Regulation des Säure-induzierten Cad-Systems von Escherichia coli durch den membranintegrierten Transkriptionsaktivator CadC und die Lysin-spezifische Permease LysP

Das Cad-System von E. coli gehört zu den Säure-induzierbaren Aminosäure-Decarboxylase-Systemen und spielt eine wichtige Rolle bei der Säureschutzantwort. In diesem System erfolgt die Reizwahr-nehmung, Signaltranslokation über die Membran und Transkriptionsregulation durch ein einziges Protein, nämlich CadC. In Gegenwart von induzierenden Bedingungen (niedriger pH-Wert und Lysin) aktiviert CadC die Expression des cadBA-Operons, dadurch kommt es zur Decarboxylierung von Lysin zu Cadaverin und CO2 durch die Lysin-Decarboxylase CadA und zum Export von Cadaverin durch den Lysin/ Cadaverin-Antiporter CadB. Dabei werden Protonen aus dem Cytoplasma entfernt und der extrazelluläre pH-Wert durch das basische Cadaverin erhöht. Das Lysin-Transportprotein LysP inhibiert die cadBA-Expression bei nicht-induzierenden Bedingungen, das bei der Decarboxylierung gebildete Cadaverin übt einen negativen Rückkopplungseffekt auf die Expression von cadBA aus. Im Rahmen der vorgelegten Arbeit wurden die Regulation des Cad-Systems durch CadC und die Lysin-spezifische Permease LysP, die Reizwahrnehmung durch CadC und die Struktur von CadC untersucht. Mittels Transkriptions- und Translationsanalysen wurde das Cad- System u. a. in E. coli MG1655 (Wildtyp) und E. coli MG1655-lysP211 (lysP-negativ) untersucht. Die cadBA- Expression war in E. coli MG1655-lysP211 deutlich höher als im Wildtyp, als Folge dessen war auch die relative Menge an CadA und die Konzentration an extrazellulärem Cadaverin erhöht. Des Weiteren war in dieser Mutante durch den Wegfall des Repressors LysP der Bedarf an extrazellulärem Lysin als Induktor der cadBA-Expression vollständig eliminiert. In Zusammenarbeit mit dem Institut für Theoretische Physik der Universität zu Köln wurde anhand der für E. coli MG1655 (Wildtyp) gemessenen Daten ein mathematisches Modell erstellt und mit Hilfe dieser Daten die Dynamik des Cad-Systems in E. coli MG1655-lysP211 berechnet. Die in vivo bestimmten Ergebnisse wurden durch die in silico erhaltenen Daten sehr gut wiedergegeben. In der vorliegenden Arbeit konnte auch gezeigt werden, dass CadC kein Sensor für Lysin ist. In vitro Experimente (ITC-Messungen, Tryptophanfluoreszenz-Messungen) ergaben, dass CadC nur eine extrem niedrige Affinität für Lysin aufweist, in vivo war ein bestimmter Schwellenwert für die Induktion der cadBA-Expression nötig, ansonsten hatte die Lysin- Konzentration keinen Einfluss. Da Lysin somit nicht durch CadC wahrgenommen wird und deshalb die Interaktion zwischen CadC und dem Transportprotein LysP nicht über die Konkurrenz beider Proteine um das Substrat Lysin erfolgt, beruht die Lysin-Abhängigkeit der cadBA-Expression vermutlich auf einer direkten Interaktion zwischen CadC und LysP. In vitro konnten CadC und LysP in Proteoliposomen quervernetzt werden, dies indizierte eine Affinität beider Proteine zueinander. Das Vorhandensein von Lysin löst die Interaktion zwischen CadC und LysP auf und ist somit ein wichtiger Schritt für die Aktivierung von CadC. Durch Messungen der intrinsischen Tryptophanfluoreszenz mit der periplasmatischen Domäne von CadC (CadC188-512) konnte gezeigt werden, dass diese eine Affinität für den Inhibitor Cadaverin aufweist, der KD-Wert hierfür betrug 96 µM. In vivo Experimente bestätigten, dass das Vorhandensein der periplasmatischen Domäne für die Hemmung der cadBA-Expression durch Cadaverin essentiell ist. Somit scheint die Inhibierung der cadBA-Expression durch Cadaverin über eine direkte Interaktion mit der periplasmatischen Domäne von CadC zu erfolgen In Zusammenarbeit mit dem Lehrstuhl für Biologische Chemie der Technischen Universität München wurden Strukturuntersuchungen zu CadC188-512 und CadC mittels 3D-Kristallisation durchgeführt. CadC188-512 konnte sehr gut überproduziert und gereinigt werden, und es entstanden bereits Kristalle, von denen einer am Synchrotron vermessen werden konnte. Die Auflösung (2,5 Å) war jedoch für eine Strukturaufklärung noch zu gering. In der periplasmatischen Domäne von CadC befinden sich zwei Cysteinreste. Mit Hilfe von in vitro Experimenten konnte die Ausbildung einer Disulfidbrücke in CadC188-512 bestätigt werden. Durch die Reduktion der Disulfidbrücke war die Affinität für den Ligand Cadaverin geringfügig vermindert. Das Auflösen der Disulfidbrücke könnte ein wichtiger Mechanismus für die Aktivierung von CadC darstellen. Wurde die Ausbildung einer Disulfidbrücke durch die Substitution der Cysteinreste durch Alanin verhindert, so wurde die cadBA-Expression in vivo Lysin-unabhängig aktiviert. Möglicherweise repräsentieren diese CadC-Derivate ein „semi-aktives“ CadC, das für eine volle Aktivierung nur noch einen Reiz benötigt

Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli

Background: In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. Results: Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. Conclusion: A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor

Age-dependent expression of DNMT1 and DNMT3B in PBMCs from a large European population enrolled in the MARK-AGE study

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated

SwissGenVar: A platform for clinical grade interpretation of genetic variants to foster personalized health care in Switzerland

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows including the extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUS) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of standards used to describe genetic variants and associated phenotypes when searching for relevant information to inform clinical decision-making. For this purpose, all five Swiss academic Medical Genetics Institutions joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its objective is to provide a protected environment for expert evidence sharing about individual variants to harmonize and up-scale their significance interpretation at clinical grade following international standards. To corroborate the clinical assessment, the variant-related data are combined with consented high-quality clinical information. Broader visibility will be gained by interfacing with international databases, thus supporting global initiatives in personalized health care

SwissGenVar: A Platform for Clinical-Grade Interpretation of Genetic Variants to Foster Personalized Healthcare in Switzerland

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows. This includes extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUSs) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of the standards used to describe genetic variants and the associated phenotypes when searching for relevant information to support clinical decision making. To address this, all five Swiss academic institutions for Medical Genetics joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its aim is to provide a protected environment for expert evidence sharing about individual variants to harmonize and upscale their significance interpretation at the clinical grade according to international standards. To corroborate the clinical assessment, the variant-related data will be combined with consented high-quality clinical information. Broader visibility will be achieved by interfacing with international databases, thus supporting global initiatives in personalized healthcare

Development of a high sensitivity label free waveguide interferometry instrument : a project of Creoptix GmbH with the center for Biochemistry ZHAW as main research partner

Creoptix GmbH has developed a novel and innovative technology for label-free detection of molecules based on grating-coupled interferometry (GCI). GCI is a proprietary technology characterized by a very high sensitivity at low technical complexity. One main application will be the measurement of binding affinities in research and development projects such as drug discovery. Goal of an interdisciplinary CTI project with the partners from ZHAW, FHNW and CSEM together with Creoptix is the development of a first GCI instrument including disposables and the optimization of its functionality, followed by field tests to pave the way to market introduction

Improved diagnosis by automated macro‐ and micro‐anatomical region mapping of skin photographs

Background: The exact location of skin lesions is key in clinical dermatology. On one hand, it supports differential diagnosis (DD) since most skin conditions have specific predilection sites. On the other hand, location matters for dermatosurgical interventions. In practice, lesion evaluation is not well standardized and anatomical descriptions vary or lack altogether. Automated determination of anatomical location could benefit both situations. Objective: Establish an automated method to determine anatomical regions in clinical patient pictures and evaluate the gain in DD performance of a deep learning model (DLM) when trained with lesion locations and images. Methods: Retrospective study based on three datasets: macro-anatomy for the main body regions with 6000 patient pictures partially labelled by a student, micro-anatomy for the ear region with 182 pictures labelled by a student and DD with 3347 pictures of 16 diseases determined by dermatologists in clinical settings. For each dataset, a DLM was trained and evaluated on an independent test set. The primary outcome measures were the precision and sensitivity with 95% CI. For DD, we compared the performance of a DLM trained with lesion pictures only with a DLM trained with both pictures and locations. Results: The average precision and sensitivity were 85% (CI 84-86), 84% (CI 83-85) for macro-anatomy, 81% (CI 80-83), 80% (CI 77-83) for micro-anatomy and 82% (CI 78-85), 81% (CI 77-84) for DD. We observed an improvement in DD performance of 6% (McNemar test P-value 0.0009) for both average precision and sensitivity when training with both lesion pictures and locations. Conclusion: Including location can be beneficial for DD DLM performance. The proposed method can generate body region maps from patient pictures and even reach surgery relevant anatomical precision, e.g. the ear region. Our method enables automated search of large clinical databases and make targeted anatomical image retrieval possible

Bottom-up effects of plant diversity on multitrophic interactions in a biodiversity experiment

Biodiversity is rapidly declining1, and this may negatively affect ecosystem processes, including economically important ecosystem services. Previous studies have shown that biodiversity has positive effects on organisms and processes4 across trophic levels. However, only a few studies have so far incorporated an explicit food-web perspective. In an eight-year biodiversity experiment, we studied an unprecedented range of above- and below-ground organisms and multitrophic interactions. A multitrophic data set originating from a single long-term experiment allows mechanistic insights that would not be gained from meta-analysis of different experiments. Here we show that plant diversity effects dampen with increasing trophic level and degree of omnivory. This was true both for abundance and species richness of organisms. Furthermore, we present comprehensive above-ground/below-ground biodiversity food webs. Both above ground and below ground, herbivores responded more strongly to changes in plant diversity than did carnivores or omnivores. Density and richness of carnivorous taxa was independent of vegetation structure. Below-ground responses to plant diversity were consistently weaker than above-ground responses. Responses to increasing plant diversity were generally positive, but were negative for biological invasion, pathogen infestation and hyperparasitism. Our results suggest that plant diversity has strong bottom-up effects on multitrophic interaction networks, with particularly strong effects on lower trophic levels. Effects on higher trophic levels are indirectly mediated through bottom-up trophic cascades

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348