TY - JOUR AB - We consider the problem of renal mass risk classification to support doctors in adjuvant treatment decisions following nephrectomy. Recommendation of adjuvant therapy based on the mass appearance poses two major challenges: first, morphologic patterns may sometimes overlap across subtypes of varying risks. Second, interobserver variability is large. These complexities encourage the use of computational models as accurate noninvasive tools to find relevant relationships between individual perioperative renal mass characteristics and patient risk. In addition, recent evidence highlights the importance of clinical context as a promising direction to inform treatment decisions post-nephrectomy. In this work, we aim to identify relevant clinical markers that can be predictive of renal cancer prognosis. As a starting point, we perform a clinical feature ablation study by training a logistic regression baseline model to predict renal cancer patients' eligibility for adjuvant therapy. The training dataset consisted of medical records of 300 individuals with renal tumors who underwent partial or radical nephrectomy between 2011 and 2020. In addition, we evaluate the same task using a transformer-based model pretrained on a much larger dataset of over 300,000 clinical records of individuals from the UK Biobank. Our findings demonstrate the pretrained model's efficacy in knowledge transfer across different populations, with radiographic data from preoperative cross-sectional imaging playing an important role in informing renal risk and treatment decisions. AU - Barros, V.* AU - Abdallah, N.* AU - Ozery-Flato, M.* AU - Dekel, A.* AU - Raboh, M.* AU - Heller, N.* AU - Rabinovici-Cohen, S.* AU - Golts, A.* AU - Gentili, A.* AU - Lang, D.M. AU - Chaudhary, S.* AU - Satish, V.* AU - Tejpaul, R.* AU - Eggel, I.* AU - Guez, I.* AU - Barkan, E.* AU - Müller, H.* AU - Hexter, E.* AU - Rosen-Zvi, M.* AU - Weight, C.* C1 - 74837 C2 - 57624 TI - Preoperative kidney tumor risk estimation with AI: From logistic regression to transformer. JO - PLoS ONE VL - 20 IS - 5 PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: This paper introduces the UK Biobank (UKB) second mental health questionnaire (MHQ2), describes its design, the respondents and some notable findings. UKB is a large cohort study with over 500,000 volunteer participants aged 40-69 years when recruited in 2006-2010. It is an important resource of extensive health, genetic and biomarker data. Enhancements to UKB enrich the data available. MHQ2 is an enhancement designed to enable and facilitate research with psychosocial and mental health aspects. METHODS: UKB sent participants a link to MHQ2 by email in October-November 2022. The MHQ2 was designed by a multi-institutional consortium to build on MHQ1. It characterises lifetime depression further, adds data on panic disorder and eating disorders, repeats 'current' mental health measures and updates information about social circumstances. It includes established measures, such as the PHQ-9 for current depression and CIDI-SF for lifetime panic, as well as bespoke questions. Algorithms and R code were developed to facilitate analysis. RESULTS: At the time of analysis, MHQ2 results were available for 169,253 UKB participants, of whom 111,275 had also completed the earlier MHQ1. Characteristics of respondents and the whole UKB cohort are compared. The major phenotypes are lifetime: depression (18%); panic disorder (4.0%); a specific eating disorder (2.8%); and bipolar affective disorder I (0.4%). All mental disorders are found less with older age and also seem to be related to selected social factors. In those participants who answered both MHQ1 (2016) and MHQ2 (2022), current mental health measure showed that fewer respondents have harmful alcohol use than in 2016 (relative risk 0.84), but current depression (RR 1.07) and anxiety (RR 0.98) have not fallen, as might have been expected given the relationship with age. We also compare lifetime concepts for test-retest reliability. CONCLUSIONS: There are some drawbacks to UKB due to its lack of population representativeness, but where the research question does not depend on this, it offers exceptional resources that any researcher can apply to access. This paper has just scratched the surface of the results from MHQ2 and how this can be combined with other tranches of UKB data, but we predict it will enable many future discoveries about mental health and health in general. AU - Davis, K.A.S.* AU - Coleman, J.R.I.* AU - Adams, M.* AU - Breen, G.* AU - Cai, N. AU - Davies, H.L.* AU - Davies, K.J.A.* AU - Dregan, A.* AU - Eley, T.C.* AU - Fox, E.* AU - Holliday, J.* AU - Hübel, C.* AU - John, A.* AU - Kassam, A.S.* AU - Kempton, M.J.* AU - Lee, W.* AU - Li, D.* AU - Maina, J.G.* AU - McCabe, R.* AU - McIntosh, A.M.* AU - Oram, S.* AU - Richards, M.* AU - Skelton, M.* AU - Starkey, F.* AU - Ter Kuile, A.R.* AU - Thornton, L.M.* AU - Wang, R.* AU - Yu, Z.* AU - Zvrskovec, J.* AU - Hotopf, M.* C1 - 74811 C2 - 57602 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The UK Biobank mental health enhancement 2022: Methods and results. JO - PLoS ONE VL - 20 IS - 5 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - Biodiversity loss is a global challenge of the 21st century. Environmental DNA (eDNA)-based metabarcoding offers a cost- and time-efficient alternative to conventional biodiversity surveys, enabling detection of rare, cryptic, and elusive species from environmental samples. However, limited access to genomic technologies restricts the application of eDNA metabarcoding in highly biodiverse remote regions and low- and middle-income countries (LMICs). Here, we directly compared the latest portable nanopore sequencing methods with established Illumina sequencing for vertebrate eDNA metabarcoding of Zambian water samples. Our results show that due to recent improvements in sequencing chemistry and optimized basecalling, nanopore sequencing data can recapitulate or even surpass established protocols, demonstrating the feasibility of in situ biodiversity assessments. eDNA- and camera trap-based species detections had minimal overlap in species detections, suggesting a complementary rather than substituting application of these biodiversity monitoring technologies. We finally demonstrate that our entire eDNA workflow can be successfully implemented in a mobile laboratory under remote field conditions by completing all steps-from sample collection to data analysis-within the Luambe National Park in Zambia. This approach has important implications for capacity building in LMICs and for overcoming limitations associated with sample export. AU - Gygax, D. AU - Ramirez, S.* AU - Chibesa, M.* AU - Simpamba, T.* AU - Riffel, M.* AU - Riffel, T.* AU - Srivathsan, A.* AU - Nijland, R.* AU - Urban, L. C1 - 75796 C2 - 58154 TI - Evaluation of nanopore sequencing for increasing accessibility of eDNA studies in biodiverse countries. JO - PLoS ONE VL - 20 IS - 10 PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Pancreatic surgery remains associated with significant morbidity. Pancreatoduodenectomy (PD) with high-risk stigmata for postoperative pancreatic fistula (POPF) may delay or hinder administration of adjuvant therapy. Total pancreatectomy (TP) prevents POPF-associated complications but implies permanent exocrine and endocrine insufficiency. Islet autotransplantation (IAT) has the potential to compensate endocrine function. METHODS AND ANALYSIS: XANDTX is a single-centre randomized controlled pilot trial comparing high-risk PD with TP and simultaneous IAT in patients with periampullary cancer. After screening for eligibility and obtaining informed consent, a total of 32 adult patients will be intraoperatively randomized in a 1:1 ratio. The primary hypothesis is that TP with IAT prevents POPF-associated complications and leads to a shorter period to initiation of adjuvant therapy and a higher overall rate of adjuvant therapy administration. Secondary endpoints include perioperative morbidity and mortality, metabolic outcome, quality of life (QoL) and oncological long-term outcome. Each patient will be followed up for 5 years. DISCUSSION: The XANDTX pilot trial will aim to provide surgical and oncological feasibility and safety data of total pancreatectomy with simultaneous islet autotransplantation in management of resectable periampullary cancer. The results will form the basis for a further confirmatory controlled study. TRIAL REGISTRATION: This study was registered on ClinicalTrials.gov (NCT05843877) on February 27, 2023 and EudraCT (2023-507773-17-00) on April 18, 2024. AU - Hempel, S.* AU - Kolbinger, F.R.* AU - Oehme, F.* AU - Radulova-Mauersberger, O.* AU - Schmid, J. AU - Schubert, U. AU - Schepp, F.* AU - Bornstein, S.R. AU - Korn, S.* AU - Trips, E.* AU - Weitz, J.* AU - Distler, M. AU - Ludwig, B. C1 - 75242 C2 - 57877 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Pancreatoduodenectomy versus total pancreatectomy and simultaneous intraportal islet autotransplantation for periampullary cancer at high-risk of postoperative pancreatic fistula (XANDTX-trial): Protocol of a randomized controlled pilot trial. JO - PLoS ONE VL - 20 IS - 7 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - The health status of laboratory animals plays a decisive role not only for the health and welfare of the animals but also for the validity of study results. In recent years, there has been an increasing number of publications on environmental health monitoring (EHM), which uses molecular biological methods to detect nucleic acids of infectious agents in individually ventilated cage systems, e.g. in exhaust air dust. This monitoring strategy can reduce the number of mice used for health monitoring in conformity with the 3Rs. Numerous studies have shown that EHM is reliable and sensitive and is, therefore, a useful method for health monitoring of mice. An online survey was created to assess the prevalence of the use of EHM in Germany, Austria, and Switzerland and to better understand the factors influencing its use in animal facilities. The survey revealed that the majority of facilities primarily equipped with individually ventilated cage systems already use EHM to varying degrees, replacing between 8 and 1200 animals per year and facility. However, the predominant strategy is still the use of (sentinel) animals for health monitoring. Beliefs on factors such as cost, reliability and the number of false-positive results differ significantly between facilities that predominantly use either animals or EHM. Additionally, the choice of monitoring strategy may be influenced by the existing cage system and the availability of a decontamination option for the equipment. The evaluation of the survey showed that there is still a gap in knowledge and a demand for specific training on the topic of health monitoring and especially on EHM. AU - Mahabir, E.* AU - Schmidt, K.* AU - Kolbe, T.* AU - Buchheister, S.* AU - Miller, M. C1 - 75834 C2 - 58133 TI - A survey on the implementation of environmental health monitoring in mouse facilities in German-speaking countries. JO - PLoS ONE VL - 20 IS - 10 PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: We described waning in anti-SARS-CoV-2 IgG in adult general populations infected during the first wave of the COVID-19 pandemic in 2020 across three European countries. METHODS: Coordinated analyses were conducted separately in three population-based cohorts with complementary follow-up schedules: the KoCo19 (Germany), EpiCov (France), and CON-VINCE (Luxembourg) cohorts. Serological follow-up was based on the anti-SARS-CoV-2 ELISA-S IgG (Euroimmun) assay. We selected all adults aged 18-79 who had a positive serology (IgG optical density (OD) ratio ≥1.1) between February and July 2020, and at least one subsequent IgG measurement within the following 12 months, while still non-vaccinated. RESULTS: The proportion of seroreversion was 0% within the four first months, based on Koco19 data (n = 65 participants). In the longer term, 31.3% of participants had seroreverted at 6 months (95%CI: 24.4-39.1) (based on EpiCov data, n = 599), 31.3% (95%CI: 11.0-58.7) at 12 months (based on CON-VINCE data, n = 16). From EpiCov data, both baseline low IgG levels and seroneutralization negativity remained predictive of seroreversion in multivariable analysis. CONCLUSION: From population-based cohorts, anti-S IgG levels remained stable during the first 4 months following SARS-CoV-2 infection. Most of the decay occurred afterward; nearly one-third of people seroreverted 6 and 12 months later. Low IgG levels and seroneutralization negativity were independent predictors of seroreversion. AU - Novelli, S.* AU - Reinkemeyer, C.* AU - Bulaev, D.* AU - O'Sullivan, M.P.* AU - Snoeck, C.J.* AU - Rauschenberger, A.* AU - Manto, C.* AU - Kolodkin, A.L.* AU - Ghosh, S.* AU - Satagopam, V.* AU - le Chenadec, J.* AU - Barthelemy, K.* AU - Priet, S.* AU - de Lamballerie, X.* AU - Wieser, A.* AU - Kroidl, I.* AU - Vaillant, M.* AU - Meyer, L.* AU - Hoelscher, M. AU - Castelletti, N. AU - Krüger, R.* AU - Warszawski, J.* C1 - 74361 C2 - 57481 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Waning of anti-SARS-CoV-2 antibodies after the first wave of the COVID-19 pandemic in 2020: A 12-month-evaluation in three population-based European studies. JO - PLoS ONE VL - 20 IS - 5 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: It has been hypothesized but seldom tested that the winter excess in cardiovascular disease (CVD) is related to hypovitaminosis D. The present study examined the association between CVD and (i) seasonality of 25-hydroxyvitamin D (25[OH]D) and (ii) individual 25(OH)D concentrations. METHODS AND FINDINGS: Harmonized 25(OH)D data were obtained from the Biomarkers for Cardiovascular Risk Assessment in Europe (BiomarCaRE) project, including 79,570 participants examined between 1984 and 2010. One 25(OH)D measurement was available per participant. Primary endpoints were CVD incidence (coronary heart disease or stroke; n = 6006) and CVD mortality (n = 2985). To study (i), Poisson regression-derived rate ratios were compared according to two-month categories, ordered by baseline 25(OH)D concentrations. To study (ii), Cox regression-derived hazard ratios were compared according to quarters of baseline 25(OH)D concentrations. With respect to (i), despite a median 25(OH)D concentration ratio of 1:1.79, the trough months of 25(OH)D in March and April had a similar CVD incidence as the peak months of 25(OH)D in August and September (rate ratio: 1.07, 95% CI: 0.98-1.17). CVD mortality was slightly higher in the trough months compared to the peak months (rate ratio: 1.27, 95% CI: 1.12-1.44) but not compared to the other months (despite median 25[OH]D concentration ratios up to 1:1.62; p ≥ 0.077). The CVD mortality peak in January preceded the 25(OH)D trough, not adhering to the temporality criterion of Bradford Hill. With respect to (ii), compared to the lowest quarter, the highest quarter of 25(OH)D was associated with lower CVD incidence (hazard ratio: 0.82, 95% CI: 0.76-0.89) and CVD mortality (hazard ratio: 0.64, 95% CI: 0.57-0.72). CONCLUSION: The present study does not support the hypothesis that seasonal increases in CVD are driven by short-term reductions in 25(OH)D. As in most observational studies, higher 25(OH)D concentrations were inversely associated with CVD. AU - Oskarsson, V.* AU - Salomaa, V.* AU - Jousilahti, P.* AU - Palmieri, L.* AU - Donfrancesco, C.* AU - Sans, S.* AU - Iacoviello, L.* AU - Costanzo, S.* AU - Ferrario, M.M.* AU - Cesana, G.* AU - Thorand, B. AU - Peters, A. AU - Tunstall-Pedoe, H.* AU - Woodward, M.* AU - Zeller, T.* AU - Blankenberg, S.* AU - Kuulasmaa, K.* AU - Söderberg, S.* C1 - 74191 C2 - 57362 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Cardiovascular disease outcomes in relation to 25-hydroxyvitamin D and its seasonal variation: Results from the BiomarCaRE consortium. JO - PLoS ONE VL - 20 IS - 4 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: The aim of the study was to derive median age- and sex-specific respiratory rates in a population-based sample of adults and to identify disease and lifestyle factors associated with elevated respiratory rates. METHODS: In the population-based KORA FF4 study conducted in Augsburg, Germany, 5-minute 12-lead resting electrocardiograms (ECGpro-system, AMEDTEC) were recorded in 2,224 participants from 39 to 88 years. Respiratory rate was derived from these electrocardiograms. Sex- and age-specific medians, IQRs, and percentiles were calculated. Associations of sociodemographic, disease, and lifestyle variables with elevated resting respiratory rate were assessed by univariable and multivariable logistic regression analyses. RESULTS: Respiratory rate decreased slightly from youngest to middle-aged women and men and increased in old age. Overall, median (IQR) was 15.80 (3.16) breaths per minute (brpm). Five percent of the participants had values lower than 12.06 brpm, and five percent had values above 20.06 brpm (95th percentile). Elevated respiratory rates of ≥  18.6 brpm were found in 13.8% (n =  308). In an adjusted logistic regression model, age, abdominal obesity, diabetes, COPD, smoking, and low education were significantly associated with elevated respiratory rate. Stratified analyses showed that education appeared to be more relevant in women, while the effect of diabetes was more pronounced in men. CONCLUSIONS: High respiratory rate may be an indicator of impaired health in the general population, especially regarding pulmonary and metabolic characteristics, and unfavorable lifestyle and living conditions. Individuals with an increased respiratory rate should therefore be examined and followed up more closely to recognize diseases and adverse progressions at an early stage and to possibly prevent them. AU - Rückert-Eheberg, I.-M. AU - Steger, A.* AU - Müller, A.* AU - Linkohr, B. AU - Barthel, P.* AU - Maier, M.* AU - Allescher, J.* AU - Sinner, M.F.* AU - Rizas, K.D.* AU - Rathmann, W.* AU - Laugwitz, K.L.* AU - Kääb, S.* AU - Peters, A. AU - Schmidt, G.* C1 - 73668 C2 - 56863 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Respiratory rate and its associations with disease and lifestyle factors in the general population - results from the KORA-FF4 study. JO - PLoS ONE VL - 20 IS - 3 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Glycated hemoglobin (HbA1c) measures the average blood sugar level over the past three months. As a vital biomarker of blood glucose levels, it is used to diagnose Type-2 diabetes mellitus (T2D) and monitor glycemic control. A heritability estimate of 47% to 59% suggests that about half of the variation in HbA1c levels can be attributed to genetic factors. Despite African populations being the most genetically diverse and unique for fine-mapping, there is a paucity of data on the genetic drivers of HbA1c in African individuals. In this study, we performed functional annotation and a Phenome-Wide Association Study (PheWAS) of HbA1c-associated variants in two African populations. METHOD: In this study, we utilized summary statistics of the HbA1c GWAS meta-analysis of 7,526 individuals from South Africa and Uganda to conduct a PheWAS using GWASATLAS. We also performed a functional analysis using the functional mapping and annotation (FUMA) tool. Single nucleotide polymorphisms (SNPs) were prioritized using the SNP2GENE function, while the gene expression patterns and shared molecular functions were explored in the GENE2FUNC. RESULT: Three genome-wide significant loci were identified with the lead SNPs: rs6724428, rs148228241, and rs8045544 - mapped to GULP1, HBA1, and ITFG3 genes, respectively. The minor allele frequencies of rs148228241 (0.07) and rs8045544 (0.19) are rare or non-existent in non-African populations. Both rs8045544 and rs148228241 are significantly associated with the mean corpuscular hemoglobin concentration (MCHC). A lower MCHC is associated with alpha thalassaemia, resulting from deletions in HBA1 and HBA2 genes. Such deletions are prevalent in malaria-endemic regions of Africa due to their selective survival advantage. The rs6724428 variant is associated with skeletal functions, reflecting the link between glucose metabolism and bone mineral density. DISCUSSION: Our findings highlight the interplay between glucose metabolism, erythropoiesis, and skeletal health. The significant associations of HbA1c-variants with both skeletal function and MCHC underscore the potential of these variants to impact broader physiological processes. A large-scale study of African individuals will be essential to unravel genetic variants influencing HbA1c. AU - Soremekun, C. AU - Ojewunmi, O.* AU - Nwagbata, A.* AU - Bazireh, H. AU - Sharma, S. AU - Jjingo, D.* AU - Kateete, D.P.* AU - Nash, O.* AU - Grallert, H. AU - Batini, C.* AU - Peters, A. AU - Fatumo, S.* C1 - 74836 C2 - 57623 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Phenome-wide association study and functional annotation of hemoglobin A1c-associated variants in African populations. JO - PLoS ONE VL - 20 IS - 5 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - Memory T cells play an important role in mediating long-lasting adaptive immune responses to viral infections, such as SARS-CoV-2. In the context of the latter, much of our current knowledge stems from studies in vaccinated individuals or repeatedly infected individuals. However, limited knowledge is available on these responses in fully naive individuals in German communities. We performed immunophenotyping of a previously naive SARS-CoV-2 cohort in convalescent individuals after asymptomatic to moderate COVID-19. The samples were collected median 250 days post infection during the first wave of the COVID pandemic in Germany (March - May 2020). In this cohort of 174 individuals, we phenotyped different leukocyte cell populations in peripheral blood (B, T and Natural Killer cells). We then assessed the serostatus against the SARS-CoV-2 antigens Nucleocapsid (N) and Spike subunit (S1) with its receptor binding domain (RBD), as these are important correlates of protection, by testing for presence of immunoglobulin G (IgG) antibodies. We also measured IgG antibody responses against the N antigen of the common cold coronaviruses HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E, to determine possible cross-reactivity. In a subset of the cohort (n = 76), we performed intracellular staining assays (ICS) after stimulation with SARS-CoV-2 and HCoV antigens. Key findings are significant differences in frequency of CD4+ memory T cell populations, notably CD4+ TEM and CD4+ TEMRA cells, between the group of SARS-CoV-2 positive individuals and the control group. These differences correlated with cytokine production (TNFα, IFNγ) after stimulation with SARS-CoV-2 peptides, indicating a specific T cell immune response. In conclusion, a clear memory T cell and humoral response can be detected up to 250 days post mild to moderate COVID-19 disease. Our results underline findings reported by others indicating a lasting cellular immune response even in a population which previously had not been exposed to SARS-CoV-2. AU - Weigl, N.* AU - Pleimelding, C.* AU - Gilberg, L.* AU - Huynh, D.* AU - Brand, I.* AU - Bruger, J.* AU - Frese, J.* AU - Eser, T.M.* AU - Ahmed, M.I.M.* AU - Guggenbuehl-Noller, J.M.* AU - Stirner, R.* AU - Hoelscher, M.* AU - Pritsch, M.* AU - Geldmacher, C.* AU - Kobold, S. AU - Roider, J.* C1 - 74912 C2 - 57716 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Detectable SARS-CoV-2 specific immune responses in recovered unvaccinated individuals 250 days post wild type infection. JO - PLoS ONE VL - 20 IS - 6 PB - Public Library Science PY - 2025 SN - 1932-6203 ER - TY - JOUR AB - Approximate Bayesian Computation (ABC) is a widely applicable and popular approach to estimating unknown parameters of mechanistic models. As ABC analyses are computationally expensive, parallelization on high-performance infrastructure is often necessary. However, the existing parallelization strategies leave computing resources unused at times and thus do not optimally leverage them yet. We present look-ahead scheduling, a wall-time minimizing parallelization strategy for ABC Sequential Monte Carlo algorithms, which avoids idle times of computing units by preemptive sampling of subsequent generations. This allows to utilize all available resources. The strategy can be integrated with e.g. adaptive distance function and summary statistic selection schemes, which is essential in practice. Our key contribution is the theoretical assessment of the strategy of preemptive sampling and the proof of unbiasedness. Complementary, we provide an implementation and evaluate the strategy on different problems and numbers of parallel cores, showing speed-ups of typically 10-20% and up to 50% compared to the best established approach, with some variability. Thus, the proposed strategy allows to improve the cost and run-time efficiency of ABC methods on high-performance infrastructure. AU - Alamoudi, E.* AU - Reck, F.* AU - Bundgaard, N.* AU - Graw, F.* AU - Brusch, L.* AU - Hasenauer, J. AU - Schälte, Y. C1 - 70044 C2 - 55378 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A wall-time minimizing parallelization strategy for approximate Bayesian computation. JO - PLoS ONE VL - 19 IS - 2 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Acute lymphoblastic leukemia (ALL) is the most common type of malignancy in children. ALL prognosis after initial diagnosis is generally good; however, patients suffering from relapse have a poor outcome. The tumor microenvironment is recognized as an important contributor to relapse, yet the cell-cell interactions involved are complex and difficult to study in traditional experimental models. In the present study, we established an innovative larval zebrafish xenotransplantation model, that allows the analysis of leukemic cells (LCs) within an orthotopic niche using time-lapse microscopic and flow cytometric approaches. LCs homed, engrafted and proliferated within the hematopoietic niche at the time of transplant, the caudal hematopoietic tissue (CHT). A specific dissemination pattern of LCs within the CHT was recorded, as they extravasated over time and formed clusters close to the dorsal aorta. Interactions of LCs with macrophages and endothelial cells could be quantitatively characterized. This zebrafish model will allow the quantitative analysis of LCs in a functional and complex microenvironment, to study mechanisms of niche mediated leukemogenesis, leukemia maintenance and relapse development. AU - Arner, A.* AU - Ettinger, A. AU - Blaser, B.W.* AU - Schmid, B.* AU - Jeremias, I. AU - Rostam, N.* AU - Binder-Blaser, V.* C1 - 71538 C2 - 56265 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - In vivo monitoring of leukemia-niche interactions in a zebrafish xenograft model. JO - PLoS ONE VL - 19 IS - 8 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Risk stratification scores such as the European Systematic COronary Risk Evaluation (SCORE) are used to guide individuals on cardiovascular disease (CVD) prevention. Adding high-sensitivity troponin I (hsTnI) to such risk scores has the potential to improve accuracy of CVD prediction. We investigated how applying hsTnI in addition to SCORE may impact management, outcome, and cost-effectiveness. METHODS: Characteristics of 72,190 apparently healthy individuals from the Biomarker for Cardiovascular Risk Assessment in Europe (BiomarCaRE) project were included into a discrete-event simulation comparing two strategies for assessing CVD risk. The standard strategy reflecting current practice employed SCORE (SCORE); the alternative strategy involved adding hsTnI information for further stratifying SCORE risk categories (S-SCORE). Individuals were followed over ten years from baseline examination to CVD event, death or end of follow-up. The model tracked the occurrence of events and calculated direct costs of screening, prevention, and treatment from a European health system perspective. Cost-effectiveness was expressed as incremental cost-effectiveness ratio (ICER) in € per quality-adjusted life year (QALYs) gained during 10 years of follow-up. Outputs were validated against observed rates, and results were tested in deterministic and probabilistic sensitivity analyses. RESULTS: S-SCORE yielded a change in management for 10.0% of individuals, and a reduction in CVD events (4.85% vs. 5.38%, p<0.001) and mortality (6.80% vs. 7.04%, p<0.001). S-SCORE led to 23 (95%CI: 20-26) additional event-free years and 7 (95%CI: 5-9) additional QALYs per 1,000 subjects screened, and resulted in a relative risk reduction for CVD of 9.9% (95%CI: 7.3-13.5%) with a number needed to screen to prevent one event of 183 (95%CI: 172 to 203). S-SCORE increased costs per subject by 187€ (95%CI: 177 € to 196 €), leading to an ICER of 27,440€/QALY gained. Sensitivity analysis was performed with eligibility for treatment being the most sensitive. CONCLUSION: Adding a person's hsTnI value to SCORE can impact clinical decision making and eventually improves QALYs and is cost-effective compared to CVD prevention strategies using SCORE alone. Stratifying SCORE risk classes for hsTnI would likely offer cost-effective alternatives, particularly when targeting higher risk groups. AU - Jülicher, P.* AU - Makarova, N.* AU - Ojeda, F.* AU - Giusepi, I.* AU - Peters, A. AU - Thorand, B. AU - Cesana, G.* AU - Jørgensen, T.* AU - Linneberg, A.* AU - Salomaa, V.* AU - Iacoviello, L.* AU - Costanzo, S.* AU - Söderberg, S.* AU - Kee, F.* AU - Giampaoli, S.* AU - Palmieri, L.* AU - Donfrancesco, C.* AU - Zeller, T.* AU - Kuulasmaa, K.* AU - Tuovinen, T.* AU - Lamrock, F.* AU - Conrads-Frank, A.* AU - Brambilla, P.* AU - Blankenberg, S.* AU - Siebert, U.* C1 - 71227 C2 - 56023 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Cost-effectiveness of applying high-sensitivity troponin I to a score for cardiovascular risk prediction in asymptomatic population. JO - PLoS ONE VL - 19 IS - 7 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Paper mulberry pollen, declared a pest in several countries including Pakistan, can trigger severe allergies and cause asthma attacks. We aimed to develop an algorithm that could accurately predict high pollen days to underpin an alert system that would allow patients to take timely precautionary measures. We developed and validated two prediction models that take historical pollen and weather data as their input to predict the start date and peak date of the pollen season in Islamabad, the capital city of Pakistan. The first model is based on linear regression and the second one is based on phenological modelling. We tested our models on an original and comprehensive dataset from Islamabad. The mean absolute errors (MAEs) for the start day are 2.3 and 3.7 days for the linear and phenological models, respectively, while for the peak day, the MAEs are 3.3 and 4.0 days, respectively. These encouraging results could be used in a website or app to notify patients and healthcare providers to start preparing for the paper mulberry pollen season. Timely action could reduce the burden of symptoms, mitigate the risk of acute attacks and potentially prevent deaths due to acute pollen-induced allergy. AU - Kakakhail, A.* AU - Rextin, A.* AU - Buters, J.T.M. AU - Lin, C.* AU - Maya-Manzano, J.M. AU - Nasim, M.* AU - Oteros, J.* AU - Picornell, A.* AU - Pinnock, H.* AU - Schwarze, J.* AU - Yusuf, O.* C1 - 69888 C2 - 55306 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Predicting the main pollen season of Broussonetia Papyrifera (paper mulberry) tree. JO - PLoS ONE VL - 19 IS - 2 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Estimating parameters of dynamic models from experimental data is a challenging, and often computationally-demanding task. It requires a large number of model simulations and objective function gradient computations, if gradient-based optimization is used. In many cases, steady-state computation is a part of model simulation, either due to steady-state data or an assumption that the system is at steady state at the initial time point. Various methods are available for steady-state and gradient computation. Yet, the most efficient pair of methods (one for steady states, one for gradients) for a particular model is often not clear. In order to facilitate the selection of methods, we explore six method pairs for computing the steady state and sensitivities at steady state using six real-world problems. The method pairs involve numerical integration or Newton's method to compute the steady-state, and-for both forward and adjoint sensitivity analysis-numerical integration or a tailored method to compute the sensitivities at steady-state. Our evaluation shows that all method pairs provide accurate steady-state and gradient values, and that the two method pairs that combine numerical integration for the steady-state with a tailored method for the sensitivities at steady-state were the most robust, and amongst the most computationally-efficient. We also observed that while Newton's method for steady-state computation yields a substantial speedup compared to numerical integration, it may lead to a large number of simulation failures. Overall, our study provides a concise overview across current methods for computing sensitivities at steady state. While our study shows that there is no universally-best method pair, it also provides guidance to modelers in choosing the right methods for a problem at hand. AU - Lakrisenko, P. AU - Pathirana, D.* AU - Weindl, D. AU - Hasenauer, J. C1 - 72124 C2 - 56506 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Benchmarking methods for computing local sensitivities in ordinary differential equation models at dynamic and steady states. JO - PLoS ONE VL - 19 IS - 10 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Understanding the athlete's movements and the restrictions incurred by protective equipment is crucial for improving the equipment and subsequently, the athlete's performance. The task of equipment improvement is especially challenging in sports including advanced manoeuvres such as ice hockey and requires a holistic approach guiding the researcher's attention toward the right variables. The purposes of this study were (a) to quantify the effects of protective equipment in ice hockey on player's performance and (b) to identify the restrictions incurred by it. Twenty male hockey players performed four different drills with and without protective equipment while their performance was quantified. A neural network accompanied by layer-wise relevance propagation was applied to the 3D kinematic data to identify variables and time points that were most relevant for the neural network to distinguish between the equipment and no equipment condition, and therefore presumable result from restrictions incurred by the protective equipment. The study indicated that wearing the protective equipment, significantly reduced performance. Further, using the 3D kinematics, an artificial neural network could accurately distinguish between the movements performed with and without the equipment. The variables contributing the most to distinguishing between the equipment conditions were related to the upper extremities and movements in the sagittal plane. The presented methodology consisting of artificial neural networks and layer-wise relevance propagation contributed to insights without prior knowledge of how and to which extent joint angles are affected in complex maneuvers in ice hockey in the presence of protective equipment. It was shown that changes to the equipment should support the flexion movements of the knee and hip and should allow players to keep their upper extremities closer to the torso. AU - Lennartz, R.* AU - Khassetarash, A.* AU - Nigg, S.R.* AU - Eskofier, B.M. AU - Nigg, B.M.* C1 - 72015 C2 - 56548 TI - Neural network and layer-wise relevance propagation reveal how ice hockey protective equipment restricts players' motion. JO - PLoS ONE VL - 19 IS - 10 PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Butyrate has been proposed as a drug therapy by acting as a lysine deacetylase (KDAC) inhibitor and elevating protein acetylation, in particular on histones. Nonetheless, recent studies suggest that tissues such as the gut can utilize butyrate as a metabolite. We have previously shown that the addition of butyrate induces a rapid increase of oxygen consumption in whole Drosophila melanogaster heads. Here we show that while head oxygen consumption is increased by the addition of butyrate, no apparent changes are observed on the proteome and acetylome. Instead, we show that butyrate is metabolized and incorporated into the tricarboxylic acid (TCA) cycle. Collectively our data supports the notion that the therapeutic benefits of acute butyrate treatment may be also mediated by improving metabolic rates, rather than solely targeting the epigenome or acetylome. AU - Müller-Eigner, A.* AU - Gille, B.* AU - Dethloff, F.* AU - Meng, C.* AU - Ludwig, C.* AU - Heiker, J.T. AU - Giavalisco, P.* AU - Peleg, S.* C1 - 72855 C2 - 56755 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Sodium butyrate is incorporated into central metabolism in fly head while inducing oxygen consumption increase. JO - PLoS ONE VL - 19 IS - 12 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Multiple risk factors contribute jointly to the development and progression of cardiometabolic diseases. Therefore, joint longitudinal trajectories of multiple risk factors might represent different degrees of cardiometabolic risk. METHODS: We analyzed population-based data comprising three examinations (Exam 1: 1999-2001, Exam 2: 2006-2008, Exam 3: 2013-2014) of 976 male and 1004 female participants of the KORA cohort (Southern Germany). Participants were followed up for cardiometabolic diseases, including cardiovascular mortality, myocardial infarction and stroke, or a diagnosis of type 2 diabetes, until 2016. Longitudinal multivariate k-means clustering identified sex-specific trajectory clusters based on nine cardiometabolic risk factors (age, systolic and diastolic blood pressure, body-mass-index, waist circumference, Hemoglobin-A1c, total cholesterol, high- and low-density lipoprotein cholesterol). Associations between clusters and cardiometabolic events were assessed by logistic regression models. RESULTS: We identified three trajectory clusters for men and women, respectively. Trajectory clusters reflected a distinct distribution of cardiometabolic risk burden and were associated with prevalent cardiometabolic disease at Exam 3 (men: odds ratio (OR)ClusterII = 2.0, 95% confidence interval: (0.9-4.5); ORClusterIII = 10.5 (4.8-22.9); women: ORClusterII = 1.7 (0.6-4.7); ORClusterIII = 5.8 (2.6-12.9)). Trajectory clusters were furthermore associated with incident cardiometabolic cases after Exam 3 (men: ORClusterII = 3.5 (1.1-15.6); ORClusterIII = 7.5 (2.4-32.7); women: ORClusterII = 5.0 (1.1-34.1); ORClusterIII = 8.0 (2.2-51.7)). Associations remained significant after adjusting for a single time point cardiovascular risk score (Framingham). CONCLUSIONS: On a population-based level, distinct longitudinal risk profiles over a 14-year time period are differentially associated with cardiometabolic events. Our results suggest that longitudinal data may provide additional information beyond single time-point measures. Their inclusion in cardiometabolic risk assessment might improve early identification of individuals at risk. AU - Niedermayer, F. AU - Schauberger, G.* AU - Rathmann, W.* AU - Klug, S.J.* AU - Thorand, B. AU - Peters, A. AU - Rospleszcz, S. C1 - 70374 C2 - 55239 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Clusters of longitudinal risk profile trajectories are associated with cardiometabolic diseases: Results from the population-based KORA cohort. JO - PLoS ONE VL - 19 IS - 3 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - Short telomeres are associated with cardiovascular disease (CVD). We aimed to investigate, if genetically determined telomere-length effects CVD-risk in the Heinz-Nixdorf-Recall study (HNRS) population. We selected 14 single-nucleotide polymorphisms (SNPs) associated with telomere-length (p<10-8) from the literature and after exclusion 9 SNPs were included in the analyses. Additionally, a genetic risk score (GRS) using these 9 SNPs was calculated. Incident CVD was defined as fatal and non-fatal myocardial infarction, stroke, and coronary death. We included 3874 HNRS participants with available genetic data and had no known history of CVD at baseline. Cox proportional-hazards regression was used to test the association between the SNPs/GRS and incident CVD-risk adjusting for common CVD risk-factors. The analyses were further stratified by CVD risk-factors. During follow-up (12.1±4.31 years), 466 participants experienced CVD-events. No association between SNPs/GRS and CVD was observed in the adjusted analyses. However, the GRS, rs10936599, rs2487999 and rs8105767 increase the CVD-risk in current smoker. Few SNPs (rs10936599, rs2487999, and rs7675998) showed an increased CVD-risk, whereas rs10936599, rs677228 and rs4387287 a decreased CVD-risk, in further strata. The results of our study suggest different effects of SNPs/GRS on CVD-risk depending on the CVD risk-factor strata, highlighting the importance of stratified analyses in CVD risk-factors. AU - Tannemann, N.* AU - Erbel, R.* AU - Nöthen, M.M.* AU - Jöckel, K.H.* AU - Pechlivanis, S. C1 - 70681 C2 - 55813 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Genetic polymorphisms affecting telomere length and their association with cardiovascular disease in the Heinz-Nixdorf-Recall study. JO - PLoS ONE VL - 19 IS - 5 PB - Public Library Science PY - 2024 SN - 1932-6203 ER - TY - JOUR AB - In ecotoxicology, evaluation of toxicities and no observed effect concentrations (NOEC) of test compounds in experimental fish is commonly based on molecular-, biochemical- and analytical chemistry analyses of organ/tissue samples and the assessment of (histo-) pathological lesions. Standardization of organ/tissue sampling locations, sample numbers, and sample processing contributes to warrant the reproducibility and inter- and intra-study comparability of analysis results. The present article provides the first comprehensive tissue sampling guidelines specifically adapted to rainbow trout (Oncorhynchus mykiss) as a frequently used fish species in ecotoxicological studies. A broad spectrum of ~40 different organs and tissues is covered. Appropriate sampling locations, sample sizes and sample numbers for subsequent routine histopathological evaluation (all organs/tissue) and for molecular analyses (~30 organs/tissues) are described in detail and illustrated with schematic drawings and representative macroscopic and histological images. These field-proven sampling guidelines were developed based on the pertinent literature and practical experience in ecotoxicological fish studies. They are intended to serve as a standard reference for any routine ecotoxicological study using rainbow trout as a test system. A broad application of the featured tissue sampling procedures will help to improve the reproducibility of analyses and to reduce inter- and intra-study variability induced by sampling bias and (normal) inter-sample morphological variation, and will therefore provide a robust basis for reliable characterization of toxicity and NOEC identification of diverse test substances and aquatic pollutants. AU - Fiedler, S.* AU - Schrader, H.* AU - Theobalt, N.* AU - Hofmann, I.* AU - Geiger, T.* AU - Arndt, D.* AU - Wanke, R.* AU - Schwaiger, J.* AU - Blutke, A. C1 - 68111 C2 - 54589 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Standardized tissue sampling guidelines for histopathological and molecular analyses of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies. JO - PLoS ONE VL - 18 IS - 7 PB - Public Library Science PY - 2023 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The low five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) and the low diagnostic rate of early-stage PDAC via imaging highlight the need to discover novel biomarkers and improve the current screening procedures for early diagnosis. Familial pancreatic cancer (FPC) describes the cases of PDAC that are present in two or more individuals within a circle of first-degree relatives. Using innovative high-throughput proteomics, we were able to quantify the protein profiles of individuals at risk from FPC families in different potential pre-cancer stages. However, the high-dimensional proteomics data structure challenges the use of traditional statistical analysis tools. Hence, we applied advanced statistical learning methods to enhance the analysis and improve the results' interpretability. METHODS: We applied model-based gradient boosting and adaptive lasso to deal with the small, unbalanced study design via simultaneous variable selection and model fitting. In addition, we used stability selection to identify a stable subset of selected biomarkers and, as a result, obtain even more interpretable results. In each step, we compared the performance of the different analytical pipelines and validated our approaches via simulation scenarios. RESULTS: In the simulation study, model-based gradient boosting showed a more accurate prediction performance in the small, unbalanced, and high-dimensional datasets than adaptive lasso and could identify more relevant variables. Furthermore, using model-based gradient boosting, we discovered a subset of promising serum biomarkers that may potentially improve the current screening procedure of FPC. CONCLUSION: Advanced statistical learning methods helped us overcome the shortcomings of an unbalanced study design in a valuable clinical dataset. The discovered serum biomarkers provide us with a clear direction for further investigations and more precise clinical hypotheses regarding the development of FPC and optimal strategies for its early detection. AU - Ha, C.S.R. AU - Müller-Nurasyid, M. AU - Petrera, A. AU - Hauck, S.M. AU - Marini, F.* AU - Bartsch, D.K.* AU - Slater, E.P.* AU - Strauch, K. C1 - 67352 C2 - 54188 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Proteomics biomarker discovery for individualized prevention of familial pancreatic cancer using statistical learning. JO - PLoS ONE VL - 18 IS - 1 PB - Public Library Science PY - 2023 SN - 1932-6203 ER - TY - JOUR AB - Young adults with a later chronotype are vulnerable for a discrepancy in sleep rhythm between work- and free days, called social jet lag (SJL). This study analysed (i) chronotype/SJL association with visceral fat/skeletal muscle mass, (ii) the attribution to physical activity behaviour, and (iii) chronotype-specific changes in physical activity behaviour in young adults during the Covid-19 pandemic lockdown. Chronotype and SJL were derived from the Munich-Chrono-Type-Questionnaire in 320 German students (age 18-25 years) from September 2019 to January 2020, 156 of these participated in an online follow-up survey in June 2020. Body composition was assessed by bioimpedance analysis at baseline. Multivariable linear regression analyses were used to relate chronotype/SJL to body composition; the contribution of self-reported physical activity was tested by mediation analysis. At baseline, a later chronotype and a larger SJL were associated with a higher visceral fat mass (P<0.05), this relation was notably mediated by the attention to physical activity (P<0.05). Chronotype (P = 0.02) but not SJL (P = 0.87) was inversely associated with skeletal muscle mass. During the pandemic lockdown, chronotype hardly changed, but SJL was reduced. Timing and physical activity behaviour remained in most participants and changes were unrelated to chronotype (all P>0.07). A later chronotype/higher SJL may increase the risk of a higher visceral fat mass even in this relatively healthy sample, which may be partly due to their physical activity behaviour. Despite a reduction in SJL during the pandemic lockdown, later chronotypes did not change their physical activity behaviour more than earlier chronotypes. AU - Krueger, B.* AU - Stutz, B.* AU - Jankovic, N.* AU - Alexy, U.* AU - Kilanowski, A. AU - Libuda, L.* AU - Buyken, A.E.* C1 - 67215 C2 - 54226 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The association of chronotype and social jet lag with body composition in German students: The role of physical activity behaviour and the impact of the pandemic lockdown. JO - PLoS ONE VL - 18 IS - 1 PB - Public Library Science PY - 2023 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Celiac disease has an increasing incidence worldwide and is treated with lifelong adherence to a gluten-free diet. We aimed to describe gluten-free diet adherence rates in children with screening-identified celiac disease, determine adherence-related factors, and compare adherence to food records in a multinational prospective birth cohort study. METHODS: Children in The Environmental Determinants of Diabetes in the Young study with celiac disease were included. Subjects had at least annual measurement of adherence (parent-report) and completed 3-day food records. Descriptive statistics, t-tests, Kruskal-Wallis tests and multivariable logistic and linear regression were employed. RESULTS: Two hundred ninety (73%) and 199 (67%) of subjects were always adherent to a gluten-free diet at 2 and 5 years post celiac disease diagnosis respectively. The percentage of children with variable adherence increased from 1% at 2 years to 15% at 5 years. Children with a first-degree relative with celiac disease were more likely to be adherent to the gluten-free diet. Gluten intake on food records could not differentiate adherent from nonadherent subjects. Adherent children from the United States had more gluten intake based on food records than European children (P < .001 and P = .007 at 2 and 5 years respectively). CONCLUSION: Approximately three-quarters of children with screening-identified celiac disease remain strictly adherent to a gluten-free diet over time. There are no identifiable features associated with adherence aside from having a first-degree relative with celiac disease. Despite good parent-reported adherence, children from the United States have more gluten intake when assessed by food records. Studies on markers of gluten-free diet adherence, sources of gluten exposure (particularly in the United States), and effects of adherence on mucosal healing are needed. AU - Mehta, P.* AU - Li, Q.* AU - Stahl, M.* AU - Uusitalo, U.* AU - Lindfors, K.* AU - Butterworth, M.D.* AU - Kurppa, K.* AU - Virtanen, S.* AU - Koletzko, S.* AU - Aronsson, C.* AU - Hagopian, W.A.* AU - Rewers, M.J.* AU - Toppari, J.* AU - Ziegler, A.-G. AU - Akolkar, B.* AU - Krischer, J.P.* AU - Agardh, D.* AU - Liu, E.* C1 - 67399 C2 - 54165 TI - Gluten-free diet adherence in children with screening-detected celiac disease using a prospective birth cohort study. JO - PLoS ONE VL - 18 IS - 2 PY - 2023 SN - 1932-6203 ER - TY - JOUR AB - Calibrating model parameters on heterogeneous data can be challenging and inefficient. This holds especially for likelihood-free methods such as approximate Bayesian computation (ABC), which rely on the comparison of relevant features in simulated and observed data and are popular for otherwise intractable problems. To address this problem, methods have been developed to scale-normalize data, and to derive informative low-dimensional summary statistics using inverse regression models of parameters on data. However, while approaches only correcting for scale can be inefficient on partly uninformative data, the use of summary statistics can lead to information loss and relies on the accuracy of employed methods. In this work, we first show that the combination of adaptive scale normalization with regression-based summary statistics is advantageous on heterogeneous parameter scales. Second, we present an approach employing regression models not to transform data, but to inform sensitivity weights quantifying data informativeness. Third, we discuss problems for regression models under non-identifiability, and present a solution using target augmentation. We demonstrate improved accuracy and efficiency of the presented approach on various problems, in particular robustness and wide applicability of the sensitivity weights. Our findings demonstrate the potential of the adaptive approach. The developed algorithms have been made available in the open-source Python toolbox pyABC. AU - Schälte, Y. AU - Hasenauer, J. C1 - 67910 C2 - 54388 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Informative and adaptive distances and summary statistics in sequential approximate Bayesian computation. JO - PLoS ONE VL - 18 IS - 5 PB - Public Library Science PY - 2023 SN - 1932-6203 ER - TY - JOUR AB - PURPOSE: The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. MATERIALS AND METHODS: Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. RESULTS: Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. CONCLUSIONS: This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence. AU - Babini, G.* AU - Baiocco, G.* AU - Barbieri, S.* AU - Morini, J.* AU - Sangsuwan, T.* AU - Haghdoost, S.* AU - Yentrapalli, R. AU - Azimzadeh, O. AU - Rombouts, C.* AU - Aerts, A.* AU - Quintens, R.* AU - Ebrahimian, T.* AU - Benotmane, M.A.* AU - Ramadan, R.* AU - Baatout, S.* AU - Tapio, S. AU - Harms-Ringdahl, M.* AU - Ottolenghi, A.* C1 - 64643 C2 - 52367 TI - A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells. JO - PLoS ONE VL - 17 IS - 3 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND AND AIM: Despite its vasodilatory effect, adrenomedullin and its surrogate mid-regional pro-adrenomedullin (MR-proADM) have been found to be positively associated with all-cause and cardiovascular mortality. However, the underlying mechanisms thereof remain unclear and the associations were mostly shown in geriatric cohorts or in patients with chronic diseases. Therefore, we aimed to investigate the possible involvement of abdominal obesity, selected adipokines, and biomarkers of subclinical inflammation in the association of MR-proADM with mortality in a population based study cohort. METHODS: Prospective analysis of the KORA F4 study; median follow-up 9.1 (8.8-9.4) years. Complete data on MR-proADM and mortality was available for 1551 participants, aged 56.9±12.9 years (mean±SD). Correlation and regression analyses of MR-proADM with overall (BMI) and abdominal obesity (waist circumference), selected adipokines and biomarkers of subclinical inflammation. Cox proportional hazard models on the association of MR-proADM with all-cause and cardiovascular mortality with adjustment for cardiovascular risk factors and selected biomarkers in study subgroups (n = 603-1551). RESULTS: MR-proADM associated with all-cause (HR (95%CI): 2.37 (1.72-3.26) and 2.31 (1.67-3.20)) and cardiovascular mortality (4.28 (2.19-8.39) and 4.44 (2.25-8.76)) after adjustment for traditional cardiovascular risk factors including BMI or waist circumference, respectively. MR-proADM was further associated with four out of seven examined adipokines (leptin, retinol-binding protein-4, chemerin, and adiponectin) and with five out of eleven examined biomarkers of subclinical inflammation (high-sensitivity C-reactive protein, interleukin-6, myeloperoxidase, interleukin-22, and interleukin-1 receptor antagonist) after multivariable adjustment and correction for multiple testing. However, only IL-6 substantially attenuated the association of MR-proADM with all-cause mortality. CONCLUSIONS: We found an association of MR-proADM with (abdominal) obesity, selected adipokines, and biomarkers of subclinical inflammation. However, the association of MR-proADM with mortality was independent of these parameters. Future studies should investigate the role of IL-6 and further characteristics of subclinical inflammation in the association between MR-proADM and all-cause mortality. AU - Gar, C. AU - Thorand, B. AU - Herder, C.* AU - Sujana, C. AU - Heier, M. AU - Meisinger, C. AU - Peters, A. AU - Koenig, W.* AU - Rathmann, W.* AU - Roden, M.* AU - Stumvoll, M.* AU - Maalmi, H.* AU - Meitinger, T.* AU - Then, H.* AU - Seissler, J. AU - Then, C. C1 - 64009 C2 - 51984 TI - Association of circulating MR-proADM with all-cause and cardiovascular mortality in the general population: Results from the KORA F4 cohort study. JO - PLoS ONE VL - 17 IS - 1 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Since the beginning of the pandemic in 2020, COVID-19 has changed the medical landscape. International recommendations for localized prostate cancer (PCa) include deferred treatment and adjusted therapeutic routines. MATERIALS AND METHODS: To longitudinally evaluate changes in PCa treatment strategies in urological and radiotherapy departments in Germany, a link to a survey was sent to 134 institutions covering two representative baseline weeks prior to the pandemic and 13 weeks from March 2020 to February 2021. The questionnaire captured the numbers of radical prostatectomies, prostate biopsies and case numbers for conventional and hypofractionation radiotherapy. The results were evaluated using descriptive analyses. RESULTS: A total of 35% of the questionnaires were completed. PCa therapy increased by 6% in 2020 compared to 2019. At baseline, a total of 69 radiotherapy series and 164 radical prostatectomies (RPs) were documented. The decrease to 60% during the first wave of COVID-19 particularly affected low-risk PCa. The recovery throughout the summer months was followed by a renewed reduction to 58% at the end of 2020. After a gradual decline to 61% until July 2020, the number of prostate biopsies remained stable (89% to 98%) during the second wave. The use of RP fluctuated after an initial decrease without apparent prioritization of risk groups. Conventional fractionation was used in 66% of patients, followed by moderate hypofractionation (30%) and ultrahypofractionation (4%). One limitation was a potential selection bias of the selected weeks and the low response rate. CONCLUSION: While the diagnosis and therapy of PCa were affected in both waves of the pandemic, the interim increase between the peaks led to a higher total number of patients in 2020 than in 2019. Recommendations regarding prioritization and fractionation routines were implemented heterogeneously, leaving unexplored potential for future pandemic challenges. AU - Harke, N.N.* AU - Wagner, C.* AU - Hermann, R.M.* AU - Hadaschik, B.A.* AU - Radtke, J.P.* AU - Altay-Langguth, A.* AU - Aufderklamm, S.* AU - Bach, C.* AU - Becker-Schiebe, M.* AU - Blana, A.* AU - Bruns, F.* AU - Buse, S.* AU - Combs, S.E. AU - Engels, C.L.* AU - Ezzibdeh, E.* AU - Fiedler, M.* AU - Fischer, L.A.* AU - Farzat, M.* AU - Frismann, A.* AU - Heck, M.M.* AU - Henkenberens, C.* AU - Roesch, M.C.* AU - Käding, C.* AU - Klautke, G.* AU - Krausewitz, P.* AU - Kuczyk, M.A.* AU - Leitsmann, C.* AU - Lettmaier, S.* AU - Mahjoub, S.* AU - Manseck, A.* AU - Medenwald, D.* AU - Meyer, A.* AU - Micke, O.* AU - Moritz, R.* AU - Ott, M.* AU - Peters, I.* AU - Pokupic, S.* AU - Porres, D.* AU - Preisser, F.* AU - Reichel, K.* AU - Schneider, A.* AU - Schwentner, C.* AU - Scobioala, S.* AU - Truss, M.* AU - Wegener, D.* AU - Wezel, F.* AU - Willborn, K.* AU - Witt, J.H.* AU - Wittig, A.* AU - Wittlinger, M.* AU - Wolff, H.A.* AU - Zimmermanns, V.* AU - Christiansen, H.* C1 - 65429 C2 - 52676 TI - Lessons learned after one year of COVID-19 from a urologist and radiotherapist view: A German survey on prostate cancer diagnosis and treatment. JO - PLoS ONE VL - 17 IS - 6 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - Targeting the tumor-associated carbonic anhydrase XII (CA XII) is considered a promising strategy to improve cancer treatment. As such progress is highly demanded for ovarian carcinomas, the present study aimed to provide deeper information about their CA XII expression profile. A large collection of tissue specimens was stained immunohistochemically with a specific anti-CA XII antibody to evaluate the expression in neoplastic and non-neoplastic epithelial ovarian cells. In addition, flow cytometry was used to measure CA XII expression on tumor cells from malignant ascites fluid. Binding of the antibody revealed a significant CA XII expression in most ovarian carcinoma tissue samples and ascites-derived ovarian carcinoma cells. Moreover, CA XII was expressed at higher levels in ovarian carcinomas as compared to borderline ovarian tumors and non-neoplastic ovarian epithelia. Within the carcinoma tissues, high expression of CA XII was associated with higher tumor grading and a trend towards shorter overall survival. Our results indicate that CA XII plays a crucial role for the malignancy of ovarian carcinoma cells and emphasize the potential of CA XII as a diagnostic marker and therapeutic target in the management of ovarian carcinomas. AU - Hiepp, L.* AU - Mayr, D.* AU - Gärtner, K. AU - Schmoeckel, E.* AU - Klauschen, F.* AU - Burges, A.* AU - Mahner, S.* AU - Zeidler, R. AU - Czogalla, B.* C1 - 65816 C2 - 52916 TI - Carbonic anhydrase XII as biomarker and therapeutic target in ovarian carcinomas. JO - PLoS ONE VL - 17 IS - 7 July PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - PURPOSE: Galectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro. METHODS: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis. RESULTS: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively. CONCLUSION: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins. AU - Hillenmayer, A.* AU - Wertheimer, C.M.* AU - Geerlof, A. AU - Eibl, K.H.* AU - Priglinger, S.* AU - Priglinger, C.* AU - Ohlmann, A.* C1 - 64688 C2 - 52407 TI - Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro. JO - PLoS ONE VL - 17 IS - 3 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - Substantial genetic correlations have been reported across psychiatric disorders and numerous cross-disorder genetic variants have been detected. To identify the genetic variants underlying general psychopathology in childhood, we performed a genome-wide association study using a total psychiatric problem score. We analyzed 6,844,199 common SNPs in 38,418 school-aged children from 20 population-based cohorts participating in the EAGLE consortium. The SNP heritability of total psychiatric problems was 5.4% (SE = 0.01) and two loci reached genome-wide significance: rs10767094 and rs202005905. We also observed an association of SBF2, a gene associated with neuroticism in previous GWAS, with total psychiatric problems. The genetic effects underlying the total score were shared with common psychiatric disorders only (attention-deficit/hyperactivity disorder, anxiety, depression, insomnia) (rG > 0.49), but not with autism or the less common adult disorders (schizophrenia, bipolar disorder, or eating disorders) (rG < 0.01). Importantly, the total psychiatric problem score also showed at least a moderate genetic correlation with intelligence, educational attainment, wellbeing, smoking, and body fat (rG > 0.29). The results suggest that many common genetic variants are associated with childhood psychiatric symptoms and related phenotypes in general instead of with specific symptoms. Further research is needed to establish causality and pleiotropic mechanisms between related traits. AU - Neumann, A.* AU - Nolte, I.M.* AU - Pappa, I.* AU - Ahluwalia, T.S.* AU - Pettersson, E.* AU - Rodriguez, A.* AU - Whitehouse, A.* AU - van Beijsterveldt, C.E.M.* AU - Benyamin, B.* AU - Hammerschlag, A.R.* AU - Helmer, Q.* AU - Karhunen, V.* AU - Krapohl, E.* AU - Lu, Y.* AU - van der Most, P.J.* AU - Palviainen, T.* AU - St Pourcain, B.* AU - Seppälä, I.* AU - Suarez, A.* AU - Vilor-Tejedor, N.* AU - Tiesler, C.M. AU - Wang, C.* AU - Wills, A.* AU - Zhou, A.* AU - Alemany, S.* AU - Bisgaard, H.* AU - Bønnelykke, K.* AU - Davies, G.E.* AU - Hakulinen, C.* AU - Henders, A.K.* AU - Hyppönen, E.* AU - Stokholm, J.* AU - Bartels, M.* AU - Hottenga, J.J.* AU - Heinrich, J.* AU - Hewitt, J.K.* AU - Keltikangas-Järvinen, L.* AU - Korhonen, T.* AU - Kaprio, J.* AU - Lahti, J.* AU - Lahti-Pulkkinen, M.* AU - Lehtimäki, T.* AU - Middeldorp, C.M.* AU - Najman, J.M.* AU - Pennell, C.E.* AU - Power, C.* AU - Oldehinkel, A.J.* AU - Plomin, R.* AU - Räikkönen, K.* AU - Raitakari, O.T.* AU - Rimfeld, K.* AU - Sass, L.* AU - Snieder, H.* AU - Standl, M.* AU - Sunyer, J.* AU - Williams, G.M.* AU - Bakermans-Kranenburg, M.J.* AU - Boomsma, D.I.* AU - van IJzendoorn, M.H.* AU - Hartman, C.A.* AU - Tiemeier, H.* C1 - 65948 C2 - 52562 TI - A genome-wide association study of total child psychiatric problems scores. JO - PLoS ONE VL - 17 IS - 8 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Although gene-expression (GE) and protein levels are typically strongly genetically regulated, their correlation is known to be low. Here we investigate this phenomenon by focusing on the genetic background of this correlation in order to understand the similarities and differences in the genetic regulation of these omics layers. METHODS AND RESULTS: We performed locus-wide association studies of 92 protein levels measured in whole blood for 2,014 samples of European ancestry and found that 66 are genetically regulated. Three female- and one male-specific effects were detected. We estimated the genetically regulated GE for all significant genes in 49 GTEx v8 tissues. A total of 7 proteins showed negative correlations with their respective GE across multiple tissues. Finally, we tested for causal links of GE on protein expression via Mendelian Randomization, and confirmed a negative causal effect of GE on protein level for five of these genes in a total of 63 gene-tissue pairs: BLMH, CASP3, CXCL16, IL6R, and SFTPD. For IL6R, we replicated the negative causal effect on coronary-artery disease (CAD), while its GE was positively linked to CAD. CONCLUSION: While total GE and protein levels are only weakly correlated, we found high correlations between their genetically regulated components across multiple tissues. Of note, strong negative causal effects of tissue-specific GE on five protein levels were detected. Causal network analyses revealed that GE effects on CAD risks was in general mediated by protein levels. AU - Pott, J.* AU - Garcia, T.* AU - Hauck, S.M. AU - Petrera, A. AU - Wirkner, K.* AU - Loeffler, M.* AU - Kirsten, H.* AU - Peters, A. AU - Scholz, M.* C1 - 65069 C2 - 52151 TI - Genetically regulated gene expression and proteins revealed discordant effects. JO - PLoS ONE VL - 17 IS - 5 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - AIMS: Women after gestational diabetes mellitus (GDM) are a risk group for cardiometabolic diseases but are hard to reach by conventional lifestyle programs. Therefore, we tested whether a novel, smartphone-delivered intervention, TRIANGLE, is accepted by women after GDM and alters cardiometabolic risk behaviors and outcomes. TRIANGLE targets gradual habit change of mind and emotion, physical activity, nutrition, and sleep. METHODS: We conducted a 6-month multicenter, randomized-controlled trial of TRIANGLE versus standard care with 66 women 3-18 months after GDM in Germany. The primary outcome was the proportion of women achieving ≥3 out of 5 Diabetes Prevention Program goals, i.e. physical activity ≥150 min/week (moderate to high intensity), fiber intake ≥15 g/1,000 kcal, fat intake <30% of total energy intake, saturated fat intake <10% of total energy intake, and weight reduction ≥5% if BMI ≥23 kg/m2 or weight maintenance if BMI <23 kg/m2. Intervention participants also rated the TRIANGLE app in the Mobile Application Rating Scale (uMARS). RESULTS: In the predefined, modified intention-to-treat analysis including 64 women, 6 out of 27 women in the intervention group [22%(10-40)] and 3 out of 27 women in the control group [11%(3-27)] reached the primary outcome (p = 0.47). In the predefined per-protocol intervention subgroup, the proportion was 4 out of 14 women [29%(11-55); p = 0.20 vs. control]. TRIANGLE app users were active on 42% of days and rated the app's quality and perceived impact with 4.3±0.8 out of 5 uMARS points. CONCLUSIONS: This first trial did not show the efficacy of the TRIANGLE intervention. However, the app was well accepted and considered helpful by most users. Therefore, this trial supports further development and testing of TRIANGLE and other app interventions for women after GDM. Additionally, it identifies necessary adaptations in trial design to better accommodate non-intensive lifestyle interventions for this target group. TRIAL REGISTRATION: Trial registration at drks.de (DRKS00012996). AU - Potzel, A. AU - Gar, C. AU - Banning, F. AU - Sacco, V. AU - Fritsche, A. AU - Fritsche, L. AU - Müssig, K.* AU - Dauben, L.* AU - Seissler, J. AU - Lechner, A. C1 - 64940 C2 - 52531 TI - A novel smartphone app to change risk behaviors of women after gestational diabetes: A randomized controlled trial. JO - PLoS ONE VL - 17 IS - 4 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016–2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally. AU - Scheck, M.K.* AU - Lehmann, L.* AU - Zaucha, M.* AU - Schwarzlmueller, P.* AU - Huber, K.* AU - Pritsch, M.* AU - Barba-Spaeth, G.* AU - Thorn-Seshold, O.* AU - Krug, A.B.* AU - Endres, S. AU - Rothenfußer, S. AU - Thorn-Seshold, J. C1 - 64316 C2 - 52180 TI - FluoRNT: A robust, efficient assay for the detection of neutralising antibodies against yellow fever virus 17D. JO - PLoS ONE VL - 17 IS - 2 February PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Much is known about the association between physical activity and the occurrence of stroke. However, the evidence about the correlation between pre-stroke physical activity and post-stroke quality of life remains inconsistent. Thus, there is a high public health relevance to the topic. AIM: The aim of this study was to investigate the association between pre-stroke physical activity and physical quality of life after three months. METHODS: Data arises from 858 patients with stroke included a prospective single-centre observational cohort study in Augsburg, Germany, between September 2018 and November 2019. The participants were recruited at the Department of Neurology and Clinical Neurophysiology, University Hospital of Augsburg after their stroke event. The level of physical activity was determined following the short form of the International Physical Activity Questionnaire at baseline. Physical quality of life was assessed three months after hospital discharge using the German version of the Stroke Impact Scale (SIS). A multiple linear regression model and a quantile regression were carried out. RESULTS: A total of 497 patients were included in the analysis (mean age 69.6, 58.8% male), 26.2% had a high, 18.9% a moderate and 54.9% a low level of pre-stroke physical activity. Patients with high pre-stroke physical activity had a significantly better physical quality of life three months after stroke in the SIS physical domain (beta = 4.1) and in the SIS subdomains hand function (beta = 5.6), mobility (beta = 4.1) and activities of daily living (beta = 3.7). In the physical domain and the subdomain mobility, the effect was especially strong for persons with low physical quality of life after three months. CONCLUSION: Pre-stroke physical activity seems to have an important and positive association with physical quality of life after three months in patients with mild disability. Further studies are needed to confirm these results. AU - Zirnsak, M.* AU - Meisinger, C. AU - Linseisen, J. AU - Ertl, M.* AU - Zickler, P.* AU - Naumann, M.* AU - Kirchberger, I. C1 - 65584 C2 - 52386 TI - Associations between pre-stroke physical activity and physical quality of life three months after stroke in patients with mild disability. JO - PLoS ONE VL - 17 IS - 6 PY - 2022 SN - 1932-6203 ER - TY - JOUR AB - In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed. AU - Candeias, J. AU - Schmidt-Weber, C.B. AU - Buters, J.T.M. C1 - 63578 C2 - 51597 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Dosing intact birch pollen grains at the airliquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B. JO - PLoS ONE VL - 16 IS - 11 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - PURPOSE: OCT-angiography (OCT-A) offers a non-invasive method to visualize retinochoroidal microvasculature. As glaucoma disease affects retinal ganglion cells in the macula, macular microcirculation is of interest. The purpose of the study was to investigate regional macular vascular characteristics in patients with ocular hypertension (OHT), pre-perimetric primary open-angle glaucoma (pre-POAG) and controls by OCT-A in three microvascular layers. MATERIAL AND METHODS: 180 subjects were recruited from the Erlangen Glaucoma Registry, the Department of Ophthalmology, University of Erlangen and residents: 38 OHT, 20 pre-POAG, 122 controls. All subjects received an ophthalmological examination including measurements of retinal nerve fibre layer (RNFL), retinal ganglion cell layer (RGC), inner nuclear layer (INL), and Bruch's Membrane Opening-Minimum Rim Width (BMO-MRW). Macular vascular characteristics (vessel density, VD, foveal avascular zone, FAZ) were measured by OCT-A (Spectralis OCT II) in superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). RESULTS: With age correction of VD data, type 3 tests on fixed effects showed a significant interaction between diagnosis and sectorial VD in SVP (p = 0.0004), ICP (p = 0.0073), and DCP (p = 0.0003). Moreover, a significance in sectorial VD was observed within each layer (p<0.0001) and for the covariate age (p<0.0001). FAZ differed significantly between patients' groups only in ICP (p = 0.03), not in SVP and DCP. For VD the AUC values of SVP, ICP, and DCP were highest among diagnostic modalities (AUC: 0.88, 95%-CI: 0.75-1.0, p<0.001). CONCLUSION: Regional reduced macula VD was observed in all three retinal vascular layers of eyes with OHT and pre-POAG compared to controls, indicating localized microvascular changes as early marker in glaucoma pathogenesis. AU - Hohberger, B.* AU - Lucio, M. AU - Schlick, S.* AU - Wollborn, A.* AU - Hosari, S.* AU - Mardin, C.* C1 - 61317 C2 - 50123 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - OCT-angiography: Regional reduced macula microcirculation in ocular hypertensive and pre-perimetric glaucoma patients. JO - PLoS ONE VL - 16 IS - 2 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Aims Insulin sensitivity and insulin secretion can be estimated by multiple indices from fasting blood samples or blood samples obtained during oral glucose tolerance tests. The test-retest reliability of these indices in repeated measurements within the same individuals can strongly vary. Methods We analyzed data of persons without diabetes who underwent two repeated OGTTs. For each measurement pair, we calculated multiple commonly used indices for the assessment of insulin secretion and insulin sensitivity. We then evaluated the coefficient of variation (standard deviation/mean) and discriminant ratio for each index. Results 89 persons underwent two OGTTs with a median interval of 86 days (IQR 64–249). Among indices of insulin sensitivity derived from fasting blood samples, the revised quantitative insulin sensitivity check index had the smallest coefficient of variation (2.8 ± 2.1%) whereas the C-peptide based homeostasis model assessment 2 had the highest discriminant ratio (1.97 (1.65–2.39)). As for insulin sensitivity indices that are based on OGTT, the oral glucose insulin sensitivity index had the smallest coefficient of variation (6.5 ± 5.1%). The highest discriminant ratio was found for the non-esterified fatty acids-based insulin sensitivity index (NEFA-ISI, 2.70 (2.30–3.22)). For the assessment of insulin secretion from fasting variables, the lowest mean coefficient of variation was found for C-peptide based homeostasis model assessment 2 beta with 10.8 ± 8% and the highest discriminant ratio for the C-peptide / Glucose-Ratio (2.18 (1.84–2.63)). Among indices assessing insulin secretion from an OGTT, the lowest coefficient of variation was found for the ratio of the areas under the C-peptide and glucose curves from 0 to 120 minutes with 11.3 ± 9.7%. Conclusion The data reveal large differences in the reproducibility and the discrimination capability of different indices that assess insulin sensitivity or insulin secretion. Our findings can aid the selection of an appropriate index in clinical studies. AU - Hudak, S. AU - Huber, P. AU - Lamprinou, A. AU - Fritsche, L. AU - Stefan, N. AU - Peter, A. AU - Birkenfeld, A.L. AU - Fritsche, A. AU - Heni, M. AU - Wagner, R. C1 - 63375 C2 - 51493 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Reproducibility and discrimination of different indices of insulin sensitivity and insulin secretion. JO - PLoS ONE VL - 16 IS - 10 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Although investigation of the brains of criminals began quite early in the history of psychophysiological research, little is known about brain plasticity of offenders with psychopathy. Building on our preliminary study reporting successful brain self-regulation using slow cortical potential (SCP) neurofeedback in offenders with psychopathy, we investigated the central nervous and autonomic peripheral changes occurring after brain self-regulation in a group of severe male offenders with psychopathy. Regarding the central nervous system, an overall suppression of the psychopathic overrepresentation of slow frequency bands was found, such as delta and theta band activity, after EEG neurofeedback. In addition, an increase in alpha band activity could be observed after the SCP self-regulation training. Electrodermal activity adaptively changed according to the regulation task, and this flexibility improved over training time. The results of this study point towards a constructive learning process and plasticity in neural and peripheral measures of offenders with psychopathy. AU - Konicar, L.* AU - Radev, S.* AU - Silvoni, S.* AU - Bolinger, E.* AU - Veit, R. AU - Strehl, U.* AU - Vesely, C.* AU - Plener, P.L.* AU - Poustka, L.* AU - Birbaumer, N.* C1 - 60948 C2 - 49999 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Balancing the brain of offenders with psychopathy? Resting state EEG and electrodermal activity after a pilot study of brain self-regulation training. JO - PLoS ONE VL - 16 IS - 1 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - The MERS-CoV, SARS-CoV, and SARS-CoV-2 are highly pathogenic viruses that can cause severe pneumonic diseases in humans. Unfortunately, there is a non-available effective treatment to combat these viruses. Domain-motif interactions (DMIs) are an essential means by which viruses mimic and hijack the biological processes of host cells. To disentangle how viruses achieve this process can help to develop new rational therapies. Data mining was performed to obtain DMIs stored as regular expressions (regexp) in 3DID and ELM databases. The mined regexp information was mapped on the coronaviruses’ proteomes. Most motifs on viral protein that could interact with human proteins are shared across the coronavirus species, indicating that molecular mimicry is a common strategy for coronavirus infection. Enrichment ontology analysis for protein domains showed a shared biological process and molecular function terms related to carbon source utilization and potassium channel regulation. Some of the mapped motifs were nested on B, and T cell epitopes, suggesting that it could be as an alternative way for reverse vaccinology. The information obtained in this study could be used for further theoretic and experimental explorations on coronavirus infection mechanism and development of medicines for treatment. AU - Martínez, Y.A.* AU - Guo, X.* AU - Portales-Pérez, D.P.* AU - Rivera, G.* AU - Castañeda-Delgado, J.E.* AU - Garcia Perez, C. AU - Enciso-Moreno, J.A.* AU - Lara-Ramírez, E.E.* C1 - 61414 C2 - 50237 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The analysis on the human protein domain targets and host-like interacting motifs for the MERS-CoV and SARS-CoV/CoV-2 infers the molecular mimicry of coronavirus. JO - PLoS ONE VL - 16 IS - 2 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - To strengthen the coordinating function of general practitioners (GPs) in the German healthcare system, a copayment of €10 was introduced in 2004. Due to a perceived lack of efficacy and a high administrative burden, it was abolished in 2012. The present cohort study investigates characteristics and differences of GP-coordinated and uncoordinated patients in Bavaria, Germany, concerning morbidity and ambulatory specialist costs and whether these differences have changed after the abolition of the copayment. We performed a retrospective routine data analysis, using claims data of the Bavarian Association of the Statutory Health Insurance Physicians during the period 2011–2012 (with copayment) and 2013–2016 (without copayment), covering 24 quarters. Coordinated care was defined as specialist contact only with referral. Multinomial regression modelling, including inverse probability of treatment weighting, was used for the cohort analysis of 500 000 randomly selected patients. Longitudinal regression models were calculated for cost estimation. Coordination of care decreased substantially after the abolition of the copayment, accompanied by increasing proportions of patients with chronic and mental diseases in the uncoordinated group, and a corresponding decrease in the coordinated group. In the presence of the copayment, uncoordinated patients had €21.78 higher specialist costs than coordinated patients, increasing to €24.94 after its abolition. The results indicate that patients incur higher healthcare costs for specialist ambulatory care when their care is uncoordinated. This effect slightly increased after abolition of the copayment. Beyond that, the abolition of the copayment led to a substantial reduction in primary care coordination, particularly affecting vulnerable patients. Therefore, coordination of care in the ambulatory setting should be strengthened. AU - Olm, M.* AU - Donnachie, E.* AU - Tauscher, M.* AU - Gerlach, R.* AU - Linde, K.* AU - Maier, W. AU - Schwettmann, L. AU - Schneider, A.* C1 - 62399 C2 - 50792 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Ambulatory specialist costs and morbidity of coordinated and uncoordinated patients before and after abolition of copayment: A cohort analysis. JO - PLoS ONE VL - 16 IS - 6 June PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Accumulating evidence links dietary intake to inflammatory processes involved in non-communicable disease (NCD) development. The dietary inflammatory index (DII) designed by Shivappa et al. has been shown to capture the inflammatory potential of dietary behavior in a large number of epidemiological studies. Thus, the DII may serve as future tool to assess someone's nutritional inflammatory capacities and hence, the individual risks for NCD development later in life. The calculation method of the DII, however, can benefit from alternative mathematical steps, particularly regarding the transformation from standardized daily food consumption to percentile scores. Here, we provide novel approaches, the scaling-formula (SF) and scaling-formula with outlier detection (SFOD) methods, with the aim to optimize the DII calculation method proposed by Shivappa and colleagues. We illustrate on simulated data specific limitations of the original DII calculation and show the benefits of the SF/SFOD by using simulated data and data from the prospective TEENDIAB study cohort, which supports the application of SF/SFOD in future epidemiological and clinical studies. AU - Pawlow, X. AU - Ott, R. AU - Winkler, C. AU - Ziegler, A.-G. AU - Hummel, S. C1 - 63506 C2 - 51572 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A new mathematical approach to improve the original dietary inflammatory index (DII) calculation. JO - PLoS ONE VL - 16 IS - 11 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Background Working in the nursing sector is accompanied by great physical and mental health burdens. Consequently, it is necessary to develop target-oriented, sustainable profession-specific support and health promotion measures for nurses. Objectives The present review aims to give an overview of existing major health problems and violence experiences of nurses in different settings (acute care hospitals, long-term care facilities, and home-based long-term care) in Germany. Methods A systematic literature search was conducted in PubMed and PubPsych and completed by a manual search upon included studies' references and health insurance reports. Articles were included if they had been published after 2010 and provided data on health problems or violence experiences of nurses in at least one care setting. Results A total of 29 studies providing data on nurses health problems and/or violence experience were included. Of these, five studies allowed for direct comparison of nurses in the settings. In addition, 14 studies provided data on nursing working in acute care hospitals, ten on nurses working in long-term care facilities, and four studies on home-based long-term care. The studies either conducted a setting-specific approach or provided subgroup data from setting-unspecific studies. The remaining studies did not allow setting-related differentiation of the results. The available results indicate that mental health problems are the highest for nurses in acute care hospitals. Regarding violence experience, nurses working in long-term care facilities appear to be most frequently affected. Conclusion The state of research on setting-specific differences of nurses' health problems and violence experiences is insufficient. Setting-specific data are necessesary to develop target-group specific and feasible interventions to support the nurses' health and prevention of violence, as well as dealing with violence experiences of nurses. AU - Schaller, A.* AU - Klas, T.* AU - Gernert, M.* AU - Steinbeisser, K. C1 - 63606 C2 - 51600 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Health problems and violence experiences of nurses working in acute care hospitals, longterm care facilities, and home-based longterm care in Germany: A systematic review. JO - PLoS ONE VL - 16 IS - 11 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: As base excess had shown superiority over lactate as a prognostic parameter in intensive care unit (ICU) surgical patients we aimed to evaluate course of lactate, base excess and pH for prediction of mortality of medical ICU patients. MATERIALS AND METHODS: For lactate, pH and base excess, values at the admission to ICU, at 24 ± 4 hours, maximum or minimum in the first 24 hours and in 24-48 hours after admission were collected from all patients admitted to the Medical ICU of the University Hospital Tübingen between January 2016 until December 2018 (N = 4067 at admission, N = 1715 with ICU treatment > 48 h) and investigated for prediction of in-hospital-mortality. RESULTS: Mortality was 22% and significantly correlated with all evaluated parameters. Strongest predictors of mortality determined by ROC were maximum lactate in 24 h (AUROC 0.74, cut off 2.7 mmol/L, hazard ratio of risk group with value > cut off 3.20) and minimum pH in 24 h (AUROC 0.71, cut off 7.31, hazard ratio for risk group 2.94). Kaplan Meier Curves stratified across these cut offs showed early and clear separation. Hazard ratios per standard deviation increase were highest for maximum lactate in 24 h (HR 1.65), minimum base excess in 24 h (HR 1.56) and minimum pH in 24 h (HR 0.75). CONCLUSION: Lactate, pH and base excess were all suitable predictors of mortality in internal ICU patients, with maximum / minimum values in 24 and 24-48 h after admission altogether stronger predictors than values at admission. Base excess and pH were not superior to lactate for prediction of mortality. AU - Schork, A. AU - Moll, K.* AU - Haap, M.* AU - Riessen, R.* AU - Wagner, R. C1 - 63897 C2 - 51784 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Course of lactate, pH and base excess for prediction of mortality in medical intensive care patients. JO - PLoS ONE VL - 16 IS - 12 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Computational reproducibility is a corner stone for sound and credible research. Especially in complex statistical analyses-such as the analysis of longitudinal data-reproducing results is far from simple, especially if no source code is available. In this work we aimed to reproduce analyses of longitudinal data of 11 articles published in PLOS ONE. Inclusion criteria were the availability of data and author consent. We investigated the types of methods and software used and whether we were able to reproduce the data analysis using open source software. Most articles provided overview tables and simple visualisations. Generalised Estimating Equations (GEEs) were the most popular statistical models among the selected articles. Only one article used open source software and only one published part of the analysis code. Replication was difficult in most cases and required reverse engineering of results or contacting the authors. For three articles we were not able to reproduce the results, for another two only parts of them. For all but two articles we had to contact the authors to be able to reproduce the results. Our main learning is that reproducing papers is difficult if no code is supplied and leads to a high burden for those conducting the reproductions. Open data policies in journals are good, but to truly boost reproducibility we suggest adding open code policies. AU - Seibold, H. AU - Czerny, S.* AU - Decke, S.* AU - Dieterle, R.* AU - Eder, T.* AU - Fohr, S.* AU - Hahn, N.* AU - Hartmann, R.* AU - Heindl, C.* AU - Kopper, P.* AU - Lepke, D.* AU - Loidl, V.* AU - Mandl, M.* AU - Musiol, S.* AU - Peter, J.* AU - Piehler, A.* AU - Rojas, E.* AU - Schmid, S.* AU - Schmidt, H.* AU - Schmoll, M.* AU - Schneider, L.* AU - To, X.Y.* AU - Tran, V.* AU - Völker, A.* AU - Wagner, M.* AU - Wagner, J.* AU - Waize, M.* AU - Wecker, H.* AU - Yang, R.* AU - Zellner, S.* AU - Nalenz, M.* C1 - 62314 C2 - 50767 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A computational reproducibility study of PLOS ONE articles featuring longitudinal data analyses. JO - PLoS ONE VL - 16 IS - 6 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Investment Case is a participatory approach that has been used over the years for better strategic actions and planning in the health sector. Based on this approach, a District Investment Case (DIC) program was launched to improve maternal, neonatal and child health services in partnership with government, non-government sectors and UNICEF Nepal. In the meantime, this study aimed to explore perceptions and experiences of local stakeholders regarding health planning and budgeting and explore the role of the DIC program in ensuring equity in access to maternal and child health services. METHODS: This study adopted an exploratory phenomenography design with a purposive sampling technique for data collection. Three DIC implemented districts and three comparison districts were selected and total 30 key informant interviews with district level stakeholders and six focus groups with community stakeholders were carried out. A deductive approach was used to explore the perception of local stakeholders of health planning and budgeting of the health care expenses on the local level. RESULTS: Investment Case approach helped stakeholders in planning systematically based on evidence through collaborative and participatory approach while in comparison areas previous year plan was mainly primarily considered as reference. Resource constraints and geographical difficulty were key barriers in executing the desired plan in both intervention and comparison districts. Positive changes were observed in coverage of maternal and child health services in both groups. A few participants reported no difference due to the DIC program. The participants specified the improvement in access to information, access and utilization of health services by women. This has influenced the positive health care seeking behavior. CONCLUSIONS: The decentralized planning and management approach at the district level helps to ensure equity in access to maternal, newborn and child health care. However, quality evidence, inclusiveness, functional feedback and support system and local resource utilization should be the key consideration. AU - Thapa, J.K.* AU - Stöckl, D. AU - Sangroula, R.K.* AU - Pun, A.* AU - Thapa, M.* AU - Maskey, M.K.* AU - Delius, M.* C1 - 63170 C2 - 51364 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Investment case approach for equitable access to maternal neonatal and child health services: Stakeholders' perspective in Nepal. JO - PLoS ONE VL - 16 IS - 10 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species. AU - Theobalt, N.* AU - Hofmann, I.* AU - Fiedler, S.* AU - Renner, S.* AU - Dhom, G.* AU - Feuchtinger, A. AU - Walch, A.K. AU - Hrabě de Angelis, M. AU - Wolf, E.* AU - Wanke, R.* AU - Blutke, A. C1 - 61646 C2 - 50132 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy. JO - PLoS ONE VL - 16 IS - 3 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - Objective This study aims to investigate the correlation between spinopelvic parameters in supine position (pelvic incidence (PI), sacral slope (SS), pelvic tilt (PT), lumbar lordosis (LL)), disc degeneration and herniation of the thoracolumbar spine, as well as cardiovascular risk factors and back pain in a southern German cohort from the general population. Methods This study is a cross-sectional, case-control study drawn from a prospective cohort of the "Cooperative Health Research in the Region of Augsburg/Kooperative Gesundheitsforschung in der Region Augsburg" study (KORA). In total, 374 participants (mean age 56.4 ± 9.2 years; 57.8% male) from the whole-body MRI cohort (FF4) were included. All participants underwent a standardized whole-body MRI on which disc degeneration of the thoracic and lumbar spine was evaluated using a sequence adapted Pfirrmann score. PI, PT, SS and LL were measured according to the description in the literature, using sagittal imaging. Furthermore, disc bulging and protrusion were assessed. Correlations were estimated by logistic regression models providing odds ratios. Results Mean PI was 54.0° ± 11.1°, PT 13.0° ± 5.8°, SS 40.2° ± 8.8° and LL 36.2° ± 9.6°. SS was greater in men (p<0.05) and lumbar lordosis in women (p<0.001). PT increased by 0.09° per age-year with rising age. Age was not associated with PI, SS and LL. Neither BMI, hypertension, cholesterol, lipid levels, nor physical activity were associated with PI, PT, SS or LL. Diabetes mellitus negatively correlated with SS (β =-4.19; 95%CI-7.31-1.06, p<0.01). Smaller spinopelvic parameters (PI, SS and LL) where significantly (p<0.05) correlated with an increased frequency of disc bulging, as well as a local clustering in the lumbar, but not the thoracic spine. Conclusion In conclusion, spinopelvic parameters, measured in supine position, are significantly correlated with disc bulging alone; there is no significant correlation between supine spinopelvic parameters and disc degeneration, back pain or cardiovascular risk factors. AU - Walter, S.S.* AU - Lorbeer, R.* AU - Hefferman, G.* AU - Schlett, C.L.* AU - Peters, A. AU - Rospleszcz, S. AU - Nikolaou, K.* AU - Bamberg, F.* AU - Notohamiprodjo, M.* AU - Maurer, E.* C1 - 62265 C2 - 50614 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Correlation between thoracolumbar disc degeneration and anatomical spinopelvic parameters in supine position on MRI. JO - PLoS ONE VL - 16 IS - 6 PB - Public Library Science PY - 2021 SN - 1932-6203 ER - TY - JOUR AB - The novel coronavirus (SARS-CoV-2), identified in China at the end of December 2019 and causing the disease COVID-19, has meanwhile led to outbreaks all over the globe with about 2.2 million confirmed cases and more than 150,000 deaths as of April 17, 2020. In this work, mathematical models are used to reproduce data of the early evolution of the COVID-19 outbreak in Germany, taking into account the effect of actual and hypothetical non-pharmaceutical interventions. Systems of differential equations of SEIR type are extended to account for undetected infections, stages of infection, and age groups. The models are calibrated on data until April 5. Data from April 6 to 14 are used for model validation. We simulate different possible strategies for the mitigation of the current outbreak, slowing down the spread of the virus and thus reducing the peak in daily diagnosed cases, the demand for hospitalization or intensive care units admissions, and eventually the number of fatalities. Our results suggest that a partial (and gradual) lifting of introduced control measures could soon be possible if accompanied by further increased testing activity, strict isolation of detected cases, and reduced contact to risk groups. AU - Barbarossa, M.V.* AU - Fuhrmann, J.* AU - Meinke, J.H.* AU - Krieg, S.* AU - Varma, H.V.* AU - Castelletti, N. AU - Lippert, T.* C1 - 60054 C2 - 49198 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Modeling the spread of COVID-19 in Germany: Early assessment and possible scenarios. JO - PLoS ONE VL - 15 IS - 9 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Background We investigated associations of area-level deprivation with obstetric and perinatal outcomes in a large population-based routine dataset. Methods We used the data of n = 827,105 deliveries who were born in hospitals between 2009 to 2016 in Bavaria, Germany. The Bavarian Index of Multiple Deprivation (BIMD) on district level was assigned to each mother by the zip code of her residential address. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) for preterm deliveries, Caesarian sections (CS), stillbirths, small for gestational age (SGA) births and low 5-minute Apgar scores by BIMD quintiles with and without adjustment for potential confounders. Results We observed a significantly increased risk for preterm deliveries in mothers from the most deprived compared to the least deprived districts (e.g. OR [95% CI] for highest compared to lowest deprivation quintile: 1.06 [1.03, 1.09]) in adjusted analyses. Increased deprivation was also associated with higher SGA and secondary CS rates, but with lower proportions of stillbirths, primary CS and low Apgar scores. When one large clinic with an unusually high stillbirth rate was excluded, the association of BIMD with stillbirths was attenuated and almost disappeared. Conclusions We found that area-level deprivation in Bavaria was positively associated with preterm and SGA births, confirming previous studies. In contrast, the finding of an inverse association between deprivation and both stillbirth rates and low Apgar score came somewhat surprising. However, we conclude that the stillbirths finding is spurious and reflects regional bias due to a clinic which seems to specialize in termination of pregnancies. AU - Beyerlein, A. AU - Lack, N.* AU - Maier, W. C1 - 59781 C2 - 48962 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Associations of area-level deprivation with adverse obstetric and perinatal outcomes in Bavaria, Germany: Results from a cross-sectional study. JO - PLoS ONE VL - 15 IS - 7 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - RationaleProbe-based confocal endomicroscopy provides real time videos of autoflourescent elastin structures within the alveoli. With it, multiple changes in the elastin structure due to different diffuse parenchymal lung diseases have previously been described. However, these evaluations have mainly relied on qualitative evaluation by the examiner and manually selected parts post-examination.ObjectivesTo develop a fully automatic method for quantifying structural properties of the imaged alveoli elastin and to perform a preliminary assessment of their diagnostic potential.Methods46 patients underwent probe-based confocal endomicroscopy, of which 38 were divided into 4 groups categorizing different diffuse parenchymal lung diseases. 8 patients were imaged in representative healthy lung areas and used as control group. Alveolar elastin structures were automatically segmented with a trained machine learning algorithm and subsequently evaluated with two methods developed for quantifying the local thickness and structural connectivity.Measurements and main resultsThe automatic segmentation algorithm performed generally well and all 4 patient groups showed statistically significant differences with median elastin thickness, standard deviation of thickness and connectivity compared to the control group.ConclusionAlveoli elastin structures can be quantified based on their structural connectivity and thickness statistics with a fully-automated algorithm and initial results highlight its potential for distinguishing parenchymal lung diseases from normal alveoli. AU - Bondesson, D. AU - Silbernagel, E.* AU - Behr, J. AU - Reichenberger, F.* AU - Dinkel, J.* C1 - 59035 C2 - 48546 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Automated evaluation of probe-based confocal laser endomicroscopy in the lung. JO - PLoS ONE VL - 15 IS - 5 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Lipoprotein(a) [Lp(a)] is a major cardiovascular risk factor, which is largely genetically determined by one major gene locus, the LPA gene. Many aspects of the transcriptional regulation of LPA are poorly understood and the role of epigenetics has not been addressed yet. Therefore, we conducted an epigenome-wide analysis of DNA methylation on Lp(a) levels in two population-based studies (total n = 2208). We identified a CpG site in the LPA promoter which was significantly associated with Lp(a) concentrations. Surprisingly, the identified CpG site was found to overlap the SNP rs76735376. We genotyped this SNP de-novo in three studies (total n = 7512). The minor allele of rs76735376 (1.1% minor allele frequency) was associated with increased Lp(a) values (p = 1.01e-59) and explained 3.5% of the variation of Lp(a). Statistical mediation analysis showed that the effect on Lp(a) is rather originating from the base change itself and is not mediated by DNA methylation levels. This finding is supported by eQTL data from 208 liver tissue samples from the GTEx project, which shows a significant association of the rs76735376 minor allele with increased LPA expression. To evaluate, whether the association signal at rs76735376 may actually be derived from a stronger eQTL signal in LD with this SNP, eQTL association results of all correlated SNPs (r(2)>= 0.1) were integrated with genetic association results. This analysis pinpointed to rs10455872 as the potential trigger of the effect of rs76735376. Furthermore, both SNPs coincide with short apo(a) isoforms. Adjusting for both, rs10455872 and the apo(a) isoforms diminished the effect size of rs76735376 to 5.38 mg/dL (p = 0.0463). This indicates that the effect of rs76735376 can be explained by both an independent effect of the SNP and a strong correlation with rs10455872 and apo(a) isoforms. AU - Coassin, S.* AU - Hermann-Kleiter, N.* AU - Haun, M.* AU - Wahl, S. AU - Wilson, R. AU - Paulweber, B.* AU - Kunze, S. AU - Meitinger, T. AU - Strauch, K. AU - Peters, A. AU - Waldenberger, M. AU - Kronenberg, F.* AU - Lamina, C.* C1 - 58970 C2 - 48483 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A genome-wide analysis of DNA methylation identifies a novel association signal for Lp(a) concentrations in the LPA promoter. JO - PLoS ONE VL - 15 IS - 4 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - [This corrects the article DOI: 10.1371/journal.pone.0172995.]. AU - Esslinger, U.* AU - Garnier, S.* AU - Korniat, A.* AU - Proust, C.* AU - Kararigas, G.* AU - Müller-Nurasyid, M. AU - Empana, J.P.* AU - Morley, M.P.* AU - Perret, C.* AU - Stark, K.* AU - Bick, A.G.* AU - Prasad, S.K.* AU - Kriebel, J. AU - Li, J.* AU - Tiret, L.* AU - Strauch, K. AU - O'Regan, D.P.* AU - Marguiles, K.B.* AU - Seidman, J.G.* AU - Boutouyrie, P.* AU - Lacolley, P.* AU - Jouven, X.* AU - Hengstenberg, C.* AU - Komajda, M.* AU - Hakonarson, H.* AU - Isnard, R.* AU - Arbustini, E.* AU - Grallert, H. AU - Cook, S.A.* AU - Seidman, C.E.* AU - Regitz-Zagrosek, V.* AU - Cappola, T.P.* AU - Charron, P.* AU - Cambien, F.* AU - Villard, E.* C1 - 58634 C2 - 48188 TI - Correction: Exome-wide association study reveals novel susceptibility genes to sporadic dilated cardiomyopathy. JO - PLoS ONE VL - 15 IS - 2 PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments. AU - Fiedler, S.* AU - Wünnemann, H.* AU - Hofmann, I.* AU - Theobalt, N.* AU - Feuchtinger, A. AU - Walch, A.K. AU - Schwaiger, J.* AU - Wanke, R.* AU - Blutke, A. C1 - 60839 C2 - 49711 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies. JO - PLoS ONE VL - 15 IS - 12 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - The impact of sex-specific body fat distribution on the susceptibility to five chronic infections, helicobacter pylori and human herpesviruses 3 to 6 (i.e. varicella-zoster, Epstein-Barr, cytomegalo- and human herpesvirus 6), has not previously been examined. In the present study, seropositivity was determined via multiplex serology in serum samples of study participants collected in 2006/08 and 2013/14 during the follow-up examinations F4 (n = 3080) and FF4 (n = 2279) of the German population-based baseline KORA S4 survey. We quantified the severity of overall and abdominal obesity by body mass index, body adiposity index, waist circumference, waist-to-hip ratio, and waist-to-height ratio. Using sex-specific logistic spline-models, cross-sectional and longitudinal associations between obesity measures and seropositivity of the previously mentioned infections were investigated. Overall and abdominal fat content were significantly associated with seropositivity of varicella-zoster virus in both cross-sectional and longitudinal analyses among women. In addition, a non-significant inverse relationship with Epstein-Barr virus seroprevalence in both sexes and a trend towards a positive association with human herpesvirus 6 seropositivity in women were observed. Therefore, in women total body fat may be associated with VZV-seropositivity and may influence the reactivation of the varicella-zoster virus, independent of adipose tissue distribution. AU - Freuer, D. AU - Linseisen, J. AU - Waterboer, T.* AU - Pessler, F.* AU - Guzmán, C.A.* AU - Wawro, N. AU - Peters, A.* AU - Meisinger, C. C1 - 58898 C2 - 48440 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Seropositivity of selected chronic infections and different measures of obesity. JO - PLoS ONE VL - 15 IS - 4 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed. AU - Herbert, E.* AU - Stewart, M.* AU - Hutchison, M.* AU - Flenniken, A.M.* AU - Qu, D.* AU - Nutter, L.M.J.* AU - McKerlie, C.* AU - Hobson, L.* AU - Kick, B.* AU - Lyons, B.* AU - Wiegand, J.-P.* AU - Doty, R.* AU - Aguilar-Pimentel, J.A. AU - Hrabě de Angelis, M. AU - Dickinson, M.* AU - Seavitt, J.* AU - White, J.K.* AU - Scudamore, C.L.* AU - Wells, S.* C1 - 59372 C2 - 48767 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities. JO - PLoS ONE VL - 15 IS - 6 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1β, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1β, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants' contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study. AU - Lapuente, D.* AU - Stab, V.* AU - Genannt Bonsmann, M.S.* AU - Maaske, A. AU - Köster, M.* AU - Xiao, H.* AU - Ehrhardt, C.* AU - Tenbusch, M.* C1 - 58846 C2 - 48631 TI - Innate signalling molecules as genetic adjuvants do not alter the efficacy of a DNA-based influenza A vaccine. JO - PLoS ONE VL - 15 IS - 4 PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Background Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor. Methods We investigated the contribution of rare and low-frequency variation to EwS susceptibility in the largest EwS genome-wide association study to date (733 EwS cases and 1,346 unaffected controls of European ancestry). Results We identified two low-frequency variants, rs112837127 and rs2296730, on chromosome 20 that were associated with EwS risk (OR = 0.186 and 2.038, respectively; P-value < 5x10(-8)) and located near previously reported common susceptibility loci. After adjusting for the most associated common variant at the locus, only rs112837127 remained a statistically significant independent signal (OR = 0.200, P-value = 5.84x10(-8)). Conclusions These findings suggest rare variation residing on common haplotypes are important contributors to EwS risk. Impact Motivate future targeted sequencing studies for a comprehensive evaluation of low-frequency and rare variation around common EwS susceptibility loci. AU - Lin, S.H.* AU - Sampson, J.N.* AU - Grünewald, T.G.P.* AU - Surdez, D.* AU - Reynaud, S.* AU - Mirabeau, O.* AU - Karlins, E.* AU - Rubio, R.A.* AU - Zaidi, S.* AU - Grossetête-Lalami, S.* AU - Ballet, S.* AU - Lapouble, E.* AU - Laurence, V.* AU - Michon, J.* AU - Pierron, G.* AU - Kovar, H.* AU - Kontny, U.* AU - González-Neira, A.* AU - Alonso, J.* AU - Patino-Garcia, A.* AU - Corradini, N.* AU - Bérard, P.M.* AU - Miller, J.* AU - Freedman, N.D.* AU - Rothman, N.* AU - Carter, B.D.* AU - Dagnall, C.L.* AU - Burdett, L.* AU - Jones, K.* AU - Manning, M.* AU - Wyatt, K.* AU - Zhou, W.* AU - Yeager, M.* AU - Cox, D.G.* AU - Hoover, R.N.* AU - Khan, J.* AU - Armstrong, G.T.* AU - Leisenring, W.M.* AU - Bhatia, S.* AU - Robison, L.L.* AU - Kulozik, A.E.* AU - Kriebel, J. AU - Meitinger, T. AU - Metzler, M.* AU - Krumbholz, M.* AU - Hartmann, W.* AU - Strauch, K.* AU - Kirchner, T.* AU - Dirksen, U.* AU - Mirabello, L.* AU - Tucker, M.A.* AU - Tirode, F.* AU - Morton, L.M.* AU - Chanock, S.J.* AU - Delattre, O.* AU - Machiela, M.J.* C1 - 60038 C2 - 49075 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Low-frequency variation near common germline susceptibility loci are associated with risk of Ewing sarcoma. JO - PLoS ONE VL - 15 IS - 9 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4 degrees C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freezethaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples. AU - Matias-Garcia, P.R. AU - Wilson, R. AU - Mussack, V.* AU - Reischl, E. AU - Waldenberger, M. AU - Gieger, C. AU - Anton, G. AU - Peters, A. AU - Kühn-Steven, A. C1 - 57830 C2 - 48097 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples. JO - PLoS ONE VL - 15 IS - 1 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - In this proof-of-concept study, we tested whether placebo effects can be monitored and predicted by plasma proteins. In a randomized controlled design, 90 participants were exposed to a nauseating stimulus on two separate days and were randomly allocated to placebo treatment or no treatment on the second day. Significant placebo effects on nausea, motion sickness, and (in females) gastric activity could be verified. Using label-free tandem mass spectrometry, 74 differentially regulated proteins were identified as correlates of the placebo effect. Gene ontology (GO) enrichment analyses identified acute-phase proteins and microinflammatory proteins to be involved, and the identified GO signatures predicted day-adjusted scores of nausea indices in the placebo group. We also performed GO enrichment analyses of specific plasma proteins predictable by the experimental factors or their interactions and identified 'grooming behavior' as a prominent hit. Finally, Receiver Operator Characteristics (ROC) allowed to identify plasma proteins differentiating placebo responders from non-responders, comprising immunoglobulins and proteins involved in oxidation reduction processes and complement activation. Plasma proteomics is a promising tool to identify molecular correlates and predictors of the placebo effect in humans. AU - Meissner, K.* AU - Lutter, D. AU - von Toerne, C. AU - Haile, A.* AU - Woods, S.C.* AU - Hoffmann, V.* AU - Ohmayer, U. AU - Hauck, S.M. AU - Tschöp, M.H. C1 - 60163 C2 - 49263 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Molecular classification of the placebo effect in nausea. JO - PLoS ONE VL - 15 IS - 9 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an unexplained chronic, debilitating illness characterized by fatigue, sleep disturbances, cognitive dysfunction, orthostatic intolerance and gastrointestinal problems. Using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we analyzed the plasma proteomes of 39 ME/CFS patients and 41 healthy controls. Logistic regression models, with both linear and quadratic terms of the protein levels as independent variables, revealed a significant association between ME/CFS and the immunoglobulin heavy variable (IGHV) region 3-23/30. Stratifying the ME/CFS group based on self-reported irritable bowel syndrome (sr-IBS) status revealed a significant quadratic effect of immunoglobulin lambda constant region 7 on its association with ME/CFS with sr-IBS whilst IGHV3-23/30 and immunoglobulin kappa variable region 3-11 were significantly associated with ME/CFS without sr-IBS. In addition, we were able to predict ME/CFS status with a high degree of accuracy (AUC = 0.774-0.838) using a panel of proteins selected by 3 different machine learning algorithms: Lasso, Random Forests, and XGBoost. These algorithms also identified proteomic profiles that predicted the status of ME/CFS patients with sr-IBS (AUC = 0.806-0.846) and ME/CFS without sr-IBS (AUC = 0.754-0.780). Our findings are consistent with a significant association of ME/CFS with immune dysregulation and highlight the potential use of the plasma proteome as a source of biomarkers for disease. AU - Milivojevic, M.* AU - Che, X.* AU - Bateman, L.* AU - Cheng, A.* AU - Garcia, B.A.* AU - Hornig, M.* AU - Huber, M.B. AU - Klimas, N.G.* AU - Lee, B.* AU - Lee, H.* AU - Levine, S.* AU - Montoya, J.G.* AU - Peterson, D.L.* AU - Komaroff, A.L.* AU - Lipkin, W.I.* C1 - 59821 C2 - 49057 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Plasma proteomic profiling suggests an association between antigen driven clonal B cell expansion and ME/CFS. JO - PLoS ONE VL - 15 IS - 7 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Enhancer redundancy has been postulated to provide a buffer for gene expression against genetic and environmental perturbations. While work in Drosophila has identified functionally overlapping enhancers, work in mammalian models has been limited. Recently, we have identified two partially redundant enhancers, nPE1 and nPE2, that drive proopiomelanocortin gene expression in the hypothalamus. Here we demonstrate that deletion of nPE1 produces mild obesity while knockout of nPE2 has no discernible metabolic phenotypes. Additionally, we show that acute leptin administration has significant effects on nPE1 knockout mice, with food intake and body weight change significantly impacted by peripheral leptin treatment. nPE1 knockout mice became less responsive to leptin treatment over time as percent body weight change increased over 2 week exposure to peripheral leptin. Both Pomc and Agrp mRNA were not differentially affected by chronic leptin treatment however we did see a decrease in Pomc and Agrp mRNA in both nPE1 and nPE2 knockout calorie restricted mice as compared to calorie restricted PBS-treated WT mice. Collectively, these data suggest dynamic regulation of Pomc by nPE1 such that mice with nPE1 knockout become less responsive to the anorectic effects of leptin treatment over time. Our results also support our earlier findings in which nPE2 may only be critical in adult mice that lack nPE1, indicating that these neural enhancers work synergistically to influence metabolism. AU - Na, E.S.* AU - Lam, D.D. AU - Yokosawa, E.* AU - Adams, J.M.* AU - Olson, D.P.* AU - Low, M.J.* C1 - 60954 C2 - 49651 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Decreased sensitivity to the anorectic effects of leptin in mice that lack a Pomc-specific neural enhancer. JO - PLoS ONE VL - 15 IS - 12 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - BackgroundAuditory steady-state responses (ASSRs) are ongoing evoked brain responses to continuous auditory stimuli that play a role for auditory processing of complex sounds and speech perception. Transient auditory event-related responses (AERRs) have previously been recorded using fetal magnetoencephalography (fMEG) but involve different neurological pathways. Previous studies in children and adults demonstrated that the cortical components of the ASSR are significantly affected by state of consciousness and by maturational changes in neonates and young infants. To our knowledge, this is the first study to investigate ASSRs in human fetuses.Methods47 fMEG sessions were conducted with 24 healthy pregnant women in three gestational age groups (30-32 weeks, 33-35 weeks and 36-39 weeks). The stimulation consisted of amplitude-modulated (AM) tones with a duration of one second, a carrier frequency (CF) of 500 Hz and a modulation frequency (MF) of 27 Hz or 42 Hz. Both tones were presented in a random order with equal probability adding up to 80-100 repetitions per tone. The ASSR across trials was quantified by assessing phase synchrony in the cortical signals at the stimulation frequency.Results and conclusionTen out of 47 recordings were excluded due to technical problems or maternal movements. Analysis of the included 37 fetal recordings revealed a statistically significant response for the phase coherence between trials for the MF of 27 Hz but not for 42 Hz. An exploratory subgroup analysis moreover suggested an advantage in detectability for fetal behavioral state 2F (active asleep) compared to 1F (quiet asleep) detected using fetal heart rate. In conclusion, this pilot study is the first description of a method to detect human ASSRs in fetuses. The findings warrant further investigations of the developing fetal brain. AU - Niepel, D.* AU - Krishna, B.* AU - Siegel, E.R.* AU - Draganova, R.* AU - Preissl, H. AU - Govindan, R.B.* AU - Eswaran, H.* C1 - 59796 C2 - 48991 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A pilot study: Auditory steady-state responses (ASSR) can be measured in human fetuses using fetal magnetoencephalography (fMEG). JO - PLoS ONE VL - 15 IS - 7 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - PurposeWe hypothesize that MRI-based renal compartment volumes, particularly renal sinus fat as locally and potentially independently acting perivascular fat tissue, increase with glucose intolerance. We therefore analyze the distribution of renal volumes in individuals with normal glucose levels and prediabetic and diabetic individuals and investigate potential associations with other typical cardiometabolic biomarkers.Material and methodsThe sample comprised N = 366 participants who were either normoglycemic (N = 230), had prediabetes (N = 87) or diabetes (N = 49), as determined by Oral Glucose Tolerance Test. Other covariates were obtained by standardized measurements and interviews. Whole-body MR measurements were performed on a 3 Tesla scanner. For assessment of the kidneys, a coronal T1w dual-echo Dixon and a coronal T2w single shot fast spin echo sequence were employed. Stepwise semi-automated segmentation of the kidneys on the Dixon-sequences was based on thresholding and geometric assumptions generating volumes for the kidneys and sinus fat. Inter- and intra-reader variability were determined on a subset of 40 subjects. Associations between glycemic status and renal volumes were evaluated by linear regression models, adjusted for other potential confounding variables. Furthermore, the association of renal volumes with visceral adipose tissue was assessed by linear regression models and Pearson's correlation coefficient.ResultsRenal volume, renal sinus volume and renal sinus fat increased gradually from normoglycemic controls to individuals with prediabetes to individuals with diabetes (renal volume: 280.3 +/- 64.7 ml vs 303.7 +/- 67.4 ml vs 320.6 +/- 77.7ml, respectively, p < 0.001). After adjustment for age and sex, prediabetes and diabetes were significantly associated to increased renal volume, sinus volume (e.g. beta(Prediabetes) = 10.1, 95% CI: [6.5, 13.7]; p<0.01, beta(Diabetes) = 11.86, 95% CI: [7.2, 16.5]; p<0.01) and sinus fat (e.g. beta(Prediabetes) = 7.13, 95% CI: [4.5, 9.8]; p<0.001, beta(Diabetes) = 7.34, 95% CI: [4.0, 10.7]; p<0.001). Associations attenuated after adjustment for additional confounders were only significant for prediabetes and sinus volume (beta = 4.0 95% CI [0.4, 7.6]; p<0.05). Hypertension was significantly associated with increased sinus volume (beta = 3.7, 95% CI: [0.4, 7.0; p<0.05]) and absolute sinus fat volume (beta = 3.0, 95% CI: [0.7, 5.3]; p<0.05). GFR and all renal volumes were significantly associated as well as urine creatinine levels and renal sinus volume (beta = 1.6, 95% CI: [0.1, 2.9]; p<0.05).ConclusionRenal volume and particularly renal sinus fat volume already increases significantly in prediabetic subjects and is significantly associated with VAT. This shows, that renal sinus fat is a perivascular adipose tissue, which early undergoes changes in the development of metabolic disease. Our findings underpin that renal sinus fat is a link between metabolic disease and associated chronic kidney disease, making it a potential imaging biomarker when assessing perivascular adipose tissue. AU - Notohamiprodjo, M.* AU - Goepfert, M.* AU - Will, S.* AU - Lorbeer, R.* AU - Schick, F.* AU - Rathmann, W.* AU - Martirosian, P.* AU - Peters, A. AU - Müller-Peltzer, K.* AU - Helck, A.* AU - Rospleszcz, S. AU - Bamberg, F.* C1 - 58326 C2 - 48180 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Renal and renal sinus fat volumes as quantified by magnetic resonance imaging in subjects with prediabetes, diabetes, and normal glucose tolerance. JO - PLoS ONE VL - 15 IS - 2 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - AIM: Subclasses of different glycaemic disturbances could explain the variation in characteristics of individuals with type 2 diabetes (T2D). We aimed to examine the association between subgroups based on their glucose curves during a five-point mixed-meal tolerance test (MMT) and metabolic traits at baseline and glycaemic deterioration in individuals with T2D. METHODS: The study included 787 individuals with newly diagnosed T2D from the Diabetes Research on Patient Stratification (IMI-DIRECT) Study. Latent class trajectory analysis (LCTA) was used to identify distinct glucose curve subgroups during a five-point MMT. Using general linear models, these subgroups were associated with metabolic traits at baseline and after 18 months of follow up, adjusted for potential confounders. RESULTS: At baseline, we identified three glucose curve subgroups, labelled in order of increasing glucose peak levels as subgroup 1-3. Individuals in subgroup 2 and 3 were more likely to have higher levels of HbA1c, triglycerides and BMI at baseline, compared to those in subgroup 1. At 18 months (n = 651), the beta coefficients (95% CI) for change in HbA1c (mmol/mol) increased across subgroups with 0.37 (-0.18-1.92) for subgroup 2 and 1.88 (-0.08-3.85) for subgroup 3, relative to subgroup 1. The same trend was observed for change in levels of triglycerides and fasting glucose. CONCLUSIONS: Different glycaemic profiles with different metabolic traits and different degrees of subsequent glycaemic deterioration can be identified using data from a frequently sampled mixed-meal tolerance test in individuals with T2D. Subgroups with the highest peaks had greater metabolic risk. AU - Obura, M.* AU - Beulens, J.W.J.* AU - Slieker, R.* AU - Koopman, A.D.M.* AU - Hoekstra, T.* AU - Nijpels, G.* AU - Elders, P.* AU - Koivula, R.W.* AU - Kurbasic, A.* AU - Laakso, M.* AU - Hansen, T.H.* AU - Ridderstråle, M.* AU - Hansen, T.* AU - Pavo, I.* AU - Forgie, I.* AU - Jablonka, B.* AU - Ruetten, H.* AU - Mari, A.* AU - McCarthy, M.I.* AU - Walker, M.* AU - Heggie, A.* AU - McDonald, T.J.* AU - Perry, M.H.* AU - De Masi, F.* AU - Brunak, S.* AU - Mahajan, A.* AU - Giordano, G.N.* AU - Kokkola, T.* AU - Dermitzakis, E.* AU - Viñuela, A.* AU - Pedersen, O.* AU - Schwenk, J.M.* AU - Adamski, J. AU - Teare, H.J.A.* AU - Pearson, E.R.* AU - Franks, P.W.* AU - 't Hart, L.M.* AU - Rutters, F.* C1 - 60631 C2 - 49506 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Post-load glucose subgroups and associated metabolic traits in individuals with type 2 diabetes: An IMI-DIRECT study. JO - PLoS ONE VL - 15 IS - 11 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Purpose The concept of dysphagia/aspiration-related structures (DARS) was developed against the background of severe late side effects of radiotherapy (RT) for head and neck cancer (HNC). DARS can be delineated on CT scans, but with a better morphological discrimination on magnetic resonance imaging (MRI). Swallowing function was analyzed by use of patient charts and prospective investigations and questionnaires. Method Seventeen HNC patients treated with intensity-modulated radiotherapy (IMRT) +/- chemotherapy between 5/2012 - 8/2015 were included. Planning CT (computed tomography) scans and MRIs (magnetic resonance imaging) prior, during 40 Gray (Gy) radiotherapy and posttreatment were available and co-registered to delineate DARS. The RT dose of each DARS was calculated. Five patients were investigated posttreatment for swallowing function and assessed by means of various questionnaires for quality of life (QoL), swallowing, and voice function. Results By retrospective comparison of DARS volume, a significant change in four of eight DARS was detected over time. Three increased and one diminished. The risk of posttreatment dysphagia rose by every 1Gy above the mean dose (D mean) of RT to DARS. 7.5 was the risk factor for dysphagia in the first 6 months, reducing to 4.7 for months 6-12 posttreatment. For all five patients of the prospective part of swallowing investigations, a function disturbance was detected. These results were in contrast to the self-assessment of patients by questionnaires. There was neither a dose dependency of D mean DARS volume changes over time nor of dysphonia and no correlation between volume changes, dysphagia or dysphonia. Conclusion Delineation of DARS on MRI co-registered to planning CT gave the opportunity to differentiate morphology better than by CT alone. Due to the small number of patients with complete MRI scans over time, we failed to detect a dose dependency of DARS and swallowing and voice disorder posttreatment. AU - Pigorsch, S.U.* AU - May, C.* AU - Kessel, K.A. AU - Graf, S.* AU - Bier, H.* AU - Nüsslin, F.* AU - Waschulzik, B.* AU - Combs, S.E. C1 - 60019 C2 - 49168 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - MRI- and CT-determined changes of dysphagia / aspiration-related structures (DARS) during and after radiotherapy. JO - PLoS ONE VL - 15 IS - 9 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: The occurrence of pneumonia separates severe cases of COVID-19 from the majority of cases with mild disease. However, the factors determining whether or not pneumonia develops remain to be fully uncovered. We therefore explored the associations of several lifestyle factors with signs of pneumonia in COVID-19. METHODS: Between May and July 2020, we conducted an online survey of 201 adults in Germany who had recently gone through COVID-19, predominantly as outpatients. Of these, 165 had a PCR-based diagnosis and 36 had a retrospective diagnosis by antibody testing. The survey covered demographic information, eight lifestyle factors, comorbidities and medication use. We defined the main outcome as the presence vs. the absence of signs of pneumonia, represented by dyspnea, the requirement for oxygen therapy or intubation. RESULTS: Signs of pneumonia occurred in 39 of the 165 individuals with a PCR-based diagnosis of COVID-19 (23.6%). Among the lifestyle factors examined, only overweight/obesity was associated with signs of pneumonia (odds ratio 2.68 (1.29-5.59) p = 0.008). The observed association remained significant after multivariate adjustment, with BMI as a metric variable, and also after including the antibody-positive individuals into the analysis. CONCLUSIONS: This exploratory study finds an association of overweight/obesity with signs of pneumonia in COVID-19. This finding suggests that a signal proportional to body fat mass, such as the hormone leptin, impairs the body's ability to clear SARS-CoV-2 before pneumonia develops. This hypothesis concurs with previous work and should be investigated further to possibly reduce the proportion of severe cases of COVID-19. AU - Sacco, V. AU - Rauch, B. AU - Gar, C. AU - Haschka, S. AU - Potzel, A. AU - Kern-Matschilles, S. AU - Banning, F. AU - Benz, I. AU - Meisel, M. AU - Seissler, J. AU - Lechner, A. C1 - 60592 C2 - 49395 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Overweight/obesity as the potentially most important lifestyle factor associated with signs of pneumonia in COVID-19. JO - PLoS ONE VL - 15 IS - 11 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - Objectives Uromodulin has been associated with arterial hypertension in genome-wide association studies, but data from clinical and preclinical studies are inconsistent. We here analyzed the association of serum uromodulin (sUmod) with arterial hypertension and vasoactive hormones in a population-based study. Methods In 1108 participants of the KORA F4 study aged 62-81 years, sUmod was measured and the association of sUmod with arterial hypertension was assessed using logistic regression models. The associations of sUmod with renin and aldosterone and with the vasoconstrictive prohormone C-terminal pro-endothelin-1 (CT-proET-1) were analyzed in 1079 participants and in 618 participants, respectively, using linear regression models. Results After multivariable adjustment including sex, age, eGFR, BMI, fasting glucose, current smoking, previous stroke and myocardial infarction, sUmod was inversely associated with arterial hypertension (OR 0.78; 95% CI 0.68-0.91; p = 0.001). SUmod was not significantly associated with renin and aldosterone after adjustment for sex, age and eGFR. However, sUmod was inversely associated with CT-proET-1 (beta -0.19 +/- 0.04; p < 0.001) after adjustment for sex, age, eGFR, BMI, arterial hypertension, fasting glucose, current smoking, previous stroke and myocardial infarction. The association with CT-proET-1 was stronger in participants with hypertension (beta -0.22 +/- 0.04) than in normotensive participants (beta -0.13 +/- 0.06; p for interaction hypertension = 0.003 in the model adjusted for hypertension). Conclusions SUmod was inversely associated with arterial hypertension and the vasoconstrictive prohormone CT-proET-1, suggesting direct or indirect effects of sUmod on blood pressure regulation. AU - Then, C. AU - Thorand, B. AU - Then, H.L.* AU - Meisinger, C. AU - Heier, M. AU - Peters, A. AU - Koenig, W.* AU - Rathmann, W.* AU - Bidlingmaier, M.* AU - Lechner, A. AU - Reincke, M.* AU - Scherberich, J.E.* AU - Seissler, J. C1 - 59881 C2 - 48954 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Serum uromodulin is inversely associated with arterial hypertension and the vasoconstrictive prohormone CT-proET-1 in the population-based KORA F4 study. JO - PLoS ONE VL - 15 IS - 8 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - ObjectiveTo investigate the impact of metabolic syndrome and its components on osteoarthritis of the hip joints compared to a healthy cohort in the KORA MRI-study.MethodsRandomly selected men and women from the general population were classified as having metabolic syndrome, defined as presence of central obesity plus two of the following four components: elevated blood pressure (BP), elevated fasting glucose, elevated triglycerides (TG) and low HDL-cholesterol (HDL-c), or as controls without metabolic syndrome. Therefore, each subject underwent detailed assessment of waist circumference as well as fasting glucose, systolic and diastolic BP, TG, and HDL-c concentrations as well as a full-body MR scan. MR measurements were performed on a 3 Tesla scanner (Magnetom Skyra, Siemens) including a dual-echo Dixon and a T2 SS-FSE sequence for anatomical structures. In order to quantify osteoarthritis of the hip, assessment was performed by two independent, experienced radiologists for joint gap narrowing, osteophytic lipping and subchondral changes (e.g. sclerosis, pseudocysts). Associations between metabolic syndrome components and hip degeneration were estimated by logistic regression models providing odds ratios.ResultsAmong 354 included participants (mean age: 56.1 +/- 9.2 years; 55.4% male), 119 (34%) had metabolic syndrome, while 235 (66%) were part of the control group. Except for elevated blood glucose (p = 0.02), none of the metabolic syndromes' component was independently associated with osteoarthritis. Multivariable adjusted ORs for osteoarthritis of the right hip were 1.00 (95% CI 0.98;1.03), 1.00 (95% CI 0.99;1.00), 1.01 (95% CI 0.99;1.03), 1.00 (95% CI 0.97;1.04) and 1.01 (95% CI 0.96;1.06), and for the left hip 1.00 (95% CI 0.98;1.03), 1.00 (95% CI 1.00;1.01), 1.01 (95% CI 0.99;1.03), 0.99 (95% CI 0.96;1.02) and 1.04 (95% CI 0.99;1.09) for waist circumference, triglyceride, HDL-c and systolic and diastolic BP, respectively. Blood glucose was a borderline non-dependent factor for osteoarthritis of the right hip (OR: 1.02 (95% CI 1.0;1.04); p = 0.05). Furthermore, the compound metabolic syndrome was not significantly associated (OR left hip: 1.53 (95% CI 0.8;2.92), p = 0.20; OR right hip: 1.33 (95% CI 0.72;2.45), p = 0.37) with osteoarthritis of the hip joint. Age as well as gender (left hip) were the only parameters in univariate and multivariate analysis to be significantly associated with osteoarthritis of the hip joint.ConclusionThe compound metabolic syndrome showed no association with osteoarthritis of the hip joint. Age was the only parameter to be dependently and independently associated to osteoarthritis of both hip joints, while elevated blood glucose was independently associated with degeneration of the right hip joint. AU - Walter, S.S.* AU - Wintermeyer, E.* AU - Klinger, C.* AU - Lorbeer, R.* AU - Rathmann, W.* AU - Peters, A. AU - Schlett, C.L.* AU - Thorand, B. AU - Gatidis, S.* AU - Nikolaou, K.* AU - Bamberg, F.* AU - Notohamiprodjo, M.* C1 - 58545 C2 - 48310 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Association between metabolic syndrome and hip osteoarthritis in middle-aged men and women from the general population. JO - PLoS ONE VL - 15 IS - 3 PB - Public Library Science PY - 2020 SN - 1932-6203 ER - TY - JOUR AB - ObjectivesRisk stratification has so far been evaluated under the assumption that women fully adhere to screening recommendations. However, the participation in German cancer screening programs remains low at 54%. The question arises whether risk-stratified screening is economically efficient under the assumption that adherence is not perfect.MethodWe have adapted a micro-simulation Markov model to the German context. Annual, biennial, and triennial routine screening are compared with five risk-adapted strategies using thresholds of relative risk to stratify screening frequencies. We used three outcome variables (mortality reduction, quality-adjusted life years, and false-positive results) under the assumption of full adherence vs. an adherence rate of 54%. Strategies are evaluated using efficiency frontiers and probabilistic sensitivity analysis (PSA).ResultsThe reduced adherence rate affects both performance and cost; incremental cost-effectiveness ratios remain constant. The results of PSA show that risk-stratified screening strategies are more efficient than biennial routine screening under certain conditions. At any willingness-to-pay (WTP), there is a risk-stratified alternative with a higher likelihood of being the best choice. However, without explicit decision criteria and WTP, risk-stratified screening is not more efficient than biennial routine screening. Potential improvements in the adherence rates have significant health gains and budgetary implications.ConclusionIf the participation rate for mammographic screening is as low as in Germany, stratified screening is not clearly more efficient than routine screening but dependent on the WTP. A more promising design for future stratified strategies is the combination of risk stratification mechanisms with interventions to improve the low adherence in selected high-risk groups. AU - Arnold, M. AU - Pfeifer, K.* AU - Quante, A.S.* C1 - 56130 C2 - 46846 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Is risk-stratified breast cancer screening economically efficient in Germany? JO - PLoS ONE VL - 14 IS - 5 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - ObjectiveRegular physical activity may be associated with improved lung function via reduced systemic inflammation, although studies exploring this mechanism are rare. We evaluated the role of C-reactive protein in blood, which is a common marker of systemic inflammation, on the association of physical activity with forced expiratory volume in one second and forced vital capacity.MethodsCross-sectional data on spirometry, C-reactive protein levels and self-reported physical activity (yes/no; >= 2 times and.1hr per week of vigorous physical activity) were available in the European Community Respiratory Health Survey (N = 2347 adults, 49.3% male, 28-56 years-old). A subsample was also assessed 10 years later using the International Physical Activity Questionnaire, and tertiles of Metabolic Equivalent of Task-minutes per week spent in vigorous, moderate and walking activities were calculated (N = 671, 49.6% male, 40-67 years-old). Adjusted cross-sectional mixed linear regression models and the "mediate" package in "R" were used to assess the presence of mediation.ResultsDespite positive significant associations between nearly all physical activity metrics with forced expiratory volume in one second and forced vital capacity, there was no evidence that C-reactive protein levels played a role. An influence of C-reactive protein levels was only apparent in the smaller subsample when comparing the medium to low tertiles of moderate activity (mean difference [95% CIs]: 21.1ml [5.2, 41.9] for forced expiratory volume in one second and 17.3ml [2.6, 38.0] for forced vital capacity).ConclusionsIn a population of adults, we found no consistent evidence that the association of physical activity with forced expiratory volume in one second or forced vital capacity is influenced by the level of C-reactive protein in blood. AU - Fuertes, E.* AU - Carsin, A.E.* AU - Garcia-Larsen, V.* AU - Guerra, S.* AU - Pin, I.* AU - Leynaert, B.* AU - Accordini, S.* AU - Martinez-Moratalla, J.* AU - Antò, J.M.* AU - Urrutia, I.* AU - Le Gouellec, A.* AU - Heinrich, J. AU - Gislason, T.* AU - Jõgi, R.* AU - Janson, C.* AU - Jarvis, D.* AU - Garcia-Aymerich, J.* C1 - 56953 C2 - 47372 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The role of C-reactive protein levels on the association of physical activity with lung function in adults. JO - PLoS ONE VL - 14 IS - 9 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - © 2019 Gaertner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. But experimental studies of related situations, such as those involving bankruptcies or bequests in which the sum of the legal claims that can be made against a bank or firm or estate are greater than their values, have produced strong support for the proportionality principle. To test whether this principle would find support in other situations involving losses we designed an experimental game in which four players start out with differing initial endowments of real money. They are then informed that a certain amount of this resource has to be given back to the experimenter. How should the loss be shared among the agents? This game was run at different locations and under different treatments over a period of almost three years. We found that the proportionality principle was rarely proposed and even less frequently accepted as a solution to this problem. One of the main reasons for this result was that the two players with the smallest endowments opposed most of the proposals which asked them to contribute at least some positive amount of their own initial resource. AU - Gaertner, W.* AU - Bradley, R.* AU - Xu, Y.* AU - Schwettmann, L. C1 - 56609 C2 - 47164 TI - Against the proportionality principle: Experimental findings on bargaining over losses. JO - PLoS ONE VL - 14 IS - 7 PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Asprosin is a counter-regulatory hormone to insulin which plays a role in fasting. It may therefore also play a role in hypoglycaemia unawareness, which has been subsequently examined in this pilot study. Intravenous glucose tolerance test was used to induce controlled hyperglycemia whereas a hyperinsulinemic clamp test was used to induce a controlled hypoglycaemia in 15 patients with diabetes type 1, with and without hypoglycaemia unawareness. Changes in asprosin plasma levels did not differ between patients with and without hypoglycaemia unawareness. However, nine patients with insulin resistance as well as higher liver stiffness values and low-density lipoprotein but lower high-density lipoprotein levels did not show the expected increase in asprosin plasma levels during hypoglycemia. Therefore, insulin resistance and alterations in liver structure, most likely early stages of non-alcoholic fatty liver disease, seem to be relevant in type 1 diabetes and do not only lead to elevated plasma levels of asprosin, but also to a blunted asprosin response in hypoglycemia. AU - Groener, J.B.* AU - Valkanou, A.* AU - Kender, Z.* AU - Pfeiffenberger, J.* AU - Kihm, L.* AU - Fleming, T.* AU - Nawroth, P.P. AU - Kopf, S.* C1 - 56939 C2 - 47366 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Asprosin response in hypoglycemia is not related to hypoglycemia unawareness but rather to insulin resistance in type 1 diabetes. JO - PLoS ONE VL - 14 IS - 9 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Purpose and methodsA meta-analysis using data from seven German population-based cohorts was performed by the German Epidemiological consortium of Peripheral Arterial Disease (GEPArD) to investigate whether one question about claudication is more efficient for PAD screening than established questionnaires. Claudication was defined on the basis of the answer to one question asking for pain in the leg during normal walking. This simple question was compared with established questionnaires, including the Edinburgh questionnaire. The associations of claudication with continuous ABI values and decreased ABI were analyzed by linear and logistic regression analysis, respectively. The results of the studies were pooled in a random effect meta-analysis, which included data from 27,945 individuals (14,052 women, age range 20-84 years).ResultsMeta-analysis revealed a significant negative association between claudication and ABI, which was stronger in men (beta = -0.07; 95%CI -0.10, -0.04) than in women (beta = -0.02; 95%CI -0.02, -0.01). Likewise, the presence of claudication symptoms was related to an increased odds of a decreased ABI in both men (Odds ratio = 5.40; 95%CI 4.20, 6.96) and women (Odds ratio = 1.99; 95%CI 1.58, 2.51).ConclusionsAsking only one question about claudication was able to identify many individuals with a high likelihood of a reduced ABI with markedly higher sensitivity and only slightly reduced specificity compared to more complex questionnaires. At least in men, this question should be established as first screening step. AU - Kieback, A.G.* AU - Espinola-Klein, C.* AU - Lamina, C.* AU - Moebus, S.* AU - Tiller, D.* AU - Lorbeer, R.* AU - Schulz, A.* AU - Meisinger, C. AU - Medenwald, D.* AU - Erbel, R.* AU - Kluttig, A.* AU - Wild, P.S.* AU - Kronenberg, F.* AU - Kröger, K.* AU - Ittermann, T.* AU - Dörr, M.* C1 - 57256 C2 - 47636 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - One simple claudication question as first step in Peripheral Arterial Disease (PAD) screening: A meta-analysis of the association with reduced Ankle Brachial Index (ABI) in 27,945 subjects. JO - PLoS ONE VL - 14 IS - 11 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Doxorubicin (DOX) is a widely used chemotherapeutic anticancer drug. Its intrinsic fluorescence properties enable investigation of tumor response, drug distribution and metabolism. First phantom studies in vitro showed optoacoustic property of DOX. We therefore aimed to further investigate the optoacoustic properties of DOX in biological tissue in order to explore its potential as theranostic agent. We analysed doxorubicin hydrochloride (Dox center dot HCl) and liposomal encapsulated doxorubicin hydrochloride (Dox center dot Lipo), two common drugs for anticancer treatment in clinical medicine. Optoacoustic measurements revealed a strong signal of both doxorubicin substrates at 488 nm excitation wavelength. Post mortem analysis of intra-tumoral injections of DOX revealed a detectable optoacoustic signal even at three days after the injection. We thereby demonstrate the general feasibility of doxorubicin detection in biological tissue by means of optoacoustic tomography, which could be applied for high resolution imaging at mesoscopic depths dictated by effective penetration of visible light into the biological tissues. AU - Kimm, M.A.* AU - Gross, C.* AU - Déan-Ben, X.L.* AU - Ron, A. AU - Rummeny, E.J.* AU - Lin, H.-C. AU - Höltke, C.* AU - Razansky, D. AU - Wildgruber, M.* C1 - 56195 C2 - 46887 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Optoacoustic properties of Doxorubicin - A pilot study. JO - PLoS ONE VL - 14 IS - 5 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Murine Astrovirus is one of the most prevalent viral agents in laboratory rodent facilities worldwide, but its influence on biomedical research results is poorly examined. Due to possible influence on research results and high seroprevalence rates in mice, it appears useful to include this virus into routine health monitoring programs. In order to establish exhaust air particle PCR as a reliable detection method for Murine Astrovirus infections in mice kept in individually ventilated cages (IVC) and compare the method to sentinel mice monitoring regarding reproducibility and detection limit, we conducted a study with defined Murine Astrovirus cage prevalence. In parallel, the efficacy of both detection strategies (soiled-bedding sentinel (SBS) and exhaust air dust (EAD) analysis) was tested for Myocoptes musculinus. The fur mite was used as a reference organism during the whole study period to ensure the validity of this method. Because some publications already demonstrated successful detection of several pathogens, including murine fur mite species, via EAP-PCR. Detection of Murine Astrovirus infections at low prevalence is possible with both methods tested. Detection by exhaust air particles (EAP) is faster, more sensitive and more reliable compared to soiled bedding sentinels (SBS). Exhaust air particle PCR also detected the reference organism Myocoptes musculinus, which was not detected at all by sentinel mice, not even by high sensitivity fur swab qPCR. In conclusion, Murine Astrovirus can be detected by both exhaust air particle PCR and soiled bedding sentinels. We recommend exhaust air particle PCR as the better detection technique for Murine Astrovirus, because it is more reliable. Environmental samples are the method of choice for detection of Myocoptes musculinus because relying on soiled bedding sentinels harbors a big risk of missing existing infestations. AU - Körner, C. AU - Miller, M. AU - Brielmeier, M. C1 - 56770 C2 - 47385 TI - Detection of Murine Astrovirus and Myocoptes musculinus in individually ventilated caging systems: Investigations to expose suitable detection methods for routine hygienic monitoring. JO - PLoS ONE VL - 14 IS - 8 PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - BackgroundLittle is known about outpatient health services use following critical illness and intensive care. We examined the association of intensive care with outpatient consultations and quality of life in a population-based sample.MethodsCross-sectional analysis of data from 6,686 participants of the Study of Health in Pomerania (SHIP), which consists of two independent population-based cohorts. Statistical modeling was done using Poisson regression, negative binomial and generalized linear models for consultations, and a fractional response model for quality of life (EQ-5D-3L index value), with results expressed as prevalence ratios (PR) or percent change (PC). Entropy balancing was used to adjust for observed confounding.ResultsICU treatment in the previous year was reported by 139 of 6,686 (2,1%) participants, and was associated with a higher probability (PR 1.05 [CI:1.03;1.07]), number (PC +58.0% [CI:22.8;103.2]) and costs (PC +64.1% [CI:32.0;103.9]) of annual outpatient consultations, as well as with a higher number of medications (PC +37.8% [CI:17.7;61.5]). Participants with ICU treatment were more likely to visit a specialist (PR 1.13 [CI:1.09; 1.16]), specifically internal medicine (PR 1.67 [CI:1.45;1.92]), surgery (PR 2.42 [CI:1.92;3.05]), psychiatry (PR 2.25 [CI:1.30;3.90]), and orthopedics (PR 1.54 [CI:1.11;2.14]). There was no significant effect regarding general practitioner consultations. ICU treatment was also associated with lower health-related quality of life (EQ-5D index value: PC -13.7% [CI:-27.0;-0.3]). Furthermore, quality of life was inversely associated with outpatient consultations in the previous month, more so for participants with ICU treatment.ConclusionsOur findings suggest that ICU treatment is associated with an increased utilization of outpatient specialist services, higher medication intake, and impaired quality of life. AU - Kosilek, R.P.* AU - Baumeister, S. AU - Ittermann, T.* AU - Gründling, M.* AU - Brunkhorst, F.M.* AU - Felix, S.B.* AU - Abel, P.* AU - Friesecke, S.* AU - Apfelbacher, C.* AU - Brandl, M.* AU - Schmidt, K.* AU - Hoffmann, W.* AU - Schmidt, C.O.* AU - Chenot, J.F.* AU - Völzke, H.* AU - Gensichen, J.S.* C1 - 56944 C2 - 47370 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The association of intensive care with utilization and costs of outpatient healthcare services and quality of life. JO - PLoS ONE VL - 14 IS - 9 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Introduction The major stress-inducible heat shock protein 70 (Hsp70) is induced after different stress stimuli. In tumors, elevated intracellular Hsp70 levels were associated on the one hand with radio- and chemotherapy resistance and on the other hand with a favorable outcome for patients. This study was undertaken to investigate cytosolic Hsp70 (cHsp70) as a potential biomarker for progression free (PFS) and overall survival (OS) in patients with primary glioblastomas (GBM). Methods The cHsp70 expression in tumor tissue of 60 patients diagnosed with primary GBM was analyzed by immunohistochemistry. The cHsp70 expression was correlated to the PFS and OS of the patients. Results A high cHsp70 expression was associated with a prolonged PFS (hazard ratio = 0.374, p = 0.001) and OS (hazard ratio = 0.416, p = 0.014) in GBM patients treated according to the standard Stupp protocol with surgery, radiotherapy and temozolomide. Conclusions These data suggest that the intracellular Hsp70 expression might serve as a prognostic marker in patients with primary GBM. AU - Lämmer, F.* AU - Delbridge, C.* AU - Würstle, S.* AU - Neff, F.* AU - Meyer, B.* AU - Schlegel, J.* AU - Kessel, K.A. AU - Schmid, T.E. AU - Schilling, D. AU - Combs, S.E. C1 - 56771 C2 - 47386 TI - Cytosolic Hsp70 as a biomarker to predict clinical outcome in patients with glioblastoma. JO - PLoS ONE VL - 14 IS - 8 PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - BackgroundGenome-wide association studies of common diseases or metabolite quantitative traits often identify common variants of small effect size, which may contribute to phenotypes by modulation of gene expression. Thus, there is growing demand for cellular models enabling to assess the impact of gene regulatory variants with moderate effects on gene expression. Mitochondrial fatty acid oxidation is an important energy metabolism pathway. Common noncoding acyl-CoA dehydrogenase short chain (ACADS) gene variants are associated with plasma C4-acylcarnitine levels and allele-specific modulation of ACADS expression may contribute to the observed phenotype.Methods and findingsWe assessed ACADS expression and intracellular acylcarnitine levels in human lymphoblastoid cell lines (LCL) genotyped for a common ACADS variant associated with plasma C4-acylcarnitine and found a significant genotype-dependent decrease of ACADS mRNA and protein. Next, we modelled gradual decrease of ACADS expression using a tetracy-cline- regulated shRNA-knockdown of ACADS in Huh7 hepatocytes, a cell line with high fatty acid oxidation-(FAO)-capacity. Assessing acylcarnitine flux in both models, we found increased C4-acylcarnitine levels with decreased ACADS expression levels. Moreover, assessing time-dependent changes of acylcarnitine levels in shRNA-hepatocytes with altered ACADS expression levels revealed an unexpected effect on long-and medium-chain fatty acid intermediates. ConclusionsBoth, genotyped LCL and regulated shRNA-knockdown are valuable tools to model moderate, gradual gene-regulatory effects of common variants on cellular phenotypes. Decreasing ACADS expression levels modulate short and surprisingly also long/medium chain acylcarnitines, and may contribute to increased plasma acylcarnitine levels. AU - Matejka, K. AU - Stückler, F. AU - Salomon, M.* AU - Ensenauer, R.* AU - Reischl, E. AU - Hoerburger, L.* AU - Grallert, H. AU - Kastenmüller, G. AU - Peters, A. AU - Daniel, H.* AU - Krumsiek, J. AU - Theis, F.J. AU - Hauner, H. AU - Laumen, H. C1 - 56131 C2 - 46845 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Dynamic modelling of an ACADS genotype in fatty acid oxidation - Application of cellular models for the analysis of common genetic variants. JO - PLoS ONE VL - 14 IS - 5 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - ObjectiveTransfemoral Transcatheter Aortic Valve Implantation (TAVI) has become a standard therapy for patients with aortic valve stenosis. Fluoroscopic imaging is essential for TAVI with the anesthesiologist's workplace close to patient's head side. While the use of lead-caps has been shown to be useful for interventional cardiologists, data are lacking for anesthesiologists.MethodsA protective cap with a 0.35 lead-equivalent was worn on 15 working days by one anesthesiologist. Six detectors (three outside, three inside) were analyzed to determine the reduction of radiation. Literature search was conducted between April and October 2018.ResultsIn the observational period, 32 TAVI procedures were conducted. A maximum radiation dose of 0.55 mSv was detected by the dosimeters at the outside of the cap. The dosimeters inside the cap, in contrast, displayed a constant radiation dose of 0.08 mSv.ConclusionThe anesthesiologist's head is exposed to significant radiation during TAVI and it can be protected by wearing a lead-cap. AU - Mayr, N.P.* AU - Wiesner, G.* AU - Kretschmer, A.* AU - Brönner, J. AU - Hödlmoser, H. AU - Husser, O.* AU - Kasel, A.M.* AU - Lange, R.* AU - Tassani-Prell, P.* C1 - 55361 C2 - 46296 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Assessing the level of radiation experienced by anesthesiologists during transfemoral Transcatheter Aortic Valve Implantation and protection by a lead cap. JO - PLoS ONE VL - 14 IS - 1 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology. AU - Miller, M. AU - Sabrautzki, S. AU - Beyerlein, A. AU - Brielmeier, M. C1 - 56919 C2 - 47425 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Combining fish and environmental PCR for diagnostics of diseased laboratory zebrafish in recirculating systems. JO - PLoS ONE VL - 14 IS - 9 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Intestinal integrity is maintained by balanced numbers of CD103(+) Dendritic cells (DCs), which generate peripherally induced regulatory T cells (iTregs). We have developed a mouse model where DC-specific constitutive CD40 signals caused a strong reduction of CD103(+) DCs in the lamina propria (LP) and intestinal lymph nodes (LN). As a consequence, also iTregs were strongly reduced and transgenic mice on the C57Bl/6-background (B6) developed fatal colitis. Here we describe that transgenic mice on a pure Balb/c-background (B/c) do not show any pathologies, while transgenic C57Bl/6 x Balb/c (F1) mice develop weak colon inflammation, without fatal colitis. This graded pathology correlated with the effects of CD40-signalling on DCs in each background, with striking loss of CD103(+) DCs in B6, but reduced in F1 and diminished in B/c background. We further show direct correlation of CD103(+) DC-numbers with numbers of iTregs, the frequencies of which behave correspondingly. Striking effects on B6-DCs reflected robust loss of surface MHCII, known to be crucial for iTreg induction. Furthermore, elevated levels of IL-23 together with IL-1, found only in B6 mice, support generation of intestinal IFN-gamma(+) IL-17(+) Th17 cells and IFN-gamma(+) Th1 cells, responsible for onset of disease. Together, this demonstrates a novel aspect of colitis-control, depending on genetic background. Moreover, strain-specific environmental sensing might alter the CD103(+) DC/iTreg-axis to tip intestinal homeostatic balance to pathology. AU - Ogrinc Wagner, A.* AU - Friedrich, V.* AU - Barthels, C.* AU - Marconi, P.* AU - Blutke, A. AU - Brombacher, F.* AU - Brocker, T.* C1 - 55248 C2 - 46300 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Strain specific maturation of Dendritic cells and production of IL-1β controls CD40-driven colitis. JO - PLoS ONE VL - 14 IS - 1 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - The era of next-generation sequencing has mounted the foundation of many gene expression studies. In rheumatoid arthritis research, this has led to the discovery of important candidate genes which offered novel insights into mechanisms and their possible roles in the cure of the disease. In the last years, data generation has outstripped data analysis and while many studies focused on specific aspects of the disease, a global picture of the disease is not yet accomplished. Here, we analyzed and compared a collection of gene expression information from healthy individuals and from patients suffering under different arthritis conditions from published studies containing the following clinical conditions: early and established rheumatoid arthritis, osteoarthritis and arthralgia. We show comprehensive overviews of this data collection and give new insights specifically on gene expression in the early stage, into sex-dependent gene expression, and we describe general differences in expression of different biotypes of genes. Many genes that are related to cytoskeleton changes (actin filament related genes) are differently expressed in early rheumatoid arthritis in comparison to healthy subjects; interestingly, eight of these genes reverse their expression ratio significantly between men and women compared early rheumatoid arthritis and healthy subjects. There are some slighter changes between men and woman between the conditions early and established rheumatoid arthritis. Another aspect are miRNAs and other gene biotypes which are not only promising candidates for diagnoses but also change their expression grossly in average at rheumatoid arthritis and arthralgia compared to the healthy condition. With a selection of intersecting genes, we were able to generate simple classification models to distinguish between healthy and rheumatoid arthritis as well as between early rheumatoid arthritis to other arthritides based on gene expression. AU - Platzer, A.* AU - Nussbaumer, T. AU - Karonitsch, T.* AU - Smolen, J.S.* AU - Aletaha, D.* C1 - 56642 C2 - 47189 TI - Analysis of gene expression in rheumatoid arthritis and related conditions offers insights into sex-bias, gene biotypes and co-expression patterns. JO - PLoS ONE VL - 14 IS - 7 PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams >= 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Sub-millimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation. AU - Sammer, M.* AU - Teiluf, K. AU - Girst, S.* AU - Greubel, C.* AU - Reindl, J.* AU - Ilicic, K. AU - Walsh, D.W.M.* AU - Aichler, M. AU - Walch, A.K. AU - Combs, S.E. AU - Wilkens, J.J. AU - Dollinger, G.* AU - Schmid, T.E. C1 - 56845 C2 - 47384 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Beam size limit for pencil minibeam radiotherapy determined from side effects in an in-vivo mouse ear model. JO - PLoS ONE VL - 14 IS - 9 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Proton radiotherapy using minibeams of sub-millimeter dimensions reduces side effects in comparison to conventional proton therapy due to spatial fractionation. Since the proton minibeams widen with depth, the homogeneous irradiation of a tumor can be ensured by adjusting the beam distances to tumor size and depth to maintain tumor control as in conventional proton therapy. The inherent advantages of protons in comparison to photons like a limited range that prevents a dosage of distal tissues are maintained by proton minibeams and can even be exploited for interlacing from different beam directions. A first animal study was conducted to systematically investigate and quantify the tissue-sparing effects of proton pencil minibeams as a function of beam size and dose distributions, using beam widths between s = 95, 199, 306, 411, 561 and 883 mu m (standard deviation) at a defined center-to-center beam distance (ctc) of 1.8 mm. The average dose of 60 Gy was distributed in 4x4 minibeams using 20 MeV protons (LET similar to 2.7 keV/mu m). The induced radiation toxicities were measured by visible skin reactions and ear swelling for 90 days after irradiation. The largest applied beam size to ctc ratio (s/ctc = 0.49) is similar to a homogeneous irradiation and leads to a significant 3-fold ear thickness increase compared to the control group. Erythema and desquamation was also increased significantly 3-4 weeks after irradiation. With decreasing beam sizes and thus decreasing s/ctc, the maximum skin reactions are strongly reduced until no ear swelling or other visible skin reactions should occur for s/ctc < 0.032 (extrapolated from data). These results demonstrate that proton pencil minibeam radiotherapy has better tissue-sparing for smaller s/ctc, corresponding to larger peak-to-valley dose ratios PVDR, with the best effect for s/ctc < 0.032. However, even quite large s/ctc (e.g. s/ctc = 0.23 or 0.31, i.e. PVDR = 10 or 2.7) show less acute side effects than a homogeneous dose distribution. This suggests that proton minibeam therapy spares healthy tissue not only in the skin but even for dose distributions appearing in deeper layers close to the tumor enhancing its benefits for clinical proton therapy. AU - Sammer, M.* AU - Zahnbrecher, E. AU - Dobiasch, S. AU - Girst, S.* AU - Greubel, C.* AU - Ilicic, K. AU - Reindl, J.* AU - Schwarz, B.* AU - Siebenwirth, C.* AU - Walsh, D.W.M.* AU - Combs, S.E. AU - Dollinger, G.* AU - Schmid, T.E. C1 - 57417 C2 - 47789 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Proton pencil minibeam irradiation of an in-vivo mouse ear model spares healthy tissue dependent on beam size. JO - PLoS ONE VL - 14 IS - 11 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - Background and objectiveThe revised World Health Organization classification of 2016 for myeloid neoplasms and acute leukemia added a section of myeloid neoplasms with germline predisposition. The main objective of our study was to evaluate the frequency of hematologic and solid malignancies in the family history of patients with acute myeloid leukemia (AML) by using a systemic pedigree interview. The family history was taken of 50 patients between 24 and 80 years.Findings8/50 (16%) patients with AML had family members with hematologic malignancies. 2/50 (4%) patients had family members of first degree with hematologic malignancies. Furthermore in 42/50 (84%) of AML patients solid malignancies were documented in family members of any degree and in 31/50 (62%) in family members of first degree. The most commonly occurring malignancies in our cohort were breast and colorectal cancer. We analyzed the pedigrees for cancer syndromes that can be associated with acute leukemia like Li-Fraumeni syndrome, Lynch syndrome and hereditary breast cancer. 2/50 (4%) patients fulfilled the criteria for familial breast and ovarian cancer from the German consortium and 1/50 (2%) patients fulfilled the Bethesda Guidelines criteria for hereditary nonpolyposis colorectal cancer. No pedigree met the criteria for Li-Fraumeni syndrome. In 29 cases we compared the patient history obtained in the routine work-up with our data. The accuracy of the obtained family history was 23%, outlining that in the clinical routine information about family histories often escapes notice.ConclusionOur study shows that though generally considered a sporadic disease, the presence of hematologic and solid malignancies in the family history of AML patients is relatively high. One should keep in mind that cancer syndromes like hereditary breast cancer are associated with a higher incidence of leukemia. These data are relevant in the context of family donor search for allogeneic stem cell transplantation, genetic counseling and testing as well as cancer prevention. AU - Sandner, A.S.* AU - Weggel, R.* AU - Mehraein, Y.* AU - Schneider, S.* AU - Hiddemann, W.* AU - Spiekermann, K. C1 - 55907 C2 - 48070 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Frequency of hematologic and solid malignancies in the family history of 50 patients with acute myeloid leukemia - a single center analysis. JO - PLoS ONE VL - 14 IS - 4 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - The E1B 55kDa produced by human adenovirus type 5 is a multifunctional protein that participates in the regulation of several steps during the viral replication cycle. Previous studies suggest this protein plays an important role in postranscriptional regulation of viral and cellular gene expression, as it is required for the selective accumulation of maximal levels of viral late mRNA in the cytoplasm of the infected cell; however the molecular mechanisms that are altered or regulated by this protein have not been elucidated. A ribonucleoprotein motif that could implicate the direct interaction of the protein with RNA was initially predicted and tested in vitro, but the interaction with RNA could not be detected in infected cells, suggesting the interaction may be weak or transient. Here it was determined that the E1B 55kDa interacts with RNA in the context of the viral infection in non-transformed human cells, and its contribution to the adenovirus replication cycle was evaluated. Using recombinant adeno-viruses with amino acid substitutions or a deletion in the ribonucleoprotein motif the interaction of E1B 55kDa with RNA was found to correlate with timely and efficient viral DNA replication and viral late mRNA accumulation and splicing. AU - Tejera, B.* AU - López, R.E.* AU - Hidalgo, P.* AU - Cárdenas, R.* AU - Ballesteros, G.* AU - Rivillas, L.* AU - French, L.* AU - Amero, C.* AU - Pastor, N.* AU - Santiago, Á.* AU - Groitl, P. AU - Dobner, T.* AU - Gonzalez, R.A.* C1 - 55802 C2 - 46584 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The human adenovirus type 5 E1B 55kDa protein interacts with RNA promoting timely DNA replication and viral late mRNA metabolism. JO - PLoS ONE VL - 14 IS - 4 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - BackgroundAlthough lung cancer is most commonly diagnosed in elderly patients, evidence about tumor-directed therapy in elderly patients is sparse, and it is unclear to what extent this affects treatment and care. Our study aimed to discover potential disparities in care between elderly patients and those under 65 years of age.MethodsWe studied claims from 13 283 German patients diagnosed with lung cancer in 2009 who survived for at least 90 days after diagnosis. We classified patients as "non-elderly" (<= 65), "young-old" (65-74), "middle-old" (75-84), and "old-old" (>= 85). We compared receipt of tumor-directed therapy (6 months after diagnosis), palliative care, opioids, antidepressants, and pathologic diagnosis confirmation via logistic regression. We used generalized linear regression (gamma distribution) to compare group-specific costs of care for 3 months after diagnosis. We adjusted all models by age, nursing home residency, nursing care need, comorbidity burden, and area of residence (urban, rural). The age group "non-elderly" served as reference group.ResultsCompared with the reference group "non-elderly", the likelihood of receiving any tumor-directed treatment was significantly lower in all age groups with a decreasing gradient with advancing age. Elderly lung cancer patients received significantly fewer resections and radiotherapy than non-elderly patients. In particular, treatment with antineoplastic therapy declined with increasing age ("young-old" (OR = 0.76, CI = [0.70,0.83]), "middle-old" (OR = 0.45, CI = [0.36,0.50]), and "old-old" (OR = 0.13, CI = [0.10,0.17])). Patients in all age groups were less likely to receive structured palliative care than "non-elderly" ("young-old" (OR = 0.84, CI = [0.76,0.92]), "middle-old" (OR = 0.71, CI = [0.63,0.79]), and "old-old" (OR = 0.57, CI = [0.44,0.73])). Moreover, increased age was significantly associated with reduced quotas for outpatient treatment with opioids and antidepressants. Costs of care decreased significantly with increasing age.ConclusionThis study suggests the existence of age-dependent care disparities in lung cancer patients, where elderly patients are at risk of potential undertreatment. To support equal access to care, adjustments to public health policies seem to be urgently required. AU - Walter, J. AU - Tufman, A.* AU - Holle, R. AU - Schwarzkopf, L. C1 - 56290 C2 - 46966 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - "Age matters"- German claims data indicate disparities in lung cancer care between elderly and young patients. JO - PLoS ONE VL - 14 IS - 6 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - BackgroundFibrinogen is an essential hemostatic factor and cardiovascular disease risk factor. Early attempts at evaluating the causal effect of fibrinogen on coronary heart disease (CHD) and myocardial infraction (MI) using Mendelian randomization (MR) used single variant approaches, and did not take advantage of recent genome-wide association studies (GWAS) or multi-variant, pleiotropy robust MR methodologies.Methods and findingsWe evaluated evidence for a causal effect of fibrinogen on both CHD and MI using MR. We used both an allele score approach and pleiotropy robust MR models. The allele score was composed of 38 fibrinogen-associated variants from recent GWAS. Initial analyses using the allele score used a meta-analysis of 11 European-ancestry prospective cohorts, free of CHD and MI at baseline, to examine incidence CHD and MI. We also applied 2 sample MR methods with data from a prevalent CHD and MI GWAS. Results are given in terms of the hazard ratio (HR) or odds ratio (OR), depending on the study design, and associated 95% confidence interval (CI).In single variant analyses no causal effect of fibrinogen on CHD or MI was observed. In multi-variant analyses using incidence CHD cases and the allele score approach, the estimated causal effect (HR) of a 1 g/L higher fibrinogen concentration was 1.62 (CI = 1.12, 2.36) when using incident cases and the allele score approach. In 2 sample MR analyses that accounted for pleiotropy, the causal estimate (OR) was reduced to 1.18 (CI = 0.98, 1.42) and 1.09 (CI = 0.89, 1.33) in the 2 most precise (smallest CI) models, out of 4 models evaluated. In the 2 sample MR analyses for MI, there was only very weak evidence of a causal effect in only 1 out of 4 models.ConclusionsA small causal effect of fibrinogen on CHD is observed using multi-variant MR approaches which account for pleiotropy, but not single variant MR approaches. Taken together, results indicate that even with large sample sizes and multi-variant approaches MR analyses still cannot exclude the null when estimating the causal effect of fibrinogen on CHD, but that any potential causal effect is likely to be much smaller than observed in epidemiological studies. AU - Ward-Caviness, C.K. AU - de Vries, P.S.* AU - Wiggins, K.L.* AU - Huffman, J.E.* AU - Yanek, L.R.* AU - Bielak, L.F.* AU - Giulianini, F.* AU - Guo, X.* AU - Kleber, M.E.* AU - Kacprowski, T.* AU - Groß, S.* AU - Petersman, A.* AU - Davey Smith, G.* AU - Hartwig, F.P.* AU - Bowden, J.* AU - Hemani, G.* AU - Müller-Nurasyid, M. AU - Strauch, K. AU - Koenig, W.* AU - Waldenberger, M. AU - Meitinger, T. AU - Pankratz, N.* AU - Boerwinkle, E.* AU - Tang, W.* AU - Fu, Y.P.* AU - Johnson, A.D.* AU - Song, C.* AU - de Maat, M.P.M.* AU - Uitterlinden, A.G.* AU - Franco, O.H.* AU - Brody, J.A.* AU - McKnight, B.* AU - Chen, Y.I.* AU - Psaty, B.M.* AU - Mathias, R.A.* AU - Becker, D.M.* AU - Peyser, P.A.* AU - Smith, J.A.* AU - Bielinski, S.J.* AU - Ridker, P.M.* AU - Taylor, K.D.* AU - Yao, J.* AU - Tracy, R.* AU - Delgado, G.* AU - Trompet, S.* AU - Sattar, N.* AU - Jukema, J.W.* AU - Becker, L.C.* AU - Kardia, S.L.R.* AU - Rotter, J.I.* AU - März, W.* AU - Dörr, M.* AU - Chasman, D.I.* AU - Dehghan, A.* AU - O'Donnell, C.J.* AU - Smith, N.L.* AU - Peters, A. AU - Morrison, A.C.* C1 - 56028 C2 - 46790 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Mendelian randomization evaluation of causal effects of fibrinogen on incident coronary heart disease. JO - PLoS ONE VL - 14 IS - 5 PB - Public Library Science PY - 2019 SN - 1932-6203 ER - TY - JOUR AB - The emissions of BVOCs from oilseed rape (Brassica napus), both when the plant is exposed to clean air and when it is fumigated with ozone at environmentally-relevant mixing ratios (ca. 135 ppbv), were measured under controlled laboratory conditions. Emissions of BVOCs were recorded from combined leaf and root chambers using a recently developed Selective Reagent Ionisation-Time of Flight-Mass Spectrometer (SRI-ToF-MS) enabling BVOC detection with high time and mass resolution, together with the ability to identify certain molecular functionality. Emissions of BVOCs from below-ground were found to be dominated by sulfur compounds including methanethiol, dimethyl disulfide and dimethyl sulfide, and these emissions did not change following fumigation of the plant with ozone. Emissions from above-ground plant organs exposed to clean air were dominated by methanol, monoterpenes, 4-oxopentanal and methanethiol. Ozone fumigation of the plants caused a rapid decrease in monoterpene and sesquiterpene concentrations in the leaf chamber and increased concentrations of ca. 20 oxygenated species, almost doubling the total carbon lost by the plant leaves as volatiles. The drop in sesquiterpenes concentrations was attributed to ozonolysis occurring to a major extent on the leaf surface. The drop in monoterpene concentrations was attributed to gas phase reactions with OH radicals deriving from ozonolysis reactions. As plant-emitted terpenoids have been shown to play a role in plant-plant and plant-insect signalling, the rapid loss of these species in the air surrounding the plants during photochemical pollution episodes may have a significant impact on plant-plant and plant-insect communications. AU - Acton, W.J.F.* AU - Jud, W. AU - Ghirardo, A. AU - Wohlfahrt, G.* AU - Hewitt, C.N.* AU - Taylor, J.E.* AU - Hansel, A.* C1 - 54931 C2 - 45972 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - The effect of ozone fumigation on the biogenic volatile organic compounds (BVOCs) emitted from Brassica napus above- and below-ground. JO - PLoS ONE VL - 13 IS - 12 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Aims This population-based study sought to extend knowledge on factors explaining regional differences in type 2 diabetes mellitus medication patterns in Germany. Methods Individual baseline and follow-up data from four regional population-based German cohort studies (SHIP [northeast], CARLA [east], HNR [west], KORA [south]) conducted between 1997 and 2010 were pooled and merged with both data on regional deprivation and regional health care services. To analyze regional differences in any or newer anti-hyperglycemic medication, medication prevalence ratios (PRs) were estimated using multivariable Poisson regression models with a robust error variance adjusted gradually for individual and regional variables. Results The study population consisted of 1,437 people aged 45 to 74 years at baseline, (corresponding to 49 to 83 years at follow-up) with self-reported type 2 diabetes. The prevalence of receiving any anti-hyperglycemic medication was 16% higher in KORA (PR 1.16 [1.08 +/- 1.25]), 10% higher in CARLA (1.10 [1.01 +/- 1.18]), and 7% higher in SHIP (PR 1.07 [1.00 +/- 1.15]) than in HNR. The prevalence of receiving newer anti-hyperglycemic medication was 49% higher in KORA (1.49 [1.09 +/- 2.05]), 41% higher in CARLA (1.41 [1.02 +/- 1.96]) and 1% higher in SHIP (1.01 [0.72 +/- 1.41]) than in HNR, respectively. After gradual adjustment for individual variables, regional deprivation and health care services, the effects only changed slightly. Conclusions Neither comprehensive individual factors including socioeconomic status nor regional deprivation or indicators of regional health care services were able to sufficiently explain regional differences in anti-hyperglycemic treatment in Germany. To understand the underlying causes, further research is needed. AU - Bächle, C.* AU - Claessen, H.* AU - Maier, W. AU - Tamayo, T.* AU - Schunk, M. AU - Rückert-Eheberg, I.-M. AU - Holle, R. AU - Meisinger, C. AU - Moebus, S.* AU - Jöckel, K.H.* AU - Schipf, S.* AU - Völzke, H.* AU - Hartwig, S.* AU - Kluttig, A.* AU - Kroll, L.E.* AU - Linnenkamp, U.* AU - Icks, A.* C1 - 52873 C2 - 44305 CY - San Francisco TI - Regional differences in antihyperglycemic medication are not explained by individual socioeconomic status, regional deprivation, and regional health care services. Observational results from the German DIAB-CORE consortium. JO - PLoS ONE VL - 13 IS - 1 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Encapsulation of primary bovine adrenocortical cells in alginate is an efficacious model of a bioartificial adrenal cortex. Such a bioartificial adrenal cortex can be used for the restoration of lost adrenal function in vivo as well as for in vitro modeling of the adrenal microenvironment and for investigation of cell-cell interactions in the adrenals. The aim of this work was the optimization of a bioartificial adrenal cortex, that is the generation of a highly productive, self-regenerating, long-term functioning and immune tolerant bioartificial organ. To achieve this, it is necessary that adrenocortical stem and progenitor cells are present in the bioartificial gland, as these undifferentiated cells play important roles in the function of the mature gland. Here, we verified the presence of adrenocortical progenitors in cultures of bovine adrenocortical cells, studied the dynamics of their appearance and growth and determined the optimal time point for cell encapsulation. These procedures increased the functional life span and reduced the immunogenicity of the bioartificial adrenal cortex. This model allows the use of the luteinizing hormone-releasing hormone (LHRH) agonist triptorelin, the neuropeptide bombesin, and retinoic acid to alter cell number and the release of cortisol over long periods of time. AU - Balyura, M.* AU - Gelfgat, E.* AU - Steenblock, C.* AU - Androutsellis-Theotokis, A.* AU - Ruiz-Babot, G.* AU - Guasti, L.* AU - Werdermann, M.* AU - Ludwig, B. AU - Bornstein, T.D.* AU - Schally, A.V.* AU - Brennand, A.* AU - Bornstein, S.R. C1 - 53424 C2 - 44725 CY - San Francisco TI - Expression of progenitor markers is associated with the functionality of a bioartificial adrenal cortex. JO - PLoS ONE VL - 13 IS - 3 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: To evaluate the impact of preoperative anemia and perioperative blood transfusion (PBT) on disease free (DFS) and overall survival (OS) of patients with head and neck squamous cell carcinoma (HNSCC).METHODS: Retrospective study of 354 patients primarily treated with surgery between 2006 and 2016. Cases were selected according to completeness and accuracy of available clinical data. Thus, a selection bias cannot be excluded. Patients who received PBT were identified by our controlling department and verified by our blood bank data base.RESULTS: Both, preoperative anemia and PBT significantly decreased OS in univariate analysis. Although PBT was needed more frequently by older patients in worse physical conditions with more advanced HNSCC, subgroup analysis also demonstrate a profoundly negative effect of PBT on OS in younger patients and early stage HNSCC. According to a restrictive transfusion policy at our hospital the transfusion rate was comparably low. We could not verify increasing effects of PBT on cancer recurrence rates as it was previously shown.DISCUSSION: Preoperative anemia is the most common paraneoplastic syndrome in HNSCC. Despite its devastating prognostic effect we suggest a restrictive transfusion policy whenever possible. Our data also show that anemia as an independent prognostic factor in head and neck surgical oncology is defined not only by low hemoglobin concentrations but low red blood cell counts as well. AU - Baumeister, P. AU - Canis, M.* AU - Reiter, M.* C1 - 54613 C2 - 45708 TI - Preoperative anemia and perioperative blood transfusion in head and neck squamous cell carcinoma. JO - PLoS ONE VL - 13 IS - 10 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background The aim of our study was to determine the relation of alcohol consumption and cigarette smoking on continuous-measured hepatic fat fraction (HFF) in a population free of cardiovascular disease. We suggested a direct correlation of alcohol consumption with HFF and increased HFF in former smokers compared to current smokers. Methods Data from 384 subjects (mean age: 56 years, 58% men) of a population-based cohort study (KORA) were included in a cross-sectional design. Liver fat was assessed by 3 Tesla magnetic resonance imaging (MRI) using a multi-echo Dixon sequence and T2-corrected single voxel multi-echo spectroscopy (H-1-MRS). Smoking status was classified as never, former or current smoker and alcohol consumption as non-, moderate (0.1-39.9 g/day for men and 0.1-19.9 g/day for women), or heavy drinker (>= 40 g/day for men and >= 20 g/day for women). Fatty liver disease was defined as HFF >= 5.56%. Results Average HFF was 8.8% by H-1-MRS and 8.5% by MRI. Former smokers showed a higher HFF (MRI: beta = 2.64; p = 0.006) and a higher FLD prevalence (MRI: OR = 1.91; p = 0.006) compared to never smokers. Current smokers showed decreased odds for FLD measured by H-1-MRS after multivariable adjustment (OR = 0.37; p = 0.007) with never smoker as reference. Heavy drinking was positively associated with HFF (H-1-MRS: beta = 2.99; p = 0.003) and showed highest odds for FLD (H-1-MRS: OR = 3.05; p = 0.008) with non-drinker as reference. Moderate drinking showed a positive association with HFF (H-1-MRS: beta = 1.54; p = 0.061 and MRI: beta = 1.75; p = 0.050). Conclusions Our data revealed lowest odds for FLD in current smokers, moderate drinkers showing higher HFF than non-drinkers and heavy drinkers showing highest HFF and odds for FLD. These findings partly conflict with former literature and underline the importance of further studies to investigate the complex effects on liver metabolism. AU - Bayerl, C.* AU - Lorbeer, R.* AU - Heier, M. AU - Meisinger, C. AU - Rospleszcz, S. AU - Schafnitzel, A.* AU - Patscheider, H.* AU - Auweter, S.* AU - Peters, A. AU - Ertl-Wagner, B.* AU - Reiser, M.* AU - Bamberg, F.* AU - Hetterich, H.* C1 - 52877 C2 - 44306 CY - San Francisco TI - Alcohol consumption, but not smoking is associated with higher MR-derived liver fat in an asymptomatic study population. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo. AU - Breunig, C. AU - Durovic, T. AU - Neuner, A.M.* AU - Baumann, V.* AU - Wiesbeck, M.F.* AU - Köferle, A.* AU - Götz, M. AU - Ninkovic, J. AU - Stricker, S.H. C1 - 53481 C2 - 44589 TI - One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR). JO - PLoS ONE VL - 13 IS - 4 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Heavy alcohol consumption is an established risk factor for hypertension; the mechanism by which alcohol consumption impact blood pressure (BP) regulation remains unknown. We hypothesized that a genome-wide association study accounting for gene-alcohol consumption interaction for BP might identify additional BP loci and contribute to the understanding of alcohol-related BP regulation. We conducted a large two-stage investigation incorporating joint testing of main genetic effects and single nucleotide variant (SNV)-alcohol consumption interactions. In Stage 1, genome-wide discovery meta-analyses in approximate to 131 K individuals across several ancestry groups yielded 3,514 SNVs (245 loci) with suggestive evidence of association (P <1.0 x 10(-5)). In Stage 2, these SNVs were tested for independent external replication in individuals across multiple ancestries. We identified and replicated (at Bonferroni correction threshold) five novel BP loci (380 SNVs in 21 genes) and 49 previously reported BP loci (2,159 SNVs in 109 genes) in European ancestry, and in multi-ancestry meta-analyses (P < 5.0 x 10(-8)). For African ancestry samples, we detected 18 potentially novel BP loci (P< 5.0 x 10(-8)) in Stage 1 that warrant further replication. Additionally, correlated meta-analysis identified eight novel BP loci (11 genes). Several genes in these loci (e.g., PINX1, GATA4, BLK, FTO and GABBR2 have been previously reported to be associated with alcohol consumption. These findings provide insights into the role of alcohol consumption in the genetic architecture of hypertension. AU - Feitosa, M.F.* AU - Kraja, A.T.* AU - Chasman, D.I.* AU - Sung, Y.J.* AU - Winkler, T.W.* AU - Ntalla, I.* AU - Guo, X.* AU - Franceschini, N.* AU - Cheng, C.Y.* AU - Sim, X.* AU - Vojinovic, D.* AU - Marten, J.* AU - Musani, S.K.* AU - Li, C.* AU - Bentley, A.R.* AU - Brown, M.R.* AU - Schwander, K.* AU - Richard, M.A.* AU - Noordam, R.* AU - Aschard, H.* AU - Bartz, T.M.* AU - Bielak, L.F.* AU - Dorajoo, R.* AU - Fisher, V.A.* AU - Hartwig, F.P.* AU - Horimoto, A.R.V.R.* AU - Lohman, K.K.* AU - Manning, A.K.* AU - Rankinen, T.* AU - Smith, A.V.* AU - Tajuddin, S.M.* AU - Wojczynski, M.K.* AU - Alver, M.* AU - Boissel, M.* AU - Cai, Q.* AU - Campbell, A.* AU - Chai, J.F.* AU - Chen, X.* AU - Divers, J.* AU - Gao, C.* AU - Goel, A.* AU - Hagemeijer, Y.* AU - Harris, S.E.* AU - He, M.* AU - Hsu, F.C.* AU - Jackson, A.U.* AU - Kähönen, M.* AU - Kasturiratne, A.* AU - Komulainen, P.* AU - Kühnel, B. AU - Laguzzi, F.* AU - Luan, J.* AU - Matoba, N.* AU - Nolte, I.M.* AU - Padmanabhan, S.* AU - Riaz, M.* AU - Rueedi, R.* AU - Robino, A.* AU - Said, M.A.* AU - Scott, R.A.* AU - Sofer, T.* AU - Stancáková, A.* AU - Takeuchi, F.* AU - Tayo, B.O.* AU - van der Most, P.J.* AU - Varga, T.V.* AU - Vitart, V.* AU - Meitinger, T. AU - Peters, A. AU - Strauch, K. AU - Province, M.A.* AU - Wen, W.* AU - Yanek, L.R.* AU - Zhang, W.* AU - Zhao, J.H.* AU - Afaq, S.* AU - Amin, N.* AU - Amini, M.* AU - Arking, D.E.* AU - Aung, T.* AU - Boerwinkle, E.* AU - Waldenberger, M. AU - Levy, D.* C1 - 53872 C2 - 45020 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Novel genetic associations for blood pressure identified via gene-alcohol interaction in up to 570K individuals across multiple ancestries. JO - PLoS ONE VL - 13 IS - 6 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Lifestyle interventions in type 2 diabetes (DM2) prevention implementation studies can be effective and lasting. Long-term weight loss maintenance enhances the intervention effect through a significant decrease in diabetes incidence over time. Our objective was to identify factors predicting long-term successful weight reduction maintenance achieved during a DM2 prevention program in patients with high DM2 risk in primary health care. Study participants (n = 263), middle-aged, slightly obese with baseline increased DM2 risk (Finnish Diabetes Risk Score (FINDRISC)>14), but no diabetes were invited to receive 11 lifestyle counselling sessions, guided physical activity sessions and motivational support during 10-months. The study participants had three clinical examinations during the study (baseline, one and three years). Stepwise regression analysis was used to determine demographic, clinical, and lifestyle predictors of weight reduction maintenance two years after the discontinuation of the intervention. Out of 105 patients who completed all three examinations (baseline age 56.6 (standard deviation (SD) = 10.7), body mass index 31.1 kg/m2 (SD = 4.9), FINDRISC 18.6 (SD = 3.1)), 73 patients (70%) showed weight loss during the intervention (mean weight loss 4.2 kg, SD = 5.1). The total weight loss achieved in the maintainers (27 of 73 study participants) two years after the intervention had finished was 6.54 kg (4.47 kg+2.0 kg). The non-maintainers, on the other hand, returned to their initial weight at the start of the intervention (+0.21 kg). In multivariable analysis baseline history of increased glucose (odds ratio (OR) = 3.7; 95% confidence interval (CI) 1.0-13.6) and reduction of total fat in diet during follow-up (OR = 4.3; 95% CI 1.5-12.2) were independent predictors of successful weight loss. Further studies exploring predictors of weight loss maintenance in diabetes prevention are needed to help health care providers to redesign interventions and improve long-term outcomes of real life interventions. AU - Gilis-Januszewska, A.* AU - Barengo, N.C.* AU - Lindstrom, J.* AU - Wójtowicz, E.* AU - Acosta, T.* AU - Tuomilehto, J.* AU - Schwarz, P.E. AU - Piwońska-Solska, B.* AU - Szybinski, Z.* AU - Windak, A.* AU - Hubalewska-Dydejczyk, A.* C1 - 53472 C2 - 44744 TI - Predictors of long term weight loss maintenance in patients at high risk of type 2 diabetes participating in a lifestyle intervention program in primary health care: The DE-PLAN study. JO - PLoS ONE VL - 13 IS - 3 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background An association between low socioeconomic status (SES) and lung cancer has been observed in several studies, but often without adequate control for smoking behavior. We studied the association between lung cancer and occupationally derived SES, using data from the international pooled SYNERGY study. Methods Twelve case-control studies from Europe and Canada were included in the analysis. Based on occupational histories of study participants we measured SES using the International Socio-Economic Index of Occupational Status (ISEI) and the European Socio-economic Classification (ESeC). We divided the ISEI range into categories, using various criteria. Stratifying by gender, we calculated odds ratios (OR) and 95% confidence intervals (CI) by unconditional logistic regression, adjusting for age, study, and smoking behavior. We conducted analyses by histological subtypes of lung cancer and subgroup analyses by study region, birth cohort, education and occupational exposure to known lung carcinogens. Results The analysis dataset included 17,021 cases and 20,885 controls. There was a strong elevated OR between lung cancer and low SES, which was attenuated substantially after adjustment for smoking, however a social gradient persisted. SES differences in lung cancer risk were higher among men (lowest vs. highest SES category: ISEI OR 1.84 (95% CI 1.61-2.09); ESeC OR 1.53 (95% CI 1.44-1.63)), than among women (lowest vs. highest SES category: ISEI OR 1.54 (95% CI 1.20-1.98); ESeC OR 1.34 (95% CI 1.19-1.52)). Conclusion SES remained a risk factor for lung cancer after adjustment for smoking behavior. AU - Hovanec, J.* AU - Siemiatycki, J.* AU - Conway, D.I.* AU - Olsson, A.* AU - Stücker, I.* AU - Guida, F.* AU - Jöckel, K.H.* AU - Pohlabeln, H.* AU - Ahrens, W.* AU - Brüske, I. AU - Wichmann, H.-E. AU - Gustavsson, P.* AU - Consonni, D.* AU - Merletti, F.* AU - Richiardi, L.* AU - Simonato, L.* AU - Fortes, C.* AU - Parent, M.E.* AU - McLaughlin, J.* AU - Demers, P.* AU - Landi, M.T.* AU - Caporaso, N.* AU - Tardón, A.* AU - Zaridze, D.* AU - Szeszenia-Dabrowska, N.* AU - Rudnai, P.* AU - Lissowska, J.* AU - Fabianova, E.* AU - Field, J.* AU - Dumitru, R.S.* AU - Bencko, V.* AU - Foretova, L.* AU - Janout, V.* AU - Kromhout, H.* AU - Vermeulen, R.* AU - Boffetta, P.* AU - Straif, K.* AU - Schüz, J.* AU - Kendzia, B.* AU - Pesch, B.* AU - Brüning, T.* AU - Behrens, T.* C1 - 53015 C2 - 44399 CY - San Francisco TI - Lung cancer and socioeconomic status in a pooled analysis of case-control studies. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono-and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. AU - Kasurinen, S. AU - Happo, M.S.* AU - Rönkkö, T.J.* AU - Orasche, J. AU - Jokiniemi, J.* AU - Kortelainen, M.* AU - Tissari, J.* AU - Zimmermann, R. AU - Hirvonen, M.R.* AU - Jalava, P.I.* C1 - 53072 C2 - 44331 CY - San Francisco TI - Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background Chronic obstructive pulmonary disease (COPD) has a high prevalence rate in Germany and a further increase is expected within the next years. Although risk factors on an individual level are widely understood, only little is known about the spatial heterogeneity and population-based risk factors of COPD. Background knowledge about broader, population-based processes could help to plan the future provision of healthcare and prevention strategies more aligned to the expected demand. The aim of this study is to analyze how the prevalence of COPD varies across northeastern Germany on the smallest spatial-scale possible and to identify the location-specific population-based risk factors using health insurance claims of the AOK Nordost. Methods To visualize the spatial distribution of COPD prevalence at the level of municipalities and urban districts, we used the conditional autoregressive Besag-York-Mollie A (BYM) model. Geographically weighted regression modelling (GWR) was applied to analyze the location-specific ecological risk factors for COPD. Results The sex-and age-adjusted prevalence of COPD was 6.5% in 2012 and varied widely across northeastern Germany. Population-based risk factors consist of the proportions of insurants aged 65 and older, insurants with migration background, household size and area deprivation. The results of the GWR model revealed that the population at risk for COPD varies considerably across northeastern Germany. Conclusion Area deprivation has a direct and an indirect influence on the prevalence of COPD. Persons ageing in socially disadvantaged areas have a higher chance of developing COPD, even when they are not necessarily directly affected by deprivation on an individual level. This underlines the importance of considering the impact of area deprivation on health for planning of healthcare. Additionally, our results reveal that in some parts of the study area, insurants with migration background and persons living in multi-persons households are at elevated risk of COPD. AU - Kauhl, B.* AU - Maier, W. AU - Schweikart, J.* AU - Keste, A.* AU - Moskwyn, M.* C1 - 52894 C2 - 44236 CY - San Francisco TI - Who is where at risk for Chronic Obstructive Pulmonary Disease? A spatial epidemiological analysis of health insurance claims for COPD in Northeastern Germany. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background To date, the scientific literature on socioeconomic correlates and determinants of physical activity behaviours has been dispersed throughout a number of systematic reviews, often focusing on one factor (e.g. education or parental income) in one specific age group (e.g. pre-school children or adults). The aim of this umbrella review is to provide a comprehensive and systematic overview of the scientific literature from previously conducted research by summarising and synthesising the importance and strength of the evidence related to socioeconomic correlates and determinants of PA behaviours across the life course. Methods Medline, Embase, ISI Web of Science, Scopus and SPORTDiscus were searched for systematic literature reviews and meta-analyses of observational studies investigating the association between socioeconomic determinants of PA and PA itself (from January 2004 to September 2017). Data extraction evaluated the importance of determinants, strength of evidence, and methodological quality of the selected papers. The full protocol is available from PROSPERO (PROSPERO2014: CRD42015010616). Results Nineteen reviews were included. Moderate methodological quality emerged. For adults, convincing evidence supports a relationship between PA and socioeconomic status (SES), especially in relation to leisure time (positive relationship) and occupational PA (negative relationship). Conversely, no association between PA and SES or parental SES was found for pre-school, school-aged children and adolescents. Conclusions Available evidence on the socioeconomic determinants of PA behaviour across the life course is probable (shows fairly consistent associations) at best. While some evidence is available for adults, less was available for youth. This is mainly due to a limited quantity of primary studies, weak research designs and lack of accuracy in the PA and SES assessment methods employed. Further PA domain specific studies using longitudinal design and clear measures of SES and PA assessment are required. AU - O'Donoghue, G.* AU - Kennedy, A.* AU - Puggina, A.* AU - Aleksovska, K.* AU - Buck, C.* AU - Burns, C.* AU - Cardon, G.* AU - Carlin, A.* AU - Ciarapica, D.* AU - Colotto, M.* AU - Condello, G.* AU - Coppinger, T.C.* AU - Cortis, C.* AU - D'Haese, S.* AU - De Craemer, M.* AU - di Blasio, A.M.* AU - Hansen, S.* AU - Iacoviello, L.* AU - Issartel, J.* AU - Izzicupo, P.* AU - Jaeschke, L.* AU - Kanning, M.* AU - Ling, F.C.M.* AU - Luzak, A. AU - Napolitano, G.* AU - Nazare, J.A.* AU - Perchoux, C.* AU - Pesce, C.* AU - Pischon, T.* AU - Polito, A.* AU - Sannella, A.* AU - Schulz, H. AU - Simon, C.* AU - Sohun, R.* AU - Steinbrecher, A.* AU - Schlicht, W.* AU - MacDonncha, C.* AU - Capranica, L.* AU - Boccia, S.* C1 - 52754 C2 - 44308 CY - San Francisco TI - Socio-economic determinants of physical activity across the life course: A "DEterminants of DIet and Physical ACtivity" (DEDIPAC) umbrella literature review. JO - PLoS ONE VL - 13 IS - 1 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies. AU - Panse, M.* AU - Kluth, O.* AU - Lorza-Gil, E. AU - Kaiser, G. AU - Muehlbauer, E.* AU - Schuermann, A.* AU - Häring, H.-U. AU - Ullrich, S. AU - Gerst, F. C1 - 53780 C2 - 45035 CY - Elsevier House, Brookvale Plaza, East Park Shannon, Co, Clare, 00000, Ireland TI - Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms. JO - PLoS ONE VL - 13 IS - 5 PB - Elsevier Ireland Ltd PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Previous studies have suggested that exposure to ionizing radiation increases the risk of ischemic heart disease (IHD). The data from the Mayak nuclear worker cohort have indicated enhanced risk for IHD incidence. The goal of this study was to elucidate molecular mechanisms of radiation-induced IHD by integrating proteomics data with a transcriptomics study on post mortem cardiac left ventricle samples from Mayak workers categorized in four radiation dose groups (0 Gy, < 100 mGy, 100-500 mGy, > 500 mGy). The proteomics data that were newly analysed here, originated from a label-free analysis of cardiac samples. The transcriptomics analysis was performed on a subset of these samples. Stepwise linear regression analyses were used to correct the age-dependent changes in protein expression, enabling the separation of proteins, the expression of which was dependent only on the radiation dose, age or both of these factors. Importantly, the majority of the proteins showed only dose-dependent expression changes. Hierarchical clustering of the proteome and transcriptome profiles confirmed the separation of control and high-dose samples. Restrictive (separate p-values) and integrative (combined p-value) approaches were used to investigate the enrichment of biological pathways. The integrative method proved superior in the validation of the key biological pathways found in the proteomics analysis, namely PPAR signalling, TCA cycle and glycolysis/gluconeogenesis. This study presents a novel, improved, and comprehensive statistical approach of analysing biological effects on a limited number of samples. AU - Papiez, A.* AU - Azimzadeh, O. AU - Azizova, T.* AU - Moseeva, M.* AU - Anastasov, N. AU - Smida, J. AU - Tapio, S. AU - Polanska, J.* C1 - 55073 C2 - 46083 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Integrative multiomics study for validation of mechanisms in radiation-induced ischemic heart disease in Mayak workers. JO - PLoS ONE VL - 13 IS - 12 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Successful breast conserving cancer surgeries come along with tumor free resection margins and account for cosmetic outcome. Positive margins increase the likelihood of tumor recurrence. Intra-operative fluorescence molecular imaging (IFMI) aims to focus surgery on malignant tissue thus substantially lowering the presence of positive margins as compared with standard techniques of breast conservation (ST). A goal of this paper is to assess the incremental number of surgeries and costs of IFMI vs. ST. METHODS: We developed a decision analytical model and applied it for an early evaluation approach. Given uncertainty we considered that IFMI might reduce the proportion of positive margins found by ST from all to none and this proportion is assumed to be reduced to 10% for the base case. Inputs included data from the literature and a range of effect estimates. For the costs of IFMI, respective cost components were added to those of ST. RESULTS: The base case reduction lowered number of surgeries (mean [95% confidence interval]) by 0.22 [0.15; 0.30] and changed costs (mean [95% confidence interval]) by €-663 [€-1,584; €50]. A tornado diagram identified the Diagnosis Related Group (DRG) costs, the proportion of positive margins of ST, the staff time saving factor and the duration of frozen section analysis (FSA) as important determinants of this cost. CONCLUSIONS: These early results indicate that IFMI may be more effective than ST and through the reduction of positive margins it is possible to save follow-up surgeries-indicating further health risk-and to save costs through this margin reduction and the avoidance of FSA. AU - Präger, M. AU - Kiechle, M.* AU - Stollenwerk, B. AU - Hinzen, C. AU - Glatz, J. AU - Vogl, M. AU - Leidl, R. C1 - 53592 C2 - 44916 CY - Po Box 211, 1000 Ae Amsterdam, Netherlands TI - Costs and effects of intra-operative fluorescence molecular imaging - A model-based, early assessment. JO - PLoS ONE VL - 13 IS - 6 PB - Elsevier Science Bv PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07: 02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer(+) T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFN gamma(+); median = 5.02%) in healthy individuals. However, MHC-multimer(+) and IFN gamma-secreting T cell frequencies showed a relatively weak correlation (r(2) = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C* 07: 02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer(+) T cells were still high (median = 6.86%) and correlated now strongly (r(2) = 0.96) with IFN gamma-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C* 07: 02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8(+) T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HL-AC* 07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HL-AC* 07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT. AU - Schlott, F.* AU - Steubl, D.* AU - Ameres, S. AU - Moosmann, A. AU - Dreher, S.* AU - Heemann, U.* AU - Hösel, V.* AU - Busch, D.H.* AU - Neuenhahn, M.* C1 - 53075 C2 - 44633 CY - San Francisco TI - Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - BackgroundYoung children are susceptible to enterovirus (EV) infections, which cause significant morbidity in this age group. However, the current knowledge regarding the epidemiology of EVs and the circulating virus strains is mostly based on viruses detected in children with severe diseases leading to contact with the health care system, while the vast reservoir of EVs that circulate in the general population is less characterized.MethodologyThe present study investigates the types and the prevalence of EVs circulating in the young children of the background population in Georgia, Colorado, and Washington State in the USA, and Germany, Sweden, and Finland in Europe.A total of 4018 stool samples, collected monthly from 300 healthy and non-hospitalized children at the age of 3 +/- 18 months in 2005 +/- 2009, were analyzed for the presence of EVs using RT-PCR, followed by sequencing of the VP1-2A region of the viral genome to type the EV(s) present. All of the children carried type HLA-DQ2 or -DQ8 alleles associated with type 1 diabetes.Principal findingsAltogether 201 children (67%) were found to be EV positive. The prevalence was much lower in Finnish children (26%) than in the children of the other counties combined (75%). Infections increased by age and showed a nadir during the winter months. Children who carried both the HLA-DQ2 and -DQ8 alleles had less infections than children who were homozygous for these alleles. Coxsackieviruses type A were the most frequently detected viruses in all geographical regions. Coxsackievirus type A4, Echovirus type 18, and Echovirus type 25 were shed for longer time periods than the other EV types.ConclusionsCompared to prevalence data from symptomatic patients requiring medical attention, this study provides a better view of EVs circulating in young children in the USA and in Europe. The observations may prove useful for the selection of strategies for designing EV vaccines in the future. The study also confirms our previous serological findings suggesting that EV infections are relatively rare in Finland. AU - Sioofy-Khojine, A.* AU - Oikarinen, S.* AU - Honkanen, H.* AU - Huhtala, H.* AU - Lehtonen, J.P.* AU - Briese, T.* AU - Hyoty, H.* AU - TEDDY Study Group (Ziegler, A.-G. AU - Beyerlein, A. AU - Hummel, M. AU - Hummel, S. AU - Knopff, A. AU - Scholz, M. AU - Stock, J. AU - Warncke, K. AU - Wendel, L. AU - Winkler, C.) C1 - 54453 C2 - 45603 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Molecular epidemiology of enteroviruses in young children at increased risk of type 1 diabetes. JO - PLoS ONE VL - 13 IS - 9 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Pulmonary rehabilitation, including aerobic exercise and strength training, improves function, such as spirometric indices, in lung disease. However, we found spirometry did not correlate with physical activity (PA) in healthy adolescents (Smith ERJ: 42(4), 2016). To address whether muscle strength did, we measured these adolescents' handgrip strength and correlated it with spirometry. METHODS: In 1846 non-smoking, non-asthmatic Germans (age 15.2 years, 47% male), we modeled spirometric indices as functions of handgrip strength by linear regression in each sex, corrected for factors including age, height, and lean body mass. RESULTS: Handgrip averaged 35.4 (SD 7.3) kg in boys, 26.6 (4.2) in girls. Spirometric volumes and flows increased linearly with handgrip. In boys each kg handgrip was associated with about 28 mL greater FEV1 and FVC; 60 mL/sec faster PEF; and 38 mL/sec faster FEF2575. Effects were 10-30% smaller in girls (all p<0.0001) and stable when Z-scores for spirometry and grip were modeled, after further correction for environment and/or other exposures, and consistent across stages of puberty. CONCLUSIONS: Grip strength was associated with spirometry in a cohort of healthy adolescents whose PA was not. Thus, research into PA's relationship with lung function should consider strength as well as total PA. Strength training may benefit healthy lungs; interventions are needed to prove causality. AU - Smith, M. AU - Standl, M. AU - Berdel, D.* AU - von Berg, A.* AU - Bauer, C.P.* AU - Schikowski, T.* AU - Koletzko, S.* AU - Lehmann, I.* AU - Krämer, U.* AU - Heinrich, J. AU - Schulz, H. C1 - 53390 C2 - 44562 TI - Handgrip strength is associated with improved spirometry in adolescents. JO - PLoS ONE VL - 13 IS - 4 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - The underlying mechanisms of Parkinson's disease are not completely revealed. Especially, early diagnostic biomarkers are lacking. To characterize early pathophysiological events, research is focusing on metabolomics. In this case-control study we investigated the metabolic profile of 31 Parkinson's disease-patients in comparison to 95 neurologically healthy controls. The investigation of metabolites in CSF was performed by a 12 Tesla SolariX Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). Multivariate statistical analysis sorted the most important biomarkers in relation to their ability to differentiate Parkinson versus control. The affected metabolites, their connection and their conversion pathways are described by means of network analysis. The metabolic profiling by FT-ICR-MS in CSF yielded in a good group separation, giving insights into the disease mechanisms. A total number of 243 metabolites showed an affected intensity in Parkinson's disease, whereas 15 of these metabolites seem to be the main biological contributors. The network analysis showed a connection to the tricarboxylic cycle (TCA cycle) and therefore to mitochondrial dysfunction and increased oxidative stress within mitochondria. The metabolomic analysis of CSF in Parkinson's disease showed an association to pathways which are involved in lipid/ fatty acid metabolism, energy metabolism, glutathione metabolism and mitochondrial dysfunction. AU - Willkommen, D. AU - Lucio, M. AU - Moritz, F. AU - Forcisi, S. AU - Kanawati, B. AU - Smirnov, K. AU - Schroeter, M.* AU - Sigaroudi, A.* AU - Schmitt-Kopplin, P. AU - Michalke, B. C1 - 54932 C2 - 45942 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa SP - e0208752 TI - Metabolomic investigations in cerebrospinal fluid of Parkinson's disease. JO - PLoS ONE VL - 13 IS - 12 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background mTOR-Is positively influence the occurrence and course of certain tumors after solid organ transplantation. The effect of mTOR-Is on the overall incidence of tumors irrespective of their origin is not entirely clear. Furthermore, conflicting data have been shown on mortality under mTOR-Is. Methods The current literature was searched for prospective randomized controlled renal transplantation trials. There were 1415 trials screened of which 13 could be included (pts. = 5924). A minimum follow-up of 24 months was mandatory for inclusion. Incidence of malignancies and patient survival was assessed in meta-analyses. Results The average follow-up of all trials was 40.6 months. Malignancy was significantly reduced under mTOR-Is compared to CNIs (RR 0.70, CI 0.49–0.99, p = 0.046). This effect remained stable when combined with CNIs (RR 0.58, CI 0.34–1.00, p = 0.05). When NMSCs were excluded the risk for malignancy remained significantly reduced under mTOR-I therapy (mono and combi) (RR 0.43, CI 0.24–0.77, p = 0.0046). Graft survival was minimally decreased under mTOR-Is (RR 0.99, CI 0.98–1.00, p = 0.054). This effect was abrogated when mTOR-Is were combined with CNIs (RR 0.99, CI 0.97–1.02, p = 0.50). Patient survival was not different (RR 1.00, CI 0.99–1.01, p = 0.54). Conclusions Posttransplant patients have a lower incidence of malignancy when treated with an mTOR-I no matter if it is used in combination with CNIs or not. This beneficial effect remains significant even when NMSCs are excluded. With currently used mTOR-I-based regimen patient and graft survival is not different compared to CNI therapies. AU - Wolf, S.* AU - Hoffmann, V. AU - Habicht, A.* AU - Kauke, T.* AU - Bucher, J.B.* AU - Schoenberg, M.H.* AU - Werner, J.* AU - Guba, M.* AU - Andrassy, J.* C1 - 53539 C2 - 44741 TI - Effects of mTOR-Is on malignancy and survival following renal transplantation: A systematic review and meta-analysis of randomized trials with a minimum follow-up of 24 months. JO - PLoS ONE VL - 13 IS - 4 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - The accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow the estimation of different bioenergetic measures by monitoring the oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of the statistical best practices for determining aggregated OCR measurements and comparisons have been lacking. Therefore, to understand how OCR behaves across different biological samples, wells, and plates, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than the intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plate variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 45% and of maximal respiration by 29%. Moreover, using positive and negative controls, we show that our statistical test outperforms the existing methods, which suffer from an excess of either false positives (within-plate methods), or false negatives (between-plate methods). Altogether, this study provides statistical good practices to support experimentalists in designing, analyzing, testing, and reporting the results of mitochondrial stress tests using this high throughput platform. AU - Yepez, V.* AU - Kremer, L.S. AU - Iuso, A. AU - Gusic, M. AU - Kopajtich, R. AU - Konarikova, E. AU - Nadel, A. AU - Wachutka, L.* AU - Prokisch, H. AU - Gagneur, J.* C1 - 53949 C2 - 45130 TI - OCR-Stats: Robust estimation and statistical testing of mitochondrial respiration activities using seahorse XF analyzer. JO - PLoS ONE VL - 13 IS - 7 PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - Background It remains controversial whether measures of general or abdominal adiposity are better risk predictors for ischemic stroke. Furthermore, so far it is unclear whether body fat mass index (BFMI) and fat free mass index (FFMI) are risk predictors for ischemic stroke. This study examined the sex-specific relevance of body mass index (BMI), BROCA Index, waist circumference (WC), waist-height ratio (WHtR), BFMI and FFMI for the development of ischemic stroke in a Caucasian population. Material and methods The prospective population-based cohort study was based on 1917 men and 1832 women (aged 50 to 74 years) who participated in the third (1994/95) or fourth (1999/2001) MONICA/KORA Augsburg survey. Subjects were free of stroke at baseline. Standardized anthropometric and bioelectric impedance measurements were obtained at baseline. Hazard ratios (HR) were estimated from Cox proportional hazard models. Results During a median follow-up of 9.3 years 128 ischemic strokes occurred in men and 81 in women, respectively. Coded as quartiles WC and WHtR were significantly associated with incident stroke in multivariable analyses in women (comparing the 4th vs. the bottom quartile), but none of the adiposity measures was significantly associated with incident stroke in multivariable adjusted analyses in men. When anthropometric measures were used as continuous variables, these findings were confirmed. After multivariable adjustment the associations between obesity measures and incident ischemic stroke were statistically significant only for WC (HR 1.39, 95% CI 1.12-1.72) and WHtR in women (HR 1.39, 95% CI 1.12-1.73) per increase of 1 standard deviation. In both sexes the measures BFMI and FFMI were no independent predictors for incident ischemic stroke. Conclusions Abdominal obesity measures are independent predictors of incident ischemic stroke in women but not in men from the general adult population. Thus, it may be of particular importance for women to prevent central obesity in order to reduce their risk of ischemic stroke. AU - Zahn, K. AU - Linseisen, J. AU - Heier, M. AU - Peters, A. AU - Thorand, B. AU - Nairz, F. AU - Meisinger, C. C1 - 52878 C2 - 44302 CY - San Francisco TI - Body fat distribution and risk of incident ischemic stroke in men and women aged 50 to 74 years from the general population. The KORA Augsburg cohort study. JO - PLoS ONE VL - 13 IS - 2 PB - Public Library Science PY - 2018 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. OBJECTIVE: Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. METHODS: In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. RESULTS: AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. CONCLUSIONS: This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis. AU - Aguilar-Pimentel, J.A. AU - Graessel, A. AU - Alessandrini, F. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Russkamp, D. AU - Chaker, A. AU - Ollert, M.* AU - Blank, S. AU - Gutermuth, J.* AU - Schmidt-Weber, C.B. C1 - 51231 C2 - 42792 CY - San Francisco TI - Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma. JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Maternal obesity has a major impact on pregnancy outcomes. There is growing evidence that maternal obesity has a negative influence on placental development and function, thereby adversely influencing offspring programming and health outcomes. However, the molecular mechanisms underlying these processes are poorly understood. We analysed ten term placenta's whole transcriptomes in obese (n = 5) and normal weight women (n = 5), using the Affymetrix microarray platform. Analyses of expression data were carried out using non-parametric methods. Hierarchical clustering and principal component analysis showed a clear distinction in placental transcriptome between obese and normal weight women. We identified 72 differentially regulated genes, with most being down-regulated in obesity (n = 61). Functional analyses of the targets using DAVID and IPA confirm the dysregulation of previously identified processes and pathways in the placenta from obese women, including inflammation and immune responses, lipid metabolism, cancer pathways, and angiogenesis. In addition, we detected new molecular aspects of obesity-derived effects on the placenta, involving the glucocorticoid receptor signalling pathway and dysregulation of several genes including CCL2, FSTL3, IGFBP1, MMP12, PRG2, PRL, QSOX1, SERPINE2 and TAC3. Our global gene expression profiling approach demonstrates that maternal obesity creates a unique in utero environment that impairs the placental transcriptome. AU - Altmäe, S.* AU - Segura, M.T.* AU - Esteban, F.J.* AU - Bartel, S.* AU - Brandi, P.* AU - Irmler, M. AU - Beckers, J. AU - Demmelmair, H.* AU - López-Sabater, C.* AU - Koletzko, B.* AU - Krauss-Etschmann, S. AU - Campoy, C.* C1 - 50512 C2 - 42385 CY - San Francisco TI - Maternal pre-pregnancy obesity is associated with altered placental transcriptome. JO - PLoS ONE VL - 12 IS - 1 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP- N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP- N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP- N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety. AU - Appelman, M.D.* AU - Chakraborty, A. AU - Protzer, U. AU - McKeating, J.A.* AU - van de Graaf, S.F.J.* C1 - 50773 C2 - 42872 CY - San Francisco TI - N-glycosylation of the Na+-taurocholate cotransporting polypeptide (NTCP) determines its trafficking and stability and is required for hepatitis B Vvrus infection. JO - PLoS ONE VL - 12 IS - 1 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - In Germany, orthopedic workforce planning relies on population-to-provider-ratios represented by the 'official degree of care provision'. However, with geographic information systems (GIS), more sophisticated measurements are available. By utilizing GIS-based technologies we analyzed the current state of demand and supply of the orthopedic workforce in Germany (orthopedic accessibility) with the integrated Floating Catchment Area method. The analysis of n = 153,352,220 distances revealed significant geographical variations on national scale: 5,617,595 people (6.9% of total population) lived in an area with significant low orthopedic accessibility (average z-score = -4.0), whereas 31,748,161 people (39.0% of total population) lived in an area with significant high orthopedic accessibility (average z-score = 8.0). Accessibility was positively correlated with the degree of urbanization (r = 0.49; p<0.001) and the official degree of care provision (r = 0.33; p<0.001) and negatively correlated with regional social deprivation (r = -0.47; p<0.001). Despite advantages of simpler measures regarding implementation and acceptance in health policy, more sophisticated measures of accessibility have the potential to reduce costs as well as improve health care. With this study, significant geographical variations were revealed that show the need to reduce oversupply in less deprived urban areas in order to enable adequate care in more deprived rural areas. AU - Bauer, J.* AU - Müller, P.* AU - Maier, W. AU - Groneberg, D.* C1 - 50475 C2 - 42304 CY - San Francisco TI - Orthopedic workforce planning in Germany – an analysis of orthopedic accessibility. JO - PLoS ONE VL - 12 IS - 2 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states. Escherichia coli contains two LytS/LytTR-type histidine kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT) and YpdA/ YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment. AU - Behr, S.* AU - Brameyer, S.* AU - Witting, M. AU - Schmitt-Kopplin, P. AU - Jung, K.* C1 - 51738 C2 - 43423 CY - San Francisco TI - Comparative analysis of LytS/LytTR-type histidine kinase/response regulator systems in γ-proteobacteria. JO - PLoS ONE VL - 12 IS - 8 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Objective Dipeptidyl-peptidase 4 (DPP-4) cleaves and inactivates the insulinotropic hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide, collectively termed incretins. DPP-4 inhibitors entered clinical practice as approved therapeutics for type-2 diabetes in 2006. However, inter-individual variance in the responsiveness to DPP-4 inhibitors was reported. Thus, we asked whether genetic variation in the DPP4 gene affects incretin levels, insulin secretion, and glucose tolerance in participants of the TUbingen Family study for type-2 diabetes (TUF). Research design and methods Fourteen common (minor allele frequencies >= 0.05) DPP4 tagging single nucleotide polymorphisms (SNPs) were genotyped in 1,976 non-diabetic TUF participants characterized by oral glucose tolerance tests and bioimpedance measurements. In a subgroup of 168 subjects, plasma incretin levels were determined. Results We identified a variant, i.e., SNP rs6741949, in intron 2 of the DPP4 gene that, after correction for multiple comparisons and appropriate adjustment, revealed a significant genotype-body fat interaction effect on glucose-stimulated plasma GLP-1 levels (p = 0.0021). Notably, no genotype-BMI interaction effects were detected (p = 0.8). After stratification for body fat content, the SNP negatively affected glucose-stimulated GLP-1 levels (p = 0.0229), insulin secretion (p = 0.0061), and glucose tolerance (p = 0.0208) in subjects with high body fat content only. Conclusions A common variant, i.e., SNP rs6741949, in the DPP4 gene interacts with body adiposity and negatively affects glucose-stimulated GLP-1 levels, insulin secretion, and glucose tolerance. Whether this SNP underlies the reported inter-individual variance in responsiveness to DPP-4 inhibitors, at least in subjects with high body fat content, remains to be shown. AU - Böhm, A. AU - Wagner, R. AU - Machicao, F. AU - Holst, J.J.* AU - Gallwitz, B.* AU - Stefan, N. AU - Fritsche, A. AU - Häring, H.-U. AU - Staiger, H. C1 - 51745 C2 - 43381 CY - San Francisco TI - DPP4 gene variation affects GLP-1 secretion, insulin secretion, and glucose tolerance in humans with high body adiposity. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Many natural proteins are, as a whole or in part, intrinsically disordered. Frequently, such intrinsically disordered regions (IDRs) undergo a transition to a defined and often helical conformation upon binding to partner molecules. The intrinsic propensity of an IDR sequence to fold into a helical conformation already in the absence of a binding partner can have a decisive influence on the binding process and affinity. Using a combination of NMR spectroscopy and molecular dynamics (MD) simulations we have investigated the tendency of regions of Axin-1, an intrinsically disordered scaffolding protein of the WNT signaling pathway, to form helices in segments interacting with binding partners. Secondary chemical shifts from NMR measurements show an increased helical population in these regions. Systematic application of MD advanced sampling approaches on peptide segments of Axin-1 reproduces the experimentally observed tendency and allows insights into the distribution of segment conformations and free energies of helix formation. The results, however, were found to dependent on the force field water model. Recent water models specifically designed for IDRs significantly reduce the predicted helical content and do not improve the agreement with experiment. AU - Bomblies, R.* AU - Luitz, M.P.* AU - Scanu, S.* AU - Madl, T. AU - Zacharias, M.* C1 - 50897 C2 - 42641 CY - San Francisco TI - Transient helicity in intrinsically disordered Axin-1 studied by NMR spectroscopy and molecular dynamics simulations. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Glatiramer acetate is used therapeutically in multiple sclerosis but also known for adverse effects including elevated coronary artery disease (CAD) risk. The mechanisms underlying the cardiovascular side effects of the medication are unclear. Here, we made use of the chromosomal variation in the genes that are known to be affected by glatiramer treatment. Focusing on genes and gene products reported by drug-gene interaction database to interact with glatiramer acetate we explored a large meta-analysis on CAD genome-wide association studies aiming firstly, to investigate whether variants in these genes also affect cardiovascular risk and secondly, to identify new CAD risk genes. We traced association signals in a 200-kb region around genomic positions of genes interacting with glatiramer in up to 60 801 CAD cases and 123 504 controls. We validated the identified association in additional 21 934 CAD cases and 76 087 controls. We identified three new CAD risk alleles within the TGFB1 region on chromosome 19 that independently affect CAD risk. The lead SNP rs12459996 was genome-wide significantly associated with CAD in the extended meta-analysis (odds ratio 1.09, p = 1.58x10(-12)). The other two SNPs at the locus were not in linkage disequilibrium with the lead SNP and by a conditional analysis showed p-values of 4.05 x 10(-10) and 2.21 x 10(-6). Thus, studying genes reported to interact with glatiramer acetate we identified genetic variants that concordantly with the drug increase the risk of CAD. Of these, TGFB1 displayed signal for association. Indeed, the gene has been associated with CAD previously in both in vivo and in vitro studies. Here we establish genome-wide significant association with CAD in large human samples. AU - Braenne, I.* AU - Zeng, L.* AU - Willenborg, C.* AU - Tragante, V.* AU - Kessler, T.* AU - Willer, C.J.* AU - Laakso, M.* AU - Wallentin, L.* AU - Franks, P.W.* AU - Salomaa, V.* AU - Dehghan, A.* AU - CARDIoGRAM Consortium (Meitinger, T.) AU - Samani, N.J.* AU - Asselbergs, F.W.* AU - Erdmann, J.* AU - Schunkert, H.* C1 - 51826 C2 - 43393 CY - San Francisco TI - Genomic correlates of glatiramer acetate adverse cardiovascular effects lead to a novel locus mediating coronary risk. JO - PLoS ONE VL - 12 IS - 8 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - X-ray microbeam radiotherapy can potentially widen the therapeutic window due to a geometrical redistribution of the dose. However, high requirements on photon flux, beam collimation, and system stability restrict its application mainly to large-scale, cost-intensive synchrotron facilities. With a unique laser-based Compact Light Source using inverse Compton scattering, we investigated the translation of this promising radiotherapy technique to a machine of future clinical relevance. We performed in vitro colony-forming assays and chromosome aberration tests in normal tissue cells after microbeam irradiation compared to homogeneous irradiation at the same mean dose using 25 keV X-rays. The microplanar pattern was achieved with a tungsten slit array of 50 μm slit size and a spacing of 350 μm. Applying microbeams significantly increased cell survival for a mean dose above 2 Gy, which indicates fewer normal tissue complications. The observation of significantly less chromosome aberrations suggests a lower risk of second cancer development. Our findings provide valuable insight into the mechanisms of microbeam radiotherapy and prove its applicability at a compact synchrotron, which contributes to its future clinical translation. AU - Burger, K.* AU - Ilicic, K.* AU - Dierolf, M.* AU - Günther, B.* AU - Walsh, D.W.M.* AU - Schmid, E.* AU - Eggl, E.* AU - Achterhold, K.* AU - Gleich, B.* AU - Combs, S.E. AU - Molls, M. AU - Schmid, T.E. AU - Pfeiffer, F.* AU - Wilkens, J.J. C1 - 52223 C2 - 43813 CY - San Francisco TI - Increased cell survival and cytogenetic integrity by spatial dose redistribution at a compact synchrotron X-ray source. JO - PLoS ONE VL - 12 IS - 10 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Participation in regular physical activity is associated with a multitude of health benefits across the life course. However, many people fail to meet PA recommendations. Despite a plethora of studies, the evidence regarding the environmental (physical) determinants of physical activity remains inconclusive. OBJECTIVE: To identify the physical environmental determinants that influence PA across the life course. METHODS: An online systematic literature search was conducted using MEDLINE, ISI Web of Science, Scopus and SPORTDiscus. The search was limited to studies published in English (January 2004 to April 2016). Only systematic literature reviews (SLRs) and meta-analyses (MAs) of observational studies, that investigated the association between physical determinants and physical activity outcomes, were eligible for inclusion. The extracted data were assessed on the importance of determinants, strength of evidence and methodological quality. RESULTS: The literature search identified 28 SLRs and 3 MAs on 67 physical environmental characteristics potentially related to physical activity that were eligible for inclusion. Among preschool children, a positive association was reported between availability of backyard space and outdoor toys/equipment in the home and overall physical activity. The availability of physical activity programs and equipment within schools, and neighbourhood features such as pedestrian and cyclist safety structure were positively associated with physical activity in children and adolescents. Negative street characteristics, for example, lack of sidewalks and streetlights, were negatively associated with physical activity in adults. Inconsistent associations were reported for the majority of reviewed determinants in adults. CONCLUSION: This umbrella SLR provided a comprehensive overview of the physical environment determinants of physical activity across the life course and has highlighted, particularly amongst youth, a number of key determinants that may be associated with overall physical activity. Given the limited evidence drawn mostly from cross-sectional studies, longitudinal studies are needed to further explore these associations. AU - Carlin, A.* AU - Perchoux, C.* AU - Puggina, A.* AU - Aleksovska, K.* AU - Buck, C.* AU - Burns, C.* AU - Cardon, G.* AU - Chantal, S.* AU - Ciarapica, D.* AU - Condello, G.* AU - Coppinger, T.C.* AU - Cortis, C.* AU - D'Haese, S.* AU - De Craemer, M.* AU - di Blasio, A.M.* AU - Hansen, S.* AU - Iacoviello, L.* AU - Issartel, J.* AU - Izzicupo, P.* AU - Jaeschke, L.* AU - Kanning, M.* AU - Kennedy, A.* AU - Lakerveld, J.* AU - Chun Man Ling, F.* AU - Luzak, A. AU - Napolitano, G.* AU - Nazare, J.A.* AU - Pischon, T.* AU - Polito, A.* AU - Sannella, A.* AU - Schulz, H.* AU - Sohun, R.* AU - Steinbrecher, A.* AU - Schlicht, W.* AU - Ricciardi, W.* AU - MacDonncha, C.* AU - Capranica, L.* AU - Boccia, S.* C1 - 51693 C2 - 43417 CY - San Francisco TI - A life course examination of the physical environmental determinants of physical activity behaviour: A "Determinants of Diet and Physical Activity" (DEDIPAC) umbrella systematic literature review. JO - PLoS ONE VL - 12 IS - 8 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Assessing the relationship between lung cancer and metabolic conditions is challenging because of the confounding effect of tobacco. Mendelian randomization (MR), or the use of genetic instrumental variables to assess causality, may help to identify the metabolic drivers of lung cancer. METHODS AND FINDINGS: We identified genetic instruments for potential metabolic risk factors and evaluated these in relation to risk using 29,266 lung cancer cases (including 11,273 adenocarcinomas, 7,426 squamous cell and 2,664 small cell cases) and 56,450 controls. The MR risk analysis suggested a causal effect of body mass index (BMI) on lung cancer risk for two of the three major histological subtypes, with evidence of a risk increase for squamous cell carcinoma (odds ratio (OR) [95% confidence interval (CI)] = 1.20 [1.01-1.43] and for small cell lung cancer (OR [95%CI] = 1.52 [1.15-2.00]) for each standard deviation (SD) increase in BMI [4.6 kg/m2]), but not for adenocarcinoma (OR [95%CI] = 0.93 [0.79-1.08]) (Pheterogeneity = 4.3x10-3). Additional analysis using a genetic instrument for BMI showed that each SD increase in BMI increased cigarette consumption by 1.27 cigarettes per day (P = 2.1x10-3), providing novel evidence that a genetic susceptibility to obesity influences smoking patterns. There was also evidence that low-density lipoprotein cholesterol was inversely associated with lung cancer overall risk (OR [95%CI] = 0.90 [0.84-0.97] per SD of 38 mg/dl), while fasting insulin was positively associated (OR [95%CI] = 1.63 [1.25-2.13] per SD of 44.4 pmol/l). Sensitivity analyses including a weighted-median approach and MR-Egger test did not detect other pleiotropic effects biasing the main results. CONCLUSIONS: Our results are consistent with a causal role of fasting insulin and low-density lipoprotein cholesterol in lung cancer etiology, as well as for BMI in squamous cell and small cell carcinoma. The latter relation may be mediated by a previously unrecognized effect of obesity on smoking behavior. AU - Carreras-Torres, R.* AU - Johansson, M.* AU - Haycock, P.C.* AU - Wade, K.H.* AU - Relton, C.L.* AU - Martin, R.M.* AU - Davey Smith, G.* AU - Albanes, D.* AU - Aldrich, M.C.* AU - Andrew, A.S.* AU - Arnold, S.M.* AU - Bickeböller, H.* AU - Bojesen, S.E.* AU - Brunnström, H.* AU - Manjer, J.* AU - Brüske, I. AU - Caporaso, N.E.* AU - Chen, C.* AU - Christiani, D.C.* AU - Christian, W.J.* AU - Doherty, J.A.* AU - Duell, E.J.* AU - Field, J.K.* AU - Davies, M.P.A.* AU - Marcus, M.W.* AU - Goodman, G.E.* AU - Grankvist, K.* AU - Haugen, A.* AU - Hong, Y.C.* AU - Kiemeney, L.A.* AU - van der Heijden, E.H.F.M.* AU - Kraft, P.* AU - Johansson, M.B.* AU - Lam, S.* AU - Landi, M.T.* AU - Lazarus, P.* AU - Le Marchand, L.* AU - Liu, G.* AU - Melander, O.* AU - Park, S.L.* AU - Rennert, G.* AU - Risch, A.* AU - Haura, E.B.* AU - Scelo, G.* AU - Zaridze, D.* AU - Mukeriya, A.* AU - Savić, M.* AU - Lissowska, J.* AU - Swiatkowska, B.* AU - Janout, V.* AU - Holcatova, I.* AU - Mates, D.* AU - Schabath, M.B.* AU - Shen, H.* AU - Tardón, A.* AU - Teare, M.D.* AU - Woll, P.* AU - Tsao, M.S.* AU - Wu, X.* AU - Yuan, J.M.* AU - Hung, R.J.* AU - Amos, C.I.* AU - Mckay, J.* AU - Brennan, P.* C1 - 51276 C2 - 43010 CY - San Francisco TI - Obesity, metabolic factors and risk of different histological types of lung cancer: A Mendelian randomization study. JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Low levels of physical activity (PA) are reported to contribute to the occurrence of non-communicable diseases over the life course. Although psychological factors have been identified as an important category concerning PA behavior, knowledge on psychological determinants of PA is still inconclusive. Therefore, the aim of this umbrella systematic literature review (SLR) was to summarize and synthesize the scientific evidence on psychological determinants of PA behavior across the life course. A systematic online search was conducted on MEDLINE, ISI Web of Science, Scopus, and SPORTDiscus databases. The search was limited to studies published in English from January 2004 to April 2016. SLRs and meta-analyses (MAs) of observational studies investigating the association of psychological variables and PA were considered eligible. Extracted data were evaluated based on importance of determinants, strength of evidence, and methodological quality. The full protocol is available from PROSPERO (Record ID: CRD42015010616). Twenty reviews (14 SLRs and 6 MAs), mostly of moderate methodological quality, were found eligible. Convincing evidence was found for self-efficacy (positive association with PA) in children and adolescents, and stress (negative association with PA) regardless of age. Most of the evidence revealing an association between psychological determinants and PA is probable and limited, mainly due to differences in the definition of PA and of psychological determinants across reviews. Thus, scholars are urged to reach a consensus on clear definitions of relevant psychological determinants of PA, subsuming cultural biases and allowing the possibility to obtain clear interpretations and generalizability of findings. Finally, most psychological determinants should be considered within a larger framework of other multi-level determinants that may interact or mediate some of the effects. AU - Cortis, C.* AU - Puggina, A.* AU - Pesce, C.* AU - Aleksovska, K.* AU - Buck, C.* AU - Burns, C.* AU - Cardon, G.* AU - Carlin, A.* AU - Simon, C.* AU - Ciarapica, D.* AU - Condello, G.* AU - Coppinger, T.C.* AU - D'Haese, S.* AU - De Craemer, M.* AU - di Blasio, A.M.* AU - Hansen, S.* AU - Iacoviello, L.* AU - Issartel, J.* AU - Izzicupo, P.* AU - Jaeschke, L.* AU - Kanning, M.* AU - Kennedy, A.* AU - Ling, F.C.M.* AU - Luzak, A. AU - Napolitano, G.* AU - Nazare, J.A.* AU - O'Donoghue, G.* AU - Perchoux, C.* AU - Pischon, T.* AU - Polito, A.* AU - Sannella, A.* AU - Schulz, H. AU - Sohun, R.* AU - Steinbrecher, A.* AU - Schlicht, W.* AU - Ricciardi, W.* AU - Castellani, L.* AU - MacDonncha, C.* AU - Capranica, L.* AU - Boccia, S.* C1 - 51729 C2 - 43463 CY - San Francisco TI - Psychological determinants of physical activity across the life course: A "DEterminants of DIet and Physical ACtivity" (DEDIPAC) umbrella systematic literature review. JO - PLoS ONE VL - 12 IS - 8 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Chronic low back pain (CLBP) is one of the most common medical conditions, ranking as the greatest contributor to global disability and accounting for huge societal costs based on the Global Burden of Disease 2010 study. Large genetic and -omics studies provide a promising avenue for the screening, development and validation of biomarkers useful for personalized diagnosis and treatment (precision medicine). Multicentre studies are needed for such an effort, and a standardized and homogeneous approach is vital for recruitment of large numbers of participants among different centres (clinical and laboratories) to obtain robust and reproducible results. To date, no validated standard operating procedures (SOPs) for genetic/-omics studies in chronic pain have been developed. In this study, we validated an SOP model that will be used in the multicentre (5 centres) retrospective "PainOmics" study, funded by the European Community in the 7th Framework Programme, which aims to develop new biomarkers for CLBP through three different -omics approaches: genomics, glycomics and activomics. The SOPs describe the specific procedures for (1) blood collection, (2) sample processing and storage, (3) shipping details and (4) cross-check testing and validation before assays that all the centres involved in the study have to follow. Multivariate analysis revealed the absolute specificity and homogeneity of the samples collected by the five centres for all genetics, glycomics and activomics analyses. The SOPs used in our multicenter study have been validated. Hence, they could represent an innovative tool for the correct management and collection of reliable samples in other large-omics-based multicenter studies. AU - Dagostino, C.* AU - De Gregori, M.* AU - Gieger, C. AU - Manz, J. AU - Gudelj, I.* AU - Lauc, G.* AU - Divizia, L.* AU - Wang, W.* AU - Sim, M.* AU - Pemberton, I.K.* AU - MacDougall, J.* AU - Williams, F.* AU - Van Zundert, J.* AU - Primorac, D.* AU - Aulchenko, Y.* AU - Kapural, L.* AU - Allegri, M.* C1 - 51019 C2 - 42690 CY - San Francisco TI - Validation of standard operating procedures in a multicenter retrospective study to identify -omics biomarkers for chronic low back pain. JO - PLoS ONE VL - 12 IS - 5 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases. AU - Echtler, K.* AU - Konrad, I.* AU - Lorenz, M.* AU - Schneider, S.* AU - Hofmaier, S.* AU - Plenagl, F.* AU - Stark, K.* AU - Czermak, T.* AU - Tirniceriu, A.* AU - Eichhorn, M.E.* AU - Walch, A.K. AU - Enders, G.* AU - Massberg, S.* AU - Schulz, C.* C1 - 50624 C2 - 42449 CY - San Francisco TI - Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited. METHODS: Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons. RESULTS: We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype. CONCLUSION: Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system. AU - Engel, M. AU - Endesfelder, D. AU - Schloter-Hai, B. AU - Kublik, S. AU - Granitsiotis, M.S. AU - Boschetto, P.* AU - Stendardo, M.* AU - Barta, I.* AU - Döme, B.* AU - Deleuze, J.F.* AU - Boland, A.* AU - Müller-Quernheim, J.* AU - Prasse, A.* AU - Welte, T.* AU - Hohlfeld, J.* AU - Subramanian, D.* AU - Parr, D.* AU - Gut, I.G.* AU - Greulich, T.* AU - Koczulla, A.R.* AU - Nowinski, A.* AU - Gorecka, D.* AU - Singh, D.* AU - Gupta, S.* AU - Brightling, C.E.* AU - Hoffmann, H.* AU - Frankenberger, M. AU - Hofer, T.P. AU - Burggraf, D. AU - Heiss-Neumann, M.S. AU - Ziegler-Heitbrock, L. AU - Schloter, M. AU - zu Castell, W. C1 - 51524 C2 - 43273 CY - San Francisco TI - Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Recent research suggested a metabolic implication of osteocalcin (OCN) in e.g. insulin sensitivity or steroid production. We used an untargeted metabolomics approach by analyzing plasma and urine samples of 931 participants using mass spectrometry to reveal further metabolic actions of OCN. Several detected relations between OCN and metabolites were strongly linked to renal function, however, a number of associations remained significant after adjustment for renal function. Intermediates of proline catabolism were associated with OCN reflecting the implication in bone metabolism. The association to kynurenine points towards a pro-inflammatory state with increasing OCN. Inverse relations with intermediates of branch-chained amino acid metabolism suggest a link to energy metabolism. Finally, urinary surrogate markers of smoking highlight its adverse effect on OCN metabolism. In conclusion, the present study provides a read-out of metabolic actions of OCN. However, most of the associations were weak arguing for a limited role of OCN in whole-body metabolism. AU - Entenmann, L.* AU - Pietzner, M.* AU - Artati, A. AU - Hannemann, A.* AU - Henning, A.K.* AU - Kastenmüller, G. AU - Völzke, H.* AU - Nauck, M.* AU - Adamski, J. AU - Wallaschofski, H.* AU - Friedrich, N.* C1 - 51908 C2 - 43594 CY - San Francisco TI - Comprehensive metabolic characterization of serum osteocalcin action in a large non-diabetic sample. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Aims Dilated cardiomyopathy (DCM) is an important cause of heart failure with a strong familial component. We performed an exome-wide array-based association study (EWAS) to assess the contribution of missense variants to sporadic DCM. Methods and results 116,855 single nucleotide variants (SNVs) were analyzed in 2796 DCM patients and 6877 control subjects from 6 populations of European ancestry. We confirmed two previously identified associations with SNVs in BAG3 and ZBTB17 and discovered six novel DCM-associated loci (Q-value<0.01). The lead-SNVs at novel loci are common and located in TTN, SLC39A8, MLIP, FLNC, ALPK3 and FHOD3. In silico fine mapping identified HSPB7 as the most likely candidate at the ZBTB17 locus. Rare variant analysis (MAF<0.01) demonstrated significant association for TTN variants only (P = 0.0085). All candidate genes but one (SLC39A8) exhibit preferential expression in striated muscle tissues and mutations in TTN, BAG3, FLNC and FHOD3 are known to cause familial cardiomyopathy. We also investigated a panel of 48 known cardiomyopathy genes. Collectively, rare (n = 228, P = 0.0033) or common (n = 36, P = 0.019) variants with elevated in silico severity scores were associated with DCM, indicating that the spectrum of genes contributing to sporadic DCM extends beyond those identified here. Conclusion We identified eight loci independently associated with sporadic DCM. The functions of the best candidate genes at these loci suggest that proteostasis regulation might play a role in DCM pathophysiology. AU - Esslinger, U.B.* AU - Garnier, S.* AU - Korniat, A.* AU - Proust, C.* AU - Kararigas, G.* AU - Müller-Nurasyid, M. AU - Empana, J.* AU - Morley, M.P.* AU - Perret, C.* AU - Stark, K.* AU - Bick, A.G.* AU - Prasad, S.K.* AU - Kriebel, J. AU - Li, J.* AU - Tiret, L.* AU - Strauch, K. AU - O'Regan, D.P.* AU - Marguiles, K.B.* AU - Seidman, J.G.* AU - Boutouyrie, P.* AU - Lacolley, P.* AU - Jouven, X.* AU - Hengstenberg, C.* AU - Komajda, M.* AU - Hakonarson, H.H.* AU - Isnard, R.* AU - Arbustini, E.* AU - Grallert, H. AU - Cook, S.A.* AU - Seidman, C.E.* AU - Regitz-Zagrosek, V.* AU - Cappola, T.P.* AU - Charron, P.* AU - Cambien, F.* AU - Villard, E.* C1 - 50761 C2 - 42566 CY - San Francisco TI - Exome-wide association study reveals novel susceptibility genes to sporadic dilated cardiomyopathy. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - AIMS/HYPOTHESIS: Low physical fitness (PF) is a risk factor for type 2 diabetes mellitus (T2D). Women with a history of gestational diabetes (GDM) are at risk for T2D at a young age, but the role of PF in this population is not clear. PF has also been found to correlate inversely with plasma leptin in previous studies. Here, we examine whether women who had GDM have lower PF than women after a normoglycemic pregnancy and, second, whether PF is associated with plasma leptin, independently of body fat mass. METHODS: Cross-sectional analysis of 236 participants in the PPSDiab Study (cohort study of women 3-16 months after delivery, 152 after gestational diabetes (pGDM), 84 after normoglycemic pregnancy (control subjects); consecutively recruited 2011-16); medical history, physical examination with bioelectrical impedance analysis (BIA), whole body magnetic resonance imaging (MRI) (n = 154), 5-point oral glucose tolerance test, cardiopulmonary exercise testing, clinical chemistry including fasting plasma leptin; statistical analysis with Mann-Whitney U and t -test, Spearman correlation coefficient, multiple linear regression. RESULTS: Women pGDM had lower maximally achieved oxygen uptake (VO2peak/kg: 25.7(21.3-29.9) vs. 30.0(26.6-34.1)ml/min/kg; total VO2peak: 1733(1552-2005) vs. 1970(1767-2238)ml/min; p<0.0001 for both), and maximum workload (122.5(105.5-136.5) vs. 141.0(128.5-159.5)W; p<0.0001). Fasting plasma leptin correlated inversely with PF (VO2peak/kg ρ = -0.72 p<0.0001; VO2peak ρ = -0.16 p = 0.015; max. load ρ = -0.35 p<0.0001). These associations remained significant with adjustment for body mass index, or for body fat mass (BIA and MRI). CONCLUSIONS/INTERPRETATION: Women with a recent history of GDM were less fit than control subjects. Low PF may therefore contribute to the risk for T2D after GDM. This should be tested in intervention studies. Low PF also associated with increased leptin levels-independently of body fat. PF may therefore influence leptin levels and signaling. This hypothesis requires further investigation. AU - Gar, C. AU - Rottenkolber, M. AU - Grallert, H. AU - Banning, F. AU - Freibothe, I. AU - Sacco, V. AU - Wichmann, C. AU - Reif, S. AU - Potzel, A. AU - Dauber, V. AU - Schendell, C. AU - Sommer, N.N.* AU - Wolfarth, B.* AU - Seissler, J. AU - Lechner, A. AU - Ferrari, U. C1 - 51303 C2 - 43053 CY - San Francisco TI - Physical fitness and plasma leptin in women with recent gestational diabetes. JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background Climate change affects human health. The respective consequences are predicted to increase in the future. Patients with chronic lung disease are particularly vulnerable to the involved environmental alterations. However, their subjective perception and reactions to these alterations remain unknown. Methods In this pilot study, we surveyed 172 adult patients who underwent pulmonary rehabilitation and 832 adult tourists without lung disease in the alpine region about their perception of being affected by climate change and their potential reaction to specific consequences. The patients' survey also contained the COPD Assessment Test (CAT) to rate the severity of symptoms. Results Most of the patients stated asthma (73.8%), COPD (9.3%) or both (11.0%) as underlying disease while 5.8% suffered from other chronic lung diseases. Patients and tourists feel equally affected by current climate change in general, while allergic subjects in both groups feel significantly more affected (p = 0.04). The severity of symptoms assessed by CAT correlates with the degree of feeling affected (p< 0.01). The main disturbing consequences for patients are decreased air quality, increasing numbers of ticks and mosquitos and a rising risk for allergy and extreme weather events such as thunderstroms, while tourists are less disturbed by these factors. Increasing number of heat-days is of little concern to both groups. Conclusion Overall patients are more sensitive to health-related consequences of climate change. Yet, the hazard of heat-days seems underestimated and awareness should be raised. AU - Götschke, J.* AU - Mertsch, P.* AU - Bischof, M.* AU - Kneidinger, N.* AU - Matthes, S. AU - Renner, E.D. AU - Traidl-Hoffmann, C. AU - Duchna, H.W.* AU - Behr, J.* AU - Schmude, J.* AU - Huber, R.M.* AU - Milger, K.* C1 - 52251 C2 - 43797 CY - San Francisco TI - Perception of climate change in patients with chronic lung disease. JO - PLoS ONE VL - 12 IS - 10 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Increased levels of systemic vascular endothelial growth factors (VEGFs) in patients with diabetes are associated with increased risk of microvessel disease. On the other hand, low VEGF levels after intravitreal antibody application may be associated with acute cardiovascular complications and treatment failure. Individual levels of systemic VEGF vary in a wide range depending on analytical methods and quality of diabetes control. So far only limited information exists on intraindividual fluctuations over longer periods and circadian rhythms. We analysed the intraindividual variance of VEGF-A, VEGF-C and placental growth factor (PLGF) in CTAD (citrate-theophylline-adenine-dipyridamol) plasma as well as VEGF-A in serum over a period of 6 months in patients with stable controlled type 2 diabetes (10 M, 10 F) and age and sex matched subjects with normal glucose tolerance (NGT). Furthermore, circadian levels of VEGFs were controlled hourly from 7: 30 a. m. to 7: 30 p. m. under standardized metabolic ward conditions. In addition, the relationship to metabolic, hormonal and inflammatory biomarkers was analyzed. VEGF-A, VEGF-C and PLGF remained stable in plasma and VEGF-A in serum over 6 months in both groups. No circadian change was observed in VEGF-A serum and plasma concentrations. A minor decrease of VEGF-C plasma levels was evident after 5 p. m. in both groups and a significant peak of PLGF concentrations occurred after lunch, which was more pronounced in T2DM. In multivariate analysis, only serum VEGF-A correlated to diabetes duration, whereas VEGF-C only correlated to HbA1c and fasting blood glucose. We did not observe significant intraindividual variances for VEGF-A in serum and VEGF-A, VEGF-C and PLGF in CTAD plasma over a period of 6 months. Taken together, a single morning measurement of systemic VEGF levels after 7: 30 am appears to be a reliable parameter for the individual risk associated with abnormal VEGF concentrations in blood. AU - Hanefeld, M.* AU - Engelmann, K.* AU - Appelt, D.* AU - Sandner, D.* AU - Weigmann, I.* AU - Ganz, X.* AU - Pistrosch, F.* AU - Koehler, C.M.* AU - Gasparic, A.* AU - Birkenfeld, A.L. C1 - 52208 C2 - 43833 CY - San Francisco TI - Intra-individual variability and circadian rhythm of vascular endothelial growth factors in subjects with normal glucose tolerance and type 2 diabetes. JO - PLoS ONE VL - 12 IS - 10 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background/Objective Despite the relevance of pancreatic fat content in the development of metabolic diseases, its association with impaired glucose metabolism, diabetes, and other adipose tissue compartments remains unclear. Thus, we determined differences in pancreatic fat content by magnetic resonance imaging (MRI) between subjects with prediabetes, diabetes, and normal controls in a cohort from the general population. Methods Subjects without history of cardiovascular disease with established diabetes or prediabetes as well as normal controls were included and underwent whole-body MRI on a 3T scanner. Pancreatic fat content was quantified by measuring the proton-density fat fraction (PDFFpanc) using a 3D multi-echo GRE sequence (increment: 1.23 ms, 6 echoes) by placing ROIs in the pancreatic head, body, and tail by independent readers. In addition, hepatic fat content as well as abdominal subcutaneous and visceral adipose tissue (SAT and VAT) were measured by multi-echo GRE and 3D 2-point volume-interpolated DIXON MRI, respectively. Univariate and multivariate analyses were employed to determine associations. Results A total of 385 subjects were included in the analysis (median age: 57 years, 58.2% males), of them 53 were classified as subjects with diabetes, 95 as prediabetes, and 237 as controls (13.8%, 24.7%, and 61.6%; respectively). The median PDFFpanc was 5.2% [IQR 3.3±9.4], and significantly higher in subjects with prediabetes and diabetes as compared to controls (PDFFpanc: 6.2% [IQR: 3.5±12] vs. 8.6% [IQR: 4.3±17.5] vs. 4.9% [3.1±7.4], p<0.001, respectively). After adjusting for age, gender and BMI the association was attenuated (all p>0.12). While in univariate analysis BMI, PDFFhepatic, SAT and VAT were associated with PDFFpanc (all p<0.05), only VAT predicted PDFFpanc independently (β: 0.02, 95%-confidence interval: 0.01±0.04, p<0.001). Conclusion While pancreatic fat content differs significantly between subjects with prediabetes, diabetes and controls, this association may be confounded by age, gender, and the amount of VAT in this cross-sectional study. AU - Heber, S.D.* AU - Hetterich, H.* AU - Lorbeer, R.* AU - Bayerl, C.* AU - Machann, J. AU - Auweter, S.* AU - Storz, C.* AU - Schlett, C.L.* AU - Nikolaou, K.* AU - Reiser, M.* AU - Peters, A. AU - Bamberg, F.* C1 - 51268 C2 - 42955 TI - Pancreatic fat content by magnetic resonance imaging in subjects with prediabetes, diabetes, and controls from a general population without cardiovascular disease. JO - PLoS ONE VL - 12 IS - 5 PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that V L fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms. AU - Hora, M. AU - Sarkar, R. AU - Morris, V.K. AU - Xue, K. AU - Prade, E.* AU - Harding, E.* AU - Buchner, J.* AU - Reif, B. C1 - 51667 C2 - 43395 CY - San Francisco TI - MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - In the past, short protein-coding genes were often disregarded by genome annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of annotated genes have usually been interpreted to indicate either ncRNA or pervasive transcription. Therefore, in addition to the transcriptome, the translatome (RIBOseq) of the enteric pathogen Escherichia coli O157:H7 strain Sakai was determined at two optimal growth conditions and a severe stress condition combining low temperature and high osmotic pressure. All intergenic open reading frames potentially encoding a protein of ≥ 30 amino acids were investigated with regard to coverage by transcription and translation signals and their translatability expressed by the ribosomal coverage value. This led to discovery of 465 unique, putative novel genes not yet annotated in this E. coli strain, which are evenly distributed over both DNA strands of the genome. For 255 of the novel genes, annotated homologs in other bacteria were found, and a machine-learning algorithm, trained on small protein-coding E. coli genes, predicted that 89% of these translated open reading frames represent bona fide genes. The remaining 210 putative novel genes without annotated homologs were compared to the 255 novel genes with homologs and to 250 short annotated genes of this E. coli strain. All three groups turned out to be similar with respect to their translatability distribution, fractions of differentially regulated genes, secondary structure composition, and the distribution of evolutionary constraint, suggesting that both novel groups represent legitimate genes. However, the machine-learning algorithm only recognized a small fraction of the 210 genes without annotated homologs. It is possible that these genes represent a novel group of genes, which have unusual features dissimilar to the genes of the machine-learning algorithm training set. AU - Hücker, S.M.* AU - Ardern, Z.* AU - Goldberg, T.* AU - Schafferhans, A.* AU - Bernhofer, M.* AU - Vestergaard, G. AU - Nelson, C.W.* AU - Schloter, M. AU - Rost, B.* AU - Scherer, S.* AU - Neuhaus, K.* C1 - 51872 C2 - 43550 CY - San Francisco TI - Discovery of numerous novel small genes in the intergenic regions of the Escherichia coli O157:H7 Sakai genome. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Although hyperlipidemia is common in COPD, its relationship to comorbidities, risk factors and lung function in COPD has not been studied in detail. Using the baseline data of the COSYCONET cohort we addressed this question. Data from 1746 COPD patients (GOLD stage 1-4; mean age 64.6 y, mean FEV1%pred 57%) were evaluated, focusing on the comorbidities hyperlipidemia, diabetes and cardiovascular complex (CVC; including arterial hypertension, cardiac failure, ischemic heart disease). Risk factors comprised age, gender, BMI, and packyears of smoking. The results of linear and logistic regression analyses were implemented into a path analysis model describing the multiple relationships between parameters. Hyperlipidemia (prevalence 42.9%) was associated with lower intrathoracic gas volume (ITGV) and higher forced expiratory volume in 1 second (FEV1) when adjusting for its multiple relationships to risk factors and other comorbidities. These findings were robust in various statistical analyses. The associations between comorbidities and risk factors were in accordance with previous findings, thereby underlining the validity of our data. In conclusion, hyperlipidemia was associated with less hyperinflation and airway obstruction in patients with COPD. This surprising result might be due to different COPD phenotypes in these patients or related to effects of medication. AU - Kahnert, K.* AU - Lucke, T.* AU - Huber, R.M.* AU - Behr, J.* AU - Biertz, F.* AU - Vogt, A.* AU - Watz, H.* AU - Alter, P.* AU - Fähndrich, S.* AU - Bals, R.* AU - Holle, R. AU - Karrasch, S. AU - Söhler, S.* AU - Wacker, M. AU - Ficker, J.H.* AU - Parhofer, K.G.* AU - Vogelmeier, C.* AU - Jörres, R.A.* C1 - 51121 C2 - 42683 CY - San Francisco TI - Relationship of hyperlipidemia to comorbidities and lung function in COPD: Results of the COSYCONET cohort. JO - PLoS ONE VL - 12 IS - 5 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Introduction: For about 30 years, researchers developed prognostic scores and searched for prognostic factors to predict outcomes for cancer patients. The “Combs Prognostic Score” for re-irradiation in recurrent glioma was recently validated and results showed that the score is a significant (p < .001) and reliable predictor for patients undergoing re-irradiation (re-RT). We sought to enhance the score and generated a novel scoring approach, taking into account the information on resection of recurrent tumors, KPS, and tumor volume. Patients and methods: The prognostic score was generated based on 209 patients treated between 2002 and 2016 for recurrent glioma at the department of radiation oncology at the Klinikum rechts der Isar, Munich. To further enhance the previously validated Combs Prognostic Score, which uses the prognostic factors primary histology, time between primary RT and re-RT, and age, we added KPS, tumor volume (PTV) and re-resection into the scoring scheme. Results: The median follow-up time was 3.5 months. 67.5% were WHO IV gliomas with a median OS after re-RT of 7.9 months, 17.7% were WHO III gliomas with an OS of 11.3 months and 14.8% were WHO I/II gliomas with an OS of 14.7 months. Multivariate analyses confirmed the prognostic factors KPS (p < .001) and showed a tendency to significance for tumor volume (p = .067) and re-resection (p = .064). The new prognostic score demonstrated a high significance (p < .001). Conclusion: The “New Combs Prognostics Score” is a significant and useful tool to predict the overall effect of re-RT in patients with recurrence gliomas. This modified score offers an even better way to classify patients in clinical routine and prospective clinical trials investigating reirradiation. AU - Kessel, K.A. AU - Hesse, J.* AU - Straube, C.* AU - Zimmer, C.* AU - Graf, F.S.* AU - Schlegel, J.* AU - Meyer, B.* AU - Combs, S.E. C1 - 51537 C2 - 43296 CY - San Francisco TI - Modification and optimization of an established prognostic score after reirradiation of recurrent glioma. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Objective: Early detection of diabetes and prediabetic states is beneficial for patients, but may be delayed by patientś being overly optimistic about their own health. Therefore, we assessed how persons without known diabetes perceive their risk of having or developing diabetes, and we identified factors associated with perception of diabetes risk. Research design and methods: 1,953 participants without previously known diabetes from the population-based, German KORA FF4 Study (59.1 years, 47.8% men) had an oral glucose tolerance test. They estimated their probability of having undiagnosed diabetes mellitus (UDM) on a six category scale, and assessed whether they were at risk of developing diabetes in the future. We cross-tabulated glycemic status with risk perception, and fitted robust Poisson regression models to identify determinants of diabetes risk perception. Results: 74% (95% CI: 65-82) of persons with UDM believed that their probability of having undetected diabetes was low or very low. 72% (95% CI: 69-75) of persons with prediabetes believed that they were not at risk of developing diabetes. In people with prediabetes, seeing oneself at risk of diabetes was associated with self-rated poor general health (prevalence ratio (PR) = 3.1 (95% CI: 1.4-6.8), parental diabetes (PR = 2.6, 1.9-3.4), high educational level (PR = 1.9 (1.4-2.5)), lower age (PR = 0.7, 0.6-0.8, per 1 standard deviation increase), female sex (PR = 1.2, 0.9-1.5) and obesity (PR = 1.5, 1.2-2.0). Conclusions: People with undiagnosed diabetes or prediabetes considerably underestimate their probability of having or developing diabetes. Contrary to associations with actual diabetes risk, perceived diabetes risk was lower in men, lower educated and older persons. Copyright: AU - Kowall, B.* AU - Rathmann, W.G.* AU - Stang, A.* AU - Bongaerts, B.* AU - Kuss, O.* AU - Herder, C.* AU - Roden, M.* AU - Quante, A.S. AU - Holle, R. AU - Huth, C. AU - Peters, A. AU - Meisinger, C. C1 - 50478 C2 - 42357 CY - San Francisco TI - Perceived risk of diabetes seriously underestimates actual diabetes risk: The KORA FF4 study. JO - PLoS ONE VL - 12 IS - 1 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background: Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBVspecific antibodies can be observed in another subset of patients. Methods: We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. Results: EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Conclusion: Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry. AU - Loebel, M.* AU - Eckey, M.* AU - Sotzny, F.* AU - Hahn, E.G.* AU - Bauer, S.* AU - Grabowski, P.* AU - Zerweck, J.* AU - Holenya, P.* AU - Hanitsch, L.G.* AU - Wittke, K.* AU - Borchmann, P.* AU - Rüffer, J.U.* AU - Hiepe, F.* AU - Ruprecht, K.* AU - Behrends, U. AU - Meindl, C. AU - Volk, H.D.* AU - Reimer, U.* AU - Scheibenbogen, C.* C1 - 51413 C2 - 43231 CY - San Francisco TI - Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray. JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: We investigated the association between subclinical cardiovascular diseases assessed by MRI examination and symptoms of dizziness and vertigo in participants of a population-based sample. METHODS: Data from 400 participants (169 women) aged from 39 to 73 of a cross-sectional MRI sub-study of the "Kooperative Gesundheitsforschung in der Region Augsburg" (KORA) FF4 study from the south of Germany was used. MRI determined subclinical cardiovascular diseases include left and right ventricular structure and function as well as the presence of carotid plaque and carotid wall thickness. Cerebrum diseases include white matter lesions (WML) and cerebral microbleeds (CMB). The main outcomes of dizziness and vertigo were assessed by standardized interview. Logistic regression models were applied and adjusted odds ratios (OR) with 95% confidence intervals (CI) were provided. RESULTS: Lifetime and 12-month prevalence of dizziness and vertigo were 30% (95%CI 26% to 35%) and 21% (95%CI 17% to 26%) respectively in this sample. On multivariable analysis, cardiac and carotid measurements were not associated with dizziness and vertigo excluding orthostatic vertigo (20%, 95CI 16% to 24%). Only in male participants, there was a significant association between WML and the presence of dizziness and vertigo (OR = 2.95, 95%CI 1.08 to 8.07). There was no significant association of CMB with dizziness and vertigo. However, CMB and WML were tending to associate with a higher risk of dizziness and vertigo in the whole sample (CMB: OR = 1.48, 95%CI 0.70; 3.15; WML: OR = 1.71, 95%CI 0.80 to 3.67;), in persons with prediabetes and diabetes (WML: OR = 2.71, 95%CI 0.89 to 8.23) and in men with normal glucose metabolism (CMB: OR = 2.60, 95%CI 0.56 to 12.0; WML: OR = 3.08, 95%CI 0.58 to 16.5). CONCLUSIONS: In this sample of participants without manifest cardiovascular diseases, subclinical left and right ventricular function and carotid structure were consistently not associated with dizziness and vertigo. Subclinical cerebrum measurements, however, tend to increase the risk for dizziness and vertigo, especially in men and in persons with prediabetes or diabetes. AU - Lorbeer, R.* AU - Hetterich, H.* AU - Strobl, R.* AU - Schafnitzel, A.* AU - Patscheider, H.* AU - Schindler, A.* AU - Müller-Peltzer, K.* AU - Sommer, W.* AU - Peters, A. AU - Meisinger, C. AU - Heier, M. AU - Rathmann, W.* AU - Bamberg, F.* AU - Grill, E.* C1 - 51965 C2 - 43543 CY - San Francisco TI - Lack of association of MRI determined subclinical cardiovascular disease with dizziness and vertigo in a cross-sectional population-based study. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Intensity and duration of physical activity are associated with the achievement of health benefits. Our aim was to characterize physical activity behavior in terms of intensity, duration pattern, and adherence to the WHO physical activity recommendations in a population-based sample of adults from southern Germany. Further, we investigated associations between physical activity and sex, age, and body mass index (BMI), considering also common chronic diseases. METHODS: We analyzed 475 subjects (47% males, mean age 58 years, range 48-68 years) who wore ActiGraph accelerometers for up to seven days. Measured accelerations per minute obtained from the vertical axis (uniaxial) and the vector magnitude of all three axes (triaxial) were classified as sedentary, light or moderate-to-vigorous physical activity (MVPA) according to predefined acceleration count cut-offs. The average minutes/day spent in each activity level per subject served as outcome. Associations of sex, age, BMI, and seven chronic diseases or health limitations, with the activity levels were analyzed by negative binomial regression. RESULTS: Most of the wear time was spent in sedentarism (median 61%/day), whereas the median time spent in MVPA was only 3%, with men achieving more MVPA than women (35 vs. 28 minutes/day, p<0.05). Almost two thirds of MVPA was achieved in short bouts of less than 5 minutes, and 35% of the subjects did not achieve a single 10-minute bout. Hence, only 14% adhered to the WHO recommendation of 2.5 hours of MVPA/week in at least 10-minute bouts. Females, older subjects and obese subjects spent less time in MVPA (p<0.05), but no clear association with hypertension, asthma, diabetes, chronic obstructive pulmonary disease, anxiety/depression, pain or walking difficulties was observed in regression analyses with MVPA as outcome. CONCLUSIONS: Activity behavior among middle-aged German adults was highly insufficient, indicating a further need for physical activity promotion in order to gain health benefits. AU - Luzak, A. AU - Heier, M. AU - Thorand, B. AU - Laxy, M. AU - Nowak, D.* AU - Peters, A. AU - Schulz, H. C1 - 50629 C2 - 42468 CY - San Francisco TI - Physical activity levels, duration pattern and adherence to WHO recommendations in German adults. JO - PLoS ONE VL - 12 IS - 2 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background: DNA methylation is affected by the activities of the key enzymes and intermediate metabolites of the one-carbon pathway, one of which involves homocysteine. We investigated the effect of the well-known genetic variant associated with mildly elevated homocysteine: MTHFR 677C > T independently and in combination with other homocysteine-associated variants, on genome-wide leukocyte DNA-methylation. Methods: Methylation levels were assessed using Illumina 450k arrays on 9,894 individuals of European ancestry from 12 cohort studies. Linear-mixed-models were used to study the association of additive MTHFR 677C > T and genetic-risk score (GRS) based on 18 homocysteine-associated SNPs, with genome-wide methylation. Results: Meta-analysis revealed that the MTHFR 677C > T variant was associated with 35 CpG sites in cis, and the GRS showed association with 113 CpG sites near the homocysteine-associated variants. Genome-wide analysis revealed that the MTHFR 677C > T variant was associated with 1 trans-CpG (nearest gene ZNF184), while the GRS model showed association with 5 significant trans-CpGs annotated to nearest genes PTF1A, MRPL55, CTDSP2, CRYM and FKBP5. Conclusions: Our results do not show widespread changes in DNA-methylation across the genome, and therefore do not support the hypothesis that mildly elevated homocysteine is associated with widespread methylation changes in leukocytes. AU - Mandaviya, P.R.* AU - Joehanes, R.* AU - Aïssi, D.* AU - Kühnel, B. AU - Marioni, R.E.* AU - Truong, V.* AU - Stolk, L.* AU - Beekman, M.* AU - Bonder, M.J.* AU - Franke, L.* AU - Gieger, C. AU - Huan, T.* AU - Ikram, M.A.* AU - Kunze, S. AU - Liang, L.* AU - Lindemans, J.* AU - Liu, C.* AU - McRae, A.F.* AU - Mendelson, M.M.* AU - Müller-Nurasyid, M. AU - Peters, A. AU - Slagboom, P.E.* AU - Starr, J.M.* AU - Trégouët, D.A.* AU - Uitterlinden, A.G.* AU - Van Greevenbroek, M.M.J.* AU - van Heemst, D.* AU - van Iterson, M.* AU - Wells, P.S.* AU - Yao, C.* AU - Deary, I.J.* AU - Gagnon, F.* AU - Heijmans, B.T.* AU - Levy, D.* AU - Morange, P.E.* AU - Waldenberger, M. AU - Heil, S.G.* AU - van Meurs, J.B.J.* C1 - 52314 C2 - 43853 CY - San Francisco TI - Genetically defined elevated homocysteine levels do not result in widespread changes of DNA methylation in leukocytes. JO - PLoS ONE VL - 12 IS - 10 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Mitochondrial dysfunction contributes to myriad monogenic and complex pathologies. To understand the underlying mechanisms, it is essential to define the full complement of proteins that modulate mitochondrial function. To identify such proteins, we performed a metaanalysis of publicly available gene expression data. Gene co-expression analysis of a large and heterogeneous compendium of microarray data nominated a sub-population of transcripts that whilst highly correlated with known mitochondrial protein-encoding transcripts (MPETs), are not themselves recognized as generating proteins either localized to the mitochondrion or pertinent to functions therein. To focus the analysis on a medically-important condition with a strong yet incompletely understood mitochondrial component, candidates were cross-referenced with an MPET-enriched module independently generated via genomewide co-expression network analysis of a human heart failure gene expression dataset. The strongest uncharacterized candidate in the analysis was Leucine Rich Repeat Containing 2 (LRRC2). LRRC2 was found to be localized to the mitochondria in human cells and transcriptionally-regulated by the mitochondrial master regulator Pgc-1α. We report that Lrrc2 transcript abundance correlates with that of β-MHC, a canonical marker of cardiac hypertrophy in humans and experimentally demonstrated an elevation in Lrrc2 transcript in in vitro and in vivo rodent models of cardiac hypertrophy as well as in patients with dilated cardiomyopathy. RNAi-mediated Lrrc2 knockdown in a rat-derived cardiomyocyte cell line resulted in enhanced expression of canonical hypertrophic biomarkers as well as increased mitochondrial mass in the context of increased Pgc-1α expression. In conclusion, our meta-analysis represents a simple yet powerful springboard for the nomination of putative mitochondrially-pertinent proteins relevant to cardiac function and enabled the identification of LRRC2 as a novel mitochondrially-relevant protein and regulator of the hypertrophic response. AU - McDermott-Roe, C.* AU - Leleu, M.* AU - Rowe, G.C.* AU - Palygin, O.* AU - Bukowy, J.D.* AU - Kuo, J.* AU - Rech, M.* AU - Hermans-Beijnsberger, S.* AU - Schaefer, S.* AU - Adami, E.* AU - Creemers, E.E.* AU - Heinig, M. AU - Schroen, B.* AU - Arany, Z.* AU - Petretto, E.* AU - Geurts, A.M.* C1 - 50513 C2 - 42496 CY - San Francisco TI - Transcriptome-wide co-expression analysis identifies LRRC2 as a novel mediator of mitochondrial and cardiac function. JO - PLoS ONE VL - 12 IS - 2 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background Sulforaphane (SFN), an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT). Methods In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT. Results Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in ?H2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4. Conclusion Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer. AU - Naumann, P.S.* AU - Liermann, J.* AU - Fortunato, F.* AU - Schmid, T.E.* AU - Weber, K.J.* AU - Debus, J.* AU - Combs, S.E. C1 - 51561 C2 - 43303 CY - San Francisco TI - Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Aim Inhibition of sodium/glucose cotransporter 2 (SGLT2), the key transport protein in renal glucose reabsorption, promotes glucose excretion and represents a new concept in the therapy of type-2 diabetes. In addition, SGLT2 inhibition elevates circulating glucagon concentrations and enhances hepatic glucose production. Since SGLT2 is expressed in human pancreatic α-cells and regulates glucagon release, we tested whether common variants of the SGLT2 gene SLC5A2 associate with altered plasma glucagon concentrations in the fasting state and upon glucose challenge. Methods A study population of 375 healthy subjects at increased risk for type-2 diabetes, phenotyped by a 5-point oral glucose tolerance test (OGTT) and genotyped for recently described SLC5A2 tagging single nucleotide polymorphisms (SNPs), was selected for plasma glucagon measurements. Results After adjustment for gender, age, body mass index, and insulin sensitivity, the four tagging SNPs (rs9924771, rs3116150, rs3813008, rs9934336), tested separately or as genetic score, were neither significantly nor nominally associated with plasma glucagon concentrations at any time during the OGTT, with the inverse AUC of glucagon or the glucagon fold-change during the OGTT (p 0.2, all). Testing for genotype-related differences in the time course of the glucagon response using MANOVA did also not reveal any significant or nominal associations (p 0.5, all). Conclusion We could not obtain statistically significant evidence for a role of common SLC5A2 variants in the regulation of glucagon release in the fasting state or upon glucose challenge. Moreover, the reported nominal effects of individual SLC5A2 variants on fasting and post-challenge glucose levels may probably not be mediated by altered glucagon release. AU - Ordelheide, A.-M. AU - Böhm, A. AU - Kempe-Teufel, D. AU - Wagner, R. AU - Machicao, F. AU - Heni, M. AU - Stefan, N. AU - Fritsche, A. AU - Häring, H.-U. AU - Staiger, H. C1 - 51168 C2 - 42966 TI - Common variation in the sodium/glucose cotransporter 2 gene SLC5A2 does neither affect fasting nor glucose-suppressed plasma glucagon concentrations. JO - PLoS ONE VL - 12 IS - 5 PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - The biosynthesis of multiple secondary metabolites in the phytopathogenic ascomycete Fusarium fujikuroi is strongly affected by nitrogen availability. Here, we present the first genome-wide transcriptome and proteome analysis that compared the wild type and deletion mutants of the two major nitrogen regulators AreA and AreB. We show that AreB acts not simply as an antagonist of AreA counteracting the expression of AreA target genes as suggested based on the yeast model. Both GATA transcription factors affect a large and diverse set of common as well as specific target genes and proteins, acting as activators and repressors. We demonstrate that AreA and AreB are not only involved in fungal nitrogen metabolism, but also in the control of several complex cellular processes like carbon metabolism, transport and secondary metabolism. We show that both GATA transcription factors can be considered as master regulators of secondary metabolism as they affect the expression of more than half of the 47 putative secondary metabolite clusters identified in the genome of F. fujikuroi. While AreA acts as a positive regulator of many clusters under nitrogen-limiting conditions, AreB is able to activate and repress gene clusters (e.g. bikaverin) under nitrogen limitation and sufficiency. In addition, ChIP analyses revealed that loss of AreA or AreB causes histone modifications at some of the regulated gene clusters. AU - Pfannmüller, A.* AU - Leufken, J.* AU - Studt, L.* AU - Michielse, C.B.* AU - Sieber, C.M.K. AU - Güldener, U.* AU - Hawat, S.* AU - Hippler, M.* AU - Fufezan, C.* AU - Tudzynski, B.* C1 - 50986 C2 - 43027 TI - Comparative transcriptome and proteome analysis reveals a global impact of the nitrogen regulators AreA and AreB on secondary metabolism in Fusarium fujikuroi. JO - PLoS ONE VL - 12 IS - 4 PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Here, we aimed to investigate the potential role of DUSP6, a dual specificity phosphatase, that specifically inactivates extracellular signal-regulated kinase (ERK), for the regulation of body weight and glucose homeostasis. We further assessed whether metabolic challenges affect Dusp6 expression in selected brain areas or white adipose tissue. Hypothalamic Dusp6 mRNA levels remained unchanged in chow-fed lean vs. high fat diet (HFD) fed obese C57Bl/6J mice, and in C57Bl/6J mice undergoing prolonged fasting or refeeding with fat free diet (FFD) or HFD. Similarly, Dusp6 expression levels were unchanged in selected brain regions of Lep(ob) mice treated with 1 mg/kg of leptin for 6 days, compared to pair-fed or saline-treated Lep(ob) controls. Dusp6 expression levels remained unaltered in vitro in primary adipocytes undergoing differentiation, but were increased in eWAT of HFD-fed obese C57Bl/6J mice, compared to chow-fed lean controls. Global chow-fed DUSP6 KO mice displayed reduced body weight and lean mass and slightly increased fat mass at a young age, which is indicative for early-age weight retardation. Subsequent exposure to HFD led to a significant increase in lean mass and body weight in DUSP6 deficient mice, compared to WT controls. Nevertheless, after 26 weeks of high-fat diet exposure, we observed comparable body weight, fat and lean mass in DUSP6 WT and KO mice, suggesting overall normal susceptibility to develop obesity. In line with the increased weight gain to compensate for early-age weight retardation, HFD-fed DUSP6 KO displayed increased expression levels of anabolic genes involved in lipid and cholesterol metabolism in the epididymal white adipose tissue (eWAT), compared to WT controls. Glucose tolerance was perturbed in both chow-fed lean or HFD-fed obese DUSP6 KO, compared to their respective WT controls. Overall, our data indicate that DUSP6 deficiency has limited impact on the regulation of energy metabolism, but impairs systemic glucose tolerance. Our data are in conflict to earlier reports that propose protection from diet-induced obesity and glucose intolerance in DUSP6 deficient mice. Reasons for the discrepancies remain elusive, but may entail differential genetic backgrounds, environmental factors such as the type and source of HFD, or alterations in the gut microbiome between facilities. AU - Pfuhlmann, K. AU - Pfluger, P.T. AU - Schriever, S.C. AU - Müller, T.D. AU - Tschöp, M.H. AU - Stemmer, K. C1 - 51922 C2 - 43592 CY - San Francisco TI - Dual specificity phosphatase 6 deficiency is associated with impaired systemic glucose tolerance and reversible weight retardation in mice. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Nasal mucosa and olfactory bulb are separated by the cribriform plate which is perforated by olfactory nerves. We have previously demonstrated that the cribriform plate is permissive for T cells and monocytes and that viruses can enter the bulb upon intranasal injection by axonal transportation. Therefore, we hypothesized that nasal mucosa and olfactory bulb are equipped to deal with constant infectious threats. To detect genes involved in this process, we compared gene expression in nasal mucosa and bulb of mice kept under specific pathogen free (SPF) conditions to gene expression of mice kept on non-SPF conditions using RNA deep sequencing. We found massive alterations in the expression of immune-related genes of the nasal mucosa, while the bulb did not respond immunologically. The absence of induction of immune-related genes in the olfactory bulb suggests effective defence mechanisms hindering entrance of environmental pathogens beyond the outer arachnoid layer. The genes detected in this study may include candidates conferring susceptibility to meningitis. AU - Piotrowski, C.* AU - Lede, V.* AU - Butthof, A.* AU - Kaiser, N.* AU - Hirrlinger, P.G.* AU - Tschöp, M.H. AU - Schöneberg, T.* AU - Bechmann, I.* C1 - 52230 C2 - 43859 CY - San Francisco TI - Open housing drives the expression of immune response genes in the nasal mucosa, but not the olfactory bulb. JO - PLoS ONE VL - 12 IS - 10 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Longitudinal evidence on the association between physical activity (PA) or weight and health-related quality of life (HRQL) is sparse and studies describe inconclusive results. The aim of this study was to examine longitudinal associations between change in PA and HRQL as between change in weight and HRQL respectively. METHODS: Analyses are based on data from the KORA S4 cohort study (1999-2001; n = 4,261, mean age 49.0 ± 13.3 years) and the two follow-up examinations (F4: 2006-2008; FF4: 2013-2014). Information on PA was collected in standardized interviews. Weight was measured objectively. Mental and physical components of HRQL were assessed via the SF-12 questionnaire. First, change in HRQL was regressed on change in PA and weight. Second, hierarchical linear models were fitted, which allowed estimation of between-subject and within-subject effects. Analyses were adjusted for the covariates sex, baseline diseases, and education. RESULTS: A change to a physically more active lifestyle is positively associated with physical and mental HRQL. Although weight gain is associated with impairments in physical HRQL, the data show an inverse relationship between weight gain and mental HRQL. The results were consistent for both the change score analyses and the hierarchical linear models. DISCUSSION: Our findings stress the importance of interventions on PA/weight. Nonetheless, more research is needed to reveal the causal relationship between PA/weight and HRQL. AU - Rabel, M.* AU - Meisinger, C. AU - Peters, A. AU - Holle, R. AU - Laxy, M. C1 - 52004 C2 - 43655 CY - San Francisco TI - The longitudinal association between change in physical activity, weight, and health-related quality of life: Results from the population-based KORA S4/F4/FF4 cohort study. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Aims/hypothesis To develop a prediction model that can predict HbA1c levels after six years in the non-diabetic general population, including previously used readily available predictors. Methods Data from 5,762 initially non-diabetic subjects from three population-based cohorts (Hoorn Study, Inter99, KORA S4/F4) were combined to predict HbA1c levels at six year follow-up. Using backward selection, age, BMI, waist circumference, use of anti-hypertensive medication, current smoking and parental history of diabetes remained in sex-specific linear regression models. To minimize overfitting of coefficients, we performed internal validation using bootstrapping techniques. Explained variance, discrimination and calibration were assessed using R2, classification tables (comparing highest/lowest 50% HbA1c levels) and calibration graphs. The model was externally validated in 2,765 non-diabetic subjects of the population-based cohort METSIM. Results At baseline, mean HbA1c level was 5.6% (38 mmol/mol). After a mean follow-up of six years, mean HbA1c level was 5.7% (39 mmol/mol). Calibration graphs showed that predicted HbA1c levels were somewhat underestimated in the Inter99 cohort and overestimated in the Hoorn and KORA cohorts, indicating that the model's intercept should be adjusted for each cohort to improve predictions. Sensitivity and specificity (95% CI) were 55.7% (53.9, 57.5) and 56.9% (55.1, 58.7) respectively, for women, and 54.6% (52.7, 56.5) and 54.3% (52.4, 56.2) for men. External validation showed similar performance in the METSIM cohort. Conclusions/interpretation In the non-diabetic population, our DIRECT-DETECT prediction model, including readily available predictors, has a relatively low explained variance and moderate discriminative performance, but can help to distinguish between future highest and lowest HbA1c levels. Absolute HbA1c values are cohort-dependent. AU - Rauh, S.P.* AU - Heymans, M.W.* AU - Koopman, A.D.M.* AU - Nijpels, G.* AU - Stehouwer, C.D.* AU - Thorand, B. AU - Rathmann, W.G.* AU - Meisinger, C. AU - Peters, A. AU - de Las Heras Gala, T. AU - Glümer, C.* AU - Pedersen, O.* AU - Cederberg, H.* AU - Kuusisto, J.* AU - Laakso, M.* AU - Pearson, E.R.* AU - Franks, P.W.* AU - Rutters, F.* AU - Dekker, J.M.* C1 - 50523 C2 - 42408 CY - San Francisco TI - Predicting glycated hemoglobin levels in the non-diabetic general population: Development and validation of the DIRECTDETECT prediction model-a DIRECT study. JO - PLoS ONE VL - 12 IS - 2 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Gene-set analysis (GSA) is an approach using the results of single-marker genome-wide association studies when investigating pathways as a whole with respect to the genetic basis of a disease. METHODS: We performed a meta-analysis of seven GSAs for lung cancer, applying the method META-GSA. Overall, the information taken from 11,365 cases and 22,505 controls from within the TRICL/ILCCO consortia was used to investigate a total of 234 pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. RESULTS: META-GSA reveals the systemic lupus erythematosus KEGG pathway hsa05322, driven by the gene region 6p21-22, as also implicated in lung cancer (p = 0.0306). This gene region is known to be associated with squamous cell lung carcinoma. The most important genes driving the significance of this pathway belong to the genomic areas HIST1-H4L, -1BN, -2BN, -H2AK, -H4K and C2/C4A/C4B. Within these areas, the markers most significantly associated with LC are rs13194781 (located within HIST12BN) and rs1270942 (located between C2 and C4A). CONCLUSIONS: We have discovered a pathway currently marked as specific to systemic lupus erythematosus as being significantly implicated in lung cancer. The gene region 6p21-22 in this pathway appears to be more extensively associated with lung cancer than previously assumed. Given wide-stretched linkage disequilibrium to the area APOM/BAG6/MSH5, there is currently simply not enough information or evidence to conclude whether the potential pleiotropy of lung cancer and systemic lupus erythematosus is spurious, biological, or mediated. Further research into this pathway and gene region will be necessary. AU - Rosenberger, A.* AU - Sohns, M.* AU - Friedrichs, S.* AU - Hung, R.J.* AU - Fehringer, G.* AU - McLaughlin, J.* AU - Amos, C.I.* AU - Brennan, P.* AU - Risch, A.* AU - Brüske, I. AU - Caporaso, N.E.* AU - Landi, M.T.* AU - Christiani, D.C.* AU - Wei, Y.* AU - Bickeböller, H.* C1 - 50675 C2 - 42464 CY - San Francisco TI - Gene-set meta-analysis of lung cancer identifies pathway related to systemic lupus erythematosus. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Plants adapt to the environment by either long-term genome evolution or by acclimatization processes where the cellular processes and metabolism of the plant are adjusted within the existing potential in the genome. Here we studied the adaptation strategies in date palm, Phoenix dactylifera, under mild heat, drought and combined heat and drought by transcriptomic and metabolomic profiling. In transcriptomics data, combined heat and drought resembled heat response, whereas in metabolomics data it was more similar to drought. In both conditions, soluble carbohydrates, such as fucose, and glucose derivatives, were increased, suggesting a switch to carbohydrate metabolism and cell wall biogenesis. This result is consistent with the evidence from transcriptomics and cis-motif analysis. In addition, transcriptomics data showed transcriptional activation of genes related to reactive oxygen species in all three conditions (drought, heat, and combined heat and drought), suggesting increased activity of enzymatic antioxidant systems in cytosol, chloroplast and peroxisome. Finally, the genes that were differentially expressed in heat and combined heat and drought stresses were significantly enriched for circadian and diurnal rhythm motifs, suggesting new stress avoidance strategies. AU - Safronov, O.* AU - Kreuzwieser, J.* AU - Haberer, G. AU - Alyousif, M.S.* AU - Schulze, W.* AU - Al-Harbi, N.* AU - Arab, L.* AU - Ache, P.* AU - Stempfl, T.* AU - Kruse, J.* AU - Mayer, K.X. AU - Hedrich, R.* AU - Rennenberg, H.* AU - Salojärvi, J.* AU - Kangasjärvi, J.* C1 - 51230 C2 - 43105 CY - San Francisco TI - Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm). JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Human Cytomegalovirus (CMV) can lead to primary infection or reactivation in CMV-seronegative or-seropositive kidney transplant recipients, respectively. Complications comprise severe end-organ diseases and acute or chronic transplant rejection. Risk for CMV manifestation is stratified according to the CMV-IgG-serostatus, with donor+/recipient-(D+/R-) patients carrying the highest risk for CMV-replication. However, risk factors predisposing for primary infection in CMV-seronegative recipients are still not fully elucidated. Therefore, we monitored D+/R-high-risk patients undergoing kidney transplantation in combination with antiviral prophylaxis for the incidence of CMV-viremia for a median follow-up time of 784 days (156-1155 days). In this period, we analyzed the functional CMV-specific T cell response by intracellular cytokine staining and CMV-serology by ELISA. Only four of eight D +/R-patients developed clinically relevant CMV-viremia followed by seroconversion. Viremia triggered expansion of functional CMV-specific T cells correlating with protection against secondary CMV-reactivations. In contrast, all other patients remained permanently aviremic and showed no immunological correlate of infection after discontinuation of antiviral prophylaxis for up to three years. Comparing cold ischemic times (CIT) of viremic (median = 1020 min; 720-1080 min) and aviremic patients (median = 335 min; 120-660 min) revealed significantly (p = 0.0286) protracted CIT in patients with primary CMV-infection. Taken together, primary CMV-infection affects only a subgroup of D+/R-patients correlating with length of CIT. Therefore, patients with extended CIT should be thoroughly monitored for CMV-replication well beyond discontinuation of antiviral prophylaxis. In contrast, patients with short CIT remained permanently uninfected and might benefit from shorter prophylactic treatment. AU - Schlott, F. AU - Steubl, D.* AU - Hoffmann, D. AU - Matevossian, E.* AU - Lutz, J.* AU - Heemann, U.* AU - Hösel, V.* AU - Busch, D.H.* AU - Renders, L.* AU - Neuenhahn, M.* C1 - 50771 C2 - 42862 CY - San Francisco TI - Primary cytomegalovirus infection in seronegative kidney transplant patients is associated with protracted cold ischemic time of seropositive donor organs. JO - PLoS ONE VL - 12 IS - 1 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - The objective of this analysis is to compare people with prevalent type 2 diabetes, incident type 2 diabetes and without diabetes with respect to longitudinal change in health-related quality of life (HRQOL) when adjusting for baseline determinants of HRQOL. Research design and methods Primary baseline and follow-up data from three regional and one national population-based cohort studies in Germany were pooled for analysis. HRQOL was measured using physical and mental health summary scores (PCS and MCS) from the German version of the Short Form Health Survey with 36 or 12 items. Mean score change per observation year was compared between the three groups (prevalent diabetes, incident diabetes, no diabetes) based on linear regression models. Results The analysis included pooled data from 5367 people aged 45-74 years at baseline. Of these, 85.5% reported no diabetes at baseline and follow-up, 6.3% reported diabetes at both baseline and follow-up (prevalent diabetes), and 8.2% reported diabetes only at followup (incident diabetes). Over a mean observation period of 8.7 years, annual decline in HRQOL scores is pronounced at 0.27-0.32 (PCS) and 0.34-0.38 (MCS) in the group with prevalent diabetes compared with people without diabetes. Those with incident diabetes showed intermediate values but did not differ significantly from people without diabetes after adjustment for covariates in the full model. Conclusion Compared with data from cross-sectional analysis, the HRQOL loss associated with prevalent diabetes appears to be much larger than previously assumed. AU - Schunk, M. AU - Reitmeir, P. AU - Rückert-Eheberg, I.-M. AU - Tamayo, T.* AU - Schipf, S.* AU - Meisinger, C. AU - Peters, A. AU - Scheidt-Nave, C.* AU - Ellert, U.* AU - Hartwig, S.* AU - Kluttig, A.* AU - Völzke, H.* AU - Holle, R. C1 - 51145 C2 - 42686 CY - San Francisco TI - Longitudinal change in health-related quality of life in people with prevalent and incident type 2 diabetes compared to diabetes-free controls. JO - PLoS ONE VL - 12 IS - 5 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - We propose a stochastic model for use in epidemiological analysis, describing the age-dependent development of atherosclerosis with adequate simplification. The model features the uptake of monocytes into the arterial wall, their proliferation and transition into foam cells. The number of foam cells is assumed to determine the health risk for clinically relevant events such as stroke. In a simulation study, the model was checked against the age-dependent prevalence of atherosclerotic lesions. Next, the model was applied to incidence of atherosclerotic stroke in the cohort of male workers from the Mayak nuclear facility in the Southern Urals. It describes the data as well as standard epidemiological models. Based on goodness-of-fit criteria the risk factors smoking, hypertension and radiation exposure were tested for their effect on disease development. Hypertension was identified to affect disease progression mainly in the late stage of atherosclerosis. Fitting mechanistic models to incidence data allows to integrate biological evidence on disease progression into epidemiological studies. The mechanistic approach adds to an understanding of pathogenic processes, whereas standard epidemiological methods mainly explore the statistical association between risk factors and disease outcome. Due to a more comprehensive scientific foundation, risk estimates from mechanistic models can be deemed more reliable. To the best of our knowledge, such models are applied to epidemiological data on cardiovascular diseases for the first time. AU - Simonetto, C. AU - Azizova, T.V.* AU - Barjaktarovic, Z. AU - Bauersachs, J.* AU - Jacob, P. AU - Kaiser, J.C. AU - Meckbach, R. AU - Schöllnberger, H. AU - Eidemüller, M. C1 - 50898 C2 - 42554 CY - San Francisco TI - A mechanistic model for atherosclerosis and its application to the cohort of Mayak workers. JO - PLoS ONE VL - 12 IS - 4 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - We explored the assembly of intestinal microbiota in healthy male participants during the randomized crossover design of run-in (5 day) and experimental phases (21-day normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, with balanced fluid and dietary intakes, controlled circadian rhythm, microbial ambiental burden and 24/7 medical surveillance. The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) were 0.209 and 133.1 ± 0.3 mmHg for NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg for both hypoxic variants (HBR and HAmb; ~4000 m simulated altitude), respectively. A number of parameters linked to intestinal environment such as defecation frequency, intestinal electrical conductivity (IEC), sterol and polyphenol content and diversity, indole, aromaticity and spectral characteristics of dissolved organic matter (DOM) were measured (64 variables). The structure and diversity of bacterial microbial community was assessed using 16S rRNA amplicon sequencing. Inactivity negatively affected frequency of defecation and in combination with hypoxia increased IEC (p < 0.05). In contrast, sterol and polyphenol diversity and content, various characteristics of DOM and aromatic compounds, the structure and diversity of bacterial microbial community were not significantly affected over time. A new in-house PlanHab database was established to integrate all measured variables on host physiology, diet, experiment, immune and metabolic markers (n = 231). The observed progressive decrease in defecation frequency and concomitant increase in IEC suggested that the transition from healthy physiological state towards the developed symptoms of low magnitude obesity-related syndromes was dose dependent on the extent of time spent in inactivity and preceded or took place in absence of significant rearrangements in bacterial microbial community. Species B. thetaiotamicron, B. fragilis, B. dorei and other Bacteroides with reported relevance for dysbiotic medical conditions were significantly enriched in HBR, characterized with most severe inflammation symptoms, indicating a shift towards host mucin degradation and proinflammatory immune crosstalk. AU - Šket, R.* AU - Treichel, N. AU - Kublik, S. AU - Debevec, T.* AU - Eiken, O.* AU - Mekjavić, I.* AU - Schloter, M. AU - Vital, M.* AU - Chandler, J.* AU - Tiedje, J.M.* AU - Murovec, B.* AU - Prevoršek, Z.* AU - Likar, M.* AU - Stres, B.* C1 - 52495 C2 - 44020 CY - San Francisco TI - Hypoxia and inactivity related physiological changes precede or take place in absence of significant rearrangements in bacterial community structure: The PlanHab randomized trial pilot study. JO - PLoS ONE VL - 12 IS - 12 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Because of unreliable self-report, accelerometry is increasingly used to objectively monitor physical activity (PA). However, results of accelerometric studies vary depending on the chosen cutpoints between activity intensities. Population-specific activity patterns likely affect the size of these differences. To establish their size and stability we apply three sets of cutpoints, including two calibrated to a single reference, to our accelerometric data and compare PA estimates. METHODS: 1402 German adolescents from the GINIplus and LISAplus cohorts wore triaxial accelerometers (Actigraph GT3x) for one week (mean 6.23 days, 14.7 hours per day) at the hip. After validation of wear, we applied three sets of cutpoints for youth, including the most common standard (Freedson, 2005) and two calibrated to a single reference, (Romanzini uni- and triaxial, from Romanzini, 2014) to these data, estimating daily sedentary, light, moderate, vigorous and moderate-to-vigorous PA (MPA, VPA, MVPA). Stability of differences was assessed by comparing Romanzini's two sets of cutpoints. RESULTS: Relative agreement between cutpoints was closer for activity of lower intensities (largest difference for sedentary behaviour 9%) but increased for higher intensities (largest difference for light activity 40%, MPA 102%, VPA 88%; all p<0.01). Romanzini's uniaxial and triaxial cutpoints agreed no more closely with each other than with Freedson's. CONCLUSIONS: Estimated PA differed significantly between different sets of cutpoints, even when those cutpoints agreed perfectly on another dataset (i.e. Romanzini's.) This suggests that the detected differences in estimated PA depend on population-specific activity patterns, which cannot be easily corrected for: converting activity estimates from one set of cutpoints to another may require access to raw data. This limits the utility of accelerometry for comparing populations in place and time. We suggest that accelerometric research adopt a standard for data processing, and apply and present the results of this standard in addition to those from any other method. AU - Smith, M. AU - Standl, M. AU - Heinrich, J. AU - Schulz, H. C1 - 52276 C2 - 43852 CY - San Francisco TI - Accelerometric estimates of physical activity vary unstably with data handling. JO - PLoS ONE VL - 12 IS - 11 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8; 21) or inv 16 types. Differences in lipidomes of t(8; 21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8; 21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools. AU - Stefanko, A.* AU - Thiede, C.* AU - Ehninger, G.* AU - Simons, K.* AU - Grzybek, M. C1 - 50766 C2 - 42837 CY - San Francisco TI - Lipidomic approach for stratification of acute myeloid leukemia patients. JO - PLoS ONE VL - 12 IS - 2 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - T-cell based vaccines have been considered as attractive candidates for prevention of hepatitis C virus (HCV) infections. In this study we compared the magnitude and phenotypic characteristics of CD8+ T-cells induced by three commonly used viral vectors, Adenovirus-5 (Ad5), Vaccinia virus (VV) and Modified Vaccinia Ankara (MVA) expressing the HCV NS3/4A protein. C57/BL6 mice were primed with DNA expressing NS3/4A and boosted with each of the viral vectors in individual groups of mice. We then tracked the vaccine-induced CD8+ T-cell responses using pentamer binding and cytokine production analysis. Overall, our data indicate that the memory cells induced by Ad5 were inferior to those induced by VV or MVA. We found that Ad5 boosting resulted in rapid expansion and significantly higher frequencies of NS3-specific T-cells compared to VV and MVA boosting. However, the functional profiles, assessed through analysis of the memory cell marker CD127 and the anti-apoptotic molecule Bcl-2 in the blood, spleen, and liver; and measurements of interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 production indicated significantly lower frequencies of long-lived memory T-cells following Ad5 boosting compared to VV and MVA. This same set of analyses suggested that the memory cells induced following boosting with MVA were superior to those induced by both Ad5 and VV. This superiority of the MVA-induced CD8+ T-cells was confirmed following surrogate challenge of mice with a recombinant mouse herpes virus expressing the HCV NS3 protein. Higher levels of NS3-specific CD8+ T-cells displaying the functional markers CD69, Ki67 and Granzyme B were found in the spleens of mice boosted with MVA compared to VV and Ad5, both alone and in combination. These data suggest that MVA may be a more successful viral vector for induction of effective CD8+ T-cell responses against hepatitis C virus. AU - Tan, W.G.* AU - Zubkova, I.* AU - Kachko, A.* AU - Wells, F.* AU - Adler, H. AU - Sutter, G.* AU - Major, M.E.* C1 - 51566 C2 - 43228 CY - San Francisco TI - Qualitative differences in cellular immunogenicity elicited by hepatitis C virus T-Cell vaccines employing prime-boost regimens. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background: Skin autofluorescence, a biomarker for advanced glycation end products (AGEs) accumulation, has been shown to predict diabetes-related cardiovascular complications and is associated with several environmental and lifestyle factors. In the present study, we examined the association between various smoking behaviors and skin autofluorescence, as well as the association between several cotinine biomarkers and skin autofluorescence, using both epidemiological and metabolomics data. Methods: In a cross-sectional study, we evaluated participants from the LifeLines Cohort Study and the Qatar Metabolomics Study on Diabetes (QMDiab). In the LifeLines Cohort Study smoking behavior and secondhand smoking were assessed in 8,905 individuals including 309 individuals (3.5%) with type 2 diabetes. In QMDiab, cotinine biomarkers were measured in saliva, plasma and urine in 364 individuals of whom 188 (51%) had type 2 diabetes. Skin autofluorescence was measured non-invasively in all participants using the AGE Reader. Results: Skin autofluorescence levels increased with a higher number of hours being exposed to secondhand smoking. Skin autofluorescence levels of former smokers approached levels of never smokers after around 15 years of smoking cessation. Urinary cotinine N-oxide, a biomarker of nicotine exposure, was found to be positively associated with skin autofluorescence in the QMDiab study (p = 0.03). Conclusions: In the present study, we have demonstrated that secondhand smoking is associated with higher skin autofluorescence levels whereas smoking cessation has a beneficial effect on skin autofluorescence. Finally, urinary cotinine N-oxide might be used as an alternative way for questionnaires to examine the effect of (environmental) tobacco smoking on skin autofluorescence. AU - van Waateringe, R.P.* AU - Mook-Kanamori, M.J.* AU - Slagter, S.N.* AU - van der Klauw, M.M.* AU - van Vliet-Ostaptchouk, J.V.* AU - Graaff, R.* AU - Lutgers, H.L.* AU - Suhre, K. AU - El-Din Selim, M.M.* AU - Mook-Kanamori, D.O.* AU - Wolffenbuttel, B.H.R.* C1 - 51458 C2 - 43220 CY - San Francisco TI - The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation. JO - PLoS ONE VL - 12 IS - 6 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Background Genome-wide association studies have identified novel genetic associations for asthma, but without taking into account the role of active tobacco smoking. This study aimed to identify novel genes that interact with ever active tobacco smoking in adult onset asthma. Methods We performed a genome-wide interaction analysis in six studies participating in the GABRIEL consortium following two meta-analyses approaches based on 1) the overallinteraction effect and 2) the genetic effect in subjects with and without smoking exposure. We performed a discovery meta-analysis including 4,057 subjects of European descent and replicated our findings in an independent cohort (LifeLines Cohort Study), including 12,475 subjects. Results First approach: 50 SNPs were selected based on an overall interaction effect at p<10-4. The most pronounced interaction effect was observed for rs9969775 on chromosome 9 (discovery meta-analysis: ORint = 0.50, p = 7.63∗10-5, replication: ORint = 0.65, p = 0.02). Second approach: 35 SNPs were selected based on the overall genetic effect in exposed subjects (p <10-4). The most pronounced genetic effect was observed for rs5011804 on chromosome 12 (discovery meta-analysis ORint = 1.50, p = 1.21∗10-4; replication: ORint = 1.40, p = 0.03). Conclusions Using two genome-wide interaction approaches, we identified novel polymorphisms in nonannotated intergenic regions on chromosomes 9 and 12, that showed suggestive evidence for interaction with active tobacco smoking in the onset of adult asthma. AU - Vonk, J.M.* AU - Scholtens, S.* AU - Postma, D.S.* AU - Moffatt, M.F.* AU - Jarvis, D.* AU - Ramasamy, A.* AU - Wjst, M. AU - Omenaas, E.R.* AU - Bouzigon, E.* AU - Demenais, F.* AU - Nadif, R.* AU - Siroux, V.* AU - Polonikov, A.V.* AU - Solodilova, M.* AU - Ivanov, V.P.* AU - Curjuric, I.* AU - Imboden, M.* AU - Kumar, A.* AU - Probst-Hensch, N.* AU - Ogorodova, L.M.* AU - Puzyrev, V.P.* AU - Bragina, E.Y.* AU - Freidin, M.B.* AU - Nolte, I.M.* AU - Farrall, A.M.* AU - Cookson, W.O.C.M.* AU - Strachan, D.P.* AU - Koppelman, G.H.* AU - Boezen, H.M.* C1 - 50702 C2 - 42633 CY - San Francisco TI - Adult onset asthma and interaction between genes and active tobacco smoking: The GABRIEL consortium. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Air pollution is a worldwide contributor to cardiovascular disease mortality and morbidity. Traffic-related air pollution is a widespread environmental exposure and is associated with multiple cardiovascular outcomes such as coronary atherosclerosis, peripheral arterial disease, and myocardial infarction. Despite the recognition of the importance of both genetic and environmental exposures to the pathogenesis of cardiovascular disease, studies of how these two contributors operate jointly are rare. We performed a genome-wide interaction study (GWIS) to examine gene-traffic exposure interactions associated with coronary atherosclerosis. Using race-stratified cohorts of 538 African-Americans (AA) and 1562 European-Americans (EA) from a cardiac catheterization cohort (CATHGEN), we identify gene-by-traffic exposure interactions associated with the number of significantly diseased coronary vessels as a measure of chronic atherosclerosis. We found five suggestive (P<1x10-5) interactions in the AA GWIS, of which two (rs1856746 and rs2791713) replicated in the EA cohort (P < 0.05). Both SNPs are in the PIGR-FCAMR locus and are eQTLs in lymphocytes. The protein products of both PIGRand FCAMRare implicated in inflammatory processes. In the EA GWIS, there were three suggestive interactions; none of these replicated in the AA GWIS. All three were intergenic; the most significant interaction was in a regulatory region associated with SAMSN1, a gene previously associated with atherosclerosis and B cell activation. In conclusion, we have uncovered several novel genes associated with coronary atherosclerosis in individuals chronically exposed to increased ambient concentrations of traffic air pollution. These genes point towards inflammatory pathways that may modify the effects of air pollution on cardiovascular disease risk. AU - Ward-Caviness, C.K. AU - Neas, L.M.* AU - Blach, C.* AU - Haynes, C.S.* AU - LaRocque-Abramson, K.* AU - Grass, E.* AU - Elaine Dowdy, Z.* AU - Devlin, R.B.* AU - Diaz-Sanchez, D.* AU - Cascio, W.E.* AU - Miranda, M.L.* AU - Gregory, S.G.* AU - Shah, S.H.* AU - Kraus, W.E.* AU - Hauser, E.R.* C1 - 50894 C2 - 42568 CY - San Francisco TI - A genome-wide trans-ethnic interaction study links the PIGR-FCAMR locus to coronary atherosclerosis via interactions between genetic variants and residential exposure to traffic. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Genome-wide association meta-analyses (GWAMAs) conducted separately by two strata have identified differences in genetic effects between strata, such as sex-differences for body fat distribution. However, there are several approaches to identify such differences and an uncertainty which approach to use. Assuming the availability of stratified GWAMA results, we compare various approaches to identify between-strata differences in genetic effects. We evaluate type I error and power via simulations and analytical comparisons for different scenarios of strata designs and for different types of between-strata differences. For strata of equal size, we find that the genome-wide test for difference without any filtering is the best approach to detect stratum-specific genetic effects with opposite directions, while filtering for overall association followed by the difference test is best to identify effects that are predominant in one stratum. When there is no a priori hypothesis on the type of difference, a combination of both approaches can be recommended. Some approaches violate type I error control when conducted in the same data set. For strata of unequal size, the best approach depends on whether the genetic effect is predominant in the larger or in the smaller stratum. Based on real data from GIANT (>175 000 individuals), we exemplify the impact of the approaches on the detection of sex-differences for body fat distribution (identifying up to 10 loci). Our recommendations provide tangible guidelines for future GWAMAs that aim at identifying between-strata differences. A better understanding of such effects will help pinpoint the underlying mechanisms. AU - Winkler, T.W.* AU - Justice, A.E.* AU - Cupples, L.A.* AU - Kronenberg, F.* AU - Kutalik, Z.* AU - Heid, I.M.* AU - GIANT Consortium (Wichmann, H.-E. AU - Albrecht, E. AU - Gieger, C. AU - Grallert, H. AU - Thorand, B. AU - Illig, T. AU - Müller-Nurasyid, M. AU - Peters, A.) C1 - 51746 C2 - 43422 CY - San Francisco TI - Approaches to detect genetic effects that differ between two strata in genome-wide meta-analyses: Recommendations based on a systematic evaluation. JO - PLoS ONE VL - 12 IS - 7 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma. AU - Wisskirchen, K. AU - Metzger, K. AU - Schreiber, S. AU - Asen, T. AU - Weigand, L.* AU - Dargel, C. AU - Witter, K.* AU - Kieback, E.* AU - Sprinzl, M.F. AU - Uckert, W.* AU - Schiemann, M.* AU - Busch, D.H.* AU - Krackhardt, A.M.* AU - Protzer, U. C1 - 51699 C2 - 43413 CY - San Francisco TI - Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use. JO - PLoS ONE VL - 12 IS - 8 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - Lipid identification is a major bottleneck in high-throughput lipidomics studies. However, tools for the analysis of lipid tandem MS spectra are rather limited. While the comparison against spectra in reference libraries is one of the preferred methods, these libraries are far from being complete. In order to improve identification rates, the in silico fragmentation tool MetFrag was combined with Lipid Maps and lipid-class specific classifiers which calculate probabilities for lipid class assignments. The resulting LipidFrag workflow was trained and evaluated on different commercially available lipid standard materials, measured with data dependent UPLC-Q-ToF-MS/MS acquisition. The automatic analysis was compared against manual MS/MS spectra interpretation. With the lipid class specific models, identification of the true positives was improved especially for cases where candidate lipids from different lipid classes had similar MetFrag scores by removing up to 56% of false positive results. This LipidFrag approach was then applied to MS/MS spectra of lipid extracts of the nematode Caenorhabditis elegans. Fragments explained by LipidFrag match known fragmentation pathways, e.g., neutral losses of lipid headgroups and fatty acid side chain fragments. Based on prediction models trained on standard lipid materials, high probabilities for correct annotations were achieved, which makes LipidFrag a good choice for automated lipid data analysis and reliability testing of lipid identifications. AU - Witting, M. AU - Ruttkies, C.* AU - Neumann, S.* AU - Schmitt-Kopplin, P. C1 - 50745 C2 - 42634 CY - San Francisco TI - LipidFrag: Improving reliability of in silico fragmentation of lipids and application to the Caenorhabditis elegans lipidome. JO - PLoS ONE VL - 12 IS - 3 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Assessment of health benefits associated with physical activity depend on the activity duration, intensity and frequency, therefore their correct identification is very valuable and important in epidemiological and clinical studies. The aims of this study are: to develop an algorithm for automatic identification of intended jogging periods; and to assess whether the identification performance is improved when using two accelerometers at the hip and ankle, compared to when using only one at either position. METHODS: The study used diarized jogging periods and the corresponding accelerometer data from thirty-nine, 15-year-old adolescents, collected under field conditions, as part of the GINIplus study. The data was obtained from two accelerometers placed at the hip and ankle. Automated feature engineering technique was performed to extract features from the raw accelerometer readings and to select a subset of the most significant features. Four machine learning algorithms were used for classification: Logistic regression, Support Vector Machines, Random Forest and Extremely Randomized Trees. Classification was performed using only data from the hip accelerometer, using only data from ankle accelerometer and using data from both accelerometers. RESULTS: The reported jogging periods were verified by visual inspection and used as golden standard. After the feature selection and tuning of the classification algorithms, all options provided a classification accuracy of at least 0.99, independent of the applied segmentation strategy with sliding windows of either 60s or 180s. The best matching ratio, i.e. the length of correctly identified jogging periods related to the total time including the missed ones, was up to 0.875. It could be additionally improved up to 0.967 by application of post-classification rules, which considered the duration of breaks and jogging periods. There was no obvious benefit of using two accelerometers, rather almost the same performance could be achieved from either accelerometer position. CONCLUSIONS: Machine learning techniques can be used for automatic activity recognition, as they provide very accurate activity recognition, significantly more accurate than when keeping a diary. Identification of jogging periods in adolescents can be performed using only one accelerometer. Performance-wise there is no significant benefit from using accelerometers on both locations. AU - Zdravevski, E.* AU - Risteska Stojkoska, B.* AU - Standl, M. AU - Schulz, H. C1 - 51836 C2 - 43496 CY - San Francisco TI - Automatic machine-learning based identification of jogging periods from accelerometer measurements of adolescents under field conditions. JO - PLoS ONE VL - 12 IS - 9 PB - Public Library Science PY - 2017 SN - 1932-6203 ER - TY - JOUR AB - CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. AU - Albert, T.K.* AU - Antrecht, C.* AU - Kremmer, E. AU - Meisterernst, M.* C1 - 47658 C2 - 39168 TI - The establishment of a hyperactive structure allows the tumour suppressor protein p53 to function through P-TEFb during limited CDK9 kinase inhibition. JO - PLoS ONE VL - 11 IS - 1 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Angiotensin-I-converting enzyme (ACE) inhibitors are an important class of antihypertensives whose action on the human organism is still not fully understood. Although it is known that ACE especially cleaves COOH-terminal dipeptides from active polypeptides, the whole range of substrates and products is still unknown. When analyzing the action of ACE inhibitors, effects of genetic variation on metabolism need to be considered since genetic variance in the ACE gene locus was found to be associated with ACE-concentration in blood as well as with changes in the metabolic profiles of a general population. To investigate the interactions between genetic variance at the ACE-locus and the influence of ACE-therapy on the metabolic status we analyzed 517 metabolites in 1,361 participants from the KORA F4 study. We replicated our results in 1,964 individuals from TwinsUK. We observed differences in the concentration of five dipeptides and three ratios of di- and oligopeptides between ACE inhibitor users and non-users that were genotype dependent. Such changes in the concentration affected major homozygotes, and to a lesser extent heterozygotes, while minor homozygotes showed no or only small changes in the metabolite status. Two of these resulting dipeptides, namely aspartylphenylalanine and phenylalanylserine, showed significant associations with blood pressure which qualifies them-and perhaps also the other dipeptides-as readouts of ACE-activity. Since so far ACE activity measurement is substrate specific due to the usage of only one oligopeptide, taking several dipeptides as potential products of ACE into account may provide a broader picture of the ACE activity. AU - Altmaier, E. AU - Menni, C.* AU - Heier, M. AU - Meisinger, C. AU - Thorand, B. AU - Quell, J. AU - Kobl, M. AU - Römisch-Margl, W. AU - Valdes, A.M.* AU - Mangino, M.* AU - Waldenberger, M. AU - Strauch, K. AU - Illig, T. AU - Adamski, J. AU - Spector, T.* AU - Gieger, C. AU - Suhre, K. AU - Kastenmüller, G. C1 - 48490 C2 - 41114 CY - San Francisco TI - The pharmacogenetic footprint of ACE inhibition: A population-based metabolomics study. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Introduction: The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin-induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods: BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX); (n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results: At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1; p<0.01), (IL-1β; p<0.001), (IL-5; p<0.01), (IL-6; p<0.001), (IL-13; p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion: AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model. AU - Andrade-Sousa, A.S.* AU - Rogério Pereira, P.* AU - MacKenzie, B.* AU - Oliveira-Junior, M.C.* AU - Assumpção-Neto, E.* AU - Brandão-Rangel, M.A.* AU - Damaceno-Rodrigues, N.R.* AU - Garcia Caldini, E.* AU - Velosa, A.P.* AU - Teodoro, W.R.* AU - Ligeiro de Oliveira, A.P.* AU - Dolhnikoff, M.* AU - Eickelberg, O. AU - Vieira, R.P.* C1 - 49565 C2 - 40811 CY - San Francisco TI - Aerobic exercise attenuated bleomycin-induced lung fibrosis in Th2-dominant mice. JO - PLoS ONE VL - 11 IS - 9 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Prenatal exposure to stress such as increased level of reactive oxygen species or antiviral therapy are known factors leading to adult heart defects. The risks following a radiation exposure during fetal period are unknown, as are the mechanisms of any potential cardiac damage. The aim of this study was to gather evidence for possible damage by investigating long-term changes in the mouse heart proteome after prenatal exposure to low and moderate radiation doses. Pregnant C57Bl/6J mice received on embryonic day 11 (E11) a single total body dose of ionizing radiation that ranged from 0.02 Gy to 1.0 Gy. The offspring were sacrificed at the age of 6 months or 2 years. Quantitative proteomic analysis of heart tissue was performed using Isotope Coded Protein Label technology and tandem mass spectrometry. The proteomics data were analyzed by bioinformatics and key changes were validated by immunoblotting. Persistent changes were observed in the expression of proteins representing mitochondrial respiratory complexes, redox and heat shock response, and the cytoskeleton, even at the low dose of 0.1 Gy. The level of total and active form of the kinase MAP4K4 that is essential for the embryonic development of mouse heart was persistently decreased at the radiation dose of 1.0 Gy. This study provides the first insight into the molecular mechanisms of cardiac impairment induced by ionizing radiation exposure during the prenatal period. AU - Bakshi, M.V. AU - Azimzadeh, O. AU - Merl-Pham, J. AU - Verreet, T.* AU - Hauck, S.M. AU - Benotmane, M.A.* AU - Atkinson, M.J. AU - Tapio, S. C1 - 48775 C2 - 41322 CY - San Francisco TI - In-utero low-dose irradiation leads to persistent alterations in the mouse heart proteome. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - In recent years, genome-wide association studies (GWAS) have identified many loci that are shared among common disorders and this has raised interest in pleiotropy. For performing appropriate analysis, several methods have been proposed, e.g. conducting a look-up in external sources or exploiting GWAS results by meta-analysis based methods. We recently proposed the Compare & Contrast Meta-Analysis (CCMA) approach where significance thresholds were obtained by simulation. Here we present analytical formulae for the density and cumulative distribution function of the CCMA test statistic under the null hypothesis of no pleiotropy and no association, which, conveniently for practical reasons, turns out to be exponentially distributed. This allows researchers to apply the CCMA method without having to rely on simulations. Finally, we show that CCMA demonstrates power to detect disease-specific, agonistic and antagonistic loci comparable to the frequently used Subset-Based Meta-Analysis approach, while better controlling the type I error rate. AU - Baurecht, H.* AU - Hotze, M.* AU - Rodriguez, E.* AU - Manz, J. AU - Weidinger, S.* AU - Cordell, H.J.* AU - Augustin, T.* AU - Strauch, K. C1 - 48538 C2 - 41132 CY - San Francisco TI - Compare and Contrast Meta Analysis (CCMA): A method for identification of pleiotropic loci in genome-wide association studies. JO - PLoS ONE VL - 11 IS - 5 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Dupuytren´s disease, a fibromatosis of the connective tissue in the palm, is a common complex disease with a strong genetic component. Up to date nine genetic loci have been found to be associated with the disease. Six of these loci contain genes that code for Wnt signalling proteins. In spite of this striking first insight into the genetic factors in Dupuytren´s disease, much of the inherited risk in Dupuytren´s disease still needs to be discovered. The already identified loci jointly explain ~1% of the heritability in this disease. To further elucidate the genetic basis of Dupuytren´s disease, we performed a genome-wide meta-analysis combining three genome-wide association study (GWAS) data sets, comprising 1,580 cases and 4,480 controls. We corroborated all nine previously identified loci, six of these with genome-wide significance (p-value < 5x10-8). In addition, we identified 14 new suggestive loci (p-value < 10-5). Intriguingly, several of these new loci contain genes associated with Wnt signalling and therefore represent excellent candidates for replication. Next, we compared whole-transcriptome data between patient- and control-derived tissue samples and found the Wnt/β-catenin pathway to be the top deregulated pathway in patient samples. We then conducted network and pathway analyses in order to identify protein networks that are enriched for genes highlighted in the GWAS meta-analysis and expression data sets. We found further evidence that the Wnt signalling pathways in conjunction with other pathways may play a critical role in Dupuytren´s disease. AU - Becker, K.* AU - Siegert, S.* AU - Toliat, M.R.* AU - Du, J.* AU - Casper, R.* AU - Dolmans, G.H.* AU - Werker, P.M.* AU - Tinschert, S.* AU - Franke, A.* AU - Gieger, C. AU - Strauch, K. AU - Nothnagel, M.* AU - Nürnberg, P.* AU - Hennies, H.C.* C1 - 49183 C2 - 41706 CY - San Francisco TI - Meta-analysis of genome-wide association studies and network analysis-based integration with gene expression data identify new suggestive loci and unravel a Wnt-centric network associated with Dupuytren's disease. JO - PLoS ONE VL - 11 IS - 7 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Age-related macular degeneration (AMD) is a vision impairing disease of the central retina characterized by early and late forms in individuals older than 50 years of age. However, there is little knowledge to what extent also younger adults are affected. We have thus set out to estimate the prevalence of early AMD features and late AMD in a general adult population by acquiring color fundus images in 2,840 individuals aged 25 to 74 years of the Cooperative Health Research in the Region of Augsburg project (KORA) in South Germany. Among the 2,546 participants with gradable images for each eye, 10.9% (n = 277) had early AMD features (applying the 9-step Age-Related Eye Disease Study Severity Scale), 0.2% (n = 6) had late AMD. Prevalence increased with age, reaching 26.3% for early AMD features and 1.9% for late AMD at the age 70+. However, signs of early AMD were found in subjects as young as 25 years, with the risk for early AMD features increasing linearly by years of age in men, and, less consistent with a linear increase, in women. Risk for early AMD features increased linearly by pack years of smoking in men, not in women, nor was there any association with other lifestyle or metabolic factors. By providing much soughtafter prevalence estimates for AMD from Central Europe, our data underscores a substantial proportion of the adult population with signs of early AMD, including individuals younger than 50 years. This supports the notion that early AMD features in the young might be under-acknowledged. AU - Brandl, C.* AU - Breinlich, V.* AU - Stark, K.J.* AU - Enzinger, S.* AU - Aßenmacher, M.* AU - Olden, M.* AU - Grassmann, F.* AU - Graw, J. AU - Heier, M. AU - Peters, A. AU - Helbig, H.* AU - Küchenhoff, H.* AU - Weber, B.H.F.* AU - Heid, I.M. C1 - 50076 C2 - 42009 CY - San Francisco TI - Features of age-related macular degeneration in the general adults and their dependency on age, sex, and smoking: Results from the German KORA study. JO - PLoS ONE VL - 11 IS - 11 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (similar to 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair. AU - Buchfellner, A.* AU - Yurlova, L.* AU - Nueske, S.* AU - Scholz, A.M.* AU - Bogner, J.* AU - Ruf, B.* AU - Zolghadr, K.* AU - Drexler, S.E.* AU - Drexler, G.A.* AU - Girst, S.* AU - Greubel, C.* AU - Reindl, J.* AU - Siebenwirth, C.* AU - Römer, T.* AU - Friedl, A.A. AU - Rothbauer, U.* C1 - 48254 C2 - 41010 CY - San Francisco TI - A new nanobody-based biosensor to study endogenous PARP1 in vitro and in live human cells. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Background: Chronic widespread musculoskeletal pain (CWP) is the cardinal symptom of fibromyalgia and affects about 12% of the general population. Familial aggregation of CWP has been repeatedly demonstrated with estimated heritabilities of around 50%, indicating a genetic susceptibility. The objective of the study was to explore genome-wide disease-differentially methylated positions (DMPs) for chronic widespread pain (CWP) in a sample of unrelated individuals and a subsample of discordant monozygotic (MZ) twins. Methodology/Principle Findings: A total of N = 281 twin individuals from the TwinsUK registry, including N = 33 MZ twins discordant for self-reported CWP, were part of the discovery sample. The replication sample included 729 men and 756 women from a subsample of the KORA S4 survey-an independent population-based cohort from Southern Germany. Epigenome-wide analysis of DNA methylation was conducted using the Illumina Infinium HumanMethylation 450 DNA Bead Chip in both the discovery and replication sample. Of our 40 main loci that were carried forward for replication, three CPGs reached significant p-values in the replication sample, including malate dehydrogenase 2 (MDH2; p-value 0.017), tetranectin (CLEC3B; p-value 0.039), and heat shock protein beta-6 (HSPB6; p-value 0.016). The associations between the collagen type I, alpha 2 chain (COL1A2) and monoamine oxidase B (MAOB) observed in the discovery sample-both of which have been previously reported to be biological candidates for pain-could not be replicated. Conclusion/Significance: Our results may serve as a starting point to encourage further investigation in large and independent population-based cohorts of DNA methylation and other epigenetic changes as possible disease mechanisms in CWP. Ultimately, understanding the key mechanisms underlying CWP may lead to new treatments and inform clinical practice. AU - Burri, A.* AU - Marinova, Z.* AU - Robinson, M.D.* AU - Kühnel, B. AU - Waldenberger, M. AU - Wahl, S. AU - Kunze, S. AU - Gieger, C. AU - Livshits, G.* AU - Williams, F.M.* C1 - 49953 C2 - 41939 CY - San Francisco TI - Are epigenetic factors implicated in chronic widespread pain? JO - PLoS ONE VL - 11 IS - 11 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Plant growth and development can be influenced by mutualistic and non-mutualistic microorganisms. We investigated the ability of the ericoid endomycorrhizal fungus Oidiodendron maius to influence growth and development of the non-host plant Arabidopsis thaliana. Different experimental setups (non-compartmented and compartmented co-culture plates) were used to investigate the influence of both soluble and volatile fungal molecules on the plant phenotype. O. maius promoted growth of A. thaliana in all experimental setups. In addition, a peculiar clumped root phenotype, characterized by shortening of the primary root and by an increase of lateral root length and number, was observed in A. thaliana only in the non-compartmented plates, suggesting that soluble diffusible molecules are responsible for this root morphology. Fungal auxin does not seem to be involved in plant growth promotion and in the clumped root phenotype because co-cultivation with O. maius did not change auxin accumulation in plant tissues, as assessed in plants carrying the DR5::GUS reporter construct. In addition, no correlation between the amount of fungal auxin produced and the plant root phenotype was observed in an O. maius mutant unable to induce the clumped root phenotype in A. thaliana. Addition of active charcoal, a VOC absorbant, in the compartmented plates did not modify plant growth promotion, suggesting that VOCs are not involved in this phenomenon. The low VOCs emission measured for O. maius further corroborated this hypothesis. By contrast, the addition of CO2 traps in the compartmented plates drastically reduced plant growth, suggesting involvement of fungal CO2 in plant growth promotion. Other mycorrhizal fungi, as well as a saprotrophic and a pathogenic fungus, were also tested with the same experimental setups. In the non-compartmented plates, most fungi promoted A. thaliana growth and some could induce the clumped root phenotype. In the compartmented plate experiments, a general induction of plant growth was observed for most other fungi, especially those producing higher biomass, further strengthening the role of a nonspecific mechanism, such as CO2 emission. AU - Casarrubia, S.* AU - Sapienza, S.* AU - Fritz, H.* AU - Daghino, S.* AU - Rosenkranz, M. AU - Schnitzler, J.-P. AU - Martin, F.* AU - Perotto, S.* AU - Martino, E.* C1 - 50193 C2 - 42105 CY - San Francisco TI - Ecologically different fungi affect arabidopsis development: Contribution of soluble and volatile compounds. JO - PLoS ONE VL - 11 IS - 12 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects. AU - Côme, C.* AU - Cvrljevic, A.* AU - Khan, M.M.* AU - Treise, I. AU - Adler, T. AU - Aguilar-Pimentel, J.A. AU - Au-Yeung, B.* AU - Sittig, E.* AU - Laajala, T.D.* AU - Chen, Y.* AU - Oeder, S. AU - Calzada-Wack, J. AU - Horsch, M. AU - Aittokallio, T.* AU - Busch, D.H.* AU - Ollert, M. AU - Neff, F. AU - Beckers, J. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Chen, Z.* AU - Lahesmaa, R.* AU - Westermarck, J.* C1 - 48457 C2 - 41118 CY - San Francisco TI - CIP2A promotes T-cell activation and immune response to Listeria monocytogenes infection. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - European beech forests growing on marginal calcareous soils have been proposed to be vulnerable to decreased soil water availability. This could result in a large-scale loss of ecological services and economical value in a changing climate. In order to evaluate the potential consequences of this drought-sensitivity, we investigated potential species range shifts for European beech forests on calcareous soil in the 21st century by statistical species range distribution modelling for present day and projected future climate conditions. We found a dramatic decline by 78% until 2080. Still the physiological or biogeochemical mechanisms underlying the drought sensitivity of European beech are largely unknown. Drought sensitivity of beech is commonly attributed to plant physiological constraints. Furthermore, it has also been proposed that reduced soil water availability could promote nitrogen (N) limitation of European beech due to impaired microbial N cycling in soil, but this hypothesis has not yet been tested. Hence we investigated the influence of simulated climate change (increased temperatures, reduced soil water availability) on soil gross microbial N turnover and plant N uptake in the beech-soil interface of a typical mountainous beech forest stocking on calcareous soil in SW Germany. For this purpose, triple 15N isotope labelling of intact beech seedling-soil-microbe systems was combined with a space-for-time climate change experiment. We found that nitrate was the dominant N source for beech natural regeneration. Reduced soil water content caused a persistent decline of ammonia oxidizing bacteria and therefore, a massive attenuation of gross nitrification rates and nitrate availability in the soil. Consequently, nitrate and total N uptake of beech seedlings were strongly reduced so that impaired growth of beech seedlings was observed already after one year of exposure to simulated climatic change. We conclude that the N cycle in this ecosystem and here specifically nitrification is vulnerable to reduced water availability, which can directly lead to nutritional limitations of beech seedlings. This tight link between reduced water availability, drought stress for nitrifiers, decreased gross nitrification rates and nitrate availability and finally nitrate uptake by beech seedlings could represent the Achilles' heel for beech under climate change stresses. AU - Dannenmann, M.* AU - Bimüller, C.* AU - Gschwendtner, S. AU - Leberecht, M.* AU - Tejedor, J.* AU - Bilela, S.* AU - Gasche, R.* AU - Hanewinkel, M.* AU - Baltensweiler, A.* AU - Kögel-Knabner, I.* AU - Polle, A.* AU - Schloter, M. AU - Simon, J.* AU - Rennenberg, H.* C1 - 49078 C2 - 41596 CY - San Francisco TI - Climate change impairs nitrogen cycling in European beech forests. JO - PLoS ONE VL - 11 IS - 7 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The 2013 ACC/AHA guidelines introduced an algorithm for risk assessment of atherosclerotic cardiovascular disease (ASCVD) within 10 years. In Germany, risk assessment with the ESC SCORE is limited to cardiovascular mortality. Applicability of the novel ACC/AHA risk score to the German population has not yet been assessed. We therefore sought to recalibrate and evaluate the ACC/AHA risk score in two German cohorts and to compare it to the ESC SCORE. METHODS: We studied 5,238 participants from the KORA surveys S3 (1994-1995) and S4 (1999-2001) and 4,208 subjects from the Heinz Nixdorf Recall (HNR) Study (2000-2003). There were 383 (7.3%) and 271 (6.4%) first non-fatal or fatal ASCVD events within 10 years in KORA and in HNR, respectively. Risk scores were evaluated in terms of calibration and discrimination performance. RESULTS: The original ACC/AHA risk score overestimated 10-year ASCVD rates by 37% in KORA and 66% in HNR. After recalibration, miscalibration diminished to 8% underestimation in KORA and 12% overestimation in HNR. Discrimination performance of the ACC/AHA risk score was not affected by the recalibration (KORA: C = 0.78, HNR: C = 0.74). The ESC SCORE overestimated by 5% in KORA and by 85% in HNR. The corresponding C-statistic was 0.82 in KORA and 0.76 in HNR. CONCLUSIONS: The recalibrated ACC/AHA risk score showed strongly improved calibration compared to the original ACC/AHA risk score. Predicting only cardiovascular mortality, discrimination performance of the commonly used ESC SCORE remained somewhat superior to the ACC/AHA risk score. Nevertheless, the recalibrated ACC/AHA risk score may provide a meaningful tool for estimating 10-year risk of fatal and non-fatal cardiovascular disease in Germany. AU - de Las Heras Gala, T. AU - Geisel, M.H.* AU - Peters, A. AU - Thorand, B. AU - Baumert, J.J. AU - Lehmann, N.* AU - Jöckel, K.-H.* AU - Moebus, S.* AU - Erbel, R.* AU - Meisinger, C. AU - Mahabadi, A.A.* AU - Koenig, W.* C1 - 49714 C2 - 40895 CY - San Francisco TI - Recalibration of the ACC/AHA risk score in two population-based German cohorts. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Antimicrobial peptides (AMPs) are small peptides with less than 50 amino acids and are part of the innate immune response in almost all organisms, including bacteria, vertebrates, invertebrates and plants. AMPs are active against a broad-spectrum of pathogens. The inducible expression of AMPs in plants is a promising approach to combat plant pathogens with minimal negative side effects, such as phytotoxicity or infertility. In this study, inducible expression of the de-novo designed AMP SP1-1 in Micro Tom tomato protected tomato fruits against bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria. The peptide SP1-1 was targeted to the apoplast which is the primary infection site for plant pathogens, by fusing SP1-1 peptide to the signal peptide RsAFP1 of radish (Raphanus sativus). The pathogen inducibility of the expression was enabled by using an optimized inducible 4XW2/4XS promoter. As a result, the tomato fruits of independently generated SP1-1 transgenic lines were significantly more resistant to X. campestris pv. vesicatoria than WT tomato fruits. In transgenic lines, bacterial infection was reduced up to 65% in comparison to the infection of WT plants. Our study demonstrates that the combination of the 4XW2/4XS cis-element from parsley with the synthetic antimicrobial peptide SP1-1 is a good alternative to protect tomato fruits against infections with X. campestris pv. vesicatoria. AU - Diaz, A.H. AU - Kovacs, I. AU - Lindermayr, C. C1 - 49677 C2 - 40873 CY - San Francisco TI - Inducible expression of the De-Novo designed antimicrobial peptide SP1-1 in tomato confers resistance to Xanthomonas campestris pv. vesicatoria. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - AIMS: B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) predict cardiovascular endpoints in patients and all-cause death in the general population. This was assigned to their association with clinical cardiac remodelling defined as changes in size, shape and function of the heart. The aim of this study was to evaluate whether NT-proBNP and BNP were associated with cardiovascular and overall death independent of clinical cardiac remodelling measured by echocardiography as left ventricular hypertrophy (LVH), diastolic dysfunction and left ventricular ejection fraction (EF). METHODS AND RESULTS: In a general population-based cohort study from Germany (KORA-S3) with subjects' baseline age ranging from 25 to 74 years, cardiac morphology and function were assessed as left ventricular mass (LVM), diastolic dysfunction and EF by echocardiography and circulating NT-proBNP and BNP were measured at baseline. In 1,223 subjects with mortality follow-up information, we examined the association of baseline NT-proBNP and BNP with cardiovascular mortality (number of deaths = 52, median follow-up time = 12.9years) using Cox regression without and with adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF. The risk of cardiovascular mortality increased with higher NT-proBNP levels measured at baseline (hazard ratio HR = 1.67 per unit increment in logNT-proBNP, p = 2.78*10-4, adjusted for age and sex). This increased risk persisted after adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF (HR = 1.73; p = 0.047). When excluding subjects with relevant LVH (LVM to body surface area > 149g/m2 in men / 122g/m2 in women), the NT-proBNP association with mortality was still significant (n = 1,138; number of deaths = 35; HR = 1.48; p = 0.04). We found similar results for BNP. CONCLUSION: Our data confirms NT-proBNP and BNP as predictor of cardiovascular mortality in a large general population-based study with long-term follow-up. Our study extends previously published population-based studies to younger and potentially healthier individuals without relevant LVH, diastolic dysfunction or LVD. AU - Dietl, A.* AU - Stark, K.* AU - Zimmermann, M.E.* AU - Meisinger, C. AU - Schunkert, H.* AU - Birner, C.M.* AU - Maier, L.S.* AU - Peters, A. AU - Heid, I.M.* AU - Luchner, A.* C1 - 49688 C2 - 40876 CY - San Francisco TI - NT-proBNP predicts cardiovascular death in the general population independent of left ventricular mass and function: Insights from a large population-based study with long-term follow-up. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: Early life environment is essential for lung growth and maximally attained lung function. Whether early life exposures impact on lung function decline in adulthood, an indicator of lung ageing, has scarcely been studied. METHODS: Spirometry data from two time points (follow-up time 9-11 years) and information on early life exposures, health and life-style were available from 12862 persons aged 28-73 years participating in the European population-based cohorts SAPALDIA (n = 5705) and ECRHS (n = 7157). The associations of early life exposures with lung function (FEV1) decline were analysed using mixed-effects linear regression. RESULTS: Early life exposures were significantly associated with FEV1 decline, with estimates almost as large as personal smoking. FEV1 declined more rapidly among subjects born during the winter season (adjusted difference in FEV1/year of follow-up [95%CI] -2.04ml [-3.29;-0.80]), of older mothers, (-1.82 ml [-3.14;-0.49]) of smoking mothers (-1.82ml [-3.30;-0.34] or with younger siblings (-2.61ml [-3.85;-1.38]). Less rapid FEV1-decline was found in subjects who had attended daycare (3.98ml [2.78;5.18]), and indicated in subjects with pets in childhood (0.97ml [-0.16;2.09]). High maternal age and maternal smoking appeared to potentiate effects of personal smoking. The effects were independent of asthma at any age. CONCLUSION: Early life factors predicted lung function decline decades later, suggesting that some mechanisms related lung ageing may be established early in life. Early life programming of susceptibility to adult insults could be a possible pathway that should be explored further. AU - Dratva, J.* AU - Zemp, E.* AU - Dharmage, S.C.* AU - Accordini, S.* AU - Burdet, L.* AU - Gislason, T.* AU - Heinrich, J. AU - Janson, C.* AU - Jarvis, D.* AU - de Marco, R.* AU - Norbäck, D.* AU - Pons, M.* AU - Real, F.G.* AU - Sunyer, J.* AU - Villani, S.* AU - Probst-Hensch, N.* AU - Svanes, C.* C1 - 47861 C2 - 39603 CY - San Francisco TI - Early life origins of lung ageing: Early life exposures and lung function decline in adulthood in two European cohorts aged 28-73 years. JO - PLoS ONE VL - 11 IS - 1 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Conflicting findings were observed in recent studies assessing the association between patients' area-level socio-economic status and the received number of computed tomography (CT) examinations in children. The aim was to investigate the association between area-level socio-economic status and variation in CT examination practice for pediatric patients in Germany. METHODS: Data from Radiology Information Systems for children aged 0 to < 15 years without cancer who had at least one CT examination between 2001 and 2010 were extracted in 20 hospitals across Germany. The small-area German Index of Multiple Deprivation (GIMD) was used to assess regional deprivation. The GIMD scores were classified into least, medium and most deprived areas and linked with the patient's last known postal code. A multinomial logistic regression model was used to assess the association between patients' CT numbers and regional deprivation adjusting for age, sex, and location of residence (urban/rural). RESULTS: A total of 37,810 pediatric patients received 59,571 CT scans during the study period. 27,287 (72%) children received only one CT, while n = 885 (2.3%) received six or more. Increasing numbers of CT examinations in non-cancer patients were significantly associated with higher regional deprivation, which increased, although CI overlap, for higher CT categories: '2-3 CT' odds ratio (OR) = 1.45, 95%CI: 1.40-1.50; '4-5 CT' OR = 1.48, 95%CI: 1.38-1.59; '6+CT' OR = 1.54, 95%CI: 1.41-1.69. In addition, male sex, higher age categories, and specific body regions were positively associated with increased numbers of CT examinations. CONCLUSION: We observed a positive association between regional deprivation and CT numbers in non-cancer pediatric patients. Limitations of the ecological approach and the lack of differentiation of CT details have to be acknowledged. More information on CT indications is necessary for a full assessment of this finding. In addition, further work on ways to assess socio-economic status more accurately may be required. AU - Dreger, S.* AU - Krille, L.* AU - Maier, W. AU - Pokora, R.* AU - Blettner, M.* AU - Zeeb, H.* C1 - 48421 C2 - 41074 CY - San Francisco TI - Regional deprivation and non-cancer related computed tomography use in pediatric patients in Germany: Cross-sectional analysis of cohort data. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Background: The aim of the thesis is to improve treatment plans of carbon ion irradiation by integrating the tissues' specific α/β-values for patients with locally advanced pancreatic cancer (LAPC). Material and Methods: Five patients with LAPC were included in this study. By the use of the treatment planning system Syngo RT Planning (Siemens, Erlangen, Germany) treatment plans with carbon ion beams have been created. Dose calculation was based on α/β-values for both organs at risk (OAR) and the tumor. Twenty-five treatment plans and thirty-five forward calculations were created. With reference to the anatomy five field configurations were included. Single Beam Optimization (SBO) and Intensity Modulated Particle Therapy (IMPT) were used for optimization. The plans were analyzed with respect to both dose distributions and individual anatomy. The plans were evaluated using a customized index. Results: With regard to the target, a field setup with one single posterior field achieves the highest score in our index. Field setups made up of three fields achieve good results in OAR sparing. Nevertheless, the field setup with one field is superior in complex topographic conditions. But, allocating an α/β-value of 2 Gy to the spinal cord leads to critical high maximum doses in the spinal cord. The evaluation of dose profiles showed significant dose peaks at borders of the α/β-gradient, especially in case of a single posterior field. Conclusion: Optimization with specific α/β-values allows a more accurate view on dose distribution than previously. A field setup with one single posterior field achieves good results in case of difficult topographic conditions, but leads to high maximum doses to the spinal cord. So, field setups with multiple fields seem to be more adequate in case of LAPC, being surrounded by highly radiosensitive normal tissues. AU - Dreher, C.* AU - Scholz, C.* AU - Pommer, M.* AU - Brons, S.* AU - Prokesch, H.* AU - Ecker, S.* AU - Debus, J.* AU - Jäkel, O.* AU - Combs, S.E. AU - Habermehl, D. C1 - 49807 C2 - 40960 CY - San Francisco TI - Optimization of carbon ion treatment plans by integrating tissue specific α/β-values for patients with non-resectable pancreatic cancer. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions. AU - Gerencser, A.A.* AU - Mookerjee, S.A.* AU - Jastroch, M. AU - Brand, M.D.* C1 - 49871 C2 - 41829 TI - Measurement of the absolute magnitude and time courses of mitochondrial membrane potential in primary and clonal pancreatic beta-cells. JO - PLoS ONE VL - 11 IS - 7 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - The quality and safety of raw milk still remains a worldwide challenge. Culture-dependent methods indicated that the continuous N2 gas-flushing of raw milk reduced the bacterial growth during cold storage by up to four orders of magnitude, compared to cold storage alone. This study investigated the influence of N2 gas-flushing on bacterial diversity in bovine raw-milk samples, that were either cold stored at 6°C or additionally flushed with pure N2 for up to one week. Next-generation sequencing (NGS) of the V1-V2 hypervariable regions of 16S rRNA genes, derived from amplified cDNA, which was obtained from RNA directly isolated from raw-milk samples, was performed. The reads, which were clustered into 2448 operational taxonomic units (OTUs), were phylogenetically classified. Our data revealed a drastic reduction in the diversity of OTUs in raw milk during cold storage at 6°C at 97% similarity level; but, the N2-flushing treatment alleviated this reduction and substantially limited the loss of bacterial diversity during the same cold-storage period. Compared to cold-stored milk, the initial raw-milk samples contained less Proteobacteria (mainly Pseudomonadaceae, Moraxellaceae and Enterobacteriaceae) but more Firmicutes (mainly Ruminococcaceaea, Lachnospiraceae and Oscillospiraceaea) and Bacteroidetes (mainly Bacteroidales). Significant differences between cold-stored and additionally N2-flushed milk were mainly related to higher levels of Pseudomononadaceae (including the genera Pseudomonas and Acinetobacter) in cold-stored milk samples; furthermore, rare taxa were better preserved by the N2 gas flushing compared to the cold storage alone. No major changes in bacterial composition with time were found regarding the distribution of the major 9 OTUs, that dominated the Pseudomonas genus in N2-flushed or non-flushed milk samples, other than an intriguing predominance of bacteria related to P. veronii. Overall, this study established that neither bacteria causing milk spoilage nor any well-known human pathogen or anaerobe benefited from the N2 gas flushing even though the N2-flushed and non-flushed cold-stored milk differed in bacterial counts by up to 104-fold. AU - Gschwendtner, S. AU - Alatossava, T.* AU - Kublik, S. AU - Fuka, M.M.* AU - Schloter, M. AU - Munsch-Alatossava, P.* C1 - 47626 C2 - 39378 TI - N2 gas flushing alleviates the loss of bacterial diversity and inhibits psychrotrophic Pseudomonas during the cold storage of bovine raw milk. JO - PLoS ONE VL - 11 IS - 1 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - We show the error in water-limited yields simulated by crop models which is associated with spatially aggregated soil and climate input data. Crop simulations at large scales (regional, national, continental) frequently use input data of low resolution. Therefore, climate and soil data are often generated via averaging and sampling by area majority. This may bias simulated yields at large scales, varying largely across models. Thus, we evaluated the error associated with spatially aggregated soil and climate data for 14 crop models. Yields of winter wheat and silage maize were simulated under water-limited production conditions. We calculated this error from crop yields simulated at spatial resolutions from 1 to 100 km for the state of North Rhine-Westphalia, Germany. Most models showed yields biased by <15% when aggregating only soil data. The relative mean absolute error (rMAE) of most models using aggregated soil data was in the range or larger than the inter-annual or inter-model variability in yields. This error increased further when both climate and soil data were aggregated. Distinct error patterns indicate that the rMAE may be estimated from few soil variables. Illustrating the range of these aggregation effects across models, this study is a first step towards an ex-ante assessment of aggregation errors in large-scale simulations. AU - Hoffmann, H.* AU - Zhao, G.* AU - Asseng, S.* AU - Bindi, M.* AU - Biernath, C.J. AU - Constantin, J.* AU - Coucheney, E.* AU - Dechow, R.* AU - Doro, L.* AU - Eckersten, H.* AU - Gaiser, T.* AU - Grosz, B.* AU - Heinlein, F. AU - Kassie, B.T.* AU - Kersebaum, K.C.* AU - Klein, C. AU - Kuhnert, M.* AU - Lewan, E.* AU - Moriondo, M.* AU - Nendel, C.* AU - Priesack, E. AU - Raynal, H.* AU - Roggero, P.P.* AU - Rötter, R.P.* AU - Siebert, S.* AU - Specka, X.* AU - Tao, F.* AU - Teixeira, E.* AU - Trombi, G.* AU - Wallach, D.* AU - Weihermüller, L.* AU - Yeluripati, J.* AU - Ewert, F.* C1 - 48292 C2 - 41003 CY - San Francisco TI - Impact of spatial soil and climate input data aggregation on regional yield simulations. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). An accumulation of transition-type changes was found to be responsible for higher evolution rates. The percentages of unedited and edited genomes and the quasispecies complexity showed no relationships with the evolution rate, but the fluctuations in the percentages of genomes and in complexity suggest continuous adaptation of HDV to the host conditions. AU - Homs, M.* AU - Rodriguez-Frias, F.* AU - Gregori, J.* AU - Ruiz, A.* AU - Reimundo, P.* AU - Casillas, R.* AU - Tabernero, D.* AU - Godoy, C.* AU - Barakat, S.* AU - Quer, J.* AU - Riveiro-Barciela, M.* AU - Roggendorf, M. AU - Esteban, R.* AU - Buti, M.* C1 - 48974 C2 - 41508 CY - San Francisco TI - Evidence of an exponential decay pattern of the hepatitis delta virus evolution rate and fluctuations in quasispecies complexity in long-term studies of chronic delta infection. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - A hallmark of the newly published GLI (Global Lungs Initiative) spirometric reference values is their "all-age" (3-95yr) predictive power, accomplished by incorporating non-linear age dependencies into modelling parameters. This modelling strategy is especially promising for the age range of puberty; however, the performance of GLI-values for adolescents is currently unknown. We calculated GLI-based z-scores for children/adolescents without apparent respiratory diseases from two different German studies, LUNOKID (N = 1943, 4-19 years) and GINIplus (N = 1042, 15 years) and determined the goodness of fit for specific age groups. We defined fit sufficient if the absolute mean of z-scores was <0.5. For children (<10yr) the mean GLI-based z-scores for FEV1 and FVC reached a good fit with mean z-scores for FEV1 between -0.11 and 0.01 and mean z-scores for FVC between 0.01 and 0.16, but larger deviations were observed in adolescents, especially boys (mean z-score -0.58 for FEV1 and -0.57 for FVC in GINIplus). The fit for FEV1/FVC was sufficient. GLI reference values provided reasonable estimates for the individuals enrolled in our studies, which span the age range of lung growth and development. However, we found that GLI-predictions overestimated lung volumes, especially those for German adolescent boys, which may, left unrecognised, lead to erroneous diagnosis of lung disease. Caution should be taken when applying these reference values to epidemiologic studies. AU - Hüls, A.* AU - Krämer, U.* AU - Gappa, M.* AU - Müller-Brandes, C.* AU - Schikowski, T.* AU - von Berg, A.* AU - Hoffmann, B.* AU - Schuster, A.* AU - Wisbauer, M.* AU - Flexeder, C. AU - Heinrich, J. AU - Schulz, H. AU - Berdel, D.* C1 - 49129 C2 - 41629 CY - San Francisco TI - Age dependency of GLI reference values compared with paediatric lung function data in two German studies (GINIplus and LUNOKID). JO - PLoS ONE VL - 11 IS - 7 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation. AU - Jung, B. AU - Padula, D. AU - Burtscher, I. AU - Landerer, C. AU - Lutter, D. AU - Theis, F.J. AU - Messias, A.C. AU - Geerlof, A. AU - Sattler, M. AU - Kremmer, E. AU - Boldt, K. AU - Ueffing, M. AU - Lickert, H. C1 - 47950 C2 - 39777 CY - San Francisco TI - Pitchfork and Gprasp2 target smoothened to the primary cilium for Hedgehog pathway activation. JO - PLoS ONE VL - 11 IS - 2 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Phosphorylation is the most widespread and well studied reversible posttranslational modification. Discovering tissue-specific preferences of phosphorylation sites is important as phosphorylation plays a role in regulating almost every cellular activity and disease state. Here we present a comprehensive analysis of global and tissue-specific sequence and structure properties of phosphorylation sites utilizing recent proteomics data. We identified tissue-specific motifs in both sequence and spatial environments of phosphorylation sites. Target site preferences of kinases across tissues indicate that, while many kinases mediate phosphorylation in all tissues, there are also kinases that exhibit more tissue-specific preferences which, notably, are not caused by tissue-specific kinase expression. We also demonstrate that many metabolic pathways are differentially regulated by phosphorylation in different tissues. AU - Karabulut, N.P.* AU - Frishman, D. C1 - 48923 C2 - 41497 CY - San Francisco TI - Sequence- and structure-based analysis of tissue-specific phosphorylation sites. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Gene expression, signal transduction and many other cellular processes are subject to stochastic fluctuations. The analysis of these stochastic chemical kinetics is important for understanding cell-to-cell variability and its functional implications, but it is also challenging. A multitude of exact and approximate descriptions of stochastic chemical kinetics have been developed, however, tools to automatically generate the descriptions and compare their accuracy and computational efficiency are missing. In this manuscript we introduced CERENA, a toolbox for the analysis of stochastic chemical kinetics using Approximations of the Chemical Master Equation solution statistics. CERENA implements stochastic simulation algorithms and the finite state projection for microscopic descriptions of processes, the system size expansion and moment equations for meso- and macroscopic descriptions, as well as the novel conditional moment equations for a hybrid description. This unique collection of descriptions in a single toolbox facilitates the selection of appropriate modeling approaches. Unlike other software packages, the implementation of CERENA is completely general and allows, e.g., for time-dependent propensities and non-mass action kinetics. By providing SBML import, symbolic model generation and simulation using MEX-files, CERENA is user-friendly and computationally efficient. The availability of forward and adjoint sensitivity analyses allows for further studies such as parameter estimation and uncertainty analysis. The MATLAB code implementing CERENA is freely available from http://cerenadevelopers.github.io/CERENA/. AU - Kazeroonian, A. AU - Fröhlich, F. AU - Raue, A.* AU - Theis, F.J. AU - Hasenauer, J. C1 - 47992 C2 - 39817 CY - San Francisco TI - CERENA: ChEmical REaction Network Analyzer - a toolbox for the simulation and analysis of stochastic chemical kinetics. JO - PLoS ONE VL - 11 IS - 1 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Introduction: To understand if and which patients would be open-minded to Complementary and Alternative Medicine (CAM) use parallel to their oncological treatment. Moreover, we sought to determine which methods are most accepted and which are the primary motivators to use CAM. Methods: We developed and anonymously conducted a questionnaire for patients in the oncology center (TU Munich). Questions focus on different CAM methods, previous experiences, and willingness to apply or use CAM when offered in a university-based setting. Results: A total of 171 of 376 patients (37.4% women, 62.0% men, 0.6% unknown) participated. This corresponds to a return rate of 45%. Median age was 64 years (17-87 years). Of all participants, 15.2% used CAM during their oncological therapy; 32.7% have used it in the past. The majority (81.9%) was not using CAM during therapy; 55.5% have not used CAM in the past respectively. The analysis revealed a significant correlation between education and CAM use during therapy (r = 0.18; p = 0.02), and CAM use in the past (r = 0.17; p = 0.04). Of all patients using CAM during therapy, favored methods were food supplements (42.3%), vitamins/minerals (42.3%), massage (34.6%). Motivations are especially the reduction of side effect and stress, the positive effect of certain CAM-treatments on the immune system and tumor therapy. Results showed no difference between women and men. Most patients not having had any experience with CAM complain about the deficiency of information by their treating oncologist (31.4%) as well as missing treatment possibilities (54.3%). Conclusion: Since many patients believe in study results demonstrating the efficacy of CAM, it stresses our task to develop innovative study protocols to investigate the outcomes of certain CAM on symptom reduction or other endpoints. Thus, prospective trials and innovative evidence-based treatment concepts to include CAM into high-end oncology is what patients demand and what a modern oncology center should offer. AU - Kessel, K.A. AU - Lettner, S.* AU - Kessel, C.* AU - Bier, H.* AU - Biedermann, T.* AU - Friess, H.* AU - Herrschbach, P.* AU - Gschwend, J.E.* AU - Meyer, B.* AU - Peschel, C.* AU - Schmid, R.* AU - Schwaiger, M.* AU - Wolff, K.D.* AU - Combs, S.E. C1 - 49926 C2 - 41900 CY - San Francisco TI - Use of Complementary and Alternative Medicine (CAM) as part of the oncological treatment: Survey about patients' attitude towards CAM in a university-based oncology center in Germany. JO - PLoS ONE VL - 11 IS - 11 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Cardiomyopathies represent a rare group of disorders often of genetic origin. While approximately 50% of genetic causes are known for other types of cardiomyopathies, the genetic spectrum of restrictive cardiomyopathy (RCM) is largely unknown. The aim of the present study was to identify the genetic background of idiopathic RCM and to compile the obtained genetic variants to the novel signalling pathways using in silico protein network analysis. PATIENTS AND METHODS: We used Illumina MiSeq setup to screen for 108 cardiomyopathy and arrhythmia-associated genes in 24 patients with idiopathic RCM. Pathogenicity of genetic variants was classified according to American College of Medical Genetics and Genomics classification. RESULTS: Pathogenic and likely-pathogenic variants were detected in 13 of 24 patients resulting in an overall genotype-positive rate of 54%. Half of the genotype-positive patients carried a combination of pathogenic, likely-pathogenic variants and variants of unknown significance. The most frequent combination included mutations in sarcomeric and cytoskeletal genes (38%). A bioinformatics approach underlined the mechanotransducing protein networks important for RCM pathogenesis. CONCLUSIONS: Multiple gene mutations were detected in half of the RCM cases, with a combination of sarcomeric and cytoskeletal gene mutations being the most common. Mutations of genes encoding sarcomeric, cytoskeletal, and Z-line-associated proteins appear to have a predominant role in the development of RCM. AU - Kostareva, A.* AU - Kiselev, A.* AU - Gudkova, A.* AU - Frishman, G. AU - Ruepp, A. AU - Frishman, D. AU - Smolina, N.* AU - Tarnovskaya, S.* AU - Nilsson, D.* AU - Zlotina, A.* AU - Khodyuchenko, T.* AU - Vershinina, T.* AU - Pervunina, T.* AU - Klyushina, A.* AU - Kozlenok, A.* AU - Sjoberg, G.* AU - Golovljova, I.* AU - Sejersen, T.* AU - Shlyakhto, E.* C1 - 49543 C2 - 40765 CY - San Francisco TI - Genetic spectrum of idiopathic restrictive cardiomyopathy uncovered by next-generation sequencing. JO - PLoS ONE VL - 11 IS - 9 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina Human Methylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2×10-5 and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2×10-5 and 3.0×10-3). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-Hadjusted p-value = 1.5×10-9). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes-related traits are associated and that these associations are partially BMI-dependent. Furthermore, the interaction of ABCG1 with glucose metabolism is modulated by epigenetic processes. AU - Kriebel, J. AU - Herder, C.* AU - Rathmann, W.* AU - Wahl, S. AU - Kunze, S. AU - Molnos, S. AU - Volkova, N. AU - Schramm, K. AU - Carstense-Kirberg, M.* AU - Waldenberger, M. AU - Gieger, C. AU - Peters, A. AU - Illig, T.* AU - Prokisch, H. AU - Roden, M.* AU - Grallert, H. C1 - 48413 C2 - 41062 CY - San Francisco TI - Association between DNA methylation in whole blood and measures of glucose metabolism: Kora F4 study. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls. AU - Kumar, S.* AU - Rathkolb, B.* AU - Kemter, E.* AU - Sabrautzki, S. AU - Michel, D. AU - Adler, T. AU - Becker, L. AU - Beckers, J. AU - Busch, D.H.* AU - Garrett, L. AU - Hans, W. AU - Hölter, S.M. AU - Horsch, M. AU - Klingenspor, M.* AU - Klopstock, T.* AU - Rácz, I.* AU - Rozman, J. AU - Vargas Panesso, I.L. AU - Vernaleken, A. AU - Zimmer, A.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Wolf, E.* AU - Aigner, B.* C1 - 48187 C2 - 39979 CY - San Francisco TI - Generation and standardized, systemic phenotypic analysis of Pou3f3L423P mutant mice. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Little is known about the development of the quality of diabetes care in Germany. The aim of this study is to analyze time trends in patient self-management, physician-delivered care, medication, risk factor control, complications and quality of life from 2000 to 2014. METHODS: Analyses are based on data from individuals with type 2 diabetes of the population-based KORA S4 (1999-2001, n = 150), F4 (2006-2008, n = 203), FF4 (2013/14, n = 212) cohort study. Information on patient self-management, physician-delivered care, medication, risk factor control and quality of life were assessed in standardized questionnaires and examinations. The 10-year coronary heart disease (CHD) risk was calculated using the UKPDS risk engine. Time trends were analyzed using multivariable linear and logistic regression models adjusted for age, sex, education, diabetes duration, and history of cardiovascular disease. RESULTS: From 2000 to 2014 the proportion of participants with type 2 diabetes receiving oral antidiabetic/cardio-protective medication and of those reaching treatment goals for glycemic control (HbA1c<7%, 60% to 71%, p = 0.09), blood pressure (<140/80 mmHg, 25% to 69%, p<0.001) and LDL cholesterol (<2.6 mmol/l, 13% to 27%, p<0.001) increased significantly. However, improvements were generally smaller from 2007 to 2014 than from 2000 to 2007. Modeled 10-year CHD risk decreased from 30% in 2000 to 24% in 2007 to 19% in 2014 (p<0.01). From 2007 to 2014, the prevalence of microvascular complications decreased and quality of life increased, but no improvements were observed for the majority of indicators of self-management. CONCLUSION: Despite improvements, medication and risk factor control has remained suboptimal. The flattening of improvements and deteriorations in quality of (self-) care since 2007 indicate that more effort is needed to improve quality of care and patient self-management. Due to selection or lead time bias an overestimation of quality of care improvements cannot be ruled out. AU - Laxy, M. AU - Knoll, G. AU - Schunk, M. AU - Meisinger, C. AU - Huth, C. AU - Holle, R. C1 - 49754 C2 - 40923 CY - San Francisco TI - Quality of diabetes care in Germany improved from 2000 to 2007 to 2014, but improvements diminished since 2007. Evidence from the population-based KORA Studies. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - The construct of sarcopenia is still discussed with regard to best appropriate measures of muscle volume and muscle function. The aim of this post-hoc analysis of a cross-sectional experimental study was to investigate and describe the hierarchy of the association between thigh muscle volume and measurements of functional performance in older women. Thigh muscle volume of 68 independently living older women (mean age 77.6 years) was measured via magnetic resonance imaging. Isometric strength was assessed for leg extension in a movement laboratory in sitting position with the knee flexed at 90 degrees and for hand grip. Maximum and habitual gait speed was measured on an electronic walk way. Leg muscle power was measured during single leg push and during sit-to-stand performance. Thigh muscle volume was associated with sit-to-stand performance power (r = 0.628), leg push power (r = 0.550), isometric quadriceps strength (r = 0.442), hand grip strength (r = 0.367), fast gait speed (r = 0.291), habitual gait speed (r = 0.256), body mass index (r = 0.411) and age (r = -0.392). Muscle power showed the highest association with thigh muscle volume in healthy older women. Sit-to-stand performance power showed an even higher association with thigh muscle volume compared to single leg push power. AU - Lindemann, U.* AU - Mohr, C.* AU - Machann, J. AU - Blatzonis, K.* AU - Rapp, K.* AU - Becker, C.* C1 - 49068 C2 - 41614 CY - San Francisco TI - Association between thigh muscle volume and leg muscle power in older women. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Objective In large cohort studies comorbidities are usually self-reported by the patients. This way to collect health information only represents conditions known, memorized and openly reported by the patients. Several studies addressed the relationship between self-reported comorbidities and medical records or pharmacy data, but none of them provided a structured, documented method of evaluation. We thus developed a detailed procedure to compare self-reported comorbidities with information on comorbidities derived from medication inspection. This was applied to the data of the German COPD cohort COSYCONET. Methods Approach I was based solely on ICD10-Codes for the diseases and the indications of medications. To overcome the limitations due to potential non-specificity of medications, Approach II was developed using more detailed information, such as ATC-Codes specific for one disease. The relationship between reported comorbidities and medication was expressed by a four-level concordance score. Results Approaches I and II demonstrated that the patterns of concordance scores markedly differed between comorbidities in the COSYCONET data. On average, Approach I resulted in more than 50% concordance of all reported diseases to at least one medication. The more specific Approach II showed larger differences in the matching with medications, due to large differences in the disease-specificity of drugs. The highest concordance was achieved for diabetes and three combined cardiovascular disorders, while it was substantial for dyslipidemia and hyperuricemia, and low for asthma. Conclusion Both approaches represent feasible strategies to confirm self-reported diagnoses via medication. Approach I covers a broad spectrum of diseases and medications but is limited regarding disease-specificity. Approach II uses the information from medications specific for a single disease and therefore can reach higher concordance scores. The strategies described in a detailed and reproducible manner are generally applicable in large studies and might be useful to extract as much information as possible from the available data. AU - Lucke, T.* AU - Herrera, M.* AU - Wacker, M. AU - Holle, R. AU - Biertz, F.* AU - Nowak, D.* AU - Huber, R.* AU - Söhler, S.* AU - Vogelmeier, C.* AU - Ficker, J.H.* AU - Mückter, H.* AU - Jörres, R.A.* C1 - 49892 C2 - 41898 CY - San Francisco TI - Systematic analysis of self-reported comorbidities in large cohort studies - A novel stepwise approach by evaluation of medication. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Circadian rhythms govern vital functions. Their disruption provokes metabolic imbalance favouring obesity and type-2 diabetes. The aim of the study was to assess the role of clock genes in human prediabetes. To this end, genotype-phenotype associations of 121 common single nucleotide polymorphisms (SNPs) tagging ARNTL, ARNTL2, CLOCK, CRY1, CRY2, PER1, PER2, PER3, and TIMELESS were assessed in a study population of 1,715 non-diabetic individuals metabolically phenotyped by 5-point oral glucose tolerance tests. In subgroups, hyperinsulinaemic-euglycaemic clamps, intravenous glucose tolerance tests, and magnetic resonance imaging/spectroscopy were performed. None of the tested SNPs was associated with body fat content, insulin sensitivity, or insulin secretion. Four CRY2 SNPs were associated with fasting glycaemia, as reported earlier. Importantly, carriers of these SNPs' minor alleles revealed elevated fasting glycaemia and, concomitantly, reduced liver fat content. In human liver tissue samples, CRY2 mRNA expression was directly associated with hepatic triglyceride content. Our data may point to CRY2 as a novel switch in hepatic fuel metabolism promoting triglyceride storage and, concomitantly, limiting glucose production. The anti-steatotic effects of the glucose-raising CRY2 alleles may explain why these alleles do not increase type-2 diabetes risk. AU - Machicao, F. AU - Peter, A. AU - Machann, J. AU - Koenigsrainer, I.* AU - Böhm, A. AU - Lutz, S.Z. AU - Heni, M. AU - Fritsche, A. AU - Schick, F. AU - Koenigsrainer, A.* AU - Stefan, N. AU - Häring, H.-U. AU - Staiger, H. C1 - 47771 C2 - 39561 TI - Glucose-raising polymorphisms in the human clock gene Cryptochrome 2 (CRY2) affect hepatic lipid content. JO - PLoS ONE VL - 11 IS - 1 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Background Elevated levels of C-reactive protein (CRP, determined by a high-sensitivity assay) indicate low-grade inflammation which is implicated in many age-related disorders. Epigenetic studies on CRP might discover molecular mechanisms underlying CRP regulation. We aimed to identify DNA methylation sites related to CRP concentrations in cells and tissues regulating low-grade inflammation. Results Genome-wide DNA methylation was measured in peripheral blood in 1,741 participants of the KORA F4 study using Illumina HumanMethylation450 BeadChip arrays. Four CpG sites (located at BCL3, AQP3, SOCS3, and cg19821297 intergenic at chromosome 19p13.2, P ≤ 1.01E-07) were significantly hypomethylated at high CRP concentrations independent of various confounders including age, sex, BMI, smoking, and white blood cell composition. Findings were not sex-specific. CRP-related top genes were enriched in JAK/ STAT pathways (Benjamini-Hochberg corrected P < 0.05). Results were followed-up in three studies using DNA from peripheral blood (EPICOR, n = 503) and adipose tissue (TwinsUK, n = 368) measured as described above and from liver tissue (LMU liver cohort, n = 286) measured by MALDI-TOF mass spectrometry using EpiTYPER. CpG sites at the AQP3 locus (significant p-values in peripheral blood = 1.72E-03 and liver tissue = 1.51E-03) and the SOCS3 locus (p-values in liver < 2.82E-05) were associated with CRP in the validation panels. Conclusions Epigenetic modifications seem to engage in low-grade inflammation, possibly via JAK/ STAT mediated pathways. Results suggest a shared relevance across different tissues at the AQP3 locus and highlight a role of DNA methylation for CRP regulation at the SOCS3 locus. AU - Marzi, C. AU - Holdt, L.M.* AU - Fiorito, G.* AU - Tsai, P.C.* AU - Kretschmer, A. AU - Wahl, S. AU - Guarrera, S.* AU - Teupser, D.* AU - Spector, T.D.* AU - Iacoviello, L.* AU - Sacerdote, C.* AU - Strauch, K. AU - Lee, S.* AU - Thasler, W.E.* AU - Peters, A. AU - Thorand, B. AU - Wolf, P. AU - Prokisch, H. AU - Tumino, R.* AU - Gieger, C. AU - Krogh, V.* AU - Panico, S.* AU - Bell, J.T.* AU - Matullo, G.* AU - Waldenberger, M. AU - Grallert, H. AU - Koenig, W.* C1 - 49955 C2 - 41937 CY - San Francisco TI - Epigenetic signatures at AQP3 and SOCS3 engage in low-grade inflammation across different tissues. JO - PLoS ONE VL - 11 IS - 11 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Our study aims to identify metabolic markers associated with either a gain in abdominal (measured by waist circumference) or peripheral (measured by hip circumference) body fat mass. METHODS: Data of 4 126 weight-gaining adults (18-75 years) from three population-based, prospective German cohort studies (EPIC, KORA, DEGS) were analysed regarding a waist-gaining (WG) or hip-gaining phenotype (HG). The phenotypes were obtained by calculating the differences of annual changes in waist minus hip circumference. The difference was displayed for all cohorts. The highest 10% of this difference were defined as WG whereas the lowest 10% were defined as HG. A total of 121 concordant metabolite measurements were conducted using Biocrates AbsoluteIDQ® kits in EPIC and KORA. Sex-specific associations with metabolite concentration as independent and phenotype as the dependent variable adjusted for confounders were calculated. The Benjamini-Hochberg method was used to correct for multiple testing. RESULTS: Across studies both sexes gained on average more waist than hip circumference. We could identify 12 metabolites as being associated with the WG (n = 8) or HG (n = 4) in men, but none were significant after correction for multiple testing; 45 metabolites were associated with the WG (n = 41) or HG (n = 4) in women. For WG, n = 21 metabolites remained significant after correction for multiple testing. Respective odds ratios (OR) ranged from 0.66 to 0.73 for tryptophan, the diacyl-phosphatidylcholines (PC) C32:3, C36:0, C38:0, C38:1, C42:2, C42:5, the acyl-alkyl-PCs C32:2, C34:0, C36:0, C36:1, C36:2, C38:0, C38:2, C40:1, C40:2, C40:5, C40:6, 42:2, C42:3 and lyso-PC C17:0. CONCLUSION: Both weight-gaining men and women showed a clear tendency to gain more abdominal than peripheral fat. Gain of abdominal fat seems to be related to an initial metabolic state reflected by low concentrations of specific metabolites, at least in women. Thus, higher levels of specific PCs may play a protective role in gaining waist circumference. AU - Merz, B.* AU - Nöthlings, U.* AU - Wahl, S. AU - Haftenberger, M.* AU - Schienkiewitz, A.* AU - Adamski, J. AU - Suhre, K. AU - Wang-Sattler, R. AU - Grallert, H. AU - Thorand, B. AU - Pischon, T.* AU - Bachlechner, U.* AU - Floegel, A.* AU - Peters, A. AU - Boeing, H.* C1 - 48842 C2 - 41449 CY - San Francisco TI - Specific metabolic markers are associated with future waist-gaining phenotype in women. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR. AU - Michna, A. AU - Braselmann, H. AU - Selmansberger, M. AU - Dietz, A.* AU - Hess J. AU - Gomolka, M.* AU - Hornhardt, S.* AU - Blüthgen, N.* AU - Zitzelsberger, H. AU - Unger, K. C1 - 49243 C2 - 41783 CY - San Francisco TI - Natural cubic spline regression modeling followed by dynamic network reconstruction for the identification of radiation-sensitivity gene association networks from time-course transcriptome data. JO - PLoS ONE VL - 11 IS - 8 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Background Forced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes. Methods Per-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 children (ALSPAC). Associations were considered replicated if the replication p-value survived Bonferroni correction (p<0.002; 0.05/25), with a nominal p-value considered as suggestive evidence. For SNPs with evidence of replication, effects on the expression levels of nearby genes in lung tissue were tested in 1,111 lung samples (Lung eQTL consortium), with further functional investigation performed using public epigenomic profiling data (ENCODE). Results NCOR2-rs12708369 showed strong replication in children (p = 0.0002), with replication unavailable in adults due to low imputation quality. This intronic variant is in a strong transcriptional enhancer element in lung fibroblasts, but its eQTL effects could not be tested due to low imputation quality in the eQTL dataset. SERPINE2-rs6754561 replicated at nominal level in both adults (p = 0.036) and children (p = 0.045), while WNT16-rs2707469 replicated at nominal level only in adults (p = 0.026). The eQTL analyses showed association of WNT16-rs2707469 with expression levels of the nearby gene CPED1.We found no statistically significant eQTL effects for SERPINE2-rs6754561. Conclusions We have identified a new gene, NCOR2, in the retinoic acid signalling pathway pointing to a role of Vitamin A metabolism in the regulation of FVC. Our findings also support SERPINE2, a COPD gene with weak previous evidence of association with FVC, and suggest WNT16 as a further promising candidate. AU - Minelli, C.* AU - Dean, C.H.* AU - Hind, M.* AU - Alves, A.C.* AU - Amaral, A.F.S.* AU - Siroux, V.* AU - Huikari, V.* AU - Artigas, M.S.* AU - Evans, D.M* AU - Loth, D.W.* AU - Bossé, Y.* AU - Postma, D.S.* AU - Sin, D.* AU - Thompson, J.* AU - Demenais, F.* AU - Henderson, J.* AU - SpiroMeta Consortium (*) AU - CHARGE Consortium (Albrecht, E. AU - Flexeder, C. AU - Gieger, C. AU - Grallert, H. AU - Hampel, R. AU - Heinrich, J. AU - Karrasch, S. AU - Schulz, H. AU - Wjst, M.) AU - Bouzigon, E.* AU - Jarvis, D.* AU - Jarvelin, M.R.* AU - Burney, P.* C1 - 48080 C2 - 39903 TI - Association of forced vital capacity with the developmental gene NCOR2. JO - PLoS ONE VL - 11 IS - 2 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response. AU - Mutschelknaus, L. AU - Peters, C.* AU - Winkler, K. AU - Yentrapalli, R. AU - Heider, T. AU - Atkinson, M.J. AU - Mörtl, S. C1 - 48184 C2 - 41082 CY - San Francisco TI - Exosomes derived from squamous head and neck cancer promote cell survival after ionizing radiation. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Pollen allergies have been rapidly increasing over the last decades. Many allergenic proteins and non-allergenic adjuvant compounds of pollen are involved in the plant defense against environmental or microbial stress. The first aim of this study was to analyze and compare the colonizing microbes on allergenic pollen. The second aim was to investigate detectable correlations between pollen microbiota and parameters of air pollution or pollen allergenicity. To reach these aims, bacterial and fungal DNA was isolated from pollen samples of timothy grass (Phleum pratense, n = 20) and birch trees (Betula pendula, n = 55). With this isolated DNA, a terminal restriction fragment length polymorphism analysis was performed. One result was that the microbial diversity on birch tree and timothy grass pollen samples (Shannon/Simpson diversity indices) was partly significantly correlated to allergenicity parameters (Bet v 1/Phl p 5, pollen-associated lipid mediators). Furthermore, the microbial diversity on birch pollen samples was correlated to on-site air pollution (nitrogen dioxide (NO2), ammonia (NH3), and ozone (O3)). What is more, a significant negative correlation was observed between the microbial diversity on birch pollen and the measured NO2 concentrations on the corresponding trees. Our results showed that the microbial composition of pollen was correlated to environmental exposure parameters alongside with a differential expression of allergen and pollen-associated lipid mediators. This might translate into altered allergenicity of pollen due to environmental and microbial stress. AU - Obersteiner, A. AU - Gilles, S.* AU - Frank, U. AU - Beck, I.* AU - Häring, F.* AU - Ernst, D. AU - Rothballer, M. AU - Hartmann, A. AU - Traidl-Hoffmann, C.* AU - Schmid, M. C1 - 47969 C2 - 39812 CY - San Francisco TI - Pollen-associated microbiome correlates with pollution parameters and the allergenicity of pollen. JO - PLoS ONE VL - 11 IS - 2 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks. AU - Penterling, C.* AU - Drexler, G.A.* AU - Böhland, C.* AU - Stamp, R.* AU - Wilke, C. AU - Braselmann, H. AU - Caldwell, R.B. AU - Reindl, J.* AU - Girst, S.* AU - Greubel, C.* AU - Siebenwirth, C.* AU - Mansour, W.Y.* AU - Borgmann, K.* AU - Dollinger, G.* AU - Unger, K. AU - Friedl, A.A. C1 - 48731 C2 - 41292 CY - San Francisco TI - Depletion of histone demethylase Jarid1A resulting in histone hyperacetylation and radiation sensitivity does not affect DNA double-strand break repair. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - MicroRNAs are involved in almost all biological processes and have emerged as regulators of signaling pathways. We show that miRNA target genes and pathway genes are not uniformly expressed across human tissues. To capture tissue specific effects, we developed a novel methodology for tissue specific pathway analysis of miRNAs. We incorporated the most recent and highest quality miRNA targeting data (TargetScan and StarBase), RNA-seq based gene expression data (EBI Expression Atlas) and multiple new pathway data sources to increase the biological relevance of the predicted miRNA-pathway associations. We identified new potential roles of miR-199a-3p, miR-199b-3p and the miR-200 family in hepatocellular carcinoma, involving the regulation of metastasis through MAPK and Wnt signaling. Also, an association of miR-571 and Notch signaling in liver fibrosis was proposed. To facilitate data update and future extensions of our tool, we developed a flexible database backend using the graph database neo4j. The new backend as well as the novel methodology were included in the updated miTALOS v2, a tool that provides insights into tissue specific miRNA regulation of biological pathways. miTALOS v2 is available at http://mips.helmholtz-muenchen.de/mitalos. AU - Preusse, M. AU - Theis, F.J. AU - Müller, N.S. C1 - 48167 C2 - 39939 CY - San Francisco TI - miTALOS v2: Analyzing tissue specific microRNA function. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a therapeutic option allowing for selectively targeting RPE cells with pathogenic relevance for development of PVR. AU - Priglinger, C.S.* AU - Obermann, J. AU - Szober, C.M.* AU - Merl-Pham, J. AU - Ohmayer, U. AU - Behler, J. AU - Gruhn, F. AU - Kreutzer, T.C.* AU - Wertheimer, C.* AU - Geerlof, A. AU - Priglinger, S.G.* AU - Hauck, S.M. C1 - 47686 C2 - 39713 TI - Epithelial-to-mesenchymal transition of RPE cells in vitro confers increased β1,6-N-glycosylation and increased susceptibility to galectin-3 binding. JO - PLoS ONE VL - 11 IS - 1 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Mounting evidence links prenatal exposure to maternal tobacco smoking with disruption of DNA methylation (DNAm) profile in the blood of infants. However, data on the postnatal stability of such DNAm signatures in childhood, as assessed by Epigenome Wide Association Studies (EWAS), are scarce. Objectives of this study were to investigate DNAm signatures associated with in utero tobacco smoke exposure beyond the 12th week of gestation in whole blood of children at age 5.5 years, to replicate previous findings in young European and American children and to assess their biological role by exploring databases and enrichment analysis. DNA methylation was measured in blood of 366 children of the multicentre European Childhood Obesity Project Study using the Illumina Infinium HM450 Beadchip (HM450K). An EWAS was conducted using linear regression of methylation values at each CpG site against in utero smoke exposure, adjusted for study characteristics, biological and technical effects. Methylation levels at five HM450K probes in MYO1G (cg12803068, cg22132788, cg19089201), CNTNAP2 (cg25949550), and FRMD4A (cg11813497) showed differential methylation that reached epigenome-wide significance according to the false-discovery-rate (FDR) criteria (q-value<0.05). Whereas cg25949550 showed decreased methylation (-2% DNAm ß-value), increased methylation was observed for the other probes (9%: cg12803068; 5%: cg22132788; 4%: cg19089201 and 4%: cg11813497) in exposed relative to non-exposed subjects. This study thus replicates previous findings in children ages 3 to 5, 7 and 17 and confirms the postnatal stability of MYO1G, CNTNAP2 and FRMD4A differential methylation. The role of this differential methylation in mediating childhood phenotypes, previously associated with maternal smoking, requires further investigation. AU - Rzehak, P.* AU - Saffery, R.* AU - Reischl, E. AU - Covic, M.* AU - Wahl, S. AU - Grote, V.* AU - Xhonneux, A.* AU - Langhendries, J.P.* AU - Ferre, N.* AU - Closa-Monasterolo, R.* AU - Verduci, E.* AU - Riva, E.* AU - Socha, P.* AU - Gruszfeld, D.* AU - Koletzko, B.* C1 - 48598 C2 - 41201 CY - San Francisco TI - Maternal smoking during pregnancy and DNA-methylation in children at age 5.5 years: Epigenome-wide-analysis in the European Childhood Obesity Project (CHOP)-study. JO - PLoS ONE VL - 11 IS - 5 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols. AU - Sapcariu, S.C.* AU - Kanashova, T.* AU - Dilger, M.* AU - Diabaté, S.* AU - Oeder, S. AU - Passig, J.* AU - Radischat, C.* AU - Buters, J.T.M. AU - Sippula, O.* AU - Streibel, T. AU - Paur, H.R.* AU - Schläger, C.* AU - Mülhopt, S.* AU - Stengel, B.* AU - Rabe, R.* AU - Harndorf, H.* AU - Krebs, T.* AU - Karg, E.W. AU - Gröger, T.M. AU - Weiss, C.* AU - Dittmar, G.* AU - Hiller, K.* AU - Zimmermann, R. C1 - 48900 C2 - 41477 CY - San Francisco TI - Metabolic profiling as well as stable isotope assisted metabolic and proteomic analysis of RAW 264.7 macrophages exposed to ship engine aerosol emissions: Different effects of heavy fuel oil and refined diesel fuel. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Aims Recent studies have identified unique brain effects of insulin detemir (Levemir (R)). Due to its pharmacologic properties, insulin detemir may reach higher concentrations in the brain than regular insulin. This might explain the observed increased brain stimulation after acute insulin detemir application but it remained unclear whether chronic insulin detemir treatment causes alterations in brain activity as a consequence of overstimulation. Methods In mice, we examined insulin detemir's prolonged brain exposure by continuous subcutaneous (s.c.) application using either micro-osmotic pumps or daily s.c. injections and performed continuous radiotelemetric electrocorticography and locomotion recordings. Results Acute intracerebroventricular injection of insulin detemir activated cortical and locomotor activity significantly more than regular insulin in equimolar doses (0.94 and 5.63mU in total), suggesting an enhanced acute impact on brain networks. However, given continuously s.c., insulin detemir significantly reduced cortical activity (theta: 21.3 +/- 6.1% vs. 73.0 +/- 8.1%, P<0.001) and failed tomaintain locomotion, while regular insulin resulted in an increase of both parameters. Conclusions The data suggest that permanently-increased insulin detemir levels in the brain convert its hyperstimulatory effects and finally mediate impairments in brain activity and locomotion. This observation might be considered when human studies with insulin detemir are designed to target the brain in order to optimize treatment regimens. AU - Sartorius, T. AU - Hennige, A.M. AU - Fritsche, A. AU - Häring, H.-U. C1 - 49646 C2 - 40882 CY - San Francisco TI - Sustained treatment with insulin detemir in mice alters brain activity and locomotion. JO - PLoS ONE VL - 11 IS - 9 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Serum metabolite profiling can be used to identify pathways involved in the pathogenesis of and potential biomarkers for a given disease. Both restless legs syndrome (RLS) and Parkinson`s disease (PD) represent movement disorders for which currently no blood-based biomarkers are available and whose pathogenesis has not been uncovered conclusively. We performed unbiased serum metabolite profiling in search of signature metabolic changes for both diseases. METHODS: 456 metabolites were quantified in serum samples of 1272 general population controls belonging to the KORA cohort, 82 PD cases and 95 RLS cases by liquid-phase chromatography and gas chromatography separation coupled with tandem mass spectrometry. Genetically determined metabotypes were calculated using genome-wide genotyping data for the 1272 general population controls. RESULTS: After stringent quality control, we identified decreased levels of long-chain (polyunsaturated) fatty acids of individuals with PD compared to both RLS (PD vs. RLS: p = 0.0001 to 5.80x10-9) and general population controls (PD vs. KORA: p = 6.09x10-5 to 3.45x10-32). In RLS, inositol metabolites were increased specifically (RLS vs. KORA: p = 1.35x10-6 to 3.96x10-7). The impact of dopaminergic drugs was reflected in changes in the phenylalanine/tyrosine/dopamine metabolism observed in both individuals with RLS and PD. CONCLUSIONS: A first discovery approach using serum metabolite profiling in two dopamine-related movement disorders compared to a large general population sample identified significant alterations in the polyunsaturated fatty acid metabolism in PD and implicated the inositol metabolism in RLS. These results provide a starting point for further studies investigating new perspectives on factors involved in the pathogenesis of the two diseases as well as possible points of therapeutic intervention. AU - Schulte, E.C. AU - Altmaier, E. AU - Berger, H.S. AU - Do, K.T. AU - Kastenmüller, G. AU - Wahl, S. AU - Adamski, J. AU - Peters, A. AU - Krumsiek, J. AU - Suhre, K.* AU - Haslinger, B.* AU - Ceballos-Baumann, A.* AU - Gieger, C. AU - Winkelmann, J. C1 - 47754 C2 - 39468 CY - San Francisco TI - Alterations in lipid and inositol metabolisms in two dopaminergic disorders. JO - PLoS ONE VL - 11 IS - 1 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: There are significant geographical differences in the prevalence and incidence of celiac disease that cannot be explained by HLA alone. More than 40 loci outside of the HLA region have been associated with celiac disease. We investigated the roles of these non-HLA genes in the development of tissue transglutaminase autoantibodies (tTGA) and celiac disease in a large international prospective cohort study. METHODS: A total of 424,788 newborns from the US and European general populations and first-degree relatives with type 1 diabetes were screened for specific HLA genotypes. Of these, 21,589 carried 1 of the 9 HLA genotypes associated with increased risk for type 1 diabetes and celiac disease; we followed 8676 of the children in a 15 y prospective follow-up study. Genotype analyses were performed on 6010 children using the Illumina ImmunoChip. Levels of tTGA were measured in serum samples using radio-ligand binding assays; diagnoses of celiac disease were made based on persistent detection of tTGA and biopsy analysis. Data were analyzed using Cox proportional hazards analyses. RESULTS: We found 54 single-nucleotide polymorphisms (SNPs) in 5 genes associated with celiac disease (TAGAP, IL18R1, RGS21, PLEK, and CCR9) in time to celiac disease analyses (10-4>P>5.8x10-6). The hazard ratios (HR) for the SNPs with the smallest P values in each region were 1.59, 1.45, 2.23, 2.64, and 1.40, respectively. Outside of regions previously associated with celiac disease, we identified 10 SNPs in 8 regions that could also be associated with the disease (P<10-4). A SNP near PKIA (rs117128341, P = 6.5x10-8, HR = 2.8) and a SNP near PFKFB3 (rs117139146, P<2.8x10-7, HR = 4.9) reached the genome-wide association threshold in subjects from Sweden. Analyses of time to detection of tTGA identified 29 SNPs in 2 regions previously associated with celiac disease (CTLA4, P = 1.3x10-6, HR = 0.76 and LPP, P = 2.8x10-5, HR = .80) and 6 SNPs in 5 regions not previously associated with celiac disease (P<10-4); non-HLA genes are therefore involved in development of tTGA. CONCLUSIONS: In conclusion, using a genetic analysis of a large international cohort of children, we associated celiac disease development with 5 non-HLA regions previously associated with the disease and 8 regions not previously associated with celiac disease. We identified 5 regions associated with development of tTGA. Two loci associated with celiac disease progression reached a genome-wide association threshold in subjects from Sweden. AU - Sharma, A.* AU - Liu, X.* AU - Hadley, D.* AU - Hagopian, W.* AU - Liu, E.* AU - Chen, W.M.* AU - Onengut-Gumuscu, S.* AU - Simell, V.* AU - Rewers, M.* AU - Ziegler, A.-G. AU - Lernmark, A.* AU - Simell, O.* AU - Toppari, J.* AU - Krischer, J.P.* AU - Akolkar, B.* AU - Rich, S.S.* AU - Agardh, D.* AU - She, J.X.* AU - TEDDY Study Group (Beyerlein, A. AU - Hummel, M. AU - Hummel, S. AU - Knopff, A. AU - Peplow, C. AU - Roth, R. AU - Stock, J. AU - Strauss, E. AU - Warncke, K. AU - Winkler, C.) C1 - 48192 C2 - 41054 CY - San Francisco TI - Identification of non-HLA genes associated with celiac disease and country-specific differences in a large, international pediatric cohort. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Thyroid disorders are well known to be associated with cardiovascular diseases. Some studies have shown that the negative effects of thyroid disorders are partially reversible after adequate treatment. The aim of this analysis was to assess the risk of incident ischemic cerebrovascular diseases in study participants treated for thyroid dysfunctions in a population-based cohort study. METHODS: For the presented analyses data from 8564 male and 8714 female individuals aged 25 to 74 years of the MONICA/KORA cohort were used (median follow-up 14.0 years). A combined binary variable "thyroid disorder" (TDC) was created utilizing data on self-reported physician-treated thyroid disorders and information about medication use. To examine the association between TDC and incident ischemic cerebrovascular events, we performed multiple adjusted Cox proportional hazard regression models and calculated hazard ratios and corresponding 95% confidence intervals (HR, 95%CI). RESULTS: During follow-up between 1984 and 2008/2009, 514 incident fatal and non-fatal ischemic cerebrovascular events occurred in men and 323 in women. At baseline, 3.5% of men and 15.6% of women reported TDC. In the fully adjusted model, males who reported TDC had a significantly reduced risk of ischemic cerebrovascular events (HR = 0.52, 95%CI = 0.29-0.92). A similar result was obtained in men, when we utilized information on thyroid hormones use only. For the total study population and for women with TDC we found no association with ischemic cerebrovascular events. CONCLUSIONS: In our longitudinal analyses subjects with treated thyroid diseases had no increased risk of incident ischemic cerebrovascular events. Surprisingly in males, even a significantly reduced risk of incident ischemic cerebrovascular events was found, a result that deserves further clarification. AU - Six-Merker, J. AU - Meisinger, C. AU - Jourdan, C. AU - Heier, M. AU - Hauner, H.* AU - Peters, A. AU - Linseisen, J. C1 - 48625 C2 - 41217 CY - San Francisco TI - Treatment of thyroid dysfunctions decreases the risk of cerebrovascular events in men but not in women: Results of the MONICA/KORA cohort study. JO - PLoS ONE VL - 11 IS - 5 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Background: Physical activity (PA) is a well-known and underused protective factor for numerous health outcomes, and interventions are hampered by lack of objective data.We combined accelerometers with diaries to estimate the contributions to total activity from different domains throughout the day and week in adolescents. Methods: Accelerometric and diary data from 1403 adolescents (45% male, mean age 15.6 ± 0.5 years) were combined to evaluate daily levels and domains of sedentary, light, and moderateto-vigorous activity (MVPA) during a typical week. Freedson's cutoff points were applied to determine levels of activity. Total activity was broken down into school physical education (PE), school outside PE, transportation to school, sport, and other time. Results: About 2/3 of adolescents' time was spent sedentary, 1/3 in light activity, and about 5% in MVPA. Boys and girls averaged 46 (SD 22) and 38 (23) minutes MVPA per day. Adolescents were most active during leisure sport, spending about 30% of it in MVPA, followed by PE (about 20%) transport to school (14%) and either school class time or other time (3%). PE provided 5% of total MVPA, while leisure sport provided 16% and transportation to school 8%. School was the most sedentary part of the day with over 75% of time outside PE spent sedentary. Conclusions: These German adolescents were typical of Europeans in showing low levels of physical activity, with significant contributions from leisure sport, transportation and school PE. Leisure sport was the most active part of the day, and participation did not vary significantly by sex, study center (region of Germany) or BMI. Transportation to school was frequent and thus accounted for a significant fraction of total MVPA. This indicates that even in a population with good access to dedicated sporting activities, frequent active transportation can add significantly to total MVPA. AU - Smith, M. AU - Berdel, D.* AU - Nowak, D.* AU - Heinrich, J. AU - Schulz, H. C1 - 48416 C2 - 41059 CY - San Francisco TI - Physical activity levels and domains assessed by accelerometry in German adolescents from GINIplus and LISAplus. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Physical activity (PA) protects against most noncommunicable diseases and has been associated with decreased risk of allergic phenotype, which is increasing worldwide. However, the association is not always present; furthermore it is not clear whether it is strongest for asthma, rhinitis, symptoms of these, or atopic sensitization; which sex is most affected; or whether it can be explained by either avoidance of sport or exacerbation of symptoms by exercise. Interventions are thus difficult to target. METHODS: PA was measured by one-week accelerometry in 1137 Germans (mean age 15.6 years, 47% boys) from the GINIplus and LISAplus birth cohorts, and modeled as a correlate of allergic symptoms, sensitization, or reported doctor-diagnosed asthma or rhinitis. RESULTS: 8.3% of children had asthma, of the remainder 7.9% had rhinitis, and of the remainder 32% were sensitized to aero-allergens (atopic). 52% were lung-healthy controls. Lung-healthy boys and girls averaged 46.4 min and 37.8 min moderate-to-vigorous PA per day, of which 14.6 and 11.4 min was vigorous. PA in allergic girls was not altered, but boys with asthma got 13% less moderate and 29% less vigorous PA, and those with rhinitis with 13% less moderate PA, than lung-healthy boys. Both sexes participated comparably in sport (70 to 84%). Adolescents with wheezing (up to 68%, in asthma) and/or nose/eye symptoms (up to 88%, in rhinitis) were no less active. CONCLUSIONS: We found that asthma and rhinitis, but not atopy, were independently associated with low PA in boys, but not in girls. These results indicate that allergic boys remain a high-risk group for physical inactivity even if they participate comparably in sport. Research into the link between PA and allergy should consider population-specific and sex-specific effects, and clinicians, parents, and designers of PA interventions should specifically address PA in allergic boys to ensure full participation. AU - Smith, M. AU - Berdel, D.* AU - Bauer, C.P.* AU - Koletzko, S.* AU - Nowak, D.* AU - Heinrich, J. AU - Schulz, H. C1 - 49366 C2 - 41796 CY - San Francisco TI - Asthma and rhinitis are associated with less objectively-measured moderate and vigorous physical activity, but similar sport participation, in adolescent German boys: GINIplus and LISAplus cohorts. JO - PLoS ONE VL - 11 IS - 8 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Type 2 diabetes mellitus (T2D) shows regional differences in Germany. The purpose of the project was to compare the prevalence of prediabetes and undiagnosed T2D in two regions in Germany, the Northeast and the South, and to evaluate their associations with regional variations in lifestyle factors and hypertension. METHODS: Data from the KORA-F4 study (South Germany, 2006-2008) and the SHIP-TREND study (Northeast Germany, 2008-2012) were used. Participants aged 35-79 years without T2D with an overnight fasting of more than 8 hours and an oral glucose tolerance test were included: KORA-F4: n = 2,616 and SHIP-TREND: n = 1,968. RESULTS: The prevalence of prediabetes/newly diagnosed T2D was especially high in men (about 60%) and women (about 50%) in the Northeast, followed by men (about 50%) and women (about 30%) in the South. Lifestyle factors associated with T2D varied between the regions: more participants in the Northeast were active smokers and the percentages of people with overweight or obesity were greater than in their southern counterparts. However, these differences could not explain the striking disparity in prediabetes/newly diagnosed T2D. The frequency of hypertension was also distinctly higher in the Northeast than in the South and clearly associated with prediabetes/newly diagnosed T2D. Especially in men living in the Northeast, screening individuals with blood pressure ≥ 140/90mmHg might reveal up to 70% of those with prediabetes/newly diagnosed T2D. CONCLUSIONS: Knowledge about regional variability in T2D and related risk factors is important for the planning of diabetes prevention programs. In our analyses, common lifestyle factors did not nearly explain these variations between the northern SHIP-TREND and the southern KORA-F4 studies. Further examinations of regional socioeconomic, political, environmental and other aspects are needed. Meanwhile, targeted diabetes prevention strategies with a special focus on men living in the northern parts of Germany are reasonable. AU - Stöckl, D. AU - Rückert, I.-M. AU - Heier, M. AU - Peters, A. AU - Schipf, S.* AU - Krabbe, C.* AU - Völzke, H.* AU - Tamayo, T.* AU - Rathmann, W. AU - Meisinger, C. C1 - 48776 C2 - 41321 CY - San Francisco TI - Regional variability of lifestyle factors and hypertension with prediabetes and newly diagnosed type 2 diabetes mellitus: The population-based KORA-F4 and SHIP-TREND studies in Germany. JO - PLoS ONE VL - 11 IS - 6 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of noninvasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radio-sensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease. AU - Storch, K.* AU - Dickreuter, E.* AU - Artati, A. AU - Adamski, J. AU - Cordes, N.* C1 - 50190 C2 - 42230 CY - San Francisco TI - BEMER electromagnetic field therapy reduces cancer cell radioresistance by enhanced ROS formation and induced DNA Damage. JO - PLoS ONE VL - 11 IS - 12 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: Numerous genetic loci have been associated with measures of central fat accumulation, such as waist-to-hip ratio adjusted for body mass index (WHRadjBMI). However the mechanisms by which genetic variations influence obesity remain largely elusive. Lipolysis is a key process for regulation of lipid storage in adipocytes, thus is implicated in obesity and its metabolic complications. Here, genetic variants at 36 WHRadjBMI-associated loci were examined for their influence on abdominal subcutaneous adipocyte lipolysis. SUBJECTS AND METHODS: Fasting subcutaneous adipose tissue biopsies were collected from 789 volunteers (587 women and 202 men, body mass index (BMI) range 17.7-62.3 kg/m2). We quantified subcutaneous adipocyte lipolysis, both spontaneous and stimulated by the catecholamine isoprenaline or a cyclic AMP analogue. DNA was extracted from peripheral blood mononuclear cells and genotyping of SNPs associated with WHRadjBMI conducted. The effects on adipocyte lipolysis measures were assessed for SNPs individually and combined in a SNP score. RESULTS: The WHRadjBMI-associated loci CMIP, PLXND1, VEGFA and ZNRF3-KREMEN1 demonstrated nominal associations with spontaneous and/or stimulated lipolysis. Candidate genes in these loci have been reported to influence NFκB-signaling, fat cell size and Wnt signalling, all of which may influence lipolysis. SIGNIFICANCE: This report provides evidence for specific WHRadjBMI-associated loci as candidates to modulate adipocyte lipolysis. Additionally, our data suggests that genetically increased central fat accumulation is unlikely to be a major cause of altered lipolysis in abdominal adipocytes. AU - Strawbridge, R.J.* AU - Laumen, H. AU - Hamsten, A.* AU - Breier, M. AU - Grallert, H. AU - Hauner, H. AU - Arner, P.* AU - Dahlman, I.* C1 - 48519 C2 - 41116 CY - San Francisco TI - Effects of genetic loci associated with central obesity on adipocyte lipolysis. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - This prospective, randomized, placebo-controlled, double-blinded phase I clinical trial investigates safety and efficacy of botulinum toxin (BoNT) to preserve gland function after radiotherapy in patients with head and neck cancer. Twelve patients with advanced head and neck cancer were injected with BoNT into the submandibular glands prior to primary radiochemotherapy. Six patients received BoNT/A and 6 patients BoNT/A and B, half of each subgroup into their left and the other half into their right gland. As an internal control, sodium chloride was injected into the respective contralateral gland (placebo). For the evaluation of the salivary gland function, technetium pertechnetate salivary gland scintigraphy was performed before and after the end of radiotherapy. BoNT/A and B were well tolerated. Analysis of the scintigraphic data revealed no statistically significant difference between BoNT and placebo regarding the scintigraphic uptake difference (pBoNT/A = 0.84 and pBoNT/A-B = 0.56 for BoNT/A vs. placebo and BoNT/A-B vs. placebo, respectively). We also found no significant difference in treatment between BoNT and placebo in terms of salivary excretion fraction (pBoNT/A = 0.44; pBoNT/A-B = 0.44). This study demonstrates that BoNT can be safely combined with radiochemotherapy. Dosing and timing of BoNT injection should be further investigated for efficacy analysis. TRIAL REGISTRATION: German Registry for Clinical Trails DRKS00004595. AU - Teymoortash, A.* AU - Pfestroff, A.* AU - Wittig, A.* AU - Franke, N.* AU - Hoch, S.* AU - Harnisch, S.* AU - Schade-Brittinger, C.* AU - Hoeffken, H.* AU - Engenhart-Cabillic, R.* AU - Brugger, M. AU - Strauch, K. C1 - 48431 C2 - 41099 CY - San Francisco TI - Safety and efficacy of botulinum toxin to preserve gland function after radiotherapy in patients with head and neck cancer: A prospective, randomized, placebo-controlled, double-blinded phase I clinical trial. JO - PLoS ONE VL - 11 IS - 3 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. METHODS: We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. RESULTS AND CONCLUSION: We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted. AU - Volckmar, A.L.* AU - Han, C.T.* AU - Pütter, C.* AU - Haas, S.* AU - Vogel, C.I.* AU - Knoll, N.* AU - Struve, C.* AU - Göbel, M.* AU - Haas, K.* AU - Herrfurth, N.* AU - Jarick, I.* AU - Grallert, H. AU - Schürmann, A.* AU - Al-Hasani, H.* AU - Hebebrand, J.* AU - Sauer, S.* AU - Hinney, A.* C1 - 47797 C2 - 39506 CY - San Francisco TI - Analysis of genes involved in body weight regulation by targeted re-sequencing. JO - PLoS ONE VL - 11 IS - 2 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic ("traffic exposure")-a recognized vascular disease risk factor-on peripheral arterial disease (BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10(-8)) in European-Americans. Rs755249 is located in the 3' untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes. AU - Ward-Caviness, C.K. AU - Neas, L.M.* AU - Blach, C.* AU - Haynes, C.S.* AU - LaRocque-Abramson, K.* AU - Grass, E.* AU - Dowdy, E.* AU - Devlin, R.B.* AU - Diaz-Sanchez, D.* AU - Cascio, W.E.* AU - Miranda, M.L.* AU - Gregory, S.G.* AU - Shah, S.H.* AU - Kraus, W.E.* AU - Hauser, E.R.* C1 - 48619 C2 - 41224 CY - San Francisco TI - Genetic variants in the bone morphogenic protein gene family modify the association between residential exposure to traffic and peripheral arterial disease. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - People spend most of their time inside buildings and the indoor microbiome is a major part of our everyday environment. It affects humans' wellbeing and therefore its composition is important for use in inferring human health impacts. It is still not well understood how environmental conditions affect indoor microbial communities. Existing studies have mostly focussed on the local (e.g., building units) or continental scale and rarely on the regional scale, e.g. a specific metropolitan area. Therefore, we wanted to identify key environmental determinants for the house dust microbiome from an existing collection of spatially (area of Munich, Germany) and temporally (301 days) distributed samples and to determine changes in the community as a function of time. To that end, dust samples that had been collected once from the living room floors of 286 individual households, were profiled for fungal and bacterial community variation and diversity using microbial fingerprinting techniques. The profiles were tested for their association with occupant behaviour, building characteristics, outdoor pollution, vegetation, and urbanization. Our results showed that more environmental and particularly outdoor factors (vegetation, urbanization, airborne particulate matter) affected the community composition of indoor fungi than of bacteria. The passage of time affected fungi and, surprisingly, also strongly affected bacteria. We inferred that fungal communities in indoor dust changed semi-annually, whereas bacterial communities paralleled outdoor plant phenological periods. These differences in temporal dynamics cannot be fully explained and should be further investigated in future studies on indoor microbiomes. AU - Weikl, F. AU - Tischer, C.G. AU - Probst, A.J.* AU - Heinrich, J. AU - Markevych, I.* AU - Jochner, S.* AU - Pritsch, K. C1 - 48456 C2 - 41166 CY - San Francisco TI - Fungal and bacterial communities in indoor dust follow different environmental determinants. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes. AU - Weisschuh, N.* AU - Mayer, A.K.* AU - Strom, T.M. AU - Kohl, S.* AU - Glöckle, N.* AU - Schubach, M.* AU - Andreasson, S.* AU - Bernd, A.* AU - Birch, D.G.* AU - Hamel, C.P.* AU - Heckenlively, J.R.* AU - Jacobson, S.G.* AU - Kamme, C.* AU - Kellner, U.* AU - Kunstmann, E.* AU - Maffei, P.* AU - Reiff, C.M.* AU - Rohrschneider, K.* AU - Rosenberg, T.* AU - Rudolph, G.* AU - Vámos, R.* AU - Varsányi, B.* AU - Weleber, R.G.* AU - Wissinger, B.* C1 - 47690 C2 - 39701 TI - Mutation detection in patients with retinal dystrophies using targeted next generation sequencing. JO - PLoS ONE VL - 11 IS - 1 PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. AU - Wittmann, A. AU - Grimm, M.* AU - Scherthan, H.* AU - Horsch, M. AU - Beckers, J. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Ford, S.J. AU - Burton, N.C. AU - Razansky, D. AU - Trümbach, D. AU - Aichler, M. AU - Walch, A.K. AU - Calzada-Wack, J. AU - Neff, F. AU - Wurst, W. AU - Hartmann, T.* AU - Floß, T. C1 - 49828 C2 - 40952 CY - San Francisco TI - Sphingomyelin synthase 1 is essential for male fertility in mice. JO - PLoS ONE VL - 11 IS - 10 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Metabolomic profiling is a powerful approach to characterize human metabolism and help understand common disease risk. Although multiple high-throughput technologies have been developed to assay the human metabolome, no technique is capable of capturing the entire human metabolism. Large-scale metabolomics data are being generated in multiple cohorts, but the datasets are typically profiled using different metabolomics platforms. Here, we compared analyses across two of the most frequently used metabolomic platforms, Biocrates and Metabolon, with the aim of assessing how complimentary metabolite profiles are across platforms. We profiled serum samples from 1,001 twins using both targeted (Biocrates, n = 160 metabolites) and non-targeted (Metabolon, n = 488 metabolites) mass spectrometry platforms. We compared metabolite distributions and performed genome-wide association analyses to identify shared genetic influences on metabolites across platforms. Comparison of 43 metabolites named for the same compound on both platforms indicated strong positive correlations, with few exceptions. Genome-wide association scans with high-throughput metabolic profiles were performed for each dataset and identified genetic variants at 7 loci associated with 16 unique metabolites on both platforms. The 16 metabolites showed consistent genetic associations and appear to be robustly measured across platforms. These included both metabolites named for the same compound across platforms as well as unique metabolites, of which 2 (nonanoylcarnitine (C9) [Biocrates]/Unknown metabolite X-13431 [Metabolon] and PC aa C28:1 [Biocrates]/1-stearoylglycerol [Metabolon]) are likely to represent the same or related biochemical entities. The results demonstrate the complementary nature of both platforms, and can be informative for future studies of comparative and integrative metabolomics analyses in samples profiled on different platforms. AU - Yet, I.* AU - Menni, C.* AU - Shin, S.Y.* AU - Mangino, M.* AU - Soranzo, N.* AU - Adamski, J. AU - Suhre, K.* AU - Spector, T.D.* AU - Kastenmüller, G. AU - Bell, J.T.* C1 - 48388 C2 - 41026 CY - San Francisco TI - Genetic influences on metabolite levels: A comparison across metabolomic platforms. JO - PLoS ONE VL - 11 IS - 4 PB - Public Library Science PY - 2016 SN - 1932-6203 ER - TY - JOUR AB - Advances in the "omics" field bring about the need for a high number of good quality samples. Many omics studies take advantage of biobanked samples to meet this need. Most of the laboratory errors occur in the pre-analytical phase. Therefore evidence-based standard operating procedures for the pre-analytical phase as well as markers to distinguish between 'good' and 'bad' quality samples taking into account the desired downstream analysis are urgently needed. We studied concentration changes of metabolites in serum samples due to pre-storage handling conditions as well as due to repeated freeze-thaw cycles. We collected fasting serum samples and subjected aliquots to up to four freeze-thaw cycles and to pre-storage handling delays of 12, 24 and 36 hours at room temperature (RT) and on wet and dry ice. For each treated aliquot, we quantified 127 metabolites through a targeted metabolomics approach. We found a clear signature of degradation in samples kept at RT. Storage on wet ice led to less pronounced concentration changes. 24 metabolites showed significant concentration changes at RT. In 22 of these, changes were already visible after only 12 hours of storage delay. Especially pronounced were increases in lysophosphatidylcholines and decreases in phosphatidylcholines. We showed that the ratio between the concentrations of these molecule classes could serve as a measure to distinguish between 'good' and 'bad' quality samples in our study. In contrast, we found quite stable metabolite concentrations during up to four freeze-thaw cycles. We concluded that pre-analytical RT handling of serum samples should be strictly avoided and serum samples should always be handled on wet ice or in cooling devices after centrifugation. Moreover, serum samples should be frozen at or below -80°C as soon as possible after centrifugation. AU - Anton, G. AU - Wilson, R. AU - Yu, Z. AU - Prehn, C. AU - Zukunft, S. AU - Adamski, J. AU - Heier, M. AU - Meisinger, C. AU - Römisch-Margl, W. AU - Wang-Sattler, R. AU - Hveem, K.* AU - Wolfenbuttel, B.H.R.* AU - Peters, A. AU - Kastenmüller, G. AU - Waldenberger, M. C1 - 44330 C2 - 36770 CY - San Francisco TI - Pre-analytical sample quality: Metabolite ratios as an intrinsic marker for prolonged room temperature exposure of serum samples. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function. AU - Bender, J.* AU - Engeholm, M.* AU - Ederer, M.S.* AU - Breu, J.* AU - Møller, T.C.* AU - Michalakis, S.* AU - Rasko, T.* AU - Wanker, E.E.* AU - Biel, M.* AU - Martinez, K.L.* AU - Wurst, W. AU - Deussing, J.M.* C1 - 46782 C2 - 37798 TI - Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) clustering with MAGUKs is mediated via its C-terminal PDZ binding motif. JO - PLoS ONE VL - 10 IS - 9 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis. AU - Braun, P.* AU - Grass, G.* AU - Aceti, A.* AU - Serrecchia, L.* AU - Affuso, A.* AU - Marino, L.* AU - Grimaldi, S.* AU - Pagano, S.* AU - Hanczaruk, M.* AU - Georgi, E.* AU - Northoff, B.* AU - Schöler, A. AU - Schloter, M. AU - Antwerpen, M.* AU - Fasanella, A.* C1 - 46705 C2 - 37749 CY - San Francisco TI - Microevolution of Anthrax from a Young Ancestor (MAYA) suggests a soil-borne life cycle of Bacillus anthracis. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Genomic copy number variants (CNVs) have been implicated in multiple psychiatric disorders, but not much is known about their influence on anxiety disorders specifically. Using next-generation sequencing (NGS) and two additional array-based genotyping approaches, we detected CNVs in a mouse model consisting of two inbred mouse lines showing high (HAB) and low (LAB) anxiety-related behavior, respectively. An influence of CNVs on gene expression in the central (CeA) and basolateral (BLA) amygdala, paraventricular nucleus (PVN), and cingulate cortex (Cg) was shown by a two-proportion Z-test (p = 1.6 x 10-31), with a positive correlation in the CeA (p = 0.0062), PVN (p = 0.0046) and Cg (p = 0.0114), indicating a contribution of CNVs to the genetic predisposition to trait anxiety in the specific context of HAB/LAB mice. In order to confirm anxiety-relevant CNVs and corresponding genes in a second mouse model, we further examined CD-1 outbred mice. We revealed the distribution of CNVs by genotyping 64 CD 1 individuals using a high-density genotyping array (Jackson Laboratory). 78 genes within those CNVs were identified to show nominally significant association (48 genes), or a statistical trend in their association (30 genes) with the time animals spent on the open arms of the elevated plus-maze (EPM). Fifteen of them were considered promising candidate genes of anxiety-related behavior as we could show a significant overlap (permutation test, p = 0.0051) with genes within HAB/LAB CNVs. Thus, here we provide what is to our knowledge the first extensive catalogue of CNVs in CD-1 mice and potential corresponding candidate genes linked to anxiety-related behavior in mice. AU - Brenndörfer, J.* AU - Altmann, A.* AU - Widner-Andrä, R.* AU - Pütz, B.* AU - Czamara, D.* AU - Tilch, E. AU - Kam-Thong, T.* AU - Weber, P.* AU - Rex-Haffner, M.* AU - Bettecken, T.* AU - Bültmann, A.* AU - Müller-Myhsok, B.* AU - Binder, E.E.* AU - Landgraf, R.* AU - Czibere, L.* C1 - 44977 C2 - 37134 CY - San Francisco TI - Connecting anxiety and genomic copy number variation: A genome-wide analysis in CD-1 mice. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND AND OBJECTIVE: Cat allergen concentrations higher than 8 μg/g in settled house dust, have been suggested to provoke exacerbation of allergic respiratory symptoms. However, whether the 8μg/g of indoor cat allergen concentration is indeed the minimal exposure required for triggering the asthma related respiratory symptoms or the development of sensitization has not yet been confirmed. We studied the associations between domestic cat allergen concentrations and allergic symptoms in the European Community Respiratory Health Survey II, with the aim of confirming this suggested threshold. METHODS: Cat allergen concentrations were measured in the mattress dust of 3003 participants from 22 study centres. Levels of specific immunoglobulin E to cat allergens were measured in serum samples using an immunoassay. Information on allergic symptoms, medication use, home environment and smoking was obtained from a face-to-face interview. RESULTS: Domestic cat allergen concentrations were not associated with allergic/ asthmatic symptoms in the entire study population, nor in the subset sensitized to cat allergen. We also found no association among individuals exposed to concentrations higher than 8 μg/g. However, exposure to medium cat allergen concentrations (0.24-0.63 μg/g) was positively associated with reported asthmatic respiratory symptoms in subjects who have experienced allergic symptoms when near animals. CONCLUSIONS: The proposed 8 μg/g threshold of cat allergen concentrations for the exacerbation of allergic/ respiratory symptoms was not confirmed in a general European adult population. Potential biases attributable to avoidance behaviours and an imprecise exposure assessment cannot be excluded. AU - Chen, C.M. AU - Thiering, E. AU - Zock, J.P.* AU - Villani, S.* AU - Olivieri, M.* AU - Modig, L.* AU - Jarvis, D.* AU - Norbäck, D.* AU - Verlato, G.* AU - Heinrich, J. C1 - 45097 C2 - 37249 CY - San Francisco TI - Is there a threshold concentration of cat allergen exposure on respiratory symptoms in adults? JO - PLoS ONE VL - 10 IS - 6 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of four Czech grassland soils affected by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact (17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition of archaeal and bacterial communities in each soil type and CMN. Both short- and long- term cattle impact negatively altered archaeal and bacterial diversity, leading to increase of homogenization of microbial communities in overwintering soils over time. Moreover, strong shifts in the prokaryotic communities were observed in response to cattle overwintering, with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g. Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria, and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes, Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae suggested increased abundance of soil-borne microbes under altered environmental conditions. The observed changes in prokaryotic community composition may have driven corresponding changes in soil functioning. AU - Chronakova, A.* AU - Schloter-Hai, B. AU - Radl, V. AU - Endesfelder, D. AU - Quince, C.* AU - Elhottova, D.* AU - Simek, M.* AU - Schloter, M. C1 - 46577 C2 - 37671 CY - San Francisco TI - Response of archaeal and bacterial soil communities to changes associated with outdoor cattle overwintering. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Abnormal upper arm-forearm muscle synergies after stroke are poorly understood. We investigated whether upper arm function primes paralyzed forearm muscles in chronic stroke patients after Brain-Machine Interface (BMI)-based rehabilitation. Shaping upper arm-forearm muscle synergies may support individualized motor rehabilitation strategies. METHODS: Thirty-two chronic stroke patients with no active finger extensions were randomly assigned to experimental or sham groups and underwent daily BMI training followed by physiotherapy during four weeks. BMI sessions included desynchronization of ipsilesional brain activity and a robotic orthosis to move the paretic limb (experimental group, n = 16). In the sham group (n = 16) orthosis movements were random. Motor function was evaluated with electromyography (EMG) of forearm extensors, and upper arm and hand Fugl-Meyer assessment (FMA) scores. Patients performed distinct upper arm (e.g., shoulder flexion) and hand movements (finger extensions). Forearm EMG activity significantly higher during upper arm movements as compared to finger extensions was considered facilitation of forearm EMG activity. Intraclass correlation coefficient (ICC) was used to test inter-session reliability of facilitation of forearm EMG activity. RESULTS: Facilitation of forearm EMG activity ICC ranges from 0.52 to 0.83, indicating fair to high reliability before intervention in both limbs. Facilitation of forearm muscles is higher in the paretic as compared to the healthy limb (p<0.001). Upper arm FMA scores predict facilitation of forearm muscles after intervention in both groups (significant correlations ranged from R = 0.752, p = 0.002 to R = 0.779, p = 0.001), but only in the experimental group upper arm FMA scores predict changes in facilitation of forearm muscles after intervention (R = 0.709, p = 0.002; R = 0.827, p<0.001). CONCLUSIONS: Residual upper arm motor function primes recruitment of paralyzed forearm muscles in chronic stroke patients and predicts changes in their recruitment after BMI training. This study suggests that changes in upper arm-forearm synergies contribute to stroke motor recovery, and provides candidacy guidelines for similar BMI-based clinical practice. AU - Curado, M.R.* AU - Cossio, E.G.* AU - Broetz, D.* AU - Agostini, M.* AU - Cho, W.* AU - Brasil, F.L.* AU - Yilmaz, O.* AU - Liberati, G.* AU - Lepski, G.* AU - Birbaumer, N. AU - Ramos-Murguialday, A.* C1 - 47239 C2 - 40611 TI - Residual upper arm motor function primes innervation of paretic forearm muscles in chronic stroke after Brain-Machine Interface (BMI) training. JO - PLoS ONE VL - 10 IS - 10 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Application of 3-iodothyronamine (3-T1AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T1AM. However, 3-T1AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T1AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct Gs-, Gi/o-, G12/13-, Gq/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T1AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the Gq/11 pathway but show differences in the basal activity in Gs and MAP kinase signaling. In contrast to mTaar5, 3-T1AM application at hTAAR5 resulted in significant reduction in basal IP3 formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T1AM, exhibiting inhibitory effects on IP3 formation and MAP kinase signaling pathways, but does not mediate Gs signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T1AM-mediated effects may differ between rodents and humans. AU - Dinter, J.* AU - Mühlhaus, J.* AU - Wienchol, C.L.* AU - Yi, C.-X. AU - Nürnberg, D.* AU - Morin, S. AU - Grüters, A.* AU - Köhrle, J.* AU - Schöneberg, T.* AU - Tschöp, M.H.* AU - Krude, H.* AU - Kleinau, G.* AU - Biebermann, H.* C1 - 43446 C2 - 36634 CY - San Francisco TI - Inverse agonistic action of 3-iodothyronamine at the human trace amine-associated receptor 5. JO - PLoS ONE VL - 10 IS - 2 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND AND AIM: Partial pancreatic resection is accompanied not only by a reduction in the islet cell mass but also by a variety of other factors that are likely to interfere with glucose metabolism. The aim of this work was to characterize the patient dynamics of blood glucose homeostasis during the course of partial pancreatic resection and to specify the associated clinico-pathological variables. METHODS: In total, 84 individuals undergoing elective partial pancreatic resection were consecutively recruited into this observational trial. The individuals were assigned based on their fasting glucose or oral glucose tolerance testing results into one of the following groups: (I) deteriorated, (II) stable or (III) improved glucose homeostasis three months after surgery. Co-variables associated with blood glucose dynamics were identified. RESULTS: Of the 84 participants, 25 (30%) displayed a normal oGTT, 17 (20%) showed impaired glucose tolerance, and 10 (12%) exhibited pathological glucose tolerance. Elevated fasting glucose was present in 32 (38%) individuals before partial pancreatic resection. Three months after partial pancreatic resection, 14 (17%) patients deteriorated, 16 (19%) improved, and 54 (64%) retained stable glucose homeostasis. Stability and improvement was associated with tumor resection and postoperative normalization of recently diagnosed glucose dysregulation, preoperatively elevated tumor markers and markers for common bile duct obstruction, acute pancreatitis and liver cell damage. Improvement was linked to preoperatively elevated insulin resistance, which normalized after resection and was accompanied by a decrease in fasting- and glucose-stimulated insulin secretion. CONCLUSIONS: Surgically reversible blood glucose dysregulation diagnosed concomitantly with a (peri-) pancreatic tumor appears secondary to compromised liver function due to tumor compression of the common bile duct and the subsequent increase in insulin resistance. It can be categorized as "cholestasis-induced diabetes" and thereby distinguished from other forms of hyperglycemic disorders. AU - Ehehalt, F. AU - Sturm, D. AU - Rösler, M. AU - Distler, M.* AU - Weitz, J.* AU - Kersting, S. AU - Ludwig, B. AU - Schwanebeck, U.* AU - Saeger, H.-D. AU - Solimena, M. AU - Grützmann, R.* C1 - 46546 C2 - 37718 CY - San Francisco TI - Blood glucose homeostasis in the course of partial pancreatectomy - evidence for surgically reversible diabetes induced by cholestasis. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Cell dispersal (or detachment) is part of the developmental cycle of microbial biofilms. It can be externally or internally induced, and manifests itself in discrete sloughing events, whereby many cells disperse in an instance, or in continuous slower dispersal of single cells. One suggested trigger of cell dispersal is quorum sensing, a cell-cell communication mechanism used to coordinate gene expression and behavior in groups based on population densities. METHOD: To better understand the interplay of colony growth and cell dispersal, we develop a dynamic, spatially extended mathematical model that includes biofilm growth, production of quorum sensing molecules, cell dispersal triggered by quorum sensing molecules, and re-attachment of cells. This is a highly nonlinear system of diffusion-reaction equations that we study in computer simulations. RESULTS: Our results show that quorum sensing induced cell dispersal can be an efficient mechanism for bacteria to control the size of a biofilm colony, and at the same time enhance its downstream colonization potential. In fact we find that over the lifetime of a biofilm colony the majority of cells produced are lost into the aqueous phase, supporting the notion of biofilms as cell nurseries. We find that a single quorum sensing based mechanism can explain both, discrete dispersal events and continuous shedding of cells from a colony. Moreover, quorum sensing induced cell dispersal affects the structure and architecture of the biofilm, for example it might lead to the formation of hollow inner regions in a biofilm colony. AU - Emerenini, B.O.* AU - Hense, B.A. AU - Kuttler, C.* AU - Eberl, H.J.* C1 - 46399 C2 - 37552 CY - San Francisco TI - A mathematical model of quorum sensing induced biofilm detachment. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - It has been suggested that mitochondrial dysfunction has an influence on lipid metabolism. The fact that mitochondrial defects can be accumulated over time as a normal part of aging may explain why cholesterol levels often are altered with age. To test the hypothesis whether mitochondrial variants are associated with lipid profile (total cholesterol, LDL, HDL, and triglycerides) we analyzed a total number of 978 mitochondrial single nucleotide polymorphisms (mtSNPs) in a sample of 2,815 individuals participating in the population-based KORA F4 study. To assess mtSNP association while taking the presence of heteroplasmy into account we used the raw signal intensity values measured on the microarray and applied linear regression. Ten mtSNPs (mt3285, mt3336, mt5285, mt6591, mt6671, mt9163, mt13855, mt13958, mt14000, and mt14580) were significantly associated with HDL cholesterol and one mtSNP (mt15074) with triglycerides levels. These results highlight the importance of the mitochondrial genome among the factors that contribute to the regulation of lipid levels. Focusing on mitochondrial variants may lead to further insights regarding the underlying physiological mechanisms, or even to the development of innovative treatments. Since this is the first mitochondrial genome-wide association analysis (mtGWAS) for lipid profile, further analyses are needed to follow up on the present findings. AU - Flaquer, A. AU - Rospleszcz, S. AU - Reischl, E. AU - Zeilinger, S. AU - Prokisch, H. AU - Meitinger, T. AU - Meisinger, C. AU - Peters, A. AU - Waldenberger, M. AU - Grallert, H. AU - Strauch, K. C1 - 44697 C2 - 36993 CY - San Francisco TI - Mitochondrial GWA analysis of lipid profile identifies genetic variants to be associated with HDL cholesterol and triglyceride levels. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Nonesterified fatty acids (NEFA) play pathophysiological roles in metabolic syndrome and type 2 diabetes (T2D). In this study, we analyzed the fasting NEFA profiles of normoglycemic individuals at risk for T2D (women with a recent history of gestational diabetes (GDM)) in comparison to controls (women after a normoglycemic pregnancy). We also examined the associations of NEFA species with overweight/obesity, body fat distribution and insulin sensitivity. SUBJECTS AND METHODS: Using LC-MS/MS, we analyzed 41 NEFA species in the fasting sera of 111 women (62 post-GDM, 49 controls). Clinical characterization included a five-point oral glucose tolerance test (OGTT), biomarkers and anthropometrics, magnetic resonance imaging (n = 62) and a food frequency questionnaire. Nonparametric tests with Bonferroni correction, binary logistic regression analyses and rank correlations were used for statistical analysis. RESULTS: Women after GDM had a lower molar percentage of total saturated fatty acids (SFA; 38.55% vs. 40.32%, p = 0.0002) than controls. At an explorative level of significance several NEFA species were associated with post-GDM status (with and without adjustment for body mass index (BMI) and HbA1c): The molar percentages of 14:0, 16:0, 18:0 and 18:4 were reduced, whereas those of 18:1, 18:2, 20:2, 24:4, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and total n-6 NEFA were increased. BMI and the amount of body fat correlated inversely with several SFA and MUFA and positively with various PUFA species over the whole study cohort (abs(ρ)≥0.3 for all). 14:0 was inversely and BMI-independently associated with abdominal visceral adiposity. We saw no correlations of NEFA species with insulin sensitivity and the total NEFA concentration was similar in the post-GDM and the control group. CONCLUSION: In conclusion, we found alterations in the fasting NEFA profile associated with a recent history of gestational diabetes, a risk marker for T2D. NEFA composition also varied with overweight/obesity and with body fat distribution, but not with insulin sensitivity. AU - Fugmann, M. AU - Uhl, O.* AU - Hellmuth, C.* AU - Hetterich, H.* AU - Kammer, N.N.* AU - Ferrari, U. AU - Parhofer, K.G.* AU - Koletzko, B.* AU - Seissler, J. AU - Lechner, A. C1 - 44975 C2 - 37135 CY - San Francisco TI - Differences in the serum nonesterified fatty acid profile of young women associated with a recent history of gestational diabetes and overweight/obesity. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC. AU - Giopanou, I.* AU - Lilis, I.* AU - Papaleonidopoulos, V.* AU - Marazioti, A.* AU - Spella, M.* AU - Vreka, M.* AU - Papadaki, H.* AU - Stathopoulos, G.T. C1 - 46289 C2 - 37459 CY - San Francisco TI - Comprehensive evaluation of nuclear factor-κΒ expression patterns in non-small cell lung cancer. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses. AU - Gosens, I.* AU - Kermanizadeh, A.* AU - Jacobsen, N.R.* AU - Lenz, A.-G. AU - Bokkers, B.* AU - de Jong, W.H.* AU - Krystek, P.* AU - Tran, L.* AU - Stone, V.* AU - Wallin, H.* AU - Stöger, T. AU - Cassee, F.R.* C1 - 44837 C2 - 37057 CY - San Francisco TI - Comparative hazard identification by a single dose lung exposure of zinc oxide and silver nanomaterials in mice. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Lipids account for the majority of pulmonary surfactant, which is essential for normal breathing. We asked if interstitial lung diseases (ILD) in children may disrupt alveolar surfactant and give clues for disease categorization. METHODS: Comprehensive lipidomics profiles of broncho-alveolar lavage fluid were generated in 115 children by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Two reference populations were compared to a broad range of children with ILD. RESULTS: Class and species composition in healthy children did not differ from that in children with ILD related to diffuse developmental disorders, chronic tachypnoe of infancy, ILD related to lung vessels and the heart, and ILD related to reactive lymphoid lesions. As groups, ILDs related to the alveolar surfactant region, ILD related to unclear respiratory distress syndrome in the mature neonate, or in part ILD related to growth abnormalities reflecting deficient alveolarisation, had significant alterations of some surfactant specific phospholipids. Additionally, lipids derived from inflammatory processes were identified and differentiated. In children with ABCA3-deficiency from two ILD causing mutations saturated and monounsaturated phosphatidylcholine species with 30 and 32 carbons and almost all phosphatidylglycerol species were severely reduced. In other alveolar disorders lipidomic profiles may be of less diagnostic value, but nevertheless may substantiate lack of significant involvement of mechanisms related to surfactant lipid metabolism. CONCLUSIONS: Lipidomic profiling may identify specific forms of ILD in children with surfactant alterations and characterized the molecular species pattern likely to be transported by ABCA3 in vivo. AU - Griese, M.* AU - Kirmeier, H.G.* AU - Liebisch, G. AU - Rauch, D.* AU - Stückler, F. AU - Schmitz, G.* AU - Zarbock, R.* C1 - 43380 C2 - 36571 CY - San Francisco TI - Surfactant lipidomics in healthy children and childhood interstitial lung disease. JO - PLoS ONE VL - 10 IS - 2 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: To examine the relationship between symptoms of insomnia and sleep duration and incident total (non-fatal plus fatal) strokes, non-fatal strokes, and fatal strokes in a large cohort of men and women from the general population in Germany. METHODS: In four population-based MONICA (monitoring trends and determinants in cardiovascular disease)/KORA (Cooperative Health Research in the Region of Augsburg) surveys conducted between 1984 and 2001, 17,604 men and women (aged 25 to 74 years) were asked about issues like sleep, health behavior, and medical history. In subsequent surveys and mortality follow-ups, incident stroke cases (cerebral hemorrhage, ischemic stroke, transient ischemic attack, unknown stroke type) were gathered prospectively until 2009. Sex-specific hazard ratios (HR) and their 95% confidence intervals (CI) were estimated using sequential Cox proportional hazards regression models. RESULTS: During a mean follow-up of 14 years, 917 strokes (710 non-fatal strokes and 207 fatal strokes) were observed. Trouble falling asleep and difficulty staying asleep were not significantly related to any incident stroke outcome in either sex in the multivariable models. Among men, the HR for the association between short (≤5 hours) and long (≥10 hours) daily sleep duration and total strokes were 1.44 (95% CI: 1.01-2.06) and 1.63 (95% CI: 1.16-2.29), after adjustment for basic confounding variables. As for non-fatal strokes and fatal strokes, in the analyses adjusted for age, survey, education, physical activity, alcohol consumption, smoking habits, body mass index, hypertension, diabetes, and dyslipidemia, the increased risks persisted, albeit somewhat attenuated, but no longer remained significant. Among women, in the multivariable analyses the quantity of sleep was also not related to any stroke outcome. CONCLUSION: In the present study, symptoms of insomnia and exceptional sleep duration were not significantly predictive of incident total strokes, non-fatal strokes, and fatal strokes in either sex. AU - Helbig, A.K. AU - Stöckl, D. AU - Heier, M. AU - Ladwig, K.-H. AU - Meisinger, C. C1 - 46445 C2 - 37541 CY - San Francisco TI - Symptoms of insomnia and sleep duration and their association with incident strokes: Findings from the population-based MONICA/KORA Augsburg cohort study. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - The correct wiring of neuronal circuits is of crucial importance for precise neuromuscular functionality. Therefore, guidance cues provide tight spatiotemporal control of axon growth and guidance. Mice lacking the guidance cue Semaphorin 3F (Sema3F) display very specific axon wiring deficits of motor neurons in the medial aspect of the lateral motor column (LMCm). While these deficits have been investigated extensively during embryonic development, it remained unclear how Sema3F mutant mice cope with these errors postnatally. We therefore investigated whether these animals provide a suitable model for the exploration of adaptive plasticity in a system of miswired neuronal circuitry. We show that the embryonically developed wiring deficits in Sema3F mutants persist until adulthood. As a consequence, these mutants display impairments in motor coordination that improve during normal postnatal development, but never reach wildtype levels. These improvements in motor coordination were boosted to wildtype levels by housing the animals in an enriched environment starting at birth. In contrast, a delayed start of enriched environment housing, at 4 weeks after birth, did not similarly affect motor performance of Sema3F mutants. These results, which are corroborated by neuroanatomical analyses, suggest a critical period for adaptive plasticity in neuromuscular circuitry. Interestingly, the formation of perineuronal nets, which are known to close the critical period for plastic changes in other systems, was not altered between the different housing groups. However, we found significant changes in the number of excitatory synapses on limb innervating motor neurons. Thus, we propose that during the early postnatal phase, when perineuronal nets have not yet been formed around spinal motor neurons, housing in enriched environment conditions induces adaptive plasticity in the motor system by the formation of additional synaptic contacts, in order to compensate for coordination deficits. AU - Helmbrecht, M.S. AU - Soellner, H. AU - Castiblanco-Urbina, M.A. AU - Winzeck, S. AU - Sundermeier, J. AU - Theis, F.J. AU - Fouad, K.* AU - Huber, A.B. C1 - 44420 C2 - 36871 CY - San Francisco TI - A critical period for postnatal adaptive plasticity in a model of motor axon miswiring. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype - envirotype interactions for other diseases. AU - Horsch, M. AU - Aguilar-Pimentel, J.A. AU - Bönisch, C. AU - Côme, C.* AU - Kolster-Fog, C.* AU - Jensen, K.T.* AU - Lund, A.H.* AU - Lee, I.* AU - Grossman, L.I.* AU - Sinkler, C.* AU - Hüttemann, M.* AU - Bohn, E.* AU - Fuchs, H. AU - Ollert, M.* AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 46562 C2 - 37635 CY - San Francisco TI - Cox4i2, Ifit2, and Prdm11 mutant mice: Effective selection of genes predisposing to an altered airway inflammatory response from a large compendium of mutant mouse lines. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Comorbidities are often reported in patients with COPD and may influence the cost of care. Yet, the extent by which comorbidities affect costs remains to be determined. OBJECTIVES: To review, quantify and evaluate excess costs of comorbidities in COPD. METHODS: Using a systematic review approach, Pubmed and Embase were searched for studies analyzing excess costs of comorbidities in COPD. Resulting studies were evaluated according to study characteristics, comorbidity measurement and cost indicators. Mark-up factors were calculated for respective excess costs. Furthermore, a checklist of quality criteria was applied. RESULTS: Twelve studies were included. Nine evaluated comorbidity specific costs; three examined index-based results. Pneumonia, cardiovascular disease and diabetes were associated with the highest excess costs. The mark-up factors for respective excess costs ranged between 1.5 and 2.5 in the majority of cases. On average the factors constituted a doubling of respective costs in the comorbid case. The main cost driver, among all studies, was inpatient cost. Indirect costs were not accounted for by the majority of studies. Study heterogeneity was high. CONCLUSIONS: The reviewed studies clearly show that comorbidities are associated with significant excess costs in COPD. The inclusion of comorbid costs and effects in future health economic evaluations of preventive or therapeutic COPD interventions seems highly advisable. AU - Huber, M.B. AU - Wacker, M. AU - Vogelmeier, C.F.* AU - Leidl, R. C1 - 44419 C2 - 36831 CY - San Francisco TI - Excess costs of comorbidities in chronic obstructive pulmonary disease: A systematic review. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and of loss of disability-adjusted life years worldwide. It often is accompanied by the presence of comorbidity. OBJECTIVES: To systematically review the influence of COPD comorbidity on generic health-related quality of life (HRQoL). METHODS: A systematic review approach was used to search the databases Pubmed, Embase and Cochrane Library for studies evaluating the influence of comorbidity on HRQoL in COPD. Identified studies were analyzed according to study characteristics, generic HRQoL measurement instrument, COPD severity and comorbid HRQoL impact. Studies using only non-generic instruments were excluded. RESULTS: 25 studies met the selection criteria. Seven studies utilized the EQ-5D, six studies each used the SF-36 or SF-12. The remaining studies used one of six other instruments each. Utilities were calculated by four EQ-5D studies and one 15D study. Patient populations covered both early and advanced stages of COPD and ranged from populations with mostly stage 1 and 2 to studies with patients classified mainly stage 3 and 4. Evidence was mainly created for cardiovascular disease, depression and anxiety as well as diabetes but also for quantitative comorbid associations. Strong evidence is pointing towards the significant negative association of depression and anxiety on reduced HRQoL in COPD patients. While all studies found the occurrence of specific comorbidities to decrease HRQoL in COPD patients, the orders of magnitude diverged. Due to different patient populations, different measurement tools and different concomitant diseases the study heterogeneity was high. CONCLUSIONS: Facilitating multimorbid intervention guidance, instead of applying a parsimony based single disease paradigm, should constitute an important goal for improving HRQoL of COPD patients in research and in clinical practice. AU - Huber, M.B. AU - Wacker, M. AU - Vogelmeier, C.F.* AU - Leidl, R. C1 - 46326 C2 - 37499 CY - San Francisco TI - Comorbid influences on generic health-related quality of life in COPD: A systematic review. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - We tested for interactions between body mass index (BMI) and common genetic variants affecting serum urate levels, genome-wide, in up to 42569 participants. Both stratified genome-wide association (GWAS) analyses, in lean, overweight and obese individuals, and regression-type analyses in a non BMI-stratified overall sample were performed. The former did not uncover any novel locus with a major main effect, but supported modulation of effects for some known and potentially new urate loci. The latter highlighted a SNP at RBFOX3 reaching genome-wide significant level (effect size 0.014, 95% CI 0.008-0.02, Pinter= 2.6 x 10-8). Two top loci in interaction term analyses, RBFOX3 and ERO1LB-EDARADD, also displayed suggestive differences in main effect size between the lean and obese strata. All top ranking loci for urate effect differences between BMI categories were novel and most had small magnitude but opposite direction effects between strata. They include the locus RBMS1-TANK (men, Pdifflean-overweight= 4.7 x 10-8), a region that has been associated with several obesity related traits, and TSPYL5 (men, Pdifflean-overweight= 9.1 x 10-8), regulating adipocytes-produced estradiol. The top-ranking known urate loci was ABCG2, the strongest known gout risk locus, with an effect halved in obese compared to lean men (Pdifflean-obese= 2 x 10-4). Finally, pathway analysis suggested a role for N-glycan biosynthesis as a prominent urate-associated pathway in the lean stratum. These results illustrate a potentially powerful way to monitor changes occurring in obesogenic environment. AU - Huffman, J.E.* AU - Albrecht, E. AU - Teumer, A.* AU - Mangino, M.* AU - Kapur, K.* AU - Johnson, T.* AU - Kutalik, Z.* AU - Pirastu, N.* AU - Pistis, G.* AU - Lopez, L.M.* AU - Haller, T.* AU - Salo, P.* AU - Goel, A.* AU - Li, M.* AU - Tanaka, T.* AU - Dehghan, A.* AU - Ruggiero, D.* AU - Malerba, G.* AU - Smith, A.V.* AU - Nolte, I.M.* AU - Portas, L.* AU - Phipps-Green, A.* AU - Boteva, L.* AU - Navarro, P.* AU - Johansson, A.* AU - Hicks, A.A.* AU - Polasek, O.* AU - Esko, T.* AU - Peden, J.F.* AU - Harris, S.E.* AU - Murgia, F.* AU - Wild, S.H.* AU - Tenesa, A.* AU - Tin, A.* AU - Mihailov, E.* AU - Grotevendt, A.* AU - Gislason, G.K.* AU - Coresh, J.* AU - D'Adamo, P.* AU - Ulivi, S.* AU - Vollenweider, P.* AU - Waeber, G.* AU - Campbell, S.* AU - Kolcic, I.* AU - Fisher, K.* AU - Viigimaa, M.* AU - Metter, J.E.* AU - Masciullo, C.* AU - Trabetti, E.* AU - Bombieri, C.* AU - Sorice, R.* AU - Döring, A. AU - Reischl, E. AU - Strauch, K. AU - Hofman, A.* AU - Uitterlinden, A.G.* AU - Waldenberger, M. AU - Wichmann, H.-E. AU - Davies, G.* AU - Gow, A.J.* AU - Dalbeth, N.* AU - Stamp, L.* AU - Smit, J.H.* AU - Kirin, M.* AU - Nagaraja, R.* AU - Nauck, M.* AU - Schurmann, C.* AU - Budde, K.* AU - Farrington, S.M.* AU - Theodoratou, E.* AU - Jula, A.* AU - Salomaa, V.* AU - Sala, C.* AU - Hengstenberg, C.* AU - Burnier, M.* AU - Mägi, R.* AU - Klopp, N.* AU - Kloiber, S.* AU - Schipf, S.* AU - Ripatti, S.* AU - Cabras, S.* AU - Soranzo, N.* AU - Homuth, G.* AU - Nutile, T.* AU - Munroe, P.B.* AU - Hastie, N.* AU - Campbell, H.* AU - Rudan, I.* AU - Cabrera, C.* AU - Haley, C.* AU - Franco, O.H.* AU - Merriman, T.R.* AU - Gudnason, V.* AU - Pirastu, M.* AU - Penninx, B.W.* AU - Snieder, H.* AU - Metspalu, A.* AU - Ciullo, M.* AU - Pramstaller, P.P.* AU - van Duijn, C.M.* AU - Ferrucci, L.* AU - Gambaro, G.* AU - Deary, I.J.* AU - Dunlop, M.G.* AU - Wilson, J.F.* AU - Gasparini, P.* AU - Gyllensten, U.* AU - Spector, T.D.* AU - Wright, A.F.* AU - Hayward, C.* AU - Watkins, H.* AU - Perola, M.* AU - Bochud, M.* AU - Kao, W.H.* AU - Caulfield, M.* AU - Toniolo, D.* AU - Völzke, H.* AU - Gieger, C. AU - Köttgen, A.* AU - Vitart, V.* C1 - 44019 C2 - 36731 CY - San Francisco TI - Modulation of genetic associations with serum urate levels by body-mass-index in humans. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms. AU - Hüttl, R.E. AU - Luxenhofer, G. AU - Bianchi, E. AU - Haupt, C. AU - Joshi, R.* AU - Prochiantz, A.* AU - Huber, A.B. C1 - 43458 C2 - 36659 CY - San Francisco TI - Engrailed 1 mediates correct formation of limb innervation through two distinct mechanisms. JO - PLoS ONE VL - 10 IS - 2 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Biomarker fatty acids (FAs) reflecting de novo lipogenesis (DNL) are strongly linked to the risk of cardiometabolic diseases. Liver fat accumulation could mediate this relation. There is very limited data from human population-based studies that have examined this relation. OBJECTIVE: The aim of this study was to investigate the relation between specific FAs in the DNL pathway and liver fat accumulation in a large population-based study. METHODS: We conducted a cross-sectional analysis of a subsample (n = 1,562) of the EPIC-Potsdam study, which involves 27,548 middle-aged men and women. Baseline blood samples have been analyzed for proportions of 32 FAs in erythrocyte membranes (determined by gas chromatography) and biomarker concentrations in plasma. As indicators for DNL, the DNL-index (16:0 / 18:2n-6) and proportions of individual blood FAs in the DNL pathway were used. Plasma parameters associated with liver fat content (fetuin-A, ALT, and GGT) and the algorithm-based fatty liver index (FLI) were used to reflect liver fat accumulation. RESULTS: The DNL-index tended to be positively associated with the FLI and was positively associated with GGT activity in men (p for trend: 0.12 and 0.003). Proportions of 14:0 and 16:0 in erythrocytes were positively associated with fetuin-A, whereas 16:1n-7 were positively associated with the FLI and GGT activity (all p for trends in both sexes at least 0.004). Furthermore, the proportion of 16:1n-7 was positively related to fetuin-A in women and ALT activity in men (all p for trend at least 0.03). The proportion of 16:1n-9 showed positive associations with the FLI and GGT activity in men and fetuin-A in both sexes, whereas 18:1n-7 was positively associated with GGT activity in men (all p for trend at least 0.048). CONCLUSION: Findings from this large epidemiological study suggest that liver fat accumulation could link erythrocyte FAs in the DNL pathway to the risk of cardiometabolic diseases. AU - Jacobs, S.* AU - Jager, S.* AU - Jansen, E.* AU - Peter, A. AU - Stefan, N. AU - Boeing, H.* AU - Schulze, M.B.* AU - Kröger, J.* C1 - 44888 C2 - 37172 CY - San Francisco TI - Associations of erythrocyte fatty acids in the de novo lipogenesis pathway with proxies of liver fat accumulation in the EPIC-Potsdam study. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Objective Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis. Study Design Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis. Results After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs. Conclusions We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis. AU - Kächele, M.* AU - Hennige, A.M. AU - Machann, J. AU - Hieronimus, A. AU - Lamprinou, A. AU - Machicao, F. AU - Schick, F. AU - Fritsche, A. AU - Stefan, N. AU - Nürnberg, B.* AU - Häring, H.-U. AU - Staiger, H. C1 - 47681 C2 - 39447 TI - Variation in the phosphoinositide 3-kinase gamma gene affects plasma HDL-cholesterol without modification of metabolic or inflammatory markers. JO - PLoS ONE VL - 10 IS - 12 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - The recent advent of conformation capture techniques has provided unprecedented insights into the spatial organization of chromatin. We present a large-scale investigation of the inter-chromosomal segment and gene contact networks in embryonic stem cells of two mammalian organisms: humans and mice. Both interaction networks are characterized by a high degree of clustering of genome regions and the existence of hubs. Both genomes exhibit similar structural characteristics such as increased flexibility of certain Y chromosome regions and co-localization of centromere-proximal regions. Spatial proximity is correlated with the functional similarity of genes in both species. We also found a significant association between spatial proximity and the co-expression of genes in the human genome. The structural properties of chromatin are also species specific, including the presence of two highly interactive regions in mouse chromatin and an increased contact density on short, gene-rich human chromosomes, thereby indicating their central nuclear position. Trans-interacting segments are enriched in active marks in human and had no distinct feature profile in mouse. Thus, in contrast to interactions within individual chromosomes, the inter-chromosomal interactions in human and mouse embryonic stem cells do not appear to be conserved. AU - Kaufmann, S.* AU - Fuchs, C. AU - Gonik, M.* AU - Khrameeva, E.E.* AU - Mironov, A.A.* AU - Frishman, D. C1 - 44835 C2 - 37058 CY - San Francisco TI - Inter-chromosomal contact networks provide insights into mammalian chromatin organization. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Home-based secondary prevention programs led by nurses have been proposed to facilitate patients' adjustment to acute myocardial infarction (AMI). The objective of this study was to conduct secondary analyses of the three-year follow-up of a nurse-based case management for elderly patients discharged from hospital after an AMI. METHODS: In a single-centre randomized two-armed parallel group trial of hospitalized patients with AMI ≥65 years, patients hospitalized between September 2008 and May 2010 in the Hospital of Augsburg, Germany, were randomly assigned to case management or usual care. The case-management intervention consisted of a nurse-based follow-up for three years including home visits and telephone calls. Study endpoints were time to first unplanned readmission or death, clinical parameters, functional status, depressive symptoms and malnutrition risk. Persons who assessed three-year outcomes and validated readmission data were blinded. The intention-to-treat approach was applied to the statistical analyses which included Cox Proportional Hazards models. RESULTS: Three hundred forty patients were allocated to receive case-management (n = 168) or usual care (n = 172). During three years, in the intervention group there were 80 first unplanned readmissions and 6 deaths, while the control group had 111first unplanned readmissions and 3 deaths. The intervention did not significantly affect time to first unplanned readmission or death (Hazard Ratio 0.89, 95% confidence interval (CI) 0.67-1.19; p = 0.439), blood pressure, cholesterol level, instrumental activities of daily life (IADL) (only for men), and depressive symptoms. However, patients in the intervention group had a significantly better functional status, as assessed by the HAQ Disability Index, IADL (only for women), and hand grip strength, and better SCREEN-II malnutrition risk scores than patients in the control group. CONCLUSIONS: A nurse-based management among elderly patients with AMI did not significantly affect time to unplanned readmissions or death during a three-year follow-up. However, the results indicate that functional status and malnutrition risk can be improved. TRIAL REGISTRATION: Current Controlled Trials ISRCTN02893746. AU - Kirchberger, I. AU - Hunger, M. AU - Stollenwerk, B. AU - Seidl, H. AU - Burkhardt, K. AU - Kuch, B.* AU - Meisinger, C. AU - Holle, R. C1 - 44020 C2 - 36730 CY - San Francisco TI - Effects of a 3-year nurse-based case management in aged patients with acute myocardial infarction on rehospitalisation, mortality, risk factors, physical functioning and mental health. A secondary analysis of the randomized controlled KORINNA study. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Wild house mice form social hierarchies with aggressive males defending territories, in which females, young mice and submissive adult males share nests. In contrast, socially excluded males are barred from breeding groups, have numerous bite wounds and patches of thinning fur. Since their feeding times are often disrupted, we investigated whether social exclusion leads to changes in epigenetic marks of metabolic genes in liver tissue. We used chromatin immunoprecipitation and quantitative PCR to measure enrichment of two activating histone marks at 15 candidate loci. The epigenetic profiles of healthy males sampled from nest boxes differed significantly from the profiles of ostracized males caught outside of nests and showing bite wounds indicative of social exclusion. Enrichment of histone-3 lysine-4 trimethylation (H3K4me3) changed significantly at genes Cyp4a14, Gapdh, Nr3c1, Pck1, Ppara, and Sqle. Changes at histone-3 lysine-27 acetylation (H3K27ac) marks were detected at genes Fasn, Nr3c1, and Plin5. A principal components analysis separated the socialized from the ostracized mice. This was independent of body weight for the H3K4me3 mark, and partially dependent for H3K27ac. There was no separation, however, between healthy males that had been sampled from two different nests. A hierarchical cluster analysis also separated the two phenotypes, which was independent of body weight for both markers. Our study shows that a period of social exclusion during adult life leads to quantitative changes in histone modification patterns in mouse liver tissue. Similar epigenetic changes might occur during the development of stress-induced metabolic disorders in humans. AU - Krause, L. AU - Haubold, B.* AU - Börsch-Haubold, A.G.* C1 - 46570 C2 - 37708 CY - San Francisco TI - Social exclusion changes histone modifications H3K4me3 and H3K27ac in liver tissue of wild house mice. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions. AU - Kunze, S. AU - Dalke, C. AU - Fuchs, H. AU - Klaften, M. AU - Rössler, U.* AU - Hornhardt, S.* AU - Gomolka, M.* AU - Puk, O. AU - Sabrautzki, S. AU - Kulka, U.* AU - Hrabě de Angelis, M. AU - Graw, J. C1 - 44816 C2 - 37000 CY - San Francisco TI - New mutation in the mouse Xpd/Ercc2 gene leads to recessive cataracts. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Infant formulas containing non-digestible oligosaccharides (NDO) similar to the composition in breast milk or a combination of lactic acid bacteria (LAB) and NDO have been shown to harbor preventive effects towards immune-regulatory disorders. The aim of this study was to investigate the immune-modulatory potential of non-digestible short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS) mimicking the natural distribution of oligosaccharides in human breast milk in presence or absence of certain LAB strains in human monocyte derived dendritic cells (MoDC). Immature human MoDC prepared from peripheral blood of healthy non-atopic volunteers were screened in vitro after stimulation with specific scGOS/lcFOS in presence or absence of LAB. IL-10 and IL-12p70 release was analyzed after 24 hours in cell-free supernatants by enzyme-linked immunosorbent assay (ELISA). A luminex-based assay was conducted to assess further cytokine and chemokine release by MoDC. To investigate the resulting T cell response, stimulated MoDC were co-incubated with naïve T cells in allogeneic stimulation assays and intracellular Foxp3 expression, as well as immune-suppressive capacity was determined. Oligosaccharides did not induce relevant amounts of IL-12p70 production, but did promote IL-10 release by MoDC. Furthermore, scGOS/lcFOS mixtures exerted a significant enhancing effect on LAB induced IL-10 secretion by MoDC while no increase in IL-12p70 production was observed. Blocking toll like receptor (TLR)4 abrogated the increase in IL-10 in both the direct stimulation and the LAB stimulation of MoDC, suggesting that scGOS/lcFOS act via TLR4. Finally, scGOS/lcFOS-treated MoDC were shown to upregulate the number of functional suppressive Foxp3 positive T cells following allogeneic stimulation. Our results indicate anti-inflammatory and direct, microbiota independent, immune-modulatory properties of scGOS/lcFOS mixtures on human MoDC suggesting a possible induction of regulatory T cells (Tregs). The tested combinations of LAB and scGOS/lcFOS might represent a useful dietary ingredient for the maintenance of intestinal homeostasis via the induction of Tregs. AU - Lehmann, S. AU - Hiller, J. AU - van Bergenhenegouwen, J.* AU - Knippels, L.M.* AU - Garssen, J.* AU - Traidl-Hoffmann, C.* C1 - 46285 C2 - 37463 CY - San Francisco TI - In vitro evidence for immune-modulatory properties of non-digestible oligosaccharides: Direct effect on human monocyte derived dendritic cells. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53. AU - Linzen, U.* AU - Lilischkis, R.* AU - Pandithage, R.* AU - Schilling, B.* AU - Ullius, A.* AU - Luscher-Firzlaff, J.* AU - Kremmer, E. AU - Lüscher, B.* AU - Vervoorts, J.* C1 - 44339 C2 - 36827 CY - San Francisco TI - ING5 is phosphorylated by CDK2 and controls cell proliferation independently of p53. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response. AU - Lorber, B.* AU - Chew, D.J.* AU - Hauck, S.M. AU - Chong, R.S.* AU - Fawcett, J.W.* AU - Martin, K.R.* C1 - 44079 C2 - 36787 CY - San Francisco TI - Retinal glia promote dorsal root ganglion axon regeneration. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. AU - Lorenz, F.K.* AU - Wilde, S. AU - Voigt, K.* AU - Kieback, E.* AU - Mosetter, B. AU - Schendel, D.J. AU - Uckert, W.* C1 - 43951 C2 - 36682 CY - San Francisco TI - Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. AU - Maugg, D. AU - Rothenaigner, I. AU - Schorpp, K.K. AU - Potukuchi, H.K. AU - Korsching, E.* AU - Baumhoer, D. AU - Hadian, K. AU - Smida, J. AU - Nathrath, M. C1 - 45106 C2 - 37230 CY - San Francisco TI - New small molecules targeting apoptosis and cell viability in osteosarcoma. JO - PLoS ONE VL - 10 IS - 6 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress. AU - Medrikova, D.* AU - Sijmonsma, T.P.* AU - Sowodniok, K.* AU - Richards, D.M.* AU - Delacher, M.* AU - Sticht, C.* AU - Gretz, N.* AU - Schafmeier, T. AU - Feuerer, M.* AU - Herzig, S. C1 - 43510 C2 - 36658 CY - San Francisco TI - Brown adipose tissue harbors a distinct sub-population of regulatory T cells. JO - PLoS ONE VL - 10 IS - 2 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Skeletal muscle cell differentiation is impaired by elevated levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α) with pathological significance in chronic diseases or inherited muscle disorders. Insulin like growth factor-1 (IGF1) positively regulates muscle cell differentiation. Both, TNF-α and IGF1 affect gene and microRNA (miRNA) expression in this process. However, computational prediction of miRNA-mRNA relations is challenged by false positives and targets which might be irrelevant in the respective cellular transcriptome context. Thus, this study is focused on functional information about miRNA affected target transcripts by integrating miRNA and mRNA expression profiling data. METHODOLOGY/PRINCIPAL FINDINGS: Murine skeletal myocytes PMI28 were differentiated for 24 hours with concomitant TNF-α or IGF1 treatment. Both, mRNA and miRNA expression profiling was performed. The data-driven integration of target prediction and paired mRNA/miRNA expression profiling data revealed that i) the quantity of predicted miRNA-mRNA relations was reduced, ii) miRNA targets with a function in cell cycle and axon guidance were enriched, iii) differential regulation of anti-differentiation miR-155-5p and miR-29b-3p as well as pro-differentiation miR-335-3p, miR-335-5p, miR-322-3p, and miR-322-5p seemed to be of primary importance during skeletal myoblast differentiation compared to the other miRNAs, iv) the abundance of targets and affected biological processes was miRNA specific, and v) subsets of miRNAs may collectively regulate gene expression. CONCLUSIONS: Joint analysis of mRNA and miRNA profiling data increased the process-specificity and quality of predicted relations by statistically selecting miRNA-target interactions. Moreover, this study revealed miRNA-specific predominant biological implications in skeletal muscle cell differentiation and in response to TNF-α or IGF1 treatment. Furthermore, myoblast differentiation-associated miRNAs are suggested to collectively regulate gene clusters and targets associated with enriched specific gene ontology terms or pathways. Predicted miRNA functions of this study provide novel insights into defective regulation at the transcriptomic level during myocyte proliferation and differentiation due to inflammatory stimuli. AU - Meyer, S.U.* AU - Sass, S. AU - Müller, N.S. AU - Krebs, S.* AU - Bauersachs, S.* AU - Kaiser, S.* AU - Blum, H.* AU - Thirion, C.* AU - Krause, S.* AU - Theis, F.J. AU - Pfaffl, M.W.* C1 - 46575 C2 - 37644 CY - San Francisco TI - Integrative analysis of MicroRNA and mRNA data reveals an orchestrated function of MicroRNAs in skeletal myocyte differentiation in response to TNF-α or IGF1. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice. AU - Miller, M. AU - Zorn, J. AU - Brielmeier, M. C1 - 47465 C2 - 39350 TI - High-resolution melting curve analysis for identification of Pasteurellaceae species in experimental animal facilities. JO - PLoS ONE VL - 10 IS - 11 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Exposure to high concentrations of Manganese (Mn) is known to potentially induce an accumulation in the brain, leading to a Parkinson related disease, called manganism. Versatile mechanisms of Mn-induced brain injury are discussed, with inactivation of mitochondrial defense against oxidative stress being a major one. So far, studies indicate that the main Mn-species entering the brain are low molecular mass (LMM) compounds such as Mn-citrate. Applying a single low dose MnCl2 injection in rats, we observed alterations in Mn-species pattern within the brain by analysis of aqueous brain extracts by size-exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). Additionally, electrospray ionization-ion cyclotron resonance-Fourier transform-mass spectrometry (ESI-ICR/FT-MS) measurement of methanolic brain extracts revealed a comprehensive analysis of changes in brain metabolisms after the single MnCl2 injection. Major alterations were observed for amino acid, fatty acid, glutathione, glucose and purine/pyrimidine metabolism. The power of this metabolomic approach is the broad and detailed overview of affected brain metabolisms. We also correlated results from the metallomic investigations (Mn concentrations and Mn-species in brain) with the findings from metabolomics. This strategy might help to unravel the role of different Mn-species during Mn-induced alterations in brain metabolism. AU - Neth, K. AU - Lucio, M. AU - Walker, A. AU - Zorn, J. AU - Schmitt-Kopplin, P. AU - Michalke, B. C1 - 46923 C2 - 39036 TI - Changes in brain metallome/metabolome pattern due to a single i.v. injection of manganese in rats. JO - PLoS ONE VL - 10 IS - 9 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. OBJECTIVES: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. METHODS: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. RESULTS: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. CONCLUSIONS: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices. AU - Oeder, S. AU - Kanashova, T.* AU - Sippula, O.* AU - Sapcariu, S.C.* AU - Streibel, T. AU - Arteaga-Salas, J.M. AU - Passig, J.* AU - Dilger, M.* AU - Paur, H.R.* AU - Schläger, C.* AU - Mülhopt, S.* AU - Diabaté, S.* AU - Weiss, C.* AU - Stengel, B.* AU - Rabe, R.* AU - Harndorf, H.* AU - Torvela, T.* AU - Jokiniemi, J.K.* AU - Hirvonen, M.R.* AU - Schmidt-Weber, C.B. AU - Traidl-Hoffmann, C.* AU - BéruBé, K.A.* AU - Wlodarczyk, A.J.* AU - Prytherch, Z.* AU - Michalke, B. AU - Krebs, T.* AU - Prévôt, A.S.* AU - Kelbg, M.* AU - Tiggesbäumker, J.* AU - Karg, E.W. AU - Jakobi, G. AU - Scholtes, S. AU - Schnelle-Kreis, J. AU - Lintelmann, J. AU - Matuschek, G. AU - Sklorz, M.* AU - Klingbeil, S.* AU - Orasche, J. AU - Richthammer, P. AU - Müller, L. AU - Elsasser, M. AU - Reda, A. AU - Gröger, T.M. AU - Weggler, B.A. AU - Schwemer, T.* AU - Czech, H.* AU - Rüger, C.P.* AU - Abbaszade, G. AU - Radischat, C.* AU - Hiller, K.* AU - Buters, J.T.M. AU - Dittmar, G.* AU - Zimmermann, R. C1 - 45104 C2 - 37235 CY - San Francisco TI - Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions. JO - PLoS ONE VL - 10 IS - 6 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast. AU - Pfannmüller, A.* AU - Wagner, D.E.* AU - Sieber, C.M.K. AU - Schönig, B.* AU - Boeckstaens, M.* AU - Marini, A.M.* AU - Tudzynski, B.* C1 - 44481 C2 - 36946 CY - San Francisco TI - The general amino acid permease FfGap1 of Fusarium fujikuroi Is sorted to the vacuole in a nitrogen-dependent, but Npr1 kinase-independent manner. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and can here be exemplified for OS. AU - Poos, K.* AU - Smida, J. AU - Maugg, D. AU - Eckstein, G.N. AU - Baumhoer, D. AU - Nathrath, M. AU - Korsching, E.* C1 - 44265 C2 - 36775 CY - San Francisco TI - Genomic heterogeneity of osteosarcoma - shift from single candidates to functional modules. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. AU - Purahong, W.* AU - Stempfhuber, B. AU - Lentendu, G.* AU - Francioli, D.* AU - Reitz, T.* AU - Buscot, F.* AU - Schloter, M. AU - Krüger, D.* C1 - 43714 C2 - 36799 CY - San Francisco TI - Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Recent reports suggested that chronic herpesvirus infection, as a constituent of the so-called virome, may not only exert harmful effects but may also be beneficial to the host, for example mediating increased resistance to secondary infections or to tumors. To further challenge this concept, specifically regarding increased resistance to tumors, we infected chimeric HLA-DR4-H2-E (DR4) mice, a mouse strain which spontaneously develops hematological tumors, with the rodent herpesvirus murine gammaherpesvirus 68 (MHV-68). Using this model, we observed that infection with wildtype MHV-68 completely prevented tumor formation. This happened, however, at the cost of hyposplenism. In contrast to wildtype infection, infection with a latency-deficient mutant of MHV-68 neither prevented tumor formation nor induced hyposplenism. The underlying mechanisms are not known but might be related to an infection-mediated priming of the immune response, resulting in the suppression of a tumor promoting endogenous retrovirus. Thus, under certain circumstances, chronic herpesvirus infection may prevent the development of tumors. AU - Raffegerst, S.H. AU - Steer, B. AU - Hohloch, M. AU - Adler, H. C1 - 47602 C2 - 39379 TI - Prevention of tumor formation by latent gammaherpesvirus infection. JO - PLoS ONE VL - 10 IS - 12 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Gene-set analysis (GSA) methods are used as complementary approaches to genome-wide association studies (GWASs). The single marker association estimates of a predefined set of genes are either contrasted with those of all remaining genes or with a null non-associated background. To pool the p-values from several GSAs, it is important to take into account the concordance of the observed patterns resulting from single marker association point estimates across any given gene set. Here we propose an enhanced version of Fisher's inverse χ2-method META-GSA, however weighting each study to account for imperfect correlation between association patterns. SIMULATION AND POWER: We investigated the performance of META-GSA by simulating GWASs with 500 cases and 500 controls at 100 diallelic markers in 20 different scenarios, simulating different relative risks between 1 and 1.5 in gene sets of 10 genes. Wilcoxon's rank sum test was applied as GSA for each study. We found that META-GSA has greater power to discover truly associated gene sets than simple pooling of the p-values, by e.g. 59% versus 37%, when the true relative risk for 5 of 10 genes was assume to be 1.5. Under the null hypothesis of no difference in the true association pattern between the gene set of interest and the set of remaining genes, the results of both approaches are almost uncorrelated. We recommend not relying on p-values alone when combining the results of independent GSAs. APPLICATION: We applied META-GSA to pool the results of four case-control GWASs of lung cancer risk (Central European Study and Toronto/Lunenfeld-Tanenbaum Research Institute Study; German Lung Cancer Study and MD Anderson Cancer Center Study), which had already been analyzed separately with four different GSA methods (EASE; SLAT, mSUMSTAT and GenGen). This application revealed the pathway GO0015291 "transmembrane transporter activity" as significantly enriched with associated genes (GSA-method: EASE, p = 0.0315 corrected for multiple testing). Similar results were found for GO0015464 "acetylcholine receptor activity" but only when not corrected for multiple testing (all GSA-methods applied; p≈0.02). AU - Rosenberger, A.* AU - Friedrichs, S.* AU - Amos, C.I.* AU - Brennan, P.* AU - Fehringer, G.* AU - Heinrich, J. AU - Hung, R.J.* AU - Muley, T.R.* AU - Müller-Nurasyid, M. AU - Risch, A.* AU - Bickeböller, H.* C1 - 47208 C2 - 39145 TI - META-GSA: Combining findings from gene-set analyses across several genome-wide association studies. JO - PLoS ONE VL - 10 IS - 10 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Hypertension is a very common comorbidity and major risk factor for cardiovascular complications, especially in people with Type 2 Diabetes (T2D). Nevertheless, studies in the past have shown that blood pressure is often insufficiently controlled in medical practice. For the DIAB-CARE study, we used longitudinal data based on the German DIAB-CORE Consortium to assess whether health care regarding hypertension has improved during the last decade in our participants. METHODS: Data of the three regional population-based studies CARLA (baseline 2002-2006 and follow-up 2007-2010), KORA (baseline 1999-2001 and follow-up 2006-2008) and SHIP (baseline 1997-2001 and follow-up 2002-2006) were pooled. Stratified by T2D status we analysed changes in frequencies, degrees of awareness, treatment and control. Linear mixed models were conducted to assess the influence of sex, age, study, and T2D status on changes of systolic blood pressure between the baseline and follow-up examinations (mean observation time 5.7 years). We included 4,683 participants aged 45 to 74 years with complete data and accounted for 1,256 participants who were lost to follow-up by inverse probability weighting. RESULTS: Mean systolic blood pressure decreased in all groups from baseline to follow-up (e.g. - 8.5 mmHg in those with incident T2D). Pulse pressure (PP) was markedly higher in persons with T2D than in persons without T2D (64.14 mmHg in prevalent T2D compared to 52.87 mmHg in non-T2D at baseline) and did not change much between the two examinations. Awareness, treatment and control increased considerably in all subgroups however, the percentage of those with insufficiently controlled hypertension remained high (at about 50% of those with hypertension) especially in prevalent T2D. Particularly elderly people with T2D often had both, high blood pressure ≥140/90 mmHg and a PP of ≥60 mmHg. Blood pressure in men had improved more than in women at follow-up, however, men still had higher mean SBP than women at follow-up. CONCLUSION: Blood pressure management has developed positively during past years in Germany. While hypertension prevalence, awareness and treatment were substantially higher in participants with T2D than in those without T2D at follow-up, hypertension control was achieved only in about half the number of people in each T2D group leaving much room for further improvement. AU - Rückert, I.-M. AU - Baumert, J.J. AU - Schunk, M. AU - Holle, R. AU - Schipf, S.* AU - Völzke, H.* AU - Kluttig, A.* AU - Greiser, K.H.* AU - Tamayo, T.* AU - Rathmann, W.* AU - Meisinger, C. C1 - 46441 C2 - 37537 CY - San Francisco TI - Blood pressure control has improved in people with and without type 2 diabetes but remains suboptimal: A longitudinal study based on the German DIAB-CORE consortium. JO - PLoS ONE VL - 10 IS - 7 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: It is a matter of debate whether impaired insulin action originates from a defect at the neural level or impaired transport of the hormone into the brain. In this study, we aimed to investigate the effect of aging on insulin concentrations in the periphery and the central nervous system as well as its impact on insulin-dependent brain activity. METHODS: Insulin, glucose and albumin concentrations were determined in 160 paired human serum and cerebrospinal fluid (CSF) samples. Additionally, insulin was applied in young and aged mice by subcutaneous injection or intracerebroventricularly to circumvent the blood-brain barrier. Insulin action and cortical activity were assessed by Western blotting and electrocorticography radiotelemetric measurements. RESULTS: In humans, CSF glucose and insulin concentrations were tightly correlated with the respective serum/plasma concentrations. The CSF/serum ratio for insulin was reduced in older subjects while the CSF/serum ratio for albumin increased with age like for most other proteins. Western blot analysis in murine whole brain lysates revealed impaired phosphorylation of AKT (P-AKT) in aged mice following peripheral insulin stimulation whereas P-AKT was comparable to levels in young mice after intracerebroventricular insulin application. As readout for insulin action in the brain, insulin-mediated cortical brain activity instantly increased in young mice subcutaneously injected with insulin but was significantly reduced and delayed in aged mice during the treatment period. When insulin was applied intracerebroventricularly into aged animals, brain activity was readily improved. CONCLUSIONS: This study discloses age-dependent changes in insulin CSF/serum ratios in humans. In the elderly, cerebral insulin resistance might be partially attributed to an impaired transport of insulin into the central nervous system. AU - Sartorius, T. AU - Peter, A. AU - Heni, M. AU - Maetzler, W.* AU - Fritsche, A. AU - Häring, H.-U. AU - Hennige, A.M. C1 - 44842 C2 - 37054 CY - San Francisco TI - The brain response to peripheral insulin declines with age: A contribution of the blood-brain barrier? JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients. AU - Schemionek, M.* AU - Kharabi Masouleh, B.* AU - Klaile, Y.* AU - Krug, U.* AU - Hebestreit, K.* AU - Schubert, C.* AU - Dugas, M.* AU - Büchner, T.* AU - Wörmann, B.* AU - Hiddemann, W. AU - Berdel, W.E.* AU - Brümmendorf, T.H.* AU - Müller-Tidow, C.* AU - Koschmieder, S.* C1 - 44916 C2 - 37119 CY - San Francisco TI - Identification of the adapter molecule MTSS1 as a potential oncogene-specific tumor suppressor in acute myeloid leukemia. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Lower levels of hemoglobin A1c (HbA1c) are associated with a decreased risk of cardiovascular complications in diabetic and non-diabetic individuals. The aim of the study was to longitudinally investigate the association between the use of 11 vitamins and minerals (vitamins E, C, D, B1, folic acid, carotenoids, calcium, magnesium, zinc, iron, and selenium) and change in HbA1c levels over 10 years in non-diabetic individuals drawn from the general population. METHODS: Baseline data were available from 4447 subjects included in the population-based "Monitoring of Trends and Determinants in Cardiovascular Diseases" (MONICA) Augsburg S3 survey (1994/95). Follow-up data were derived from 2774 participants in the follow-up survey named "Cooperative Health Research in the Region of Augsburg" (KORA) F3 (2004/05). Vitamin/mineral intake from supplements and medications was assessed in a personal interview, where participants were asked to bring product packages of preparations that had been ingested during the last 7 days prior to the examination. Associations between regular vitamin/mineral intake amounts and HbA1c levels measured at baseline and follow-up were investigated using generalized estimating equation models. For carotenoids, analyses were stratified by smoking status. RESULTS: None of the investigated nutrients except for carotenoids was significantly associated with changes in HbA1c levels after 10 years. Regular intake of carotenoids from supplements and medications in amounts > 6.8mg/d (upper tertile) was associated with an absolute -0.26% (95% CI: -0.43 to -0.08) lower increase in HbA1c levels compared with no intake of carotenoids. An inverse association was observed in those who never smoked but not in (former) smokers. CONCLUSION: Larger prospective and intervention studies in non-diabetic/non-smoking individuals are needed to confirm the results and to assess whether the observed associations between carotenoid intake and change in HbA1c levels are causal. If our results are confirmed, high carotenoid intake could be one strategy for the prevention of cardiovascular complications in non-diabetic people. AU - Schwab, S. AU - Zierer, A. AU - Heier, M. AU - Fischer, B.* AU - Huth, C. AU - Baumert, J.J. AU - Meisinger, C. AU - Peters, A. AU - Thorand, B. C1 - 47245 C2 - 39316 TI - Intake of vitamin and mineral supplements and longitudinal association with HbA1c levels in the general non-diabetic population - results from the MONICA/KORA S3/F3 study. JO - PLoS ONE VL - 10 IS - 10 PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution. AU - Semren, N. AU - Habel-Ungewitter, N.C. AU - Fernandez, I.E. AU - Königshoff, M. AU - Eickelberg, O. AU - Stöger, T. AU - Meiners, S. C1 - 46728 C2 - 37764 CY - San Francisco TI - Validation of the 2nd generation proteasome inhibitor oprozomib for local therapy of pulmonary fibrosis. JO - PLoS ONE VL - 10 IS - 9 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis. AU - Serth, K. AU - Schuster-Gossler, K.* AU - Kremmer, E. AU - Hansen, B.T.* AU - Marohn-Köhn, B.* AU - Gossler, A.* C1 - 44336 C2 - 36828 CY - San Francisco TI - O-fucosylation of DLL3 is required for its function during somitogenesis. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells. AU - Sezgin, E.* AU - Gutmann, T. AU - Buhl, T.* AU - Dirkx, R. AU - Grzybek, M. AU - Coskun, Ü. AU - Solimena, M. AU - Simons, K.* AU - Levental, I.* AU - Schwille, P.* C1 - 44477 C2 - 36949 CY - San Francisco TI - Adaptive lipid packing and bioactivity in membrane domains. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - A detailed analysis of cerebrovascular diseases (CeVD) for the cohort of workers at Mayak Production Association (PA) is presented. This cohort is especially suitable for the analysis of radiation induced circulatory diseases, due to the detailed medical surveillance and information on several risk factors. The risk after external, typically protracted, gamma exposure is analysed, accounting for potential additional internal alpha exposure. Three different endpoints have been investigated: incidence and mortality from all cerebrovascular diseases and incidence of stroke. Particular emphasis was given to the form of the dose-response relationship and the time dependence of the radiation induced risk. Young attained age was observed to be an important, aggravating modifier of radiation risk for incidence of CeVD and stroke. For incidence of CeVD, our analysis supports a dose response sub-linear for low doses. Finally, the excess relative risk per dose was confirmed to be significantly higher for incidence of CeVD compared to CeVD mortality and incidence of stroke. Arguments are presented for this difference to be based on a true biological effect. AU - Simonetto, C. AU - Schöllnberger, H. AU - Azizova, T.V.* AU - Grigoryeva, E.S.* AU - Pikulina, M.V.* AU - Eidemüller, M. C1 - 44540 C2 - 37012 CY - San Francisco TI - Cerebrovascular diseases in workers at Mayak PA: The difference in radiation risk between incidence and mortality. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing. AU - Sinreih, M.* AU - Zukunft, S. AU - Sosič, I.* AU - Cesar, J.* AU - Gobec, S.* AU - Adamski, J. AU - Lanisnik Rizner, T.* C1 - 43287 C2 - 36340 CY - San Francisco TI - Combined liquid chromatography - tandem mass spectrometry analysis of progesterone metabolites. JO - PLoS ONE VL - 10 IS - 2 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Sporting activities differ in their ability to promote moderate-to-vigorous physical activity (MVPA). To assess adolescents' engagement in sport under field conditions we used accelerometers to measure their MVPA levels during sport. We pay special attention to differences between team and individual sport and between common sports. METHODS: Diary data and 7-day accelerometry from 1054 Germans ages 15-17 were combined to measure physical activity. 1373 diaried episodes of more than 40 common sports were identified from 626 participants and grouped into team and individual sport. We modeled the effect of team and individual sport, and described levels of MVPA and episodes of no MVPA for all recorded sports. RESULTS: German boys and girls averaged 43 (SD 21) and 37 (SD 24) minutes MVPA per day. Boys got 2.2 times as much MVPA per minute during team compared to individual sport (p<0.0001) but there was no significant difference for girls. Percent of time spent in MVPA during sport ranged from 6% for weight training to 74% for jogging, with individual sports averaging 10-30% and team sports 30-50%. 11% of sport episodes had no MVPA: half of episodes of cycling, 5% of jogging, and none for tennis or badminton. An episode of individual sport was 17 times more likely to have no MVPA than an episode of team sport (p<0.0001). CONCLUSION: Under field condition, adolescents were active for only a fraction of diaried sporting time. As measured by accelerometry, individual sport often produced no MVPA. Characteristics of the sport, such as team vs. individual, were more predictive of MVPA than were characteristics of the participant, such as background activity levels. AU - Smith, M. AU - Berdel, D.* AU - Nowak, D.* AU - Heinrich, J. AU - Schulz, H. C1 - 46614 C2 - 37660 CY - San Francisco TI - Sport engagement by accelerometry under field conditions in German adolescents: Results from GINIPlus. JO - PLoS ONE VL - 10 IS - 8 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Loss of function mutations in granulin (GRN) are linked to two distinct neurological disorders, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). It is so far unknown how a complete loss of GRN in NCL and partial loss of GRN in FTLD can result in such distinct diseases. In zebrafish, there are two GRN homologues, Granulin A (Grna) and Granulin B (Grnb). We have generated stable Grna and Grnb loss of function zebrafish mutants by zinc finger nuclease mediated genome editing. Surprisingly, the grna and grnb single and double mutants display neither spinal motor neuron axonopathies nor a reduced number of myogenic progenitor cells as previously reported for Grna and Grnb knock down embryos. Additionally, grna-/-;grnb-/- double mutants have no obvious FTLD- and NCL-related biochemical and neuropathological phenotypes. Taken together, the Grna and Grnb single and double knock out zebrafish lack any obvious morphological, pathological and biochemical phenotypes. Loss of zebrafish Grna and Grnb might therefore either be fully compensated or only become symptomatic upon additional challenge. AU - Solchenberger, B.* AU - Russell, C.* AU - Kremmer, E. AU - Haass, C.* AU - Schmid, B.* C1 - 43871 C2 - 36621 CY - San Francisco TI - Granulin knock out zebrafish lack frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis pathology. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression. AU - Sotoodehnejadnematalahi, F.* AU - Staples, K.J.* AU - Chrysanthou, E.* AU - Pearson, H.* AU - Ziegler-Heitbrock, L. AU - Burke, B.* C1 - 45343 C2 - 37297 CY - San Francisco TI - Mechanisms of hypoxic up-regulation of versican gene expression in macrophages. JO - PLoS ONE VL - 10 IS - 6 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups. AU - Vick, B. AU - Rothenberg, M.* AU - Sandhöfer, N. AU - Carlet, M. AU - Finkenzeller, C. AU - Krupka, C. AU - Grunert, M. AU - Trumpp, A.* AU - Corbacioglu, S.* AU - Ebinger, M.* AU - Andre, M.C.* AU - Hiddemann, W. AU - Schneider, S.* AU - Subklewe, M. AU - Metzeler, K.H. AU - Spiekermann, K. AU - Jeremias, I. C1 - 43909 C2 - 36736 CY - San Francisco TI - An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Large-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r²>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls. METHODS: The coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants. RESULTS: Except for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function. CONCLUSION: Variants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted. AU - Volckmar, A.L.* AU - Song, J.Y.* AU - Jarick, I.* AU - Pütter, C.* AU - Göbel, M.* AU - Horn, L.C.* AU - Struve, C.* AU - Haas, K.* AU - Knoll, N.* AU - Grallert, H. AU - Illig, T. AU - Reinehr, T.* AU - Wang, H.J.* AU - Hebebrand, J.* AU - Hinney, A.* C1 - 44802 C2 - 37029 CY - San Francisco TI - Fine mapping of a GWAS-derived obesity candidate region on chromosome 16p11.2. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - In the recent years, short-term heart rate variability (HRV) describing complex variations of beat-to-beat interval series that are mainly controlled by the autonomic nervous system (ANS) has been increasingly analyzed to assess the ANS activity in different diseases and under various conditions. In contrast to long-term HRV analysis, short-term investigations (<30 min) provide a test result almost immediately. Thus, short-term HRV analysis is suitable for ambulatory care, patient monitoring and all those applications where the result is urgently needed. In a previous study, we could show significant variations of 5-min HRV indices according to age in almost all domains (linear and nonlinear) in 1906 healthy subjects from the KORA S4 cohort. Based on the same group of subjects, general gender-related influences on HRV indices are to be determined in this study. Short-term 5-min HRV indices from linear time and frequency domain and from nonlinear methods (compression entropy, detrended fluctuation analysis, traditional and segmented Poincaré plot analysis, irreversibility analysis, symbolic dynamics, correlation and mutual information analysis) were determined from 782 females and 1124 males. First, we examined the gender differences in two age clusters (25-49 years and 50-74 years). Secondly, we investigated the gender-specific development of HRV indices in five age decade categories, namely for ages 25-34, 35-44, 45-54, 55-64 and 65-74 years. In this study, significant modifications of the indices according to gender could be obtained, especially in the frequency domain and correlation analyses. Furthermore, there were significant modifications according to age in nearly all of the domains. The gender differences disappeared within the last two age decades and the age dependencies disappeared in the last decade. To summarize gender and age influences need to be considered when performing HRV studies even if these influences only partly differ. AU - Voss, A.* AU - Schroeder, R.* AU - Heitmann, A.* AU - Peters, A. AU - Perz, S. C1 - 44070 C2 - 36795 CY - San Francisco TI - Short-term heart rate variability-influence of gender and age in healthy subjects. JO - PLoS ONE VL - 10 IS - 3 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - GABAergic neurons are the primary inhibitory cell type in the mature brain and their dysfunction is associated with important neurological conditions like schizophrenia and anxiety. We aimed to discover the underlying mechanisms for dorsal/ventral midbrain GABAergic neurogenesis. Previous work by us and others has provided crucial insights into the key function of Mgn and Mash1 genes in determining GABAergic neurotransmitter fate. Induction of dorsal midbrain GABAergic neurons does not take place at any time during development in either of the single mutant mice. However, GABAergic neurons in the ventral midbrain remained unchanged. Thus, the similarities in MB-GABAergic phenotype observed in the Mgn and Mash1 single mutants suggest the existence of other factors that take over the function of MGN and MASH1 in the ventral midbrain or the existence of different molecular mechanisms. We show that this process essentially depends on heterodimers and homodimers formed by MGN and MASH1 and deciphered the in vivo relevance of the interaction by phenotypic analysis of Mgn/Mash1 double knockout and compound mice. Furthermore, the combination of gain- and loss-of-function experiments in the developing midbrain showed co-operative roles for Mgn and Mash1 genes in determining GABAergic identity. Transcription factors belonging to the Enhancer-of-split-related and proneural families have long been believed to counterpart each other's function. This work uncovers a synergistic cooperation between these two families, and provides a novel paradigm for how these two families cooperate for the acquisition of MB-GABAergic neuronal identity. Understanding their molecular mechanisms is essential for cell therapy strategies to amend GABAergic deficits. AU - Wende, C.-Z. AU - Zoubaa, S.* AU - Blak, A.A. AU - Echevarria, D.* AU - Martinez, S.* AU - Guillemot, F.* AU - Wurst, W. AU - Guimera, J. C1 - 44899 C2 - 37136 CY - San Francisco TI - Hairy/enhancer-of-split MEGANE and proneural MASH1 factors cooperate synergistically in midbrain GABAergic neurogenesis. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as observed in our study. AU - Widmann, P.* AU - Reverter, A.* AU - Weikard, R.* AU - Suhre, K. AU - Hammon, H.M.* AU - Albrecht, E.* AU - Kuehn, C.* C1 - 44418 C2 - 36872 CY - San Francisco TI - Systems biology analysis merging phenotype, metabolomic and genomic data identifies Non-SMC Condensin I Complex, Subunit G (NCAPG) and cellular maintenance processes as major contributors to genetic variability in bovine feed efficiency. JO - PLoS ONE VL - 10 IS - 4 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Mechanistic multi-stage models are used to analyze lung-cancer mortality after Plutonium exposure in the Mayak-workers cohort, with follow-up until 2008. Besides the established two-stage model with clonal expansion, models with three mutation stages as well as a model with two distinct pathways to cancer are studied. The results suggest that three-stage models offer an improved description of the data. The best-fitting models point to a mechanism where radiation increases the rate of clonal expansion. This is interpreted in terms of changes in cell-cycle control mediated by bystander signaling or repopulation following cell killing. No statistical evidence for a two-pathway model is found. To elucidate the implications of the different models for radiation risk, several exposure scenarios are studied. Models with a radiation effect at an early stage show a delayed response and a pronounced drop-off with older ages at exposure. Moreover, the dose-response relationship is strongly nonlinear for all three-stage models, revealing a marked increase above a critical dose. AU - Zöllner, S. AU - Sokolnikov, M.E.* AU - Eidemüller, M. C1 - 44541 C2 - 37073 CY - San Francisco TI - Beyond two-stage models for lung carcinogenesis in the Mayak workers: Implications for Plutonium risk. JO - PLoS ONE VL - 10 IS - 5 PB - Public Library Science PY - 2015 SN - 1932-6203 ER - TY - JOUR AB - Tumor heterogeneity is widely considered to be a determinant factor in tumor progression and in particular in its recurrence after therapy. Unfortunately, current medical techniques are unable to deduce clinically relevant information about tumor heterogeneity by means of non-invasive methods. As a consequence, when radiotherapy is used as a treatment of choice, radiation dosimetries are prescribed under the assumption that the malignancy targeted is of a homogeneous nature. In this work we discuss the effects of different radiation dose distributions on heterogeneous tumors by means of an individual cell-based model. To that end, a case is considered where two tumor cell phenotypes are present, which we assume to strongly differ in their respective cell cycle duration and radiosensitivity properties. We show herein that, as a result of such differences, the spatial distribution of the corresponding phenotypes, whence the resulting tumor heterogeneity can be predicted as growth proceeds. In particular, we show that if we start from a situation where a majority of ordinary cancer cells (CCs) and a minority of cancer stem cells (CSCs) are randomly distributed, and we assume that the length of CSC cycle is significantly longer than that of CCs, then CSCs become concentrated at an inner region as tumor grows. As a consequence we obtain that if CSCs are assumed to be more resistant to radiation than CCs, heterogeneous dosimetries can be selected to enhance tumor control by boosting radiation in the region occupied by the more radioresistant tumor cell phenotype. It is also shown that, when compared with homogeneous dose distributions as those being currently delivered in clinical practice, such heterogeneous radiation dosimetries fare always better than their homogeneous counterparts. Finally, limitations to our assumptions and their resulting clinical implications will be discussed. AU - Alfonso, J.C.L.* AU - Jagiella, N. AU - Nuñez, L.* AU - Herrero, M.A.* AU - Drasdo, D.* C1 - 30733 C2 - 33803 CY - San Francisco TI - Estimating dose painting effects in radiotherapy: A mathematical model. JO - PLoS ONE VL - 9 IS - 2 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The fate of polycyclic aromatic hydrocarbons (PAHs) in soil is determined by a suite of biotic and abiotic factors, and disentangling their role in the complex soil interaction network remains challenging. Here, we investigate the influence of soil composition on the microbial community structure and its response to the spiked model PAH compound phenanthrene and plant litter. We used long-term matured artificial soils differing in type of clay mineral (illite, montmorillonite) and presence of charcoal or ferrihydrite. The soils received an identical soil microbial fraction and were incubated for more than two years with two sterile manure additions. The matured artificial soils and a natural soil were subjected to the following spiking treatments: (I) phenanthrene, (II) litter, (III) litter + phenanthrene, (IV) unspiked control. Total community DNA was extracted from soil sampled on the day of spiking, 7, 21, and 63 days after spiking. Bacterial 16S rRNA gene and fungal internal transcribed spacer amplicons were quantified by qPCR and subjected to denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed that the bacterial community composition, which was strongly shaped by clay minerals after more than two years of incubation, changed in response to spiked phenanthrene and added litter. DGGE and qPCR showed that soil composition significantly influenced the microbial response to spiking. While fungal communities responded only in presence of litter to phenanthrene spiking, the response of the bacterial communities to phenanthrene was less pronounced when litter was present. Interestingly, microbial communities in all artificial soils were more strongly affected by spiking than in the natural soil, which might indicate the importance of higher microbial diversity to compensate perturbations. This study showed the influence of soil composition on the microbiota and their response to phenanthrene and litter, which may increase our understanding of complex interactions in soils for bioremediation applications. AU - Babin, D.* AU - Vogel, C.* AU - Zühlke, S.* AU - Schloter, M. AU - Pronk, G.J.* AU - Heister, K.* AU - Spiteller, M.* AU - Kögel-Knabner, I.* AU - Smalla, K.* C1 - 32188 C2 - 34985 CY - San Francisco TI - Soil mineral composition matters: Response of microbial communities to phenanthrene and plant litter addition in long-term matured artificial soils. JO - PLoS ONE VL - 9 IS - 9 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test. METHODS: sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom. RESULTS: The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum. CONCLUSIONS: BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy. AU - Balzer, L.* AU - Pennino, D. AU - Blank, S. AU - Seismann, H.* AU - Darsow, U.* AU - Schnedler, M.* AU - McIntyre, M.* AU - Ollert, M.* AU - Durham, S.R.* AU - Spillner, E.* AU - Ring, J.* AU - Cifuentes, L.* C1 - 32590 C2 - 35221 TI - Basophil activation test using recombinant allergens: Highly specific diagnostic method complementing routine tests in wasp venom allergy. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The study assesses complexity of the cardiac control directed to the sinus node and to ventricles in long QT syndrome type 1 (LQT1) patients with KCNQ1-A341V mutation. Complexity was assessed via refined multiscale entropy (RMSE) computed over the beat-to-beat variability series of heart period (HP) and QT interval. HP and QT interval were approximated respectively as the temporal distance between two consecutive R-wave peaks and between the R-wave apex and T-wave end. Both measures were automatically taken from 24-hour electrocardiographic Holter traces recorded during daily activities in non mutation carriers (NMCs, n = 14) and mutation carriers (MCs, n = 34) belonging to a South African LQT1 founder population. The MC group was divided into asymptomatic (ASYMP, n = 11) and symptomatic (SYMP, n = 23) patients according to the symptom severity. Analyses were carried out during daytime (DAY, from 2PM to 6PM) and nighttime (NIGHT, from 12PM to 4AM) off and on beta-adrenergic blockade (BBoff and BBon). We found that the complexity of the HP variability at short time scale was under vagal control, being significantly increased during NIGHT and BBon both in ASYMP and SYMP groups, while the complexity of both HP and QT variability at long time scales was under sympathetic control, being smaller during NIGHT and BBon in SYMP subjects. Complexity indexes at long time scales in ASYMP individuals were smaller than those in SYMP ones regardless of therapy (i.e. BBoff or BBon), thus suggesting that a reduced complexity of the sympathetic regulation is protective in ASYMP individuals. RMSE analysis of HP and QT interval variability derived from routine 24-hour electrocardiographic Holter recordings might provide additional insights into the physiology of the cardiac control and might be fruitfully exploited to improve risk stratification in LQT1 population. AU - Bari, V.* AU - Valencia, J.F.* AU - Vallverdú, M.* AU - Girardengo, G.* AU - Marchi, A.* AU - Bassani, T.* AU - Caminal, P.* AU - Cerutti, S.* AU - George, A.L.* AU - Brink, P.A.* AU - Crotti, L. AU - Schwartz, P.J.* AU - Porta, A.* C1 - 31010 C2 - 34114 CY - San Francisco TI - Multiscale complexity analysis of the cardiac control identifies asymptomatic and symptomatic patients in long QT syndrome type 1. JO - PLoS ONE VL - 9 IS - 4 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Plasma fibrinogen is an acute phase protein playing an important role in the blood coagulation cascade having strong associations with smoking, alcohol consumption and body mass index (BMI). Genome-wide association studies (GWAS) have identified a variety of gene regions associated with elevated plasma fibrinogen concentrations. However, little is yet known about how associations between environmental factors and fibrinogen might be modified by genetic variation. Therefore, we conducted large-scale meta-analyses of genome-wide interaction studies to identify possible interactions of genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentration. The present study included 80,607 subjects of European ancestry from 22 studies. Genome-wide interaction analyses were performed separately in each study for about 2.6 million single nucleotide polymorphisms (SNPs) across the 22 autosomal chromosomes. For each SNP and risk factor, we performed a linear regression under an additive genetic model including an interaction term between SNP and risk factor. Interaction estimates were meta-analysed using a fixed-effects model. No genome-wide significant interaction with smoking status, alcohol consumption or BMI was observed in the meta-analyses. The most suggestive interaction was found for smoking and rs10519203, located in the LOC123688 region on chromosome 15, with a p value of 6.2×10-8. This large genome-wide interaction study including 80,607 participants found no strong evidence of interaction between genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentrations. Further studies are needed to yield deeper insight in the interplay between environmental factors and gene variants on the regulation of fibrinogen concentrations. AU - Baumert, J.J. AU - Huang, J.* AU - McKnight, B.* AU - Sabater-Lleal, M.* AU - Steri, M.* AU - Chu, A.Y.* AU - Trompet, S.* AU - Lopez, L.M.* AU - Fornage, M.* AU - Teumer, A.* AU - Tang, W.* AU - Rudnicka, A.R.* AU - Mälarstig, A.* AU - Hottenga, J.J.* AU - Kavousi, M.* AU - Lahti, J.* AU - Tanaka, T.* AU - Hayward, C.* AU - Huffman, J.E.* AU - Morange, P.E.* AU - Rose, L.M.* AU - Basu, S.* AU - Rumley, A.* AU - Stott, D.J.* AU - Buckley, B.M.* AU - de Craen, A.J.* AU - Sanna, S.* AU - Masala, M.* AU - Biffar, R.* AU - Homuth, G.* AU - Silveira, A.* AU - Sennblad, B.* AU - Goel, A.* AU - Watkins, H.* AU - Müller-Nurasyid, M. AU - Rückerl, R. AU - Taylor, K.* AU - Chen, M.* AU - de Geus, E.J.* AU - Hofman, A.* AU - Witteman, J.C.* AU - de Maat, M.P.M.* AU - Palotie, A.* AU - Davies, G.* AU - Siscovick, D.S.* AU - Kolcic, I.* AU - Wild, S.H.* AU - Song, J.* AU - McArdle, W.L.* AU - Ford, I.* AU - Sattar, N.* AU - Schlessinger, D.* AU - Grotevendt, A.* AU - Franzosi, M.G.* AU - Illig, T. AU - Waldenberger, M. AU - Lumley, T.* AU - Tofler, G.H.* AU - Willemsen, G.* AU - Uitterlinden, A.G.* AU - Rivadeneira, F.* AU - Räikkönen, K.* AU - Chasman, D.I.* AU - Folsom, A.R.* AU - Lowe, G.D.* AU - Westendorp, R.G.* AU - Slagboom, P.E.* AU - Cucca, F.* AU - Wallaschofski, H.* AU - Strawbridge, R.J.* AU - Seedorf, U.* AU - Koenig, W.* AU - Bis, J.C.* AU - Mukamal, K.J.* AU - van Dongen, J.* AU - Widen, E.* AU - Franco, O.H.* AU - Starr, J.M.* AU - Liu, K.* AU - Ferrucci, L.* AU - Polasek, O.* AU - Wilson, J.F.* AU - Oudot-Mellakh, T.* AU - Campbell, H.* AU - Navarro, P.* AU - Bandinelli, S.* AU - Eriksson, J.* AU - Boomsma, D.I.* AU - Dehghan, A.* AU - Clarke, R.* AU - Hamsten, A.* AU - Boerwinkle, E.* AU - Jukema, J.W.* AU - Naitza, S.* AU - Ridker, P.M.* AU - Völzke, H.* AU - Deary, I.J.* AU - Reiner, A.P.* AU - Tregouet, D.* AU - O'Donnell, C.J.* AU - Strachan, D.P.* AU - Peters, A. AU - Smith, N.L.* C1 - 43025 C2 - 36005 TI - No evidence for genome-wide interactions on plasma fibrinogen by smoking, alcohol consumption and body mass index: Results from meta-analyses of 80,607 subjects. JO - PLoS ONE VL - 9 IS - 12 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients. AU - Becker, L. AU - Kling, E. AU - Schiller, E. AU - Zeh, R. AU - Schrewe, A. AU - Hölter, S.M. AU - Mossbrugger, I. AU - Calzada-Wack, J. AU - Strecker, V.* AU - Wittig, I.* AU - Dumitru, I.* AU - Wenz, T.* AU - Bender, A.* AU - Aichler, M. AU - Janik, D. AU - Neff, F. AU - Walch, A.K. AU - Quintanilla-Fend, L. AU - Floß, T. AU - Bekeredjian, R. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Wurst, W. AU - Meitinger, T. AU - Prokisch, H. AU - Hrabě de Angelis, M. AU - Klopstock, T. C1 - 42986 C2 - 35915 TI - MTO1-deficient mouse model mirrors the human phenotype showing complex I defect and cardiomyopathy. JO - PLoS ONE VL - 9 IS - 12 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND AND AIMS: Among obese subjects, metabolically healthy and unhealthy obesity (MHO/MUHO) can be differentiated: the latter is characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Aim of this study was, to identify adipocyte-specific metabolic signatures and functional biomarkers for MHO versus MUHO. METHODS: 10 insulin-resistant (IR) vs. 10 insulin-sensitive (IS) non-diabetic morbidly obese (BMI >40 kg/m2) Caucasians were matched for gender, age, BMI, and percentage of body fat. From subcutaneous fat biopsies, primary preadipocytes were isolated and differentiated to adipocytes in vitro. About 280 metabolites were investigated by a targeted metabolomic approach intracellularly, extracellularly, and in plasma. RESULTS/INTERPRETATION: Among others, aspartate was reduced intracellularly to one third (p = 0.0039) in IR adipocytes, pointing to a relative depletion of citric acid cycle metabolites or reduced aspartate uptake in MUHO. Other amino acids, already known to correlate with diabetes and/or obesity, were identified to differ between MUHO's and MHO's adipocytes, namely glutamine, histidine, and spermidine. Most species of phosphatidylcholines (PCs) were lower in MUHO's extracellular milieu, though simultaneously elevated intracellularly, e.g., PC aa C32∶3, pointing to increased PC synthesis and/or reduced PC release. Furthermore, altered arachidonic acid (AA) metabolism was found: 15(S)-HETE (15-hydroxy-eicosatetraenoic acid; 0 vs. 120pM; p = 0.0014), AA (1.5-fold; p = 0.0055) and docosahexaenoic acid (DHA, C22∶6; 2-fold; p = 0.0033) were higher in MUHO. This emphasizes a direct contribution of adipocytes to local adipose tissue inflammation. Elevated DHA, as an inhibitor of prostaglandin synthesis, might be a hint for counter-regulatory mechanisms in MUHO. CONCLUSION/INTERPRETATION: We identified adipocyte-inherent metabolic alterations discriminating between MHO and MUHO. AU - Böhm, A. AU - Halama, A. AU - Meile, T.* AU - Zdichavsky, M.* AU - Lehmann, R. AU - Weigert, C. AU - Fritsche, A. AU - Stefan, N. AU - Königsrainer, A.* AU - Häring, H.-U. AU - Hrabě de Angelis, M. AU - Adamski, J. AU - Staiger, H. C1 - 31000 C2 - 34079 CY - San Francisco TI - Metabolic signatures of cultured human adipocytes from metabolically healthy versus unhealthy obese individuals. JO - PLoS ONE VL - 9 IS - 4 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Chlamydia trachomatis is an obligate intracellular pathogen responsible for loss of eyesight through trachoma and for millions of cases annually of sexually transmitted diseases. The bacteria develop within a membrane-bounded inclusion. They lack enzymes for several biosynthetic pathways, including those to make some phospholipids, and exploit their host to compensate. Three-dimensional fluorescence microscopy demonstrates that small organelles of the host, peroxisomes, are translocated into the Chlamydia inclusion and are found adjacent to the bacteria. In cells deficient for peroxisome biogenesis the bacteria are able to multiply and give rise to infectious progeny, demonstrating that peroxisomes are not essential for bacterial development in vitro. Mass spectrometry-based lipidomics reveal the presence in C. trachomatis of plasmalogens, ether phospholipids whose synthesis begins in peroxisomes and have never been described in aerobic bacteria before. Some of the bacterial plasmalogens are novel structures containing bacteria-specific odd-chain fatty acids; they are not made in uninfected cells nor in peroxisome-deficient cells. Their biosynthesis is thus accomplished by the metabolic collaboration of peroxisomes and bacteria. AU - Boncompain, G.* AU - Müller, C. AU - Meas-Yedid, V.* AU - Schmitt-Kopplin, P. AU - Lazarow, P.B.* AU - Subtil, A.* C1 - 30677 C2 - 33827 CY - San Francisco TI - The intracellular bacteria Chlamydia hijack peroxisomes and utilize their enzymatic capacity to produce bacteria-specific phospholipids. JO - PLoS ONE VL - 9 IS - 1 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Background Information regarding the variability of metabolite levels over time in an individual is required to estimate the reproducibility of metabolite measurements. In intervention studies, it is critical to appropriately judge changes that are elicited by any kind of intervention. The pre-analytic phase (collection, transport and sample processing) is a particularly important component of data quality in multi-center studies. Methods Reliability of metabolites (within-and between-person variance, intraclass correlation coefficient) and stability (shipment simulation at different temperatures, use of gel-barrier collection tubes, freeze-thaw cycles) were analyzed in fasting serum and plasma samples of 22 healthy human subjects using a targeted LC-MS approach. Results Reliability of metabolite measurements was higher in serum compared to plasma samples and was good in most saturated short-and medium-chain acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids and hexose. The majority of metabolites were stable for 24 h on cool packs and at room temperature in non-centrifuged tubes. Plasma and serum metabolite stability showed good coherence. Serum metabolite concentrations were mostly unaffected by tube type and one or two freeze-thaw cycles. Conclusion A single time point measurement is assumed to be sufficient for a targeted metabolomics analysis of most metabolites. For shipment, samples should ideally be separated and frozen immediately after collection, as some amino acids and biogenic amines become unstable within 3 h on cool packs. Serum gel-barrier tubes can be used safely for this process as they have no effect on concentration in most metabolites. Shipment of non-centrifuged samples on cool packs is a cost-efficient alternative for most metabolites. AU - Breier, M. AU - Wahl, S. AU - Prehn, S. AU - Fugmann, M. AU - Ferrari, U. AU - Weise, M. AU - Banning, F. AU - Seissler, J. AU - Grallert, H. AU - Adamski, J. AU - Lechner, A. C1 - 30708 C2 - 33819 CY - San Francisco TI - Targeted metabolomics identifies reliable and stable metabolites in human serum and plasma samples. JO - PLoS ONE VL - 9 IS - 2 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. AU - Budiharjo, A.* AU - Chowdhury, S.P. AU - Dietel, K.* AU - Beator, B.* AU - Dolgova, O.* AU - Fan, B.* AU - Bleiss, W.* AU - Ziegler, J.T.* AU - Schmid, M. AU - Hartmann, A. AU - Borriss, R.* C1 - 31367 C2 - 34490 CY - San Francisco TI - Transposon mutagenesis of the plant-associated Bacillus amyloliquefaciens ssp. plantarum FZB42 revealed that the nfrA and RBAM17410 genes are involved in plant-microbe-interactions. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation-the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments "chloroplast," "CUL4-RING ubiquitin ligase complex," and "membrane" more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites. AU - Chaki, M. AU - Kovacs, I. AU - Spannagl, M. AU - Lindermayr, C. C1 - 32602 C2 - 35170 TI - Computational prediction of candidate proteins for S-nitrosylation in Arabidopsis thaliana. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease. AU - Degroote, R.L.* AU - Hauck, S.M. AU - Amann, B.* AU - Hirmer, S.* AU - Ueffing, M. AU - Deeg, C.A.* C1 - 30850 C2 - 33953 CY - San Francisco TI - Unraveling the equine lymphocyte proteome: Differential septin 7 expression associates with immune cells in equine recurrent uveitis. JO - PLoS ONE VL - 9 IS - 3 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - UNLABELLED: In chronic rhinosinusitis (CRS) different phenotypes have been reported based on cytokine profile and inflammatory cell patterns. The aim of this study was to characterize the intracytoplasmatic cytokines of T cells infiltrating the inflamed sinonasal mucosa. METHODS: Infiltrated T cells and tissue homogenates from sinonasal mucosal samples of 7 healthy subjects, 9 patients with CRS without nasal polyp (CRSsNP), 15 with CRS with nasal polyps (CRSwNP) and 5 cystic fibrosis patients (CF-NP) were analyzed for cytokine expression using flow cytometry and multiplex analysis respectively. Intracytoplasmic cytokinesin T cells were analyzed after stimulation of nasal polyps with Staphylococcus aureus enterotoxin B for 24 hours. RESULTS: The number of T cells per total living cells was significantly higher in patients with CRSwNP vs. CRSsNP and controls. 85% of the CD4(+) T cells showed to be memory T cells. The effector T cells present in all tissues have a predominant Th1 phenotype. Only in CRSwNP, a significant fraction of T cells produced the Th2 cytokines IL-4 and IL-5, while nasal polyps from CF patients were characterized by a higher CD4/CD8 T cell ratio and an increased number of Th17 cells. 24 h stimulation with SEB resulted in a significant induction of CD4(+) T cells producing IL-10 (Tr1 cells). CONCLUSION: T cell cytokine patterns in healthy and inflamed sinonasal mucosa revealed that Th2 cells (IL-4 and IL-5 producing cells) are significantly increased in CRSwNP mucosal inflammation. Exposure to SEB stimulates Tr1 cells that may contribute to the Th2 bias in CRSwNP. AU - Derycke, L.* AU - Eyerich, S. AU - van Crombruggen, K.* AU - Pérez-Novo, C.* AU - Holtappels, G.* AU - Deruyck, N.* AU - Gevaert, P.* AU - Bachert, C.* C1 - 32687 C2 - 35228 TI - Mixed T helper cell signatures in chronic rhinosinusitis with and without polyps. JO - PLoS ONE VL - 9 IS - 6 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Large amounts of manure have been applied to arable soils as fertilizer worldwide. Manure is often contaminated with veterinary antibiotics which enter the soil together with antibiotic resistant bacteria. However, little information is available regarding the main responders of bacterial communities in soil affected by repeated inputs of antibiotics via manure. In this study, a microcosm experiment was performed with two concentrations of the antibiotic sulfadiazine (SDZ) which were applied together with manure at three different time points over a period of 133 days. Samples were taken 3 and 60 days after each manure application. The effects of SDZ on soil bacterial communities were explored by barcoded pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. Samples with high concentration of SDZ were analyzed on day 193 only. Repeated inputs of SDZ, especially at a high concentration, caused pronounced changes in bacterial community compositions. By comparison with the initial soil, we could observe an increase of the disturbance and a decrease of the stability of soil bacterial communities as a result of SDZ manure application compared to the manure treatment without SDZ. The number of taxa significantly affected by the presence of SDZ increased with the times of manure application and was highest during the treatment with high SDZ-concentration. Numerous taxa, known to harbor also human pathogens, such as Devosia, Shinella, Stenotrophomonas, Clostridium, Peptostreptococcus, Leifsonia, Gemmatimonas, were enriched in the soil when SDZ was present while the abundance of bacteria which typically contribute to high soil quality belonging to the genera Pseudomonas and Lysobacter, Hydrogenophaga, and Adhaeribacter decreased in response to the repeated application of manure and SDZ. AU - Ding, G.-C.* AU - Radl, V. AU - Schloter-Hai, B. AU - Jechalke, S.* AU - Heuer, H.* AU - Smalla, K.* AU - Schloter, M. C1 - 30893 C2 - 34002 CY - San Francisco TI - Dynamics of soil bacterial communities in response to repeated application of manure containing sulfadiazine. JO - PLoS ONE VL - 9 IS - 3 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations. AU - Doppler, S.A.* AU - Werner, A.* AU - Barz, M.* AU - Lahm, H.* AU - Deutsch, M.A.* AU - Dreßen, M.* AU - Schiemann, M. AU - Voss, B.* AU - Gregoire, S.* AU - Kuppusamy, R.* AU - Wu, S.M.* AU - Lange, R.* AU - Krane, M.* C1 - 42881 C2 - 35641 TI - Myeloid zinc finger 1 (Mzf1) differentially modulates murine cardiogenesis by interacting with an Nkx2.5 cardiac enhancer. JO - PLoS ONE VL - 9 IS - 12 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - It has been suggested that mitochondrial dysfunction plays a role in metabolic disorders including obesity, diabetes, and hypertension. The fact that mitochondrial defects can be accumulated over time as a normal part of aging may explain why some individuals can eat all sorts of foods and remain at normal weight while they are young. However, around the fourth decade of life there is a trend towards "middle-age spread" with weight gain and the body's decreasing ability to metabolize calories efficiently. To test the hypothesis that mitochondrial variants are associated with BMI in adults, we analyzed a total number of 984 mitochondrial single nucleotide polymorphisms (mtSNPs) in a sample of 6,528 individuals participating in the KORA studies. To assess mtSNP association while taking heteroplasmy into account we used the raw signal intensity values measured on the microarray and applied linear regression. Significant results were obtained for 2 mtSNPs located in the Cytochrome c oxidase subunit genes (MT-CO1: Padjusted = 0.0140 and MT-CO3: Padjusted = 0.0286) and 3 mtSNPs located in the NADH dehydrogenase subunit genes (MT-ND1, MT-ND2 and MT-ND4L: Padjusted = 0.0286). Polymorphisms located in the MT-CO3 and MT-ND4L genes have not been associated with BMI or related phenotypes in the past. Our results highlight the importance of the mitochondrial genome among the factors that contribute to the risk of high BMI. Focusing on mitochondrial variants may lead to further insights regarding effects of existing medications, or even to the development of innovative treatments. AU - Flaquer, A. AU - Baumbach, C. AU - Kriebel, J. AU - Meitinger, T. AU - Peters, A. AU - Waldenberger, M. AU - Grallert, H. AU - Strauch, K. C1 - 31994 C2 - 34934 CY - San Francisco TI - Mitochondrial genetic variants identified to be associated with BMI in adults. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver. AU - Franceschi, V.* AU - Stellari, F.F.* AU - Mangia, C.* AU - Jacca, S.* AU - Lavrentiadou, S.* AU - Cavirani, S.* AU - Heikenwälder, M. AU - Donofrio, G.* C1 - 31100 C2 - 34120 CY - San Francisco TI - In vivo image analysis of BoHV-4-based vector in mice. JO - PLoS ONE VL - 9 IS - 4 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Permafrost-affected soils are among the most obvious ecosystems in which current microbial controls on organic matter decomposition are changing as a result of global warming. Warmer conditions in polygonal tundra will lead to a deepening of the seasonal active layer, provoking changes in microbial processes and possibly resulting in exacerbated carbon degradation under increasing anoxic conditions. To identify current microbial assemblages in carbon rich, water saturated permafrost environments, four polygonal tundra sites were investigated on Herschel Island and the Yukon Coast, Western Canadian Arctic. Ion Torrent sequencing of bacterial and archaeal 16S rRNA amplicons revealed the presence of all major microbial soil groups and indicated a local, vertical heterogeneity of the polygonal tundra soil community with increasing depth. Microbial diversity was found to be highest in the surface layers, decreasing towards the permafrost table. Quantitative PCR analysis of functional genes involved in carbon and nitrogen-cycling revealed a high functional potential in the surface layers, decreasing with increasing active layer depth. We observed that soil properties driving microbial diversity and functional potential varied in each study site. These results highlight the small-scale heterogeneity of geomorphologically comparable sites, greatly restricting generalizations about the fate of permafrost-affected environments in a warming Arctic. AU - Frank-Fahle, B. AU - Yergeau, E.* AU - Greer, C.W.* AU - Lantuit, H.* AU - Wagner, D.E.* C1 - 32686 C2 - 35227 TI - Microbial functional potential and community composition in permafrost-affected soils of the NW Canadian Arctic. JO - PLoS ONE VL - 9 IS - 1 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70. AU - Gehrmann, M.* AU - Stangl, S.* AU - Foulds, G.A.* AU - Oellinger, R.* AU - Breuninger, S.* AU - Rad, R.* AU - Pockley, A.G.* AU - Multhoff, G. C1 - 32050 C2 - 34943 CY - San Francisco TI - Tumor imaging and targeting potential of an Hsp70-derived 14-Mer peptide. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Ongoing climate change will lead to more extreme weather events, including severe drought periods and intense drying rewetting cycles. This will directly influence microbial nitrogen (N) turnover rates in soil by changing the water content and the oxygen partial pressure. Therefore, a space for time climate change experiment was conducted by transferring intact beech seedling-soil mesocosms from a northwest (NW) exposed site, representing today's climatic conditions, to a southwest (SW) exposed site, providing a model climate for future conditions with naturally occurring increased soil temperature (+0.8°C in average). In addition, severe drought and intense rainfall was simulated by a rainout shelter at SW and manual rewetting after 39 days drought, respectively. Soil samples were taken in June, at the end of the drought period (August), 24 and 72 hours after rewetting (August) and after a regeneration period of four weeks (September). To follow dynamics of bacterial and archaeal communities involved in N turnover, abundance and activity of nitrifiers, denitrifiers, N2-fixing microbes and N-mineralizers was analyzed based on marker genes and the related transcripts by qPCR from DNA and RNA directly extracted from soil. Abundance of the transcripts was reduced under climate change with most pronounced effects for denitrification. Our results revealed that already a transfer from NW to SW without further treatment resulted in decreased cnor and nosZ transcripts, encoding for nitric oxide reductase and nitrous oxide reductase, respectively, while nirK transcripts, encoding for nitrite reductase, remained unaffected. Severe drought additionally led to reduced nirK and cnor transcripts at SW. After rewetting, nirK transcripts increased rapidly at both sites, while cnor and nosZ transcripts increased only at NW. Our data indicate that the climate change influences activity pattern of microbial communities involved in denitrification processes to a different extend, which may impact emission rates of the greenhouse gas N2O. AU - Gschwendtner, S. AU - Tejedor, J.* AU - Bimueller, C.* AU - Dannenmann, M.* AU - Knabner, I.K.* AU - Schloter, M. C1 - 42884 C2 - 35739 TI - Climate change induces shifts in abundance and activity pattern of bacteria and archaea catalyzing major transformation steps in nitrogen turnover in a soil from a Mid-European beech forest. JO - PLoS ONE VL - 9 IS - 12 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils. CONCLUSION/SIGNIFICANCE: Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled. AU - Heimesaat, M.M.* AU - Fischer, A.* AU - Plickert, R.* AU - Wiedemann, T. AU - Loddenkemper, C.* AU - Göbel, U.B.* AU - Bereswill, S.* AU - Rieder, G.* C1 - 31630 C2 - 34623 CY - San Francisco TI - Helicobacter pylori induced gastric immunopathology is associated with distinct microbiota changes in the large intestines of long-term infected Mongolian gerbils. JO - PLoS ONE VL - 9 IS - 6 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs. AU - Helfer, M. AU - Koppensteiner, H. AU - Schneider, M. AU - Rebensburg, S. AU - Forcisi, S. AU - Müller, C. AU - Schmitt-Kopplin, P. AU - Schindler, M. AU - Brack-Werner, R. C1 - 29214 C2 - 32847 CY - San Francisco TI - The root extract of the medicinal plant Pelargonium sidoides is a potent HIV-1 attachment inhibitor. JO - PLoS ONE VL - 9 IS - 1 PB - Public Library of Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists. AU - Horsch, M. AU - Beckers, J. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Rathkolb, B. AU - Wolf, E.* AU - Aigner, B.* AU - Kemter, E.* C1 - 42794 C2 - 35348 TI - Uromodulin retention in thick ascending limb of Henle's loop affects SCD1 in neighboring proximal tubule: Renal transcriptome studies in mouse models of uromodulin-associated kidney disease. JO - PLoS ONE VL - 9 IS - 11 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Mutations in the orphan gene C19orf12 were identified as a genetic cause in a subgroup of patients with NBIA, a neurodegenerative disorder characterized by deposits of iron in the basal ganglia. C19orf12 was shown to be localized in mitochondria, however, nothing is known about its activity and no functional link exists to the clinical phenotype of the patients. This situation led us to investigate the effects of C19orf12 down-regulation in the model organism Drosophila melanogaster. Two genes are present in D. melanogaster, which are orthologs of C19orf12, CG3740 and CG11671. Here we provide evidence that transgenic flies with impaired C19orf12 homologs reflect the neurodegenerative phenotype and represent a valid tool to further analyze the pathomechanism in C19orf12-associated NBIA. AU - Iuso, A. AU - Sibon, O.C.* AU - Gorza, M. AU - Heim, K. AU - Organisti, C.* AU - Meitinger, T. AU - Prokisch, H. C1 - 30771 C2 - 33849 CY - San Francisco TI - Impairment of Drosophila orthologs of the human orphan protein c19orf12 induces bang sensitivity and neurodegeneration. JO - PLoS ONE VL - 9 IS - 2 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants. In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations - including two novel mutations - showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3. FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions. AU - Janke, H. AU - Pastore, F. AU - Schumacher, D.* AU - Herold, T. AU - Hopfner, K.P.* AU - Schneider, S.* AU - Berdel, W.E.* AU - Büchner, T.* AU - Woermann, B.J.* AU - Subklewe, M. AU - Bohlander, S.K.* AU - Hiddemann, W. AU - Spiekermann, K. AU - Polzer, H. C1 - 30875 C2 - 33955 CY - San Francisco TI - Activating FLT3 mutants show distinct gain-of-function phenotypes in vitro and a characteristic signaling pathway profile associated with prognosis in acute myeloid leukemia. JO - PLoS ONE VL - 9 IS - 3 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Colon cancer is caused by multiple genomic alterations which lead to genomic instability (GI). GI appears in molecular pathways of microsatellite instability (MSI) and chromosomal instability (CIN) with clinically observed case shares of about 15-20% and 80-85%. Radiation enhances the colon cancer risk by inducing GI, but little is known about different outcomes for MSI and CIN. Computer-based modelling can facilitate the understanding of the phenomena named above. Comprehensive biological models, which combine the two main molecular pathways to colon cancer, are fitted to incidence data of Japanese a-bomb survivors. The preferred model is selected according to statistical criteria and biological plausibility. Imprints of cell-based processes in the succession from adenoma to carcinoma are identified by the model from age dependences and secular trends of the incidence data. Model parameters show remarkable compliance with mutation rates and growth rates for adenoma, which has been reported over the last fifteen years. Model results suggest that CIN begins during fission of intestinal crypts. Chromosomal aberrations are generated at a markedly elevated rate which favors the accelerated growth of premalignant adenoma. Possibly driven by a trend of Westernization in the Japanese diet, incidence rates for the CIN pathway increased notably in subsequent birth cohorts, whereas rates pertaining to MSI remained constant. An imbalance between number of CIN and MSI cases began to emerge in the 1980s, whereas in previous decades the number of cases was almost equal. The CIN pathway exhibits a strong radio-sensitivity, probably more intensive in men. Among young birth cohorts of both sexes the excess absolute radiation risk related to CIN is larger by an order of magnitude compared to the MSI-related risk. Observance of pathway-specific risks improves the determination of the probability of causation for radiation-induced colon cancer in individual patients, if their exposure histories are known. AU - Kaiser, J.C. AU - Meckbach, R. AU - Jacob, P. C1 - 32644 C2 - 35198 TI - Genomic instability and radiation risk in molecular pathways to colon cancer. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals) and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy) and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies. AU - Karp, N.A.* AU - Speak, A.O.* AU - White, J.K.* AU - Adams, D.J.* AU - Hrabě de Angelis, M. AU - Herault, Y.* AU - Mott, R.F.* C1 - 32620 C2 - 35172 TI - Impact of temporal variation on design and analysis of mouse knockout phenotyping studies. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - High-quality mapping of genomic regions and genes between two organisms is an indispensable prerequisite for evolutionary analyses and comparative genomics. Existing approaches to this problem focus on either delineating orthologs or finding extended sequence regions of common evolutionary origin (syntenic blocks). We propose SyntenyMapper, a novel tool for refining predefined syntenic regions. SyntenyMapper creates a set of blocks with conserved gene order between two genomes and finds all minor rearrangements that occurred since the evolutionary split of the two species considered. We also present TrackMapper, a SyntenyMapper-based tool that allows users to directly compare genome features, such as histone modifications, between two organisms, and identify genes with highly conserved features. We demonstrate SyntenyMapper's advantages by conducting a large-scale analysis of micro-rearrangements within syntenic regions of 25 eukaryotic species. Unsurprisingly, the number and length of syntenic regions is correlated with evolutionary distance, while the number of micro-rearrangements depends only on the size of the harboring region. On the other hand, the size of rearranged regions remains relatively constant regardless of the evolutionary distance between the organisms, implying a length constraint in the rearrangement process. SyntenyMapper is a useful software tool for both large-scale and gene-centric genome comparisons. AU - Kaufmann, S.* AU - Frishman, D. C1 - 42767 C2 - 35298 CY - San Francisco TI - Analysis of micro-rearrangements in 25 eukaryotic species pairs by SyntenyMapper. JO - PLoS ONE VL - 9 IS - 11 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Patients suffering from brain malignancies are treated with high-dose ionising radiation. However, this may lead to severe learning and memory impairment. Preventive treatments to minimise these side effects have not been possible due to the lack of knowledge of the involved signalling pathways and molecular targets. Mouse hippocampal neuronal HT22 cells were irradiated with acute gamma doses of 0.5 Gy, 1.0 Gy and 4.0 Gy. Changes in the cellular proteome were investigated by isotope-coded protein label technology and tandem mass spectrometry after 4 and 24 hours. To compare the findings with the in vivo response, male NMRI mice were irradiated on postnatal day 10 with a gamma dose of 1.0 Gy, followed by evaluation of the cellular proteome of hippocampus and cortex 24 hours post-irradiation. Analysis of the in vitro proteome showed that signalling pathways related to synaptic actin-remodelling were significantly affected at 1.0 Gy and 4.0 Gy but not at 0.5 Gy after 4 and 24 hours. We observed radiation-induced reduction of the miR-132 and Rac1 levels; miR-132 is known to regulate Rac1 activity by blocking the GTPase-activating protein p250GAP. In the irradiated hippocampus and cortex we observed alterations in the signalling pathways similar to those in vitro. The decreased expression of miR-132 and Rac1 was associated with an increase in hippocampal cofilin and phospho-cofilin. The Rac1-Cofilin pathway is involved in the modulation of synaptic actin filament formation that is necessary for correct spine and synapse morphology to enable processes of learning and memory. We suggest that acute radiation exposure leads to rapid dendritic spine and synapse morphology alterations via aberrant cytoskeletal signalling and processing and that this is associated with the immediate neurocognitive side effects observed in patients treated with ionising radiation. AU - Kempf, S.J. AU - Buratovic, S.* AU - von Toerne, C. AU - Mörtl, S. AU - Stenerlöw, B.* AU - Hauck, S.M. AU - Atkinson, M.J. AU - Eriksson, P.* AU - Tapio, S. C1 - 32589 C2 - 35156 TI - Ionising radiation immediately impairs synaptic plasticity-associated cytoskeletal signalling pathways in HT22 cells and in mouse brain: An in vitro/in vivo comparison study. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects. AU - Khan, S.* AU - Greco, D.* AU - Michailidou, K.* AU - Milne, R.L.* AU - Muranen, T.A.* AU - Heikkinen, T.* AU - Aaltonen, K.* AU - Dennis, J.* AU - Bolla, M.K.* AU - Liu, J.* AU - Hall, P.* AU - Irwanto, A.* AU - Humphreys, K.* AU - Li, J.* AU - Czene, K.* AU - Chang-Claude, J.* AU - Hein, R.* AU - Rudolph, A.* AU - Seibold, P.* AU - Flesch-Janys, D.* AU - Fletcher, O.* AU - Peto, J.* AU - Dos Santos Silva, I.* AU - Johnson, N.* AU - Gibson, L.* AU - Aitken, Z.* AU - Hopper, J.L.* AU - Tsimiklis, H.* AU - Bui, M.* AU - Makalic, E.* AU - Schmidt, D.F.* AU - Southey, M.C.* AU - Apicella, C.* AU - Stone, J.* AU - Waisfisz, Q.* AU - Meijers-Heijboer, H.* AU - Adank, M.A.* AU - van der Luijt, R.B.* AU - Meindl, A.* AU - Schmutzler, R.K.* AU - Müller-Myhsok, B.* AU - Lichtner, P. AU - Turnbull, C.* AU - Rahman, N.* AU - Chanock, S.J.* AU - Hunter, D.J.* AU - Cox, A.* AU - Cross, S.S.* AU - Reed, M.W.* AU - Schmidt, M.K.* AU - Broeks, A.* AU - Veer, L.J.* AU - Hogervorst, F.B.* AU - Fasching, P.A.* AU - Schrauder, M.G.* AU - Ekici, A.B.* AU - Beckmann, M.W.* AU - Bojesen, S.E.* AU - Nørdestgaard, B.G.* AU - Nielsen, S.F.* AU - Flyger, H.* AU - Benítez, J.* AU - Zamora, P.M.* AU - Perez, J.I.A.* AU - Haiman, C.A.* AU - Henderson, B.E.* AU - Schumacher, F.* AU - Le Marchand, L.* AU - Pharoah, P.D.* AU - Dunning, A.M.* AU - Shah, M.* AU - Luben, R.* AU - Brown, J.* AU - Couch, F.J.* AU - Wang, X.* AU - Vachon, C.* AU - Olson, J.E.* AU - Lambrechts, D.* AU - Moisse, M.* AU - Paridaens, R.* AU - Christiaens, M.R.* AU - Guénel, P.* AU - Truong, T.* AU - Laurent-Puig, P.* AU - Mulot, C.* AU - Marme, F.* AU - Burwinkel, B.* AU - Schneeweiss, A.* AU - Sohn, C.* AU - Sawyer, E.J.* AU - Tomlinson, I.* AU - Kerin, M.J.* AU - Miller, N.* AU - Andrulis, I.L.* AU - Knight, J.A.* AU - Tchatchou, S.* AU - Mulligan, A.M.* AU - Dörk, T.* AU - Bogdanova, N.V.* AU - Antonenkova, N.N.* AU - Anton-Culver, H.* AU - Darabi, H.* AU - Eriksson, M.* AU - Garcia-Closas, M.* AU - Figueroa, J.* AU - Lissowska, J.* AU - Brinton, L.* AU - Devilee, P.* AU - Tollenaar, R.A.* AU - Seynaeve, C.* AU - van Asperen, C.J.* AU - Kristensen, V.N.* AU - Slager, S.* AU - Toland, A.E.* AU - Ambrosone, C.B.* AU - Yannoukakos, D.* AU - Lindblom, A.* AU - Margolin, S.* AU - Radice, P.* AU - Peterlongo, P.* AU - Barile, M.* AU - Mariani, P.* AU - Hooning, M.J.* AU - Martens, J.W.M.* AU - Collee, J.M.* AU - Jäger, A.* AU - Jakubowska, A.* AU - Lubinski, J.* AU - Jaworska-Bieniek, K.* AU - Durda, K.* AU - Giles, G.G.* AU - McLean, C.* AU - Brauch, H.* AU - Brüning, T* AU - Ko, Y.D.* AU - Brenner, H.* AU - Dieffenbach, A.K.* AU - Arndt, V.* AU - Stegmaier, C.* AU - Swerdlow, A.* AU - Ashworth, A.* AU - Orr, N.* AU - Jones, M.* AU - Simard, J.* AU - Goldberg, M.S.* AU - Labreche, F.* AU - Dumont, M.* AU - Winqvist, R.* AU - Pylkäs, K.* AU - Jukkola-Vuorinen, A.* AU - Grip, M.* AU - Kataja, V.* AU - Kosma, V.M.* AU - Hartikainen, J.M.* AU - Mannermaa, A.* AU - Hamann, U.* AU - Chenevix-Trench, G.* AU - Blomqvist, C.* AU - Aittomäki, K.* AU - Easton, D.F.* AU - Nevanlinna, H.* C1 - 42748 C2 - 35332 CY - San Francisco TI - MicroRNA related polymorphisms and breast cancer risk. JO - PLoS ONE VL - 9 IS - 11 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND AND PURPOSE: Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals. MATERIALS AND METHODS: The middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points. RESULTS: Post ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit. CONCLUSION: The intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area. AU - Kneipp, M. AU - Turner, J.E. AU - Hambauer, S.* AU - Krieg, S.M.* AU - Lehmberg, J.* AU - Lindauer, U.* AU - Razansky, D. C1 - 31173 C2 - 34218 TI - Functional real-time optoacoustic imaging of middle cerebral artery occlusion in mice. JO - PLoS ONE VL - 9 IS - 4 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI≥30 kg/m2) and n = 2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and their offspring could not be substantiated by the findings of the present study. AU - Knoll, N.* AU - Jarick, I.* AU - Volckmar, A.L.* AU - Klingenspor, M. AU - Illig, T. AU - Grallert, H. AU - Gieger, C. AU - Wichmann, H.-E. AU - Peters, A. AU - Wiegand, S.* AU - Biebermann, H.* AU - Fischer-Posovszky, P.* AU - Wabitsch, M.* AU - Völzke, H.* AU - Nauck, M.* AU - Teumer, A.* AU - Rosskopf, D.* AU - Rimmbach, C.* AU - Schreiber, S.* AU - Jacobs, G.* AU - Lieb, W.* AU - Franke, A.* AU - Hebebrand, J.* AU - Hinney, A.* C1 - 31188 C2 - 34283 CY - San Francisco TI - Mitochondrial DNA variants in obesity. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof. AU - Kraus, P.* AU - Sivakamasundari, V.* AU - Yu, H.B.* AU - Xing, X.* AU - Lim, S.L.* AU - Adler, T. AU - Aguilar-Pimentel, J.A. AU - Becker, L. AU - Bohla, A. AU - Garrett, L. AU - Hans, W. AU - Hölter, S.M. AU - Janas, E. AU - Moreth, K. AU - Prehn, C. AU - Puk, O. AU - Rathkolb, B. AU - Rozman, J. AU - Adamski, J. AU - Bekeredjian, R. AU - Busch, D.H. AU - Graw, J. AU - Klingenspor, M. AU - Klopstock, T. AU - Neff, F. AU - Ollert, M. AU - Stöger, T. AU - Yildirim, A.Ö. AU - Eickelberg, O. AU - Wolf, E. AU - Wurst, W. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Lufkin, T.* AU - Stanton, L.W.* C1 - 31921 C2 - 34865 CY - San Francisco TI - Pleiotropic functions for transcription factor Zscan10. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - In recent years, marine algae have emerged as a rich and promising source of molecules with potent activities against various human pathogens. The widely distributed brown alga Lobophora variegata that is often associated with tropical coral reefs exerts strong antibacterial and antiprotozoal effects, but so far has not been associated with specific anti-viral activities. This study investigated potential HIV-1 inhibitory activity of L. variegata collected from different geographical regions, using a cell-based full replication HIV-1 reporter assay. Aqueous L. variegata extracts showed strong inhibitory effects on several HIV-1 strains, including drug-resistant and primary HIV-1 isolates, and protected even primary cells (PBMC) from HIV-1-infection. Anti-viral potency was related to ecological factors and showed clear differences depending on light exposition or epiphyte growth. Assays addressing early events of the HIV-1 replication cycle indicated that L. variegata extracts inhibited entry of HIV-1 into cells at a pre-fusion step possibly by impeding mobility of virus particles. Further characterization of the aqueous extract demonstrated that even high doses had only moderate effects on viability of cultured and primary cells (PBMCs). Imaging-based techniques revealed extract effects on the plasma membrane and actin filaments as well as induction of apoptosis at concentrations exceeding EC50 of anti-HIV-1 activity by more than 400 fold. In summary, we show for the first time that L. variegata extracts inhibit HIV-1 entry, thereby suggesting this alga as promising source for the development of novel HIV-1 inhibitors. AU - Kremb, S. AU - Helfer, M. AU - Kraus, B.* AU - Wolff, H.* AU - Wild, C.* AU - Schneider, M. AU - Voolstra, C.R.* AU - Brack-Werner, R. C1 - 31962 C2 - 34916 CY - San Francisco TI - Aqueous extracts of the marine brown alga Lobophora variegata inhibit HIV-1 infection at the level of virus entry into cells. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Structural genetic variants as short tandem repeats (STRs) are not targeted in SNP-based association studies and thus, their possible association signals are missed. We systematically searched for STRs in gene regions known to contribute to total cholesterol, HDL cholesterol, LDL cholesterol and triglyceride levels in two independent studies (KORA F4, n = 2553 and SAPHIR, n = 1648), resulting in 16 STRs that were finally evaluated. In a combined dataset of both studies, the sum of STR alleles was regressed on each phenotype, adjusted for age and sex. The association analyses were repeated for SNPs in a 200 kb region surrounding the respective STRs in the KORA F4 Study. Three STRs were significantly associated with total cholesterol (within LDLR, the APOA1/C3/A4/A5/BUD13 gene region and ABCG5/8), five with HDL cholesterol (3 within CETP, one in LPL and one inAPOA1/C3/A4/A5/BUD13), three with LDL cholesterol (LDLR, ABCG5/8 and CETP) and two with triglycerides (APOA1/C3/A4/A5/BUD13 and LPL). None of the investigated STRs, however, showed a significant association after adjusting for the lead or adjacent SNPs within that gene region. The evaluated STRs were found to be well tagged by the lead SNP within the respective gene regions. Therefore, the STRs reflect the association signals based on surrounding SNPs. In conclusion, none of the STRs contributed additionally to the SNP-based association signals identified in GWAS on lipid traits. AU - Lamina, C.* AU - Haun, M.* AU - Coassin, S.* AU - Kloss-Brandstätter, A.* AU - Gieger, C. AU - Peters, A. AU - Grallert, H. AU - Strauch, K. AU - Meitinger, T. AU - Kedenko, L.* AU - Paulweber, B.* AU - Kronenberg, F.* C1 - 31801 C2 - 34785 CY - San Francisco TI - A systematic evaluation of short tandem repeats in lipid candidate genes: Riding on the SNP-wave. JO - PLoS ONE VL - 9 IS - 7 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - AIMS/HYPOTHESIS: Recently, cardiotrophin-1, a member of the interleukin-6 family of cytokines was described to protect beta-cells from apoptosis, to improve glucose-stimulated insulin secretion and insulin resistance, and to prevent streptozotocin-induced diabetes in mice. Here, we studied whether common single nucleotide polymorphisms (SNPs) in the CTF1 locus, encoding cardiotrophin-1, influence insulin secretion and insulin sensitivity in humans. METHODS: We genotyped 1,771 German subjects for three CTF1 tagging SNPs (rs1046276, rs1458201, and rs8046707). The subjects were metabolically characterized by an oral glucose tolerance test. Subgroups underwent magnetic resonance (MR) imaging/spectroscopy and hyperinsulinaemic-euglycaemic clamps. RESULTS: After appropriate adjustment, the minor allele of CTF1 SNP rs8046707 was significantly associated with decreased in vivo measures of insulin sensitivity. The other tested SNPs were not associated with OGTT-derived sensitivity parameters, nor did the three tested SNPs show any association with OGTT-derived parameters of insulin release. In the MR subgroup, SNP rs8046707 was nominally associated with lower visceral adipose tissue. Furthermore, the SNP rs1458201 showed a nominal association with increased VLDL levels. CONCLUSIONS: In conclusion, this study, even though preliminary and awaiting further confirmation by independent replication, provides first evidence that common genetic variation in CTF1 could contribute to insulin sensitivity in humans. Our SNP data indicate an insulin-desensitizing effect of cardiotrophin-1 and underline that cardiotrophin-1 represents an interesting target to influence insulin sensitivity. AU - Lutz, S.Z. AU - Franck, O.* AU - Böhm, A. AU - Machann, J. AU - Schick, F. AU - Machicao, F. AU - Fritsche, A. AU - Häring, H.-U. AU - Staiger, H. C1 - 31774 C2 - 34732 CY - San Francisco TI - Common genetic variation in the human CTF1 locus, encoding cardiotrophin-1, determines insulin sensitivity. JO - PLoS ONE VL - 9 IS - 7 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: There is increasing evidence that prevention programmes for type 2 diabetes mellitus (T2DM) and obesity need to consider individual and regional risk factors. Our objective is to assess the independent association of area level deprivation with T2DM and obesity controlling for individual risk factors in a large study covering the whole of Germany. METHODS: We combined data from two consecutive waves of the national health interview survey 'GEDA' conducted by the Robert Koch Institute in 2009 and 2010. Data collection was based on computer-assisted telephone interviews. After exclusion of participants <30 years of age and those with missing responses, we included n = 33,690 participants in our analyses. The outcome variables were the 12-month prevalence of known T2DM and the prevalence of obesity (BMI ≥30 kg/m(2)). We also controlled for age, sex, BMI, smoking, sport, living with a partner and education. Area level deprivation of the districts was defined by the German Index of Multiple Deprivation. Logistic multilevel regression models were performed using the software SAS 9.2. RESULTS: Of all men and women living in the most deprived areas, 8.6% had T2DM and 16.9% were obese (least deprived areas: 5.8% for T2DM and 13.7% for obesity). For women, higher area level deprivation and lower educational level were both independently associated with higher T2DM and obesity prevalence [highest area level deprivation: OR 1.28 (95% CI: 1.05-1.55) for T2DM and OR 1.28 (95% CI: 1.10-1.49) for obesity]. For men, a similar association was only found for obesity [OR 1.20 (95% CI: 1.02-1.41)], but not for T2DM. CONCLUSION: Area level deprivation is an independent, important determinant of T2DM and obesity prevalence in Germany. Identifying and targeting specific area-based risk factors should be considered an essential public health issue relevant to increasing the effectiveness of diabetes and obesity prevention.   AU - Maier, W. AU - Scheidt-Nave, C.* AU - Holle, R. AU - Kroll, L.E.* AU - Lampert, T.* AU - Du, Y.* AU - Heidemann, C.* AU - Mielck, A. C1 - 30711 C2 - 33790 CY - San Francisco TI - Area level deprivation is an independent determinant of prevalent type 2 diabetes and obesity at the national level in Germany. Results from the national telephone health interview surveys 'German Health Update' GEDA 2009 and 2010. JO - PLoS ONE VL - 9 IS - 2 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse. AU - Meier, F. AU - Giesert, F. AU - Delic, S. AU - Faus-Kessler, T. AU - Matheus, F. AU - Simeone, A.* AU - Hölter, S.M. AU - Kühn, R. AU - Vogt Weisenhorn, D.M. AU - Wurst, W. AU - Prakash, N. C1 - 31720 C2 - 34689 CY - San Francisco TI - FGF/FGFR2 signaling regulates the generation and correct positioning of Bergmann glia cells in the developing mouse cerebellum. JO - PLoS ONE VL - 9 IS - 7 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [18F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = -0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy. AU - Meier, R.* AU - Braren, R.* AU - Kosanke, Y.* AU - Bussemer, J.* AU - Neff, F. AU - Wildgruber, M.* AU - Schwarzenböck, S.* AU - Frank, A.* AU - Haller, B.* AU - Hohlbaum, A.M.* AU - Schwaiger, M.* AU - Gille, H.* AU - Rummeny, E.J.* AU - Beer, A.J.* C1 - 31242 C2 - 34246 CY - San Francisco TI - Multimodality multiparametric imaging of early tumor response to a novel antiangiogenic therapy based on anticalins. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - In retinitis pigmentosa - a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor's dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the understanding of the degeneration process and may guide future rescue strategies. AU - Menzler, J. AU - Channappa, L.* AU - Zeck, G.* C1 - 31995 C2 - 34933 CY - San Francisco TI - Rhythmic ganglion cell activity in bleached and blind adult mouse retinas. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The TAR DNA binding protein (TDP-43) was originally identified as a host cell factor binding to the HIV-1 LTR and thereby suppressing HIV-1 transcription and gene expression (Ou et al., J.Virol. 1995, 69(6):3584). TDP-43 is a global regulator of transcription, can influence RNA metabolism in many different ways and is ubiquitously expressed. Thus, TDP-43 could be a major factor restricting HIV-1 replication at the level of LTR transcription and gene expression. These facts prompted us to revisit the role of TDP-43 for HIV-1 replication. We utilized established HIV-1 cell culture systems as well as primary cell models and performed a comprehensive analysis of TDP-43 function and investigated its putative impact on HIV-1 gene expression. In HIV-1 infected cells TDP-43 was neither degraded nor sequestered from the nucleus. Furthermore, TDP-43 overexpression as well as siRNA mediated knockdown did not affect HIV-1 gene expression and virus production in T cells and macrophages. In summary, our experiments argue against a restricting role of TDP-43 during HIV-1 replication in immune cells. AU - Nehls, J. AU - Koppensteiner, H. AU - Brack-Werner, R. AU - Floß, T. AU - Schindler, M. C1 - 31935 C2 - 34898 CY - San Francisco TI - HIV-1 replication in human immune cells is independent of TAR DNA binding Protein 43 (TDP-43) expression. JO - PLoS ONE VL - 9 IS - 8 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The fungus F. fujikuroi is well known for its production of gibberellins causing the 'bakanae' disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding ("Api-box") motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants. AU - Niehaus, E.M.* AU - Janevska, S.* AU - von Bargen, K.W.* AU - Sieber, C.M.K. AU - Harrer, H.* AU - Humpf, H.U.* AU - Tudzynski, B.* C1 - 31811 C2 - 34782 CY - San Francisco TI - Apicidin F: Characterization and genetic manipulation of a new secondary metabolite gene cluster in the rice pathogen Fusarium fujikuroi. JO - PLoS ONE VL - 9 IS - 7 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Accelerometry is an important method for extending our knowledge about intensity, duration, frequency and patterns of physical activity needed to promote health. This study has used accelerometry to detect associations between intensity levels and related activity patterns with multimorbidity and disability. Moreover, the proportion of people meeting the physical activity recommendations for older people was assessed. METHODS: Physical activity was measured in 168 subjects (78 males; 65-89 years of age), using triaxial GT3X accelerometers for ten consecutive days. The associations between physical activity parameters and multimorbidity or disability was examined using multiple logistic regression models, which were adjusted for gender, age, education, smoking, alcohol consumption, lung function, nutrition and multimorbidity or disability. RESULTS: 35.7% of the participants met the physical activity recommendations of at least 150 minutes of moderate to vigorous activity per week. Only 11.9% reached these 150 minutes, when only bouts of at least 10 minutes were counted. Differences in moderate to vigorous activity between people with and without multimorbidity or disability were more obvious when shorter bouts instead of only longer bouts were included. Univariate analyses showed an inverse relationship between physical activity and multimorbidity or disability for light and moderate to vigorous physical activity. A higher proportion of long activity bouts spent sedentarily was associated with higher risk for multimorbidity, whereas a high proportion of long bouts in light activity seemed to prevent disability. After adjustment for covariates, there were no significant associations, anymore. CONCLUSIONS: The accumulated time in moderate to vigorous physical activity seems to have a stronger relationship with health and functioning when shorter activity bouts and not only longer bouts were counted. We could not detect an association of the intensity levels or activity patterns with multimorbidity or disability in elderly people after adjustment for covariates. AU - Ortlieb, S. AU - Gorzelniak, L.* AU - Nowak, D.* AU - Strobl, R.* AU - Grill, E.* AU - Thorand, B. AU - Peters, A. AU - Kuhn, K.A.* AU - Karrasch, S. AU - Horsch, A.* AU - Schulz, H. C1 - 34365 C2 - 35266 TI - Associations between multiple accelerometry-assessed physical activity parameters and selected health outcomes in elderly people - results from the KORA-Age study. JO - PLoS ONE VL - 9 IS - 11 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML. AU - Pastore, F. AU - Greif, P.A. AU - Schneider, S.* AU - Ksienzyk, B.* AU - Mellert, G.* AU - Zellmeier, E.* AU - Braess, J.* AU - Sauerland, C.M.* AU - Heinecke, A.* AU - Krug, U.* AU - Berdel, W.E.* AU - Buechner, T.* AU - Woermann, B.J.* AU - Hiddemann, W. AU - Spiekermann, K. C1 - 32518 C2 - 35105 TI - The NPM1 mutation type has no impact on survival in cytogenetically normal AML. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics (C, N, Mg, K, Ca, P) and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves (P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days (laccase, P<0.0001; manganese peroxidase (MnP), P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling. AU - Purahong, W.* AU - Kapturska, D.* AU - Pecyna, M.J.* AU - Schulz, E.* AU - Schloter, M. AU - Buscot, F.* AU - Hofrichter, M.* AU - Kruger, D.* C1 - 31003 C2 - 34073 CY - San Francisco TI - Influence of different forest system management practices on leaf litter decomposition rates, nutrient dynamics and the activity of ligninolytic enzymes: A case study from central European forests. JO - PLoS ONE VL - 9 IS - 4 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Psoriasis is characterized by an apoptosis-resistant and metabolic active epidermis, while a hallmark for allergic contact dermatitis (ACD) is T cell-induced keratinocyte apoptosis. Here, we induced ACD reactions in psoriasis patients sensitized to nickel (n = 14) to investigate underlying mechanisms of psoriasis and ACD simultaneously. All patients developed a clinically and histologically typical dermatitis upon nickel challenge even in close proximity to pre-existing psoriasis plaques. However, the ACD reaction was delayed as compared to non-psoriatic patients, with a maximum intensity after 7 days. Whole genome expression analysis revealed alterations in numerous pathways related to metabolism and proliferation in non-involved skin of psoriasis patients as compared to non-psoriatic individuals, indicating that even in clinically non-involved skin of psoriasis patients molecular events opposing contact dermatitis may occur. Immunohistochemical comparison of ACD reactions as well as in vitro secretion analysis of lesional T cells showed a higher Th17 and neutrophilic migration as well as epidermal proliferation in psoriasis, while ACD reactions were dominated by cytotoxic CD8+ T cells and a Th2 signature. Based on these findings, we hypothesized an ACD reaction directly on top of a pre-existing psoriasis plaque might influence the clinical course of psoriasis. We observed a strong clinical inflammation with a mixed psoriasis and eczema phenotype in histology. Surprisingly, the initial psoriasis plaque was unaltered after self-limitation of the ACD reaction. We conclude that sensitized psoriasis patients develop a typical, but delayed ACD reaction which might be relevant for patch test evaluation in clinical practice. Psoriasis and ACD are driven by distinct and independent immune mechanisms. AU - Quaranta, M. AU - Eyerich, S. AU - Knapp, B. AU - Nasorri, F.* AU - Scarponi, C.* AU - Mattii, M. AU - Garzorz-Stark, N.* AU - Harlfinger, A.T.* AU - Jaeger, T.* AU - Grosber, M.* AU - Pennino, D. AU - Mempel, M.* AU - Schnopp, C.* AU - Theis, F.J. AU - Albanesi, C.* AU - Cavani, A.* AU - Schmidt-Weber, C.B. AU - Ring, J.* AU - Eyerich, K.* C1 - 31810 C2 - 34783 TI - Allergic contact dermatitis in psoriasis patients: Typical, delayed, and non-interacting. JO - PLoS ONE VL - 9 IS - 7 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The chemical composition of grape berries is influenced by various environmental conditions often considered to be representative of a "terroir". If grapes from a given terroir are assumed to reflect this origin in their chemical compositions, the corresponding wine should also reflect it. The aim of this work was therefore to reveal the "terroir" expression within the chemodiversity of grapes and related wines, using ultrahigh-resolution mass spectrometry. Grapes and corresponding wines, from two distinct - though very proximate - terroirs of Burgundy were analyzed over three vintages (2010, 2011 and 2012). Ultrahigh-resolution mass spectrometry and ultra-high performance liquid chromatography were used as untargeted and targeted approaches to discriminate complex chemical fingerprints for vintages, classes (wines, skins or musts), and terroirs. Statistical analyses revealed that even if vintages have the most significant impact on fingerprints, the most significant terroir differences are seen in the grapes of a given vintage. AU - Roullier-Gall, C. AU - Lucio, M. AU - Noret, L.* AU - Schmitt-Kopplin, P. AU - Gougeon, R.D.* C1 - 31475 C2 - 34517 CY - San Francisco TI - How subtle is the "Terroir" effect? Chemistry-related signatures of two "Climats de Bourgogne". JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: Treatment of diabetic subjects with cinnamon demonstrated an improvement in blood glucose concentrations and insulin sensitivity but the underlying mechanisms remained unclear. This work intends to elucidate the impact of cinnamon effects on the brain by using isolated astrocytes, and an obese and diabetic mouse model. METHODS: Cinnamon components (eugenol, cinnamaldehyde) were added to astrocytes and liver cells to measure insulin signaling and glycogen synthesis. Ob/ob mice were supplemented with extract from cinnamomum zeylanicum for 6 weeks and cortical brain activity, locomotion and energy expenditure were evaluated. Insulin action was determined in brain and liver tissues. RESULTS: Treatment of primary astrocytes with eugenol promoted glycogen synthesis, whereas the effect of cinnamaldehyde was attenuated. In terms of brain function in vivo, cinnamon extract improved insulin sensitivity and brain activity in ob/ob mice, and the insulin-stimulated locomotor activity was improved. In addition, fasting blood glucose levels and glucose tolerance were greatly improved in ob/ob mice due to cinnamon extracts, while insulin secretion was unaltered. This corresponded with lower triglyceride and increased liver glycogen content and improved insulin action in liver tissues. In vitro, Fao cells exposed to cinnamon exhibited no change in insulin action. CONCLUSIONS: Together, cinnamon extract improved insulin action in the brain as well as brain activity and locomotion. This specific effect may represent an important central feature of cinnamon in improving insulin action in the brain, and mediates metabolic alterations in the periphery to decrease liver fat and improve glucose homeostasis. AU - Sartorius, T. AU - Peter, A.* AU - Schulz, N.* AU - Drescher, A.* AU - Bergheim, I.* AU - Machann, J. AU - Schick, F.* AU - Siegel-Axel, D.* AU - Schürmann, A.* AU - Weigert, C. AU - Häring, H.-U. AU - Hennige, A.M. C1 - 30862 C2 - 33994 CY - San Francisco TI - Cinnamon extract improves insulin sensitivity in the brain and lowers liver fat in mouse models of obesity. JO - PLoS ONE VL - 9 IS - 3 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model. AU - Schötz, U. AU - Deliolanis, N. AU - Ng, D.* AU - Pauli, J.* AU - Resch-Genger, U.* AU - Kühn, E. AU - Heuer, S. AU - Beisker, W. AU - Köster, R.W. AU - Zitzelsberger, H. AU - Caldwell, R.B. C1 - 32102 C2 - 34965 CY - San Francisco TI - Usefulness of a darwinian system in a biotechnological application: Evolution of optical window fluorescent protein variants under selective pressure. JO - PLoS ONE VL - 9 IS - 9 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: We aimed to assess whether whole blood expression quantitative trait loci (eQTLs) with effects in cis and trans are robust and can be used to identify regulatory pathways affecting disease susceptibility. MATERIALS AND METHODS: We performed whole-genome eQTL analyses in 890 participants of the KORA F4 study and in two independent replication samples (SHIP-TREND, N = 976 and EGCUT, N = 842) using linear regression models and Bonferroni correction. RESULTS: In the KORA F4 study, 4,116 cis-eQTLs (defined as SNP-probe pairs where the SNP is located within a 500 kb window around the transcription unit) and 94 trans-eQTLs reached genome-wide significance and overall 91% (92% of cis-, 84% of trans-eQTLs) were confirmed in at least one of the two replication studies. Different study designs including distinct laboratory reagents (PAXgene™ vs. Tempus™ tubes) did not affect reproducibility (separate overall replication overlap: 78% and 82%). Immune response pathways were enriched in cis- and trans-eQTLs and significant cis-eQTLs were partly coexistent in other tissues (cross-tissue similarity 40-70%). Furthermore, four chromosomal regions displayed simultaneous impact on multiple gene expression levels in trans, and 746 eQTL-SNPs have been previously reported to have clinical relevance. We demonstrated cross-associations between eQTL-SNPs, gene expression levels in trans, and clinical phenotypes as well as a link between eQTLs and human metabolic traits via modification of gene regulation in cis. CONCLUSIONS: Our data suggest that whole blood is a robust tissue for eQTL analysis and may be used both for biomarker studies and to enhance our understanding of molecular mechanisms underlying gene-disease associations. AU - Schramm, K. AU - Marzi, C. AU - Schurmann, C.* AU - Carstensen, M.* AU - Reinmaa, E.* AU - Biffar, R.* AU - Eckstein, G.N. AU - Gieger, C. AU - Grabe, H.J.* AU - Homuth, G.* AU - Kastenmüller, G. AU - Mägi, R.* AU - Metspalu, A.* AU - Mihailov, E.* AU - Peters, A. AU - Petersmann, A.* AU - Roden, M.* AU - Strauch, K. AU - Suhre, K. AU - Teumer, A.* AU - Völker, U.* AU - Völzke, H.* AU - Wang-Sattler, R. AU - Waldenberger, M. AU - Meitinger, T. AU - Illig, T.* AU - Herder, C.* AU - Grallert, H. AU - Prokisch, H. C1 - 31081 C2 - 36323 CY - San Francisco TI - Mapping the genetic architecture of gene regulation in whole blood. JO - PLoS ONE VL - 9 IS - 4 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Restless legs syndrome (RLS) is a common neurologic disorder characterized by nightly dysesthesias affecting the legs primarily during periods of rest and relieved by movement. RLS is a complex genetic disease and susceptibility factors in six genomic regions have been identified by means of genome-wide association studies (GWAS). For some complex genetic traits, expression quantitative trait loci (eQTLs) are enriched among trait-associated single nucleotide polymorphisms (SNPs). With the aim of identifying new genetic susceptibility factors for RLS, we assessed the 332 best-associated SNPs from the genome-wide phase of the to date largest RLS GWAS for cis-eQTL effects in peripheral blood from individuals of European descent. In 740 individuals belonging to the KORA general population cohort, 52 cis-eQTLs with pnominal<10-3 were identified, while in 976 individuals belonging to the SHIP-TREND general population study 53 cis-eQTLs with pnominal<10-3 were present. 23 of these cis-eQTLs overlapped between the two cohorts. Subsequently, the twelve of the 23 cis-eQTL SNPs, which were not located at an already published RLS-associated locus, were tested for association in 2449 RLS cases and 1462 controls. The top SNP, located in the DET1 gene, was nominally significant (p<0.05) but did not withstand correction for multiple testing (p = 0.42). Although a similar approach has been used successfully with regard to other complex diseases, we were unable to identify new genetic susceptibility factor for RLS by adding this novel level of functional assessment to RLS GWAS data. AU - Schulte, E.C. AU - Schramm, K. AU - Schurmann, C.* AU - Lichtner, P. AU - Herder, C.* AU - Roden, M.* AU - Gieger, C. AU - Peters, A. AU - Trenkwalder, C.* AU - Högl, B.* AU - Frauscher, B.* AU - Berger, K.* AU - Fietze, I.* AU - Gross, N.* AU - Stiasny-Kolster, K.* AU - Oertel, W.* AU - Bachmann, C.G.* AU - Paulus, W.* AU - * AU - Völzke, H.* AU - Schminke, U.* AU - Nauck, M.* AU - Illig, T. AU - Meitinger, T. AU - Müller-Myhsok, B.* AU - Prokisch, H. AU - Winkelmann, J. C1 - 31514 C2 - 34523 CY - San Francisco TI - Blood cis-eQTL analysis fails to identify novel association signals among sub-threshold candidates from genome-wide association studies in restless legs syndrome. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - The frequency of inherited bilateral autosomal recessive non-syndromic hearing loss (ARNSHL) in Pakistan is 1.6/1000 individuals. More than 50% of the families carry mutations in GJB2 while mutations in MYO15A account for about 5% of recessive deafness. In the present study a cohort of 30 ARNSHL families was initially screened for mutations in GJB2 and MYO15A. Homozygosity mapping was performed by employing whole genome single nucleotide polymorphism (SNP) genotyping in the families that did not carry mutations in GJB2 or MYO15A. Mutation analysis was performed for the known ARNSHL genes present in the homozygous regions to determine the causative mutations. This allowed the identification of a causative mutation in all the 30 families including 9 novel mutations, which were identified in 9 different families (GJB2 (c.598G>A, p.Gly200Arg); MYO15A (c.9948G>A, p.Gln3316Gln; c.3866+1G>A; c.8767C>T, p.Arg2923* and c.8222T>C, p.Phe2741Ser), TMC1 (c.362+18A>G), BSND (c.97G>C, p.Val33Leu), TMPRSS3 (c.726C>G, p.Cys242Trp) and MSRB3 (c.20T>G, p.Leu7Arg)). Furthermore, 12 recurrent mutations were detected in 21 other families. The 21 identified mutations included 10 (48%) missense changes, 4 (19%) nonsense mutations, 3 (14%) intronic mutations, 2 (9%) splice site mutations and 2 (9%) frameshift mutations. GJB2 accounted for 53% of the families, while mutations in MYO15A were the second most frequent (13%) cause of ARNSHL in these 30 families. The identification of novel as well as recurrent mutations in the present study increases the spectrum of mutations in known deafness genes which could lead to the identification of novel founder mutations and population specific mutated deafness genes causative of ARNSHL. These results provide detailed genetic information that has potential diagnostic implication in the establishment of cost-efficient allele-specific analysis of frequently occurring variants in combination with other reported mutations in Pakistani populations. AU - Shafique, S.* AU - Siddiqi, S.* AU - Schraders, M.* AU - Oostrik, J.* AU - Ayub, H.* AU - Bilal, A.* AU - Ajmal, M.* AU - Seco, C.Z.* AU - Strom, T.M. AU - Mansoor, A.* AU - Mazhar, K.* AU - Shah, S.T.* AU - Hussain, A.* AU - Azam, M.* AU - Kremer, H.* AU - Qamar, R.* C1 - 31642 C2 - 34617 CY - San Francisco TI - Genetic spectrum of autosomal recessive non-syndromic hearing loss in Pakistani families. JO - PLoS ONE VL - 9 IS - 6 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination. AU - Sieber, C.M.K. AU - Lee, W. AU - Wong, P. AU - Münsterkötter, M. AU - Mewes, H.-W. AU - Schmeitzl, C.* AU - Varga, E.* AU - Berthiller, F.* AU - Adam, G.* AU - Güldener, U. C1 - 32601 C2 - 35891 TI - The Fusarium graminearum genome reveals more secondary metabolite gene clusters and hints of horizontal gene transfer. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. METHODOLOGY/PRINCIPAL FINDINGS: Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. CONCLUSIONS/SIGNIFICANCE: This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature. AU - Sievert, W. AU - Tapio, S. AU - Breuninger, S. AU - Gaipl, U.* AU - Andratschke, N. AU - Trott, K.R.* AU - Multhoff, G. C1 - 30867 C2 - 33989 CY - San Francisco TI - Adhesion molecule expression and function of primary endothelial cells in benign and malignant tissues correlates with proliferation. JO - PLoS ONE VL - 9 IS - 3 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - We present an updated analysis of incidence and mortality from atherosclerotic induced ischemic heart diseases in the cohort of workers at the Mayak Production Association (PA). This cohort constitutes one of the most important sources for the assessment of radiation risk. It is exceptional because it comprises information on several other risk factors. While most of the workers have been exposed to external gamma radiation, a large proportion has additionally been exposed to internal radiation from inhaled plutonium. Compared to a previous study by Azizova et al. 2012, the updated dosimetry system MWDS-2008 has been applied and methods of analysis have been revised. We extend the analysis of the significant incidence risk and observe that main detrimental effects of external radiation exposure occur after more than about 30 years. For mortality, significant risk was found in males with an excess relative risk per dose of 0.09 (95% CI: 0.02; 0.16) [Formula: see text] while risk was insignificant for females. With respect to internal radiation exposure no association to risk could be established. AU - Simonetto, C. AU - Azizova, T.V.* AU - Grigoryeva, E.S.* AU - Kaiser, J.C. AU - Schöllnberger, H. AU - Eidemüller, M. C1 - 31283 C2 - 34404 CY - San Francisco TI - Ischemic heart disease in workers at Mayak PA: Latency of incidence risk after radiation exposure. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10-8), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10-11) and 8q12 (minimum p value 1.82×10-11) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. "Replication-level" association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution. AU - Simpson, C.L.* AU - Wojciechowski, R.* AU - Oexle, K.* AU - Murgia, F.* AU - Portas, L.* AU - Li, X.* AU - Verhoeven, V.J.M.* AU - Vitart, V.* AU - Schache, M.* AU - Hosseini, S.M.* AU - Hysi, P.G.* AU - Raffel, L.J.* AU - Cotch, M.F.* AU - Chew, E.* AU - Klein, B.E.K.* AU - Klein, R.* AU - Wong, T.Y.* AU - van Duijn, C.M.* AU - Mitchell, P.* AU - Saw, S.M.* AU - Fossarello, M.* AU - Wang, J.J.* AU - DCCT/EDIC Research Group (*) AU - Polasek, O.* AU - Campbell, H.* AU - Rudan, I.* AU - Oostra, B.A.* AU - Uitterlinden, A.G.* AU - Hofman, A.* AU - Rivadeneira, F.* AU - Amin, N.* AU - Karssen, L.C.* AU - Vingerling, J.R.* AU - Döring, A. AU - Bettecken, T. AU - Bencic, G.* AU - Gieger, C. AU - Wichmann, H.-E. AU - Wilson, J.F.* AU - Venturini, C.* AU - Fleck, B.* AU - Cumberland, P.M.* AU - Rahi, J.S.* AU - Hammond, C.J.* AU - Hayward, C.* AU - Wright, A.F.* AU - Paterson, A.D.* AU - Baird, P.N.* AU - Klaver, C.C.* AU - Rotter, J.I.* AU - Pirastu, M.* AU - Meitinger, T. AU - Bailey-Wilson, J.E.* C1 - 32262 C2 - 34992 CY - San Francisco TI - Genome-wide meta-analysis of myopia and hyperopia provides evidence for replication of 11 loci. JO - PLoS ONE VL - 9 IS - 9 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Among adipokines and hepatokines, adiponectin and fetuin-A were consistently found to predict the incidence of type 2 diabetes, both by regulating insulin sensitivity. OBJECTIVE: To determine to what extent circulating adiponectin and fetuin-A are independently associated with incident type 2 diabetes in humans, and the major mechanisms involved. METHODS: Relationships with incident diabetes were tested in two cohort studies: within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study (628 cases) and the Nurses' Health Study (NHS; 470 cases). Relationships with body fat compartments, insulin sensitivity and insulin secretion were studied in the Tübingen Lifestyle Intervention Program (TULIP; N = 358). RESULTS: Circulating adiponectin and fetuin-A, independently of several confounders and of each other, associated with risk of diabetes in EPIC-Potsdam (RR for 1 SD: adiponectin: 0.45 [95% CI 0.37-0.54], fetuin-A: 1.18 [1.05-1.32]) and the NHS (0.51 [0.42-0.62], 1.35 [1.16-1.58]). Obesity measures considerably attenuated the association of adiponectin, but not of fetuin-A. Subjects with low adiponectin and concomitantly high fetuin-A had the highest risk. Whereas both proteins were independently (both p<1.8×10-7) associated with insulin sensitivity, circulating fetuin-A (r = -0.37, p = 0.0004), but not adiponectin, associated with insulin secretion in subjects with impaired glucose tolerance. CONCLUSIONS: We provide novel information that adiponectin and fetuin-A independently of each other associate with the diabetes risk. Furthermore, we suggest that they are involved in the development of type 2 diabetes via different mechanisms, possibly by mediating effects of their source tissues, expanded adipose tissue and nonalcoholic fatty liver. AU - Stefan, N. AU - Sun, Q.* AU - Fritsche, A. AU - Machann, J. AU - Schick, F.* AU - Gerst, F. AU - Jeppesen, C. AU - Joost, H.G. AU - Hu, F.B.* AU - Boeing, H.* AU - Ullrich, S. AU - Häring, H.-U. AU - Schulze, M.B. C1 - 30905 C2 - 34003 TI - Impact of the adipokine adiponectin and the hepatokine fetuin-a on the development of type 2 diabetes: Prospective cohort- and cross-sectional phenotyping studies. JO - PLoS ONE VL - 9 IS - 3 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Diabetes treatment may differ by region and patients' socioeconomic position. This may be particularly true for newer drugs. However, data are highly limited. METHODS: We examined pooled individual data of two population-based German studies, KORA F4 (Cooperative Health Research in the Region of Augsburg, south), and the HNR (Heinz Nixdorf Recall study, west) both carried out 2006 to 2008. To ascertain the association between region and educational level with anti-hyperglycemic medication we fitted poisson regression models with robust error variance for any and newer anti-hyperglycemic medication, adjusting for age, sex, diabetes duration, BMI, cardiovascular disease, lifestyle, and insurance status. RESULTS: The examined sample comprised 662 participants with self-reported type 2 diabetes (KORA F4: 83 women, 111 men; HNR: 183 women, 285 men). The probability to receive any anti-hyperglycemic drug as well as to be treated with newer anti-hyperglycemic drugs such as insulin analogues, thiazolidinediones, or glinides was significantly increased in southern compared to western Germany (prevalence ratio (PR); 95% CI: 1.12; 1.02-1.22, 1.52;1.10-2.11 respectively). Individuals with lower educational level tended to receive anti-hyperglycemic drugs more likely than their better educated counterparts (PR; 95% CI univariable: 1.10; 0.99-1.22; fully adjusted: 1.10; 0.98-1.23). In contrast, lower education was associated with a lower estimated probability to receive newer drugs among those with any anti-hyperglycemic drugs (PR low vs. high education: 0.66; 0.48-0.91; fully adjusted: 0.68; 0.47-0.996). CONCLUSIONS: We found regional and individual social disparities in overall and newer anti-hyperglycemic medication which were not explained by other confounders. Further research is needed. AU - Tamayo, T.* AU - Claessen, H.* AU - Rückert, I.-M. AU - Maier, W. AU - Schunk, M. AU - Meisinger, C. AU - Mielck, A. AU - Holle, R. AU - Thorand, B. AU - Narres, M.* AU - Moebus, S.* AU - Mahabadi, A.A.* AU - Pundt, N.* AU - Krone, B.* AU - Slomiany, U.* AU - Erbel, R.* AU - Jöckel, K.-H.* AU - Rathmann, W.* AU - Icks, A.* C1 - 31593 C2 - 34598 CY - San Francisco TI - Treatment pattern of type 2 diabetes differs in two German regions and with patients' socioeconomic position. JO - PLoS ONE VL - 9 IS - 6 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: We have previously found regional differences in the prevalence of known type 2 diabetes between northeastern and southern Germany. We aim to also provide prevalence estimates for prediabetes (isolated impaired fasting glucose (i-IFG), isolated glucose intolerance (i-IGT), combined IFG and IGT) and unknown type 2 diabetes for both regions. METHODS: Prevalence (95%CI) of prediabetes (i-IFG: fasting glucose 5.6-6.9 mmol/l; i-IGT: 2 h postchallenge gluose 7.8-11.0 mmol/l, oral glucose tolerance test (OGTT), ≥8 h overnight fasting) and unknown diabetes were analyzed in two regional population-based surveys (age group 35-79 years): SHIP-TREND (Study of Health in Pomerania (northeast), 2008-2012) and KORA F4 (Cooperative Health Research in the region of Augsburg (south), 2006-2008). Both studies used similar methods, questionnaires, and identical protocols for OGTT. Overall, 1,980 participants from SHIP-TREND and 2,617 participants from KORA F4 were included. RESULTS: Age-sex-standardized prevalence estimates (95%CI) of prediabetes and unknown diabetes were considerably higher in the northeast (SHIP-TREND: 43.1%; 40.9-45.3% and 7.1%; 5.9-8.2%) than in the south of Germany (KORA F4: 30.1%; 28.4-31.7% and 3.9%; 3.2-4.6%), respectively. In particular, i-IFG (26.4%; 24.5-28.3% vs. 17.2%; 15.7-18.6%) and IFG+IGT (11.2%; 9.8-12.6% vs. 6.6%; 5.7-7.5%) were more frequent in SHIP-TREND than in KORA. In comparison to normal glucose tolerance, the odds of having unknown diabetes (OR, 95%CI: 2.59; 1.84-3.65) or prediabetes (1.98; 1.70-2.31) was higher in the northeast than in the south after adjustment for known risk factors (obesity, lifestyle). CONCLUSIONS: The regional differences of prediabetes and unknown diabetes are in line with the geographical pattern of known diabetes in Germany. The higher prevalences in the northeast were not explained by traditional risk factors. AU - Tamayo, T.* AU - Schipf, S.* AU - Meisinger, C. AU - Schunk, M. AU - Maier, W. AU - Herder, C.* AU - Roden, M.* AU - Nauck, M.* AU - Peters, A. AU - Völzke, H.* AU - Rathmann, W.* C1 - 42781 C2 - 35334 TI - Regional differences of undiagnosed type 2 diabetes and prediabetes prevalence are not explained by known risk factors. JO - PLoS ONE VL - 9 IS - 11 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. METHODS: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. RESULTS: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P  =  5.71 × 10-7). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P  =  2.18 × 10-8) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. CONCLUSIONS: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function. AU - Tang, W.* AU - Kowgier, M.* AU - Loth, D.W.* AU - Soler Artigas, M.* AU - Joubert, B.R.* AU - Hodge, E.* AU - Gharib, S.A.* AU - Smith, A.V.* AU - Ruczinski, I.* AU - Gudnason, V.* AU - Mathias, R.A.* AU - Harris, T.B.* AU - Hansel, N.N.* AU - Launer, L.J.* AU - Barnes, K.C.* AU - Hansen, J.G.* AU - Albrecht, E. AU - Aldrich, M.C.* AU - Allerhand, M.* AU - Barr, R.G.* AU - Brusselle, G.G.* AU - Couper, D.J.* AU - Curjuric, I.* AU - Davies, G.* AU - Deary, I.J.* AU - Dupuis, J.* AU - Fall, T.* AU - Foy, M.* AU - Franceschini, N.* AU - Gao, W.* AU - Gläser, S.* AU - Gu, X.* AU - Hancock, D.B.* AU - Heinrich, J. AU - Hofman, A.* AU - Imboden, M.* AU - Ingelsson, E.* AU - James, A.* AU - Karrasch, S. AU - Koch, B.* AU - Kritchevsky, S.B.* AU - Kumar, A.* AU - Lahousse, L.* AU - Li, G.* AU - Lind, L.* AU - Lindgren, C.* AU - Liu, Y.* AU - Lohman, K.* AU - Lumley, T.* AU - McArdle, W.L.* AU - Meibohm, B.* AU - Morris, A.P.* AU - Morrison, A.C.* AU - Musk, B.* AU - North, K.E.* AU - Palmer, L.J.* AU - Probst-Hensch, N.M.* AU - Psaty, B.M.* AU - Rivadeneira, F.* AU - Rotter, J.I.* AU - Schulz, H. AU - Smith, L.J.* AU - Sood, A.* AU - Starr, J.M.* AU - Strachan, D.P.* AU - Teumer, A.* AU - Uitterlinden, A.G.* AU - Völzke, H.* AU - Voorman, A.* AU - Wain, L.V.* AU - Wells, M.T.* AU - Wilk, J.B.* AU - Williams, O.D.* AU - Heckbert, S.R.* AU - Stricker, B.H.* AU - London, S.J.* AU - Fornage, M.* AU - Tobin, M.D.* AU - O'Connor, G.T.* AU - Hall, I.P.* AU - Cassano, P.A.* C1 - 31719 C2 - 34690 CY - San Francisco TI - Large-scale genome-wide association studies and meta-analyses of longitudinal change in adult lung function. JO - PLoS ONE VL - 9 IS - 7 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = -3.6) and caffeic acid O-methyltransferase (-3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure. AU - Vanzo, E. AU - Ghirardo, A. AU - Merl-Pham, J. AU - Lindermayr, C. AU - Heller, W. AU - Hauck, S.M. AU - Durner, J. AU - Schnitzler, J.-P. C1 - 32101 C2 - 34964 CY - San Francisco TI - S-nitroso-proteome in poplar leaves in response to acute ozone stress. JO - PLoS ONE VL - 9 IS - 9 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - PURPOSE: The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation. MATERIAL AND METHODS: The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 µm. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed. RESULTS: GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders. CONCLUSION: In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney. AU - Velroyen, A.* AU - Bech, M.* AU - Zanette, I.* AU - Schwarz, J.* AU - Rack, A.* AU - Tympner, C.* AU - Herrler, T.* AU - Staab-Weijnitz, C.A. AU - Braunagel, M.* AU - Reiser, M.* AU - Bamberg, F.* AU - Pfeiffer, F.* AU - Notohamiprodjo, M.* C1 - 32519 C2 - 35104 TI - X-ray phase-contrast tomography of renal ischemia-reperfusion damage. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2. AU - Waschbüsch, D.* AU - Michels, H.* AU - Strassheim, S.* AU - Ossendorf, E.* AU - Kessler, D.* AU - Gloeckner, C.J. AU - Barnekow, A.* C1 - 32651 C2 - 35206 TI - LRRK2 transport is regulated by its novel interacting partner Rab32. JO - PLoS ONE VL - 9 IS - 10 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Cardiovascular disease is the leading cause of morbidity and mortality in the developed world. To reduce this burden of disease, a German sickness fund ('Siemens-Betriebskrankenkasse', SBK) initiated the prevention programme 'KardioPro' including primary (risk factor reduction) and secondary (screening) prevention and guideline-based treatment. The aim of this study was to assess the effectiveness of 'KardioPro' as it is implemented in the real world. METHODS: The study is based on sickness fund routine data. The control group was selected from non-participants via propensity score matching. Study analysis was based on time-to-event analysis via Cox proportional hazards regression with the endpoint 'all-cause mortality, acute myocardial infarction (MI) and ischemic stroke (1)', 'all-cause mortality (2)' and 'non-fatal acute MI and ischemic stroke (3)'. RESULTS: A total of 26,202 insurants were included, 13,101 participants and 13,101 control subjects. 'KardioPro' enrolment was associated with risk reductions of 23.5% (95% confidence interval (CI) 13.0-32.7%) (1), 41.7% (95% CI 30.2-51.2%) (2) and 3.5% (hazard ratio 0.965, 95% CI 0.811-1.148) (3). This corresponds to an absolute risk reduction of 0.29% (1), 0.31% (2) and 0.03% (3) per year. CONCLUSION: The prevention programme initiated by a German statutory sickness fund appears to be effective with regard to all-cause mortality. The non-significant reduction in non-fatal events might result from a shift from fatal to non-fatal events. AU - Witt, S. AU - Leidl, R. AU - Becker, C. AU - Holle, R. AU - Block, M.* AU - Brachmann, J.* AU - Silber, S.* AU - Stollenwerk, B. C1 - 42919 C2 - 35863 TI - The effectiveness of the cardiovascular disease prevention programme 'KardioPro' initiated by a German sickness fund: A time-to-event analysis of routine data. JO - PLoS ONE VL - 9 IS - 12 PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND & AIMS: HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication. METHODS: HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue. RESULTS: All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease. CONCLUSIONS: Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease. AU - Wuestenberg, A.* AU - Kah, J.* AU - Singethan, K. AU - Sirma, H.* AU - Keller, A.D.* AU - Rosal, S.R.* AU - Schrader, J.* AU - Loscher, C.* AU - Volz, T.* AU - Bartenschlager, R.* AU - Lohmann, V.* AU - Protzer, U. AU - Dandri, M.* AU - Lohse, A.W.* AU - Tiegs, G.* AU - Sass, G.* C1 - 31243 C2 - 34245 CY - San Francisco TI - Matrix conditions and KLF2-dependent induction of heme oxygenase-1 modulate inhibition of HCV replication by fluvastatin. JO - PLoS ONE VL - 9 IS - 5 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2-/- mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2-/- mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments. AU - Zumbrennen-Bullough, K.B.* AU - Becker, L. AU - Garrett, L. AU - Hölter, S.M. AU - Calzada-Wack, J. AU - Mossbrugger, I. AU - Quintanilla-Fend, L. AU - Rácz, I. AU - Rathkolb, B. AU - Klopstock, T.* AU - Wurst, W. AU - Zimmer, A.* AU - Wolf, E.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Romney, S.J.* AU - Leibold, E.A.* C1 - 31542 C2 - 34585 CY - San Francisco TI - Abnormal brain iron metabolism in Irp2 deficient mice is associated with mild neurological and behavioral impairments. JO - PLoS ONE VL - 9 IS - 6 PB - Public Library Science PY - 2014 SN - 1932-6203 ER - TY - JOUR AB - Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. AU - Andréasson, C.* AU - Schick, A.J.* AU - Pfeiffer, S.M. AU - Sarov, M.* AU - Stewart, F.* AU - Wurst, W. AU - Walcher, T. C1 - 27570 C2 - 32726 TI - Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Salinibacter ruber is an extremely halophilic member of the Bacteroidetes that thrives in crystallizer ponds worldwide. Here, we have analyzed two sets of 22 and 35 co-occurring S. ruber strains, newly isolated respectively, from 100 microliters water samples from crystalizer ponds in Santa Pola and Mallorca, located in coastal and inland Mediterranean Spain and 350 km apart from each other. A set of old strains isolated from the same setting were included in the analysis. Genomic and taxonomy relatedness of the strains were analyzed by means of PFGE and MALDI-TOF, respectively, while their metabolomic potential was explored with high resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS). Overall our results show a phylogenetically very homogeneous species expressing a very diverse metabolomic pool. The combination of MALDI-TOF and PFGE provides, for the newly isolated strains, the same scenario presented by the previous studies of intra-specific diversity of S. ruber using a more restricted number of strains: the species seems to be very homogeneous at the ribosomal level while the genomic diversity encountered was rather high since no identical genome patterns could be retrieved from each of the samples. The high analytical mass resolution of ICR-FT/MS enabled the description of thousands of putative metabolites from which to date only few can be annotated in databases. Some metabolomic differences, mainly related to lipid metabolism and antibiotic-related compounds, provided enough specificity to delineate different clusters within the co-occurring strains. In addition, metabolomic differences were found between old and new strains isolated from the same ponds that could be related to extended exposure to laboratory conditions. AU - Antón, J.* AU - Lucio, M. AU - Peña, A.* AU - Cifuentes, A.* AU - Brito-Echeverría, J.* AU - Moritz, F. AU - Tziotis, D. AU - López, C.* AU - Urdiain, M.* AU - Schmitt-Kopplin, P. AU - Rosselló-Mora, R.* C1 - 24786 C2 - 31678 TI - High metabolomic microdiversity within co-occurring isolates of the extremely halophilic bacterium Salinibacter ruber. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own. AU - Barros, M.H.* AU - Hauck, F.* AU - Dreyer, J.H.* AU - Kempkes, B. AU - Niedobitek, G.* C1 - 28434 C2 - 33374 TI - Macrophage polarisation: An immunohistochemical approach for identifying M1 and M2 macrophages. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Evidence is compelling for a positive correlation between climate change, urbanisation and prevalence of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergenic effect of anthropogenic factors on susceptible individuals. OBJECTIVES: To evaluate the impact of urbanisation and climate change on pollen allergenicity. METHODS: Catkins were sampled from birch trees from different sites across the greater area of Munich, pollen were isolated and an urbanisation index, NO2 and ozone exposure were determined. To estimate pollen allergenicity, allergen content and pollen-associated lipid mediators were measured in aqueous pollen extracts. Immune stimulatory and modulatory capacity of pollen was assessed by neutrophil migration assays and the potential of pollen to inhibit dendritic cell interleukin-12 response. In vivo allergenicity was assessed by skin prick tests. RESULTS: The study revealed ozone as a prominent environmental factor influencing the allergenicity of birch pollen. Enhanced allergenicity, as assessed in skin prick tests, was mirrored by enhanced allergen content. Beyond that, ozone induced changes in lipid composition and chemotactic and immune modulatory potential of the pollen. Higher ozone-exposed pollen was characterised by less immune modulatory but higher immune stimulatory potential. CONCLUSION: It is likely that future climate change along with increasing urbanisation will lead to rising ozone concentrations in the next decades. Our study indicates that ozone is a crucial factor leading to clinically relevant enhanced allergenicity of birch pollen. Thus, with increasing temperatures and increasing ozone levels, also symptoms of pollen allergic patients may increase further.   AU - Beck, I. AU - Jochner, S.* AU - Gilles, S. AU - McIntyre, M.* AU - Buters, J.T.M. AU - Schmidt-Weber, C.B. AU - Behrendt, H. AU - Menzel, A.* AU - Traidl-Hoffmann, C. C1 - 28552 C2 - 33443 TI - High environmental ozone levels lead to enhanced allergenicity of birch pollen. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas. MATERIAL AND METHODS: Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters (UICC pTNM category, tumor grading) and survival. RESULTS: Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis (p=0.02; p=0.004; p=0.802). Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases. CONCLUSION: Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment. AU - Berezowska, S.* AU - Novotny, A.* AU - Bauer, K.* AU - Feuchtinger, A. AU - Slotta-Huspenina, J.* AU - Becker, K.* AU - Langer, R.* AU - Walch, A.K. C1 - 25999 C2 - 32015 TI - Association between HSP90 and Her2 in gastric and gastroesophageal carcinomas. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options. AU - Bermudez, O. AU - Hennen, E. AU - Koch, I.* AU - Lindner, M.* AU - Eickelberg, O. C1 - 25756 C2 - 31909 TI - Gli1 mediates lung cancer cell proliferation and Sonic hedgehog-dependent mesenchymal cell activation. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. AU - Bonzheim, I. AU - Irmler, M. AU - Klier-Richter, M. AU - Steinhilber, J.* AU - Anastasov, N. AU - Schafer, S.* AU - Adam, P.* AU - Beckers, J. AU - Raffeld, M.* AU - Fend, F.* AU - Quintanilla-Martinez, L. C1 - 24917 C2 - 31715 TI - Identification of C/EBPβ target genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by gene expression profiling and chromatin immunoprecipitation. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Applying a systematic review to identify studies eligible for meta-analysis of the association between occupational exposure to inorganic dust and the development of chronic obstructive pulmonary disease (COPD), and conducting a meta-analysis. DATA SOURCES: Searches of PubMed and Embase for the time period 1970-2010 yielded 257 cross-sectional and longitudinal studies on people exposed to inorganic dust at the workplace with data on lung function. These studies were independently abstracted and evaluated by two authors; any disagreement was resolved by a third reviewer. Of 55 publications accepted for meta-analysis, 27 investigated the effects of occupational exposure to biopersistent granular dust (bg-dust). METHODS: A random effects meta-analysis allowed us to provide an estimate of the average exposure effect on spirometric parameters presented in forest plots. Between-study heterogeneity was assessed by using I(2) statistics, with I(2)>25% indicating significant heterogeneity. Publication bias was investigated by visual inspection of funnel plots. The influence of individual studies was assessed by dropping the respective study before pooling study-specific estimates. RESULTS: The mean FEV1 of workers exposed to bg-dust was 160 ml lower or 5.7% less than predicted compared to workers with no/low exposure. The risk of an obstructive airway disease-defined as FEV1/FVC < 70%-increased by 7% per 1 mg· m(-3) respirable bg-dust. CONCLUSION: Occupational inhalative exposure to bg-dust was associated with a statistically significant decreased FEV1 and FEV1/FVC revealing airway obstruction consistent with COPD.   AU - Brüske, I. AU - Thiering, E. AU - Heinrich, J. AU - Huster, K.M.* AU - Nowak, D.* C1 - 28561 C2 - 33451 TI - Biopersistent granular dust and chronic obstructive pulmonary disease: A systematic review and meta-analysis. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states. AU - Burgstaller, G. AU - Oehrle, B. AU - Koch, I.* AU - Lindner, M.* AU - Eickelberg, O. C1 - 25983 C2 - 32040 TI - Multiplex profiling of cellular invasion in 3D cell culture models. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin(-/-)), and GOAT knockout (GOAT(-/-)) mice. Ghrelin(-/-) mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT(-/-) mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin(-/-) and GOAT(-/-) mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin(-/-) mice, yet potentiated in GOAT(-/-) mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT(-/-) mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin(-/-) and GOAT(-/-) mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities. AU - Cai, H.* AU - Cong, W.N.* AU - Daimon, C.M.* AU - Wang, R.* AU - Tschöp, M.H. AU - Sévigny, J.* AU - Martin, B.* AU - Maudsley, S.* C1 - 27929 C2 - 32863 TI - Altered lipid and salt taste responsivity in ghrelin and GOAT null mice. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The skin microbial community is regarded as essential for human health and well-being, but likewise plays an important role in the formation of body odor in, for instance, the axillae. Few molecular-based research was done on the axillary microbiome. This study typified the axillary microbiome of a group of 53 healthy subjects. A profound view was obtained of the interpersonal, intrapersonal and temporal diversity of the human axillary microbiota. Denaturing gradient gel electrophoresis (DGGE) and next generation sequencing on 16S rRNA gene region were combined and used as extent to each other. Two important clusters were characterized, where Staphylococcus and Corynebacterium species were the abundant species. Females predominantly clustered within the Staphylococcus cluster (87%, n = 17), whereas males clustered more in the Corynebacterium cluster (39%, n = 36). The axillary microbiota was unique to each individual. Left-right asymmetry occurred in about half of the human population. For the first time, an elaborate study was performed on the dynamics of the axillary microbiome. A relatively stable axillary microbiome was noticed, although a few subjects evolved towards another stable community. The deodorant usage had a proportional linear influence on the species diversity of the axillary microbiome. AU - Callewaert, C.* AU - Kerckhof, F.M.* AU - Granitsiotis, M.S. AU - van Gele, M.* AU - van de Wiele, T.* AU - Boon, N.* C1 - 27048 C2 - 32498 TI - Characterization of Staphylococcus and Corynebacterium clusters in the human axillary region. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The soil-borne pathogen Rhizoctonia solani is responsible for crop losses on a wide range of important crops worldwide. The lack of effective control strategies and the increasing demand for organically grown food has stimulated research on biological control. The aim of the present study was to evaluate the rhizosphere competence of the commercially available inoculant Bacillus amyloliquefaciens FZB42 on lettuce growth and health together with its impact on the indigenous rhizosphere bacterial community in field and pot experiments. Results of both experiments demonstrated that FZB42 is able to effectively colonize the rhizosphere (7.45 to 6.61 Log 10 CFU g−1 root dry mass) within the growth period of lettuce in the field. The disease severity (DS) of bottom rot on lettuce was significantly reduced from severe symptoms with DS category 5 to slight symptom expression with DS category 3 on average through treatment of young plants with FZB42 before and after planting. The 16S rRNA gene based fingerprinting method terminal restriction fragment length polymorphism (T-RFLP) showed that the treatment with FZB42 did not have a major impact on the indigenous rhizosphere bacterial community. However, the bacterial community showed a clear temporal shift. The results also indicated that the pathogen R. solani AG1-IB affects the rhizosphere microbial community after inoculation. Thus, we revealed that the inoculant FZB42 could establish itself successfully in the rhizosphere without showing any durable effect on the rhizosphere bacterial community. AU - Chowdhury, S.P. AU - Dietel, K.* AU - Rändeler, M.* AU - Schmid, M. AU - Junge, H.* AU - Borriss, R.* AU - Hartmann, A. AU - Grosch, R.* C1 - 26492 C2 - 32257 TI - Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Attention-deficit/hyperactivity disorder (ADHD) and dyslexia belong to the most common neuro-behavioral childhood disorders with prevalences of around 5% in school-aged children. It is estimated that 20-60% of individuals affected with ADHD also present with learning disorders. We investigated the comorbidity between ADHD symptoms and reading/spelling and math difficulties in two on-going population-based birth cohort studies. Children with ADHD symptoms were at significantly higher risk of also showing reading/spelling difficulties or disorder (Odds Ratio (OR) = 2.80, p = 6.59×10(-13)) as compared to children without ADHD symptoms. For math difficulties the association was similar (OR = 2.55, p = 3.63×10(-04)). Our results strengthen the hypothesis that ADHD and learning disorders are comorbid and share, at least partially, the same underlying process. Up to date, it is not clear, on which exact functional processes this comorbidity is based. AU - Czamara, D.* AU - Tiesler, C.M. AU - Kohlböck, G. AU - Berdel, D.* AU - Hoffmann, B.* AU - Bauer, C.P.* AU - Koletzko, S.* AU - Schaaf, B.* AU - Lehmann, I.* AU - Herbarth, O.* AU - von Berg, A.* AU - Müller-Myhsok, B.* AU - Schulte-Körne, G.* AU - Heinrich, J. C1 - 24831 C2 - 31698 TI - Children with ADHD symptoms have a higher risk for reading, spelling and math difficulties in the GINIplus and LISAplus cohort studies. JO - PLoS ONE VL - 8 IS - 5 PB - Public Libary of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner. AU - Despars, G.* AU - Carbonneau, C.L.* AU - Bardeau, P.* AU - Coutu, D.L. AU - Beauséjour, C.M.* C1 - 27473 C2 - 32687 TI - Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes. AU - Dössinger, G.* AU - Bunse, M.* AU - Bet, J.* AU - Albrecht, J. AU - Paszkiewicz, P.J.* AU - Weißbrich, B.* AU - Schiedewitz, I. AU - Henkel, L.* AU - Schiemann, M. AU - Neuenhahn, M. AU - Uckert, W.* AU - Busch, D.H. C1 - 24723 C2 - 31654 TI - MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Diffusion tensor imaging (DTI) based assessment of white matter fiber tract integrity can support the diagnosis of Alzheimer's disease (AD). The use of DTI as a biomarker, however, depends on its applicability in a multicenter setting accounting for effects of different MRI scanners. We applied multivariate machine learning (ML) to a large multicenter sample from the recently created framework of the European DTI study on Dementia (EDSD). We hypothesized that ML approaches may amend effects of multicenter acquisition. We included a sample of 137 patients with clinically probable AD (MMSE 20.6 +/- 5.3) and 143 healthy elderly controls, scanned in nine different scanners. For diagnostic classification we used the DTI indices fractional anisotropy (FA) and mean diffusivity (MD) and, for comparison, gray matter and white matter density maps from anatomical MRI. Data were classified using a Support Vector Machine (SVM) and a Naive Bayes (NB) classifier. We used two cross-validation approaches, (i) test and training samples randomly drawn from the entire data set (pooled cross-validation) and (ii) data from each scanner as test set, and the data from the remaining scanners as training set (scanner-specific cross-validation). In the pooled cross-validation, SVM achieved an accuracy of 80% for FA and 83% for MD. Accuracies for NB were significantly lower, ranging between 68% and 75%. Removing variance components arising from scanners using principal component analysis did not significantly change the classification results for both classifiers. For the scanner-specific cross-validation, the classification accuracy was reduced for both SVM and NB. After mean correction, classification accuracy reached a level comparable to the results obtained from the pooled cross-validation. Our findings support the notion that machine learning classification allows robust classification of DTI data sets arising from multiple scanners, even if a new data set comes from a scanner that was not part of the training sample. AU - Dyrba, M.* AU - Ewers, M.* AU - Wegrzyn, M.* AU - Kilimann, I.* AU - Plant, C. AU - Oswald, A.* AU - Meindl, T.* AU - Pievani, M.* AU - Bokde, A.L.W.* AU - Fellgiebel, A.* AU - Filippi, M.* AU - Hampel, H.* AU - Klöppel, S.* AU - Hauenstein, K.* AU - Kirste, T.* AU - Teipel, S.J.* AU - EDSD Study Group (*) C1 - 25923 C2 - 31984 TI - Robust automated detection of microstructural white matter degeneration in Alzheimer's disease using machine learning classification of multicenter DTI data. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface. AU - Felmy, B.* AU - Songhet, P.* AU - Slack, E.M.* AU - Müller, A.J.* AU - Kremer, M.* AU - van Maele, L.* AU - Cayet, D.* AU - Heikenwälder, M. AU - Sirard, J.C.* AU - Hardt, W.D.* C1 - 27974 C2 - 32885 TI - NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella typhimurium. JO - PLoS ONE VL - 8 IS - 10 PB - Publis Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii. AU - Frangoulidis, D.* AU - Splettstoesser, W.D.* AU - Landt, O.* AU - Dehnhardt, J.* AU - Henning, K.* AU - Hilbert, A.* AU - Bauer, T.* AU - Antwerpen, M.* AU - Meyer, H.* AU - Walter, M.C. AU - Knobloch, J.K.M.* C1 - 22747 C2 - 30922 TI - Microevolution of the chromosomal region of acute disease antigen A (adaA) in the Query (Q) fever agent Coxiella burnetii. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Context: Congenital leptin deficiency, caused by a very rare mutation in the gene encoding leptin, leads to severe obesity, hyperphagia and impaired satiety. The only systemic treatment is the substitution with metreleptin leading to weight reduction based on hormonal changes. Several studies have also shown alterations in brain function after metreleptin therapy. In a previous study, we were able to show changes in homeostatic (hypothalamus) and reward-related brain areas (striatum, orbitofrontal cortex (OFC), substantia nigra/ventral tegmental area, amygdala) 3 days and 6 months after therapy start in a leptin-deficient adolescent girl. To further access the time course of functional brain activation changes, we followed the patient for 2 years after initiation of the therapy. Design, Patient: Functional magnetic resonance imaging during visual stimulation with food (high-and low-caloric) and non-food pictures was performed 1 and 2 years after therapy start in the previously described patient. Results: The comparison of 'food vs. non-food' pictures showed a stabilization of the long-term effects in the amygdala and in the OFC. Therefore, no significant differences were observed between 6 months compared to 12 and 24 months in these regions. Additionally, a reduction of the frontopolar cortex activity over the whole time span was observed. For the comparison of high-and low-caloric pictures, long-term effects in the hypothalamus showed an assimilating pattern for the response to the food categories whereas only acute effects after 3 months were observed in hedonic brain regions. Conclusion: This follow-up study shows that the long lasting benefit of metreleptin therapy is also associated with activation changes in homeostatic, hedonic and frontal control regions in congenital leptin deficiency. AU - Frank, S.* AU - Heni, M. AU - Moss, A.* AU - von Schnurbein, J.* AU - Farooqi, S.* AU - Häring, H.-U. AU - Fritsche, A. AU - Preissl, H. AU - Wabitsch, M.* C1 - 25913 C2 - 31987 TI - Long-term stabilization effects of leptin on brain functions in a leptin-deficient patient. JO - PLoS ONE VL - 8 IS - 6 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique. AU - Frankó, A. AU - Baris, O.R.* AU - Bergschneider, E.* AU - von Toerne, C. AU - Hauck, S.M. AU - Aichler, M. AU - Walch, A.K. AU - Wurst, W. AU - Wiesner, R.J.* AU - Johnston, I.C.D.* AU - Hrabě de Angelis, M. C1 - 28721 C2 - 33526 TI - Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads. JO - PLoS ONE VL - 8 IS - 12 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe's milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU97 could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening. AU - Fuka, M.M. AU - Wallisch, S. AU - Engel, M. AU - Welzl, G. AU - Havranek, J.* AU - Schloter, M. C1 - 28488 C2 - 33422 TI - Dynamics of bacterial communities during the ripening process of different Croatian cheese types derived from raw ewe's milk cheeses. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - During HIV pathogenesis, infected macrophages behave as "viral reservoirs" that accumulate and retain virions within dedicated internal Virus-Containing Compartments (VCCs). The nature of VCCs remains ill characterized and controversial. Using wild-type HIV-1 and a replication-competent HIV-1 carrying GFP internal to the Gag precursor, we analyzed the biogenesis and evolution of VCCs in primary human macrophages. VCCs appear roughly 14 hours after viral protein synthesis is detected, initially contain few motile viral particles, and then mature to fill up with virions that become packed and immobile. The amount of intracellular Gag, the proportion of dense VCCs, and the density of viral particles in their lumen increased with time post-infection. In contrast, the secretion of virions, their infectivity and their transmission to T cells decreased overtime, suggesting that HIV-infected macrophages tend to pack and retain newly formed virions into dense compartments. A minor proportion of VCCs remains connected to the plasma membrane overtime. Surprisingly, live cell imaging combined with correlative light and electron microscopy revealed that such connections can be transient, highlighting their dynamic nature. Together, our results shed light on the late phases of the HIV-1 cycle and reveal some of its macrophage specific features. AU - Gaudin, R.* AU - Bèrre, S.* AU - Cunha de Alencar, B.* AU - Decalf, J.* AU - Schindler, M. AU - Gobert, F.X.* AU - Jouve, M.* AU - Benaroch, P.* C1 - 26542 C2 - 32288 TI - Dynamics of HIV-containing compartments in macrophages reveal sequestration of virions and transient surface connections. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development. AU - Gewies, A. AU - Castineiras-Vilarino, M.* AU - Ferch, U.* AU - Jährling, N.* AU - Heinrich, K.* AU - Hoeckendorf, U.* AU - Przemeck, G.K.H. AU - Munding, M. AU - Groß, O.* AU - Schroeder, T. AU - Horsch, M. AU - Karran, E.L.* AU - Majid, A.* AU - Antonowicz, S.* AU - Beckers, J. AU - Hrabě de Angelis, M. AU - Dodt, H.U.* AU - Peschel, C.* AU - Förster, I.* AU - Dyer, M.J.S.* AU - Ruland, J, C1 - 28429 C2 - 33407 TI - Prdm6 is essential for cardiovascular development in vivo. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Missense mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are linked to autosomal dominant forms of Parkinson's disease (PD). In order to get insights into the physiological role of Lrrk2, we examined the distribution of Lrrk2 mRNA and different splice variants in the developing murine embryo and the adult brain of Mus musculus. To analyse if the Lrrk2-paralog, Lrrk1, may have redundant functions in PD-development, we also compared Lrrk1 and Lrrk2 expression in the same tissues. Using radioactive in situ hybridization, we found ubiquitous expression of both genes at low level from embryonic stage E9.5 onward, which progressively increased up until birth. The developing central nervous system (CNS) displayed no prominent Lrrk2 mRNA signals at these time-points. However, in the entire postnatal brain Lrrk2 became detectable, showing strongest level in the striatum and the cortex of adult mice; Lrrk1 was only detectable in the mitral cell layer of the olfactory bulb. Thus, due to the non-overlapping expression patterns, a redundant function of Lrrk2 and Lrrk1 in the pathogenesis of PD seems to be unlikely. Quantification of Lrrk2 mRNA and protein level in several brain regions by real-time PCR and Western blot verified the striatum and cortex as hotspots of postnatal Lrrk2 expression. Strong expression of Lrrk2 is mainly found in neurons, specifically in the dopamine receptor 1 (DRD1a) and 2 (DRD2)-positive subpopulations of the striatal medium spiny neurons. Finally, we identified 2 new splice-variants of Lrrk2 in RNA-samples from various adult brain regions and organs: a variant with a skipped exon 5 and a truncated variant terminating in an alternative exon 42a. In order to identify the origin of these two splice variants, we also analysed primary neural cultures independently and found cell-specific expression patterns for these variants in microglia and astrocytes. AU - Giesert, F. AU - Hofmann, A. AU - Bürger, A. AU - Zerle, J. AU - Kloos, K. AU - Hafen, U. AU - Ernst, L. AU - Zhang, J. AU - Vogt Weisenhorn, D.M. AU - Wurst, W. C1 - 24558 C2 - 31573 TI - Expression analysis of lrrk1, lrrk2 and lrrk2 splice variants in mice. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The mitochondrial AAA+-ATPase ATAD3 is implicated in the regulation of mitochondrial and ER dynamics and was shown to be necessary for larval development in Caenorhabditis elegans. In order to elucidate the relevance of ATAD3 for mammalian development, the phenotype of an Atad3 deficient mouse line was analyzed. Atad3 deficient embryos die around embryonic day E7.5 due to growth retardation and a defective development of the trophoblast lineage immediately after implantation into the uterus. This indicates an essential function of Atad3 for the progression of the first steps of post-implantation development at a time point when mitochondrial biogenesis and ATP production by oxidative phosphorylation are required. Therefore, murine Atad3 plays an important role in the biogenesis of mitochondria in trophoblast stem cells and in differentiating trophoblasts. At the biochemical level, we report here that ATAD3 is present in five native mitochondrial protein complexes of different sizes, indicating complex roles of the protein in mitochondrial architecture and function. AU - Goller, T.* AU - Seibold, U.K.* AU - Kremmer, E. AU - Voos, W.* AU - Kolanus, W.* C1 - 23536 C2 - 31213 TI - Atad3 function is essential for early post-implantation development in the mouse. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Over-expression of the proto-oncogene c-MYC is frequently observed in a variety of tumors and is a hallmark of Burkitt´s lymphoma. The fact that many tumors are oncogene-addicted to c-MYC, renders c-MYC a powerful target for anti-tumor therapy. Using a xenogenic vaccination strategy by immunizing C57BL/6 mice with human c-MYC protein or non-homologous peptides, we show that the human c-MYC protein, despite its high homology between mouse and man, contains several immunogenic epitopes presented in the context of murine H2(b) haplotype. We identified an MHC class II-restricted CD4(+) T-cell epitope and therein an MHC class I-restricted CD8(+) T-cell epitope (SSPQGSPEPL) that, after prime/boost immunization, protected up to 25% of mice against a lethal lymphoma challenge. Lymphoma-rejecting animals contained MHC multimer-binding CD8(+) cell within the peripheral blood and displayed in vivo cytolytic activity with specificity for SSPQGSPEPL. Taken together these data suggest that oncogenic c-MYC can be targeted with specific T-cells. AU - Helm, F.* AU - Kammertoens, T.* AU - Lehmann, F.M. AU - Wilke, A.* AU - Bruns, H.* AU - Mautner, J. AU - Bornkamm, G.W. AU - Gerbitz, A.* C1 - 27869 C2 - 32838 TI - Targeting c-MYC with T-cells. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing. AU - Hölter, S.M. AU - Stromberg, M.* AU - Kovalenko, M.* AU - Garrett, L. AU - Glasl, L. AU - Lopez, E.* AU - Guide, J.* AU - Götz, A. AU - Hans, W. AU - Becker, L. AU - Rathkolb, B. AU - Rozman, J. AU - Schrewe, A.* AU - Klingenspor, M.* AU - Klopstock, T.* AU - Schulz, H. AU - Wolf, E.* AU - Wurst, W. AU - Gillis, T.* AU - Wakimoto, H.* AU - Seidmann, J.* AU - MacDonald, M.E.* AU - Cotman, S.L.* AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Lee, J.M.* AU - Wheeler, V.C.* C1 - 28466 C2 - 33408 TI - A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington’s diesease CAG knock-in mice. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Trace elements might play a role in the complex multifactorial pathogenesis of age-related macular degeneration (AMD). The aim of this study was to measure alterations of trace elements levels in aqueous humor of patients with non-exsudative (dry) AMD. For this pilot study, aqueous humor samples were collected from patients undergoing cataract surgery. 12 patients with dry AMD (age 77.9 +/- 6.62, female 8, male 4) and 11 patients without AMD (age 66.6 +/- 16.7, female 7, male 4) were included. Aqueous levels of cadmium, cobalt, copper, iron, manganese, selenium, and zinc were measured by use of Flow-Injection-Inductively-Coupled-Plasma-Mass-Spectrometry (FI-ICP-MS), quality controlled with certified standards. Patients with AMD had significantly higher aqueous humor levels of cadmium (median: 0.70 mu mol/L, IQR: 0.40-0.84 vs. 0.06 mu mol/L; IQR: 0.01-.018; p = 0.002), cobalt (median: 3.1 mu mol/L, IQR: 2.62-3.15 vs. 1.17 mu mol/L; IQR: 0.95-1.27; p<0.001), iron (median: 311 mu mol/L, IQR: 289-329 vs. 129 mu mol/L; IQR: 111-145; p<0.001) and zinc (median: 23.1 mu mol/L, IQR: 12.9-32.6 vs. 5.1 mu mol/L; IQR: 4.4-9.4; p = 0.020) when compared with patients without AMD. Copper levels were significantly reduced in patients with AMD (median: 16.2 mu mol/L, IQR: 11.4-31.3 vs. 49.9 mu mol/L; IQR: 32.0-. 142.0; p = 0.022) when compared to those without. No significant differences were observed in aqueous humor levels of manganese and selenium between patients with and without AMD. After an adjustment for multiple testing, cadmium, cobalt, copper and iron remained a significant factor in GLM models (adjusted for age and gender of the patients) for AMD. Alterations of trace element levels support the hypothesis that cadmium, cobalt, iron, and copper are involved in the pathogenesis of AMD. AU - Junemann, A.G.M.* AU - Stopa, P.* AU - Michalke, B. AU - Chaudhri, A.* AU - Reulbach, U.* AU - Huchzermeyer, C.* AU - Schlotzer-Schrehardt, U.* AU - Kruse, F.E.* AU - Zrenner, E.* AU - Rejdak, R.* C1 - 23650 C2 - 31262 TI - Levels of aqueous humor trace elements in patients with non-exsudative age-related macular degeneration: A case-control study. JO - PLoS ONE VL - 8 IS - 2 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Climate change and air pollution, including ozone is known to affect plants and might also influence the ragweed pollen, known to carry strong allergens. We compared the transcriptome of ragweed pollen produced under ambient and elevated ozone by 454-sequencing. An enzyme-linked immunosorbent assay (ELISA) was carried out for the major ragweed allergen Amb a 1. Pollen surface was examined by scanning electron microscopy and attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR), and phenolics were analysed by high-performance liquid chromatography. Elevated ozone had no influence on the pollen size, shape, surface structure or amount of phenolics. ATR-FTIR indicated increased pectin-like material in the exine. Transcriptomic analyses showed changes in expressed-sequence tags (ESTs), including allergens. However, ELISA indicated no significantly increased amounts of Amb a 1 under elevated ozone concentrations. The data highlight a direct influence of ozone on the exine components and transcript level of allergens. As the total protein amount of Amb a 1 was not altered, a direct correlation to an increased risk to human health could not be derived. Additional, the 454-sequencing contributes to the identification of stress-related transcripts in mature pollen that could be grouped into distinct gene ontology terms. AU - Kanter, U. AU - Heller, W. AU - Durner, J. AU - Winkler, J.B. AU - Engel, M. AU - Behrendt, H. AU - Holzinger, A.* AU - Braun, P.* AU - Hauser, M.* AU - Ferreira, F.* AU - Mayer, K.F.X. AU - Pfeifer, M. AU - Ernst, D. C1 - 24034 C2 - 31313 TI - Molecular and immunological characterization of ragweed (Ambrosia artemisiifolia L.) pollen after exposure of the plants to elevated ozone over a whole growing season. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain. Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a beta beta alpha fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins. AU - Kateb, F. AU - Perrin, H.* AU - Tripsianes, K. AU - Zou, P. AU - Spadaccini, R.* AU - Bottomley, M.* AU - Franzmann, T.M.* AU - Buchner, J.* AU - Ansieau, S.* AU - Sattler, M. C1 - 23535 C2 - 31212 TI - Structural and functional analysis of the DEAF-1 and BS69 MYND domains. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Uromodulin-associated kidney disease (UAKD) summarizes different clinical features of an autosomal dominant heritable disease syndrome in humans with a proven uromodulin (UMOD) mutation involved. It is often characterized by hyperuricemia, gout, alteration of urine concentrating ability, as well as a variable rate of disease progression inconstantly leading to renal failure and histological alterations of the kidneys. We recently established the two Umod mutant mouse lines UmodC93F and UmodA227T on the C3H inbred genetic background both showing kidney defects analogous to those found in human UAKD patients. In addition, disease symptoms were revealed that were not yet described in other published mouse models of UAKD. To examine if further organ systems and/or metabolic pathways are affected by Umod mutations as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the two mutant mouse lines UmodA227T and UmodC93F in the German Mouse Clinic. Different genotypes as well as different ages were tested. Beside the already published changes in body weight, body composition and bone metabolism, the influence of the Umod mutation on energy metabolism was confirmed. Hematological analysis revealed a moderate microcytic and erythropenic anemia in older Umod mutant mice. Data of the other analyses in 7-10 month-old mutant mice showed single small additional effects. AU - Kemter, E.* AU - Prueckl, P.* AU - Rathkolb, B. AU - Micklich, K. AU - Adler, T. AU - Becker, L. AU - Beckers, J. AU - Busch, D.H.* AU - Götz, A. AU - Hans, W. AU - Horsch, M. AU - Ivandic, B.* AU - Klingenspor, M.* AU - Klopstock, T.* AU - Rozman, J. AU - Schrewe, A. AU - Schulz, H. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Wolf, E.* AU - Aigner, B.* C1 - 28086 C2 - 32922 TI - Standardized, systemic phenotypic analysis of UmodC93F and UmodA227T mutant mice. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo. AU - Kim, J. AU - Badaloni, A.* AU - Willert, T. AU - Zimber-Strobl, U. AU - Kühn, R. AU - Wurst, W. AU - Kieslinger, M. C1 - 28431 C2 - 33371 TI - An RNAi-based approach to down-regulate a gene family in vivo. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Type 2 Diabetes is a global health burden and based on current estimates will become an even larger problem in the future. Developing new strategies to prevent and treat diabetes is a scientific challenge of high priority. The stomach hormone ghrelin has been associated with playing a role in the regulation of glucose homeostasis. However, its precise mechanism and impact on whole glucose metabolism remains to be elucidated. This study aims to clarify the role of the two ghrelin isoforms acyl- and desacyl ghrelin in regulating glucose homeostasis. Therefore ghrelin activating enzyme Ghrelin-O-acyltransferase (GOAT) was ablated in leptin-deficient ob/ob mice to study whether specific acyl ghrelin deficiency or desacyl ghrelin abundance modifies glucose tolerance on a massively obese background. As targeted deletion of acyl ghrelin does not improve glucose homeostasis in our GOAT-ob/ob mouse model we conclude that neither acyl ghrelin nor the increased ratio of desacyl/acyl ghrelin is crucial for controlling glucose homeostasis in the here presented model of massive obesity induced by leptin deficiency. AU - Kirchner, H.* AU - Heppner, K.M.* AU - Holland, J.* AU - Kabra, D. AU - Tschöp, M.H. AU - Pfluger, P.T. C1 - 24724 C2 - 31653 TI - Ablation of ghrelin O-acyltransferase does not improve glucose intolerance or body adiposity in mice on a leptin-deficient ob/ob background. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - We have detected differences in metabolite levels between doped athletes, clean athletes, and volunteers (non athletes). This outcome is obtained by comparing results of measurements from two analytical platforms: UHPLC-QTOF/MS and FT-ICR/MS. Twenty-seven urine samples tested positive for glucocorticoids or beta-2-agonists and twenty samples coming from volunteers and clean athletes were analyzed with the two different mass spectrometry approaches using both positive and negative electrospray ionization modes. Urine is a highly complex matrix containing thousands of metabolites having different chemical properties and a high dynamic range. We used multivariate analysis techniques to unravel this huge data set. Thus, the several groups we created were studied by Principal Components Analysis (PCA) and Partial Least Square regression (PLS-DA and OPLS) in the search of discriminating m/z values. The selected variables were annotated and placed on pathway by using MassTRIX. AU - Kiss, A.* AU - Lucio, M. AU - Fildier, A.* AU - Buisson, C.* AU - Schmitt-Kopplin, P. AU - Cren-Olivé, C.* C1 - 27572 C2 - 32727 TI - Doping control using high and ultra-high resolution mass spectrometry based non-targeted metabolomics-a case study of salbutamol and budesonide abuse. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50(th) and 95(th) percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50(th) percentile for the set of the 16 nuclear regulators of mitochondrial genes (p(GSEA,50) = 0.0103). This finding was not confirmed in the trios (p(GSEA,50) = 0.5991), but in KORA (p(GSEA,50) = 0.0398). The meta-analysis again indicated a trend for enrichment (p(MAGENTA,50) = 0.1052, p(MAGENTA,75) = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes. AU - Knoll, N.* AU - Jarick, I.* AU - Volckmar, A.-L.* AU - Klingenspor, M.* AU - Illig, T. AU - Grallert, H. AU - Gieger, C. AU - Wichmann, H.-E. AU - Peters, A. AU - Hebebrand, J.* AU - Scherag, A.* AU - Hinney, A.* C1 - 22833 C2 - 30955 TI - Gene set of nuclear-encoded mitochondrial regulators is enriched for common inherited variation in obesity. JO - PLoS ONE VL - 8 IS - 2 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer. PRINCIPAL FINDINGS: Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity. CONCLUSION: The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.   AU - Kraemer, A. AU - Barjaktarovic, Z. AU - Sarioglu, H. AU - Winkler, K. AU - Eckardt-Schupp, F. AU - Tapio, S. AU - Atkinson, M.J. AU - Mörtl, S. C1 - 28093 C2 - 32927 TI - Cell survival following radiation exposure requires miR-525-3p mediated suppression of ARRB1 and TXN1. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. AU - Kraemer, A. AU - Chen, I.P.* AU - Henning, S.* AU - Faust, A.* AU - Volkmer, B.* AU - Atkinson, M.J. AU - Mörtl, S. AU - Greinert, R.* C1 - 29039 C2 - 33603 TI - UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes. JO - PLoS ONE VL - 8 IS - 12 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37°C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells. AU - Kutscher, S. AU - Dembek, C.J. AU - Deckert, S. AU - Russo, C. AU - Körber, N. AU - Bogner, J.R.* AU - Geisler, F.* AU - Umgelter, A.* AU - Neuenhahn, M. AU - Albrecht, J. AU - Cosma, A.* AU - Protzer, U. AU - Bauer, T. C1 - 27964 C2 - 32880 TI - Overnight resting of PBMC changes functional signatures of antigen specific T-cell responses: Impact for immune monitoring within clinical trials. JO - PLoS ONE VL - 8 IS - 10 PB - Publis Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: A link between severe mental stress and shorter telomere length (TL) has been suggested. We analysed the impact of Posttraumatic Stress Disorder (PTSD) on TL in the general population and postulated a dose-dependent TL association in subjects suffering from partial PTSD compared to full PTSD. METHODS: Data are derived from the population-based KORA F4 study (2006-2008), located in southern Germany including 3,000 individuals (1,449 men and 1,551 women) with valid and complete TL data. Leukocyte TL was measured using a quantitative PCR-based technique. PTSD was assessed in a structured interview and by applying the Posttraumatic Diagnostic Scale (PDS) and the Impact of Event Scale (IES). A total of 262 (8.7%) subjects qualified for having partial PTSD and 51 (1.7%) for full PTSD. To assess the association of PTSD with the average TL, linear regression analyses with adjustments for potential confounding factors were performed. RESULTS: The multiple model revealed a significant association between partial PTSD and TL (beta = -0.051, p = 0.009) as well as between full PTSD and shorter TL (beta = -0.103, p = 0.014) indicating shorter TL on average for partial and full PTSD. An additional adjustment for depression and depressed mood/exhaustion gave comparable beta estimations. CONCLUSIONS: Participants with partial and full PTSD had significantly shorter leukocyte TL than participants without PTSD. The dose-dependent variation in TL of subjects with partial and full PTSD exceeded the chronological age effect, and was equivalent to an estimated 5 years in partial and 10 years in full PTSD of premature aging. AU - Ladwig, K.-H. AU - Brockhaus, A.C. AU - Baumert, J.J. AU - Lukaschek, K. AU - Emeny, R.T. AU - Kruse, J.* AU - Codd, V.* AU - Häfner, S. AU - Albrecht, E. AU - Illig, T. AU - Samani, N.J.* AU - Wichmann, H.-E. AU - Gieger, C. AU - Peters, A. C1 - 26174 C2 - 32106 TI - Posttraumatic stress disorder and not depression is associated with shorter leukocyte telomere length: Findings from 3,000 participants in the population-based KORA F4 study. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Type 2 diabetes (T2D) is a complex disease characterized by beta cell dysfunctions. Islet amyloid polypeptide (IAPP) is highly conserved and co-secreted with insulin with over 40% of autopsy cases of T2D showing islet amyloid formation due to IAPP aggregation. Dysregulation in IAPP processing, stabilization and degradation can cause excessive oligomerization with beta cell toxicity. Previous studies examining genetic associations of pathways implicated in IAPP metabolism have yielded conflicting results due to small sample size, insufficient interrogation of gene structure and gene-gene interactions. In this multi-staged study, we screened 89 tag single nucleotide polymorphisms (SNPs) in 6 candidate genes implicated in IAPP metabolism and tested for independent and joint associations with T2D and beta cell dysfunctions. Positive signals in the stage-1 were confirmed by de novo and in silico analysis in a multi-centre unrelated case-control cohort. We examined the association of significant SNPs with quantitative traits in a subset of controls and performed bioinformatics and relevant functional analyses. Amongst the tag SNPs, rs1583645 in carboxypeptidase E (CPE) and rs6583813 in insulin degrading enzyme (IDE) were associated with 1.09 to 1.28 fold increased risk of T2D (P Meta = 9.4×10(-3) and 0.02 respectively) in a meta-analysis of East Asians. Using genetic risk scores (GRS) with each risk variant scoring 1, subjects with GRS≥3 (8.2% of the cohort) had 56% higher risk of T2D than those with GRS = 0 (P = 0.01). In a subcohort of control subjects, plasma IAPP increased and beta cell function index declined with GRS (P = 0.008 and 0.03 respectively). Bioinformatics and functional analyses of CPE rs1583645 predicted regulatory elements for chromatin modification and transcription factors, suggesting differential DNA-protein interactions and gene expression. Taken together, these results support the importance of dysregulation of IAPP metabolism in T2D in East Asians. AU - Lam, V.K.* AU - Ma, R.C.* AU - Lee, H.M.* AU - Hu, C.* AU - Park, K.S.* AU - Furuta, H.* AU - Wang, Y.* AU - Tam, C.H.* AU - Sim, X.* AU - Ng, D.P.* AU - Liu, J.* AU - Wong, T.Y.* AU - Tai, E.S.* AU - Morris, A.P.* AU - DIAGRAM Consortium (Huth, C. AU - Grallert, H. AU - Gieger, C. AU - Meitinger, T. AU - Thorand, B. AU - Klopp, N. AU - Wichmann, H.-E. AU - Illig, T. AU - Petersen, A.-K.) AU - Tang, N.L.* AU - Woo, J.* AU - Leung, P.C.* AU - Kong, A.P.* AU - Ozaki, R.* AU - Jia, W.P.* AU - Lee, H.K.* AU - Nanjo, K.* AU - Xu, G.* AU - Ng, M.C.* AU - So, W.Y.* AU - Chan, J.C.* C1 - 28107 C2 - 32941 TI - Genetic associations of type 2 diabetes with islet amyloid polypeptide processing and degrading pathways in asian populations. JO - PLoS ONE VL - 8 IS - 6 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Random reductions in plant diversity can affect ecosystem functioning, but it is still unclear which components of plant diversity (species number – namely richness, presence of particular plant functional groups, or particular combinations of these) and associated biotic and abiotic drivers explain the observed relationships, particularly for soil processes. We assembled grassland communities including 1 to 16 plant species with a factorial separation of the effects of richness and functional group composition to analyze how plant diversity components influence soil nitrifying and denitrifying enzyme activities (NEA and DEA, respectively), the abundance of nitrifiers (bacterial and archaeal amoA gene number) and denitrifiers (nirK, nirS and nosZ gene number), and key soil environmental conditions. Plant diversity effects were largely due to differences in functional group composition between communities of identical richness (number of sown species), though richness also had an effect per se. NEA was positively related to the percentage of legumes in terms of sown species number, the additional effect of richness at any given legume percentage being negative. DEA was higher in plots with legumes, decreased with increasing percentage of grasses, and increased with richness. No correlation was observed between DEA and denitrifier abundance. NEA increased with the abundance of ammonia oxidizing bacteria. The effect of richness on NEA was entirely due to the build-up of nitrifying organisms, while legume effect was partly linked to modified ammonium availability and nitrifier abundance. Richness effect on DEA was entirely due to changes in soil moisture, while the effects of legumes and grasses were partly due to modified nitrate availability, which influenced the specific activity of denitrifiers. These results suggest that plant diversity-induced changes in microbial specific activity are important for facultative activities such as denitrification, whereas changes in microbial abundance play a major role for non-facultative activities such as nitrification. AU - Le Roux, X.* AU - Schmid, B.* AU - Poly, F.* AU - Barnard, R.L.* AU - Niklaus, P.A.* AU - Guillaumaud, N.* AU - Habekost, M.* AU - Oelmann, Y.* AU - Philippot, L.* AU - Salles, J.F.* AU - Schloter, M. AU - Steinbeiss, S.* AU - Weigelt, A.* C1 - 24032 C2 - 31312 TI - Soil environmental conditions and microbial build-up mediate the effect of plant diversity on soil nitrifying and denitrifying enzyme activities in temperate grasslands. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Background Several risk factors for cardiovascular disease (CVD) have been identified in recent decades. However, the association between the health system and the burden of CVD has not yet been sufficiently researched. The objective of this study was to analyse the association between health system development and the burden of CVD, in particular CVD-related disability-adjusted life–years (DALYs). Methods Univariate and multivariate generalized linear mixed models were applied to country-level data collected by the World Bank and World Health Organization. Response variables were the age-standardized CVD mortality and age-standardized CVD DALY rates. Results The amount of available health system resources, indicated by total health expenditures per capita, physician density, nurse density, dentistry density, pharmaceutical density and the density of hospital beds, was associated with reduced CVD DALY rates and CVD mortality. However, in the multivariate models, the density of nurses and midwives was positively associated with CVD. High out-of-pocket costs were associated with increased CVD mortality in both univariate and multivariate analyses. Conclusion A highly developed health system with a low level of out-of-pocket costs seems to be the most appropriate to reduce the burden of CVD. Furthermore, an efficient balance between human health resources and health technologies is essential. AU - Liu, Y. AU - Dalal, K.* AU - Stollenwerk, B. C1 - 24220 C2 - 31342 TI - The association between health system development and the burden of cardiovascular disease: An analysis of WHO country profiles. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4− NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease. AU - Lotter, H.* AU - Helk, E.* AU - Bernin, H.* AU - Jacobs, T.* AU - Prehn, C. AU - Adamski, J. AU - Gonzáles-Roldán, N.* AU - Holst, O.* AU - Tannich, E.* C1 - 22746 C2 - 30921 TI - Testosteron increases susceptibility to amebic liver abscess in mice and mediates inhibition of IFNγ secretion in natural killer T cells. JO - PLoS ONE VL - 8 IS - 2 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Structural changes of chromosomes are a primary mechanism of genome rearrangement over the course of evolution and detailed knowledge of such changes in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A, 5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species. AU - Ma, J.* AU - Stiller, J.* AU - Berkman, P.J.* AU - Wei, Y.* AU - Rogers, J.* AU - Feuillet, C.* AU - Dolezel, J.* AU - Mayer, K.F.X. AU - Eversole, K.* AU - Zheng, Y.L.* AU - Liu, C.* C1 - 28548 C2 - 33439 TI - Sequence-based analysis of translocations and inversions in bread wheat (Triticum aestivum L.). JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The pathomechanism of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphomas (CTCLs) and a malignancy of non-recirculating, skin-resident T-cells, is unknown albeit underlying viral infections have been sought for. Human endogenous retroviruses (HERVs) are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancers. We explored the transcriptional activity of HERV sequences in a total of 34 samples comprising MF and psoriasis skin lesions, as well as corresponding non-malignant skin using a retrovirus-specific microarray and quantitative RT-PCR. To identify active HERV-W loci, we cloned the HERV-W specific RT-PCR products, sequenced the cDNA clones and assigned the sequences to HERV-W loci. Finally, we used immunohistochemistry on MF patient and non-malignant inflammatory skin samples to confirm specific HERV-encoded protein expression. Firstly, a distinct, skin-specific transcription profile consisting of five constitutively active HERV groups was established. Although individual variability was common, HERV-W showed significantly increased transcription in MF lesions compared to clinically intact skin from the same patient. Predominantly transcribed HERV-W loci were found to be located in chromosomes 6q21 and 7q21.2, chromosomal regions typically altered in CTCL. Surprisingly, we also found the expression of 7q21.2/ERVWE1-encoded Syncytin-1 (Env) protein in MF biopsies and expression of Syncytin-1 was seen in malignant lymphocytes, especially in the epidermotropic ones, in 15 of 30 cases studied. Most importantly, no Syncytin-1 expression was detected in inflammatory dermatosis (Lichen ruber planus) with skin-homing, non-malignant T lymphocytes. The expression of ERVWE1 mRNA was further confirmed in 3/7 MF lesions analyzed. Our observations strengthen the association between activated HERVs and cancer. The study offers a new perspective into the pathogenesis of CTCL since we demonstrate that differences in HERV-W transcription levels between lesional MF and non-malignant skin are significant, and that ERVWE1-encoded Syncytin-1 is expressed in MF lymphoma cells. AU - Maliniemi, P.* AU - Vincendeau, M. AU - Mayer, J.* AU - Frank, O.* AU - Hahtola, S.* AU - Karenko, L.* AU - Carlsson, E.* AU - Mallet, F.* AU - Seifarth, W.* AU - Leib-Mösch, C. AU - Ranki, A.* C1 - 27931 C2 - 32864 TI - Expression of human endogenous retrovirus-W including syncytin-1 in cutaneous T-cell lymphoma. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration. AU - Manic, G.* AU - Maurin-Marlin, A.* AU - Laurent, F.* AU - Vitale, I.* AU - Thierry, S.* AU - Delelis, O.* AU - Dessen, P.* AU - Vincendeau, M. AU - Leib-Mösch, C. AU - Hazan, U.* AU - Mouscadet, J.F.* AU - Bury-Moné, S.* C1 - 26543 C2 - 32287 TI - Impact of the Ku complex on HIV-1 expression and latency. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a "candidate interactome" (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms. AU - Mechelli, R.* AU - Umeton, R.* AU - Policano, C.* AU - Annibali, V.* AU - Coarelli, G.* AU - Ricigliano, V.A.* AU - Vittori, D.* AU - Fornasiero, A.* AU - Buscarinu, M.C.* AU - International Multiple Sclerosis Genetics Consortium (*) AU - Wellcome Trust Case Control Consortium 2 (WTCCC2) (Klopp, N. AU - Rückert, I.-M. AU - Wichmann, H.-E. AU - Winkelmann, J.) AU - Romano, S.* AU - Salvetti, M.* AU - Ristori, G.* C1 - 26182 C2 - 32110 TI - A "candidate-interactome" aggregate analysis of genome-wide association data in multiple sclerosis. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Purpose: To assess whether grating-based X-ray dark-field imaging can increase the sensitivity of X-ray projection images in the diagnosis of pulmonary emphysema and allow for a more accurate assessment of emphysema distribution. Materials and Methods: Lungs from three mice with pulmonary emphysema and three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Median signal intensities of transmission (T), dark-field (V) and a combined parameter (normalized scatter) were compared between emphysema and control group. To determine the diagnostic value of each parameter in differentiating between healthy and emphysematous lung tissue, a receiver-operating-characteristic (ROC) curve analysis was performed both on a per-pixel and a per-individual basis. Parametric maps of emphysema distribution were generated using transmission, dark-field and normalized scatter signal and correlated with histopathology. Results: Transmission values relative to water were higher for emphysematous lungs than for control lungs (1.11 vs. 1.06, p<0.001). There was no difference in median dark-field signal intensities between both groups (0.66 vs. 0.66). Median normalized scatter was significantly lower in the emphysematous lungs compared to controls (4.9 vs. 10.8, p<0.001), and was the best parameter for differentiation of healthy vs. emphysematous lung tissue. In a per-pixel analysis, the area under the ROC curve (AUC) for the normalized scatter value was significantly higher than for transmission (0.86 vs. 0.78, p<0.001) and dark-field value (0.86 vs. 0.52, p<0.001) alone. Normalized scatter showed very high sensitivity for a wide range of specificity values (94% sensitivity at 75% specificity). Using the normalized scatter signal to display the regional distribution of emphysema provides color-coded parametric maps, which show the best correlation with histopathology. Conclusion: In a murine model, the complementary information provided by X-ray transmission and dark-field images adds incremental diagnostic value in detecting pulmonary emphysema and visualizing its regional distribution as compared to conventional X-ray projections. AU - Meinel, F.G.* AU - Schwab, F.* AU - Schleede, S.* AU - Bech, M.* AU - Herzen, J.* AU - Achterhold, K.* AU - Auweter, S.* AU - Bamberg, F.* AU - Yildirim, A.Ö. AU - Bohla, A. AU - Eickelberg, O. AU - Loewen, R.* AU - Gifford, M.* AU - Ruth, R.* AU - Reiser, M.F.* AU - Pfeiffer, F.* AU - Nikolaou, K.* C1 - 24455 C2 - 31553 TI - Diagnosing and mapping pulmonary emphysema on X-ray projection images: Incremental value of grating-based X-ray dark-field imaging. JO - PLoS ONE VL - 8 IS - 3 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications. AU - Merkl, C.* AU - Saalfrank, A.* AU - Riesen, N.* AU - Kühn, R. AU - Pertek, A. AU - Eser, S.* AU - Hardt, M.S.* AU - Kind, A.* AU - Saur, D.* AU - Wurst, W. AU - Iglesias, A.* AU - Schnieke, A.* C1 - 22978 C2 - 30969 TI - Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - In mammals, adult neural stem cells give rise to new hippocampal dentate granule neurons and interneurons of the olfactory bulb throughout life. The microtubule associated protein Doublecortin (DCX) is expressed by migrating neuroblasts and immature neurons, and is widely used as a stage-specific marker of adult neurogenesis and as a marker to identify neurogenic activity in the adult brain per se. Mutations in the DCX gene have been causally linked to human lissencephalic syndromes. Moreover, embryonic loss of DCX function interferes with neuronal migration and dendritic patterning in a species- and region-specific manner. A putative function of DCX in adult neurogenesis has not been directly explored. Here we show that overexpression of DCX in newly generated dentate granule neurons of the adult mouse brain has no effect on morphological maturation or migration. We also show that micro (mi) RNA-mediated retroviral knockdown of DCX does not alter morphological maturation of adult born dentate granule cells or migration of new neurons in either adult neurogenic niche. Thus, the present data indicate that DCX is dispensable for the development of new neurons in adult mice. AU - Merz, K. AU - Lie, D.C. C1 - 25757 C2 - 31908 TI - Evidence that doublecortin is dispensable for the development of adult born neurons in mice. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Understanding factors driving the ecology of N cycling microbial communities is of central importance for sustainable land use. In this study we report changes of abundance of denitrifiers, nitrifiers and nitrogen-fixing microorganisms (based on qPCR data for selected functional genes) in response to different land use intensity levels and the consequences for potential turnover rates. We investigated selected grassland sites being comparable with respect to soil type and climatic conditions, which have been continuously treated for many years as intensely used meadows (IM), intensely used mown pastures (IP) and extensively used pastures (EP), respectively. The obtained data were linked to above ground biodiversity pattern as well as water extractable fractions of nitrogen and carbon in soil. Shifts in land use intensity changed plant community composition from systems dominated by s-strategists in extensive managed grasslands to c-strategist dominated communities in intensive managed grasslands. Along the different types of land use intensity, the availability of inorganic nitrogen regulated the abundance of bacterial and archaeal ammonia oxidizers. In contrast, the amount of dissolved organic nitrogen determined the abundance of denitrifiers (nirS and nirK). The high abundance of nifH carrying bacteria at intensive managed sites gave evidence that the amounts of substrates as energy source outcompete the high availability of inorganic nitrogen in these sites. Overall, we revealed that abundance and function of microorganisms involved in key processes of inorganic N cycling (nitrification, denitrification and N fixation) might be independently regulated by different abiotic and biotic factors in response to land use intensity. AU - Meyer, A.H.* AU - Focks, A.* AU - Radl, V. AU - Keil, D.* AU - Welzl, G. AU - Schöning, I.* AU - Boch, S.* AU - Marhan, S.* AU - Kandeler, E.* AU - Schloter, M. C1 - 27241 C2 - 32584 TI - Different land use intensities in grassland ecosystems drive ecology of microbial communities involved in nitrogen turnover in soil. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5-9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17-53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12-48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct locations to avoid sampling bias. AU - Mittermeyer, G.* AU - Malinowsky, K.* AU - Beese, C.* AU - Höfler, H. AU - Schmalfeldt, B.* AU - Becker, K.F.* AU - Avril, S.* C1 - 28168 C2 - 32981 TI - Variation in cell signaling protein expression may introduce sampling bias in primary epithelial ovarian cancer. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVES: Chronic rhinosinusitis (CRS) is a common chronic disease of the upper airways and has considerable impact on quality of life. Topical delivery of drugs to the paranasal sinuses is challenging, therefore the rate of surgery is high. This study investigates the delivery efficiency of a pulsating aerosol in comparison to a nasal pump spray to the sinuses and the nose in healthy volunteers and in CRS patients before and after sinus surgery. METHODS: (99m)Tc-DTPA pulsating aerosols were applied in eleven CRSsNP patients without nasal polyps before and after sinus surgery. In addition, pulsating aerosols were studied in comparison to nasal pump sprays in eleven healthy volunteers. Total nasal and frontal, maxillary and sphenoidal sinus aerosol deposition and lung penetration were assessed by anterior and lateral planar gamma camera imaging. RESULTS: In healthy volunteers nasal pump sprays resulted in 100% nasal, non-significant sinus and lung deposition, while pulsating aerosols resulted 61.3+/-8.6% nasal deposition and 38.7% exit the other nostril. 9.7+/-2.0 % of the nasal dose penetrated into maxillary and sphenoidal sinuses. In CRS patients, total nasal deposition was 56.7+/-13.3% and 46.7+/-12.7% before and after sinus surgery, respectively (p<0.01). Accordingly, maxillary and sphenoidal sinus deposition was 4.8+/-2.2% and 8.2+/-3.8% of the nasal dose (p<0.01). Neither in healthy volunteers nor in CRS patients there was significant dose in the frontal sinuses. CONCLUSION: In contrast to nasal pump sprays, pulsating aerosols can deliver significant doses into posterior nasal spaces and paranasal sinuses, providing alternative therapy options before and after sinus surgery. Patients with chronic lung diseases based on clearance dysfunction may also benefit from pulsating aerosols, since these diseases also manifest in the upper airways. AU - Möller, W. AU - Schuschnig, U.* AU - Celik, G. AU - Münzing, W.* AU - Bartenstein, P.* AU - Häussinger, K.* AU - Kreyling, W.G. AU - Knoch, M.* AU - Canis, M.* AU - Becker, S.* C1 - 27771 C2 - 32806 TI - Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Single nucleotide polymorphisms (SNPs) in genes involved in fatty acid metabolism (FADS1 FADS2 gene cluster) are associated with plasma lipid levels. We aimed to investigate whether these associations are already present early in life and compare the relative contribution of FADS SNPs vs traditional (non-genetic) factors as determinants of plasma lipid levels. METHODS: Information on infants' plasma total cholesterol levels, genotypes of five FADS SNPs (rs174545, rs174546, rs174556, rs174561, and rs3834458), anthropometric data, maternal characteristics, and breastfeeding history was available for 521 2-year-old children from the KOALA Birth Cohort Study. For 295 of these 521 children, plasma HDLc and non-HDLc levels were also known. Multivariable linear regression analysis was used to study the associations of genetic and non-genetic determinants with cholesterol levels. RESULTS: All FADS SNPs were significantly associated with total cholesterol levels. Heterozygous and homozygous for the minor allele children had about 4% and 8% lower total cholesterol levels than major allele homozygotes. In addition, homozygous for the minor allele children had about 7% lower HDLc levels. This difference reached significance for the SNPs rs174546 and rs3834458. The associations went in the same direction for non-HDLc, but statistical significance was not reached. The percentage of total variance of total cholesterol levels explained by FADS SNPs was relatively low (lower than 3%) but of the same order as that explained by gender and the non-genetic determinants together. CONCLUSIONS: FADS SNPs are associated with plasma total cholesterol and HDLc levels in preschool children. This brings a new piece of evidence to explain how blood lipid levels may track from childhood to adulthood. Moreover, the finding that these SNPs explain a similar amount of variance in total cholesterol levels as the non-genetic determinants studied reveals the potential importance of investigating the effects of genetic variations in early life. AU - Moltó-Puigmartí, C.* AU - Jansen, E.* AU - Heinrich, J. AU - Standl, M. AU - Mensink, R.P.* AU - Plat, J.* AU - Penders, J.* AU - Mommers, M.* AU - Koppelman, G.H.* AU - Postma, D.S.* AU - Thijs, C.* C1 - 25490 C2 - 31861 TI - Genetic variation in FADS genes and plasma cholesterol levels in 2-year-old infants: KOALA Birth Cohort study. JO - PLoS ONE VL - 8 IS - 5 PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - HSC-Explorer (http://mips.helmholtz-muenchen.de/HSC/) is a publicly available, integrative database containing detailed information about the early steps of hematopoiesis. The resource aims at providing fast and easy access to relevant information, in particular to the complex network of interacting cell types and molecules, from the wealth of publications in the field through visualization interfaces. It provides structured information on more than 7000 experimentally validated interactions between molecules, bioprocesses and environmental factors. Information is manually derived by critical reading of the scientific literature from expert annotators. Hematopoiesis-relevant interactions are accompanied with context information such as model organisms and experimental methods for enabling assessment of reliability and relevance of experimental results. Usage of established vocabularies facilitates downstream bioinformatics applications and to convert the results into complex networks. Several predefined datasets (Selected topics) offer insights into stem cell behavior, the stem cell niche and signaling processes supporting hematopoietic stem cell maintenance. HSC-Explorer provides a versatile web-based resource for scientists entering the field of hematopoiesis enabling users to inspect the associated biological processes through interactive graphical presentation. AU - Montrone, C. AU - Kokkaliaris, K.D. AU - Loeffler, D. AU - Lechner, M. AU - Kastenmüller, G. AU - Schroeder, T. AU - Ruepp, A. C1 - 26540 C2 - 32291 TI - HSC-Explorer: A curated database for hematopoietic stem cells. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The G-protein coupled receptor 83 (GPR83) is an orphan G-protein coupled receptor for which the natural ligand(s) and signaling pathway(s) remain to be identified. Previous studies suggest a role of GPR83 in the regulation of thermogenesis and the control of circulating adiponectin. The aim of this study was to gain insights into the molecular underpinnings underlying GPR83 signaling. In particular, we aimed to assess the underlying G-protein activated signaling pathway of GPR83 and how this pathway is affected by mutational activation and zinc(II) challenge. Finally, we assessed the capacity of GPR83 for homodimerization. Our results show for the first time that mouse (m) GPR83 has high basal Gq/11 activity without affecting Gi or Gs signaling. Furthermore, we found that, under physiological conditions, zinc(II) (but not calcium(II) and magnesium(II)) potently activates mGPR83, thus identifying zinc(II) as an endogenous molecule with agonistic capability to activate mGPR83. In line with the observation that zinc(II)-ions activate mGPR83, we identified a cluster of ion-binding sensitive amino acids (e.g. His145, His204, Cys207, Glu217) in an activation sensitive receptor region of mGPR83. The occurrence of a constitutive activating mutant and a zinc(II)-binding residue at the N-terminal part corroborate the importance of this region in mGPR83 signal regulation. Finally, our results indicate that mGPR83 forms homodimers, which extend the current knowledge and molecular facets of GPR83 signaling. AU - Müller, A.* AU - Kleinau, G.* AU - Piechowski, C.L.* AU - Müller, T.D. AU - Finan, B. AU - Pratzka, J.* AU - Grüters, A.* AU - Krude, H.* AU - Tschöp, M.H. AU - Biebermann, H.* C1 - 23391 C2 - 31025 TI - G-Protein coupled Receptor 83 (GPR83) signaling determined by constitutive and zinc(II)-induced activity. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. METHODOLOGY/PRINCIPAL FINDINGS: A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. CONCLUSIONS: The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings. AU - Münchmeier, N.* AU - Boecker, S.* AU - Bankel, L. AU - Hinz, L.* AU - Rieth, N.* AU - Lapa, C.* AU - Mendler, A.N. AU - Nößner, E. AU - Mocikat, R. AU - Nelson, P.J. C1 - 27373 C2 - 32634 TI - A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present a quantitative characterization of the kinetics and affinity of interactions between GPCRs and one of the best characterized PDZ scaffold proteins, postsynaptic density protein 95 (PSD-95), using fluorescence polarization (FP) and surface plasmon resonance (SPR). By comparing these in vitro findings with colocalization of the full-length proteins in cells and with previous studies, we suggest that the range of relevant interactions might extend to interactions with K-i = 450 mu M in the in vitro assays. Within this range, we identify novel PSD-95 interactions with the chemokine receptor CXCR2, the neuropeptide Y receptor Y-2, and four of the somatostatin receptors (SSTRs). The interaction with SSTR1 was further investigated in mouse hippocampal neurons, where we found a clear colocalization between the endogenously expressed proteins, indicating a potential for further investigation of the role of this interaction. The approach can easily be transferred to other receptors and scaffold proteins and this could help accelerate the discovery and quantitative characterization of GPCR-PDZ interactions. AU - Møller, T.C.* AU - Wirth, V.F.* AU - Roberts, N.I.* AU - Bender, J.* AU - Bach, A.* AU - Jacky, B.P.S.* AU - Strømgaard, K.* AU - Deussing, J.M. AU - Schwartz, T.W.* AU - Martinez, K.L.* C1 - 25518 C2 - 31870 TI - PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95: Quantitative characterization of interactions. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Despite significant methodological advances in protein structure determination high-resolution structures of membrane proteins are still rare, leaving sequence-based predictions as the only option for exploring the structural variability of membrane proteins at large scale. Here, a new structural classification approach for α-helical membrane proteins is introduced based on the similarity of predicted helix interaction patterns. Its application to proteins with known 3D structure showed that it is able to reliably detect structurally similar proteins even in the absence of any sequence similarity, reproducing the SCOP and CATH classifications with a sensitivity of 65% at a specificity of 90%. We applied the new approach to enhance our comprehensive structural classification of α-helical membrane proteins (CAMPS), which is primarily based on sequence and topology similarity, in order to find protein clusters that describe the same fold in the absence of sequence similarity. The total of 151 helix architectures were delineated for proteins with more than four transmembrane segments. Interestingly, we observed that proteins with 8 and more transmembrane helices correspond to fewer different architectures than proteins with up to 7 helices, suggesting that in large membrane proteins the evolutionary tendency to re-use already available folds is more pronounced. AU - Neumann, S.* AU - Fuchs, A.* AU - Hummel, B.* AU - Frishman, D. C1 - 28160 C2 - 32975 TI - Classification of α-helical membrane proteins using predicted helix architectures. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Job strain is associated with an increased coronary heart disease risk, but few large-scale studies have examined the relationship of this psychosocial characteristic with the biological risk factors that potentially mediate the job strain - heart disease association. METHODOLOGY AND PRINCIPAL FINDINGS: We pooled cross-sectional, individual-level data from eight studies comprising 47,045 participants to investigate the association between job strain and the following cardiovascular disease risk factors: diabetes, blood pressure, pulse pressure, lipid fractions, smoking, alcohol consumption, physical inactivity, obesity, and overall cardiovascular disease risk as indexed by the Framingham Risk Score. In age-, sex-, and socioeconomic status-adjusted analyses, compared to those without job strain, people with job strain were more likely to have diabetes (odds ratio 1.29; 95% CI: 1.11-1.51), to smoke (1.14; 1.08-1.20), to be physically inactive (1.34; 1.26-1.41), and to be obese (1.12; 1.04-1.20). The association between job strain and elevated Framingham risk score (1.13; 1.03-1.25) was attributable to the higher prevalence of diabetes, smoking and physical inactivity among those reporting job strain. CONCLUSIONS: In this meta-analysis of work-related stress and cardiovascular disease risk factors, job strain was linked to adverse lifestyle and diabetes. No association was observed between job strain, clinic blood pressure or blood lipids. AU - Nyberg, S.T.* AU - Fransson, E.I.* AU - Heikkilä, K.* AU - Alfredsson, L.* AU - Casini, A.* AU - Clays, E.* AU - de Bacquer, D.* AU - Dragano, N.* AU - Erbel, R.* AU - Ferrie, J.E.* AU - Hamer, M.* AU - Jöckel, K.-H.* AU - Kittel, F.* AU - Knutsson, A.* AU - Ladwig, K.-H. AU - Lunau, T.* AU - Marmot, M.G.* AU - Nordin, M.* AU - Rugulies, R.* AU - Siegrist, J.* AU - Steptoe, A.* AU - Westerholm, P.J.* AU - Westerlund, H.* AU - Theorell, T.* AU - Brunner, E.J.* AU - Singh-Manoux, A.* AU - Batty, G.D.* AU - Kivimaki, M.* C1 - 26196 C2 - 32117 TI - Job strain and cardiovascular disease risk factors: Meta-analysis of individual-participant data from 47,000 men and women. JO - PLoS ONE VL - 8 IS - 6 PB - Public Libary of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases. AU - O'Donoghue, A.J.* AU - Jin, Y.* AU - Knudsen, G.M.* AU - Perera, N.C. AU - Jenne, D. AU - Murphy, J.E.* AU - Craik, C.S.* AU - Hermiston, T.W.* C1 - 27445 C2 - 32673 TI - Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Traditional gold mining is associated with mercury exposure. Especially vulnerable to its neurotoxic effects is the developing nervous system of a child. We aimed to investigate risk factors of mercury exposure among children in a rural mining town in Chile. METHODS: Using a validated questionnaire distributed to the parents of the children, a priori mercury risk factors, potential exposure pathways and demographics of the children were obtained. Mercury levels were measured through analyzing fingernail samples. Logistic regression modeling the effect of risk factors on mercury levels above the 75(th) percentile were made, adjusted for potential confounders. RESULTS: The 288 children had a mean age of 9.6 years (SD = 1.9). The mean mercury level in the study population was 0.13 µg/g (SD 0.11, median 0.10, range 0.001-0.86 µg/g). The strongest risk factor for children's odds of high mercury levels (>75(th) percentile, 0.165 µg/g) was to play inside a house where a family member worked with mercury (OR adjusted 3.49 95% CI 1.23-9.89). Additionally, children whose parents worked in industrial gold mining had higher odds of high mercury levels than children whose parents worked in industrial copper mining or outside mining activities. CONCLUSION: Mercury exposure through small-scale gold mining might affect children in their home environments. These results may further help to convince the local population of banning mercury burning inside the households.   AU - Ohlander, J.* AU - Huber, S.M.* AU - Schomaker, M.* AU - Heumann, C.* AU - Schierl, R.* AU - Michalke, B. AU - Jenni, O.G.* AU - Caflisch, J.* AU - Muñoz, D.M.* AU - von Ehrenstein, O.S.* AU - Radon, K.* C1 - 28562 C2 - 33452 TI - Risk factors for mercury exposure of children in a rural mining town in Northern Chile. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3. AU - Paul, K.* AU - Wang, M.L.* AU - Mladenov, E.* AU - Bencsik-Theilen, A.* AU - Bednar, T.* AU - Wu, W.Q.* AU - Arakawa, H. AU - Iliakis, G.* C1 - 24452 C2 - 31554 TI - DNA ligases I and III cooperate in alternative non-homologous end-joining in vertebrates. JO - PLoS ONE VL - 8 IS - 3 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Background Despite the fact that the fixation of nitrogen is one of the most significant nutrient processes in the terrestrial ecosystem, a thorough study of the spatial and temporal patterns in the abundance and distribution of N-fixing communities has been missing so far. Methodology/Principal Findings In order to understand the dynamics of diazotrophic communities and their resilience to external changes, we quantified the abundance and characterized the bacterial community structures based on the nifH gene, using real-time PCR, PCR-DGGE and 454-pyrosequencing, across four representative Dutch soils during one growing season. In general, higher nifH gene copy numbers were observed in soils with higher pH than in those with lower pH, but lower numbers were related to increased nitrate and ammonium levels. Results from nifH gene pyrosequencing confirmed the observed PCR-DGGE patterns, which indicated that the N fixers are highly dynamic across time, shifting around 60%. Forward selection on CCA analysis identified N availability as the main driver of these variations, as well as of the evenness of the communities, leading to very unequal communities. Moreover, deep sequencing of the nifH gene revealed that sandy soils (B and D) had the lowest percentage of shared OTUs across time, compared with clayey soils (G and K), indicating the presence of a community under constant change. Cosmopolitan nifH species (present throughout the season) were affiliated with Bradyrhizobium, Azospirillum and Methylocistis, whereas other species increased their abundances progressively over time, when appropriate conditions were met, as was notably the case for Paenibacilus and Burkholderia. Conclusions Our study provides the first in-depth pyrosequencing analysis of the N-fixing community at both spatial and temporal scales, providing insights into the cosmopolitan and specific portions of the nitrogen fixing bacterial communities in soil. AU - Pereira e Silva, M.C.* AU - Schloter-Hai, B. AU - Schloter, M. AU - van Elsas, J.D.* AU - Falcao Salles, J.* C1 - 27268 C2 - 32591 TI - Temporal dynamics of abundance and composition of nitrogen-fixing communities across agricultural soils. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Surveillance of physical activity (PA) is increasingly based on accelerometry. However, data management guidelines are lacking. We propose an approach for combining accelerometry and diary based PA information for assessment of PA in adolescents and provide an example of this approach using data from German adolescents. METHODS: The 15-year-old participants comprised a subsample the GINIplus birth cohort (n = 328, 42.4% male). Data on PA was obtained from hip-worn accelerometers (ActiGraph GT3X) for seven consecutive days, combined with a prospective activity diary. Major aspects of data management were validity of wear time, handling of non-wear time and diary comments. After data cleaning, PA and percentage of adolescents meeting the recommendations for moderate-to-vigorous activity (MVPA) per day were determined. RESULTS: From the 2224 recorded days 493 days (25%) were invalid, mainly due to uncertainties relating to non-wear time (322 days). Ultimately, 269 of 328 subjects (82%) with valid data for at least three weekdays and one weekend day were included in the analysis. Mean MVPA per day was 39.1 minutes (SD ±25.0), with boys being more active than girls (41.8±21.5 minutes vs. 37.1±27.8 minutes, p<0.001). Accordingly, 24.7% of boys and 17.2% of girls (p<0.01) met the WHO recommendations for PA. School sport accounted for only 6% of weekly MVPA. In fact, most MVPA was performed during leisure time, with the majority of adolescents engaging in ball sports (25.4%) and endurance sports (19.7%). Girls also frequently reported dancing and gymnastics (23%). CONCLUSION: For assessment of PA in adolescents, collecting both accelerometry and diary-based information is recommended. The diary is vital for the identification of invalid data and non-compliant participants. Preliminary results suggest that four out of five German adolescents do not meet WHO recommendations for PA and that school sport contributes only little to MVPA. AU - Pfitzner, R. AU - Gorzelniak, L.* AU - Heinrich, J. AU - von Berg, A.* AU - Klümper, C.* AU - Bauer, C.P.* AU - Koletzko, S.* AU - Berdel, D.* AU - Horsch, A.* AU - Schulz, H. AU - GINIplus Study Group (Wichmann, H.-E. AU - Heinrich, J.) C1 - 25484 C2 - 31858 TI - Physical activity in German adolescents measured by accelerometry and activity diary: Introducing a comprehensive approach for data management and preliminary results. JO - PLoS ONE VL - 8 IS - 6 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR. AU - Priglinger, C.S.* AU - Szober, C.M.* AU - Priglinger, S.G.* AU - Merl, J. AU - Euler, K.N.* AU - Kernt, M.* AU - Gondi, G. AU - Behler, J. AU - Geerlof, A. AU - Kampik, A.* AU - Ueffing, M. AU - Hauck, S.M. C1 - 26541 C2 - 32289 TI - Galectin-3 induces clustering of CD147 and integrin-β1 transmembrane glycoprotein receptors on the RPE cell surface. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Due to the high complexity of biological data it is difficult to disentangle cellular processes relying only on intuitive interpretation of measurements. A Systems Biology approach that combines quantitative experimental data with dynamic mathematical modeling promises to yield deeper insights into these processes. Nevertheless, with growing complexity and increasing amount of quantitative experimental data, building realistic and reliable mathematical models can become a challenging task: the quality of experimental data has to be assessed objectively, unknown model parameters need to be estimated from the experimental data, and numerical calculations need to be precise and efficient. Here, we discuss, compare and characterize the performance of computational methods throughout the process of quantitative dynamic modeling using two previously established examples, for which quantitative, dose- and time-resolved experimental data are available. In particular, we present an approach that allows to determine the quality of experimental data in an efficient, objective and automated manner. Using this approach data generated by different measurement techniques and even in single replicates can be reliably used for mathematical modeling. For the estimation of unknown model parameters, the performance of different optimization algorithms was compared systematically. Our results show that deterministic derivative-based optimization employing the sensitivity equations in combination with a multi-start strategy based on latin hypercube sampling outperforms the other methods by orders of magnitude in accuracy and speed. Finally, we investigated transformations that yield a more efficient parameterization of the model and therefore lead to a further enhancement in optimization performance. We provide a freely available open source software package that implements the algorithms and examples compared here. AU - Raue, A. AU - Schilling, M.* AU - Bachmann, J.* AU - Matteson, A.* AU - Schelker, M.* AU - Kaschek, D.* AU - Hug, S. AU - Kreutz, C.* AU - Harms, B.D.* AU - Theis, F.J. AU - Klingmüller, U.* AU - Timmer, J.* C1 - 27899 C2 - 32848 TI - Lessons learned from quantitative dynamical modeling in systems biology. JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The Notch signaling pathway mediates the direct communication between adjacent cells and regulates multiple developmental processes. Interaction of the Notch receptor with its ligands induces the liberation of the intracellular portion of Notch (NICD) referred to as regulated intramembraneous proteolysis (RIP). NICD translocates to the nucleus, and by complexing with the DNA binding protein RBPjκ and other cofactors activates transcription of bHLH genes. RIP-like processing of various mammalian Notch ligands (DLL1, JAG1 and JAG2) and the translocation of their intracellular domains (ICDs) to the nucleus has also been observed. These observations together with effects of over-expressed ligand ICDs in cultured cells on cell proliferation, differentiation, and Notch activity and target gene expression have led to the idea that the intracellular domains of Notch ligands have signaling functions. To test this hypothesis in vivo we have generated ES cells and transgenic mice that constitutively express various versions of the intracellular domain of mouse DLL1. In contrast to other cell lines, expression of DICDs in ES cells did not block proliferation or stimulate neuronal differentiation. Embryos with ubiquitous DICD expression developed to term without any apparent phenotype and grew up to viable and fertile adults. Early Notch-dependent processes or expression of selected Notch target genes were unaltered in transgenic embryos. In addition, we show that mouse DICD enters the nucleus inefficiently. Collectively, our results argue against a signaling activity of the intracellular domain of DLL1 in mouse embryos in vivo. AU - Redeker, C.* AU - Schuster-Gossler, K.* AU - Kremmer, E. AU - Gossler, A.* C1 - 28103 C2 - 32937 TI - Normal development in mice over-expressing the intracellular domain of DLL1 argues against reverse signaling by DLL1 in vivo. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Multiphoton microscopy (MPM) offers a unique approach for addressing both the function and structure of an organ in near-real time in the live animal. The method however is limited by the tissue-specific penetration depth of the excitation laser. In the kidney, structures in the range of 100 µm from the surface are accessible for MPM. This limitation of MPM aggravates the investigation of the function of structures located deeper in the renal cortex, like the glomerulus and the juxtaglomerular apparatus. In view of the relevance of gene-targeted mice for investigating the function of these structures, we aimed to identify a mouse strain with a high percentage of superficially located glomeruli. The mean distance of the 30 most superficial glomeruli from the kidney surface was determined in 10 commonly used mouse strains. The mean depth of glomeruli was 118.4±3.4, 123.0±2.7, 133.7±3.0, 132.3±2.6, 141.0±4.0, 145.3±4.3, 148.9±4.2, 151.6±2.7, 167.7±3.9, and 207.8±3.2 µm in kidney sections from 4-week-old C3H/HeN, BALB/cAnN, SJL/J, C57BL/6N, DBA/2N, CD1 (CRI), 129S2/SvPas, CB6F1, FVB/N and NMRI (Han) mice, respectively (n = 5 animals from each strain). The mean distance from the kidney surface of the most superficial glomeruli was significantly lower in the strains C3H/HeN Crl, BALB/cAnN, DBA/2NCrl, and C57BL/6N when compared to a peer group consisting of all the other strains (p<.0001). In 10-week-old mice, the most superficial glomeruli were located deeper in the cortex when compared to 4-week-old animals, with BALB/cAnN and C57BL/6N being the strains with the highest percentage of superficial glomeruli (25% percentile 116.7 and 121.9 µm, respectively). In summary, due to significantly more superficial glomeruli compared to other commonly used strains, BALB/cAnN and C57BL/6N mice appear to be particularly suitable for the investigation of glomerular function using MPM. AU - Schießl, I.M.* AU - Bardehle, S. AU - Castrop, H.* C1 - 26234 C2 - 32136 TI - Superficial nephrons in BALB/c and C57BL/6 mice facilitate in vivo multiphoton microscopy of the kidney. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Approximately 20% of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance. AU - Schulte, E.C. AU - Stahl, I. AU - Czamara, D.* AU - Ellwanger, D.C.* AU - Eck, S. AU - Graf, E. AU - Mollenhauer, B.* AU - * AU - Lichtner, P. AU - Haubenberger, D.* AU - Pirker, W.* AU - Brücke, T.* AU - Bereznai, B.* AU - Molnar, M.J.* AU - Peters, A. AU - Gieger, C. AU - Müller-Myhsok, B.* AU - Trenkwalder, C.* AU - Winkelmann, J. C1 - 28393 C2 - 33355 TI - Rare variants in PLXNA4 and Parkinson's disease. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Impulse oscillometry (IOS) is a non-demanding lung function test. Its diagnostic use may be particularly useful in patients of advanced age with physical or mental limitations unable to perform spirometry. Only few reference equations are available for Caucasians, none of them covering the old age. Here, we provide reference equations up to advanced age and compare them with currently available equations. METHODS: IOS was performed in a population-based sample of 1990 subjects, aged 45-91 years, from KORA cohorts (Augsburg, Germany). From those, 397 never-smoking, lung healthy subjects with normal spirometry were identified and sex-specific quantile regression models with age, height and body weight as predictors for respiratory system impedance, resistance, reactance, and other parameters of IOS applied. RESULTS: Women (n = 243) showed higher resistance values than men (n = 154), while reactance at low frequencies (up to 20 Hz) was lower (p<0.05). A significant age dependency was observed for the difference between resistance values at 5 Hz and 20 Hz (R5-R20), the integrated area of low-frequency reactance (AX), and resonant frequency (Fres) in both sexes whereas reactance at 5 Hz (X5) was age dependent only in females. In the healthy subjects (n = 397), mean differences between observed values and predictions for resistance (5 Hz and 20 Hz) and reactance (5 Hz) ranged between -1% and 5% when using the present model. In contrast, differences based on the currently applied equations (Vogel & Smidt 1994) ranged between -34% and 76%. Regarding our equations the indices were beyond the limits of normal in 8.1% to 18.6% of the entire KORA cohort (n = 1990), and in 0.7% to 9.4% with the currently applied equations. CONCLUSIONS: Our study provides up-to-date reference equations for IOS in Caucasians aged 45 to 85 years. We suggest the use of the present equations particularly in advanced age in order to detect airway dysfunction. AU - Schulz, H. AU - Flexeder, C. AU - Behr, J.* AU - Heier, M. AU - Holle, R. AU - Huber, R.M.* AU - Jörres, R.A.* AU - Nowak, D.* AU - Peters, A. AU - Wichmann, H.-E. AU - Heinrich, J. AU - Karrasch, S.* C1 - 24829 C2 - 31697 TI - Reference values of impulse oscillometric lung function indices in adults of advanced age. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted. AIM: We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes. METHODS: The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV). RESULTS: Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD. CONCLUSION: We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.   AU - Siegert, S.* AU - Yu, Z. AU - Wang-Sattler, R. AU - Illig, T. AU - Adamski, J. AU - Hampe, J.* AU - Nikolaus, S.* AU - Schreiber, S.* AU - Krawczak, M.* AU - Nothnagel, M.* AU - Nöthlings, U.* C1 - 27862 C2 - 32840 TI - Diagnosing fatty liver disease: A comparative evaluation of metabolic markers, phenotypes, genotypes and established biomarkers. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration. AU - Siegl, C.* AU - Hamminger, P.* AU - Jank, H.* AU - Ahting, U.* AU - Bader, B.* AU - Danek, A.* AU - Gregory, A.* AU - Hartig, M.* AU - Hayflick, S.* AU - Hermann, A.* AU - Prokisch, H. AU - Sammler, E.M.* AU - Yapici, Z.* AU - Prohaska, R.* AU - Salzer, U.* C1 - 27903 C2 - 32850 TI - Alterations of red cell membrane properties in neuroacanthocytosis. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family. AU - Silvar, C.* AU - Perovic, D.* AU - Nussbaumer, T. AU - Spannagl, M. AU - Usadel, B.* AU - Casas, A.* AU - Igartua, E.* AU - Ordon, F.* C1 - 26240 C2 - 32140 TI - Towards positional isolation of three quantitative trait loci conferring resistance to powdery mildew in two Spanish barley landraces. JO - PLoS ONE VL - 8 IS - 6 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Narrow arterioles in the retina have been shown to predict hypertension as well as other vascular diseases, likely through an increase in the peripheral resistance of the microcirculatory flow. In this study, we performed a genome-wide association study in 18,722 unrelated individuals of European ancestry from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the Blue Mountain Eye Study, to identify genetic determinants associated with variations in retinal arteriolar caliber. Retinal vascular calibers were measured on digitized retinal photographs using a standardized protocol. One variant (rs2194025 on chromosome 5q14 near the myocyte enhancer factor 2C MEF2C gene) was associated with retinal arteriolar caliber in the meta-analysis of the discovery cohorts at genome-wide significance of P-value <5×10(-8). This variant was replicated in an additional 3,939 individuals of European ancestry from the Australian Twins Study and Multi-Ethnic Study of Atherosclerosis (rs2194025, P-value = 2.11×10(-12) in combined meta-analysis of discovery and replication cohorts). In independent studies of modest sample sizes, no significant association was found between this variant and clinical outcomes including coronary artery disease, stroke, myocardial infarction or hypertension. In conclusion, we found one novel loci which underlie genetic variation in microvasculature which may be relevant to vascular disease. The relevance of these findings to clinical outcomes remains to be determined. AU - Sim, X.* AU - Jensen, R.A.* AU - Ikram, M.K.* AU - Cotch, M.F.* AU - Li, X.* AU - MacGregor, S.* AU - Xie, J.* AU - Smith, A.V.* AU - Boerwinkle, E.* AU - Mitchell, P.* AU - Klein, R.* AU - Klein, B.E.* AU - Glazer, N.L.* AU - Lumley, T.* AU - McKnight, B.* AU - Psaty, B.M.* AU - de Jong, P.T.* AU - Hofman, A.* AU - Rivadeneira, F.* AU - Uitterlinden, A.G.* AU - van Duijn, C.M.* AU - Aspelund, T.* AU - Eiriksdottir, G.* AU - Harris, T.B.* AU - Jonasson, F.* AU - Launer, L.J.* AU - Wellcome Trust Case Control Consortium 2 (WTCCC2) (*) AU - Attia, J.* AU - Baird, P.N.* AU - Harrap, S.* AU - Holliday, E.G.* AU - Inouye, M.* AU - Rochtchina, E.* AU - Scott, R.J.* AU - Viswanathan, A.* AU - Global BPgen Consortium (Eyheramendy, S. AU - Döring, A. AU - Gieger, C. AU - Illig, T. AU - Meitinger, T. AU - Pfeufer, A. AU - Wichmann, H.-E.) AU - Li, G.* AU - Smith, N.L.* AU - Wiggins, K.L.* AU - Kuo, J.Z.* AU - Taylor, K.D.* AU - Hewitt, A.W.* AU - Martin, N.G.* AU - Montgomery, G.W.* AU - Sun, C.* AU - Young, T.L.* AU - Mackey, D.A.* AU - van Zuydam, N.* AU - Doney, A.S.* AU - Palmer, C.N.* AU - Morris, A.D.* AU - Rotter, J.I.* AU - Tai, E.S.* AU - Gudnason, V.* AU - Vingerling, J.R.* AU - Siscovick, D.S.* AU - Wang, J.J.* AU - Wong, T.Y.* C1 - 28181 C2 - 32994 TI - Genetic loci for retinal arteriolar microcirculation. JO - PLoS ONE VL - 8 IS - 6 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The effects of systolic blood pressure (SBP), serum total cholesterol (TC), fasting plasma glucose (FPG), and body mass index (BMI) on the risk of cardiovascular diseases (CVD) have been established in epidemiological studies, but consistent estimates of effect sizes by age and sex are not available. METHODS: We reviewed large cohort pooling projects, evaluating effects of baseline or usual exposure to metabolic risks on ischemic heart disease (IHD), hypertensive heart disease (HHD), stroke, diabetes, and, as relevant selected other CVDs, after adjusting for important confounders. We pooled all data to estimate relative risks (RRs) for each risk factor and examined effect modification by age or other factors, using random effects models. RESULTS: Across all risk factors, an average of 123 cohorts provided data on 1.4 million individuals and 52,000 CVD events. Each metabolic risk factor was robustly related to CVD. At the baseline age of 55-64 years, the RR for 10 mmHg higher SBP was largest for HHD (2.16; 95% CI 2.09-2.24), followed by effects on both stroke subtypes (1.66; 1.39-1.98 for hemorrhagic stroke and 1.63; 1.57-1.69 for ischemic stroke). In the same age group, RRs for 1 mmol/L higher TC were 1.44 (1.29-1.61) for IHD and 1.20 (1.15-1.25) for ischemic stroke. The RRs for 5 kg/m(2) higher BMI for ages 55-64 ranged from 2.32 (2.04-2.63) for diabetes, to 1.44 (1.40-1.48) for IHD. For 1 mmol/L higher FPG, RRs in this age group were 1.18 (1.08-1.29) for IHD and 1.14 (1.01-1.29) for total stroke. For all risk factors, proportional effects declined with age, were generally consistent by sex, and differed by region in only a few age groups for certain risk factor-disease pairs. CONCLUSION: Our results provide robust, comparable and precise estimates of the effects of major metabolic risk factors on CVD and diabetes by age group.   AU - Singh, G.M.* AU - Danaei, G.* AU - Farzadfar, F.* AU - Stevens, G.A. AU - Woodward, M.* AU - Wormser, D.* AU - Kaptoge, S.* AU - Whitlock, G.* AU - Qiao, Q.* AU - Lewington, S.* AU - di Angelantonio, E.* AU - vander Hoorn, S.* AU - Lawes, C.M.* AU - Ali, M.K.* AU - Mozaffarian, D.* AU - Ezzati, M.* AU - Global Burden of Metabolic Risk Factors of Chronic Diseases Collaborating Group (*) AU - Asia-Pacific Cohort Studies Collaboration (APCSC) (*) AU - Diabetes Epidemiology Consortium (*) AU - Collaborative Analysis of Diagnostic Criteria in Europe Consortium (*) AU - Emerging Risk Factors Collaboration (Döring, A. AU - Meisinger, C.) AU - Prospective Studies Collaboration (*) C1 - 28121 C2 - 32954 TI - The age-specific quantitative effects of metabolic risk factors on cardiovascular diseases and diabetes: A pooled analysis. JO - PLoS ONE VL - 8 IS - 7 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Background: Circulating lipids levels, as well as several familial lipid metabolism disorders, are strongly associated with initiation and progression of atherosclerosis and incidence of myocardial infarction (MI). Objectives: We hypothesized that genetic variants associated with circulating lipid levels would also be associated with MI incidence, and have tested this in three independent samples. Setting and Subjects: Using age- and sex-adjusted additive genetic models, we analyzed 554 single nucleotide polymorphisms (SNPs) in 41 candidate gene regions proposed to be involved in lipid-related pathways potentially predisposing to incidence of MI in 2,602 participants of the Swedish Twin Register (STR; 57% women). All associations with nominal P<0.01 were further investigated in the Uppsala Longitudinal Study of Adult Men (ULSAM; N = 1,142). Results: In the present study, we report associations of lipid-related SNPs with incident MI in two community-based longitudinal studies with in silico replication in a meta-analysis of genome-wide association studies. Overall, there were 9 SNPs in STR with nominal P-value <0.01 that were successfully genotyped in ULSAM. rs4149313 located in ABCA1 was associated with MI incidence in both longitudinal study samples with nominal significance (hazard ratio, 1.36 and 1.40; P-value, 0.004 and 0.015 in STR and ULSAM, respectively). In silico replication supported the association of rs4149313 with coronary artery disease in an independent meta-analysis including 173,975 individuals of European descent from the CARDIoGRAMplusC4D consortium (odds ratio, 1.03; P-value, 0.048). Conclusions: rs4149313 is one of the few amino acid changing variants in ABCA1 known to associate with reduced cholesterol efflux. Our results are suggestive of a weak association between this variant and the development of atherosclerosis and MI. AU - Song, C.* AU - Pedersen, N.L.* AU - Reynolds, C.A.* AU - Sabater-Lleal, M.* AU - Kanoni, S.* AU - Willenborg, C.* AU - CARDIoGRAMplusC4D Consortium (Gieger, C. AU - Meitinger, T. AU - Peters, A.) AU - Syvanen, A.C.* AU - Watkins, H.* AU - Hamsten, A.* AU - Prince, J.A.* AU - Ingelsson, E.* C1 - 46919 C2 - 39074 TI - Genetic variants from lipid-related pathways and risk for incident myocardial infarction. JO - PLoS ONE VL - 8 IS - 3 PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Aims/hypothesis Recently, the novel myokine irisin was described to drive adipose tissue ‘browning’, to increase energy expenditure, and to improve obesity and insulin resistance in high fat-fed mice. Here, we assessed whether common single nucleotide polymorphisms (SNPs) in the FNDC5 locus, encoding the irisin precursor, contribute to human prediabetic phenotypes (overweight, glucose intolerance, insulin resistance, impaired insulin release). Methods A population of 1,976 individuals was characterized by oral glucose tolerance tests and genotyped for FNDC5 tagging SNPs. Subgroups underwent hyperinsulinaemic-euglycaemic clamps, magnetic resonance imaging/spectroscopy, and intravenous glucose tolerance tests. From 37 young and 14 elderly participants recruited in two different centres, muscle biopsies were obtained for the preparation of human myotube cultures. Results After appropriate adjustment and Bonferroni correction for the number of tested variants, SNPs rs16835198 and rs726344 were associated with in vivo measures of insulin sensitivity. Via interrogation of publicly available data from the Meta-Analyses of Glucose and Insulin-related traits Consortium, rs726344’s effect on insulin sensitivity was replicated. Moreover, novel data from human myotubes revealed a negative association between FNDC5 expression and appropriately adjusted in vivo measures of insulin sensitivity in young donors. This finding was replicated in myotubes from elderly men. AU - Staiger, H. AU - Böhm, A. AU - Scheler, M. AU - Berti, L. AU - Machann, J. AU - Schick, F. AU - Machicao, F. AU - Fritsche, A. AU - Stefan, N. AU - Weigert, C. AU - Krook, A.* AU - Häring, H.-U. AU - Hrabě de Angelis, M. C1 - 24268 C2 - 31491 TI - Common genetic variation in the human FNDC5 locus, encoding the novel muscle-derived 'browning’ factor Irisin, determines insulin sensitivity. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The abstraction of groundwater is a global phenomenon that directly threatens groundwater ecosystems. Despite the global significance of this issue, the impact of groundwater abstraction and the lowering of groundwater tables on biota is poorly known. The aim of this study is to determine the impacts of groundwater drawdown in unconfined aquifers on the distribution of fauna close to the water table, and the tolerance of groundwater fauna to sediment drying once water levels have declined. A series of column experiments were conducted to investigate the depth distribution of different stygofauna (Syncarida and Copepoda) under saturated conditions and after fast and slow water table declines. Further, the survival of stygofauna under conditions of reduced sediment water content was tested. The distribution and response of stygofauna to water drawdown was taxon specific, but with the common response of some fauna being stranded by water level decline. So too, the survival of stygofauna under different levels of sediment saturation was variable. Syncarida were better able to tolerate drying conditions than the Copepoda, but mortality of all groups increased with decreasing sediment water content. The results of this work provide new understanding of the response of fauna to water table drawdown. Such improved understanding is necessary for sustainable use of groundwater, and allows for targeted strategies to better manage groundwater abstraction and maintain groundwater biodiversity. AU - Stumpp, C. AU - Hose, G.C.* C1 - 28436 C2 - 33376 TI - The impact of water table drawdown and drying on subterranean aquatic fauna in in-vitro experiments. JO - PLoS ONE VL - 8 IS - 11 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - To explore the potential of grating-based x-ray phase-contrast computed tomography (CT) for preclinical research, a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) was investigated. One ex-vivo mouse specimen was scanned with different grating-based phase-contrast CT imaging setups covering two different settings: i) high-resolution synchrotron radiation (SR) imaging and ii) dose-reduced imaging using either synchrotron radiation or a conventional x-ray tube source. These experimental settings were chosen to assess the potential of phase-contrast imaging for two different types of application: i) high-performance imaging for virtual microscopy applications and ii) biomedical imaging with increased soft-tissue contrast for in-vivo applications. For validation and as a reference, histological slicing and magnetic resonance imaging (MRI) were performed on the same mouse specimen. For each x-ray imaging setup, attenuation and phase-contrast images were compared visually with regard to contrast in general, and specifically concerning the recognizability of lesions and cancerous tissue. To quantitatively assess contrast, the contrast-to-noise ratios (CNR) of selected regions of interest (ROI) in the attenuation images and the phase images were analyzed and compared. It was found that both for virtual microscopy and for in-vivo applications, there is great potential for phase-contrast imaging: in the SR-based benchmarking data, fine details about tissue composition are accessible in the phase images and the visibility of solid tumor tissue under dose-reduced conditions is markedly superior in the phase images. The present study hence demonstrates improved diagnostic value with phase-contrast CT in a mouse model of a complex endogenous cancer, promoting the use and further development of grating-based phase-contrast CT for biomedical imaging applications. AU - Tapfer, A.* AU - Braren, R.* AU - Bech, M.* AU - Willner, M.* AU - Zanette, I.* AU - Weitkamp, T.* AU - Trajkovic-Arsic, M.* AU - Siveke, J.T.* AU - Settles, M.* AU - Aichler, M. AU - Walch, A.K. AU - Pfeiffer, F.* C1 - 24109 C2 - 31328 TI - X-ray phase-contrast CT of a pancreatic ductal adenocarcinoma mouse model. JO - PLoS ONE VL - 8 IS - 3 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Aims/Hypothesis Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene have been shown to display a powerful association with type 2 diabetes. The aim of the present study was to evaluate metabolic alterations in carriers of a common TCF7L2 risk variant. Methods Seventeen non-diabetic subjects carrying the T risk allele at the rs7903146 TCF7L2 locus and 24 subjects carrying no risk allele were submitted to intravenous glucose tolerance test and euglycemic-hyperinsulinemic clamp. Plasma samples were analysed for concentrations of 163 metabolites through targeted mass spectrometry. Results TCF7L2 risk allele carriers had a reduced first-phase insulin response and normal insulin sensitivity. Under fasting conditions, carriers of TCF7L2 rs7903146 exhibited a non-significant increase of plasma sphingomyelins (SMs), phosphatidylcholines (PCs) and lysophosphatidylcholines (lysoPCs) species. A significant genotype effect was detected in response to challenge tests in 6 SMs (C16:0, C16:1, C18:0, C18:1, C24:0, C24:1), 5 hydroxy-SMs (C14:1, C16:1, C22:1, C22:2, C24:1), 4 lysoPCs (C14:0, C16:0, C16:1, C17:0), 3 diacyl-PCs (C28:1, C36:6, C40:4) and 4 long-chain acyl-alkyl-PCs (C40:2, C40:5, C44:5, C44:6). AU - Then, C. AU - Wahl, S. AU - Kirchhofer, A.* AU - Grallert, H. AU - Krug, S. AU - Kastenmüller, G. AU - Römisch-Margl, W. AU - Claussnitzer, M. AU - Illig, T.* AU - Heier, M. AU - Meisinger, C. AU - Adamski, J. AU - Thorand, B. AU - Huth, C. AU - Peters, A. AU - Prehn, C. AU - Heukamp, I.* AU - Laumen, H. AU - Lechner, A. AU - Hauner, H. AU - Seissler, J. C1 - 28082 C2 - 32920 TI - Plasma metabolomics reveal alterations of sphingo- and glycerophospholipid levels in non-diabetic carriers of the transcription factor 7-like 2 polymorphism rs7903146. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Stress, the physiological reaction to a stressor, is initiated in teleost fish by hormone cascades along the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol is the major stress hormone and contributes to the appropriate stress response by regulating gene expression after binding to the glucocorticoid receptor. Cortisol is inactivated when 11β-hydroxysteroid dehydrogenase (HSD) type 2 catalyzes its oxidation to cortisone. In zebrafish, Danio rerio, cortisone can be further reduced to 20β-hydroxycortisone. This reaction is catalyzed by 20β-HSD type 2, recently discovered by us. Here, we substantiate the hypothesis that 20β-HSD type 2 is involved in cortisol catabolism and stress response. We found that hsd11b2 and hsd20b2 transcripts were up-regulated upon cortisol treatment. Moreover, a cortisol-independent, short-term physical stressor led to the up-regulation of hsd11b2 and hsd20b2 along with several HPI axis genes. The morpholino-induced knock down of hsd20b2 in zebrafish embryos revealed no developmental phenotype under normal culture conditions, but prominent effects were observed after a cortisol challenge. Reporter gene experiments demonstrated that 20β-hydroxycortisone was not a physiological ligand for the zebrafish glucocorticoid or mineralocorticoid receptor but was excreted into the fish holding water. Our experiments show that 20β-HSD type 2, together with 11β-HSD type 2, represents a short pathway in zebrafish to rapidly inactivate and excrete cortisol. Therefore, 20β-HSD type 2 is an important enzyme in stress response. AU - Tokarz, J. AU - Norton, W.H.J.* AU - Möller, G. AU - Hrabě de Angelis, M. AU - Adamski, J. C1 - 11913 C2 - 30848 TI - Zebrafish 20β-hydroxysteroid dehydrogenase type 2 is important for glucocorticoid catabolism in stress response. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Association studies have identified several signals at the LRRK2 locus for Parkinson's disease (PD), Crohn's disease (CD) and leprosy. However, little is known about the molecular mechanisms mediating these effects. To further characterize this locus, we fine-mapped the risk association in 5,802 PD and 5,556 controls using a dense genotyping array (ImmunoChip). Using samples from 134 post-mortem control adult human brains (UK Human Brain Expression Consortium), where up to ten brain regions were available per individual, we studied the regional variation, splicing and regulation of LRRK2. We found convincing evidence for a common variant PD association located outside of the LRRK2 protein coding region (rs117762348, A>G, P = 2.56×10(-8), case/control MAF 0.083/0.074, odds ratio 0.86 for the minor allele with 95% confidence interval [0.80-0.91]). We show that mRNA expression levels are highest in cortical regions and lowest in cerebellum. We find an exon quantitative trait locus (QTL) in brain samples that localizes to exons 32-33 and investigate the molecular basis of this eQTL using RNA-Seq data in n = 8 brain samples. The genotype underlying this eQTL is in strong linkage disequilibrium with the CD associated non-synonymous SNP rs3761863 (M2397T). We found two additional QTLs in liver and monocyte samples but none of these explained the common variant PD association at rs117762348. Our results characterize the LRRK2 locus, and highlight the importance and difficulties of fine-mapping and integration of multiple datasets to delineate pathogenic variants and thus develop an understanding of disease mechanisms. AU - Trabzuni, D.* AU - Ryten, M.* AU - Emmett, W.* AU - Ramasamy, A.* AU - Lackner, K.J.* AU - Zeller, T.* AU - Walker, R.* AU - Smith, C.* AU - Lewis, P.A.* AU - Mamais, A.* AU - de Silva, R.* AU - Vandrovcova, J.* AU - International Parkinson's Disease Genomics Consortium (IPDGC) (Illig, T. AU - Lichtner, P.) AU - Hernandez, D.* AU - Nalls, M.A.* AU - Sharma, M.* AU - Garnier, S.* AU - Lesage, S.* AU - Simon-Sanchez, J.* AU - Gasser, T.* AU - Heutink, P.* AU - Brice, A.* AU - Singleton, A.* AU - Cai, H.* AU - Schadt, E.* AU - Wood, N.W.* AU - Bandopadhyay, R.* AU - Weale, M.E.* AU - Hardy, J.* AU - Plagnol, V.* C1 - 28179 C2 - 32992 TI - Fine-mapping, gene expression and splicing analysis of the disease associated LRRK2 locus. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Genome-wide association studies (GWAS) comprise a powerful tool for mapping genes of complex traits. However, an inflation of the test statistic can occur because of population substructure or cryptic relatedness, which could cause spurious associations. If information on a large number of genetic markers is available, adjusting the analysis results by using the method of genomic control (GC) is possible. GC was originally proposed to correct the Cochran-Armitage additive trend test. For non-additive models, correction has been shown to depend on allele frequencies. Therefore, usage of GC is limited to situations where allele frequencies of null markers and candidate markers are matched. In this work, we extended the capabilities of the GC method for non-additive models, which allows us to use null markers with arbitrary allele frequencies for GC. Analytical expressions for the inflation of a test statistic describing its dependency on allele frequency and several population parameters were obtained for recessive, dominant, and over-dominant models of inheritance. We proposed a method to estimate these required population parameters. Furthermore, we suggested a GC method based on approximation of the correction coefficient by a polynomial of allele frequency and described procedures to correct the genotypic (two degrees of freedom) test for cases when the model of inheritance is unknown. Statistical properties of the described methods were investigated using simulated and real data. We demonstrated that all considered methods were effective in controlling type 1 error in the presence of genetic substructure. The proposed GC methods can be applied to statistical tests for GWAS with various models of inheritance. All methods developed and tested in this work were implemented using R language as a part of the GenABEL package. AU - Tsepilov, Y.A.* AU - Ried, J.S. AU - Strauch, K. AU - Grallert, H. AU - van Duijn, C.M.* AU - Axenovich, T.I.* AU - Aulchenko, Y.S.* C1 - 28986 C2 - 33600 TI - Development and application of genomic control methods for genome-wide association studies using non-additive models. JO - PLoS ONE VL - 8 IS - 12 PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Economic variables such as income, education, and occupation are known to affect mortality and morbidity, such as cardiovascular disease, and have also been shown to be partly heritable. However, very little is known about which genes influence economic variables, although these genes may have both a direct and an indirect effect on health. We report results from the first large-scale collaboration that studies the molecular genetic architecture of an economic variable-entrepreneurship-that was operationalized using self-employment, a widely-available proxy. Our results suggest that common SNPs when considered jointly explain about half of the narrow-sense heritability of self-employment estimated in twin data (σg (2)/σP (2) = 25%, h (2) = 55%). However, a meta-analysis of genome-wide association studies across sixteen studies comprising 50,627 participants did not identify genome-wide significant SNPs. 58 SNPs with p<10(-5) were tested in a replication sample (n = 3,271), but none replicated. Furthermore, a gene-based test shows that none of the genes that were previously suggested in the literature to influence entrepreneurship reveal significant associations. Finally, SNP-based genetic scores that use results from the meta-analysis capture less than 0.2% of the variance in self-employment in an independent sample (p≥0.039). Our results are consistent with a highly polygenic molecular genetic architecture of self-employment, with many genetic variants of small effect. Although self-employment is a multi-faceted, heavily environmentally influenced, and biologically distal trait, our results are similar to those for other genetically complex and biologically more proximate outcomes, such as height, intelligence, personality, and several diseases. AU - van der Loos, M.J.* AU - Rietveld, C.A.* AU - Eklund, N.* AU - Koellinger, P.D.* AU - Rivadeneira, F.* AU - Abecasis, G.R.* AU - Ankra-Badu, G.A.* AU - Baumeister, S.E.* AU - Benjamin, D.J.* AU - Biffar, R.* AU - Blankenberg, S.* AU - Boomsma, D.I.* AU - Cesarini, D.* AU - Cucca, F.* AU - de Geus, E.J.* AU - Dedoussis, G.* AU - Deloukas, P.* AU - Dimitriou, M.* AU - Eiriksdottir, G.* AU - Eriksson, J.* AU - Gieger, C. AU - Gudnason, V.* AU - Höhne, B. AU - Holle, R. AU - Hottenga, J.J.* AU - Isaacs, A.* AU - Jarvelin, M.R.* AU - Johannesson, M.* AU - Kaakinen, M.* AU - Kähönen, M.* AU - Kanoni, S.* AU - Laaksonen, M.A.* AU - Lahti, J.* AU - Launer, L.J.* AU - Lehtimäki, T.* AU - Loitfelder, M.* AU - Magnusson, P.K.* AU - Naitza, S.* AU - Oostra, B.A.* AU - Perola, M.* AU - Petrovic, K.* AU - Quaye, L.* AU - Raitakari, O.* AU - Ripatti, S.* AU - Scheet, P.* AU - Schlessinger, D.* AU - Schmidt, C.O.* AU - Schmidt, H.* AU - Schmidt, R.* AU - Senft, A.* AU - Smith, A.V.* AU - Spector, T.D.* AU - Surakka, I.* AU - Svento, R.* AU - Terracciano, A.* AU - Tikkanen, E.* AU - van Duijn, C.M.* AU - Viikari, J.* AU - Völzke, H.* AU - Wichmann, H.-E. AU - Wild, P.S.* AU - Willems, S.M.* AU - Willemsen, G.* AU - van Rooij, F.J.* AU - Groenen, P.J.* AU - Uitterlinden, A.G.* AU - Hofman, A.* AU - Thurik, A.R.* C1 - 24224 C2 - 31344 TI - The molecular genetic architecture of self-employment. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Background: Evidence for an association between calcium intake and risk of cardiovascular death remains controversial. By assessing dietary intake, use of supplements, and serum levels of calcium, we aimed to disentangle this link in the third National Health and Nutrition Examination Survey (NHANES III). Methods: Mortality linkage of NHANES III to death certificate data for those aged 17 years or older (n = 20,024) was used to estimate risk of overall cardiovascular death as well as death from ischemic heart disease (IHD), acute myocardial infarction (AMI), heart failure (HF), and cerebrovascular disease (CD) with multivariate Cox proportional hazards regression analysis. Results: About 10.0% of the population died of cardiovascular disease and the majority (5.4%) died of IHD. There was increased risk of overall CVD death for those in the bottom 5% of serum calcium compared to those in the mid 90% (HR: 1.51 (95% CI: 1.03-2.22)). For women there was a statistically significant increased risk of IHD death for those with serum calcium levels in the top 5% compared to those in the mid 90% (HR: 1.72 (95% CI: 1.13-2.61)), whereas in men, low serum calcium was related to increased IHD mortality (HR: 2.32 (95% CI 1.14-3.01), P-interaction: 0.306). No clear association with CVD death was observed for dietary or supplemental calcium intake. Conclusions: Calcium as assessed by serum concentrations is involved in cardiovascular health, though differential effects by sex may exist. No clear evidence was found for an association between dietary or supplementary intake of calcium and cardiovascular death. AU - van Hemelrijck, M.* AU - Michaelsson, K.* AU - Linseisen, J. AU - Rohrmann, S.* C1 - 24449 C2 - 31556 TI - Calcium intake and serum concentration in relation to risk of cardiovascular death in NHANES III. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states. AU - Velvarska, H. AU - Niessing, D. C1 - 28766 C2 - 33544 TI - Structural insights into the globular tails of the human type V myosins Myo5a, Myo5b, and Myo5c. JO - PLoS ONE VL - 8 IS - 12 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Background: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes. AU - Venit, T.* AU - Dzijak, R.* AU - Kalendova, A.* AU - Kahle-Stephan, M.* AU - Rohozkova, J.* AU - Schmidt, V.* AU - Rülicke, T.* AU - Rathkolb, B. AU - Hans, W. AU - Bohla, A. AU - Eickelberg, O. AU - Stöger, T. AU - Wolf, E.* AU - Yildirim, A.Ö. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Hozak, P.* C1 - 24019 C2 - 31308 TI - Mouse nuclear myosin 1 knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus. JO - PLoS ONE VL - 8 IS - 4 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: The diabetes mellitus Incidence Cohort Registry (DiMelli) aims to characterize diabetes phenotypes by immunologic, metabolic, and genetic markers. We classified patients into three groups according to islet autoantibody status and examined whether patients with multiple diabetes-associated autoantibodies, one autoantibody, or without autoantibodies differed with respect to clinical, metabolic, and genetic parameters, including an insulin sensitivity (IS) score based on waist, HbA1c, and triglycerides. We also assessed whether metabolic markers predicted the immune status. MATERIALS AND METHODS: As of June 2012, 630 patients in Bavaria, Germany, aged <20 years diagnosed with any type of diabetes within the preceding 6 months were registered in DiMelli. We compared the clinical and laboratory parameters between islet autoantibody status defined patient groups. Parameters showing the strongest associations were included in principal component analysis. Receiver operating characteristic curves were used to assess the ability of the IS Score to predict islet autoantibody status. RESULTS: Patients with multiple islet autoantibodies, one autoantibody, or without autoantibodies were significantly different in terms of BMI percentile, weight loss before diagnosis, fasting C-peptide (all, P<0.001), and IS Score (P=0.034). However, principal component analysis revealed no distinct patterns according to autoantibody status. At the optimal IS Score cut-off for predicting islet autoantibody positivity (single compared to none), the specificity was 52.0% and the sensitivity was 86.8%. With respect to prediction of multiple autoantibodies (compared to none), specificity and sensitivity were slightly lower and in combination inferior to those obtained using the BMI percentile and fasting C-peptide. DISCUSSION: The DiMelli study indicated that patients with and without islet autoantibodies differed with respect to metabolic and genetic markers but there was considerable overlap of phenotypes, and autoantibody status could not be predicted by these parameters. Thus, our results suggest that refined diabetes classification may require both immune and metabolic phenotyping. AU - Warncke, K. AU - Krasmann, M. AU - Puff, R. AU - Dunstheimer, D.* AU - Ziegler, A.-G. AU - Beyerlein, A. C1 - 27263 C2 - 32590 TI - Does diabetes appear in distinct phenotypes in young people? Results of the diabetes mellitus Incidence Cohort Registry (DiMelli). JO - PLoS ONE VL - 8 IS - 9 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of alpha-, beta- and delta-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and beta-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6(+/-) lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6(-/-) lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues. AU - Xie, Q.* AU - Yang, Y.* AU - Huang, J.* AU - Ninkovic, J. AU - Walcher, T. AU - Wolf, L.* AU - Vitenzon, A.* AU - Zheng, D.Y.* AU - Götz, M. AU - Beebe, D.C.* AU - Zavadil, J.* AU - Cvekl, A.* C1 - 23153 C2 - 31009 TI - Pax6 Interactions with chromatin and identification of its novel direct target genes in lens and forebrain. JO - PLoS ONE VL - 8 IS - 1 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation. AU - Yentrapalli, R. AU - Azimzadeh, O. AU - Sriharshan, A. AU - Malinowsky, K.* AU - Merl, J. AU - Wojcik, A.* AU - Harms-Ringdahl, M.* AU - Atkinson, M.J. AU - Becker, K.-F.* AU - Haghdoost, S.* AU - Tapio, S. C1 - 26603 C2 - 32297 TI - The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Niemann-Pick type C (NPC) disease is a rare autosomal-recessively inherited lysosomal storage disorder caused by mutations in NPC1 (95%) or NPC2. Given the highly variable phenotype, diagnosis is challenging and particularly late-onset forms with predominantly neuropsychiatric presentations are likely underdiagnosed. Pathophysiologically, genetic alterations compromising the endosomal/lysosomal system are linked with age-related neurodegenerative disorders. We sought to examine a possible association of rare sequence variants in NPC1 and NPC2 with Parkinson's disease (PD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), and to genetically determine the proportion of potentially misdiagnosed NPC patients in these neurodegenerative conditions. By means of high-resolution melting, we screened the coding regions of NPC1 and NPC2 for rare genetic variation in a homogenous German sample of patients clinically diagnosed with PD (n = 563), FTLD (n = 133) and PSP (n = 94), and 846 population-based controls. The frequencies of rare sequence variants in NPC1/2 did not differ significantly between patients and controls. Disease-associated NPC1/2 mutations were found in six PD patients (1.1%) and seven control subjects (0.8%), but not in FTLD or PSP. All rare variation was detected in the heterozygous state and no compound heterozygotes were observed. Our data do not support the hypothesis that rare NPC1/2 variants confer susceptibility for PD, FTLD, or PSP in the German population. Misdiagnosed NPC patients were not present in our samples. However, further assessment of NPC disease genes in age-related neurodegeneration is warranted. AU - Zech, M. AU - Nübling, G.* AU - Castrop, F.* AU - Jochim, A.* AU - Schulte, E.C. AU - Mollenhauer, B.* AU - Lichtner, P. AU - Peters, A. AU - Gieger, C. AU - Marquardt, T.* AU - Vanier, M.T.* AU - Latour, P.* AU - Klünemann, H.H.* AU - Trenkwalder, C.* AU - Diehl-Schmid, J.* AU - Perneczky, R.* AU - Meitinger, T. AU - Oexle, K. AU - Haslinger, B.* AU - Lorenzl, S.* AU - Winkelmann, J. C1 - 29161 C2 - 31508 TI - Niemann-pick C disease gene mutations and age-related neurodegenerative disorders. JO - PLoS ONE VL - 8 IS - 12 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs) comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182) as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (-24.40% methylation; p = 2.54E-182), suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers. AU - Zeilinger, S. AU - Kühnel, B. AU - Klopp, N. AU - Baurecht, H.* AU - Kleinschmidt, A. AU - Gieger, C. AU - Weidinger, S.* AU - Lattka, E. AU - Adamski, J. AU - Peters, A. AU - Strauch, K. AU - Waldenberger, M. AU - Illig, T. C1 - 24549 C2 - 31569 TI - Tobacco smoking leads to extensive genome-wide changes in DNA methylation. JO - PLoS ONE VL - 8 IS - 5 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 mg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 mg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of "healthy" food, these peptides might serve as templates for novel antibacterial and antifungal agents. AU - Zeitler, B. AU - Diaz, A.H. AU - Dangel, A. AU - Thellmann, M. AU - Meyer, H. AU - Sattler, M. AU - Lindermayr, C. C1 - 27251 C2 - 32587 TI - De-novo design of antimicrobial peptides for plant protection. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Multiple co-occurring environmental changes are affecting soil nitrogen cycling processes, which are mainly mediated by microbes. While it is likely that various nitrogen-cycling functional groups will respond differently to such environmental changes, very little is known about their relative responsiveness. Here we conducted four long-term experiments in a steppe ecosystem by removing plant functional groups, mowing, adding nitrogen, adding phosphorus, watering, warming, and manipulating some of their combinations. We quantified the abundance of seven nitrogen-cycling genes, including those for fixation (nifH), mineralization (chiA), nitrification (amoA of ammonia-oxidizing bacteria (AOB) or archaea (AOA)), and denitrification (nirS, nirK and nosZ). First, for each gene, we compared its sensitivities to different environmental changes and found that the abundances of various genes were sensitive to distinct and different factors. Overall, the abundances of nearly all genes were sensitive to nitrogen enrichment. In addition, the abundances of the chiA and nosZ genes were sensitive to plant functional group removal, the AOB-amoA gene abundance to phosphorus enrichment when nitrogen was added simultaneously, and the nirS and nirK gene abundances responded to watering. Second, for each single- or multi-factorial environmental change, we compared the sensitivities of the abundances of different genes and found that different environmental changes primarily affected different gene abundances. Overall, AOB-amoA gene abundance was most responsive, followed by the two denitrifying genes nosZ and nirS, while the other genes were less sensitive. These results provide, for the first time, systematic insights into how the abundance of each type of nitrogen-cycling gene and the equilibrium state of all these nitrogen-cycling gene abundances would shift under each single- or multi-factorial global change. AU - Zhang, X.* AU - Liu, W.* AU - Schloter, M. AU - Zhang, G.* AU - Chen, Q.* AU - Huang, J.* AU - Li, L.* AU - Elser, J.J.* AU - Han, X.* C1 - 27917 C2 - 32857 TI - Response of the abundance of key soil microbial nitrogen-cycling genes to multi-factorial global changes. JO - PLoS ONE VL - 8 IS - 10 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Many protein-protein docking protocols are based on a shotgun approach, in which thousands of independent random-start trajectories minimize the rigid-body degrees of freedom. Another strategy is enumerative sampling as used in ZDOCK. Here, we introduce an alternative strategy, ReplicaDock, using a small number of long trajectories of temperature replica exchange. We compare replica exchange sampling as low-resolution stage of RosettaDock with RosettaDock's original shotgun sampling as well as with ZDOCK. A benchmark of 30 complexes starting from structures of the unbound binding partners shows improved performance for ReplicaDock and ZDOCK when compared to shotgun sampling at equal or less computational expense. ReplicaDock and ZDOCK consistently reach lower energies and generate significantly more near-native conformations than shotgun sampling. Accordingly, they both improve typical metrics of prediction quality of complex structures after refinement. Additionally, the refined ReplicaDock ensembles reach significantly lower interface energies and many previously hidden features of the docking energy landscape become visible when ReplicaDock is applied. AU - Zhang, Z.* AU - Lange, O.F. C1 - 27471 C2 - 32686 TI - Replica exchange improves sampling in low-resolution docking stage of RosettaDock. JO - PLoS ONE VL - 8 IS - 8 PB - Public Library of Science PY - 2013 SN - 1932-6203 ER - TY - JOUR AB - Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and -independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis. AU - Anderson, A.C.* AU - Hellwig, E.* AU - Vespermann, R.* AU - Wittmer, A.* AU - Schmid, M. AU - Karygianni, L.* AU - Al-Ahmad, A.* C1 - 11090 C2 - 30505 TI - Comprehensive analysis of secondary dental root canal infections: A combination of culture and culture-independent approaches reveals new insights. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Genome-wide association studies (GWAS) have become an effective tool to map genes and regions contributing to multifactorial human diseases and traits. A comparably small number of variants identified by GWAS are known to have a direct effect on protein structure whereas the majority of variants is thought to exert their moderate influences on the phenotype through regulatory changes in mRNA expression. MicroRNAs (miRNAs) have been identified as powerful posttranscriptional regulators of mRNAs. Binding to their target sites, which are mostly located within the 3'-untranslated region (3'-UTR) of mRNA transcripts, they modulate mRNA expression and stability. Until today almost all human mRNA transcripts are known to harbor at least one miRNA target site with an average of over 20 miRNA target sites per transcript. Among 5,101 GWAS-identified sentinel single nucleotide polymorphisms (SNPs) that correspond to 18,884 SNPs in linkage disequilibrium (LD) with the sentinels (r(2) >= 0.8) we identified a significant overrepresentation of SNPs that affect the 3'-UTR of genes (OR = 2.33, 95% CI = 2.12-2.57, P AU - Arnold, M. AU - Ellwanger, D.C. AU - Hartsperger, M.L. AU - Pfeufer, A. AU - Stuempflen, V. C1 - 7685 C2 - 29891 TI - Cis-acting polymorphisms affect complex traits through modifications of microRNA regulation pathways. JO - PLoS ONE VL - 7 IS - 5 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H2O2 release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H2O2 release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00x10(-)8) and 54 suggestive associations (p<1.00x10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and similar to 2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H2O2 release was observed in Down Syndrome (DS) individuals (p<2.88x10-12). Taken together, our results show strong evidence of genetic control of H2O2 in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders. AU - Attar, H.* AU - Bedard, K.* AU - Migliavacca, E.* AU - Gagnebin, M.* AU - Dupré, Y.* AU - Descombes, P.* AU - Borel, C.* AU - Deutsch, S.* AU - Prokisch, H. AU - Meitinger, T. AU - Mehta, D. AU - Wichmann, H.-E. AU - Delabar, J.M.* AU - Dermitzakis, E.T.* AU - Krause, K.-H.* AU - Antonarakis, S.E.* C1 - 10411 C2 - 30255 TI - Extensive natural variation for cellular hydrogen peroxide release is genetically controlled. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The neuromuscular junctions are the specialized synapses whereby spinal motor neurons control the contraction of skeletal muscles. The formation of the neuromuscular junctions is controlled by a complex interplay of multiple mechanisms coordinately activated in motor nerve terminals and in their target myotubes. However, the transcriptional regulators that control in motor neurons the genetic programs involved in neuromuscular junction development remain unknown. Here, we provide evidence that the Onecut transcription factor HNF-6 regulates in motor neurons the formation of the neuromuscular junctions. Indeed, adult Hnf6 mutant mice exhibit hindlimb muscle weakness and abnormal locomotion. This results from defects of hindlimb neuromuscular junctions characterized by an abnormal morphology and defective localization of the synaptic vesicle protein synaptophysin at the motor nerve terminals. These defects are consequences of altered and delayed formation of the neuromuscular junctions in newborn mutant animals. Furthermore, we show that the expression level of numerous regulators of neuromuscular junction formation, namely agrin, neuregulin-2 and TGF-ß receptor II, is downregulated in the spinal motor neurons of Hnf6 mutant newborn animals. Finally, altered formation of neuromuscular junction-like structures in a co-culture model of wildtype myotubes with mutant embryonic spinal cord slices is rescued by recombinant agrin and neuregulin, indicating that depletion in these factors contributes to defective neuromuscular junction development in the absence of HNF-6. Thus, HNF-6 controls in spinal motor neurons a genetic program that coordinates the formation of hindlimb neuromuscular junctions. AU - Audouard, E.* AU - Schakman, O.* AU - René, F.* AU - Hüttl, R.E. AU - Huber, A.B. AU - Loeffler, J.-P.* AU - Gailly, P.* AU - Clotman, F.* C1 - 11328 C2 - 30614 TI - The onecut transcription factor HNF-6 regulates in motor neurons the formation of the neuromuscular junctions. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), beta-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p < 0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pretherapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells. AU - Bauer, L.* AU - Langer, R.* AU - Becker, K.* AU - Hapfelmeier, A.* AU - Ott, K.* AU - Novotny, A.* AU - Höfler, H. AU - Keller, G.* C1 - 10443 C2 - 30248 TI - Expression profiling of stem cell-related genes in neoadjuvant-treated gastric cancer: A NOTCH2, GSK3B and β-catenin gene signature predicts survival. JO - PLoS ONE VL - 7 IS - 9 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Low-carbohydrate, high-fat (LC-HF) diets are popular for inducing weight loss in overweighed adults. Adaptive thermogenesis increased by specific effects of macronutrients on energy expenditure has been postulated to induce this weight loss. We studied brown adipose tissue (BAT) morphology and function following exposure to different LC-HF diets. Methods: Male Wistar rats were fed a standard control diet ad libitum or pair-fed isoenergetic amounts of three experimental diets for 4 weeks. The diets had the following macronutrient composition (% metabolizable energy: carbohydrates, fat, protein): control (64.3/16.7/19), LC-HF-low protein (LC-HF-LP, 1.7/92.8/5.5), LC-HF-normal-protein (LC-HF-NP, 2.2/78.7/19.1), and a high fat diet with carbohydrates ("high fat", 19.4/61.9/18.7). Results: Body weight gain was reduced in all pair-fed experimental groups as compared to rats fed the control diet, with more pronounced effect in rats on LC-HF diets than on the high fat diet with carbohydrates. High fat diets increased expression of PGC1 alpha and ADRB3 in BAT indicating higher SNS outflow. However, UCP1 mRNA expression and expression of UCP1 assessed by immunohistochemistry was not different between diet groups. In accordance, analysis of mitochondrial function in-vitro by extracellular flux analyser (Seahorse Bioscience) and measurement of inducible thermogenesis in vivo (primary endpoint), explored by indirect calorimetry following norepinephrine injection, did not show significant differences between groups. Histology of BAT revealed increased lipid droplet size in rats fed the high-fat diet and both LC-HF diets. Conclusion: All experimental diets upregulated expression of genes which are indicative for increased BAT activity. However, the functional measurements in vivo revealed no increase of inducible BAT thermogenesis. This indicates that lower body weight gain with LC-HF diets and a high fat diet in a pair-feeding setting is not caused by increased adaptive thermogenesis in BAT. AU - Betz, M.J.* AU - Bielohuby, M.* AU - Mauracher, B.* AU - Abplanalp, W.* AU - Müller, H.H.* AU - Pieper, K.* AU - Ramisch, J.* AU - Tschöp, M.H. AU - Beuschlein, F.* AU - Bidlingmaier, M.* AU - Slawik, M.* C1 - 7986 C2 - 29999 TI - Isoenergetic feeding of low carbohydrate-high fat diets does not increase brown adipose tissue thermogenic capacity in rats. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Previous studies suggested potential priming effects of gestational weight gain (GWG) on offspring's body composition in later life. However, consistency of these effects in normal weight, overweight and obese mothers is less clear. METHODS: We combined the individual data of three German cohorts and assessed associations of total and excessive GWG (as defined by criteria of the Institute of Medicine) with offspring's mean body mass index (BMI) standard deviation scores (SDS) and overweight at the age of 5-6 years (total: n = 6,254). Quantile regression was used to examine potentially different effects on different parts of the BMI SDS distribution. All models were adjusted for birth weight, maternal age and maternal smoking during pregnancy and stratified by maternal pre-pregnancy weight status. RESULTS: In adjusted models, positive associations of total and excessive GWG with mean BMI SDS and overweight were observed only in children of non- overweight mothers. For example, excessive GWG was associated with a mean increase of 0.08 (95% CI: 0.01, 0.15) units of BMI SDS (0.13 (0.02, 0.24) kg/m(2) of 'real' BMI) in children of normal-weight mothers. The effects of total and excessive GWG on BMI SDS increased for higher- BMI children of normal-weight mothers. DISCUSSION: Increased GWG is likely to be associated with overweight in offspring of non-overweight mothers. AU - Beyerlein, A.* AU - Nehring, I.* AU - Rzehak, P. AU - Heinrich, J. AU - Müller, M.J.* AU - Plachta-Danielzik, S.* AU - Wabitsch, M.* AU - Weck, M.* AU - Brenner, H.* AU - Rothenbacher, D.* AU - von Kries, R.* C1 - 7309 C2 - 29670 TI - Gestational weight gain and body mass index in children: Results from three German cohort studies. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Very few principles have been unraveled that explain the relationship between soil properties and soil biota across large spatial scales and different land-use types. Here, we seek these general relationships using data from 52 differently managed grassland and forest soils in three study regions spanning a latitudinal gradient in Germany. We hypothesize that, after extraction of variation that is explained by location and land-use type, soil properties still explain significant proportions of variation in the abundance and diversity of soil biota. If the relationships between predictors and soil organisms were analyzed individually for each predictor group, soil properties explained the highest amount of variation in soil biota abundance and diversity, followed by land-use type and sampling location. After extraction of variation that originated from location or land-use, abiotic soil properties explained significant amounts of variation in fungal, meso- and macrofauna, but not in yeast or bacterial biomass or diversity. Nitrate or nitrogen concentration and fungal biomass were positively related, but nitrate concentration was negatively related to the abundances of Collembola and mites and to the myriapod species richness across a range of forest and grassland soils. The species richness of earthworms was positively correlated with clay content of soils independent of sample location and land-use type. Our study indicates that after accounting for heterogeneity resulting from large scale differences among sampling locations and land-use types, soil properties still explain significant proportions of variation in fungal and soil fauna abundance or diversity. However, soil biota was also related to processes that act at larger spatial scales and bacteria or soil yeasts only showed weak relationships to soil properties. We therefore argue that more general relationships between soil properties and soil biota can only be derived from future studies that consider larger spatial scales and different land-use types. AU - Birkhofer, K.* AU - Schöning, I.* AU - Alt, F.* AU - Herold, N.* AU - Klarner, B.* AU - Maraun, M.* AU - Marhan, S.* AU - Oelmann, Y.* AU - Wubet, T.* AU - Yurkov, A.* AU - Begerow, D.* AU - Berner, D.* AU - Buscot, F.* AU - Daniel, R.* AU - Diekötter, T.* AU - Ehnes, R. B.* AU - Erdmann, G.* AU - Fischer, C.* AU - Fösel, B.* AU - Groh, J.* AU - Gutknecht, J.* AU - Kandeler, E.* AU - Lang, C.* AU - Lohaus, G.* AU - Meyer, A.H.* AU - Nacke, H.* AU - Näther, A.* AU - Overmann, J.* AU - Polle, A.* AU - Pollierer, M. M.* AU - Scheu, S.* AU - Schloter, M. AU - Schulze, E.-D.* AU - Schulze, W.* AU - Weinert, J.* AU - Weisser, W. W.* AU - Wolters, V.* AU - Schrumpf, M.* C1 - 8446 C2 - 30126 TI - General relationships between abiotic soil properties and soil biota across spatial scales and different land-use types. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Objective: Pigment epithelium-derived factor (PEDF) belongs to the serpin family of peptidase inhibitors (serpin F1) and is among the most abundant glycoproteins secreted by adipocytes. In vitro and mouse in vivo data revealed PEDF as a candidate mediator of obesity-induced insulin resistance. Therefore, we assessed whether common genetic variation within the SERPINF1 locus contributes to adipose tissue-related prediabetic phenotypes in humans. Subjects/Methods: A population of 1,974 White European individuals at increased risk for type 2 diabetes was characterized by an oral glucose tolerance test with glucose and insulin measurements (1,409 leptin measurements) and genotyped for five tagging SNPs covering 100% of common genetic variation (minor allele frequency >= 0.05) in the SERPINF1 locus. In addition, a subgroup of 486 subjects underwent a hyperinsulinaemic-euglycaemic clamp and a subgroup of 340 magnetic resonance imaging (MRI) and spectroscopy (MRS). Results: After adjustment for gender and age and Bonferroni correction for the number of SNPs tested, SNP rs12603825 revealed significant association with MRI-derived total adipose tissue mass (p = 0.0094) and fasting leptin concentrations (p = 0.0035) as well as nominal associations with bioelectrical impedance-derived percentage of body fat (p = 0.0182) and clamp-derived insulin sensitivity (p = 0.0251). The association with insulin sensitivity was completely abolished by additional adjustment for body fat (p = 0.8). Moreover, the fat mass-increasing allele of SNP rs12603825 was significantly associated with elevated fasting PEDF concentrations (p = 0.0436), and the PEDF levels were robustly and positively associated with all body fat parameters measured and with fasting leptin concentrations (p<0.0001, all). Conclusion: In humans at increased risk for type 2 diabetes, a functional common genetic variant in the gene locus encoding PEDF contributes to overall body adiposity, obesity-related insulin resistance, and circulating leptin levels. AU - Böhm, A. AU - Ordelheide, A.-M. AU - Machann, J. AU - Heni, M.* AU - Ketterer, C.* AU - Machicao, F. AU - Schick, F. AU - Stefan, N. AU - Fritsche, A. AU - Häring, H.-U. AU - Staiger, H. C1 - 7962 C2 - 29969 TI - Common genetic variation in the SERPINF1 locus determines overall adiposity, obesity-related insulin resistance, and circulating leptin levels. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Brachial circumference (BC), also known as upper arm or mid arm circumference, can be used as an indicator of muscle mass and fat tissue, which are distributed differently in men and women. Analysis of anthropometric measures of peripheral fat distribution such as BC could help in understanding the complex pathophysiology behind overweight and obesity. The purpose of this study is to identify genetic variants associated with BC through a large-scale genome-wide association scan (GWAS) meta-analysis. We used fixed-effects meta-analysis to synthesise summary results across 14 GWAS discovery and 4 replication cohorts comprising overall 22,376 individuals (12,031 women and 10,345 men) of European ancestry. Individual analyses were carried out for men, women, and combined across sexes using linear regression and an additive genetic model: adjusted for age and adjusted for age and BMI. We prioritised signals for follow-up in two-stages. We did not detect any signals reaching genome-wide significance. The FTO rs9939609 SNP showed nominal evidence for association (p<0.05) in the age-adjusted strata for men and across both sexes. In this first GWAS meta-analysis for BC to date, we have not identified any genome-wide significant signals and do not observe robust association of previously established obesity loci with BC. Large-scale collaborations will be necessary to achieve higher power to detect loci underlying BC. AU - Boraska, V.* AU - Day-Williams, A.* AU - Franklin, C.S.* AU - Elliott, K.S.* AU - Panoutsopoulou, K.* AU - Tachmazidou, I.* AU - Albrecht, E. AU - Bandinelli, S.* AU - Beilin, L.J.* AU - Bochud, M.* AU - Cadby, G.* AU - Ernst, F.* AU - Evans, D.M* AU - Hayward, C.* AU - Hicks, A.A.* AU - Huffman, J.* AU - Huth, C. AU - James, A.L.* AU - Klopp, N. AU - Kolcic, I.* AU - Kutalik, Z.* AU - Lawlor, D.A.* AU - Musk, A.W.* AU - Pehlic, M.* AU - Pennell, C.E.* AU - Perry, J.R.* AU - Peters, A. AU - Polasek, O.* AU - Pourcain, B.S.* AU - Ring, S.M.* AU - Salvi, E.* AU - Schipf, S.* AU - Staessen, J.A.* AU - Teumer, A.* AU - Timpson, N.* AU - Vitart, V.* AU - Warrington, N.M.* AU - Yaghootkar, H.* AU - Zemunik, T.* AU - Zgaga, L.* AU - An, P.* AU - Anttila, V.* AU - Borecki, I.B.* AU - Holmen, J.* AU - Ntalla, I.* AU - Palotie, A.* AU - Pietiläinen, K.H.* AU - Wedenoja, J.* AU - Winsvold, B.S.* AU - Dedoussis, G.V.* AU - Kaprio, J.* AU - Province, M.A.* AU - Zwart, J.A.* AU - Burnier, M.* AU - Campbell, H.* AU - Cusi, D.* AU - Davey Smith, G.* AU - Frayling, T.M.* AU - Gieger, C.* AU - Palmer, L.J.* AU - Pramstaller, P.P.* AU - Rudan, I.* AU - Völzke, H.* AU - Wichmann, H.-E. AU - Wright, A.F.* AU - Zeggini, E.* C1 - 7321 C2 - 29683 TI - Genome-wide association study to identify common variants associated with brachial circumference: A meta-analysis of 14 cohorts. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo. AU - Böttger, E.* AU - Multhoff, G. AU - Kun, J.F.* AU - Esen, M.* C1 - 7275 C2 - 29635 TI - Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Targeted deletion of S6 kinase (S6K) 1 in mice leads to higher energy expenditure and improved glucose metabolism. However, the molecular mechanisms controlling these effects remain to be fully elucidated. Here, we analyze the potential role of dietary lipids in regulating the mTORC1/S6K system. Analysis of S6K phosphorylation in vivo and in vitro showed that dietary lipids activate S6K, and this effect is not dependent upon amino acids. Comparison of male mice lacking S6K1 and 2 (S6K-dko) with wt controls showed that S6K-dko mice are protected against obesity and glucose intolerance induced by a high-fat diet. S6K-dko mice fed a high-fat diet had increased energy expenditure, improved glucose tolerance, lower fat mass gain, and changes in markers of lipid metabolism. Importantly, however, these metabolic phenotypes were dependent upon dietary lipids, with no such effects observed in S6K-dko mice fed a fat-free diet. These changes appear to be mediated via modulation of cellular metabolism in skeletal muscle, as shown by the expression of genes involved in energy metabolism. Taken together, our results suggest that the metabolic functions of S6K in vivo play a key role as a molecular interface connecting dietary lipids to the endogenous control of energy metabolism. AU - Castañeda, T.R.* AU - Abplanalp, W.* AU - Um, S.H.* AU - Pfluger, P.T. AU - Schrott, B.* AU - Brown, K.* AU - Grant, E.* AU - Carnevalli, L.* AU - Benoit, S.C.* AU - Morgan, D.A.* AU - Gilham, D.* AU - Hui, D.Y.* AU - Rahmouni, K.* AU - Thomas, G.* AU - Kozma, S.C.* AU - Clegg, D.J.* AU - Tschöp, M.H. C1 - 7498 C2 - 29758 TI - Metabolic control by S6 kinases depends on dietary lipids. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG), but not undifferentiated neuronal progenitor cells (NPCs) from ventral subventricular zone (SVZ), results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2). NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control). By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+), whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+). At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative). Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78%) expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG. AU - Chen, X.* AU - Lepier, A. AU - Berninger, B. AU - Tolkovsky, A.M.* AU - Herbert, J.* C1 - 7463 C2 - 29723 TI - Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Human endogenous retroviruses (HERVs) have been associated with various neurological and neuropsychiatric disorders. Transcripts and proteins of at least three HERV groups, HERV-W, ERV9 and HERV-K(HML-2) have been detected repeatedly in brain samples or cerebrospinal fluid of patients with schizophrenia suggesting that alterations in HERV activity may play a role in etiopathogenesis. Current therapies otherwise include neuroleptics and/or antidepressants that may induce epigenetic alterations and thus influence HERV expression. To investigate the effects of these drugs on HERV transcriptional activity, HERV expression profiles of a broad range of human brain cell lines treated with valproic acid (VPA), haloperidol, risperidone, and clozapine were analyzed using a retrovirus-specific microarray and qRT-PCR. Investigation of 52 HERV subgroups revealed upregulation of several class I and class II HERV elements by VPA in a dose-dependent manner. The strongest effect was observed on HERV-W and ERV9 groups in the human glioblastoma cell lines SK-N-SH and SK-N-MC, respectively. The transcript level of HERV-K(HML-2) elements was not influenced. Transcription of HERV-W, ERV9 and HERVK(HML-2) taxa was further quantified in postmortem brain samples of patients with schizophrenia, bipolar disorders and a healthy control group with regard to their medication. Patients with schizophrenia showed a significantly higher HERV-W transcription associated with VPA treatment. However in case of ERV9, enhanced transcript levels could not be explained solely by VPA treatment, since a slight increase was also found in untreated patients compared to healthy controls. HERVK( HML-2) elements appeared to be upregulated in some patients with bipolar disorders independent from medication. In conclusion, these results suggest that antipsychotic medication may contribute to increased expression of distinct HERV taxa in patients with neuropsychiatric diseases. AU - Diem, O. AU - Schäffner, M. AU - Seifarth, W.* AU - Leib-Mösch, C. C1 - 7982 C2 - 29948 TI - Influence of antipsychotic drugs on Human Endogenous Retrovirus (HERV) transcription in brain cells. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background: Mesothelial cells are critical in the pathogenesis of post-surgical intraabdominal adhesions as well as in the deterioration of the peritoneal membrane associated with long-term peritoneal dialysis. Mesothelial denudation is a pathophysiolocigally important finding in these processes. Matrix metalloproteinase (MMP) biology underlies aspects of mesothelial homeostasis as well as wound repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) moderate MMP activity. Methods and Finding: By modifying human TIMP-1 through the addition of a glycosylphosphatidylinositol (GPI) anchor, a recombinant protein was generated that efficiently focuses TIMP-1 on the cell surface. Treatment of primary mesothelial cells with TIMP-1-GPI facilitates their mobilization and migration leading to a dramatic increase in the rate of wound experimental closure. Mesothelial cells treated with TIMP-1-GPI showed a dose dependent increase in cell proliferation, reduced secretion of MMP-2, MMP-9, TNF-alpha and urokinase-type plasminogen activator (uPA), but increased tissue plasminogen activator (t-PA). Treatment resulted in reduced expression and processing of latent TGF-beta 1. Conclusions: TIMP-1-GPI stimulated rapid and efficient in vitro wound closure. The agent enhanced mesothelial cell proliferation and migration and was bioactive in the nanogram range. The application of TIMP-1-GPI may represent a new approach for limiting or repairing damaged mesothelium. AU - Djafarzadeh, R.* AU - Sauter, M.* AU - Notohamiprodjo, S.* AU - Nößner, E. AU - Goyal, P.* AU - Siess, W.* AU - Wörnle, M.* AU - Ribeiro, A.* AU - Himmelein, S.* AU - Sitter, T.* AU - Nelson, P.J.* C1 - 7689 C2 - 29887 TI - Recombinant GPI-anchored TIMP-1 stimulates growth and migration of peritoneal mesothelial cells. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Lung cancer mortality after exposure to radon decay products (RDP) among 16,236 male Eldorado uranium workers was analyzed. Male workers from the Beaverlodge and Port Radium uranium mines and the Port Hope radium and uranium refinery and processing facility who were first employed between 1932 and 1980 were followed up from 1950 to 1999. A total of 618 lung cancer deaths were observed. The analysis compared the results of the biologically-based two-stage clonal expansion (TSCE) model to the empirical excess risk model. The spontaneous clonal expansion rate of pre-malignant cells was reduced at older ages under the assumptions of the TSCE model. Exposure to RDP was associated with increase in the clonal expansion rate during exposure but not afterwards. The increase was stronger for lower exposure rates. A radiation-induced bystander effect could be a possible explanation for such an exposure response. Results on excess risks were compared to a linear dose-response parametric excess risk model with attained age, time since exposure and dose rate as effect modifiers. In all models the excess relative risk decreased with increasing attained age, increasing time since exposure and increasing exposure rate. Large model uncertainties were found in particular for small exposure rates. AU - Eidemüller, M. AU - Jacob, P. AU - Lane, R.S.* AU - Frost, S.E.* AU - Zablotska, L.B.* C1 - 8526 C2 - 30159 TI - Lung cancer mortality (1950-1999) among Eldorado uranium workers: A comparison of models of carcinogenesis and empirical excess risk models. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Pathway analysis has been proposed as a complement to single SNP analyses in GWAS. This study compared pathway analysis methods using two lung cancer GWAS data sets based on four studies: one a combined data set from Central Europe and Toronto (CETO); the other a combined data set from Germany and MD Anderson (GRMD). We searched the literature for pathway analysis methods that were widely used, representative of other methods, and had available software for performing analysis. We selected the programs EASE, which uses a modified Fishers Exact calculation to test for pathway associations, GenGen (a version of Gene Set Enrichment Analysis (GSEA)), which uses a Kolmogorov-Smirnov-like running sum statistic as the test statistic, and SLAT, which uses a p-value combination approach. We also included a modified version of the SUMSTAT method (mSUMSTAT), which tests for association by averaging χ(2) statistics from genotype association tests. There were nearly 18000 genes available for analysis, following mapping of more than 300,000 SNPs from each data set. These were mapped to 421 GO level 4 gene sets for pathway analysis. Among the methods designed to be robust to biases related to gene size and pathway SNP correlation (GenGen, mSUMSTAT and SLAT), the mSUMSTAT approach identified the most significant pathways (8 in CETO and 1 in GRMD). This included a highly plausible association for the acetylcholine receptor activity pathway in both CETO (FDR≤0.001) and GRMD (FDR = 0.009), although two strong association signals at a single gene cluster (CHRNA3-CHRNA5-CHRNB4) drive this result, complicating its interpretation. Few other replicated associations were found using any of these methods. Difficulty in replicating associations hindered our comparison, but results suggest mSUMSTAT has advantages over the other approaches, and may be a useful pathway analysis tool to use alongside other methods such as the commonly used GSEA (GenGen) approach. AU - Fehringer, G.* AU - Liu, G.* AU - Briollais, L.* AU - Brennan, P.* AU - Amos, C.I.* AU - Spitz, M.R.* AU - Bickeböller, H.* AU - Wichmann, H.-E. AU - Risch, A.* AU - Hung, R.J.* C1 - 7310 C2 - 29671 TI - Comparison of pathway analysis approaches using lung cancer GWAS data sets. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner. METHODOLOGY/PRINCIPAL FINDINGS: Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes. CONCLUSIONS/SIGNIFICANCE: These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors. AU - Gehrmann, M. AU - Stangl, S. AU - Kirschner, A. AU - Foulds, G.A. AU - Sievert, W. AU - Doß, B.T. AU - Walch, A.K. AU - Pockley, A.G.* AU - Multhoff, G. C1 - 8343 C2 - 30065 TI - Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60-70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80-100% of STAT1-, IFN-gamma-, or IFN-gamma receptor-deficient recipients died of lymphoma, indicating that host IFN-gamma signaling is critical for rejection. Lymphomas arising in IFN-gamma- and IFN-gamma-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches. AU - Gerbitz, A.* AU - Sukumar, M. AU - Helm, F.* AU - Wilke, A.* AU - Friese, C.* AU - Fahrenwaldt, C.* AU - Lehmann, F.M. AU - Loddenkemper, C.* AU - Kammertoens, T.* AU - Mautner, J. AU - Schmitt, C.A.* AU - Blankenstein, T.* AU - Bornkamm, G.W. C1 - 7695 C2 - 29884 TI - Stromal interferon-γ signaling and cross-presentation are required to eliminate antigen-loss variants of Β cell lymphomas in mice. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Human endogenous retroviruses (HERV) and related elements account for more than 8% of the human genome and significantly contribute to the human transcriptome by long terminal repeat (LTR) promoter activity. In this context, HERVs are thought to intervene in the expression of adjacent genes by providing regulatory sequences (cis-effect) or via noncoding RNA including natural antisense transcripts. To address the potential impact of HERV activity in urothelial carcinoma, we comparatively analyzed the HERV transcription profiles in paired samples of non-malignant urothelium and urothelial carcinoma derived from 13 patients with bladder cancer by means of a retrovirus-specific microarray (RetroArray). We established a characteristic HERV signature consisting of six ubiquitously active HERV subgroups (E4-1, HERV-Rb, ERV9, HERV-K-T47D, NMWV3, HERV-KC4). The transcription pattern is largely identical in human urothelial carcinoma, non-malignant urothelial tissue, four tumor-derived cell lines and in a non-malignant urothelial cell line (UROtsa). Quantitative reverse transcriptase PCR (qRT-PCR) of HERV-E4-1, HERV-K(HML-6) and HERV-T(S71-TK1) revealed a bias to lower HERV activity in carcinoma samples compared to non-malignant tissue. Determination of active HERV-E4-1 loci by cloning and sequencing revealed six HERV-E4-1 proviral loci that are differentially regulated in urothelial carcinoma cells and normal tissue. Two full-length HERV-E4-1 proviruses, HERV-Ec1 and HERV-Ec6, are located in antisense orientation in introns of the genes PLA2G4A and RNGTT, respectively. PLA2G4A encodes a cytosolic phospholipase A2 (cPLA2) that is dysregulated in many human tumors. PLA2G4A and HERV-Ec1 displayed reciprocal transcript levels in 7 of 11 urothelial carcinoma patients. Moreover, reciprocal shifts were observed after treatment of UROtsa cells with HERV-Ec1 and PLA2G4A-directed siRNAs or 5-aza-2'-deoxycytidine (aza-dC) pointing to an antagonistic regulation of PLA2G4A and HERV-Ec1 transcription in human urothelial cells. We suggest that transcription of HERV-Ec1 contributes to fine tuning of cPLA2 expression, thereby facilitating tumorigenesis. AU - Gosenca, D.* AU - Gabriel, U.* AU - Steidler, A.* AU - Mayer, J.* AU - Diem, O. AU - Erben, P.* AU - Fabarius, A.* AU - Leib-Mösch, C. AU - Hofmann, W.-K.* AU - Seifarth, W.* C1 - 11355 C2 - 30626 TI - HERV-E-mediated modulation of PLA2G4A transcription in urothelial carcinoma. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Late-stage age-related macular degeneration (AMD) is a common sight-threatening disease of the central retina affecting approximately 1 in 30 Caucasians. Besides age and smoking, genetic variants from several gene loci have reproducibly been associated with this condition and likely explain a large proportion of disease. Here, we developed a genetic risk score (GRS) for AMD based on 13 risk variants from eight gene loci. The model exhibited good discriminative accuracy, area-under-curve (AUC) of the receiver-operating characteristic of 0.820, which was confirmed in a cross-validation approach. Noteworthy, younger AMD patients aged below 75 had a significantly higher mean GRS (1.87, 95% CI: 1.69-2.05) than patients aged 75 and above (1.45, 95% CI: 1.36-1.54). Based on five equally sized GRS intervals, we present a risk classification with a relative AMD risk of 64.0 (95% CI: 14.11-1131.96) for individuals in the highest category (GRS 3.44-5.18, 0.5% of the general population) compared to subjects with the most common genetic background (GRS -0.05-1.70, 40.2% of general population). The highest GRS category identifies AMD patients with a sensitivity of 7.9% and a specificity of 99.9% when compared to the four lower categories. Modeling a general population around 85 years of age, 87.4% of individuals in the highest GRS category would be expected to develop AMD by that age. In contrast, only 2.2% of individuals in the two lowest GRS categories which represent almost 50% of the general population are expected to manifest AMD. Our findings underscore the large proportion of AMD cases explained by genetics particularly for younger AMD patients. The five-category risk classification could be useful for therapeutic stratification or for diagnostic testing purposes once preventive treatment is available. AU - Grassmann, F.* AU - Fritsche, L.G.* AU - Keilhauer, C.N.* AU - Heid, I.M. AU - Weber, B.H.F.* C1 - 7683 C2 - 29892 TI - Modelling the genetic risk in age-related macular degeneration. JO - PLoS ONE VL - 7 IS - 5 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity. AU - Greve, B.* AU - Bölling, T.* AU - Amler, S.* AU - Rössler, U.* AU - Gomolka, M.* AU - Mayer, C.* AU - Popanda, O.* AU - Dreffke, K.* AU - Rickinger, A. AU - Fritz, E.* AU - Eckardt-Schupp, F. AU - Sauerland, C.* AU - Braselmann, H. AU - Sauter, W. AU - Illig, T. AU - Riesenbeck, D.* AU - Könemann, S.* AU - Willich, N.* AU - Mörtl, S.* AU - Eich, H.T.* AU - Schmezer, P.* C1 - 11024 C2 - 30468 TI - Evaluation of different biomarkers to predict individual radiosensitivity in an inter-laboratory comparison - lessons for future studies. JO - PLoS ONE VL - 7 IS - 10 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties. AU - Gross, H.* AU - Hennard, C. AU - Masouris, I. AU - Cassel, C.* AU - Barth, S.* AU - Stober-Grässer, U.* AU - Mamiani, A.* AU - Moritz, B.* AU - Ostareck, D.* AU - Ostareck-Lederer, A.* AU - Neuenkirchen, N.* AU - Fischer, U.* AU - Deng, W.* AU - Leonhardt, H.* AU - Nößner, E. AU - Kremmer, E. AU - Grässer, F.A.* C1 - 8567 C2 - 30182 TI - Binding of the heterogeneous ribonucleoprotein K (hnRNP K) to the Epstein-Barr virus nuclear antigen 2 (EBNA2) enhances viral LMP2A expression. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background Breast cancer is the leading cause of death from cancer among women in Germany. Despite its clinical and economic relevance, no attributable costs for breast cancer have been reported for Germany so far. The objective of this study is to estimate age-specific breast cancer attributable health expenditures for Germany. Methods Sickness fund data from 1999 representing about 26 million insured (i.e. 32% of the total German population) have been analysed using generalized additive models and the error propagation law. Costs have been inflated to 2010. Results Breast cancer attributable costs decreased with age. Among breast cancer patients aged 30–45 years, about 90% of all health expenditures were due to breast cancer, whereas in breast cancer patients aged 80–90 years, about 50% were due to breast cancer. Breast cancer attributable costs amounted to about €9,000 annually for patients below 55 years of age and declined to about €3,000 in 90-year-old breast cancer patients. Conclusion This analysis provides estimates of attributable breast cancer costs in Germany. Compared with the international literature, the estimates were plausible but had a tendency to underestimate breast cancer attributable costs. AU - Gruber, E.V. AU - Stock, S.* AU - Stollenwerk, B. C1 - 11414 C2 - 30656 TI - Breast cancer attributable costs in Germany: A top-down approach based on sickness funds data. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosinbeta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins. AU - Grüner, B.M.* AU - Hahne, H.* AU - Mazur, P.K.* AU - Trajkovic-Arsic, M.* AU - Maier, S.K. AU - Esposito, I. AU - Kalideris, E.* AU - Michalski, C.W.* AU - Kleeff, J.* AU - Rauser, S. AU - Schmid, R.M.* AU - Kuster, B.* AU - Walch, A.K. AU - Siveke, J.T.* C1 - 7758 C2 - 29879 TI - MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Aims/Hypothesis: In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation. Methods: We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA-and protein expression of the cell cycle regulator p27(Kip1) was quantified by real-time RT-PCR and immunohistochemistry. Results: Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27(Kip1) content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019). Conclusions/Interpretation: We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy. AU - Heni, M.* AU - Hennenlotter, J.* AU - Scharpf, M.* AU - Lutz, S.Z.* AU - Schwentner, C.* AU - Todenhöfer, T.* AU - Schilling, D.* AU - Kühs, U.* AU - Gerber, V.* AU - Machicao, F. AU - Staiger, H. AU - Häring, H.-U. AU - Stenzl, A.* C1 - 11638 C2 - 30735 TI - Insulin receptor isoforms A and B as well as insulin receptor substrates-1 and -2 are differentially expressed in prostate cancer. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Polypyrimidine tract-binding protein 1 (PTBP1) promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in β-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion. METHODS: We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs) within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698) covering 100% of genetic variation with an r(2)≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT. RESULTS: PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04). The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103). Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108). Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018), but the rs351974 was not. CONCLUSIONS: We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo. AU - Heni, M.* AU - Ketterer, C.* AU - Wagner, R.* AU - Linder, K.* AU - Böhm, A.* AU - Herzberg-Schäfer, S.A.* AU - Machicao, F. AU - Knoch, K.P.* AU - Fritsche, A. AU - Staiger, H. AU - Häring, H.-U. AU - Solimena, M.* C1 - 43365 C2 - 36360 TI - Polymorphism rs11085226 in the gene encoding polypyrimidine tract-binding protein 1 negatively affects glucose-stimulated insulin secretion. JO - PLoS ONE VL - 7 IS - 10 PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients. AU - Horakova, O.* AU - Medrikova, D.* AU - van Schothorst, E.M.* AU - Bunschoten, A.* AU - Flachs, P.* AU - Kus, V.* AU - Kuda, O.* AU - Bardova, K.* AU - Janovska, P.* AU - Hensler, M.* AU - Rossmeisl, M.* AU - Wang-Sattler, R. AU - Prehn, C. AU - Adamski, J. AU - Illig, T. AU - Keijer, J.* AU - Kopecky, J.* C1 - 10409 C2 - 30237 TI - Preservation of metabolic flexibility in skeletal muscle by a combined use of n-3 PUFA and rosiglitazone in dietary obese mice. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: More accurate coronary heart disease (CHD) prediction, specifically in middle-aged men, is needed to reduce the burden of disease more effectively. We hypothesised that a multilocus genetic risk score could refine CHD prediction beyond classic risk scores and obtain more precise risk estimates using a prospective cohort design. METHODS: Using data from nine prospective European cohorts, including 26,221 men, we selected in a case-cohort setting 4,818 healthy men at baseline, and used Cox proportional hazards models to examine associations between CHD and risk scores based on genetic variants representing 13 genomic regions. Over follow-up (range: 5-18 years), 1,736 incident CHD events occurred. Genetic risk scores were validated in men with at least 10 years of follow-up (632 cases, 1361 non-cases). Genetic risk score 1 (GRS1) combined 11 SNPs and two haplotypes, with effect estimates from previous genome-wide association studies. GRS2 combined 11 SNPs plus 4 SNPs from the haplotypes with coefficients estimated from these prospective cohorts using 10-fold cross-validation. Scores were added to a model adjusted for classic risk factors comprising the Framingham risk score and 10-year risks were derived. RESULTS: Both scores improved net reclassification (NRI) over the Framingham score (7.5%, p = 0.017 for GRS1, 6.5%, p = 0.044 for GRS2) but GRS2 also improved discrimination (c-index improvement 1.11%, p = 0.048). Subgroup analysis on men aged 50-59 (436 cases, 603 non-cases) improved net reclassification for GRS1 (13.8%) and GRS2 (12.5%). Net reclassification improvement remained significant for both scores when family history of CHD was added to the baseline model for this male subgroup improving prediction of early onset CHD events. CONCLUSIONS: Genetic risk scores add precision to risk estimates for CHD and improve prediction beyond classic risk factors, particularly for middle aged men. AU - Hughes, M.F.* AU - Saarela, O.* AU - Stritzke, J.* AU - Kee, F.* AU - Silander, K.* AU - Klopp, N. AU - Kontto, J.* AU - Karvanen, J.* AU - Willenborg, C.* AU - Salomaa, V.* AU - Virtamo, J.* AU - Amouyel, P.* AU - Arveiler, D.* AU - Ferrieres, J.* AU - Wiklund, P.G.* AU - Baumert, J.J. AU - Thorand, B. AU - Diemert, P.* AU - Tregouet, D.A.* AU - Hengstenberg, C.* AU - Peters, A. AU - Evans, A.* AU - Koenig, W.* AU - Erdmann, J.* AU - Samani, N.J.* AU - Kuulasmaa, K.* AU - Schunkert, H.* C1 - 8455 C2 - 30117 TI - Genetic markers enhance coronary risk prediction in men: The MORGAM prospective cohorts. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - During development, fibroblast growth factors (FGF) are essential for early patterning events along the anterior-posterior axis, conferring positional identity to spinal motor neurons by activation of different Hox codes. In the periphery, signaling through one of four fibroblast growth factor receptors supports the development of the skeleton, as well as induction and maintenance of extremities. In previous studies, FGF receptor 2 (FGFR2) was found to interact with axon bound molecules involved in axon fasciculation and extension, thus rendering this receptor an interesting candidate for the promotion of proper peripheral innervation. However, while the involvement of FGFR2 in limb bud induction has been extensively studied, its role during axon elongation and formation of distinct nervous projections has not been addressed so far. We show here that motor neurons in the spinal cord express FGFR2 and other family members during the establishment of motor connections to the forelimb and axial musculature. Employing a conditional genetic approach to selectively ablate FGFR2 from motor neurons we found that the patterning of motor columns and the expression patterns of other FGF receptors and Sema3A in the motor columns of mutant embryos are not altered. In the absence of FGFR2 signaling, pathfinding of motor axons is intact, and also fasciculation, distal advancement of motor nerves and gross morphology and positioning of axonal projections are not altered. Our findings therefore show that FGFR2 is not required cell-autonomously in motor neurons during the formation of initial motor projections towards limb and axial musculature. AU - Hüttl, R.E. AU - Haehl, T. AU - Huber, A.B. C1 - 8311 C2 - 30103 TI - Fasciculation and guidance of spinal motor axons in the absence of FGFR2 signaling. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro. AU - Jost, K.L.* AU - Rottach, A.* AU - Milden, M.* AU - Bertulat, B.* AU - Becker, A.* AU - Wolf, P.* AU - Sandoval, J.* AU - Petazzi, P.* AU - Huertas, D.* AU - Esteller, M.* AU - Kremmer, E. AU - Leonhardt, H.* AU - Cardoso, M.C.* C1 - 10590 C2 - 30294 TI - Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies. JO - PLoS ONE VL - 6 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Objective: To characterise the influence of the fat free mass on the metabolite profile in serum samples from participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) S4 study. Subjects and Methods: Analyses were based on metabolite profile from 965 participants of the S4 and 890 weight-stable subjects of its seven-year follow-up study (KORA F4). 190 different serum metabolites were quantified in a targeted approach including amino acids, acylcarnitines, phosphatidylcholines (PCs), sphingomyelins and hexose. Associations between metabolite concentrations and the fat free mass index (FFMI) were analysed using adjusted linear regression models. To draw conclusions on enzymatic reactions, intra-metabolite class ratios were explored. Pairwise relationships among metabolites were investigated and illustrated by means of Gaussian graphical models (GGMs). Results: We found 339 significant associations between FFMI and various metabolites in KORA S4. Among the most prominent associations (p-values 4.75 x 10(-16) -8.95 x 10(-06)) with higher FFMI were increasing concentrations of the branched chained amino acids (BCAAs), ratios of BCAAs to glucogenic amino acids, and carnitine concentrations. For various PCs, a decrease in chain length or in saturation of the fatty acid moieties could be observed with increasing FFMI, as well as an overall shift from acyl-alkyl PCs to diacyl PCs. These findings were reproduced in KORA F4. The established GGMs supported the regression results and provided a comprehensive picture of the relationships between metabolites. In a sub-analysis, most of the discovered associations did not exist in obese subjects in contrast to non-obese subjects, possibly indicating derangements in skeletal muscle metabolism. Conclusion: A set of serum metabolites strongly associated with FFMI was identified and a network explaining the relationships among metabolites was established. These results offer a novel and more complete picture of the FFMI effects on serum metabolites in a data-driven network. AU - Jourdan, C. AU - Petersen, A.-K. AU - Gieger, C. AU - Döring, A. AU - Illig, T. AU - Wang-Sattler, R. AU - Meisinger, C. AU - Peters, A. AU - Adamski, J. AU - Prehn, C. AU - Suhre, K. AU - Altmaier, E. AU - Kastenmüller, G. AU - Römisch-Margl, W. AU - Theis, F.J. AU - Krumsiek, J. AU - Wichmann, H.-E. AU - Linseisen, J. C1 - 8193 C2 - 29947 TI - Body fat free mass is associated with the serum metabolite profile in a population-based study. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - 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. AU - Karakas, M.* AU - Baumert, J.J. AU - Kleber, M.E.* AU - Thorand, B. AU - Dallmeier, D.* AU - Silbernagel, G.* AU - Grammer, T.B.* AU - Rottbauer, W.* AU - Meisinger, C. AU - Illig, T. AU - Marz, W.* AU - Koenig, W.* C1 - 11751 C2 - 30805 TI - A variant in the abo gene explains the variation in soluble e-selectin levels - results from dense genotyping in two independent populations. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Adiponectin serum concentrations are an important biomarker in cardiovascular epidemiology with heritability etimates of 30-70%. However, known genetic variants in the adiponectin gene locus (ADIPOQ) account for only 2%-8% of its variance. As transcription factors are thought to play an under-acknowledged role in carrying functional variants, we hypothesized that genetic polymorphisms in genes coding for the main transcription factors for the ADIPOQ promoter influence adiponectin levels. Single nucleotide polymorphisms (SNPs) at these genes were selected based on the haplotype block structure and previously published evidence to be associated with adiponectin levels. We performed association analyses of the 24 selected SNPs at forkhead box O1 (FOXO1), sterol-regulatory-element-binding transcription factor 1 (SREBF1), sirtuin 1 (SIRT1), peroxisome-proliferator-activated receptor gamma (PPARG) and transcription factor activating enhancer binding protein 2 beta (TFAP2B) gene loci with adiponectin levels in three different European cohorts: SAPHIR (n = 1742), KORA F3 (n = 1636) and CoLaus (n = 5355). In each study population, the association of SNPs with adiponectin levels on log-scale was tested using linear regression adjusted for age, sex and body mass index, applying both an additive and a recessive genetic model. A pooled effect size was obtained by meta-analysis assuming a fixed effects model. We applied a significance threshold of 0.0033 accounting for the multiple testing situation. A significant association was only found for variants within SREBF1 applying an additive genetic model (smallest p-value for rs1889018 on log(adiponectin) = 0.002, β on original scale = -0.217 µg/ml), explaining ∼0.4% of variation of adiponectin levels. Recessive genetic models or haplotype analyses of the FOXO1, SREBF1, SIRT1, TFAPB2B genes or sex-stratified analyses did not reveal additional information on the regulation of adiponectin levels. The role of genetic variations at the SREBF1 gene in regulating adiponectin needs further investigation by functional studies. AU - Kedenko, L.* AU - Lamina, C.* AU - Kiesslich, T.* AU - Kapur, K.* AU - Bergmann, S.* AU - Waterworth, D.* AU - Heid, I.M. AU - Wichmann, H.-E. AU - Kedenko, I.* AU - Kronenberg, F.* AU - Paulweber, B.* C1 - 11780 C2 - 30807 TI - Genetic polymorphisms of the main transcription factors for adiponectin gene promoter in regulation of adiponectin levels: Association analysis in three European cohorts. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Disruption of endogenous circadian rhythms has been shown to increase the risk of developing type 2 diabetes, suggesting that circadian genes might play a role in determining disease susceptibility. We present the results of a pilot study investigating the association between type 2 diabetes and selected single nucleotide polymorphisms (SNPs) in/near nine circadian genes. The variants were chosen based on their previously reported association with prostate cancer, a disease that has been suggested to have a genetic link with type 2 diabetes through a number of shared inherited risk determinants. METHODOLOGY/PRINCIPAL FINDINGS: The pilot study was performed using two genetically homogeneous Punjabi cohorts, one resident in the United Kingdom and one indigenous to Pakistan. Subjects with (N = 1732) and without (N = 1780) type 2 diabetes were genotyped for thirteen circadian variants using a competitive allele-specific polymerase chain reaction method. Associations between the SNPs and type 2 diabetes were investigated using logistic regression. The results were also combined with in silico data from other South Asian datasets (SAT2D consortium) and white European cohorts (DIAGRAM+) using meta-analysis. The rs7602358G allele near PER2 was negatively associated with type 2 diabetes in our Punjabi cohorts (combined odds ratio [OR] = 0.75 [0.66-0.86], p = 3.18 × 10(-5)), while the BMAL1 rs11022775T allele was associated with an increased risk of the disease (combined OR = 1.22 [1.07-1.39], p = 0.003). Neither of these associations was replicated in the SAT2D or DIAGRAM+ datasets, however. Meta-analysis of all the cohorts identified disease associations with two variants, rs2292912 in CRY2 and rs12315175 near CRY1, although statistical significance was nominal (combined OR = 1.05 [1.01-1.08], p = 0.008 and OR = 0.95 [0.91-0.99], p = 0.015 respectively). CONCLUSIONS/SIGNIFICANCE: None of the selected circadian gene variants was associated with type 2 diabetes with study-wide significance after meta-analysis. The nominal association observed with the CRY2 SNP, however, complements previous findings and confirms a role for this locus in disease susceptibility. AU - Kelly, M.A.* AU - Rees, S.D.* AU - Hydrie, M.Z.* AU - Shera, A.S.* AU - Bellary, S.* AU - O'Hare, J.P.* AU - Kumar, S.* AU - Taheri, S.* AU - Basit, A.* AU - Barnett, A.H.* AU - DIAGRAM Consortium (Gieger, C. AU - Grallert, H. AU - Huth, C. AU - Illig, T. AU - Klopp, N. AU - Meitinger, T. AU - Petersen, A.-K. AU - Thorand, B. AU - Wichmann, H.-E.) AU - SAT2D Consortium (*) C1 - 11160 C2 - 30527 TI - Circadian gene variants and susceptibility to type 2 diabetes: A pilot study. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica. AU - Khatabi, B.* AU - Molitor, A.* AU - Lindermayr, C. AU - Pfiffi, S.* AU - Durner, J. AU - von Wettstein, D.* AU - Kogel, K.-H.* AU - Schäfer, P.* C1 - 7692 C2 - 29885 TI - Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Multimorbidity is a common problem in aged populations with a wide range of individual and societal consequences. The objective of the study was to explore patterns of comorbidity and multimorbidity in an elderly population using different analytical approaches. Data were gathered from the population-based KORA-Age project, which included 4,127 persons aged 65-94 years living in the city of Augsburg and its two surrounding counties in Southern Germany. Information on the presence of 13 chronic conditions was collected in a standardized telephone interview and a self-administered questionnaire. Patterns of comorbidity and multimorbidity were analyzed using prevalence figures, logistic regression models and exploratory tetrachoric factor analysis. The prevalence of multimorbidity (≥2 diseases) was 58.6% in the total sample. Hypertension and diabetes (Odds Ratio [OR] 2.95, 99.58% confidence interval [CI] [2.19-3.96]), as well as hypertension and stroke (OR 2.00, 99.58% CI [1.26-3.16]) most often occurred in combination. This association was independent of age, sex and the presence of other conditions. Using factor analysis, we identified four patterns of multimorbidity: the first pattern includes cardiovascular and metabolic diseases, the second includes joint, liver, lung and eye diseases, the third covers mental and neurologic diseases and the fourth pattern includes gastrointestinal diseases and cancer. 44% of the persons were assigned to at least one of the four multimorbidity patterns; 14% could be assigned to both the cardiovascular/metabolic and the joint/liver/lung/eye pattern. Further common pairs were the mental/neurologic pattern combined with the cardiovascular/metabolic pattern (7.2%) or the joint/liver/lung/eye pattern (5.3%), respectively. Our results confirmed the existence of co-occurrence of certain diseases in elderly persons, which is not caused by chance. Some of the identified patterns of multimorbidity and their overlap may indicate common underlying pathological mechanisms. AU - Kirchberger, I. AU - Meisinger, C. AU - Heier, M. AU - Zimmermann, A.-K. AU - Thorand, B. AU - Autenrieth, C.S. AU - Peters, A. AU - Ladwig, K.-H. AU - Döring, A. C1 - 7216 C2 - 29553 TI - Patterns of multimorbidity in the aged population. Results from the KORA-Age study. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The mammalian Interferon induced transmembrane protein 1 (Ifitm1) gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation. AU - Klymiuk, I. AU - Kenner, L.* AU - Adler, T. AU - Busch, D.H.* AU - Boersma, A. AU - Irmler, M. AU - Fridrich, B. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Leitner, N.* AU - Müller, M.* AU - Kühn, R. AU - Schlederer, M.* AU - Treise, I. AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 10678 C2 - 30402 TI - In vivo functional requirement of the mouse Ifitm1 gene for germ cell development, interferon mediated immune response and somitogenesis. JO - PLoS ONE VL - 7 IS - 10 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Objective: The association between blood glucose and carotid intima-media thickness (CIMT) is considered to be established knowledge. We aimed to assess whether associations between different measures of glycaemia and CIMT are actually independent of anthropometric variables and metabolic risk factors. Moreover, we checked published studies for the adjustment for shared risk factors of blood glucose and CIMT. Methods: Fasting glucose, 2-hour glucose, HbA1c, and CIMT were measured in 31-81-years-old participants of the population-based Cooperative Health Research in the Region of Augsburg (KORA) F4 study in Southern Germany (n = 2,663). CIMT was assessed according to the Rotterdam protocol. Linear and logistic regression models with adjustment for age, sex, anthropometric measures, hypertension, and dyslipidaemia were fitted to assess the association between continuous measures of glycaemia, and categories of glucose regulation, respectively, with CIMT. Results: We found a 0.10 mm increase (95%-confidence interval: 0.08-0.12) in CIMT in subjects with compared to subjects without diabetes in crude analysis. This increase was not significant in age-sex adjusted models (p = 0.17). Likewise, neither impaired fasting glucose (p = 0.22) nor impaired glucose tolerance (p = 0.93) were associated with CIMT after adjustment for age, sex, and waist circumference. In multivariable adjusted models, age, sex, hypertension, waist circumference, HDL and LDL cholesterol, but neither fasting glucose nor 2-hour glucose nor HbA1c were associated with elevated CIMT. Literature findings are inconclusive regarding an independent association of glucose levels and CIMT. Conclusion: CIMT is highly dependent on traditional cardiovascular risk factors, but no relationships between blood glucose and CIMT were found after adjustment for age, sex, and anthropometric variables. AU - Kowall, B.* AU - Ebert, N.* AU - Then, C.* AU - Thiery, J.* AU - Koenig, W.* AU - Meisinger, C. AU - Rathmann, W.* AU - Seissler, J.* C1 - 22668 C2 - 30926 TI - Associations between blood glucose and carotid intima-media thickness disappear after adjustment for shared risk factors: The KORA F4 study. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control. AU - Lee, A.W.* AU - Hengstler, H.* AU - Schwald, K.* AU - Berriel Diaz, M.* AU - Loreth, D.* AU - Kirsch, M.* AU - Kretz, O.* AU - Haas, C.A.* AU - Hrabě de Angelis, M. AU - Herzig, S.* AU - Brümmendorf, T.* AU - Klingenspor, M.* AU - Rathjen, F.G.* AU - Rozman, J. AU - Nicholson, G.* AU - Cox, R.D.* AU - Schäfer, M.K.* C1 - 8335 C2 - 29881 TI - Functional inactivation of the genome-wide association study obesity gene neuronal growth regulator 1 in mice causes a body mass phenotype. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background: A given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC. Principal Findings: We generated C57BL/6-derived embryonic stem cells that are transgenic for a humanized c-Myc gene and established a mouse strain (hc-Myc) that expresses human c-MYC instead of the murine ortholog. These transgenic animals harbor the humanized c-Myc gene integrated into the endogenous murine c-Myc locus. Despite the lack of the endogenous murine c-Myc gene, homozygous mice show a normal phenotype indicating that human c-MYC can replace its murine ortholog. Conclusions: The newly established hc-Myc mouse strain provides a model system to study in detail the adverse effects of therapies that target the human c-MYC protein. To mimic the clinical situation, hc-Myc mice may be cross-bred to mice that develop tumors due to overexpression of human c-MYC. With these double transgenic mice it will be possible to study simultaneously the therapeutic efficiency and adverse side effects of MYC-specific therapies in the same mouse. AU - Lehmann, F.M. AU - Feicht, S. AU - Helm, F.* AU - Maurberger, A.* AU - Ladinig, C. AU - Zimber-Strobl, U. AU - Kühn, R. AU - Mautner, J.* AU - Gerbitz, A.* AU - Bornkamm, G.W. C1 - 8635 C2 - 30352 TI - Humanized c-Myc mouse. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background: Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used. Methodology/Principal Findings: We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-gamma secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells. Conclusions/Significance: TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion. AU - Lichtenegger, F.S.* AU - Mueller, K.* AU - Otte, B.* AU - Beck, B.* AU - Hiddemann, W.* AU - Schendel, D.J. AU - Subklewe, M.* C1 - 10630 C2 - 30345 TI - CD86 and IL-12p70 are key players for T helper 1 polarization and natural killer cell activation by toll-like receptor-induced dendritic cells. JO - PLoS ONE VL - 7 IS - 9 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background: Atopic dermatitis (AD) is a common chronic inflammatory skin disorder where epidermal barrier dysfunction is a major factor in the pathogenesis. The identification of AD susceptibility genes related to barrier dysfunction is therefore of importance. The epidermal transglutaminases (TGM1, TGM3 and TGM5) encodes essential cross-linking enzymes in the epidermis. Objective: To determine whether genetic variability in the epidermal transglutaminases contributes to AD susceptibility. Methods: Forty-seven single nucleotide polymorphisms (SNPs) in the TGM1, TGM3 and TGM5 gene region were tested for genetic association with AD, independently and in relation to FLG genotype, using a pedigree disequilibrium test (PDT) in a Swedish material consisting of 1753 individuals from 539 families. In addition, a German case-control material, consisting of 533 AD cases and 1996 controls, was used for in silico analysis of the epidermal TGM regions. Gene expression of the TGM1, TGM3 and TGM5 gene was investigated by relative quantification with Real Time PCR (qRT-PCR). Immunohistochemical (IHC) analysis was performed to detect TG1, TG3 and TG5 protein expression in the skin of patients and healthy controls. Results: PDT analysis identified a significant association between the TGM1 SNP rs941505 and AD with allergen-specific IgE in the Swedish AD family material. However, the association was not replicated in the German case-control material. No significant association was detected for analyzed SNPs in relation to FLG genotype. TG1, TG3 and TG5 protein expression was detected in AD skin and a significantly increased TGM3 mRNA expression was observed in lesional skin by qRT-PCR. Conclusion: Although TGM1 and TGM3 may be differentially expressed in AD skin, the results from the genetic analysis suggest that genetic variation in the epidermal transglutaminases is not an important factor in AD susceptibility. AU - Liedén, A.* AU - Winge, M.C.G.* AU - Saaf, A.* AU - Kockum, I.* AU - Ekelund, E.* AU - Rodriguez, E.* AU - Fölster-Holst, R.* AU - Franke, A.* AU - Illig, T. AU - Tengvall-Linder, M.* AU - Baurecht, H.* AU - Weidinger, S.* AU - Wahlgren, C.-F.* AU - Nordenskjöld, M.* AU - Bradley, M.* C1 - 11649 C2 - 30730 TI - Genetic variation in the epidermal transglutaminase genes is not associated with atopic dermatitis. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Background: Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model. Results: Upon S. Tm-wt infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This "fast cycling" through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. Conclusion: In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut.e34812e34812 AU - Loetscher, Y.* AU - Wieser, A.* AU - Lengefeld, J.* AU - Kaiser, P.* AU - Schubert, S.* AU - Heikenwälder, M. AU - Hardt, W.D.* AU - Stecher, B.* C1 - 7615 C2 - 29869 TI - Salmonella transiently reside in luminal neutrophils in the inflamed gut. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Objective: To examine the associations between pet keeping in early childhood and asthma and allergies in children aged 6-10 years. Design: Pooled analysis of individual participant data of 11 prospective European birth cohorts that recruited a total of over 22,000 children in the 1990s. Exposure definition: Ownership of only cats, dogs, birds, rodents, or cats/dogs combined during the first 2 years of life. Outcome definition: Current asthma (primary outcome), allergic asthma, allergic rhinitis and allergic sensitization during 610 years of age. Data synthesis: Three-step approach: (i) Common definition of outcome and exposure variables across cohorts; (ii) calculation of adjusted effect estimates for each cohort; (iii) pooling of effect estimates by using random effects meta-analysis models. Results: We found no association between furry and feathered pet keeping early in life and asthma in school age. For example, the odds ratio for asthma comparing cat ownership with "no pets" (10 studies, 11489 participants) was 1.00 (95% confidence interval 0.78 to 1.28) (I 2 = 9%; p = 0.36). The odds ratio for asthma comparing dog ownership with "no pets" (9 studies, 11433 participants) was 0.77 (0.58 to 1.03) (I-2 = 0%, p = 0.89). Owning both cat(s) and dog(s) compared to "no pets" resulted in an odds ratio of 1.04 (0.59 to 1.84) (I-2 = 33%, p = 0.18). Similarly, for allergic asthma and for allergic rhinitis we did not find associations regarding any type of pet ownership early in life. However, we found some evidence for an association between ownership of furry pets during the first 2 years of life and reduced likelihood of becoming sensitized to aero-allergens. Conclusions: Pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged 6-10. Advice from health care practitioners to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given. AU - Lødrup Carlsen, K.C.* AU - Roll, S.* AU - Carlsen, K.-H.* AU - Mowinckel, P.* AU - Wijga, A.H.* AU - Brunekreef, B.* AU - Torrent, M.* AU - Roberts, G.* AU - Arshad, S.H.* AU - Kull, I.* AU - Krämer, U.* AU - von Berg, A.* AU - Eller, E.* AU - Host, A.* AU - Kuehni, C.* AU - Spycher, B.* AU - Sunyer, J.* AU - Chen, C.-M. AU - Reich, A.* AU - Asarnoj, A.* AU - Puig, C.* AU - Herbarth, O.* AU - John, J.M.M.* AU - van Steen, K.* AU - Willich, S.N.* AU - Wahn, U.* AU - Lau, S.* AU - Keil, T.* C1 - 10410 C2 - 30236 TI - Does pet ownership in infancy lead to asthma or allergy at school age? Pooled analysis of individual participant data from 11 European birth cohorts. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF(65)-SF1-RNA complex which occurs at the 3' end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions. AU - Manceau, V.* AU - Kremmer, E. AU - Nabel, E.G.* AU - Maucuer, A.* C1 - 10452 C2 - 30258 TI - The protein kinase KIS impacts gene expression during development and fear conditioning in adult mice. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [(11)C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [(11)C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [(11)C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice. AU - Manook, A.* AU - Yousefi, B.H.* AU - Willuweit, A.* AU - Platzer, S.* AU - Reder, S.* AU - Voss, A. AU - Huisman, M.* AU - Settles, M.* AU - Neff, F.* AU - Velden, J.* AU - Schoor, M.* AU - von der Kammer, H.* AU - Wester, H.J.* AU - Schwaiger, M.* AU - Henriksen, G.* AU - Drzezga, A..* C1 - 7380 C2 - 29689 TI - Small-animal PET imaging of amyloid-beta plaques with [11C]PiB and its multi-modal validation in an APP/PS1 mouse model of Alzheimer's disease. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: High serum uric acid (UA) levels are associated with the metabolic syndrome, type 2 diabetes and cardiovascular disease. It is largely unknown whether there are gender-specific differences regarding the association between UA and prediabetic states. We examined the possible association between UA levels and known as well as newly diagnosed diabetes (NDD), isolated impaired fasting glucose (i-IFG), isolated impaired glucose tolerance (i-IGT), and combined IFG/IGT in a population-based sample of 32-to-81-year-old men and women. RESEARCH DESIGN AND METHODS: An oral glucose tolerance test was carried out in all 2,740 participants without known diabetes of the Cooperative Health Research in the Region of Augsburg (KORA) F4 Study conducted between 2006 and 2008 in Southern Germany. Serum UA was analysed by the uricase method. RESULTS: In women after multivariable adjustment the associations between UA and i-IFG (OR 1.57, 95% CI 1.15-2.14), IFG/IGT (OR 1.52, 1.07-2.16), NDD (OR 1.67, 95% CI 1.28-2.17), and known diabetes (OR 1.47, 95% CI 1.18-1.82) remained significant, but the association with i-IGT (OR 1.14, 95% CI 0.95-1.36) lost significance. In contrast in men, after multivariable adjustment there was only a significant association between UA levels and i-IFG (OR 1.49, 95% CI 1.21-1.84), all other associations were non-significant (i-IGT: OR 1.09, IFG/IGT: OR 1.06, NDD: OR 0.91, known diabetes: OR 1.04; all p-values>0.05). CONCLUSIONS: Serum UA concentrations were associated with different categories of impaired glucose regulation in individuals from the general population, particularly in women. Further studies investigating the role of UA in the development of derangements in glucose metabolism are needed. AU - Meisinger, C. AU - Döring, A. AU - Stöckl, D. AU - Thorand, B. AU - Kowall, B.* AU - Rathmann, W.* C1 - 7684 C2 - 29797 TI - Uric acid is more strongly associated with impaired glucose regulation in women than in men from the general population: The KORA F4-study. JO - PLoS ONE VL - 7 IS - 5 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - We identified a congenital mechanobullous skin disorder in six calves on a single farm of an endangered German cattle breed in 2010. The condition presented as a large loss of skin distal to the fetlocks and at the mucosa of the muzzle. All affected calves were euthanized on humane grounds due to the severity, extent and progression of the skin and oral lesions. Examination of skin samples under light microscopy revealed detachment of the epidermis from the dermis at the level of the dermo epidermal junction, leading to the diagnosis of a subepidermal bullous dermatosis such as epidermolysis bullosa. The pedigree was consistent with monogenic autosomal recessive inheritance. We localized the causative mutation to an 18 Mb interval on chromosome 22 by homozygosity mapping. The COL7A1 gene encoding collagen type VII alpha 1 is located within this interval and COL7A1 mutations have been shown to cause inherited dystrophic epidermolysis bullosa (DEB) in humans. A SNP in the bovine COL7A1 exon 49 (c.4756C>T) was perfectly associated with the observed disease. The homozygous mutant T/T genotype was exclusively present in affected calves and their parents were heterozygous C/T confirming the assumed recessive mode of inheritance. All known cases and genotyped carriers were related to a single cow, which is supposed to be the founder animal. The mutant T allele was absent in 63 animals from 24 cattle breeds. The identified mutation causes a premature stop codon which leads to a truncated protein representing a complete loss of COL7A1 function (p.R1586*). We thus have identified a candidate causative mutation for this genetic disease using only three cases to unravel its molecular basis. Selection against this mutation can now be used to eliminate the mutant allele from the Rotes Hohenvieh breed. AU - Menoud, A.* AU - Welle, M.* AU - Tetens, J.* AU - Lichtner, P. AU - Drögemüller, C.* C1 - 7679 C2 - 29899 TI - A COL7A1 mutation causes dystrophic epidermolysis bullosa in Rotes Höhenvieh cattle. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Increased dietary intake of Selenium (Se) has been suggested to lower prostate cancer mortality, but supplementation trials have produced conflicting results. Se is incorporated into 25 selenoproteins. The aim of this work was to assess whether risk of prostate cancer is affected by genetic variants in genes coding for selenoproteins, either alone or in combination with Se status. 248 cases and 492 controls from an EPIC-Heidelberg nested case-control study were subjected to two-stage genotyping with an initial screening phase in which 384 tagging-SNPs covering 72 Se-related genes were determined in 94 cases and 94 controls using the Illumina Goldengate methodology. This analysis was followed by a second phase in which genotyping for candidate SNPs identified in the first phase was carried out in the full study using Sequenom. Risk of high-grade or advanced stage prostate cancer was modified by interactions between serum markers of Se status and genotypes for rs9880056 in SELK, rs9605030 and rs9605031 in TXNRD2, and rs7310505 in TXNRD1. No significant effects of SNPs on prostate cancer risk were observed when grade or Se status was not taken into account. In conclusion, the risk of high-grade or advanced-stage prostate cancer is significantly altered by a combination of genotype for SNPs in selenoprotein genes and Se status. The findings contribute to explaining the biological effects of selenium intake and genetic factors in prostate cancer development and highlight potential roles of thioredoxin reductases and selenoprotein K in tumour progression. AU - Méplan, C.* AU - Rohrmann, S.* AU - Steinbrecher, A.* AU - Schomburg, L.* AU - Jansen, E.* AU - Linseisen, J. AU - Hesketh, J.* C1 - 11378 C2 - 30644 TI - Polymorphisms in thioredoxin reductase and selenoprotein k genes and selenium status modulate risk of prostate cancer. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. METHODOLOGY/PRINCIPAL FINDINGS: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca(2+) ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca(2+) levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. CONCLUSIONS/SIGNIFICANCE: Since Ca(2+) and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract. AU - Meyer, D.* AU - Voigt, A.* AU - Widmayer, P.* AU - Borth, H.* AU - Huebner, S.* AU - Breit, A.* AU - Marschall, S. AU - Hrabě de Angelis, M. AU - Boehm, U.* AU - Meyerhof, W.* AU - Gudermann, T.* AU - Boekhoff, I.* C1 - 7104 C2 - 29608 TI - Expression of Tas1 taste receptors in mammalian spermatozoa: Functional role of Tas1r1 in regulating basal Ca2+ and cAMP concentrations in spermatozoa. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Tissue-specific transcripts are likely to be of importance for the corresponding organ. While attempting to define the specific transcriptome of the human lung, we identified the transcript of a yet uncharacterized protein, SFTA2. In silico analyses, biochemical methods, fluorescence imaging and animal challenge experiments were employed to characterize SFTA2. Human SFTA2 is located on Chr. 6p21.33, a disease-susceptibility locus for diffuse panbronchiolitis. RT-PCR verified the abundance of SFTA2-specific transcripts in human and mouse lung. SFTA2 is synthesized as a hydrophilic precursor releasing a 59 amino acid mature peptide after cleavage of an N-terminal secretory signal. SFTA2 has no recognizable homology to other proteins while orthologues are present in all mammals. SFTA2 is a glycosylated protein and specifically expressed in nonciliated bronchiolar epithelium and type II pneumocytes. In accordance with other hydrophilic surfactant proteins, SFTA2 did not colocalize with lamellar bodies but colocalized with golgin97 and clathrin-labelled vesicles, suggesting a classical secretory pathway for its expression and secretion. In the mouse lung, Sfta2 was significantly downregulated after induction of an inflammatory reaction by intratracheal lipopolysaccharides paralleling surfactant proteins B and C but not D. Hyperoxia, however, did not alter SFTA2 mRNA levels. We have characterized SFTA2 and present it as a novel unique secretory peptide highly expressed in the lung. AU - Mittal, R.A.* AU - Hammel, M.* AU - Schwarz, J. AU - Heschl, K.M.* AU - Bretschneider, N.* AU - Flemmer, A.W.* AU - Herber-Jonat, S.* AU - Königshoff, M. AU - Eickelberg, O. AU - Holzinger, A.* C1 - 7912 C2 - 29912 TI - SFTA2 - a novel secretory peptide highly expressed in the lung - is modulated by lipopolysaccharide but not hyperoxia. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 µg LPS to the airways and 5 µg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 µg LPS targeted to the alveoli caused significantly stronger effects compared to 5 µg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs. AU - Möller, W. AU - Heimbeck, I. AU - Hofer, T.P. AU - Khadem-Saba, G. AU - Neiswirth, M.* AU - Frankenberger, M. AU - Ziegler-Heitbrock, L. C1 - 7388 C2 - 29695 TI - Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Genetics of the variability of normal and diseased brain structure largely remains to be elucidated. Expansions of certain trinucleotide repeats cause neurodegenerative disorders of which Huntington's disease constitutes the most common example. Here, we test the hypothesis that variation within the IT15 gene on chromosome 4, whose expansion causes Huntington's disease, influences normal human brain structure. In 278 normal subjects, we determined CAG repeat length within the IT15 gene on chromosome 4 and analyzed high-resolution T1-weighted magnetic resonance images by the use of voxel-based morphometry. We found an increase of GM with increasing long CAG repeat and its interaction with age within the pallidum, which is involved in Huntington's disease. Our study demonstrates that a certain trinucleotide repeat influences normal brain structure in humans. This result may have important implications for the understanding of both the healthy and diseased brain. AU - Mühlau, M.* AU - Winkelmann, J. AU - Rujescu, D.* AU - Giegling, I.* AU - Koutsouleris, N.* AU - Gaser, C.* AU - Arsic, M.* AU - Weindl, A.* AU - Reiser, M.* AU - Meisenzahl, E.M.* C1 - 7139 C2 - 29472 TI - Variation within the Huntington's disease gene influences normal brain structure. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300-600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573. AU - Neukamm, S.S. AU - Toth, R.* AU - Morrice, N.* AU - Campbell, D.G.* AU - MacKintosh, C.* AU - Lehmann, R. AU - Häring, H.-U. AU - Schleicher, E.D. AU - Weigert, C. C1 - 10643 C2 - 30316 TI - Identification of the amino acids 300-600 of IRS-2 as 14-3-3 binding region with the importance of IGF-1/insulin-regulated phosphorylation of Ser-573. JO - PLoS ONE VL - 7 IS - 8 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice. AU - Noblanc, A.* AU - Peltier, M.* AU - Damon-Soubeyrand, C.* AU - Kerchkove, N.* AU - Chabory, E.* AU - Vernet, P.* AU - Saez, F.* AU - Cadet, R.* AU - Janny, L.* AU - Pons-Rejraji, H.* AU - Conrad, M. AU - Drevet, J.R.* AU - Kocer, A.* C1 - 7677 C2 - 29900 TI - Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations. AU - Palmer, N.D.* AU - McDonough, C.W.* AU - Hicks, P.J.* AU - Roh, B.H.* AU - Wing, M.R.* AU - An, S.S.* AU - Hester, J.M.* AU - Cooke, J.N.* AU - Bostrom, M.A.* AU - Rudock, M.E.* AU - Talbert, M.E.* AU - Lewis, J.P* AU - DIAGRAM Consortium (Huth, C. AU - Grallert, H. AU - Gieger, C. AU - Klopp, N. AU - Meitinger, T. AU - Petersen, A.-K. AU - Wichmann, H.-E. AU - Illig, T.) AU - MAGIC Investigators (Grallert, H. AU - Gieger, C. AU - Meisinger, C. AU - Wichmann, H.-E. AU - Illig, T. AU - Thorand, B.) AU - Ferrara, A.* AU - Lu, L.* AU - Ziegler, J.T.* AU - Sale, M.M.* AU - Divers, J.* AU - Shriner, D.* AU - Adeyemo, A.* AU - Rotimi, C.N.* AU - Ng, M.C.* AU - Langefeld, C.D.* AU - Freedman, B.I.* AU - Bowden, D.W* C1 - 7319 C2 - 29681 TI - A genome-wide association search for type 2 diabetes genes in African Americans. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ∼4% read abundance across biological, and ∼2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities. AU - Pilloni, G. AU - Granitsiotis, M.S. AU - Engel, M. AU - Lüders, T. C1 - 7923 C2 - 29921 TI - Testing the limits of 454 pyrotag sequencing: Reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220(+) bone marrow cells, CD19(-) thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis. AU - Pozzi, B.* AU - Amodio, S.* AU - Lucano, C.* AU - Sciullo, A.* AU - Ronzoni, S.* AU - Castelletti, D.* AU - Adler, T. AU - Treise, I. AU - Holmberg-Betsholtz, I.* AU - Rathkolb, B. AU - Busch, D.H.* AU - Wolf, E.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Betsholtz, C.* AU - Casola, S.* AU - di Fiore, P.P.* AU - Offenhäuser, N.* C1 - 11477 C2 - 30688 TI - The endocytic adaptor Eps15 controls marginal zone B cell numbers. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - In most studies aimed at localizing footprints of past selection, outliers at tails of the empirical distribution of a given test statistic are assumed to reflect locus-specific selective forces. Significance cutoffs are subjectively determined, rather than being related to a clear set of hypotheses. Here, we define an empirical p-value for the summary statistic by means of a permutation method that uses the observed SNP structure in the real data. To illustrate the methodology, we applied our approach to a panel of 2.9 million autosomal SNPs identified from re-sequencing a pool of 15 individuals from a brown egg layer line. We scanned the genome for local reductions in heterozygosity, suggestive of selective sweeps. We also employed a modified sliding window approach that accounts for gaps in the sequence and increases scanning resolution by moving the overlapping windows by steps of one SNP only, and suggest to call this a "creeping window'' strategy. The approach confirmed selective sweeps in the region of previously described candidate genes, i.e. TSHR, PRL, PRLHR, INSR, LEPR, IGF1, and NRAMP1 when used as positive controls. The genome scan revealed 82 distinct regions with strong evidence of selection (genome-wide p-value<0.001), including genes known to be associated with eggshell structure and immune system such as CALB1 and GAL cluster, respectively. A substantial proportion of signals was found in poor gene content regions including the most extreme signal on chromosome 1. The observation of multiple signals in a highly selected layer line of chicken is consistent with the hypothesis that egg production is a complex trait controlled by many genes. AU - Qanbari, S.* AU - Strom, T.M. AU - Haberer, G. AU - Weigend, S.* AU - Gheyas, A.A.* AU - Turner, F.* AU - Burt, D.W.* AU - Preisinger, R.* AU - Gianola, D.* AU - Simianer, H.* C1 - 11648 C2 - 30731 TI - A high resolution genome-wide scan for significant selective sweeps: An application to pooled sequence data in laying chickens. JO - PLoS ONE VL - 7 IS - 11 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Asthma has substantial morbidity and mortality and a strong genetic component, but identification of genetic risk factors is limited by availability of suitable studies. OBJECTIVES: To test if population-based cohorts with self-reported physician-diagnosed asthma and genome-wide association (GWA) data could be used to validate known associations with asthma and identify novel associations. METHODS: The APCAT (Analysis in Population-based Cohorts of Asthma Traits) consortium consists of 1,716 individuals with asthma and 16,888 healthy controls from six European-descent population-based cohorts. We examined associations in APCAT of thirteen variants previously reported as genome-wide significant (P<5 x 10(-8)) and three variants reported as suggestive (P<5× 10(-7)). We also searched for novel associations in APCAT (Stage 1) and followed-up the most promising variants in 4,035 asthmatics and 11,251 healthy controls (Stage 2). Finally, we conducted the first genome-wide screen for interactions with smoking or hay fever. MAIN RESULTS: We observed association in the same direction for all thirteen previously reported variants and nominally replicated ten of them. One variant that was previously suggestive, rs11071559 in RORA, now reaches genome-wide significance when combined with our data (P = 2.4 × 10(-9)). We also identified two genome-wide significant associations: rs13408661 near IL1RL1/IL18R1 (P(Stage1+Stage2) = 1.1x10(-9)), which is correlated with a variant recently shown to be associated with asthma (rs3771180), and rs9268516 in the HLA region (P(Stage1+Stage2) = 1.1x10(-8)), which appears to be independent of previously reported associations in this locus. Finally, we found no strong evidence for gene-environment interactions with smoking or hay fever status. CONCLUSIONS: Population-based cohorts with simple asthma phenotypes represent a valuable and largely untapped resource for genetic studies of asthma. AU - Ramasamy, A.* AU - Kuokkanen, M.* AU - Vedantam, S.* AU - Gajdos, Z.K.* AU - Couto Alves, A.* AU - Lyon, H.N.* AU - Ferreira, M.A.* AU - Strachan, D.P.* AU - Zhao, J.H.* AU - Abramson, M.J.* AU - Brown, M.A.* AU - Coin, L.* AU - Dharmage, S.C.* AU - Duffy, D.L.* AU - Haahtela, T.* AU - Heath, A.C.* AU - Janson, C.* AU - Kähönen, M.* AU - Khaw, K.T.* AU - Laitinen, J.* AU - Le Souef, P.* AU - Lehtimäki, T.* AU - Australian Asthma Genetics Consortium (AAGC) (*) AU - Madden, P.A.* AU - Marks, G.B.* AU - Martin, N.G.* AU - Matheson, M.C.* AU - Palmer, C.D.* AU - Palotie, A.* AU - Pouta, A.* AU - Robertson, C.F.* AU - Viikari, J.* AU - Widen, E.* AU - Wjst, M. AU - Jarvis, D.L.* AU - Montgomery, G.W.* AU - Thompson, P.J.* AU - Wareham, N.J.* AU - Eriksson, J.* AU - Jousilahti, P.* AU - Laitinen, T.* AU - Pekkanen, J.* AU - Raitakari, O.T.* AU - O'Connor, G.T.* AU - Salomaa, V.* AU - Jarvelin, M.R.* AU - Hirschhorn, J.N.* C1 - 11212 C2 - 30551 TI - Genome-wide association studies of asthma in population-based cohorts confirm known and suggested loci and identify an additional association near HLA. JO - PLoS ONE VL - 7 IS - 9 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Acute mountain sickness (AMS) is characterized by headache often accompanied by gastrointestinal complaints that vary from anorexia through nausea to vomiting. The aim of this study was to investigate the influence of high altitude on plasma levels of gastroenteropancreatic (GEP) peptides and their association to AMS symptoms. Plasma levels of 6 GEP peptides were measured by radioimmunoassay in 11 subjects at 490 m (Munich, Germany) and, after rapid passive ascent to 3454 m (Jungfraujoch, Switzerland), over the course of three days. In a second study (n = 5), the same peptides and ghrelin were measured in subjects who consumed standardized liquid meals at these two elevations. AMS symptoms and oxygen saturation were monitored. In the first study, both fasting (morning 8 a. m.) and stimulated (evening 8 p. m.) plasma levels of pancreatic polypeptide (PP) and cholecystokinin (CCK) were significantly lower at high altitude as compared to baseline, whereas gastrin and motilin concentrations were significantly increased. Fasting plasma neurotensin was significantly enhanced whereas stimulated levels were reduced. Both fasting and stimulated plasma motilin levels correlated with gastrointestinal symptom severity (r = 0.294, p = 0.05, and r = 0.41, p = 0.006, respectively). Mean O2-saturation dropped from 96% to 88% at high altitude. In the second study, meal-stimulated integrated (= area under curve) plasma CCK, PP, and neurotensin values were significantly suppressed at high altitude, whereas integrated levels of gastrin were increased and integrated VIP and ghrelin levels were unchanged. In summary, our data show that acute exposure to a hypobaric hypoxic environment causes significant changes in fasting and stimulated plasma levels of GEP peptides over consecutive days and after a standardized meal. The changes of peptide levels were not uniform. Based on the inhibition of PP and neurotensin release a reduction of the cholinergic tone can be postulated. AU - Riepl, R.L.* AU - Fischer, R.* AU - Hautmann, H.* AU - Hartmann, G.* AU - Müller, T.D. AU - Tschöp, M.H. AU - Toepfer, M.* AU - Otto, B.* C1 - 10444 C2 - 30246 TI - Influence of acute exposure to high altitude on basal and postprandial plasma levels of gastroenteropancreatic peptides. JO - PLoS ONE VL - 7 IS - 9 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is expressed in all diabetes-relevant tissues and mediates cytokine-induced insulin resistance. We investigated whether common single nucleotide polymorphisms (SNPs) in the MAP4K4 locus associate with glucose intolerance, insulin resistance, impaired insulin release, or elevated plasma cytokines. The best hit was tested for association with type 2 diabetes. Subjects (N = 1,769) were recruited from the Tübingen Family (TÜF) study for type 2 diabetes and genotyped for tagging SNPs. In a subgroup, cytokines were measured. Association with type 2 diabetes was tested in a prospective case-cohort study (N = 2,971) derived from the EPIC-Potsdam study. Three SNPs (rs6543087, rs17801985, rs1003376) revealed nominal and two SNPs (rs11674694, rs11678405) significant associations with 2-hour glucose levels. SNPs rs6543087 and rs11674694 were also nominally associated with decreased insulin sensitivity. Another two SNPs (rs2236936, rs2236935) showed associations with reduced insulin release, driven by effects in lean subjects only. Three SNPs (rs11674694, rs13003883, rs2236936) revealed nominal associations with IL-6 levels. SNP rs11674694 was significantly associated with type 2 diabetes. In conclusion, common variation in MAP4K4 is associated with insulin resistance and β-cell dysfunction, possibly via this gene's role in inflammatory signalling. This variation's impact on insulin sensitivity may be more important since its effect on insulin release vanishes with increasing BMI. AU - Sartorius, T. AU - Staiger, H. AU - Ketterer, C. AU - Heni, M.* AU - Machicao, F. AU - Guilherme, A.* AU - Grallert, H. AU - Schulze, M.B. AU - Boeing, H. AU - Stefan, N. AU - Fritsche, A. AU - Czech, M.P.* AU - Häring, H.-U. C1 - 11410 C2 - 30654 TI - Association of common genetic variants in the MAP4K4 locus with prediabetic traits in humans. JO - PLoS ONE VL - 7 IS - 10 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity. CONCLUSIONS/SIGNIFICANCE: In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition. AU - Schilling, D.* AU - Bayer, C.* AU - Li, W.* AU - Molls, M.* AU - Vaupel, P.* AU - Multhoff, G. C1 - 7170 C2 - 29514 TI - Radiosensitization of normoxic and hypoxic h1339 lung tumor cells by heat shock protein 90 inhibition is independent of hypoxia inducible factor-1α. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array. Gene expression signal intensities were similar after applying the log(2) or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33-48% of the variance), the RNA amplification batch (12-24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2-3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1-2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses. AU - Schurmann, C.* AU - Heim, K. AU - Schillert, A.* AU - Blankenberg, S.* AU - Carstensen, M.* AU - Dörr, M.* AU - Endlich, K.* AU - Felix, S.B.* AU - Gieger, C. AU - Grallert, H. AU - Herder, C.* AU - Hoffmann, W. AU - Homuth, G.* AU - Illig, T. AU - Kruppa, J.* AU - Meitinger, T. AU - Müller, C.* AU - Nauck, M.* AU - Peters, A. AU - Rettig, R.* AU - Roden, M.* AU - Strauch, K. AU - Völker, U.* AU - Völzke, H.* AU - Wahl, S. AU - Wallaschofski, H.* AU - Wild, P.S.* AU - Zeller, T.* AU - Teumer, A.* AU - Prokisch, H. AU - Ziegler, A.* C1 - 11641 C2 - 30734 TI - Analyzing illumina gene expression microarray data from different tissues: Methodological aspects of data analysis in the MetaXpress consortium. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies. AU - Slotta-Huspenina, J.* AU - Berg, D.* AU - Bauer, K.* AU - Wolff, C.* AU - Malinowsky, K.* AU - Bauer, L.* AU - Drecoll, E.* AU - Bettstetter, M.* AU - Feith, M.* AU - Walch, A.K. AU - Höfler, H. AU - Becker, K.F.* AU - Langer, R.* C1 - 8448 C2 - 30124 TI - Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Elevated cholesterol levels in children can be a risk factor for cardiovascular diseases in later life. In adults, it has been shown that blood lipid levels are strongly influenced by polymorphisms in the fatty acid desaturase (FADS) gene cluster in addition to nutritional and other exogenous and endogenous determinants. Our aim was to investigate whether lipid levels are determined by the FADS genotype already in children and whether this association interacts with dietary intake of n-3 fatty acids. METHODS: The analysis was based on data of 2006 children from two German prospective birth cohort studies. Total cholesterol, HDL, LDL and triglycerides were measured at 10 years of age. Six single nucleotide polymorphisms (SNPs) of the FADS gene cluster were genotyped. Dietary n-3 fatty acid intake was assessed by food frequency questionnaire. Linear regression modeling was used to assess the association between lipid levels, n-3 fatty acid intake and FADS genotype. RESULTS: Individuals carrying the homozygous minor allele had lower levels of total cholesterol [means ratio (MR) ranging from 0.96 (p = 0.0093) to 0.98 (p = 0.2949), depending on SNPs] and LDL [MR between 0.94 (p = 0.0179) and 0.97 (p = 0.2963)] compared to homozygous major allele carriers. Carriers of the heterozygous allele showed lower HDL levels [β between -0.04 (p = 0.0074) to -0.01 (p = 0.3318)] and higher triglyceride levels [MR ranging from 1.06 (p = 0.0065) to 1.07 (p = 0.0028)] compared to homozygous major allele carriers. A higher n-3 PUFA intake was associated with higher concentrations of total cholesterol, LDL, HDL and lower triglyceride levels, but these associations did not interact with the FADS1 FADS2 genotype. CONCLUSION: Total cholesterol, HDL, LDL and triglyceride concentrations may be influenced by the FADS1 FADS2 genotype already in 10 year old children. Genetically determined blood lipid levels during childhood might differentially predispose individuals to the development of cardiovascular diseases later in life. AU - Standl, M. AU - Lattka, E. AU - Stach, B.* AU - Koletzko, S.* AU - Bauer, C.P.* AU - von Berg, A.* AU - Berdel, D.* AU - Krämer, U.* AU - Schaaf, B.* AU - Roder, S.* AU - Herbarth, O.* AU - Buyken, A.* AU - Drogies, T.* AU - Thiery, J.* AU - Koletzko, B.* AU - Heinrich, J. AU - GINIplus Study Group (Heinrich, J. AU - Wichmann, H.-E. AU - Popescu, M. AU - Schoetzau, A. AU - Franke, K. AU - Mosetter, M. AU - Sausenthaler, S. AU - Schindler, J. AU - Zirngibl, A. AU - Thaqi, A. AU - Zutavern, A. AU - Laubereau, B.) AU - LISAplus Study Group (Wichmann, H.-E. AU - Bolte, G. AU - Belcredi, P. AU - Jacob, B. AU - Schoetzau, A. AU - Schindler, J. AU - Höhnke, A. AU - Heinrich, J. AU - Mosetter, M. AU - Franke, K. AU - Laubereau, B. AU - Sausenthaler, S. AU - Thaqi, A. AU - Zirngibl, A. AU - Zutavern, A.) C1 - 7544 C2 - 29790 TI - FADS1 FADS2 gene cluster, PUFA intake and blood lipids in children: Results from the GINIplus and LISAplus studies. JO - PLoS ONE VL - 7 IS - 5 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Cln3(Delta ex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Delta ex7/8) mice. Homozygous Cln3(Delta ex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Delta ex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post- receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12- 13 week old homozygous Cln3(Delta ex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Delta ex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Delta ex7/8) mice, and male homozygotes had a relative T- cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Delta ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Delta ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Delta ex7/8) mice that merit further study for JNCL biomarker development. AU - Staropoli, J.F.* AU - Haliw, L.* AU - Biswas, S.* AU - Garrett, L. AU - Hölter, S.M. AU - Becker, L. AU - Skosyrski, S.* AU - Da Silva-Buttkus, P.* AU - Calzada-Wack, J.* AU - Neff, F.* AU - Rathkolb, B. AU - Rozman, J. AU - Schrewe, A. AU - Adler, T. AU - Puk, O. AU - Sun, M. AU - Favor, J.* AU - Rácz, I.* AU - Bekeredjian, R.* AU - Busch, D.H.* AU - Graw, J. AU - Klingenspor, M.* AU - Klopstock, T.* AU - Wolf, E.* AU - Wurst, W. AU - Zimmer, A.* AU - Lopez, E.* AU - Harati, H.* AU - Hill, E.* AU - Krause, D.S.* AU - Guide, J.* AU - Dragileva, E.* AU - Gale, E.* AU - Wheeler, V.C.* AU - Boustany, R.M.* AU - Brown, D.E.* AU - Breton, S.* AU - Ruether, K.* AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Cotman, S.L.* C1 - 7680 C2 - 30144 TI - Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system. JO - PLoS ONE VL - 7 IS - 6 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve - especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research. AU - Stemberger, C.* AU - Dreher, S.* AU - Tschulik, C.* AU - Piossek, C.* AU - Bet, J.* AU - Yamamoto, T.N.* AU - Schiemann, M. AU - Neuenhahn, M.* AU - Martin, K.* AU - Schlapschy, M.* AU - Skerra, A.* AU - Schmidt, T.* AU - Edinger, M.* AU - Riddell, S.R.* AU - Germeroth, L.* AU - Busch, D.H. C1 - 7691 C2 - 29886 TI - Novel serial positive enrichment technology enables clinical multiparameter cell sorting. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: Hyperuricemia is associated with an increased risk of metabolic and cardiovascular diseases. There are pronounced sex differences in the levels of uric acid. It is largely unknown whether or not reproductive parameters which induce hormonal changes are responsible for this. We examined if there are associations between reproductive parameters and uric acid levels in a female population-based sample. METHODS: In this cross-sectional analysis, data of 1530 women aged 32 to 81 years participating in the KORA F4 study, conducted between 2006 and 2008 in Southern Germany were used. Reproductive parameters were obtained by standardized interviews. Uric acid levels were tested by the uricase method. The whole study sample and stratified in pre- and postmenopausal women was analyzed. RESULTS: Menopausal status and earlier age at menarche were associated with higher serum uric acid levels (age-adjusted: p-values 0.003, <0.001 respectively; after multivariable adjustment, including BMI: p-values 0.002, 0.036). A history of oral contraceptive use showed an association with uric acid levels only after multivariable adjustment (p-value 0.009). Hot flushes showed an association with uric acid levels only after age-adjustment (p-value 0.038), but lost significance after adding other confounders. Other reproductive factors, including parity, current or ever use of hormone replacement therapy, current use of oral contraceptives, hysterectomy, bilateral oophorectomy, or depressive mood related to menopausal transition were not associated with uric acid levels. CONCLUSIONS: Postmenopausal status, earlier age at menarche and a history of oral contraceptive use were independently associated with higher serum uric acid concentrations in women from the general population. Further studies, especially longitudinal population-based studies investigating the relationship of female reproductive parameters with uric acid levels are necessary to confirm our findings. AU - Stöckl, D. AU - Döring, A. AU - Thorand, B. AU - Heier, M. AU - Belcredi, P. AU - Meisinger, C. C1 - 7299 C2 - 29660 TI - Reproductive factors and serum uric acid levels in females from the general population: The KORA F4 study. JO - PLoS ONE VL - 7 IS - 3 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology. AU - Swadzba, M.E.* AU - Hauck, S.M. AU - Naim, H.Y.* AU - Amann, B.* AU - Deeg, C.A.* C1 - 11390 C2 - 30639 TI - Retinal glycoprotein enrichment by concanavalin A enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research. AU - Taruttis, A. AU - Morscher, S. AU - Burton, N.C. AU - Razansky, D. AU - Ntziachristos, V. C1 - 7140 C2 - 29473 TI - Fast multispectral optoacoustic tomography (MSOT) for dynamic imaging of pharmacokinetics and biodistribution in multiple organs. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter. DESIGN AND METHODS: Patients' acute lymphoblastic leukemia cells were lentivirally transduced to express the membrane-bound form of Gaussia luciferase. In vivo imaging was established in individual patients' leukemias and extensively validated. RESULTS: Bioluminescence in vivo imaging enabled reliable and continuous follow-up of individual mice. Light emission strictly correlated to post mortem quantification of leukemic burden and revealed a logarithmic, time and cell number dependent growth pattern. Imaging conveniently quantified frequencies of leukemia initiating cells in limiting dilution transplantation assays. Upon detecting a single leukemia cell within more than 10,000 bone marrow cells, imaging enabled monitoring minimal residual disease, time to tumor re-growth and relapse. Imaging quantified therapy effects precisely and with low variances, discriminating treatment failure from partial and complete responses. CONCLUSIONS: For the first time, we characterized in detail how in vivo imaging reforms preclinical studies on patient-derived tumors upon increasing monitoring resolution. In the future, in vivo imaging will enable performing precise preclinical studies on a broad range of highly demanding clinical challenges, such as treatment failure, resistance in leukemia initiating cells, minimal residual disease and relapse. AU - Terziyska, N. AU - Alves, C.C. AU - Groiss, V. AU - Schneider, K. AU - Farkasova, K.* AU - Ogris, M.* AU - Wagner, E.* AU - Ehrhardt, H. AU - Brentjens, R.J.* AU - Zur Stadt, U.* AU - Horstmann, M.* AU - Quintanilla-Martinez, L.* AU - Jeremias, I. C1 - 11899 C2 - 30844 TI - In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - In the last decade breeders of Romagnola cattle observed an outbreak of a new congenital anomaly. This lethal multi-organ developmental dysplasia is mainly characterized by facial deformities, ascites and hepatic fibrosis. Affected stillborn calves were inbred to a single founder sire suggesting autosomal monogenic recessive inheritance. We localized the causative mutation to a 1.2 Mb interval on BTA 17 by genome-wide association and identical by descent mapping. A solution-based method for targeted DNA capture combined with massively parallel sequencing was used to analyze the entire critical region containing 24 genes. Homozygosity for two non-synonymous coding sequence variants affecting the RNF34 and KDM2B genes was detected by evaluating one affected calf. Here we show that the disease phenotype is associated with a KDM2B missense mutation (c.2503G>A) leading to an amino acid exchange (p.D835N) in an evolutionary strongly conserved domain. In addition, the genetic makeup of three inbred cattle strongly supports the causality of the KDM2B mutation. This report of a naturally-occurring spontaneous mutation of a JmjC domain containing histone demethylase gene provides evidence for their important role in the endo-and mesodermal organ development. AU - Testoni, S.* AU - Bartolone, E.* AU - Rossi, M.* AU - Patrignani, A.* AU - Bruggmann, R.* AU - Lichtner, P. AU - Tetens, J.* AU - Gentile, A.* AU - Drögemüller, C.* C1 - 10826 C2 - 30404 TI - KDM2B is implicated in bovine lethal multi-organic developmental dysplasia. JO - PLoS ONE VL - 7 IS - 9 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future. AU - Treutlein, G.* AU - Dorsch, R.* AU - Euler, K.N.* AU - Hauck, S.M. AU - Amann, B.* AU - Hartmann, K.* AU - Deeg, C.A.* C1 - 11468 C2 - 30677 TI - Novel potential interacting partners of fibronectin in spontaneous animal model of interstitial cystitis. JO - PLoS ONE VL - 7 IS - 12 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Biogenically released isoprene plays important roles in both tropospheric photochemistry and plant metabolism. We performed a (13)CO(2)-labeling study using proton-transfer-reaction mass spectrometry (PTR-MS) to examine the kinetics of recently assimilated photosynthate into isoprene emitted from poplar (Populus × canescens) trees grown and measured at different atmospheric CO(2) concentrations. This is the first study to explicitly consider the effects of altered atmospheric CO(2) concentration on carbon partitioning to isoprene biosynthesis. We studied changes in the proportion of labeled carbon as a function of time in two mass fragments, M41(+), which represents, in part, substrate derived from pyruvate, and M69(+), which represents the whole unlabeled isoprene molecule. We observed a trend of slower (13)C incorporation into isoprene carbon derived from pyruvate, consistent with the previously hypothesized origin of chloroplastic pyruvate from cytosolic phosphenolpyruvate (PEP). Trees grown under sub-ambient CO(2) (190 ppmv) had rates of isoprene emission and rates of labeling of M41(+) and M69(+) that were nearly twice those observed in trees grown under elevated CO(2) (590 ppmv). However, they also demonstrated the lowest proportion of completely labeled isoprene molecules. These results suggest that under reduced atmospheric CO(2) availability, more carbon from stored/older carbon sources is involved in isoprene biosynthesis, and this carbon most likely enters the isoprene biosynthesis pathway through the pyruvate substrate. We offer direct evidence that extra-chloroplastic rather than chloroplastic carbon sources are mobilized to increase the availability of pyruvate required to up-regulate the isoprene biosynthesis pathway when trees are grown under sub-ambient CO(2). AU - Trowbridge, A.M.* AU - Asensio, D.* AU - Eller, A.S.D.* AU - Way, D.A.* AU - Wilkinson, M.J.* AU - Schnitzler, J.-P. AU - Jackson, R.B.* AU - Monson, R.K.* C1 - 7508 C2 - 29767 TI - Contribution of various carbon sources toward isoprene biosynthesis in poplar leaves mediated by altered atmospheric CO2 concentrations. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - o date there is a lack of tools to map the spatio-temporal dynamics of diverse cells in experimental heart models. Conventional histology is labor intensive with limited coverage, whereas many imaging techniques do not have sufficiently high enough spatial resolution to map cell distributions. We have designed and built a high resolution, dual channel Born-normalized near-infrared fluorescence optical projection tomography system to quantitatively and spatially resolve molecular agents distribution within whole murine heart. We validated the use of the system in a mouse model of monocytes/macrophages recruitment during myocardial infarction. While acquired, data were processed and reconstructed in real time. Tomographic analysis and visualization of the key inflammatory components were obtained via a mathematical formalism based on left ventricular modeling. We observed extensive monocyte recruitment within and around the infarcted areas and discovered that monocytes were also extensively recruited into non-ischemic myocardium, beyond that of injured tissue, such as the septum. AU - Vinegoni, C.* AU - Fumene Feruglio, P.* AU - Razansky, D. AU - Gorbatov, R.* AU - Ntziachristos, V. AU - Sbarbati, A.* AU - Nahrendorf, M.* AU - Weissleder, R.* C1 - 7496 C2 - 29756 TI - Mapping molecular agents distributions in whole mice hearts using Born-normalized optical projection tomography. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Systems Biology is a field in biological science that focuses on the combination of several or all "omics"-approaches in order to find out how genes, transcripts, proteins and metabolites act together in the network of life. Metabolomics as analog to genomics, transcriptomics and proteomics is more and more integrated into biological studies and often transcriptomic and metabolomic experiments are combined in one setup. At a first glance both data types seem to be completely different, but both produce information on biological entities, either transcripts or metabolites. Both types can be overlaid on metabolic pathways to obtain biological information on the studied system. For the joint analysis of both data types the MassTRIX webserver was updated. MassTRIX is freely available at www.masstrix.org. AU - Wägele, B. AU - Witting, M. AU - Schmitt-Kopplin, P. AU - Suhre, K. C1 - 8442 C2 - 30171 TI - MassTRIX reloaded: Combined analysis and visualization of transcriptome and metabolome data. JO - PLoS ONE VL - 7 IS - 7 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform. AU - Walker, S.L.* AU - Ariga, J.* AU - Mathias, J.R.* AU - Coothankandaswamy, V.* AU - Xie, X.* AU - Distel, M. AU - Köster, R.W. AU - Parsons, M.J.* AU - Bhalla, K.N.* AU - Saxena, M.T.* AU - Mumm, J.S.* C1 - 7135 C2 - 29469 TI - Automated reporter quantification in vivo: High-throughput screening method for reporter-based assays in zebrafish. JO - PLoS ONE VL - 7 IS - 1 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype.Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain. AU - Wefers, B. AU - Hitz, C. AU - Hölter, S.M. AU - Trümbach, D. AU - Hansen, J. AU - Weber, P.* AU - Pütz, B.* AU - Deussing, J.M.* AU - Hrabě de Angelis, M. AU - Roenneberg, T.* AU - Zheng, F.* AU - Alzheimer, C.* AU - Silva, A.* AU - Wurst, W. AU - Kühn, R. C1 - 7495 C2 - 29755 TI - MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - AIM: To investigate whether type 2 diabetes susceptibility genes and body weight influence the development of islet autoantibodies and the rate of progression to type 1 diabetes. METHODS: Genotyping for single nucleotide polymorphisms (SNP) of the type 2 diabetes susceptibility genes CDKAL1, CDKN2A/2B, FTO, HHEX-IDE, HMGA2, IGF2BP2, KCNJ11, KCNQ1, MTNR1B, PPARG, SLC30A8 and TCF7L2 was obtained in 1350 children from parents with type 1 diabetes participating in the BABYDIAB study. Children were prospectively followed from birth for islet autoantibodies and type 1 diabetes. Data on weight and height were obtained at 9 months, 2, 5, 8, 11, and 14 years of age. RESULTS: None of type 2 diabetes risk alleles at the CDKAL1, CDKN2A/2B, FTO, HHEX-IDE, HMGA2, IGF2BP2, KCNJ11, KCNQ1, MTNR1B, PPARG and SLC30A8 loci were associated with the development of islet autoantibodies or diabetes. The type 2 diabetes susceptible genotype of TCF7L2 was associated with a lower risk of islet autoantibodies (7% vs. 12% by age of 10 years, P = 0.015, P(corrected) = 0.18). Overweight children at seroconversion did not progress to diabetes faster than non-overweight children (HR: 1.08; 95% CI: 0.48-2.45, P>0.05). CONCLUSIONS: These findings do not support an association of type 2 diabetes risk factors with islet autoimmunity or acceleration of diabetes in children with a family history of type 1 diabetes. AU - Winkler, C. AU - Raab, J.* AU - Grallert, H. AU - Ziegler, A.-G. C1 - 7494 C2 - 29754 TI - Lack of association of type 2 diabetes susceptibility genotypes and body weight on the development of islet autoimmunity and type 1 diabetes. JO - PLoS ONE VL - 7 IS - 4 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - Ghrelin acylation by ghrelin O-acyltransferase (GOAT) has recently been reported to be essential for the prevention of hypoglycemia during prolonged negative energy balance. Using a unique set of four different genetic loss-of-function models for the GOAT/ghrelin/growth hormone secretagogue receptor (GHSR) system, we thoroughly tested the hypothesis that lack-of-ghrelin activation or signaling would lead to hypoglycemia during caloric deprivation. METHODOLOGY: Male and female knockout (KO) mice for GOAT, ghrelin, GHSR, or both ghrelin and GHSR (dKO) were subjected to prolonged calorie restriction (40% of ad libitum chow intake). Body weight, fat mass, and glucose levels were recorded daily and compared to wildtype (WT) controls. Forty-eight hour blood glucose profiles were generated for each individual mouse when 2% or less body fat mass was reached. Blood samples were obtained for analysis of circulating levels of acyl- and desacyl-ghrelin, IGF-1, and insulin. PRINCIPAL FINDINGS: Chronic calorie restriction progressively decreased body weight and body fat mass in all mice regardless of genotype. When fat mass was depleted to 2% or less of body weight for 2 consecutive days, random hypoglycemic events occurred in some mice across all genotypes. There was no increase in the incidence of hypoglycemia in any of the four loss-of-function models for ghrelin signaling including GOAT KO mice. Furthermore, no differences in insulin or IGF-1 levels were observed between genotypes. CONCLUSION: The endogenous GOAT-ghrelin-GHSR system is not essential for the maintenance of euglycemia during prolonged calorie restriction. AU - Yi, C.-X. AU - Heppner, K.M.* AU - Kirchner, H.* AU - Tong, J.* AU - Bielohuby, M.* AU - Gaylinn, B.D.* AU - Müller, T.D. AU - Bartley, E.* AU - Davis, H.W.* AU - Zhao, Y.* AU - Joseph, A.* AU - Kruthaupt, T.* AU - Ottaway, N.* AU - Kabra, D.G. AU - Habegger, K.M.* AU - Benoit, S.C.* AU - Bidlingmaier, M.* AU - Thorner, M.O.* AU - Perez-Tilve, D.* AU - Tschöp, M.H. AU - Pfluger, P.T. C1 - 7936 C2 - 29923 TI - The GOAT-ghrelin system is not essential for hypoglycemia prevention during prolonged calorie restriction. JO - PLoS ONE VL - 7 IS - 2 PB - Public Library of Science PY - 2012 SN - 1932-6203 ER - TY - JOUR AB - BACKROUND: Radiation therapy treatment of breast cancer, Hodgkin's disease or childhood cancers expose the heart to high local radiation doses, causing an increased risk of cardiovascular disease in the survivors decades after the treatment. The mechanisms that underlie the radiation damage remain poorly understood so far. Previous data show that impairment of mitochondrial oxidative metabolism is directly linked to the development of cardiovascular disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the radiation-induced in vivo effects on cardiac mitochondrial proteome and function were investigated. C57BL/6N mice were exposed to local irradiation of the heart with doses of 0.2 Gy or 2 Gy (X-ray, 200 kV) at the age of eight weeks, the control mice were sham-irradiated. After four weeks the cardiac mitochondria were isolated and tested for proteomic and functional alterations. Two complementary proteomics approaches using both peptide and protein quantification strategies showed radiation-induced deregulation of 25 proteins in total. Three main biological categories were affected: the oxidative phophorylation, the pyruvate metabolism, and the cytoskeletal structure. The mitochondria exposed to high-dose irradiation showed functional impairment reflected as partial deactivation of Complex I (32%) and Complex III (11%), decreased succinate-driven respiratory capacity (13%), increased level of reactive oxygen species and enhanced oxidation of mitochondrial proteins. The changes in the pyruvate metabolism and structural proteins were seen with both low and high radiation doses. CONCLUSION/SIGNIFICANCE: This is the first study showing the biological alterations in the murine heart mitochondria several weeks after the exposure to low- and high-dose of ionizing radiation. Our results show that doses, equivalent to a single dose in radiotherapy, cause long-lasting changes in mitochondrial oxidative metabolism and mitochondria-associated cytoskeleton. This prompts us to propose that these first pathological changes lead to an increased risk of cardiovascular disease after radiation exposure. AU - Barjaktarovic, Z. AU - Schmaltz, D. AU - Shyla, A. AU - Azimzadeh, O. AU - Schulz, S. AU - Haagen, J.* AU - Dörr, W.* AU - Sarioglu, H. AU - Schäfer, A. AU - Atkinson, M.J. AU - Zischka, H. AU - Tapio, S. C1 - 6842 C2 - 29346 TI - Radiation-induced signaling results in mitochondrial impairment in mouse heart at 4 weeks after exposure to X-rays. JO - PLoS ONE VL - 6 IS - 12 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range. AU - Boeke, J.* AU - Bag, I.* AU - Ramaiah, M.J.* AU - Vetter, I.* AU - Kremmer, E. AU - Pal-Bhadra, M.* AU - Bhadra, U.* AU - Imhof, A. C1 - 6236 C2 - 28651 TI - The RNA helicase Rm62 cooperates with SU(VAR)3-9 to re-silence active transcription in Drosophila melanogaster. JO - PLoS ONE VL - 6 IS - 6 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - AIMS: A study of 222 candidate genes in type 2 diabetes reported association of variants in RAPGEF1, ENPP1, TP53, NRF1, SLC2A2, SLC2A4 and FOXC2 with type 2 diabetes in 4,805 Finnish individuals. We aimed to replicate these associations in a Danish case-control study and to substantiate any replicated associations in meta-analyses. Furthermore, we evaluated the impact on diabetes-related intermediate traits in a population-based sample of middle-aged Danes. METHODS: We genotyped nine lead variants in the seven genes in 4,973 glucose-tolerant and 3,612 type 2 diabetes Danish individuals. In meta-analyses we combined case-control data from the DIAGRAM+ Consortium (n = 47,117) and the present genotyping results. The quantitative trait studies involved 5,882 treatment-naive individuals from the Danish Inter99 study. RESULTS: None of the nine investigated variants were significantly associated with type 2 diabetes in the Danish samples. However, for all nine variants the estimate of increase in type 2 diabetes risk was observed for the same allele as previously reported. In a meta-analysis of published and online data including 55,521 Europeans the G-allele of rs1042522 in TP53 showed significant association with type 2 diabetes (OR = 1.06 95% CI 1.02-1.11, p = 0.0032). No substantial associations with diabetes-related intermediary phenotypes were found. CONCLUSION: The G-allele of TP53 rs1042522 is associated with an increased prevalence of type 2 diabetes in a combined analysis of 55,521 Europeans. AU - Burgdorf, K.S.* AU - Grarup, N.* AU - Justesen, J.M.* AU - Harder, M.N.* AU - Witte, D.R.* AU - Jørgensen, T.* AU - Sandbæk, A.* AU - Lauritzen, T.* AU - Madsbad, S.* AU - Hansen, T.* AU - DIAGRAM Consortium (Huth, C. AU - Grallert, H. AU - Klopp, N. AU - Petersen, A.-K. AU - Thorand, B. AU - Illig, T. AU - Meitinger, T. AU - Gieger, C.) AU - Pedersen, O.* C1 - 6776 C2 - 29264 TI - Studies of the association of Arg72Pro of tumor suppressor protein p53 with type 2 diabetes in a combined analysis of 55,521 Europeans. JO - PLoS ONE VL - 6 IS - 1 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed. AU - Deeg, C.A.* AU - Eberhardt, C.* AU - Hofmaier, F.* AU - Amann, B.* AU - Hauck, S.M. C1 - 22925 C2 - 30960 TI - Osteopontin and fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal expression of both proteins indicates ECM re-modeling. JO - PLoS ONE VL - 6 IS - 12 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions, integrating miRNA, transcriptome and proteome profiles and advanced in silico analysis using the FAME algorithm. Since miRNAs play a crucial role in the inner ear, demonstrated by the discovery of mutations in a miRNA leading to human and mouse deafness, we applied this approach to microdissected auditory and vestibular sensory epithelia. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. Functionally important miRNAs were determined by searching for enriched or depleted targets in the transcript and protein datasets with an expression consistent with the dogma of miRNA regulation. Importantly, quite a few of the targets were detected only in the protein datasets, attributable to regulation by translational suppression. We identified and experimentally validated the regulation of PSIP1-P75, a transcriptional co-activator previously unknown in the inner ear, by miR-135b, in vestibular hair cells. Our findings suggest that miR-135b serves as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. AU - Elkan-Miller, T.* AU - Ulitsky, I.* AU - Hertzano, R.* AU - Rudnicki, A.* AU - Dror, A.A.* AU - Lenz, D.R.* AU - Elkon, R.* AU - Irmler, M. AU - Beckers, J. AU - Shamir, R.* AU - Avraham, K.B. C1 - 5965 C2 - 28449 TI - Integration of transcriptomics, proteomics, and microRNA analyses reveals novel microRNA regulation of targets in the mammalian inner ear. JO - PLoS ONE VL - 6 IS - 4 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Metabolomics is a promising tool for discovery of novel biomarkers of chronic disease risk in prospective epidemiologic studies. We investigated the between- and within-person variation of the concentrations of 163 serum metabolites over a period of 4 months to evaluate the metabolite reliability expressed by the intraclass-correlation coefficient (ICC: the ratio of between-person variance and total variance). The analyses were performed with the BIOCRATES AbsoluteIDQ™ targeted metabolomics technology, including acylcarnitines, amino acids, glycerophospholipids, sphingolipids and hexose in 100 healthy individuals from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study who had provided two fasting blood samples 4 months apart. Overall, serum reliability of metabolites over a 4-month period was good. The median ICC of the 163 metabolites was 0.57. The highest ICC was observed for hydroxysphingomyelin C14:1 (ICC = 0.85) and the lowest was found for acylcarnitine C3:1 (ICC = 0). Reliability was high for hexose (ICC = 0.76), sphingolipids (median ICC = 0.66; range: 0.24-0.85), amino acids (median ICC = 0.58; range: 0.41-0.72) and glycerophospholipids (median ICC = 0.58; range: 0.03-0.81). Among acylcarnitines, reliability of short and medium chain saturated compounds was good to excellent (ICC range: 0.50-0.81). Serum reliability was lower for most hydroxyacylcarnitines and monounsaturated acylcarnitines (ICC range: 0.11-0.45 and 0.00-0.63, respectively). For most of the metabolites a single measurement may be sufficient for risk assessment in epidemiologic studies with healthy subjects. AU - Floegel, A.* AU - Drogan, D.* AU - Wang-Sattler, R. AU - Prehn, C. AU - Illig, T. AU - Adamski, J. AU - Joost, H.G.* AU - Boeing, H.* AU - Pischon, T.* C1 - 4833 C2 - 28545 TI - Reliability of serum metabolite concentrations over a 4-month period using a targeted metabolomic approach. JO - PLoS ONE VL - 6 IS - 6 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Globally plants are the primary sink of atmospheric CO(2), but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important. METHODOLOGY: We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either (13)CO(2) to leaves or (13)C-glucose to shoots via xylem uptake. The translocation of (13)CO(2) from the source to other plant parts could be traced by (13)C-labeled isoprene and respiratory (13)CO(2) emission.PRINCIPAL FINDING: In intact plants, assimilated (13)CO(2) was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h(-1). (13)C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76-78%) from recently fixed CO(2), to a minor extent from xylem-transported sugars (7-11%) and from photosynthetic intermediates with slower turnover rates (8-11%).CONCLUSION: We quantified the plants' C loss as respiratory CO(2) and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux. AU - Ghirardo, A. AU - Gutknecht, J. AU - Zimmer, I. AU - Brüggemann, N.* AU - Schnitzler, J.-P. C1 - 5966 C2 - 28719 TI - Biogenic volatile organic compound and respiratory CO2 emissions after 13C-labeling: Online tracing of C translocation dynamics in poplar plants. JO - PLoS ONE VL - 6 IS - 2 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Tracking of fatty acid (FA) contribution to plasma or serum lipids over time was shown in children and adults. However, the potential role of FADS gene variants has not been investigated. METHODS AND PRINCIPAL FINDINGS: Serum GP FA composition of 331 children aged 2 and 6 years, participating in an ongoing birth cohort study, was analyzed. Correlation coefficients were estimated to describe FA tracking over 4 years and to assess the influence of FADS variants on tracking. We found low to moderate tracking (r = 0.12-0.49) of FA compositions and concentration between 2 and 6 years. Concentration changes of total monounsaturated FA and total saturated FA over time correlated closely (r = 0.79) but percentage values were unrelated (r = -0.02). Tracking for n-6 long chain polyunsaturated fatty acid (LC-PUFA) concentrations was lower in subjects homozygous for the major allele of FADS variants and higher in carriers of at least one minor allele, whereas for total n-3 LC-PUFA concentrations and compositions this was vice versa. For individual n-3 PUFA inconsistent results were found. CONCLUSIONS AND SIGNIFICANCE: Serum GP FA composition shows low to moderate tracking over 4 years with a higher tracking for LC-PUFA metabolites than for their precursor FA. Serum PUFA levels and their tracking seem to be more influenced by lipid and lipoprotein metabolism than by FA specific pathways. AU - Glaser, C.* AU - Rzehak, P. AU - Demmelmair, H.* AU - Klopp, N. AU - Heinrich, J. AU - Koletzko, B.* AU - LISAplus Study Group (Wichmann, H.-E. AU - Heinrich, J. AU - Bolte, G. AU - Belcredi, P. AU - Jacob, B. AU - Schoetzau, A. AU - Mosetter, M. AU - Schindler, J. AU - Höhnke, A.) C1 - 6518 C2 - 28843 TI - Influence of FADS polymorphisms on tracking of serum glycerophospholipid fatty acid concentrations and percentage composition in children. JO - PLoS ONE VL - 6 IS - 7 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+)Vβ11(+) NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+) subsets.RESULTS: Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3(+) and CCR5(+) cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4(neg)CD8(neg) (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3(+), CCR4(+), CCR5(+), CCR6(+), CCR7(+), CCR8(+) and CXCR4(+) DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3(+), CCR6(+) and CCR8(+) cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells. CONCLUSIONS: Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens. AU - Harner, S. AU - Nößner, E. AU - Nadas, K.* AU - Leumann-Runge, A.* AU - Schiemann, M. AU - Faber, F.L.* AU - Heinrich, J. AU - Krauss-Etschmann, S. C1 - 6373 C2 - 28428 TI - Cord blood Vα24-vβ11 natural killer T cells display a Th2-chemokine receptor profile and cytokine responses. JO - PLoS ONE VL - 9 IS - 1 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease. AU - Hector, A.* AU - Kormann, M.S.* AU - Mack, I.* AU - Latzin, P.* AU - Casaulta, C.* AU - Kieninger, E.* AU - Zhou, Z.* AU - Yildirim, A.Ö. AU - Bohla, A. AU - Rieber, N.* AU - Kappler, M.* AU - Koller, B.* AU - Eber, E.* AU - Eickmeier, O.* AU - Zielen, S.* AU - Eickelberg, O. AU - Griese, M.* AU - Mall, M.A.* AU - Hartl, D.* C1 - 4704 C2 - 28995 TI - The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease. JO - PLoS ONE VL - 6 IS - 9 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Although human musical performances represent one of the most valuable achievements of mankind, the best musicians perform imperfectly. Musical rhythms are not entirely accurate and thus inevitably deviate from the ideal beat pattern. Nevertheless, computer generated perfect beat patterns are frequently devalued by listeners due to a perceived lack of human touch. Professional audio editing software therefore offers a humanizing feature which artificially generates rhythmic fluctuations. However, the built-in humanizing units are essentially random number generators producing only simple uncorrelated fluctuations. Here, for the first time, we establish long-range fluctuations as an inevitable natural companion of both simple and complex human rhythmic performances. Moreover, we demonstrate that listeners strongly prefer long-range correlated fluctuations in musical rhythms. Thus, the favorable fluctuation type for humanizing interbeat intervals coincides with the one generically inherent in human musical performances. AU - Hennig, H.* AU - Fleischmann, R.* AU - Fredebohm, A.* AU - Hagmayer, Y.* AU - Nagler, J.* AU - Witt, A.* AU - Theis, F.J. AU - Geisel, T.* C1 - 6663 C2 - 29067 TI - The nature and perception of fluctuations in human musical rhythms. JO - PLoS ONE VL - 6 IS - 10 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. METHODS AND FINDINGS: We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75-1.42] and 1.11 [0.81-1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years). CONCLUSIONS: High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies. AU - Herder, C.* AU - Peeters, W.* AU - Illig, T. AU - Baumert, J.J. AU - de Kleijn, D.P.* AU - Moll, F.L.* AU - Poschen, U.* AU - Klopp, N. AU - Müller-Nurasyid, M. AU - Roden, M.* AU - Preuss, M* AU - CARDIoGRAM Consortium (Illig, T. AU - Wichmann, H.-E. AU - Döring, A. AU - Peters, A. AU - Meitinger, T. AU - Klopp, N. AU - Meisinger, C.) AU - Karakas, M.* AU - Meisinger, C. AU - Thorand, B. AU - Pasterkamp, G.* AU - Koenig, W.* C1 - 3967 C2 - 29398 TI - RANTES/CCL5 and risk for coronary events: Results from the MONICA/KORA Augsburg case-cohort, Athero-Express and CARDIoGRAM studies. JO - PLoS ONE VL - 6 IS - 12 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - This study compares inflammation-related biomarkers with established cardiometabolic risk factors in the prediction of incident type 2 diabetes and incident coronary events in a prospective case-cohort study within the population-based MONICA/KORA Augsburg cohort. Analyses for type 2 diabetes are based on 436 individuals with and 1410 individuals without incident diabetes. Analyses for coronary events are based on 314 individuals with and 1659 individuals without incident coronary events. Mean follow-up times were almost 11 years. Areas under the receiver-operating characteristic curve (AUC), changes in Akaike's information criterion (ΔAIC), integrated discrimination improvement (IDI) and net reclassification index (NRI) were calculated for different models. A basic model consisting of age, sex and survey predicted type 2 diabetes with an AUC of 0.690. Addition of 13 inflammation-related biomarkers (CRP, IL-6, IL-18, MIF, MCP-1/CCL2, IL-8/CXCL8, IP-10/CXCL10, adiponectin, leptin, RANTES/CCL5, TGF-β1, sE-selectin, sICAM-1; all measured in nonfasting serum) increased the AUC to 0.801, whereas addition of cardiometabolic risk factors (BMI, systolic blood pressure, ratio total/HDL-cholesterol, smoking, alcohol, physical activity, parental diabetes) increased the AUC to 0.803 (ΔAUC [95% CI] 0.111 [0.092-0.149] and 0.113 [0.093-0.149], respectively, compared to the basic model). The combination of all inflammation-related biomarkers and cardiometabolic risk factors yielded a further increase in AUC to 0.847 (ΔAUC [95% CI] 0.044 [0.028-0.066] compared to the cardiometabolic risk model). Corresponding AUCs for incident coronary events were 0.807, 0.825 (ΔAUC [95% CI] 0.018 [0.013-0.038] compared to the basic model), 0.845 (ΔAUC [95% CI] 0.038 [0.028-0.059] compared to the basic model) and 0.851 (ΔAUC [95% CI] 0.006 [0.003-0.021] compared to the cardiometabolic risk model, respectively. Inclusion of multiple inflammation-related biomarkers into a basic model and into a model including cardiometabolic risk factors significantly improved the prediction of type 2 diabetes and coronary events, although the improvement was less pronounced for the latter endpoint. AU - Herder, C.* AU - Baumert, J.J. AU - Zierer, A. AU - Roden, M.* AU - Meisinger, C. AU - Karakas, M.* AU - Chambless, L.* AU - Rathmann, W.* AU - Peters, A. AU - Koenig, W.* AU - Thorand, B. C1 - 5051 C2 - 28816 TI - Immunological and cardiometabolic risk factors in the prediction of type 2 diabetes and coronary events: MONICA/KORA Augsburg case-cohort study. JO - PLoS ONE VL - 6 IS - 6 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - INTRODUCTION: Analysis of exhaled breath condensate (EBC) is a noninvasive method to access the epithelial lining fluid of the lungs. Due to standardization problems the method has not entered clinical practice. The aim of the study was to assess the comparability for two commercially available devices in healthy controls. In addition, we assessed different breathing patterns in healthy controls with protein markers to analyze the source of the EBC. METHODS: EBC was collected from ten subjects using the RTube and ECoScreen Turbo in a randomized crossover design, twice with every device--once in tidal breathing and once in hyperventilation. EBC conductivity, pH, surfactant protein A, Clara cell secretory protein and total protein were assessed. Bland-Altman plots were constructed to display the influence of different devices or breathing patterns and the intra-class correlation coefficient (ICC) was calculated. The volatile organic compound profile was measured using the electronic nose Cyranose 320. For the analysis of these data, the linear discriminant analysis, the Mahalanobis distances and the cross-validation values (CVV) were calculated. RESULTS: Neither the device nor the breathing pattern significantly altered EBC pH or conductivity. ICCs ranged from 0.61 to 0.92 demonstrating moderate to very good agreement. Protein measurements were greatly influenced by breathing pattern, the device used, and the way in which the results were reported. The electronic nose could distinguish between different breathing patterns and devices, resulting in Mahalanobis distances greater than 2 and CVVs ranging from 64% to 87%. CONCLUSION: EBC pH and (to a lesser extent) EBC conductivity are stable parameters that are not influenced by either the device or the breathing patterns. Protein measurements remain uncertain due to problems of standardization. We conclude that the influence of the breathing maneuver translates into the necessity to keep the volume of ventilated air constant in further studies. AU - Hüttmann, E.-M.* AU - Greulich, T.* AU - Hattesohl, A.* AU - Schmid, S.* AU - Noeske, S.* AU - Herr, C.* AU - John, G. AU - Jörres, R.A.* AU - Müller, B.* AU - Vogelmeier, C.* AU - Koczulla, A.R.* C1 - 7195 C2 - 29540 TI - Comparison of two devices and two breathing patterns for exhaled breath condensate sampling. JO - PLoS ONE VL - 6 IS - 11 PB - Public Library Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Hematopoiesis is an ideal model system for stem cell biology with advanced experimental access. A systems view on the interactions of core transcription factors is important for understanding differentiation mechanisms and dynamics. In this manuscript, we construct a Boolean network to model myeloid differentiation, specifically from common myeloid progenitors to megakaryocytes, erythrocytes, granulocytes and monocytes. By interpreting the hematopoietic literature and translating experimental evidence into Boolean rules, we implement binary dynamics on the resulting 11-factor regulatory network. Our network contains interesting functional modules and a concatenation of mutual antagonistic pairs. The state space of our model is a hierarchical, acyclic graph, typifying the principles of myeloid differentiation. We observe excellent agreement between the steady states of our model and microarray expression profiles of two different studies. Moreover, perturbations of the network topology correctly reproduce reported knockout phenotypes in silico. We predict previously uncharacterized regulatory interactions and alterations of the differentiation process, and line out reprogramming strategies. AU - Krumsiek, J. AU - Marr, C. AU - Schroeder, T. AU - Theis, F.J. C1 - 6621 C2 - 28984 TI - Hierarchical differentiation of myeloid progenitors is encoded in the transcription factor network. JO - PLoS ONE VL - 6 IS - 8 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Combining pharmacological treatments and life style interventions is necessary for effective therapy of major diseases associated with obesity, which are clustered in the metabolic syndrome. Acting via multiple mechanisms, combination treatments may reduce dose requirements and, therefore, lower the risk of adverse side effects, which are usually associated with long-term pharmacological interventions. Our previous study in mice fed high-fat diet indicated additivity in preservation of insulin sensitivity and in amelioration of major metabolic syndrome phenotypes by the combination treatment using n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and rosiglitazone, i.e. an anti-diabetic drug of the thiazolidinedione (TZD) family. We investigated here whether pioglitazone, a TZD-drug in clinical use, could elicit the additive beneficial effects when combined with n-3 LC-PUFA. Adult male mice (C57BL/6N) were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various dietary treatments (i) cHF+F, cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids; (ii) cHF+ROSI, cHF with 10 mg rosiglitazone/kg diet; (iii) cHF+F+ROSI; (iv) cHF+PIO, cHF with 50 mg pioglitazone/kg diet; and (v) cHF+F+PIO, or chow-fed. Plasma concentrations of 163 metabolites were evaluated using a targeted metabolomics approach. Both TZDs preserved glucose homeostasis and normal plasma lipid levels while inducing adiponectin, with pioglitazone showing better effectiveness. The beneficial effects of TZDs were further augmented by the combination treatments. cHF+F+ROSI but not cHF+F+PIO counteracted development of obesity, in correlation with inducibility of fatty acid β-oxidation, as revealed by the metabolomic analysis. By contrast, only cHF+F+PIO eliminated hepatic steatosis and this treatment also reversed insulin resistance in dietary obese mice. Our results reveal differential effects of rosiglitazone and pioglitazone, unmasked in the combination treatment with n-3 LC-PUFA, and support the notion that n-3 LC-PUFA could be used as add-on treatment to TZDs in order to improve diabetic patient's therapy. AU - Kus, V.* AU - Flachs, P.* AU - Kuda, O.* AU - Bardova, K.* AU - Janovska, P.* AU - Svobodova, M.* AU - Jilkova, Z.M.* AU - Rossmeisl, M.* AU - Wang-Sattler, R. AU - Yu, Z. AU - Illig, T. AU - Kopecky, J.* C1 - 6771 C2 - 29250 TI - Unmasking differential effects of rosiglitazone and pioglitazone in the combination treatment with n-3 fatty acids in mice fed a high-fat diet. JO - PLoS ONE VL - 6 IS - 11 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097-16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future. AU - Min, J.L.* AU - Taylor, J.M.* AU - Richards, J.B.* AU - Watts, T.* AU - Pettersson, F.H.* AU - Broxholme, J.* AU - Ahmadi, K.R.* AU - Surdulescu, G.L.* AU - Lowy, E.* AU - Gieger, C. AU - Newton-Cheh, C.* AU - Perola, M.* AU - Soranzo, N.* AU - Surakka, I.* AU - Lindgren, C.M.* AU - Ragoussis, J.* AU - Morris, A.P.* AU - Cardon, L.R.* AU - Spector, T.D.* AU - Zondervan, K.T.* C1 - 6162 C2 - 28849 TI - The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits. JO - PLoS ONE VL - 6 IS - 7 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium). OBJECTIVES: To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample. METHODS: We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/-10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations. The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV(1) or FEV(1)/FVC traits using a carefully defined significance threshold of 1.3×10(-5). The most significant loci associated with FEV(1) include SNPs tagging MACROD2 (P = 6.81×10(-5)), CNTN5 (P = 4.37×10(-4)), and TRPV4 (P = 1.58×10(-3)). Among ever-smokers, SERPINA1 showed the most significant association with FEV(1) (P = 8.41×10(-5)), followed by PDE4D (P = 1.22×10(-4)). The strongest association with FEV(1)/FVC ratio was observed with ABCC1 (P = 4.38×10(-4)), and ESR1 (P = 5.42×10(-4)) among ever-smokers.CONCLUSIONS: Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV(1) among smokers in the general population. AU - Obeidat, M.* AU - Wain, L.V.* AU - Shrine, N.* AU - Kalsheker, N.* AU - Artigas, M.S.* AU - Repapi, E.* AU - Burton, P.R* AU - Johnson, T.* AU - Ramasamy, A.* AU - Zhao, J.H.* AU - Zhai, G.* AU - Huffman, J.E.* AU - Vitart, V.* AU - Albrecht, E. AU - Igl, W.* AU - Hartikainen, A.L.* AU - Pouta, A.* AU - Cadby, G.* AU - Hui, J.* AU - Palmer, L.J.* AU - Hadley, D.* AU - McArdle, W.L.* AU - Rudnicka, A.R.* AU - Barroso, I.* AU - Loos, R.J.* AU - Wareham, N.J.* AU - Mangino, M.* AU - Soranzo, N.* AU - Spector, T.D.* AU - Gläser, S.* AU - Homuth, G.* AU - Völzke, H.* AU - Deloukas, P.* AU - Granell, R.* AU - Henderson, J.* AU - Grkovic, I.* AU - Jankovic, S.* AU - Zgaga, L.* AU - Polaek, O.* AU - Rudan, I.* AU - Wright, A.F.* AU - Campbell, H.* AU - Wild, S.H.* AU - Wilson, J.F.* AU - Heinrich, J. AU - Imboden, M.* AU - Probst-Hensch, N.M.* AU - Gyllensten, U.* AU - Johansson, A.* AU - Zaboli, G.* AU - Mustelin, L.* AU - Rantanen, T.* AU - Surakka, I.* AU - Kaprio, J.* AU - Jarvelin, M.R.* AU - Hayward, C.* AU - Evans, D.M* AU - Koch, B.* AU - Musk, A.W.* AU - Elliott, P.* AU - Strachan, D.P.* AU - Tobin, M.D.* AU - Sayers, I.* AU - Hall, I.P.* AU - SpiroMeta Consortium (*) C1 - 6481 C2 - 28798 TI - A comprehensive evaluation of potential lung function associated genes in the SpiroMeta general population sample. JO - PLoS ONE VL - 6 IS - 5 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - OriP, the latent origin of Epstein-Barr virus (EBV), consists of two essential elements: the dyad symmetry (DS) and the family of repeats (FR). The function of these elements has been predominantly analyzed in plasmids transfected into transformed cells. Here, we examined the molecular functions of DS in its native genomic context and at an ectopic position in the mini-EBV episome. Mini-EBV plasmids contain 41% of the EBV genome including all information required for the proliferation of human B cells. Both FR and DS function independently of their genomic context. We show that DS is the most active origin of replication present in the mini-EBV genome regardless of its location, and it is characterized by the binding of the origin recognition complex (ORC) allowing subsequent replication initiation. Surprisingly, the integrity of oriP is not required for the formation of the pre-replicative complex (pre-RC) at or near DS. In addition we show that initiation events occurring at sites other than the DS are also limited to once per cell cycle and that they are ORC-dependent. The deletion of DS increases initiation from alternative origins, which are normally used very infrequently in the mini-EBV genome. The sequence-independent distribution of ORC-binding, pre-RC-assembly, and initiation patterns indicates that a large number of silent origins are present in the mini-EBV genome. We conclude that, in mini-EBV genomes lacking the DS element, the absence of a strong ORC binding site results in an increase of ORC binding at dispersed sites. AU - Ott, E. AU - Norio, P.* AU - Ritzi, M. AU - Schildkraut, C.* AU - Schepers, A. C1 - 6484 C2 - 28760 TI - The dyad symmetry element of Epstein-Barr virus is a dominant but dispensable replication origin. JO - PLoS ONE VL - 6 IS - 5 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. METHODS: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. RESULTS: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. CONCLUSION: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo. AU - Paron, I. AU - Berchtold, S. AU - Vörös, J. AU - Shamarla, M. AU - Erkan, M.* AU - Höfler, H. AU - Esposito, I. C1 - 6506 C2 - 28831 TI - Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway. JO - PLoS ONE VL - 6 IS - 6 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Fibroblast growth factor (Fgf) signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130) has been demonstrated to participate in maturation of various organs and tissues including skeleton, testes, lung, heart, and eye. Here we establish a novel Fgf9 allele, discovered in a dominant N-ethyl-N-nitrosourea (ENU) screen for eye-size abnormalities using the optical low coherence interferometry technique. The underlying mouse mutant line Aca12 was originally identified because of its significantly reduced lens thickness. Linkage studies located Aca12 to chromosome 14 within a 3.6 Mb spanning interval containing the positional candidate genes Fgf9 (MGI: 104723), Gja3 (MGI: 95714), and Ift88 (MGI: 98715). While no sequence differences were found in Gja3 and Ift88, we identified an A→G missense mutation at cDNA position 770 of the Fgf9 gene leading to an Y162C amino acid exchange. In contrast to previously described Fgf9 mutants, Fgf9(Y162C) carriers were fully viable and did not reveal reduced body-size, male-to-female sexual reversal or skeletal malformations. The histological analysis of the retina as well as its basic functional characterization by electroretinography (ERG) did not show any abnormality. However, the analysis of head-tracking response of the Fgf9(Y162C) mutants in a virtual drum indicated a gene-dosage dependent vision loss of almost 50%. The smaller lenses in Fgf9(Y162C) suggested a role of Fgf9 during lens development. Histological investigations showed that lens growth retardation starts during embryogenesis and continues after birth. Young Fgf9(Y162C) lenses remained transparent but developed age-related cataracts. Taken together, Fgf9(Y162C) is a novel neomorphic allele that initiates microphakia and reduced vision without effects on organs and tissues outside the eye. Our data point to a role of Fgf9 signalling in primary and secondary lens fiber cell growth. The results underline the importance of allelic series to fully understand multiple functions of a gene. AU - Puk, O. AU - Möller, G. AU - Geerlof, A. AU - Krowiorz, K. AU - Ahmad, N. AU - Wagner, S. AU - Adamski, J. AU - Hrabě de Angelis, M. AU - Graw, J. C1 - 5564 C2 - 28796 TI - The pathologic effect of a novel neomorphic Fgf9Y162C allele is restricted to decreased vision and retarded lens growth. JO - PLoS ONE VL - 6 IS - 8 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - To investigate whether the elevated liver enzymes gamma-glutamyltransferase (GGT), glutamate-pyruvate transaminase (GPT), glutamate-oxalacetate transaminase (GOT) and alkaline phosphatase (AP) and non-alcoholic fatty liver disease (NAFLD) respectively are independently associated with pre-diabetic states, namely impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) or known and newly diagnosed diabetes (NDD), in men and women from the general German population. METHODS: The study was based on 3009 subjects (1556 females, 1453 males) aged 32 to 81 years who participated in the KORA-F4-Study in 2006/2008 in Augsburg, Southern Germany. All non-diabetic participants underwent an oral glucose tolerance test to assess disturbances in glucose metabolism. NAFLD was estimated by liver enzyme concentrations and the Bedogni Fatty Liver Index (FLI) RESULTS: 229 participants (7.6%) reported known diabetes, 106 had NDD (3.5%), 107 (3.6%) had IFG, 309 (10.3%) had IGT, 69 (2.3%) were affected with both metabolic disorders (IFG/IGT) and 74 (2.5%) could not be classified. GGT and GPT were significantly elevated in persons with pre-diabetes and diabetes (GGT in diabetic persons OR = 1.76, [1.47-2.09], in IFG OR = 1.79 [1.50-2.13], GPT in diabetic persons OR = 1.51, [1.30-1.74], in NDD OR = 1.77 [1.52-2.06]), GOT and AP only inconsistently in some pre-diabetes groups. The effects were sharpened in models using an increase of two or three out of three enzymes as an estimate of fatty liver and especially in models using the FLI. Overall frequency of NAFLD applying the index was 39.8% (women: 27.3% and men: 53.2%). In participants with fatty liver disease, the OR for NDD adjusted for sex and age was 8.48 [5.13-14.00], 6.70 [3.74-12.01] for combined IFG and IGT and 4.78 [3.47-6.59] for known diabetes respectively. CONCLUSIONS: Elevated GGT and GPT-values as well as estimates of fatty liver disease are significantly associated with pre-diabetes and diabetes and thus very useful first indicators of a disturbed glucose metabolism. AU - Rückert, I.-M. AU - Heier, M. AU - Rathmann, W.* AU - Baumeister, S.E.* AU - Döring, A. AU - Meisinger, C. C1 - 5445 C2 - 28781 TI - Association between markers of fatty liver disease and impaired glucose regulation in men and women from the general population: The KORA-F4-study. JO - PLoS ONE VL - 6 IS - 8 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies. AU - Ruiss, R. AU - Jochum, S. AU - Mocikat, R. AU - Hammerschmidt, W. AU - Zeidler, R.* C1 - 6641 C2 - 29021 TI - EBV-gp350 confers B-cell tropism to tailored exosomes and is a neo-antigen in normal and malignant B cells - a new option for the treatment of B-CLL. JO - PLoS ONE VL - 6 IS - 10 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Heavy metals such as cadmium (Cd(2+)) affect microbial metabolic processes. Consequently, bacteria adapt by adjusting their cellular machinery. We have investigated the dose-dependent growth effects of Cd(2+) on Rhizobium alamii, an exopolysaccharide (EPS)-producing bacterium that forms a biofilm on plant roots. Adsorption isotherms show that the EPS of R. alamii binds cadmium in competition with calcium. A metabonomics approach based on ion cyclotron resonance Fourier transform mass spectrometry has showed that cadmium alters mainly the bacterial metabolism in pathways implying sugars, purine, phosphate, calcium signalling and cell respiration. We determined the influence of EPS on the bacterium response to cadmium, using a mutant of R. alamii impaired in EPS production (MSΔGT). Cadmium dose-dependent effects on the bacterial growth were not significantly different between the R. alamii wild type (wt) and MSΔGT strains. Although cadmium did not modify the quantity of EPS isolated from R. alamii, it triggered the formation of biofilm vs planktonic cells, both by R. alamii wt and by MSΔGT. Thus, it appears that cadmium toxicity could be managed by switching to a biofilm way of life, rather than producing EPS. We conclude that modulations of the bacterial metabolism and switching to biofilms prevails in the adaptation of R. alamii to cadmium. These results are original with regard to the conventional role attributed to EPS in a biofilm matrix, and the bacterial response to cadmium. AU - Schue, M.* AU - Fekete, A. AU - Ortet, P.* AU - Brutesco, C.* AU - Heulin, T.* AU - Schmitt-Kopplin, P. AU - Achouak, W.* AU - Santaella, C.* C1 - 6743 C2 - 29197 TI - Modulation of metabolism and switching to biofilm prevail over exopolysaccharide production in the response of Rhizobium alamii to cadmium. JO - PLoS ONE VL - 6 IS - 11 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - To explore the capacity of human CD1⁺CD16⁺⁺ and CD14⁺⁺CD16⁻ monocytes to phagocyte iron-oxide nanoparticles in vitro. Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers. Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16⁻ monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16⁻ monocytes differently. Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties. AU - Settles, M.* AU - Etzrodt, M.* AU - Kosanke, K.* AU - Schiemann, M. AU - Zimmermann, A.* AU - Meier, R.* AU - Braren, R.* AU - Huber, A.* AU - Rummeny, E.J.* AU - Weissleder, R.* AU - Swirski, F.K.* AU - Wildgruber, M.* C1 - 6928 C2 - 29435 TI - Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles. JO - PLoS ONE VL - 6 IS - 10 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/-) mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation AU - Siegert, S.* AU - Huang, H.Y.* AU - Yang, C.Y.* AU - Scarpellino, L.* AU - Carrie, L.* AU - Essex, S.* AU - Nelson, P.J. AU - Heikenwälder, M.* AU - Acha-Orbea, H.* AU - Buckley, C.D.* AU - Marsland, B.J.* AU - Zehn, D.* AU - Luther, S.A.* C1 - 6897 C2 - 29423 TI - Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide. JO - PLoS ONE VL - 6 IS - 11 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - OBJECTIVE: The metabolic syndrome is a major public health challenge and identifies persons at risk for diabetes and cardiovascular disease. The aim of this study was to examine the association between age at menarche and the metabolic syndrome (IDF and NCEP ATP III classification) and its components.DESIGN: 1536 women aged 32 to 81 years of the German population based KORA F4 study were investigated. Data was collected by standardized interviews, physical examinations, and whole blood and serum measurements. RESULTS: Young age at menarche was significantly associated with elevated body mass index (BMI), greater waist circumference, higher fasting glucose levels, and 2 hour glucose (oral glucose tolerance test), even after adjusting for the difference between current BMI and BMI at age 25. The significant effect on elevated triglycerides and systolic blood pressure was attenuated after adjustment for the BMI change. Age at menarche was inversely associated with the metabolic syndrome adjusting for age (p-values: <0.001 IDF, 0.003 NCEP classification) and additional potential confounders including lifestyle and reproductive history factors (p-values: 0.001, 0.005). Associations remain significant when additionally controlling for recollected BMI at age 25 (p-values: 0.008, 0.033) or the BMI change since age 25 (p-values: 0.005, 0.022). CONCLUSION: Young age at menarche might play a role in the development of the metabolic syndrome. This association is only partially mediated by weight gain and increased BMI. A history of early menarche may help to identify women at risk for the metabolic syndrome. AU - Stöckl, D. AU - Meisinger, C. AU - Peters, A. AU - Thorand, B. AU - Huth, C. AU - Heier, M. AU - Rathmann, W.* AU - Kowall, B.* AU - Stöckl, H.* AU - Döring, A. C1 - 6760 C2 - 29223 TI - Age at menarche and its association with the metabolic syndrome and its components: Results from the KORA F4 study. JO - PLoS ONE VL - 6 IS - 10 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Plasmacytoid dendritic cells (pDCs) play a key role in detecting pathogens by producing large amounts of type I interferon (IFN) by sensing the presence of viral infections through the Toll-Like Receptor (TLR) pathway. TLR9 is a sensor of viral and bacterial DNA motifs and activates the IRF7 transcription factor which leads to type I IFN secretion by pDCs. However, during chronic hepatitis B virus (HBV) infection, pDCs display an impaired ability to secrete IFN-α following ex vivo stimulation with TLR9 ligands. Here we highlight several strategies used by HBV to block IFN-α production through a specific impairment of the TLR9 signaling. Our results show that HBV particle internalisation could inhibit TLR9- but not TLR7-mediated secretion of IFN-α by pDCs. We observed that HBV down-regulated TLR9 transcriptional activity in pDCs and B cells in which TLR9 mRNA and protein levels were reduced. HBV can interfere with TLR9 activity by blocking the MyD88-IRAK4 axis and Sendai virus targeting IRF7 to block IFN-α production. Neutralising CpG motif sequences were identified within HBV DNA genome of genotypes A to H which displayed a suppressive effect on TLR9-immune activation. Moreover, TLR9 mRNA and protein were downregulated in PBMCs from patients with HBV-associated chronic hepatitis and hepatocellular carcinoma. Thus HBV has developed several escape mechanisms to avoid TLR9 activation in both pDCs and B lymphocytes, which may in turn contribute to the establishment and/or persistence of chronic infection. AU - Vincent, I.E.* AU - Zannetti, C.* AU - Lucifora, J. AU - Norder, H.* AU - Protzer, U. AU - Hainaut, P.* AU - Zoulim, F.* AU - Tommasino, M.* AU - Trépo, C.* AU - Hasan, U.* AU - Chemin, I.* C1 - 6695 C2 - 29132 TI - Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells. JO - PLoS ONE VL - 6 IS - 10 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: In the recently published meta-analysis of multiple sclerosis genome-wide association studies De Jager et al. identified three single nucleotide polymorphisms associated to MS: rs17824933 (CD6), rs1800693 (TNFRSF1A) and rs17445836 (61.5 kb from IRF8). To refine our understanding of these associations we sought to replicate these findings in a large more extensive independent sample set of 11 populations of European origin. PRINCIPAL FINDINGS: We calculated individual and combined associations using a meta-analysis method by Kazeem and Farral (2005). We confirmed the association of rs1800693 in TNFRSF1A (p 4.19 × 10-7, OR 1.12, 7,665 cases, 8,051 controls) and rs17445836 near IRF8 (p 5.35 × 10-10, OR 0.84, 6,895 cases, 7,580 controls and 596 case-parent trios) The SNP rs17824933 in CD6 also showed nominally significant evidence for association (p 2.19 × 10-5, OR 1.11, 8,047 cases, 9,174 controls, 604 case-parent trios). CONCLUSIONS: Variants in TNFRSF1A and in the vicinity of IRF8 were confirmed to be associated in these independent cohorts, which supports the role of these loci in etiology of multiple sclerosis. The variant in CD6 reached genome-wide significance after combining the data with the original meta-analysis. Fine mapping is required to identify the predisposing variants in the loci and future functional studies will refine their molecular role in MS pathogenesis. AU - International Multiple Sclerosis Genetics Consortium (Winkelmann, J. AU - Wichmann, H.-E.) C1 - 6054 C2 - 28786 TI - The genetic association of variants in CD6, TNFRSF1A and IRF8 to multiple sclerosis: A multicenter case-control study. JO - PLoS ONE VL - 6 IS - 4 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r) of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01). Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10), which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D) patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices. CONCLUSIONS/SIGNIFICANCE: Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is used, either matrix should generate similar results in clinical and biological studies. The higher metabolite concentrations in serum, however, make it possible to provide more sensitive results in biomarker detection. AU - Yu, Z. AU - Kastenmüller, G. AU - He, Y. AU - Belcredi, P. AU - Möller, G. AU - Prehn, C. AU - Mendes, J. AU - Wahl, S. AU - Römisch-Margl, W. AU - Ceglarek, U.* AU - Polonikov, A.* AU - Dahmen, N.* AU - Prokisch, H. AU - Xie, L.* AU - Li, Y.* AU - Wichmann, H.-E. AU - Peters, A. AU - Kronenberg, F.* AU - Suhre, K. AU - Adamski, J. AU - Illig, T. AU - Wang-Sattler, R. C1 - 6442 C2 - 28696 TI - Differences between human plasma and serum metabolite profiles. JO - PLoS ONE VL - 6 IS - 7 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Transforming growth factor beta 1 (TGFβ1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells. METHODOLOGY: We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. RESULTS AND CONCLUSIONS: Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2. AU - Zhang, Y.* AU - Handley, D.* AU - Kaplan, T.* AU - Yu, H. AU - Bais, A.S.* AU - Richards, T.* AU - Pandit, K.V.* AU - Zeng, Q.* AU - Benos, P.V.* AU - Friedman, N.* AU - Eickelberg, O. AU - Kaminski, N.* C1 - 6022 C2 - 28575 TI - High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells. JO - PLoS ONE VL - 6 IS - 5 PB - Public Library of Science PY - 2011 SN - 1932-6203 ER - TY - JOUR AB - Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins. AU - Graebsch, A. AU - Roche, S. AU - Kostrewa, D.* AU - Söding, J.* AU - Niessing, D. C1 - 5546 C2 - 27602 TI - Of bits and bugs - on the use of bioinformatics and a bacterial crystal structure to solve a eukaryotic repeat-protein structure. JO - PLoS ONE VL - 5 IS - 10 PB - Public Library Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. Methodology/Principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. Conclusions/Significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis. AU - Haebig, K.* AU - Gloeckner, C.J. AU - Miralles, M.G.* AU - Gillardon, F.* AU - Schulte, C.* AU - Riess, O.* AU - Ueffing, M. AU - Biskup, S.* AU - Bonin, M.* C1 - 5996 C2 - 27985 TI - ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2. JO - PLoS ONE VL - 5 IS - 10 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Background: Gene expression as governed by the interplay of the components of regulatory networks is indeed one of the most complex fundamental processes in biological systems. Although several methods have been published to unravel the hierarchical structure of regulatory networks, weaknesses such as the incorrect or inconsistent assignment of elements to their hierarchical levels, the incapability to cope with cyclic dependencies within the networks or the need for a manual curation to retrieve non-overlapping levels remain unsolved. Methodology/Results: We developed HiNO as a significant improvement of the so-called breadth-first-search (BFS) method. While BFS is capable of determining the overall hierarchical structures from gene regulatory networks, it especially has problems solving feed-forward type of loops leading to conflicts within the level assignments. We resolved these problems by adding a recursive correction approach consisting of two steps. First each vertex is placed on the lowest level that this vertex and its regulating vertices are assigned to (downgrade procedure). Second, vertices are assigned to the next higher level (upgrade procedure) if they have successors with the same level assignment and have themselves no regulators. We evaluated HiNO by comparing it with the BFS method by applying them to the regulatory networks from Saccharomyces cerevisiae and Escherichia coli, respectively. The comparison shows clearly how conflicts in level assignment are resolved in HiNO in order to produce correct hierarchical structures even on the local levels in an automated fashion. Conclusions: We showed that the resolution of conflicting assignments clearly improves the BFS-method. While we restricted our analysis to gene regulatory networks, our approach is suitable to deal with any directed hierarchical networks structure such as the interaction of microRNAs or the action of non-coding RNAs in general. Furthermore we provide a user-friendly web-interface for HiNO that enables the extraction of the hierarchical structure of any directed regulatory network. AU - Hartsperger, M.L. AU - Strache, R. AU - Stuempflen, V. C1 - 5670 C2 - 27935 TI - HiNO: An approach for inferring hierarchical organization from regulatory networks. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Background: Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes. Methodology: We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset. Principal Findings: We found a significant overrepresentation of association signals in pathways related to cholesterol metabolism and the immune response in both of the two largest genome-wide association studies for LOAD. Significance: Processes related to cholesterol metabolism and the innate immune response have previously been implicated by pathological and epidemiological studies of Alzheimer's disease, but it has been unclear whether those findings reflected primary aetiological events or consequences of the disease process. Our independent evidence from two large studies now demonstrates that these processes are aetiologically relevant, and suggests that they may be suitable targets for novel and existing therapeutic approaches. AU - Jones, L.* AU - Holmans, P.A.* AU - Hamshere, M.L.* AU - Harold, D.* AU - Moskvina, V.* AU - Ivanov, D.* AU - Pocklington, A.* AU - Abraham, R.* AU - Hollingworth, P.* AU - Sims, R.* AU - Gerrish, A.* AU - Pahwa, J.S.* AU - Jones, N.* AU - Stretton, A.* AU - Morgan, A.R.* AU - Lovestone, S.* AU - Powell, J.* AU - Proitsi, P.* AU - Lupton, M.K.* AU - Brayne, C.* AU - Rubinsztein, D.C.* AU - Gill, M.* AU - Lawlor, B.* AU - Lynch, A.* AU - Morgan, K.* AU - Brown, KS.* AU - Passmore, P.A.* AU - Craig, D.* AU - McGuinness, B.* AU - Todd, S.* AU - Holmes, C.* AU - Mann, D.* AU - Smith, A.D.* AU - Love, S.* AU - Kehoe, P.G.* AU - Mead, S.* AU - Fox, N.* AU - Rossor, M.* AU - Collinge, J.* AU - Maier, W.* AU - Jessen, F.* AU - Schürmann, B.* AU - van den Bussche, H.* AU - Heuser, I.* AU - Peters, O.* AU - Kornhuber, J.* AU - Wiltfang, J.* AU - Dichgans, M.* AU - Frölich, L.* AU - Hampel, H.* AU - Hüll, M.* AU - Rujescu, D.* AU - Goate, A.M.* AU - Kauwe, J.S.* AU - Cruchaga, C.* AU - Nowotny, P.* AU - Morris, J.C.* AU - Mayo, K.* AU - Livingston, G.* AU - Bass, N.J.* AU - Gurling, H.* AU - McQuillin, A.* AU - Gwilliam, R.* AU - Deloukas, P.* AU - Al-Chalabi, A.* AU - Shaw, C.E.* AU - Singleton, A.B.* AU - Guerreiro, R.* AU - Mühleisen, T.W.* AU - Nöthen, M.M.* AU - Moebus, S.* AU - Jöckel, K.-H.* AU - Klopp, N. AU - Wichmann, H.-E. AU - Rüther, E.* AU - Carrasquillo, M.M.* AU - Pankratz, V.S.* AU - Younkin, S.G.* AU - Hardy, J.* AU - O'Donovan, M.C.* AU - Owen, M.J.* AU - Williams, J.* A2 - Joseph El, K.h.o.u.r.y.* ; Massachusetts General Hospital and Harvard Medical, S.c.h.o.o.l.* ; United States of, A.m.e.r.i.c.a.* C1 - 5672 C2 - 27941 TI - Genetic evidence implicates the immune system and cholesterol metabolism in the aetiology of Alzheimer's disease. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - MicroRNAs are a large class of post-transcriptional regulators that bind to the 3' untranslated region of messenger RNAs. They play a critical role in many cellular processes and have been linked to the control of signal transduction pathways. Recent studies indicate that microRNAs can function as tumor suppressors or even as oncogenes when aberrantly expressed. For more general insights of disease-associated microRNAs, we analyzed their impact on human signaling pathways from two perspectives. On a global scale, we found a core set of signaling pathways with enriched tissue-specific microRNA targets across diseases. The function of these pathways reflects the affinity of microRNAs to regulate cellular processes associated with apoptosis, proliferation or development. Comparing cancer and non-cancer related microRNAs, we found no significant differences between both groups. To unveil the interaction and regulation of microRNAs on signaling pathways locally, we analyzed the cellular location and process type of disease-associated microRNA targets and proteins. While disease-associated proteins are highly enriched in extracellular components of the pathway, microRNA targets are preferentially located in the nucleus. Moreover, targets of disease-associated microRNAs preferentially exhibit an inhibitory effect within the pathways in contrast to disease proteins. Our analysis provides systematic insights into the interaction of disease-associated microRNAs and signaling pathways and uncovers differences in cellular locations and process types of microRNA targets and disease-associated proteins. AU - Kowarsch, A. AU - Marr, C. AU - Schmidl, D. AU - Ruepp, A. AU - Theis, F.J. C1 - 4679 C2 - 28140 TI - Tissue-specific target analysis of disease-associated microRNAs in human signaling pathways. JO - PLoS ONE VL - 5 IS - 6 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Background: A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISIMatsuda) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns. Methodology and Principal Findings: By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISIMatsuda of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISIMatsuda value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI > 15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX. Conclusions: Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISIMatsuda are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase. AU - Lucio, M. AU - Fekete, A. AU - Weigert, C.* AU - Wägele, B. AU - Zhao, X.J.* AU - Chen, J.* AU - Fritsche, A.* AU - Häring, H.-U.* AU - Schleicher, E.D.* AU - Xu, G.W.* AU - Schmitt-Kopplin, P. AU - Lehmann, R.* C1 - 3106 C2 - 28010 CY - San Francisco TI - Insulin sensitivity is reflected by characteristic metabolic fingerprints - a Fourier transform mass spectrometric non-targeted metabolomics approach. JO - PLoS ONE VL - 5 IS - 10 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities. CONCLUSIONS/SIGNIFICANCE: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors. AU - Mazur, P.K.* AU - Grüner, B.M.* AU - Nakhai, H.* AU - Sipos, B.* AU - Zimber-Strobl, U.* AU - Strobl, L.J. AU - Radtke, F.* AU - Schmid, R.M.* AU - Siveke, J.T.* C1 - 5542 C2 - 27580 TI - Identification of epidermal pdx1 expression discloses different roles of notch1 and notch2 in murine KrasG12D-induced skin carcinogenesis in vivo. JO - PLoS ONE VL - 5 IS - 10 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - The first genome wide association study (GWAS) for childhood asthma identified a novel major susceptibility locus on chromosome 17q21 harboring the ORMDL3 gene, but the role of previous asthma candidate genes was not specifically analyzed in this GWAS. We systematically identified 89 SNPs in 14 candidate genes previously associated with asthma in >3 independent study populations. We re-genotyped 39 SNPs in these genes not covered by GWAS performed in 703 asthmatics and 658 reference children. Genotyping data were compared to imputation data derived from Illumina HumanHap300 chip genotyping. Results were combined to analyze 566 SNPs covering all 14 candidate gene loci. Genotyped polymorphisms in ADAM33, GSTP1 and VDR showed effects with p-values <0.0035 (corrected for multiple testing). Combining genotyping and imputation, polymorphisms in DPP10, EDN1, IL12B, IL13, IL4, IL4R and TNF showed associations at a significance level between p = 0.05 and p = 0.0035. These data indicate that (a) GWAS coverage is insufficient for many asthma candidate genes, (b) imputation based on these data is reliable but incomplete, and (c) SNPs in three previously identified asthma candidate genes replicate in our GWAS population with significance after correction for multiple testing in 14 genes. AU - Michel, S.* AU - Liang, L.M.* AU - Depner, M.* AU - Klopp, N. AU - Ruether, A.* AU - Kumar, A.* AU - Schedel, M.* AU - Vogelberg, C.* AU - von Mutius, E.* AU - von Berg, A.* AU - Bufe, A.* AU - Rietschel, E.* AU - Heinzmann, A.* AU - Laub, O.* AU - Simma, B.* AU - Frischer, T.* AU - Genuneit, J.* AU - Gut, I.G.* AU - Schreiber, S.* AU - Lathrop, M* AU - Illig, T. AU - Kabesch, M.* C1 - 5673 C2 - 27940 TI - Unifying candidate gene and GWAS approaches in asthma. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution. AU - Möller, G. AU - Husen, B.* AU - Kowalik, D. AU - Hirvelä, L.* AU - Plewczynski, D.* AU - Rychlewski, L.* AU - Messinger, J.* AU - Thole, H.* AU - Adamski, J. C1 - 848 C2 - 27177 TI - Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1. JO - PLoS ONE VL - 5 IS - 6 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se. METHODOLOGY/PRINCIPAL FINDINGS: We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses. CONCLUSIONS/SIGNIFICANCE: These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses. AU - Nörder, M.* AU - Becker, P.D.* AU - Drexler, I. AU - Link, C.* AU - Erfle, V. AU - Guzman, C.A.* C1 - 5976 C2 - 27721 TI - Modified vaccinia virus Ankara exerts potent immune modulatory activities in a murine model. JO - PLoS ONE VL - 5 IS - 6 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. METHODS AND FINDINGS: UCB-derived mononuclear cells (MNC) or selected CD34(+) cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34(+) selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin(+), CD133(+) and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. CONCLUSIONS: This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice. AU - Peters, R.* AU - Wolf, M.J.* AU - van den Broek, M.* AU - Nuvolone, M.* AU - Dannenmann, S.* AU - Stieger, B.* AU - Rapold, R.* AU - Konrad, D.* AU - Rubin, A.* AU - Bertino, J.R.* AU - Aguzzi, A.* AU - Heikenwälder, M. AU - Knuth, A.K.* C1 - 4917 C2 - 28086 TI - Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture. JO - PLoS ONE VL - 5 IS - 12 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Circulating cytokine patterns may be relevant for the diagnosis of asthma, for the discrimination of certain phenotypes, and prognostic factors for exacerbation of disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated serum samples from 944 individuals of 218 asthma-affected families by a multiplex, microsphere based system detecting at high sensitivity eleven asthma associated mediators: eotaxin (CCL11), granulocyte macrophage stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-4 (IL-4), IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17 and tumor necrosis factor alpha (TNFα). Single cytokine levels were largely similar between asthmatic and healthy individuals when analysing asthma as single disease entity. Regulatory differences between parental and pediatric asthma were reflected by six of the eleven mediators analyzed (eotaxin, IL-4, IL-5, IL-10, IL-12, TNFα). IL-12 (p40) and IL-5 were the best predictor for extrinsic asthma in children with an increased odds ratio of 2.85 and 1.96 per log pg/ml increase (IL-12 (p40): 1.2-6.8, p=0.019, and IL-5: 1.2-2.5, p=0.025). Frequent asthma attacks in children are associated with elevated IL-5 serum levels (p=0.013). Cytokine patterns seem to be individually balanced in both, healthy and diseased adults and children, with various cytokines correlating among each other (IL-17 and IFNγ (rs=0.67), IL-4 and IL-5 (rs=0.55), IFNγ and GM-CSF (rs=0.54)). CONCLUSION/SIGNIFICANCE: Our data support mainly an age- but also an asthma phenotype-dependent systemic immune regulation. AU - Pukelsheim, K. AU - Stöger, T. AU - Kutschke, D. AU - Ganguly, K. AU - Wjst, M. C1 - 5028 C2 - 27831 TI - Cytokine profiles in asthma families depend on age and phenotype. JO - PLoS ONE VL - 5 IS - 12 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury. AU - Riederer, I. AU - Sievert, W. AU - Eissner, G.* AU - Molls, M. AU - Multhoff, G. C1 - 5708 C2 - 27862 TI - Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells. JO - PLoS ONE VL - 5 IS - 12 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Association of genetic-variants in the FADS1-FADS2-gene-cluster with fatty-acid-composition in blood of adult-populations is well established. We analyze this genetic-association in two children-cohort-studies. In addition, the association between variants in the FADS-gene-cluster and blood-fatty-acid-composition with eczema was studied. METHODS AND PRINCIPAL FINDINGS: Data of two population-based-birth-cohorts in The Netherlands and Germany (KOALA, LISA) were pooled (n = 879) and analyzed by (logistic) regression regarding the mutual influence of single-nucleotide-polymorphisms (SNPs) in the FADS-gene-cluster (rs174545, rs174546, rs174556, rs174561, rs3834458), on polyunsaturated fatty acids (PUFA) in blood and parent-reported eczema until the age of 2 years. All SNPs were highly significantly associated with all PUFAs except for alpha-linolenic-acid and eicosapentaenoic-acid, also after correction for multiple-testing. All tested SNPs showed associations with eczema in the LISA-study, but not in the KOALA-study. None of the PUFAs was significantly associated with eczema neither in the pooled nor in the analyses stratified by study-cohort. CONCLUSIONS AND SIGNIFICANCE: PUFA-composition in young children's blood is under strong control of the FADS-gene-cluster. Inconsistent results were found for a link between these genetic-variants with eczema. PUFA in blood was not associated with eczema. Thus the hypothesis of an inflammatory-link between PUFA and eczema by the metabolic-pathway of LC-PUFAs as precursors for inflammatory prostaglandins and leukotrienes could not be confirmed by these data. AU - Rzehak, P. AU - Thijs, C.* AU - Standl, M. AU - Mommers, M.* AU - Glaser, C.* AU - Jansen, E.* AU - Klopp, N. AU - Koppelman, G.H.* AU - Singmann, P. AU - Postma, D.S.* AU - Sausenthaler, S. AU - Dagnelie, P.C.* AU - van den Brandt, P.A.* AU - Koletzko, B.* AU - Heinrich, J. C1 - 1474 C2 - 27540 TI - Variants of the FADS1 FADS2 gene cluster, blood levels of polyunsaturated fatty acids and eczema in children within the first 2 years of life. JO - PLoS ONE VL - 5 IS - 10 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed. Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period. This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens. AU - Santoriello, C.* AU - Gennaro, E.* AU - Anelli, V.* AU - Distel, M. AU - Kelly, A.* AU - Köster, R.W. AU - Hurlstone, A.* AU - Mione, M.* C1 - 6082 C2 - 27829 CY - San Francisco SP - 1-11 TI - Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish. JO - PLoS ONE VL - 5 IS - 12 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Evidence increasingly shows that stream ecosystems greatly contribute to global carbon fluxes. This involves a tight coupling between biofilms, the dominant form of microbial life in streams, and dissolved organic carbon (DOC), a very significant pool of organic carbon on Earth. Yet, the interactions between microbial biodiversity and the molecular diversity of resource use are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using six 40-m-long streamside flumes, we created a gradient of streambed landscapes with increasing spatial flow heterogeneity to assess how physical heterogeneity, inherent to streams, affects biofilm diversity and DOC use. We determined bacterial biodiversity in all six landscapes using 16S-rRNA fingerprinting and measured carbon uptake from glucose and DOC experimentally injected to all six flumes. The diversity of DOC molecules removed from the water was determined from ultrahigh-resolution Fourier Transform Ion Cyclotron Resonance mass spectrometry (FTICR-MS). Bacterial beta diversity, glucose and DOC uptake, and the molecular diversity of DOC use all increased with increasing flow heterogeneity. Causal modeling and path analyses of the experimental data revealed that the uptake of glucose was largely driven by physical processes related to flow heterogeneity, whereas biodiversity effects, such as complementarity, most likely contributed to the enhanced uptake of putatively recalcitrant DOC compounds in the streambeds with higher flow heterogeneity. CONCLUSIONS/SIGNIFICANCE: Our results suggest biophysical mechanisms, including hydrodynamics and microbial complementarity effects, through which physical heterogeneity induces changes of resource use and carbon fluxes in streams. These findings highlight the importance of fine-scale streambed heterogeneity for microbial biodiversity and ecosystem functioning in streams, where homogenization and loss of habitats increasingly reduce biodiversity. AU - Singer, G.* AU - Besemer, K.* AU - Schmitt-Kopplin, P. AU - Hödl, I.* AU - Battin, T.J.* C1 - 1482 C2 - 27180 TI - Physical heterogeneity increases biofilm resource use and its molecular diversity in stream mesocosms. JO - PLoS ONE VL - 5 IS - 4 PB - Pubilc Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. AU - Steer, B. AU - Adler, B.* AU - Stipan, J.* AU - Stewart, J.P.* AU - Adler, H. C1 - 981 C2 - 27346 TI - A gammaherpesvirus complement regulatory protein promotes initiation of infection by activation of protein kinase Akt/PKB. JO - PLoS ONE VL - 5 IS - 7 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Metabolomics is the rapidly evolving field of the comprehensive measurement of ideally all endogenous metabolites in a biological fluid. However, no single analytic technique covers the entire spectrum of the human metabolome. Here we present results from a multiplatform study, in which we investigate what kind of results can presently be obtained in the field of diabetes research when combining metabolomics data collected on a complementary set of analytical platforms in the framework of an epidemiological study. METHODOLOGY/PRINCIPAL FINDINGS: 40 individuals with self-reported diabetes and 60 controls (male, over 54 years) were randomly selected from the participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) study, representing an extensively phenotyped sample of the general German population. Concentrations of over 420 unique small molecules were determined in overnight-fasting blood using three different techniques, covering nuclear magnetic resonance and tandem mass spectrometry. Known biomarkers of diabetes could be replicated by this multiple metabolomic platform approach, including sugar metabolites (1,5-anhydroglucoitol), ketone bodies (3-hydroxybutyrate), and branched chain amino acids. In some cases, diabetes-related medication can be detected (pioglitazone, salicylic acid). CONCLUSIONS/SIGNIFICANCE: Our study depicts the promising potential of metabolomics in diabetes research by identification of a series of known and also novel, deregulated metabolites that associate with diabetes. Key observations include perturbations of metabolic pathways linked to kidney dysfunction (3-indoxyl sulfate), lipid metabolism (glycerophospholipids, free fatty acids), and interaction with the gut microflora (bile acids). Our study suggests that metabolic markers hold the potential to detect diabetes-related complications already under sub-clinical conditions in the general population. AU - Suhre, K. AU - Meisinger, C. AU - Döring, A. AU - Altmaier, E. AU - Belcredi, P. AU - Gieger, C. AU - Chang, D.* AU - Milburn, M.V.* AU - Gall, W.E.* AU - Weinberger, K.M.* AU - Mewes, H.-W. AU - Hrabě de Angelis, M. AU - Wichmann, H.-E. AU - Kronenberg, F.* AU - Adamski, J. AU - Illig, T. C1 - 4996 C2 - 27742 TI - Metabolic footprint of diabetes: A multiplatform metabolomics study in an epidemiological setting. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Territorial subdivisions and geographic borders are essential for understanding phenomena in sociology, political science, history, and economics. They influence the interregional flow of information and cross-border trade and affect the diffusion of innovation and technology. However, it is unclear if existing administrative subdivisions that typically evolved decades ago still reflect the most plausible organizational structure of today. The complexity of modern human communication, the ease of long-distance movement, and increased interaction across political borders complicate the operational definition and assessment of geographic borders that optimally reflect the multi-scale nature of today's human connectivity patterns. What border structures emerge directly from the interplay of scales in human interactions is an open question. Based on a massive proxy dataset, we analyze a multi-scale human mobility network and compute effective geographic borders inherent to human mobility patterns in the United States. We propose two computational techniques for extracting these borders and for quantifying their strength. We find that effective borders only partially overlap with existing administrative borders, and show that some of the strongest mobility borders exist in unexpected regions. We show that the observed structures cannot be generated by gravity models for human traffic. Finally, we introduce the concept of link significance that clarifies the observed structure of effective borders. Our approach represents a novel type of quantitative, comparative analysis framework for spatially embedded multi-scale interaction networks in general and may yield important insight into a multitude of spatiotemporal phenomena generated by human activity. AU - Thiemann, C.* AU - Theis, F.J. AU - Grady, D.* AU - Brune, R.* AU - Brockmann, D.* C1 - 3903 C2 - 28029 TI - The structure of borders in a small world. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that knockout mice for Eps8, a regulator of actin dynamics, display reduced body weight, partial resistance to age- or diet-induced obesity, and overall improved metabolic status. Alteration in the liver gene expression profile, in behavior and metabolism point to a calorie restriction-like phenotype in Eps8 knockout mice. Additionally, and consistent with a calorie restricted metabolism, Eps8 knockout mice show increased lifespan. The metabolic alterations in Eps8 knockout mice correlated with a significant reduction in intestinal fat absorption presumably caused by a 25% reduction in intestinal microvilli length. CONCLUSIONS/SIGNIFICANCE: Our findings implicate actin dynamics as a novel variable in the determination of longevity. Additionally, our observations suggest that subtle differences in energy balance can, over time, significantly affect bodyweight and metabolic status in mice. AU - Tocchetti, A.* AU - Soppo, C.B.E.* AU - Zani, F.* AU - Bianchi, F.* AU - Gagliani, M.C.* AU - Pozzi, B.* AU - Rozman, J. AU - Elvert, R. AU - Ehrhardt, N. AU - Rathkolb, B. AU - Mörth, C. AU - Horsch, M. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Beckers, J. AU - Klingenspor, M.* AU - Wolf, E.* AU - Hrabě de Angelis, M. AU - Scanziani, E.* AU - Tacchetti, C.* AU - Scita, G.* AU - di Fiore, P.P.* AU - Offenhäuser, N.* C1 - 302 C2 - 27193 CY - San Francisco TI - Loss of the actin remodeler Eps8 causes intestinal defects and improved metabolic status in mice. JO - PLoS ONE VL - 5 IS - 3 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines. Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell. AU - White, R.E.* AU - Groves, I.J.* AU - Turro, E.* AU - Yee, J.* AU - Kremmer, E. AU - Allday, M.J.* A2 - Masucci, M.G.* C1 - 5671 C2 - 27934 TI - Extensive co-operation between the Epstein-Barr virus EBNA3 proteins in the manipulation of host gene expression and epigenetic chromatin modification. JO - PLoS ONE VL - 5 IS - 11 PB - Public Library of Science PY - 2010 SN - 1932-6203 ER - TY - JOUR AB - HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so far focused on the HP1a isoform and its role in transcriptional regulation. As HP1a requires additional factors to bind methylated chromatin in vitro, we wondered whether another isoform might also require additional targeting factors. Indeed, we found that HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to chromatin in vivo. Moreover, we show that the interaction between HP1c and Woc constitutes a transcriptional feedback loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to methylated heterochromatin. AU - Abel, J.* AU - Eskeland, R.* AU - Raffa, G.D.* AU - Kremmer, E. AU - Imhof, A.* C1 - 867 C2 - 26710 TI - Drosophila HP1c is regulated by an auto-regulatory feedback loop through its binding partner Woc. JO - PLoS ONE VL - 4 IS - 4 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Control of gammaherpesvirus infections requires a complex, well orchestrated immune response regulated by positive and negative co-signaling molecules. While the impact of co-stimulatory molecules has been addressed in various studies, the role of co-inhibitory receptors has not been tested. The ITIM-bearing CEACAM1 is an inhibitory receptor expressed by a variety of immune cells, including B, T and NK cells. Using Ceacam1(-/-) mice, we analyzed the in vivo function of CEACAM1 during acute and latent murine gammaherpesvirus 68 (MHV-68) infection. During acute lytic replication, we observed lower virus titers in the lungs of Ceacam1(-/-) mice than in WT mice. In contrast, during latency amplification, Ceacam1(-/-) mice displayed increased splenomegaly and a higher latent viral load in the spleen. Analysis of the immune response revealed increased virus-specific antibody levels in Ceacam1(-/-) mice, while the magnitude of the T cell-mediated antiviral immune response was reduced. These findings suggest that inhibitory receptors can modulate the efficacy of immune responses against gammaherpesvirus infections. AU - Adler, H. AU - El-Gogo, S.* AU - Guggemoos, S.* AU - Zimmermann, W.* AU - Beauchemin, N.* AU - Kammerer, R.* C1 - 1498 C2 - 26369 TI - Perturbation of lytic and latent gammaherpesvirus infection in the absence of the inhibitory receptor CEACAM1. JO - PLoS ONE VL - 4 IS - 7 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - To reduce the increasing amount of time spent on literature search in the life sciences, several methods for automated knowledge extraction have been developed. Co-occurrence based approaches can deal with large text corpora like MEDLINE in an acceptable time but are not able to extract any specific type of semantic relation. Semantic relation extraction methods based on syntax trees, on the other hand, are computationally expensive and the interpretation of the generated trees is difficult. Several natural language processing (NLP) approaches for the biomedical domain exist focusing specifically on the detection of a limited set of relation types. For systems biology, generic approaches for the detection of a multitude of relation types which in addition are able to process large text corpora are needed but the number of systems meeting both requirements is very limited. We introduce the use of SENNA ("Semantic Extraction using a Neural Network Architecture"), a fast and accurate neural network based Semantic Role Labeling (SRL) program, for the large scale extraction of semantic relations from the biomedical literature. A comparison of processing times of SENNA and other SRL systems or syntactical parsers used in the biomedical domain revealed that SENNA is the fastest Proposition Bank (PropBank) conforming SRL program currently available. 89 million biomedical sentences were tagged with SENNA on a 100 node cluster within three days. The accuracy of the presented relation extraction approach was evaluated on two test sets of annotated sentences resulting in precision/recall values of 0.71/0.43. We show that the accuracy as well as processing speed of the proposed semantic relation extraction approach is sufficient for its large scale application on biomedical text. The proposed approach is highly generalizable regarding the supported relation types and appears to be especially suited for general-purpose, broad-scale text mining systems. The presented approach bridges the gap between fast, co-occurrence-based approaches lacking semantic relations and highly specialized and computationally demanding NLP approaches. AU - Barnickel, T. AU - Weston, J.* AU - Collobert, R.* AU - Mewes, H.-W. AU - Stuempflen, V. C1 - 313 C2 - 26468 TI - Large scale application of neural network based semantic role labeling for automated relation extraction from biomedical texts. JO - PLoS ONE VL - 4 IS - 7 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Background : Atherosclerosis is the primary cause of coronary artery disease (CAD). There is increasing recognition that lesion composition rather than size determines the acute complications of atherosclerotic disease. Low serum adiponectin levels were reported to be associated with coronary artery disease and future incidence of acute coronary syndrome (ACS). The impact of adiponectin on lesion composition still remains to be determined. Methodology/Principal Findings: We measured serum adiponectin levels in 303 patients with stable typical or atypical chest pain, who underwent dual-source multi-slice CT-angiography to exclude coronary artery stenosis. Atherosclerotic plaques were classified as calcified, mixed or non-calcified. In bivariate analysis adiponectin levels were inversely correlated with total coronary plaque burden (r = −0.21, p = 0.0004), mixed (r = −0.20, p = 0.0007) and non-calcified plaques (r = −0.18, p = 0.003). No correlation was seen with calcified plaques (r = −0.05, p = 0.39). In a fully adjusted multivariate model adiponectin levels remained predictive of total plaque burden (estimate: −0.036, 95%CI: −0.052 to −0.020, p<0.0001), mixed (estimate: −0.087, 95%CI: −0.132 to −0.042, p = 0.0001) and non-calcified plaques (estimate: −0.076, 95%CI: −0.115 to −0.038, p = 0.0001). Adiponectin levels were not associated with calcified plaques (estimate: −0.021, 95% CI: −0.043 to −0.001, p = 0.06). Since the majority of coronary plaques was calcified, adiponectin levels account for only 3% of the variability in total plaque number. In contrast, adiponectin accounts for approximately 20% of the variability in mixed and non-calcified plaque burden. Conclusions/Significance : Adiponectin levels predict mixed and non-calcified coronary atherosclerotic plaque burden. Low adiponectin levels may contribute to coronary plaque vulnerability and may thus play a role in the pathophysiology of ACS. AU - Broedl, U.C.* AU - Lebherz, C.* AU - Lehrke, M.* AU - Stark, R.G. AU - Greif, M.* AU - Becker, F.* AU - von Ziegler, A.* AU - Tittus, J.* AU - Reiser, M.* AU - Becker, C.* AU - Göke, B.* AU - Parhofer, K.G.* AU - Leber, A.W.* C1 - 1616 C2 - 26453 TI - Low adiponectin levels are an independent predictor of mixed and non-calcified coronary atherosclerotic plaques. JO - PLoS ONE VL - 4 IS - 3 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC). METHODOLOGY/PRINCIPAL FINDINGS: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF(DeltaARE) and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells. CONCLUSIONS/SIGNIFICANCE: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect. AU - Hormannsperger, G.* AU - Clavel, T.* AU - Hoffmann, M.* AU - Reiff, C.* AU - Kelly, D.* AU - Loh, G.* AU - Blaut, M.* AU - Hölzlwimmer, G. AU - Laschinger, M.* AU - Haller, D.* C1 - 4345 C2 - 28341 TI - Post-translational inhibition of IP-10 secretion in IEC by probiotic bacteria: impact on chronic inflammation. JO - PLoS ONE VL - 4 IS - 2 PB - Public Library Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - The causes and etiology of Crohn's disease (CD) are currently unknown although both host genetics and environmental factors play a role. Here we used non-targeted metabolic profiling to determine the contribution of metabolites produced by the gut microbiota towards disease status of the host. Ion Cyclotron Resonance Fourier Transform Mass Spectrometry (ICR-FT/MS) was used to discern the masses of thousands of metabolites in fecal samples collected from 17 identical twin pairs, including healthy individuals and those with CD. Pathways with differentiating metabolites included those involved in the metabolism and or synthesis of amino acids, fatty acids, bile acids and arachidonic acid. Several metabolites were positively or negatively correlated to the disease phenotype and to specific microbes previously characterized in the same samples. Our data reveal novel differentiating metabolites for CD that may provide diagnostic biomarkers and/or monitoring tools as well as insight into potential targets for disease therapy and prevention. AU - Jansson, J.* AU - Willing, B.* AU - Lucio, M. AU - Fekete, A. AU - Dicksved, J.* AU - Halfvarson, J.* AU - Tysk, C.* AU - Schmitt-Kopplin, P. C1 - 84 C2 - 26351 TI - Metabolomics reveals metabolic biomarkers of Crohn's disease. JO - PLoS ONE VL - 4 IS - 7 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - In the post-genome era, insufficient functional annotation of predicted genes greatly restricts the potential of mining genome data. We demonstrate that an evolutionary approach, which is independent of functional annotation, has great potential as a tool for genome analysis. We chose the genome of a model filamentous fungus Neurospora crassa as an example. Phylogenetic distribution of each predicted protein coding gene (PCG) in the N. crassa genome was used to classify genes into six mutually exclusive lineage specificity (LS) groups, i.e. Eukaryote/Prokaryote-core, Dikarya-core, Ascomycota-core, Pezizomycotina-specific, N. crassa-orphans and Others. Functional category analysis revealed that only approximately 23% of PCGs in the two most highly lineage-specific grouping, Pezizomycotina-specific and N. crassa-orphans, have functional annotation. In contrast, approximately 76% of PCGs in the remaining four LS groups have functional annotation. Analysis of chromosomal localization of N. crassa-orphan PCGs and genes encoding for secreted proteins showed enrichment in subtelomeric regions. The origin of N. crassa-orphans is not known. We found that 11% of N. crassa-orphans have paralogous N. crassa-orphan genes. Of the paralogous N. crassa-orphan gene pairs, 33% were tandemly located in the genome, implying a duplication origin of N. crassa-orphan PCGs in the past. LS grouping is thus a useful tool to explore and understand genome organization, evolution and gene function in fungi. AU - Kasuga, T.* AU - Mannhaupt, G. AU - Glass, N.L.* C1 - 2711 C2 - 26081 TI - Relationship between phylogenetic distribution and genomic features in Neurospora crassa. JO - PLoS ONE VL - 4 IS - 4 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. METHODOLOGY/PRINCIPAL FINDINGS: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. CONCLUSIONS/SIGNIFICANCE: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events. AU - Levin, A.* AU - Hayouka, Z.* AU - Helfer, M.* AU - Brack-Werner, R. AU - Friedler, A.* AU - Loyter, A.* C1 - 510 C2 - 25971 TI - Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration. JO - PLoS ONE VL - 4 IS - 1 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Background: Heidenreich et al. ( Risk Anal 1997 17 391-399) considered parameter identifiability in the context of the two-mutation cancer model and demonstrated that combinations of all but two of the model parameters are identifiable. We consider the problem of identifiability in the recently developed carcinogenesis models of Little and Wright ( Math Biosci 2003 183 111-134) and Little et al. (J Theoret Biol 2008 254 229-238). These models, which incorporate genomic instability, generalize a large number of other quasi-biological cancer models, in particular those of Armitage and Doll (Br J Cancer 1954 8 1-12), the two-mutation model (Moolgavkar et al. Math Biosci 1979 47 55-77), the generalized multistage model of Little ( Biometrics 1995 51 1278-1291), and a recently developed cancer model of Nowak et al. (PNAS 2002 99 16226-16231). Methodology/Principal Findings: We show that in the simpler model proposed by Little and Wright ( Math Biosci 2003 183 111-134) the number of identifiable combinations of parameters is at most two less than the number of biological parameters, thereby generalizing previous results of Heidenreich et al. ( Risk Anal 1997 17 391-399) for the two-mutation model. For the more general model of Little et al. ( J Theoret Biol 2008 254 229-238) the number of identifiable combinations of parameters is at most r + 1 less than the number of biological parameters, where r is the number of destabilization types, thereby also generalizing all these results. Numerical evaluations suggest that these bounds are sharp. We also identify particular combinations of identifiable parameters. Conclusions/Significance: We have shown that the previous results on parameter identifiability can be generalized to much larger classes of quasi-biological carcinogenesis model, and also identify particular combinations of identifiable parameters. These results are of theoretical interest, but also of practical significance to anyone attempting to estimate parameters for this large class of cancer models. AU - Little, M.P.* AU - Heidenreich, W.F. AU - Li, G.Q.* C1 - 1319 C2 - 26748 TI - Parameter identifiability and redundancy in a general class of stochastic carcinogenesis models. JO - PLoS ONE VL - 4 IS - 12 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Using principal component (PC) analysis, we studied the genetic constitution of 3,112 individuals from Europe as portrayed by more than 270,000 single nucleotide polymorphisms (SNPs) genotyped with the Illumina Infinium platform. In cohorts where the sample size was >100, one hundred randomly chosen samples were used for analysis to minimize the sample size effect, resulting in a total of 1,564 samples. This analysis revealed that the genetic structure of the European population correlates closely with geography. The first two PCs highlight the genetic diversity corresponding to the northwest to southeast gradient and position the populations according to their approximate geographic origin. The resulting genetic map forms a triangular structure with a) Finland, b) the Baltic region, Poland and Western Russia, and c) Italy as its vertexes, and with d) Central- and Western Europe in its centre. Inter- and intra- population genetic differences were quantified by the inflation factor lambda (lambda) (ranging from 1.00 to 4.21), fixation index (F(st)) (ranging from 0.000 to 0.023), and by the number of markers exhibiting significant allele frequency differences in pair-wise population comparisons. The estimated lambda was used to assess the real diminishing impact to association statistics when two distinct populations are merged directly in an analysis. When the PC analysis was confined to the 1,019 Estonian individuals (0.1% of the Estonian population), a fine structure emerged that correlated with the geography of individual counties. With at least two cohorts available from several countries, genetic substructures were investigated in Czech, Finnish, German, Estonian and Italian populations. Together with previously published data, our results allow the creation of a comprehensive European genetic map that will greatly facilitate inter-population genetic studies including genome wide association studies (GWAS). AU - Nelis, M.* AU - Esko, T.* AU - Mägi, R.* AU - Zimprich, F.* AU - * AU - Toncheva, D.* AU - Karachanak, S.* AU - Piskackova, T.* AU - Balascák, I.* AU - Peltonen, L.* AU - Jakkula, E.* AU - Rehnström, K.* AU - Lathrop, M* AU - Heath, S.* AU - Galan, P.* AU - Schreiber, S.* AU - Meitinger, T. AU - Pfeufer, A. AU - Wichmann, H.-E. AU - Melegh, B.* AU - Polgár, N.* AU - Toniolo, D.* AU - Gasparini, P.* AU - D'Adamo, P.* AU - Klovins, J.* AU - Nikitina-Zake, L.* AU - Kucinskas, V.* AU - Kasnauskiene, J.* AU - Lubinski, J.* AU - Debniak, T.* AU - Limborska, S.* AU - Khrunin, A.* AU - Estivill, X.* AU - Rabionet, R.* AU - Marsal, S.* AU - Julià, A.* AU - Antonarakis, S.E.* AU - Deutsch, S.* AU - Borel, C.* AU - Attar, H.* AU - Gagnebin, M.* AU - Macek, M.* AU - Krawczak, M.* AU - Remm, M.* AU - Metspalu, A.* C1 - 1638 C2 - 26302 TI - Genetic structure of Europeans: A view from the North-East. JO - PLoS ONE VL - 4 IS - 5 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - To identify loci affecting the electrocardiographic QT interval, a measure of cardiac repolarisation associated with risk of ventricular arrhythmias and sudden cardiac death, we conducted a meta-analysis of three genome-wide association studies (GWAS) including 3,558 subjects from the TwinsUK and BRIGHT cohorts in the UK and the DCCT/EDIC cohort from North America. Five loci were significantly associated with QT interval at P<1x10(-6). To validate these findings we performed an in silico comparison with data from two QT consortia: QTSCD (n = 15,842) and QTGEN (n = 13,685). Analysis confirmed the association between common variants near NOS1AP (P = 1.4x10(-83)) and the phospholamban (PLN) gene (P = 1.9x10(-29)). The most associated SNP near NOS1AP (rs12143842) explains 0.82% variance; the SNP near PLN (rs11153730) explains 0.74% variance of QT interval duration. We found no evidence for interaction between these two SNPs (P = 0.99). PLN is a key regulator of cardiac diastolic function and is involved in regulating intracellular calcium cycling, it has only recently been identified as a susceptibility locus for QT interval. These data offer further mechanistic insights into genetic influence on the QT interval which may predispose to life threatening arrhythmias and sudden cardiac death. AU - Nolte, I.M.* AU - Wallace,C.* AU - Newhouse, S.J.* AU - Waggott, D.* AU - Fu, J.* AU - Soranzo, N.* AU - Gwilliam, R.* AU - Deloukas, P.* AU - Savelieva, I.* AU - Zheng, D.* AU - Dalageorgou, C.* AU - Farrall, M.* AU - Samani, N.J.* AU - Connell, J.* AU - Brown, M.* AU - Dominiczak, A.* AU - Lathrop, M* AU - Zeggini, E.* AU - Wain, L.V.* AU - Newton-Cheh, C.* AU - Eijgelsheim, M.* AU - Rice, K.* AU - de Bakker, P.I.* AU - Pfeufer, A.* AU - Sanna, S.* AU - Arking, D.E.* AU - Asselbergs, F.W.* AU - Spector, T.D.* AU - Carter, N.D.* AU - Jeffery, S.* AU - Tobin, M.* AU - Caulfield, M.* AU - Snieder, H.* AU - Paterson, A.D.* AU - Munroe, P.B.* AU - Jamshidi, Y.* C1 - 680 C2 - 26454 TI - Common genetic variation near the Phospholamban gene is associated with cardiac repolarisation: Meta-analysis of three genome-wide association studies. JO - PLoS ONE VL - 4 IS - 7 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. METHODOLOGY/PRINCIPAL FINDINGS: The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. CONCLUSIONS/SIGNIFICANCE: The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection. AU - Noyes, H.A.* AU - Alimohammadian, M.H.* AU - Agaba, M.* AU - Brass, A.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hulme, H.* AU - Iraqi, F.* AU - Kemp, S.* AU - Rathkolb, B. AU - Wolf, E.* AU - Hrabě de Angelis, M.* AU - Roshandel, D.* AU - Naessens, J.* C1 - 1134 C2 - 26338 TI - Mechanisms controlling anaemia in Trypanosoma congolense infected mice. JO - PLoS ONE VL - 4 IS - 4 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - A chimeric HLA-DR4-H2-E (DR4) homozygous transgenic mouse line spontaneously develops diverse hematological malignancies with high frequency (70%). The majority of malignancies were distributed equally between T and B cell neoplasms and included lymphoblastic T cell lymphoma (LTCL), lymphoblastic B cell lymphoma (LBCL), diffuse large B cell lymphoma (DLBCL), the histiocyte/T cell rich variant of DLBCL (DLBCL-HA/T cell rich DLBCL), splenic marginal zone lymphoma (SMZL), follicular B cell lymphoma (FBL) and plasmacytoma (PCT). Most of these neoplasms were highly similar to human diseases. Also, some non-lymphoid malignancies such as acute myeloid leukemia (AML) and histiocytic sarcoma were found. Interestingly, composite lymphomas, including Hodgkin-like lymphomas, were also detected that had CD30(+) Hodgkin/Reed-Sternberg (H/RS)-like cells, representing a tumor type not previously described in mice. Analysis of microdissected H/RS-like cells revealed their origin as germinal center B cells bearing somatic hypermutations and, in some instances, crippled mutations, as described for human Hodgkin lymphoma (HL). Transgene integration in an oncogene was excluded as an exclusive driving force of tumorigenesis and age-related lymphoma development suggests a multi-step process. Thus, this DR4 line is a useful model to investigate common molecular mechanisms that may contribute to important neoplastic diseases in man. AU - Raffegerst, S.H. AU - Hölzlwimmer, G. AU - Kunder, S. AU - Mysliwietz, J. AU - Quintanilla-Martinez, L. AU - Schendel, D.J. C1 - 1658 C2 - 26737 TI - Diverse hematological malignancies including hodgkin-like lymphomas develop in chimeric MHC class II transgenic mice. JO - PLoS ONE VL - 4 IS - 12 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Disruption of the postsynaptic density (PSD), a network of scaffold proteins located in dendritic spines, is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer's disease (AD). Extending our previous demonstration that derangement of the PSD by soluble amyloid-beta (Abeta) involves proteasomal degradation of PSD-95, a protein important for ionotropic glutamate receptor trafficking, we now show that Abeta also disrupts two other scaffold proteins, Homer1b and Shank1, that couple PSD-95 with ionotropic and metabotropic glutamate receptors. Treatment of fronto-cortical neurons with soluble Abeta results in rapid (within 1 h) and significant thinning of the PSD, decreased synaptic levels of Homer1b and Shank1, and reduced synaptic mGluR1 levels. We show that de novo protein synthesis is required for the declustering effects of Abeta on Homer1b (but not Shank1) and that, in contrast to PSD-95, Abeta-induced Homer1b and Shank1 cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Abeta diverges in two other respects: i) whereas the activity of both NMDAR and VDCC is required for Abeta-induced declustering of Homer1b, Abeta-induced declustering of Shank1 only requires NMDAR activity; and ii) whereas the effects of Abeta on Homer1b involve engagement of the PI-3K pathway and calcineurin phosphatase (PP2B) activity, those on Shank1 involve activation of the ERK pathway. In summary, soluble Abeta recruits discrete signalling pathways to rapidly reduce the synaptic localization of major components of the PSD and to regulate the availability of mGluR1 in the synapse. AU - Roselli, F.* AU - Hutzler, P. AU - Wegerich, Y.* AU - Livrea, P.* AU - Almeida, O.F.* C1 - 1095 C2 - 26350 TI - Disassembly of shank and homer synaptic clusters is driven by soluble β-amyloid₁₋₄₀ through divergent NMDAR-dependent signalling pathways. JO - PLoS ONE VL - 4 IS - 6 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes. AU - Rubio-Aliaga, I. AU - Przemeck, G.K.H. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Adler, T. AU - Hans, W. AU - Horsch, M. AU - Rathkolb, B. AU - Rozman, J. AU - Schrewe, A. AU - Wagner, S. AU - Hölter, S.M. AU - Becker, L. AU - Klopstock, T.* AU - Wurst, W. AU - Wolf, E.* AU - Klingenspor, M.* AU - Ivandic, B.T.* AU - Busch, D.H.* AU - Beckers, J. AU - Hrabě de Angelis, M. C1 - 1456 C2 - 26368 TI - Dll1 haploinsufficiency in adult mice leads to a complex phenotype affecting metabolic and immunological processes. JO - PLoS ONE VL - 4 IS - 6 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - It is known that miRNA target sites are very short and the effect of miRNA-target site interaction alone appears as being unspecific. Recent experiments suggest further context signals involved in miRNA target site recognition and regulation. Here, we present a novel GC-rich RNA motif downstream of experimentally supported miRNA target sites in human mRNAs with no similarity to previously reported functional motifs. We demonstrate that the novel motif can be found in at least one third of all transcripts regulated by miRNAs. Furthermore, we show that motif occurrence and the frequency of miRNA target sites as well as the stability of their duplex structures correlate. The finding, that the novel motif is significantly associated with miRNA target sites, suggests a functional role of the motif in miRNA target site biology. Beyond, the novel motif has the impact to improve prediction of miRNA target sites significantly. AU - Schmidt, T. AU - Mewes, H.-W. AU - Stuempflen, V. C1 - 270 C2 - 26390 TI - A novel putative miRNA target enhancer signal. JO - PLoS ONE VL - 4 IS - 7 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - The outcome of Genome-Wide Association Studies (GWAS) has challenged the field of blood pressure (BP) genetics as previous candidate genes have not been among the top loci in these scans. We used Affymetrix 500K genotyping data of KORA S3 cohort (n = 1,644; Southern-Germany) to address (i) SNP coverage in 160 BP candidate genes; (ii) the evidence for associations with BP traits in genome-wide and replication data, and haplotype analysis. In total, 160 gene regions (genic region+/-10 kb) covered 2,411 SNPs across 11.4 Mb. Marker densities in genes varied from 0 (n = 11) to 0.6 SNPs/kb. On average 52.5% of the HAPMAP SNPs per gene were captured. No evidence for association with BP was obtained for 1,449 tested SNPs. Considerable associations (P<10(-3)) were detected for the genes, where >50% of HAPMAP SNPs were tagged. In general, genes with higher marker density (>0.2 SNPs/kb) revealed a better chance to reach close to significance associations. Although, none of the detected P-values remained significant after Bonferroni correction (P<0.05/2319, P<2.15 x 10(-5)), the strength of some detected associations was close to this level: rs10889553 (LEPR) and systolic BP (SBP) (P = 4.5 x 10(-5)) as well as rs10954174 (LEP) and diastolic BP (DBP) (P = 5.20 x 10(-5)). In total, 12 markers in 7 genes (ADRA2A, LEP, LEPR, PTGER3, SLC2A1, SLC4A2, SLC8A1) revealed considerable association (P<10(-3)) either with SBP, DBP, and/or hypertension (HYP). None of these were confirmed in replication samples (KORA S4, HYPEST, BRIGHT). However, supportive evidence for the association of rs10889553 (LEPR) and rs11195419 (ADRA2A) with BP was obtained in meta-analysis across samples stratified either by body mass index, smoking or alcohol consumption. Haplotype analysis highlighted LEPR and PTGER3. In conclusion, the lack of associations in BP candidate genes may be attributed to inadequate marker coverage on the genome-wide arrays, small phenotypic effects of the loci and/or complex interaction with life-style and metabolic parameters. AU - Sõber, S.* AU - Org, E.* AU - Kepp, K.* AU - Juhanson, P.* AU - Eyheramendy, S. AU - Gieger, C. AU - Lichtner, P. AU - Klopp, N. AU - Veldre, G.* AU - Viigimaa, M.* AU - Döring, A. AU - Putku, M.* AU - Kelgo, P* AU - Shaw-Hawkins, S.* AU - Howard, P.* AU - Onipinla, A.* AU - Dobson, R.J.* AU - Newhouse, S.J.* AU - Brown, M.* AU - Dominiczak, A.* AU - Connell, J.* AU - Samani, N.* AU - Farrall, M* AU - Caulfield, M.J.* AU - Munroe, P.B.* AU - Illig, T. AU - Wichmann, H.-E. AU - Meitinger, T. AU - Laan, M.* C1 - 112 C2 - 26585 TI - Targeting 160 candidate genes for blood pressure regulation with a genome-wide genotyping array. JO - PLoS ONE VL - 4 IS - 6 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: Recently, a large meta-analysis including over 28,000 participants identified nine different loci with association to serum uric acid (UA) levels. Since elevated serum UA levels potentially cause gout and are a possible risk factor for coronary artery disease (CAD) and myocardial infarction (MI), we performed two large case-control association analyses with participants from the German MI Family Study. In the first study, we assessed the association of the qualitative trait gout and ten single nucleotide polymorphisms (SNP) markers that showed association to UA serum levels. In the second study, the same genetic polymorphisms were analyzed for association with CAD. METHODS AND FINDINGS: A total of 683 patients suffering from gout and 1,563 healthy controls from the German MI Family Study were genotyped. Nine SNPs were identified from a recently performed genome-wide meta-analysis on serum UA levels (rs12129861, rs780094, rs734553, rs2231142, rs742132, rs1183201, rs12356193, rs17300741 and rs505802). Additionally, the marker rs6855911 was included which has been associated with gout in our cohort in a previous study. SNPs rs734553 and rs6855911, located in SLC2A9, and SNP rs2231142, known to be a missense polymorphism in ABCG2, were associated with gout (p=5.6*10(-7), p=1.1*10(-7), and p=1.3*10(-3), respectively). Other SNPs in the genes PDZK1, GCKR, LRRC16A, SLC17A1-SLC17A3, SLC16A9, SLC22A11 and SLC22A12 failed the significance level. None of the ten markers were associated with risk to CAD in our study sample of 1,473 CAD cases and 1,241 CAD-free controls. CONCLUSION: SNP markers in SLC2A9 and ABCG2 genes were found to be strongly associated with the phenotype gout. However, not all SNP markers influencing serum UA levels were also directly associated with the clinical manifestation of gout in our study sample. In addition, none of these SNPs showed association with the risk to CAD in the German MI Family Study. AU - Stark, K.* AU - Reinhard, W.* AU - Grassl, M.* AU - Erdmann, J.* AU - Schunkert, H.* AU - Illig, T. AU - Hengstenberg, C.* C1 - 1174 C2 - 26491 TI - Common polymorphisms influencing serum uric acid levels contribute to susceptibility to gout, but not to coronary artery disease. JO - PLoS ONE VL - 4 IS - 11 PB - Public Library of Science PY - 2009 SN - 1932-6203 ER - TY - JOUR AB - Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a-/- mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a-/- mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors. AU - Andersson, E.R.* AU - Prakash, N. AU - Cajanek, L.* AU - Minina, E. AU - Bryja, V.* AU - Bryjova, L.* AU - Yamaguchi, T.P.* AU - Hall, A.C.* AU - Wurst, W. AU - Arenas, E.* C1 - 1962 C2 - 25890 TI - Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo. JO - PLoS ONE VL - 3 IS - 10 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Autoimmune pancreatocholangitis (AIPC) is an emerging, not completely characterized disease. Aim of this study was the comprehensive evaluation of a series of AIPC patients, who were diagnosed and treated in a European institution between January 2003 and July 2006. METHODOLOGY/PRINCIPAL FINDINGS: Thirty-three patients with histologically confirmed AIPC were analyzed and compared to 20 patients with non-autoimmune chronic pancreatitis (CP) and 14 patients with primary sclerosing cholangitis (PSC). Clinical features and conventional histopathology were taken into account. Immunohistochemistry and real-time quantitative PCR were used for the characterization of the inflammatory infiltrate and the stromal reaction. AIPC was localized in the pancreatic head in 94% of the patients. Intra- and/or extrapancreatic biliary tract involvement was present in 64% of the cases. The number of infiltrating T-lymphocytes, macrophages and total plasma cells was significantly higher in AIPC than in CP (3-, 4- and 8-fold increase, respectively). The absolute number of IgG4-positive plasma cells was higher in AIPC than in CP and PSC (7-fold and 35-fold increase, respectively), but significance was only reached in comparison with PSC. CXCR5- and CXCL13-positive cells were almost exclusively detected in AIPC. CONCLUSIONS/SIGNIFICANCE: AIPC is mainly a disease of the pancreatic head with possible extension into the periphery of the gland and/or into the biliary tract/gallbladder. The morphology of AIPC, as well as the immune- and stromal reaction is characteristic and comparable between cases with and without biliary tract involvement. Immunological markers (IgG4, CXCR5, CXCL13) can be of diagnostic relevance in specific settings. AU - Esposito, I. AU - Born, D.* AU - Bergmann, F.* AU - Longerich, T.* AU - Welsch, T.* AU - Giese, N.A.* AU - Büchler, M.W.* AU - Kleeff, J.* AU - Friess, H.* AU - Schirmacher, P.* C1 - 3541 C2 - 25666 TI - Autoimmune pancreatocholangitis, non-autoimmune pancreatitis and primary sclerosing cholangitis: A comparative morphological and immunological analysis. JO - PLoS ONE VL - 3 IS - 7 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Human tumors differ from normal tissues in their capacity to present Hsp70, the major stress-inducible member of the HSP70 family, on their plasma membrane. Membrane Hsp70 has been found to serve as a prognostic indicator of overall patient survival in leukemia, lower rectal and non small cell lung carcinomas. Why tumors, but not normal cells, present Hsp70 on their cell surface and the impact of membrane Hsp70 on cancer progression remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Although Hsp70 has been reported to be associated with cholesterol rich microdomains (CRMs), the partner in the plasma membrane with which Hsp70 interacts has yet to be identified. Herein, global lipid profiling demonstrates that Hsp70 membrane-positive tumors differ from their membrane-negative counterparts by containing significantly higher amounts of globotriaoslyceramide (Gb3), but not of other lipids such as lactosylceramide (LacCer), dodecasaccharideceramide (DoCer), galactosylceramide (GalCer), ceramide (Cer), or the ganglioside GM1. Apart from germinal center B cells, normal tissues are Gb3 membrane-negative. Co-localization of Hsp70 and Gb3 was selectively determined in Gb3 membrane-positive tumor cells, and these cells were also shown to bind soluble Hsp70-FITC protein from outside in a concentration-dependent manner. Given that the latter interaction can be blocked by a Gb3-specific antibody, and that the depletion of globotriaosides from tumors reduces the amount of membrane-bound Hsp70, we propose that Gb3 is a binding partner for Hsp70. The in vitro finding that Hsp70 predominantly binds to artificial liposomes containing Gb3 (PC/SM/Chol/Gb3, 17/45/33/5) confirms that Gb3 is an interaction partner for Hsp70. CONCLUSIONS/SIGNIFICANCE: These data indicate that the presence of Gb3 enables anchorage of Hsp70 in the plasma membrane of tumors and thus they might explain tumor-specific membrane localization of Hsp70. AU - Gehrmann, M.* AU - Liebisch, G.* AU - Schmitz, G.* AU - Anderson, R.* AU - Steinem, C.* AU - de Maio, A.* AU - Pockley, G.* AU - Multhoff, G. C1 - 2941 C2 - 25194 TI - Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. JO - PLoS ONE VL - 3 IS - 4 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Statistically reconstructing haplotypes from single nucleotide polymorphism (SNP) genotypes, can lead to falsely classified haplotypes. This can be an issue when interpreting haplotype association results or when selecting subjects with certain haplotypes for subsequent functional studies. It was our aim to quantify haplotype reconstruction error and to provide tools for it. METHODS AND RESULTS: By numerous simulation scenarios, we systematically investigated several error measures, including discrepancy, error rate, and R(2), and introduced the sensitivity and specificity to this context. We exemplified several measures in the KORA study, a large population-based study from Southern Germany. We find that the specificity is slightly reduced only for common haplotypes, while the sensitivity was decreased for some, but not all rare haplotypes. The overall error rate was generally increasing with increasing number of loci, increasing minor allele frequency of SNPs, decreasing correlation between the alleles and increasing ambiguity. CONCLUSIONS: We conclude that, with the analytical approach presented here, haplotype-specific error measures can be computed to gain insight into the haplotype uncertainty. This method provides the information, if a specific risk haplotype can be expected to be reconstructed with rather no or high misclassification and thus on the magnitude of expected bias in association estimates. We also illustrate that sensitivity and specificity separate two dimensions of the haplotype reconstruction error, which completely describe the misclassification matrix and thus provide the prerequisite for methods accounting for misclassification. AU - Lamina, C. AU - Bongardt, F.* AU - Küchenhoff, H.* AU - Heid, I.M. C1 - 4042 C2 - 25260 TI - Haplotype reconstruction error as a classical misclassification problem: Introducing sensitivity and specificity as error measures. JO - PLoS ONE VL - 3 IS - 3 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Rare mutations of the low-density lipoprotein receptor gene (LDLR) cause familial hypercholesterolemia, which increases the risk for coronary artery disease (CAD). Less is known about the implications of common genetic variation in the LDLR gene regarding the variability of cholesterol levels and risk of CAD. METHODS: Imputed genotype data at the LDLR locus on 1 644 individuals of a population-based sample were explored for association with LDL-C level. Replication of association with LDL-C level was sought for the most significant single nucleotide polymorphism (SNP) within the LDLR gene in three European samples comprising 6 642 adults and 533 children. Association of this SNP with CAD was examined in six case-control studies involving more than 15 000 individuals. FINDINGS: Each copy of the minor T allele of SNP rs2228671 within LDLR (frequency 11%) was related to a decrease of LDL-C levels by 0.19 mmol/L (95% confidence interval (CI) [0.13-0.24] mmol/L, p = 1.5x10(-10)). This association with LDL-C was uniformly found in children, men, and women of all samples studied. In parallel, the T allele of rs2228671 was associated with a significantly lower risk of CAD (Odds Ratio per copy of the T allele: 0.82, 95% CI [0.76-0.89], p = 2.1x10(-7)). Adjustment for LDL-C levels by logistic regression or Mendelian Randomisation models abolished the significant association between rs2228671 with CAD completely, indicating a functional link between the genetic variant at the LDLR gene locus, change in LDL-C and risk of CAD. CONCLUSION: A common variant at the LDLR gene locus affects LDL-C levels and, thereby, the risk for CAD. AU - Linsel-Nitschke, P.* AU - Götz, A.* AU - Erdmann, J.* AU - Braenne, I.* AU - Braund, P.* AU - Hengstenberg, C.* AU - Stark, K.* AU - Fischer, M.* AU - Schreiber, S.* AU - El Mokhtari, N.E.* AU - Schaefer, A.* AU - Schrezenmeir, J.* AU - Rubin, D.* AU - Hinney, A.* AU - Reinehr, T.* AU - Roth, C.* AU - Ortlepp, J.* AU - Hanrath, P.* AU - Hall, A.S.* AU - Mangino, M.* AU - Lieb, W.* AU - Lamina, C. AU - Heid, I.M. AU - Döring, A. AU - Gieger, C. AU - Peters, A. AU - Meitinger, T. AU - Wichmann, H.-E. AU - König, I.R.* AU - Ziegler, A.* AU - Kronenberg, F.* AU - Samani, N.J.* AU - Schunkert, H.* AU - Wellcome Trust Case Consortium (WTCCC) (*) AU - CARDIOGENICS Consortium (*) C1 - 2360 C2 - 25566 TI - Lifelong reduction of LDL-cholesterol related to a common variant in the LDL-receptor gene decreases the risk of coronary artery disease--a Mendelian Randomisation study. JO - PLoS ONE VL - 3 IS - 8 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - BACKGROUND: CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia. METHODS: Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from - to +++ (score = % x intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC. RESULTS: In normal epithelia CD44s and CD44v6 were expressed in 60-95% and 50-80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5+/-14.5 and 198+/-11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9+/-7.16 and 242+/-11.7; 291.8+/-5.88 and 287.3+/-6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6. CONCLUSION: CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. AU - Mack, B.* AU - Gires, O. C1 - 2609 C2 - 25885 TI - CD44s and CD44v6 expression in head and neck epithelia. JO - PLoS ONE VL - 3 IS - 10 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Clinical and pathological changes in familial Creutzfeldt-Jakob disease (CJD) cases may be similar or indistinguishable from sporadic CJD. Therefore determination of novel mutations in PRNP remains of major importance.We identified two different rare mutations in codon 188 of the prion protein gene (PRNP) in four patients suffering from a disease clinically very similar to the major subtype of sporadic CJD. Both mutations result in an exchange of the amino acid residue threonine for a highly basic residue, either arginine (T188R) or lysine (T188K). The T188R mutation was found in one patient and the T188K mutation in three patients. The prevalence of mutations at codon 188 of PRNP was tested in 593 sporadic CJD cases and 735 healthy individuals. Neither mutation was found. The data presented here argue in favor of T188K being a pathogenic mutation causing genetic CJD. Since one individual with this mutation, who is the father of a clinically affected patient with T188K mutation, is now 79 years old and shows no signs of disease, this mutation is likely associated with a penetrance under 100%. Further observations will have to show whether T188R is a pathogenic mutation. AU - Roeber, S.* AU - Grasbon-Frodl, E.M.* AU - Windl, O.* AU - Krebs, B.* AU - Xiang, W.* AU - Vollmert, C. AU - Illig, T. AU - Schröter, A.* AU - Arzberger, T.* AU - Weber, P.* AU - Zerr, I.* AU - Kretzschmar, H.A.* C1 - 793 C2 - 25505 TI - Evidence for a pathogenic role of different mutations at codon 188 of PRNP. JO - PLoS ONE VL - 3 IS - 5 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells. AU - Soerensen, J. AU - Jakupoglu, C. AU - Beck, H.* AU - Förster, H. AU - Schmidt, J. AU - Schmahl, W.* AU - Schweizer, U.* AU - Conrad, M. AU - Brielmeier, M. C1 - 2633 C2 - 25449 TI - The role of thioredoxin reductases in brain development. JO - PLoS ONE VL - 3 IS - 3 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Exposure to nicotine during smoking causes a multitude of metabolic changes that are poorly understood. We quantified and analyzed 198 metabolites in 283 serum samples from the human cohort KORA (Cooperative Health Research in the Region of Augsburg). Multivariate analysis of metabolic profiles revealed that the group of smokers could be clearly differentiated from the groups of former smokers and non-smokers. Moreover, 23 lipid metabolites were identified as nicotine-dependent biomarkers. The levels of these biomarkers are all up-regulated in smokers compared to those in former and non-smokers, except for three acyl-alkyl-phosphatidylcholines (e.g. plasmalogens). Consistently significant results were further found for the ratios of plasmalogens to diacyl-phosphatidylcolines, which are reduced in smokers and regulated by the enzyme alkylglycerone phosphate synthase (alkyl-DHAP) in both ether lipid and glycerophospholipid pathways. Notably, our metabolite profiles are consistent with the strong down-regulation of the gene for alkyl-DHAP (AGPS) in smokers that has been found in a study analyzing gene expression in human lung tissues. Our data suggest that smoking is associated with plasmalogen-deficiency disorders, caused by reduced or lack of activity of the peroxisomal enzyme alkyl-DHAP. Our findings provide new insight into the pathophysiology of smoking addiction. Activation of the enzyme alkyl-DHAP by small molecules may provide novel routes for therapy. AU - Wang-Sattler, R. AU - Yu, Y.* AU - Mittelstraß, K. AU - Lattka, E. AU - Altmaier, E. AU - Gieger, C. AU - Ladwig, K.-H. AU - Dahmen, N.* AU - Weinberger, K.M.* AU - Hao, P.* AU - Liu, L.* AU - Li, Y.* AU - Wichmann, H.-E. AU - Adamski, J. AU - Suhre, K. AU - Illig, T. C1 - 3188 C2 - 25909 TI - Metabolic profiling reveals distinct variations linked to nicotine consumption in humans--first results from the KORA study. JO - PLoS ONE VL - 3 IS - 12 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function. AU - Wiesner, M. AU - Zentz, C. AU - Mayr, C.* AU - Wimmer, R.* AU - Hammerschmidt, W. AU - Zeidler, R.* AU - Moosmann, A. C1 - 2563 C2 - 25413 TI - Conditional immortalization of human B cells by CD40 ligation. JO - PLoS ONE VL - 3 IS - 1 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95% of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV's pathogenesis and latency in a suitable and amenable host. METHODOLOGY/PRINCIPAL FINDINGS: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV's nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that fundamental differences in gene regulation between mouse and man might block the route towards a tractable murine model for EBV. AU - Zychlinska, M. AU - Herrmann, H. AU - Zimber-Strobl, U. AU - Hammerschmidt, W. C1 - 748 C2 - 25417 TI - Restricted expression of Epstein-Barr virus latent genes in murine B cells derived from embryonic stem cells. JO - PLoS ONE VL - 3 IS - 4 PB - Public Library of Science PY - 2008 SN - 1932-6203 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is associated with a number of human malignancies. EBV-positive post-transplant lymphoproliferative disease in solid organ and hematopoietic stem cell transplant recipients has been successfully treated by the adoptive transfer of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components. Although patients receiving T cell preparations with a higher CD4+ T cell proportion show better clinical responses, the specificity of the infused CD4+ component has remained completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We generated LCL-stimulated T cell lines from 21 donors according to clinical protocols, and analyzed the antigen specificity of the CD4+ component in EBV-specific T cell preparations using a genetically engineered EBV mutant that is unable to enter the lytic cycle, and recombinantly expressed and purified EBV proteins. Surprisingly, CD4+ T cell lines from acutely and persistently EBV-infected donors consistently responded against EBV lytic cycle antigens and autoantigens, but barely against latent cycle antigens of EBV hitherto considered principal immunotherapeutic targets. Lytic cycle antigens were predominantly derived from structural proteins of the virus presented on MHC II via receptor-mediated uptake of released viral particles, but also included abundant infected cell proteins whose presentation involved intercellular protein transfer. Importantly, presentation of virion antigens was severely impaired by acyclovir treatment of stimulator cells, as currently performed in most clinical protocols. CONCLUSIONS/SIGNIFICANCE: These results indicate that structural antigens of EBV are the immunodominant targets of CD4+ T cells in LCL-stimulated T cell preparations. These findings add to our understanding of the immune response against this human tumor-virus and have important implications for the improvement of immunotherapeutic strategies against EBV. AU - Adhikary, D. AU - Behrends, U. AU - Boerschmann, H.* AU - Pfünder, A.* AU - Burdach, S.* AU - Moosmann, A. AU - Witter, K.* AU - Bornkamm, G.W. AU - Mautner, J. C1 - 1915 C2 - 24650 TI - Immunodominance of lytic cycle antigens in epstein-barr virus-specific CD4+ T cell preparations for therapy. JO - PLoS ONE VL - 2 IS - 7 PB - Public Library of Science PY - 2007 SN - 1932-6203 ER - TY - JOUR AB - Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces. AU - Gillet, L.* AU - Adler, H. AU - Stevenson, P.G.* C1 - 1760 C2 - 24428 TI - Glycosaminoglycan interactions in murine gammaherpesvirus-68 infection. JO - PLoS ONE VL - 2 IS - 4 PB - Public Library of Science PY - 2007 SN - 1932-6203 ER - TY - JOUR AB - Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity. METHODOLOGY/PRINCIPAL FINDINGS: a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency >/=10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13x10(-7), corrected p = 0.0494; odds ratio (OR)(CT) 1.67, 95% confidence interval (CI) 1.22-2.27; OR(TT) 2.76, 95% CI 1.88-4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium. CONCLUSIONS/SIGNIFICANCE: Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings. AU - Hinney, A.* AU - Nguyen, T.T.* AU - Scherag, A.* AU - Friedel, S.* AU - Brönner, G.* AU - Müller, T.D.* AU - Grallert, H. AU - Illig, T. AU - Wichmann, H.-E. AU - Rief, W.* AU - Schäfer, H.* AU - Hebebrand, J.* C1 - 4885 C2 - 25013 TI - Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants. JO - PLoS ONE VL - 2 IS - 12 PB - Public Library of Science PY - 2007 SN - 1932-6203 ER - TY - JOUR AB - Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression. AU - Thakur, N.N. AU - El-Gogo, S.* AU - Steer, B. AU - Freimüller, B.* AU - Waha, A.* AU - Adler, H. C1 - 3431 C2 - 24524 SP - e733 TI - A gammaherpesviral internal repeat contributes to latency amplification. JO - PLoS ONE VL - 2 IS - 8 PB - Public Library of Science PY - 2007 SN - 1932-6203 ER -