miRNAs und bioreaktive Substanzen mit Einfluss auf Legionella pneumophila verursachte Infektionen

Legionella pneumophila ist ein gramnegatives Bakterium, das sich als intrazelluläres Pathogen, neben Amöben als natürlichen Wirt, auch in alveolaren Makrophagen vermehren kann. Dort löst es das Pontiac-Fieber, oder in schwerwiegenderen Fällen die Legionärskrankheit aus, welche durch eine schwere Pneumonie charakterisiert ist. Das Ziel dieser Arbeit war es ein Hochdurchsatzverfahren zu entwickeln. Durch dieses sollte es ermöglicht werden, eine große Anzahl an Substanzen zu untersuchen und so eine erweiterte Einsicht in die Interaktion zwischen Legionellen und Makrophagen zu gewinnen. Mit dieser Methode wurden Proben einer miRNA-Library und Proben einer selbst zusammengestellten Sammlung aus bioreaktiven Substanzen untersucht. Dabei wurde das Verhältnis zwischen infizierten und nicht-infizierten Zellen sowie die Infektionsstärke abhängig von der Behandlung bestimmt. Als Versuchsmodell dienten differenzierte THP-1 Zellen um Makrophagen zu imitieren, die in Folge mit Legionella pneumophila des Stammes Corby infiziert wurden. Obwohl die Analyse der miRNA-Library technische Schwierigkeiten offenbarte, konnten mit miRNA-101-3p, miRNA-106a-5p und miRNA-107, drei miRNAs identifiziert und validiert werden, die die Infektion der Makrophagen steigerten. Über Massenspektrometrie wurden, durch die miRNAs regulierte Proteine detektiert, um Aufschluss darüber zu geben, wodurch die Steigerung der Infektion bedingt ist. In Folge zeigten sich mehrere Proteinklassen sowie Signalwege, die durch die Transfektion der miRNAs, eine veränderte Proteinkonzentration aufwiesen und eine potentielle Beteiligung in dieser Infektion haben. Durch die Analyse der bioreaktiven Substanzen wurden mit 25-hydroxy Cholesterol, H-89, Fluoxetin, Forskolin und W-7, fünf Substanzen gefunden, die in diesem Infektionskontext unbekannt waren. Da die Kontrolle Amitriptylin und Fluoxetin als Inhibitoren der sauren Sphingomyelinase agieren, wurden mit Benztropin und Perhexilin zwei weitere Substanzen getestet, die dieses Protein inhibieren. Erneut zeigte sich eine starke Reduktion der Infektionsrate, was auf die Relevanz dieses Protein während des Infektionsgeschehens hindeutet. Dies wird auch dadurch bestärkt, dass sowohl Makrophagen als auch Legionellen die Aktivität der sauren Sphingomyelinase zu regulieren scheinen. Zusammengefasst wurde in dieser Arbeit eine Screening-Methode etabliert die es, trotz Limitationen, erlaubt eine erhöhte Anzahl an Proben auf deren Einfluss auf die Legionellen-Makrophagen Interaktion zu testen. Die von den drei miRNAs sowie fünf Substanzen regulierten Proteinklassen und Signalwege geben Anhaltspunkte für die weitere Forschung

Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Detection and segmentation of morphologically complex eukaryotic cells in fluorescence microscopy images via feature pyramid fusion.

Detection and segmentation of macrophage cells in fluorescence microscopy images is a challenging problem, mainly due to crowded cells, variation in shapes, and morphological complexity. We present a new deep learning approach for cell detection and segmentation that incorporates previously learned nucleus features. A novel fusion of feature pyramids for nucleus detection and segmentation with feature pyramids for cell detection and segmentation is used to improve performance on a microscopic image dataset created by us and provided for public use, containing both nucleus and cell signals. Our experimental results indicate that cell detection and segmentation performance significantly benefit from the fusion of previously learned nucleus features. The proposed feature pyramid fusion architecture clearly outperforms a state-of-the-art Mask R-CNN approach for cell detection and segmentation with relative mean average precision improvements of up to 23.88% and 23.17%, respectively

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

Abstract Background Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections. Methods Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid. Results In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment. Conclusion Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstrac

Additional file 7: Figure S3. of Functional analysis and transcriptional output of the GĂśttingen minipig genome

Schematic description of the in silico workflow for selection of Sus scrofa specific lncRNAs. * 16 genomes: cow, horse, dog, human, orang utan, chimpanzee, cynomolgus monkey, rhesus monkey, marmoset, rabbit, guinea pig, hamster, mouse, rat, opossum, chicken. See text for more detailed description. (JPEG 854 kb

Additional file 2: Table S2. of Functional analysis and transcriptional output of the GĂśttingen minipig genome

Contig assembly statistics. Roche-454 reads that were not incorporated into the minipig genome were assembled de-novo with Roche-Newbler software. (DOCX 13 kb