Literature optimized integration of gene expression for organ-specific evaluation of toxicogenomics datasets

The study of drug toxicity in human organs is complicated by their complex inter-relations and by the obvious difficulty to testing drug effects on biologically relevant material. Animal models and human cell cultures offer alternatives for systematic and large-scale profiling of drug effects on gene expression level, as typically found in the so-called toxicogenomics datasets. However, the complexity of these data, which includes variable drug doses, time points, and experimental setups, makes it difficult to choose and integrate the data, and to evaluate the appropriateness of one or another model system to study drug toxicity (of particular drugs) of particular human organs. Here, we define a protocol to integrate drug-wise rankings of gene expression changes in toxicogenomics data, which we apply to the TG-GATEs dataset, to prioritize genes for association to drug toxicity in liver or kidney. Contrast of the results with sets of known human genes associated to drug toxicity in the literature allows to compare different rank aggregation approaches for the task at hand. Collectively, ranks from multiple models point to genes not previously associated to toxicity, notably, the PCNA clamp associated factor (PCLAF), and genes regulated by the master regulator of the antioxidant response NFE2L2, such as NQO1 and SRXN1. In addition, comparing gene ranks from different models allowed us to evaluate striking differences in terms of toxicity-associated genes between human and rat hepatocytes or between rat liver and rat hepatocytes. We interpret these results to point to the different molecular functions associated to organ toxicity that are best described by each model. We conclude that the expected production of toxicogenomics panels with larger numbers of drugs and models, in combination with the ongoing increase of the experimental literature in organ toxicity, will lead to increasingly better associations of genes for organism toxicity

Annexin A1 exerts renoprotective effects in experimental crescentic glomerulonephritis

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45 + leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence

Dissecting the action of an evolutionary conserved non-coding region on renin promoter activity

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of ∼775 bp in size, ∼14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score

Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal

Numerical Analysis and Modelling of the Heart Rhythm

Titelseite und Inhaltsverzeichnis 1\. Numerische Analyse und Modellierung des Herzrhythmus 1.1 Zusammenfassung 1.2 Zusammenfassende Einleitung und Herleitung der Problemstellungen 1.3 Problemstellungen 2\. Zusammenstellung der Forschungsergebnisse 2.1 Lineare und nichtlineare Eigenschaften der Herzfrequenz in der postnatalen Entwicklung 2.2 Die Komplexität des Herzrhythmus in der postnatalen Entwicklung 2.3 Quantitative Analyse der Synchronisation von Herz- und Atemrhythmus bei Kindern 2.4 Quantifizierung der Kopplungsrichtung von Herz- und Atemrhythmus bei Kindern 2.5 Ein abgeschwächter Barorezeptorreflex verursacht eine pathologische Herzrhythmus-Turbulenz 3\. Zusammenfassende Diskussion 4\. Literaturverzeichnis 5\. Danksagung 6\. Eidesstattliche VersicherungDie Analyse des Herzrhythmus liefert wichtige Aufschlüsse über Eigenschaften des kardiovaskulären Regulationssystems. So findet man zum Beispiel eine verminderte Herzfrequenzvariabilität bei pathologischen Zuständen des Herzens oder des autonomen Nervensystems. Die Herzfrequenzvariabilität wurde bisher unter verschiedenen physiologischen Bedingungen wie Schlaf, körperlicher Belastung und im Altersgang analysiert. Es ist jedoch wenig über die normale Entwicklung von Herzfrequenzparametern unmittelbar nach der Geburt bekannt. Es konnte im ersten Schwerpunkt der vorliegenden Schrift gezeigt werden, dass sich lineare und nichtlineare Parameter des Herzrhythmus jeweils schlafstadienspezifisch in charakteristischer Weise nach der Geburt verändern. Die altersbezogene Veränderung kann als Widerspiegelung mehrerer Reifungsprozesse angesehen werden. Besonders wichtig ist, dass keiner der Parameter einen geradlinigen Altersverlauf aufzeigt. Diese Tatsache deutet darauf hin, dass mehrere der Reifung unterworfene Faktoren die Parameter des Herzrhythmus beeinflussen. Einen weiteren Schwerpunkt stellt die Analyse der Interaktion von Atemrhythmus und Herzrhythmus in der postnatalen Periode dar. Zwei wichtige Aspekte wurden hier berücksichtigt: zum einen die Charakterisierung und Quantifizierung der Synchronisation beider Rhythmen und zum anderen die Frage nach der Richtung der Kopplung dieser Rhythmen. Es wurde zunächst ein numerisches Verfahren entwickelt, das eine Analyse großer Datenmengen zulässt. Damit konnte nachgewiesen werden, dass eine Synchronisation von Herz- und Atemrhythmus über mehrere Minuten hinweg auch innerhalb des ersten Lebenshalbjahres vorkommt. Die Häufigkeit ganzzahliger Verhältnisse der Synchronisation (z.B. 3:1, 4:1) zeigte in unseren Untersuchungen einen charakteristischen Altersverlauf. Um die Interaktion von rhythmischen Systemen zu verstehen, muss die Frage nach der Kausalität der Interaktion geklärt werden. Die Ergebnisse der Analyse dazu zeigen, dass keine eindeutige Dominanz des Informationstransfers vom Atemrhythmus auf den Herzrhythmus gegeben ist. Vielmehr unterliegt die Richtung der Kopplung einer Altersentwicklung innerhalb der ersten sechs Lebensmonate. Es konnte weiterhin gezeigt werden, dass die Atemfrequenz deutlich die Dominanz der Richtung der Interaktion beeinflusst. Die atemfrequenzabhängige Richtung der Interaktion ist statistisch unabhängig vom Schlafstadium. Daraus lässt sich ableiten, dass die Strukturen, die maßgeblich für die atemfrequenzabhängige Beziehung sind, nicht in entscheidendem Maße funktionell moduliert werden. Die in dieser Arbeit erprobten Verfahren können auch auf andere Systeme oszillatorischer Natur übertragen werden. Ein dritter Schwerpunkt beschäftigt sich mit der Frage nach dem Hintergrund zweier neuartiger auf dem Herzrhythmus basierender Prädiktoren für den plötzlichen Herztod. Die Analyse stützt sich auf ein neu entwickeltes numerisch-mathematisches Kreislaufmodell. Dieser Ansatz war nötig, da sich das Verhalten eines Systems beim zeitlichen Ineinandergreifen verschiedener Prozesse nicht ohne dieses Hilfsmittel analysieren lässt. Es konnte mittels der Simulation gezeigt werden, dass bei abgeschwächter Barorezeptorreflex-Antwort genau die Herzfrequenz-Muster nach einer ventrikulären Extrasystole auftreten, die den pathologischen Werten der Prädiktoren für den plötzlichen Herztod entsprechen.This habilitation thesis deals with the numerical analysis and modelling of various aspects of heart rate. It is presented in a cumulative form, i.e. multiple journal articles of the author related to the subject are bundled together

c ○ World Scientific Publishing Company QUANTITATIVE ANALYSIS OF CARDIORESPIRATORY SYNCHRONIZATION IN INFANTS

We investigate the phase synchronization of heartbeat and respiration in a group of healthy infants. Having presented and compared two quantitative measures of synchronization, we conclude that one of these measures — the conditional probability index — allows reliable detection of synchronous epochs of different order n:m and, thus, makes possible an automatic processing of large data sets. In our analysis of experimental time series, we have found numerous epochs of phase synchronization. It turned out that the average degree of synchronization varies with the age of the newborns. 1