An extendable, interactive visualization tool would be the method of choice to fulfill all of these visualization needs. In this paper, we have present MetaSee, an extendable toolbox that facilitates the interactive visualization of metagenomic samples of interests. The main components of MetaSee include: (I) a core visualization engine that is composed of different views for comparison of multiple samples: Global view, Phylogenetic view, Sample view and Taxa view, as well as link-out for more in-depth analysis; (II) front-end user interface with real metagenomic models that connect to the above core visualization engine and (III) open-source portal for the development of plug-ins for MetaSee. This integrative visualization tool not only provides the visualization effects, but also enables researchers to perform in-depth analysis of the metagenomic samples of interests. Moreover, its open-source portal allows for the design of plug-ins for MetaSee, which would facilitate the development of any additional visualization effects.

Description: by Letizia Zullo, Michela Chiappalone, Sergio Martinoia, Fabio Benfenati Developed biological systems are endowed with the ability of interacting with the environment; they sense the external state and react to it by changing their own internal state. Many attempts have been made to build ‘hybrids’ with the ability of perceiving, modifying and reacting to external modifications. Investigation of the rules that govern network changes in a hybrid system may lead to finding effective methods for ‘programming’ the neural tissue toward a desired task. Here we show a new perspective in the use of cortical neuronal cultures from embryonic mouse as a working platform to study targeted synaptic modifications. Differently from the common timing-based methods applied in bio-hybrids robotics, here we evaluated the importance of endogenous spike timing in the information processing. We characterized the influence of a spike-patterned stimulus in determining changes in neuronal synchronization (connectivity strength and precision) of the evoked spiking and bursting activity in the network. We show that tailoring the stimulation pattern upon a neuronal spike timing induces the network to respond stronger and more precisely to the stimulation. Interestingly, the induced modifications are conveyed more consistently in the burst timing. This increase in strength and precision may be a key in the interaction of the network with the external world and may be used to induce directional changes in bio-hybrid systems.

Description: by Yi Zhang, Chunxiong Luo, Ke Zou, Zhengwei Xie, Onn Brandman, Qi Ouyang, Hao Li A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.

Description: by The PLOS ONE Editors

Description: by Andrea Hartner, Carlos Menendez-Castro, Nada Cordasic, Ines Marek, Gudrun Volkert, Bernd Klanke, Wolfgang Rascher, Karl F. Hilgers In the normal kidney, the α8 integrin chain is expressed only on mesangial cells and vascular smooth muscle cells. α8 integrin ligates several matrix molecules including fibronectin, osteopontin and fibrillin-1. Recently, we detected de novo expression of α8 integrin on epithelial cells in renal cysts. We hypothesized that the α8 integrin chain is induced in tubular epithelia undergoing dedifferentiation and contributes to the fibrotic response in the tubulointerstitium (TI) after unilateral ureteral obstruction (UUO). After induction of UUO in rats by ligation of the right ureter, increased expression of the α8 integrin chain and its ligands was observed. In the TI, α8 integrin was localized to cytokeratin-positive epithelial cells and to interstitial fibroblasts; and colocalized with its ligands. In mice underexpressing α8 integrin UUO led to collagen deposition and fibroblast activation comparable to wild types. Mice lacking α8 integrin showed even more TI damage, fibroblast activation and collagen deposition after UUO compared to wild type mice. We conclude that the expression of the α8 integrin chain and its ligands is strongly induced in the TI after UUO, but underexpression of α8 integrin does not attenuate TI fibrosis. Mice lacking the α8 integrin chain are even more susceptible to TI damage than wild type mice. Thus, interactions of α8 integrin with its ligands do not seem to contribute to the development or progression of TI fibrosis in UUO. Targeting α8 integrin might not be a useful approach for anti-fibrotic therapy.

Description: by Miguel A. Lanaspa, Christina Cicerchi, Gabriela Garcia, Nanxing Li, Carlos A. Roncal-Jimenez, Christopher J. Rivard, Brandi Hunter, Ana Andrés-Hernando, Takuji Ishimoto, Laura G. Sánchez-Lozada, Jeffrey Thomas, Robert S. Hodges, Colin T. Mant, Richard J. Johnson Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.

Description: by Jiwei Zhang, Yanmei Zhang, Yaohua Zhong, Yinbo Qu, Tianhong Wang Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina ) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae , but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.

Description: by Diane S. Lazard, Christophe Vincent, Frédéric Venail, Paul Van de Heyning, Eric Truy, Olivier Sterkers, Piotr H. Skarzynski, Henryk Skarzynski, Karen Schauwers, Stephen O'Leary, Deborah Mawman, Bert Maat, Andrea Kleine-Punte, Alexander M. Huber, Kevin Green, Paul J. Govaerts, Bernard Fraysse, Richard Dowell, Norbert Dillier, Elaine Burke, Andy Beynon, François Bergeron, Deniz Başkent, Françoise Artières, Peter J. Blamey Objective To test the influence of multiple factors on cochlear implant (CI) speech performance in quiet and in noise for postlinguistically deaf adults, and to design a model of predicted auditory performance with a CI as a function of the significant factors. Study Design Retrospective multi-centre study. Methods Data from 2251 patients implanted since 2003 in 15 international centres were collected. Speech scores in quiet and in noise were converted into percentile ranks to remove differences between centres. The influence of 15 pre-, per- and postoperative factors, such as the duration of moderate hearing loss (mHL), the surgical approach (cochleostomy or round window approach), the angle of insertion, the percentage of active electrodes, and the brand of device were tested. The usual factors, duration of profound HL (pHL), age, etiology, duration of CI experience, that are already known to have an influence, were included in the statistical analyses. Results The significant factors were: the pure tone average threshold of the better ear, the brand of device, the percentage of active electrodes, the use of hearing aids (HAs) during the period of pHL, and the duration of mHL. Conclusions A new model was designed showing a decrease of performance that started during the period of mHL, and became faster during the period of pHL. The use of bilateral HAs slowed down the related central reorganization that is the likely cause of the decreased performance.

Description: by Daojun Yu, Yu Chen, Shenghai Wu, Baohong Wang, Yi-Wei Tang, Lanjuan Li Background Human papillomaviruses (HPV) are classified into high-risk HPV and low-risk HPV. The most common high-risk HPV types in cervical cancer are HPV 16 and 18, and the most common low-risk types causing genital warts are HPV 6 and HPV 11. In this study, applying novel AllGlo fluorescent probes, we established a quadruplex quantitative PCR method to simultaneously detect and differentiate HPV 6, 11, 16 and 18 in a single tube. Methods The specificity, the sensitivity, the detection limit, the reproducibility and the standard curve of this method were examined. Finally, clinical samples that had been tested previously by TaqMan PCR and HPV GenoArray (GA) test were used to verify the accuracy and sensitivity of the method. Results The assay has a sensitivity of 10 1 to 10 2 copies/test and a linear detection range from 10 1 to 10 8 copies/test. The mean amplification efficiencies for HPV 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r 2 ) of each standard curve was above 0.99 for plasmid templates ranging from 10 3 to 10 7 copies/test. There was 100% agreement between the AllGlo quadruplex quantitative PCR, HPV GA test and TaqMan uniplex qPCR methods. Conclusions AllGlo quadruplex quantitative PCR in a single tube has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. The convenient single tube format makes this assay a powerful tool for the studies of mixed infections by multiple pathogens, viral typing and viral load quantification.

Description: by Ilia Banakh, Leonel J. Gonez, Robyn M. Sutherland, Gaetano Naselli, Leonard C. Harrison Pancreas stem cells are a potential source of insulin-producing β cells for the therapy of diabetes. In adult tissues the ‘side population’ (SP) of cells that effluxes the DNA binding dye Hoechst 33342 through ATP-binding cassette transporters has stem cell properties. We hypothesised therefore that the SP would expand in response to β cell injury and give rise to functional β cells. SP cells were flow sorted from dissociated pancreas cells of adult mice, analysed for phenotype and cultured with growth promoting and differentiation factors before analysis for hormone expression and glucose-stimulated insulin secretion. SP cell number and colony forming potential (CFP) increased significantly in models of type diabetes, and after partial pancreatectomy, in the absence of hyperglycaemia. SP cells, ∼1% of total pancreas cells at 1 week of age, were enriched 〉10-fold for CFP compared to non-SP cells. Freshly isolated SP cells contained no insulin protein or RNA but expressed the homeobox transcription factor Pdx1 required for pancreas development and β cell function. Pdx1, along with surface expression of CD326 (Ep-Cam), was a marker of the colony forming and proliferation potential of SP cells. In serum-free medium with defined factors, SP cells proliferated and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose. Insulin expression was maintained when tissue was transplanted within vascularised chambers into diabetic mice. SP cells in the adult pancreas expand in response to β cell injury and are a source of β cell progenitors with potential for the treatment of diabetes.

Description: by Teresa Villarreal-Molina, Carlos Posadas-Romero, Sandra Romero-Hidalgo, Erika Antúnez-Argüelles, Araceli Bautista-Grande, Gilberto Vargas-Alarcón, Eric Kimura-Hayama, Samuel Canizales-Quinteros, Juan Gabriel Juárez-Rojas, Rosalinda Posadas-Sánchez, Guillermo Cardoso-Saldaña, Aída Medina-Urrutia, María del Carmen González-Salazar, Rocío Martínez-Alvarado, Esteban Jorge-Galarza, Alessandra Carnevale Background ABCA1 genetic variation is known to play a role in HDL-C levels and various studies have also implicated ABCA1 variation in cardiovascular risk. The functional ABCA1 /R230C variant is frequent in the Mexican population and has been consistently associated with low HDL-C concentrations. Although it has been associated with other cardiovascular risk factors such as obesity and type 2 diabetes mellitus, it is not known whether it is associated with coronary artery disease (CAD). Aim The purpose of the study was to analyze whether the ABCA1 /R230C variant is associated with premature CAD in a case-control association study (GEA or Genetics of Atherosclerotic Disease), and to explore whether BMI modulates the effect of the C230 allele on other metabolic traits using a population-based design. Results The C230 allele was significantly associated with both lower HDL-C levels and a lower risk of premature CAD as compared to controls (OR = 0.566; Padd  = 1.499×10 −5 ). In addition, BMI modulated the effect of R230C on body fat distribution, as the correlation between BMI and visceral to subcutaneous adipose tissue (a metric of the propensity to store fat viscerally as compared to subcutaneously) was negative in RR homozygous individuals, but positive in premenopausal women bearing the C230 allele, with a statistically significant interaction ( P  = 0.005). BMI-R230C interaction was also significant for triglyceride levels in women regardless of their menopausal status ( P =  0.036). Conclusion This is the first study assessing the effect of the R230C/ ABCA1 variant in remature CAD. C230 was associated with both decreased HDL-C levels and a lower risk of premature CAD, and gender-specific BMI-R230C interactions were observed for different metabolic traits. These interactions may help explain inconsistencies in associations, and underscore the need to further analyze interactions of this functional and frequent variant with diet, exercise and other environmental factors.

Description: by Elodie Bousquet, Min Zhao, André Ly, Guillaume Leroux les Jardins, Brigitte Goldenberg, Marie-Christine Naud, Laurent Jonet, Bernadette Besson-Lescure, Frederic Jaisser, Nicolette Farman, Yvonne De Kozak, Francine Behar-Cohen We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Description: by Sumit Malhotra, Sanjay P. Zodpey, Shivani Chandra, Ram Pal Vashist, Srinath Satyanaryana, Rony Zachariah, Anthony D. Harries Background The Indian guidelines on following up sputum smear-negative Pulmonary tuberculosis (PTB) patients differ from the current World Health Organization (WHO) guidelines in that the former recommends two follow up sputum examinations (once at the end of intensive phase and the other at the end of treatment) while the latter recommends only one follow up sputum smear microscopy examination, which is done at the end of the intensive phase. This study was conducted to examine if there was any added value in performing an additional sputum smear examination at the end of treatment within the context of a national TB program. Methods This study was a descriptive record based review conducted in nine tuberculosis (TB) units in Delhi, India. All consecutive new sputum smear-negative PTB patients registered in these nine TB units from 1 st January 2009 to 31 st December 2009 were included in the study. Results Of 2567 new sputum smear-negative TB patients, 1973 (90%) had sputum specimens examined at the end of the intensive phase, of whom 36 (2%) were smear-positive: the majority (n = 28) successfully completed treatment with either the same or a re-treatment regimen. At treatment completion, 1766 (85%) patients had sputum specimens examined, of whom 16 (0.9%) were smear-positive: all these were changed to a re-treatment regimen. Amongst the sputum-positive patients identified as a result of follow up (n = 52), four were diagnosed with multi-drug resistant TB (MDR-TB), three of whom were detected after smear examination at the end of treatment. Conclusions Given the high burden of TB in India, a 0.9% additional yield of smear-positive sputum smears at the end of treatment translates to 3,297 cases of smear-positive PTB. End-of-treatment smear is a low-yield strategy for detection of smear-positive TB cases, although further studies are needed to determine its population-level impact and cost, particularly in relation to other TB control interventions.

Description: by Youbo You, Lijun Bai, Ruwei Dai, Chongguang Zhong, Ting Xue, Hu Wang, Zhenyu Liu, Wenjuan Wei, Jie Tian As an ancient Chinese healing modality which has gained increasing popularity in modern society, acupuncture involves stimulation with fine needles inserted into acupoints. Both traditional literature and clinical data indicated that modulation effects largely depend on specific designated acupoints. However, scientific representations of acupoint specificity remain controversial. In the present study, considering the new findings on the sustained effects of acupuncture and its time-varied temporal characteristics, we employed an electrophysiological imaging modality namely magnetoencephalography with a temporal resolution on the order of milliseconds. Taken into account the differential band-limited signal modulations induced by acupuncture, we sought to explore whether or not stimulation at Stomach Meridian 36 (ST36) and a nearby non-meridian point (NAP) would evoke divergent functional connectivity alterations within delta, theta, alpha, beta and gamma bands. Whole-head scanning was performed on 28 healthy participants during an eyes-closed no-task condition both preceding and following acupuncture. Data analysis involved calculation of band-limited power (BLP) followed by pair-wise BLP correlations. Further averaging was conducted to obtain local and remote connectivity. Statistical analyses revealed the increased connection degree of the left temporal cortex within delta (0.5–4 Hz), beta (13–30 Hz) and gamma (30–48 Hz) bands following verum acupuncture. Moreover, we not only validated the closer linkage of the left temporal cortex with the prefrontal and frontal cortices, but further pinpointed that such patterns were more extensively distributed in the ST36 group in the delta and beta bands compared to the restriction only to the delta band for NAP. Psychophysical results for significant pain threshold elevation further confirmed the analgesic effect of acupuncture at ST36. In conclusion, our findings may provide a new perspective to lend support for the specificity of neural expression underlying acupuncture.

Description: by Madelyn M. Shapiro, Vandana Chakravartty, John E. Cronan Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo . Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli . Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.

Description: by Laurent Hocqueloux, Philippe Choisy, Gwenaël Le Moal, Françoise Borsa-Lebas, David Plainchamp, Eric Legac, Thierry Prazuck, Xavier de la Tribonnière, Yazdan Yazdanpanah, Jean-Jacques Parienti Background Among HIV-1 infected patients who achieved virologic suppression, the use of atazanavir without pharmacologic boosting is debated. We evaluated the efficacy and tolerance of maintenance therapy with unboosted atazanavir in clinical practice. Methods and Results This multicenter retrospective cohort study evaluated the efficacy of switching HIV-1-infected patients controlled on triple therapy to unboosted (ATV 0 , n = 98) versus ritonavir-boosted atazanavir (ATV/r, n = 254) +2 nucleos(t)ide reverse transcriptase inhibitors. The primary endpoint was time to virologic failure (VF, 〉200 copies/mL). ATV groups were compared controlling for potential confounding bias by inverse probability weighted Cox analysis and propensity-score matching. Overall and adjusted VF rates were similar for both strategies. Both strategies improved dyslipidemia and creatininemia, with less jaundice in the ATV 0 group. Conclusion In previously well-suppressed patients, within an observational cohort setting, ATV 0 –based triple-therapy appeared as effective as ATV/r- based triple-therapy to maintain virologic suppression, even if co-administered with TDF, but was better tolerated.

Description: by Carmen Amador, Miguel Ángel Toro, Jesús Fernández The maintenance of genetically differentiated populations can be important for several reasons (whether for wild species or domestic breeds of economic interest). When those populations are introgressed by foreign individuals, methods to eliminate the exogenous alleles can be implemented to recover the native genetic background. This study used computer simulations to explore the usefulness of several molecular based diagnostic approaches to recover of a native population after suffering an introgression event where some exogenous alleles were admixed for a few generations. To remove the exogenous alleles, different types of molecular markers were used in order to decide which of the available individuals contributed descendants to next generation and their number of offspring. Recovery was most efficient using diagnostic markers (i.e., with private alleles) and least efficient when using alleles present in both native and exogenous populations at different frequencies. The increased inbreeding was a side-effect of the management strategy. Both values (% of native alleles and inbreeding) were largely dependent on the amount of exogenous individuals entering the population and the number of generations of admixture that occurred prior to management.

Description: by Z. Y. Yuan, Han Y. H. Chen The production and turnover of fine roots play substantial roles in the biogeochemical cycles of terrestrial ecosystems. However, the disparity among the estimates of both production and turnover, particularly due to technical limitations, has been debated for several decades. Here, we conducted a meta-analysis to compare published estimates of fine root production and turnover rates derived from different methods at the same sites and at the same sampling time. On average, the estimates of fine root production and turnover rates were 87% and 124% higher, respectively, by indirect methods than by direct methods. The substantially higher fine root production and turnover estimated by indirect methods, on which most global carbon models are based, indicate the necessity of re-assessing the global carbon model predictions for atmospheric carbon sequestration in soils as a result of the production and turnover of fine roots.

Description: by Hua Liu, Jianyang Xu, Baoci Shan, Yongzhong Li, Lin Li, Jingquan Xue, Binbin Nie Background In acupuncture brain imaging trials, there are many non-acupuncture factors confounding the neuronal mapping. The modality of the placebo, subjects’ psychological attitude to acupuncture and their physical state are the three most confounding factors. Objective To obtain more precise and accurate cerebral fMRI mapping of acupuncture. Design and Setting A 2×2 randomized, controlled, participant-blinded cross-over factorial acupuncture trial was conducted at Xuanwu Hospital in Beijing, China. Participants Forty-one college students with myopia were recruited to participate in our study and were allocated randomly to four groups, Group A, Group B, Group C and Group D. Interventions Group A received real acupuncture (RA) and treatment instruction (TI); Group B received RA and non-treatment instruction (NI); Group C received sham acupuncture (SA) and TI; Group D received SA and NI. Results Stimulation at LR3 activated some areas of the visual cortex, and the cerebral response to non-acupuncture factors was complex and occurred in multiple areas. Conclusions The results provide more evidence regarding the credibility of acupuncture therapy and suggest that more precise experimental designs are needed to eliminate sources of bias in acupuncture controlled trials and to obtain sound results.

Description: by Veronica Veschi, Marialaura Petroni, Beatrice Cardinali, Carlo Dominici, Isabella Screpanti, Luigi Frati, Armando Bartolazzi, Alberto Gulino, Giuseppe Giannini MYCN amplification occurs in about 20–25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.

Description: by Li Wu, Dinggeng Chai, Marie E. Fraser, Steven Zimmerly Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.