ABSTRACT
Increasing amounts of biological data are accumulating in the pharmaceutical industry and academic institutions. However, data does not equal actionable information, and guidelines for appropriate data capture, harmonization, integration, mining, and visualization need to be established to fully harness its potential. Here, we describe ongoing efforts at Merck & Co. to structure data in the area of chemogenomics. We are integrating complementary data from both internal and external data sources into one chemogenomics database (Chemical Genetic Interaction Enterprise; CHEMGENIE). Here, we demonstrate how this well-curated database facilitates compound set design, tool compound selection, target deconvolution in phenotypic screening, and predictive model building.
Subject(s)
Databases, Factual , Drug Discovery , Genomics , Models, Theoretical , PhenotypeABSTRACT
BACKGROUND: Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied. RESULTS: Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. CONCLUSIONS: This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.
Subject(s)
Blood Vessels/embryology , Embryonic Development , Endothelium, Vascular/embryology , Endothelium, Vascular/metabolism , Kruppel-Like Transcription Factors/metabolism , Animals , Embryo, Mammalian , Gene Expression Regulation, Developmental , Intracranial Hemorrhages/embryology , Intracranial Hemorrhages/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Knockout , Microscopy, Electron , Morphogenesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Occludin/genetics , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Signal Transduction , Tamoxifen/pharmacologyABSTRACT
In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. C. albicans is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that C. albicans interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of C. albicans with bacteria and the impact on morphology we utilized a haploinsufficiency based C. albicans mutant screen to test for the ability of C. albicans to produce hyphae in the presence of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, CDR4, displayed increased expression when co-cultured with S. aureus, but not E. coli or P. aeruginosa. Our data demonstrates the ability to use a large scale library screen to identify genes involved in Candida-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of C. albicans morphogenesis.
