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Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed. Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206). Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68–0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76–0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed. Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation. Keywords: Kidney transplant rejection, Urinary metabolites, Biomarker, NMR-spectroscopy, Non-invasive test

Peroxisomal biogenesis factor PEX26 is a membrane anchor for the multi-subunit PEX1-PEX6 protein complex that controls ubiquitination and dislocation of PEX5 cargo receptors for peroxisomal matrix protein import. PEX26 associates with the peroxisomal translocation pore via PEX14 and a splice variant (PEX26Δex5) of unknown function has been reported. Here, we demonstrate PEX26 homooligomerization mediated by two heptad repeat domains adjacent to the transmembrane domain. We show that isoform-specific domain organization determines PEX26 oligomerization and impacts peroxisomal β-oxidation and proliferation. PEX26 and PEX26Δex5 displayed different patterns of interaction with PEX2-PEX10 or PEX13-PEX14 complexes, which relate to distinct pre-peroxisomes in the de novo synthesis pathway. Our data support an alternative PEX14-dependent mechanism of peroxisomal membrane association for the splice variant, which lacks a transmembrane domain. Structure-function relationships of PEX26 isoforms explain an extended function in peroxisomal homeostasis and these findings may improve our understanding of the broad phenotype of PEX26-associated human disorders.

(1) Background: With vaccination and new variants of SARS-CoV-2 on the horizon, efficient testing in schools may enable prevention of mass infection outbreaks, keeping schools safe places and buying time until decisions on feasibility and the necessity of vaccination in children and youth are made. We established, in the course of the WICOVIR (Where Is the COrona VIRus) study, that gargle-based pool-PCR testing offers a feasible, efficient, and safe testing system for schools in Germany when applied by central university laboratories. (2) Objectives: We evaluated whether this approach can be implemented in different rural and urban settings. (3) Methods: We assessed the arrangements required for successful implementation of the WICOVIR approach in a variety of settings in terms of transport logistics, data transfer and pre-existing laboratory set-up, as well as the time required to establish the set-up. (4) Results: We found that once regulatory issues have been overcome, all challenges pertaining to logistics, data transfer, and laboratory testing on different platforms can be solved within one month. Pooling and depooling of samples down to the individual test result were achievable within one working day in all settings. Local involvement of the community and decentralized set-ups were keys for success. (5) Conclusion: The WICOVIR gargle-based pool-PCR system is so robust and simple that it can be implemented within one month in all settings now or in future pandemics.

BACKGROUNDThe inverse relationship between HDL cholesterol and cardiovascular mortality is weakened in coronary artery disease (CAD). We aimed to investigate the associations of HDL particle concentrations with cardiovascular mortality and the impact of CAD on these associations. We also sought to comparatively evaluate HDL cholesterol and HDL particle concentrations in predicting cardiovascular mortality.METHODSTotal and subclass HDL particle concentrations were measured by nuclear magnetic resonance spectroscopy in 2290 participants of the LUdwigshafen RIsk and Cardiovascular Health study referred for coronary angiography. The participants were prospectively followed over a median (interquartile range) duration of 10.0 (6.1–10.6) years.RESULTSThe mean (SD) age of the participants (1575 males, 715 females) was 62.9 (10.4) years; body mass index, 27.6 (4.1) kg/m2; HDL cholesterol, 39 (11) mg/dL [1 (0.29) mmol/L]; and total HDL particle concentration, 24.1 (5.8) μmol/L. Of the participants, 434 died from cardiovascular diseases. In multivariate analyses, tertiles of total HDL particle concentrations were inversely related to cardiovascular mortality (hazard ratio for third vs first tertile = 0.55, P < 0.001). This association was primarily mediated by small HDL particles (P < 0.001). Adding total or small HDL particle concentrations rather than HDL cholesterol to multivariate prediction models improved performance metrics for cardiovascular mortality. The presence of CAD had no impact on the associations between HDL particle concentrations and cardiovascular mortality.CONCLUSIONSHigh HDL particle concentration is consistently and independently of CAD associated with decreased cardiovascular mortality. Whether the inverse relationship between HDL particle concentration and cardiovascular mortality may be translated into novel therapies is under investigation.

Allograft rejection is still an important complication after kidney transplantation. Currently, monitoring of these patients mostly relies on the measurement of serum creatinine and clinical evaluation. The gold standard for diagnosing allograft rejection, i.e. performing a renal biopsy is invasive and expensive. So far no adequate biomarkers are available for routine use. We aimed to develop a urine metabolite constellation that is characteristic for acute renal allograft rejection. NMR-Spectroscopy was applied to a training cohort of transplant recipients with and without acute rejection. We obtained a metabolite constellation of four metabolites that shows promising performance to detect renal allograft rejection in the cohorts used (AUC of 0.72 and 0.74, respectively). A metabolite constellation was defined with the potential for further development of an in-vitro diagnostic test that can support physicians in their clinical assessment of a kidney transplant patient.

Aims: In this study, we specifically aimed at identifying distinct miRNAs that are governing nutrientdriven adipogenesis via the mTOR pathway. Methodology: The murine pre-adipocyte cell line 3T3-L1 was differentiated in the presence and absence of mTOR inhibitor rapamycin. MiRNA profiles were generated using two different array platforms. Differential regulated miRNAs were subjected to in silico target prediction. Candidate miRNA-target pairs were studied via luciferase assays, transfection assays and functional assays. Results: This study confirmed 31 previously known and revealed 7 additional miRNAs, namely miROriginal Research Article Kaeuferle et al.; JABB, 2(2): 109-122, 2015; Article no.JABB.2015.013 110 34a, -96, -106b, -148b, -214*, -678 and -690 as being involved in adipocyte differentiation. Furthermore, we identified a subset of 16 miRNAs showing both responsiveness to rapamycin and being predicted to target genes within the mTOR pathway. Within mTOR signaling, the novel miRNA-target pairs miR183-Tsc1 (tuberous sclerosis 1), miR378-Tsc1 and miR103-Pik3r1 (phosphatidylinositol 3-kinase regulatory subunit alpha), were confirmed via luciferase-based binding assays. Accordingly we observed down-regulation of Tsc1 on transcript level in cells transfected with miR-183 or -378 (18.3 % P = .004 and 40.4 % P < .0001). However, on protein level we could detect Tsc1 up-regulation (35.7 % and 39.8 %), which is consistent with an inhibitory influence on mTOR signaling (33.6 % and 32.9 %) and on adipocyte differentiation of these miRNAs. Conclusion: Our findings reveal a novel role for a set of miRNAs during adipocyte differentiation and mTOR signaling. Especially the attenuating effect of miR-183 and -378 on adipocyte differentiation and mTOR signaling suggests their potential as biomarkers for nutrient-driven dysregulation of adipogenesis.

Background & Aims: MicroRNAs are important genetic regulators of physiological and pathophysiological processes including cancer initiation and progression of hepatoblastoma, the most common liver tumour in childhood. We aimed to identify malignant and metastasis promoting effects of miR-492, a miRNA, previously reported to be overexpressed in metastatic hepatoblastoma. Furthermore, we intended to evaluate its diagnostic and prognostic potential. Methods: Stable and transient overexpression of miR-492 in two liver tumour cell lines HepT1 and HUH7 was used to analyse features of metastatic tumour progression such as proliferation, anchorage-independent growth, migration and invasion. Via a mass spectrometry based proteomic screen, we investigated miRNA-492-dependent effects on proteome level and explored the underlying biology. One of the predicted target genes, CD44, was experimentally validated via luciferase assays. Diagnostic and prognostic properties of miR-492 were studied in hepatoblastoma tumour samples. Results: We show that miR-492 significantly enhances cell proliferation, anchorage-independent growth, migration and invasion of hepatoblastoma cells. We also identified and validated CD44, a transmembrane adhesion receptor for hyaluronan, as direct and functional target of miR-492. This miRNA has a strong direct impact on two CD44 isoforms (standard and v10). High miR-492 expression correlates with high-risk or aggressive tumours and further bears potential for predicting reduced event-free survival. Conclusions: We identified miR-492 and its target CD44 as regulators of a number of biological features important for malignancy and metastasis. Furthermore, we demonstrated the diagnostic and prognostic potential of miR-492, a promising novel therapeutic target and biomarker for hepatoblastoma.

Background Cold exposure and β3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase–mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. Objective The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. Methods Body mass index–matched young, lean men were exposed to a personalized cooling protocol for 2 hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1 H-nuclear magnetic resonance. Adenosine triphosphate–binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [ 3 H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. Results Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low–density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro . Conclusions Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Zusammenfassung Die NMR-Spektroskopie ist eine moderne analytische Methode, die sich gut zur Analyse unterschiedlichster Körperflüssigkeiten eignet. Im Serum können neben kleinen Metaboliten auch differenziert Lipoprotein-Fraktionen bestimmt werden. Die Technik ist gut standardisierbar und erlaubt im Vergleich zur Lipoproteinfraktionierung mittels Ultrazentrifugation eine umfassende Automatisierung und guten Probendurchsatz. Da in einer Messung alle auszuwertenden Parameter gleichzeitig gemessen werden, eignet sich die Methode besonders gut für Metabolomics-Ansätze, in denen auch subtile Verschiebungen in den Metabolitenverhältnissen von Bedeutung sein können. Dieser Review gibt eine Übersicht über aktuelle Ansätze der NMR-spektroskopischen Messtechnik, Auswertestrategien und praktischen Anwendungen in verschiedenen Studien, sowohl auf der Seite der Lipidanalyse, also auch hinsichtlich des Metabolitenprofils. Die NMR-Spektroskopie hat sich in den letzten Jahren von einer reinen Forschungsmethode mit großen Schritten weiterentwickelt und dürfte in Zukunft einen Platz in der Routinediagnostik einnehmen.

Motivation: Most integral membrane proteins form dimeric or oligomeric complexes. Oligomerization is frequently supported by the noncovalent interaction of transmembrane helices. It is currently not clear how many high-affinity transmembrane domains (TMD) exist in a proteome and how specific their interactions are with respect to preferred contacting faces and their underlying residue motifs. Results: We first identify a threshold of 55% sequence similarity, which demarcates the border between meaningful alignments of TMDs and chance alignments. Clustering the human single-span membrane proteome using this threshold groups � 40% of the TMDs. The homotypic interaction of the TMDs representing the 33 largest clusters was systematically investigated under standardized conditions. The results reveal a broad distribution of relative affinities. High relative affinity frequently coincides with (i) the existence of a preferred helix–helix interface and (ii) sequence specificity as indicated by reduced affinity after mutating conserved residues. Contact: langosch@tum.de Supplementary information: Supplementary data are available at Bioinformatics online.

// Sandra Diersch 1 , Patrick Wenzel 1 , Melanie Szameitat 1 , Philipp Eser 2 , Mariel C. Paul 1 , Barbara Seidler 1 , Stefan Eser 1 , Marlena Messer 1 , Maximilian Reichert 3 , Philipp Pagel 4 , Irene Esposito 5 , Roland M. Schmid 1 , Dieter Saur 1 , Gunter Schneider 1 1 II. Medizinische Klinik, Technische Universitat Munchen, Munchen, Germany 2 Gene Center Munich, Ludwig-Maximilians-Universitat (LMU) Munchen, Munchen, Germany 3 Division of Gastroenterology, Department of Medicine, Abramson Cancer Center, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania, USA 4 numares GmbH, Regensburg, Germany 5 Institut fur Allgemeine Pathologie und Pathologische Anatomie, Technische Universitat Munchen, Munchen, Germany Correspondence: Gunter Schneider, email: // Keywords : pancreatic cancer, PI3K, Efemp1, p27, Bez235 Received : February 04, 2013 Accepted : February 25, 2013 Published : February 26, 2013 Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a dismal disease with a poor prognosis and targeted therapies have failed in the clinic so far. Several evidences point to the phosphatidylinositol 3-kinase (PI3K)-mTOR pathway as a promising signaling node for targeted therapeutic intervention. Markers, which predict responsiveness of PDAC cells towards PI3K inhibitors are unknown. However, such markers are needed and critical to better stratify patients in clinical trials. We used a large murine Kras G12D - and PI3K (p110α H1047R )-driven PDAC cell line platform to unbiased define modulators of responsiveness towards the dual PI3K-mTOR inhibitor Bez235. In contrast to other tumor models, we show that Kras G12D - and PI3K (p110α H1047R )-driven PDAC cell lines are equally sensitive towards Bez235. In an unbiased approach we found that the extracellular matrix protein Efemp1 controls sensitivity of murine PDAC cells towards Bez235. We show that Efemp1 expression is connected to the cyclin-dependent kinase inhibitor p27 Kip1 . In a murine Kras G12D -driven PDAC model, p27 Kip1 haploinsufficiency accelerates cancer development in vivo. Furthermore, p27 Kip1 controls Bez235 sensitivity in a gene dose-dependent fashion in murine PDAC cells and lowering of p27 Kip1 decreases Bez235 responsiveness in murine PDAC models. Together, we define the Efemp1-p27Kip1 axis as a potential marker module of PDAC cell sensitivity towards dual PI3K-mTOR inhibitors, which might help to better stratify patients in clinical trials.

Background In this study we explored the role of microRNAs (miRNAs) as mediators of leukemogenic effects of the fusion gene MLL-AF9, which results from a frequent chromosomal translocation in infant and monoblastic acute myeloid leukemia (AML). Methods We performed a specific and efficient knockdown of endogenous MLL-AF9 in the human monoblastic AML cell line THP1. Results The knockdown associated miRNA expression profile revealed 21 MLL-AF9 dependently expressed miRNAs. Gene ontology analyses of target genes suggested an impact of these miRNAs on downstream gene regulation via targeting of transcriptional modulators as well as involvement in many functions important for leukemia maintenance as e.g. myeloid differentiation, cell cycle and stem cell maintenance. Furthermore, we identified one of the most intensely repressed miRNAs, miR-511, to raise CCL2 expression (a chemokine ligand important for immunosurveillance), directly target cyclin D1, inhibit cell cycle progression, increase cellular migration and promote monoblastic differentiation. With these effects, miR-511 may have a therapeutic potential as a pro-differentiation agent as well as in leukemia vaccination approaches. Conclusions Our study provides new insights into the understanding of miRNAs as functional mediators of the leukemogenic fusion gene MLL-AF9 and opens new opportunities to further investigate specific therapeutic options for AML via the miRNA level. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0283-5) contains supplementary material, which is available to authorized users.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.

Background: Home mechanical ventilation (HMV) is increasingly used in children with chronic respiratory insufficiency, but data on incidence and type of adverse events are limited. Setting: Pediatric HMV program at a tertiary university hospital. Methods: The authors retrospectively analyzed the type and incidence of severe emergencies in a mixed pediatric HMV program. Results: In all, 295 patient-years of HMV in 54 patients could be analyzed. A total of 26 patients had neuromuscular disease. In 16 patients, mechanical ventilation was initiated at

Domain Interaction MAp (DIMA, available at http://webclu.bio.wzw.tum.de/dima) is a database of predicted and known interactions between protein domains. It integrates 5807 structurally known interactions imported from the iPfam and 3did databases and 46,900 domain interactions predicted by four computational methods: domain phylogenetic profiling, domain pair exclusion algorithm correlated mutations and domain interaction prediction in a discriminative way. Additionally predictions are filtered to exclude those domain pairs that are reported as non-interacting by the Negatome database. The DIMA Web site allows to calculate domain interaction networks either for a domain of interest or for entire organisms, and to explore them interactively using the Flash-based Cytoscape Web software.

Campomelic dysplasia is a malformation syndrome with multiple symptoms including characteristic shortness and bowing of the long bones (campomelia). CD, often lethal due to airway malformations, is caused by heterozygous mutations in SOX9, an SRY-related gene regulating testis and chondrocyte development including expression of many cartilage genes such as type II collagen. Male to female sex reversal occurs in the majority of affected individuals with an XY karyotype. A mild form without campomelia exists, in which sex-reversal may be also absent. We report here two novel SOX9 missense mutations in a male (c.495C>G; p.His165Gln) and a female (c.337A>G; p.Met113Val) within the DNA-binding domain leading to non-lethal acampomelic CD. Functional analyses of mutant proteins demonstrate residual DNA-binding and transactivation of SOX9-regulated genes. Combining our data and reports from the literature we postulate a genotype-phenotype correlation: SOX9 mutations allowing for residual function lead to a mild form of CD in which campomelia and sex reversal may be absent. © 2010 Wiley-Liss, Inc.

Engraftment syndrome (ES) has been recognized as an inflammatory condition during neutrophil recovery after hematopoietic stem cell transplantation (HSCT) characterized by noninfectious fever and skin rash. It has been reported to occur frequently after autologous HSCT in children and adults, and has been shown to be an independent risk factor for increased transplant-related mortality (TRM). However, virtually no data exist on its occurrence after allogeneic HSCT in children. To determine incidence, predisposing factors for, and complications of ES in a pediatric transplant cohort, we analyzed 61 consecutive recipients of a myeloablative allogeneic HSCT for the occurrence of ES. Diagnosis of ES was established when children presented with ≥2 of the following symptoms within 7 days before engraftment: (1) fever >38.0°C, (2) skin rash, (3) weight gain and albumin drop, or (4) dyspnea, hypoxia, and pulmonary infiltrates. Incidence of ES in this cohort was 48% (29 of 61). In a univariate analysis, posttransplant granulocyte-colony stimulating factor (G-CSF) administration (P = .02), and high mononuclear cell count (MNC) (P = .002) were identified as significant risk factors predisposing for the development of ES. In a multiple logistic regression analysis, amphotericin B therapy (P = .009) and high MNC (P = .004) were significant explanatory variables for ES risk. There was a slight trend toward a higher rate of chronic GVHD (cGVHD) in patients with ES (P = .11). However, after a median follow-up of 9.5 years overall survival (OS) (P = .53) and TRM (P = .65) did not differ between the 2 groups. ES presenting with fever, rash, weight gain, and pulmonary symptoms should be recognized as a frequent complication of allogeneic HSCT after myeloablative conditioning in children. Treatment with G-CSF, amphotericin B, and a high nucleated cell count of the graft predisposed for the development of ES in this study. OS and TRM in this cohort were not affected by the occurrence of ES.

DIMA-the domain interaction map has evolved from a simple web server for domain phylogenetic profiling into an integrative prediction resource combining both experimental data on domain-domain interactions and predictions from two different algorithms. With this update, DIMA obtains greatly improved coverage at the level of genomes and domains as well as with respect to available prediction approaches. The domain phylogenetic profiling method now uses SIMAP as its backend for exhaustive domain hit coverage: 7038 Pfam domains were profiled over 460 completely sequenced genomes. Domain pair exclusion predictions were produced from 83 969 distinct protein-protein interactions obtained from IntAct resulting in 21 513 domain pairs with significant domain pair exclusion algorithm scores. Additional predictions applying the same algorithm to predicted protein interactions from STRING yielded 2378 high-confidence pairs. Experimental data comes from iPfam (3074) and 3did (3034 pairs), two databases identifying domain contacts in solved protein structures. Taken together, these two resources yielded 3653 distinct interacting domain pairs. DIMA is available at http://mips.gsf.de/genre/proj/dima.

Introduction: The concentration and size of HDL particles are considered to be related to their atheroprotective properties. This study in patients at increased cardiovascular risk was performed to evaluate the associations of HDL particle characteristics with all-cause and cardiovascular mortality. Methods and Results: HDL particle concentrations for all sizes and for size categories (7-8.5 nm, 8.5-10 nm, 10-13 nm, and 13-16 nm) were measured in 2,290 participants of the LURIC study (1,575 males, 715 females) by nuclear magnetic resonance spectroscopy. Mean (standard deviation) age was 62.9 (10.4) years, body mass index was 27.6 (4.1) kg/m 2 , HDL cholesterol was 39 (11) mg/dl, and HDL particle concentration for all sizes was 25.1 (6.0) μmol/l. During a median (interquartile range) follow-up of 10.0 (6.1-10.6) years, 662 participants died from any cause and 434 from cardiovascular diseases. In multivariable analyses including adjustment for HDL cholesterol tertiles of HDL particle concentrations for all sizes were inversely related to all-cause (3 rd vs. 1 st tertile: HR [95%CI] = 0.69 [0.55-0.87], P=0.001) and cardiovascular mortality (HR [95%CI] = 0.67 [0.50-0.89], P=0.007). These associations were primarily mediated by the inverse relationship between the smallest HDL particles (7-8.5 nm) and endpoints (P≤0.005). The associations of larger HDL particles (8.5-10 nm, 10-13 nm, and 13-16 nm) with endpoints were less pronounced and did not reach statistical significance in multivariable analyses (all P>0.1). Conclusions: In participants of the LURIC study high concentrations of HDL particles predict decreased cardiovascular risk. This association is mainly driven by small HDL particles.

NMR spectroscopy is a modern analytical method which is extremely suitable for the analysis of various body fluids. In addition to small metabolite quantification, the method is capable of differentiated lipoprotein subfraction measurements. The technique can be well standardized and allows extensive automation and good sample throughput compared with lipoprotein fractionation via ultracentrifugation. Because all evaluated parameters are determined simultaneously in one measurement, this method is especially suitable for metabolomics approaches in which even subtle changes in metabolite ratios may be relevant. This review gives an overview of the current methods of NMR spectroscopy, analysis strategies, and practical applications in various studies concerning lipid analysis as well as metabolomics. Over the course of the past years, NMR spectroscopy has heavily evolved from a mere research method into a tool that can be expected to play an important role in routine diagnostic testing in the future.

MfunGD (http://mips.gsf.de/genre/proj/mfungd/) 15 provides a resource for annotated mouse proteins and their occurrence in protein networks. Manual annotation concentrates on proteins which are found to interact physically with other proteins. Accordingly, manually curated information from a 20 protein–protein interaction database (MPPI) and a database of mammalian protein complexes is interconnected with MfunGD. Protein function annotation is performed using the Functional Catalogue (FunCat) annotation scheme which is widely used 25 for the analysis of protein networks. The dataset is also supplemented with information about the literature that was used in the annotation process as well as links to the SIMAP Fasta database, the Pedant protein analysis system and cross-references to 30 external resources. Proteins that so far were not manually inspected are annotated automatically by a graphical probabilistic model and/or superparamagnetic clustering. The database is continuously expanding to include the rapidly growing amount of 35 functional information about gene products from mouse. MfunGD is implemented in GenRE, a J2EEbased component-oriented multi-tier architecture following the separation of concern principle.

NFAT and NF-kappaB proteins are members of a superfamily of transcription factors whose activity plays a crucial role in the activation, proliferation and apoptosis of lymphocytes. Both types of factors share a number of properties, including similar DNA binding domains and rapid nuclear translocation upon antigenic stimulation. While NF-kappaBs control both innate and adaptive immune responses, NFATs control the adaptive immune system which emerged-in parallel with the appearance of the NFAT family-in jawed fish. However, NFATs and NF-kappaBs differ remarkably in their function. Whereas NFATs support activation-induced cell death (AICD) of T and B cells, NF-kappaB proteins frequently exert a strong anti-apoptotic effect on lymphocytes and other cells. While the anti-apoptotic activity of NF-kappaBs contributes to their oncogenic capacity, the pro-apoptotic activity favors NFATs as tumor suppressors in lymphoid cells.

Item does not contain fulltext The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase beta-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The beta-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the beta-subunit from the cell surface to CD63+ intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase beta-subunit complexed with CD63. Coexpression of the beta-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the beta-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.

Background Previously we have examined the effect of maternal dietary n-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation during pregnancy on offspring fat mass. Considering the involvement of the placenta in fetal programming, we aimed to analyze the sex-specific gene expression in human term placenta and its response to the n-3 LCPUFA intervention, as well as their correlations to offspring adiposity. Results Placental gene expression was assessed in a control and n-3 LCPUFA intervention group by DNA microarrays, biological pathway analyses and RT-qPCR validation. Expression data were correlated with sex steroid hormone levels in placenta and cord plasma, and offspring anthropometric data. Transcriptome data revealed sexually dimorphic gene expression in control placentas per se, whereas in intervention placentas sex-specific expression changed, and more n-3 LCPUFA-regulated genes were found in female than male placentas. Sexually dimorphic gene expression and n-3 LCPUFA-responsive genes were enriched in the pathway for cell cycle and its associated modulator pathways. Significant mRNA expression changes for CDK6, PCNA, and TGFB1 were confirmed by RT-qPCR. CDK6 and PCNA mRNA levels correlated with offspring birth weight and birth weight percentiles. Significantly reduced placental estradiol-17β/testosterone ratio upon intervention found in female offspring correlated with mRNA levels for the 'Wnt signaling' genes DVL1 and LRP6. Conclusions Overall, human placentas show sexually dimorphic gene expression and responsiveness to maternal n-3 LCPUFA intervention during pregnancy with more pronounced effects in female placentas. The absence of correlations of analyzed placental gene expression with offspring adipose tissue growth in the first year is not mutually exclusive with programming effects, which may manifest later in life, or in other physiological processes. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-941) contains supplementary material, which is available to authorized users.

Owing to their manifold immune regulatory functions, regulatory T cells (Treg) have received tremendous interest as targets for therapeutic intervention of diverse immunological pathologies or cancer. Directed manipulation of Treg will only be achievable with extensive knowledge about the intrinsic programs that define their regulatory function. We simultaneously analyzed miR and mRNA transcript levels in resting and activated human Treg cells in comparison with non-regulatory conventional T cells (Tcon). Based on experimentally validated miR-target information, both transcript levels were integrated into a comprehensive pathway analysis. This strategy revealed characteristic signal transduction pathways involved in Treg biology such as T-cell receptor-, Toll-like receptor-, transforming growth factor-β-, JAK/STAT (Janus kinase/signal transducers and activators of transcription)- and mammalian target of rapamycin signaling, and allowed for the prediction of specific pathway activities on the basis of miR and mRNA transcript levels in a probabilistic manner. These data encourage new concepts for targeted control of Treg cell effector functions.

The response of target cells to the steroid hormone aldosterone has been divided into acute nongenomic (10 min) and sustained genomic (10 min) action. In the light of recent experiments this nomenclature does not hold anymore and should be abandoned. By applying atomic force microscopy (AFM) we observed in living endothelial cells that aldosterone induces cell volume increase in less than 10 minutes. The cell nucleus was identified as the swelling site. Hormone-induced nuclear swelling can reach 15 to 28% of total cell volume dissipating within 30 minutes. This phenomenon could have functional impact on flow resistance in small blood vessels. AFM-investigation of the intracellular signal pathway in nuclear envelope of aldosterone-injected Xenopus laevis oocytes visualizes putative intracellular receptors (40 kD granules) bound to nuclear pores 2 minutes after hormone injection, with subsequent macromolecule translocation into the nucleus. 15 minutes later macromolecules (800 kD plugs) appear in the central channels of the nuclear pores. The plugs resemble ribonucleoproteins that carry the aldosterone-induced mRNA to the ribosomes. We postulate that steroid-induced nuclear swelling is caused by a shift of receptors/transcription factors from cytoplasm into nucleoplasm followed by gene transcription. Nuclear volume returns to normal when mRNA export through the nuclear pores is finished. Thus, steroid-induced net-movements of macromolecules between intracellular compartments initiate shifts in cell volume compensated by volume regulatory transporters and ion channels in the plasma membrane.

Previously, we reported strong influences of genetic variants on metabolic phenotypes, some of them with clinical relevance. Here, we hypothesize that DNA methylation may have an important and potentially independent effect on human metabolism. To test this hypothesis, we conducted what is to the best of our knowledge the first epigenome-wide association study (EWAS) between DNA methylation and metabolic traits (metabotypes) in human blood. We assess 649 blood metabolic traits from 1814 participants of the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) population study for association with methylation of 457 004 CpG sites, determined on the Infinium HumanMethylation450 BeadChip platform. Using the EWAS approach, we identified two types of methylome-metabotype associations. One type is driven by an underlying genetic effect; the other type is independent of genetic variation and potentially driven by common environmental and life-style-dependent factors. We report eight CpG loci at genome-wide significance that have a genetic variant as confounder (P = 3.9 × 10(-20) to 2.0 × 10(-108), r(2) = 0.036 to 0.221). Seven loci display CpG site-specific associations to metabotypes, but do not exhibit any underlying genetic signals (P = 9.2 × 10(-14) to 2.7 × 10(-27), r(2) = 0.008 to 0.107). We further identify several groups of CpG loci that associate with a same metabotype, such as 4-vinylphenol sulfate and 4-androsten-3-beta,17-beta-diol disulfate. In these cases, the association between CpG-methylation and metabotype is likely the result of a common external environmental factor, including smoking. Our study shows that analysis of EWAS with large numbers of metabolic traits in large population cohorts are, in principle, feasible. Taken together, our data suggest that DNA methylation plays an important role in regulating human metabolism.

Summary: The MIPS mammalian protein–protein interaction database (MPPI) is a new resource of high-quality experimental protein interaction data in mammals. The content is based on published experimental evidence that has been processed by human expert curators. We provide the full dataset for download and a flexible and powerful web interface for users with various requirements. Availability: The MPPI database is located at http://mips.gsf.de/proj/ppi/ Contact: d.frishman@wzw.tum.de

Background The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na+,K+-ATPase. The catalytic subunit of the Na+,K+-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na+,K+-ATPase interacting protein. PP-2A C-subunit interacted with the Na+,K+-ATPase, but not with the homologous sequences of the H+,K+-ATPase. We confirmed that the Na+,K+-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na+,K+-ATPase trafficking through direct association. PP2A inhibits association between the Na+,K+-ATPase and arrestin, and diminishes the effect of arrestin on Na+,K+-ATPase trafficking. GRK phosphorylates the Na+,K+-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.

Steroid-resistant focal segmental glomerulosclerosis (FSGS) often recurs after renal transplantation. In this international survey, we sought to identify genotype-phenotype correlations of recurrent FSGS. We surveyed 83 patients with childhood-onset primary FSGS who received at least one renal allograft and analyzed 53 of these patients for NPHS2 mutations. The mean age at diagnosis was 6.7 years, and the mean age at first renal transplantation was 13 years. FSGS recurred in 30 patients (36%) after a median of 13 days (range, 1.5 to 152 days). Twenty-three patients received a second kidney transplant, and FSGS recurred in 11 (48%) after a median of 16 days (range, 2.7 to 66 days). None of the 11 patients with homozygous or compound heterozygous NPHS2 mutations developed recurrent FSGS compared with 45% of patients without mutations. These data suggest that genetic testing for pathogenic mutations may be important for prognosis and treatment of FSGS both before and after transplantation.

MicroRNAs (miRNAs) are well-known regulators of proliferation, apoptosis, and differentiation and are recognized to play an important role in the development of cancers. Here we aimed to identify the functional contribution of miRNAs to the biology of hepatoblastoma (HB), the most common malignant liver tumor in childhood. As overexpression of the oncogene PLAG1 (pleomorphic adenoma gene 1) is a characteristic phenomenon in HB, we used RNA interference and subsequent miRNA array analysis to identify miR-492 as most strongly influenced by PLAG1. We provide novel experimental evidence that miR-492 can originate from the coding sequence of the HB marker gene keratin 19 (KRT19). In agreement with these in vitro observations, significantly elevated levels of coexpressed KRT19 and miR-492 were particularly found in metastatic HB tumor samples. Stable overexpression of miR-492 in HB cell clones served to identify a broad range of differentially expressed transcripts, including several candidate targets of miR-492 predicted by computational algorithms. Among those the liver enzyme BAAT showed significant association with miR-492 expression in HB tumor samples. Conclusion: A close functional relationship between KRT19 and miR-492 was identified that may play an important role in the progression of malignant embryonal liver tumors. Additionally, miR-492 and its associated targets might serve as new HB biomarkers of clinical utility and could assist to explore targeted therapies, especially in metastatic HB with a poor prognosis. (HEPATOLOGY 2011)

Teresa Español, [et al.], A large proportion of patients with mutations in the Wiskott-Aldrich syndrome (WAS) protein gene exhibit the milder phenotype termed X-linked thrombocytopenia (XLT). Whereas stem cell transplantation at an early age is the treatment of choice for patients with WAS, therapeutic options for patients with XLT are controversial. In a retrospective multicenter study we defined the clinical phenotype of XLT and determined the probability of severe disease-related complications in patients older than 2 years with documented WAS gene mutations and mild-to-moderate eczema or mild, infrequent infections. Enrolled were 173 patients (median age, 11.5 years) from 12 countries spanning 2830 patient-years. Serious bleeding episodes occurred in 13.9%, life-threatening infections in 6.9%, autoimmunity in 12.1%, and malignancy in 5.2% of patients. Overall and event-free survival probabilities were not significantly influenced by the type of mutation or intravenous immunoglobulin or antibiotic prophylaxis. Splenectomy resulted in increased risk of severe infections. This analysis of the clinical outcome and molecular basis of patients with XLT shows excellent long-term survival but also a high probability of severe disease-related complications. These observations will allow better decision making when considering treatment options for individual patients with XLT. © 2010 by The American Society of Hematology., We also thank the staff of the European Society for Immunodeficiencies registry for their support. This work was supported in part by a grant from Biotest AG, Dreieich, Germany (M. H. A.).

The Negatome is a collection of protein and domain pairs that are unlikely to be engaged in direct physical interactions. The database currently contains experimentally supported non-interacting protein pairs derived from two distinct sources: by manual curation of literature and by analyzing protein complexes with known 3D structure. More stringent lists of non-interacting pairs were derived from these two datasets by excluding interactions detected by high-throughput approaches. Additionally, non-interacting protein domains have been derived from the stringent manual and structural data, respectively. The Negatome is much less biased toward functionally dissimilar proteins than the negative data derived by randomly selecting proteins from different cellular locations. It can be used to evaluate protein and domain interactions from new experiments and improve the training of interaction prediction algorithms. The Negatome database is available at http://mips.helmholtz-muenchen.de/proj/ppi/negatome.

Patients with congenital adrenal hyperplasia (CAH) are at risk for early pubertal development and diminished pubertal growth. Liberal treatment with glucocorticoids will prevent early puberty but may inhibit growth outright.The aim of the study was to determine an optimal range for hydrocortisone dosing during puberty in children with classical CAH who were exclusively treated with hydrocortisone.The effects of glucocorticoid treatment for classical CAH were retrospectively analyzed in 92 patients (57 females). Growth pattern, final height (FH), and mean daily hydrocortisone dose were recorded.Pubertal growth was significantly reduced in all patients: salt-wasting (SW) females, 13.8 +/- 7.4 cm; simple virilizing (SV) females, 13.1 +/- 6.2 cm; vs. reference, 20.3 +/- 6.8 cm (P0.05); and SW males, 17.7 +/- 6.7 cm; SV males, 16.2 +/- 5.7 cm; vs. reference, 28.2 +/- 8.2 cm (P0.05). Decreased pubertal growth resulted in FH at the lower limit of genetic potential (corrected FH in SW females, -0.6 +/- 0.9; SV females, -0.3 +/- 0.9; SW males, -0.8 +/- 0.8; and SV males, -1.0 +/- 1.0). During puberty, mean daily hydrocortisone dose was 17.2 +/- 3.4 mg/m(2) in females (SW, 17.0 +/- 3.3; SV, 17.4 +/- 3.5) and 17.9 +/- 2.5 mg/m(2) in males (SW, 17.4 +/- 2.0; SV, 18.7 +/- 3.1). In a logistic regression model, a significant correlation between hydrocortisone dose and FH was found (P0.01), and the positive predictive value for short stature rose from below 30% to above 60% when hydrocortisone dose exceeded 17 mg/m(2).With conventional hydrocortisone treatment, pubertal growth is significantly reduced in both sexes, resulting in a FH at the lower limit of genetic potential. These deleterious effects on pubertal growth can be reduced if hydrocortisone does not exceed 17 mg/m(2).