Project description:Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs), able to separate anti-inflammatory effects via gene suppression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene suppression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms may therefore increase chances to find novel GR ligands with an improved therapeutic benefit.
Project description:We have employed a custom bioprospecing approach to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses we identified forty eight novel potential cationic antimicrobial peptides, with all but one of the identified peptides being derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa 9027 and Staphylococcus aureus 25983, with seven peptides exhibiting antimicrobial activity against both microbes, and one only showing significant potency against Pseudomonas aeruginosa 9027. This study demonstrates the power and promise of our bioprospecting approach to CAMP discovery and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection.
Project description:To identify novel ghost factors regulating the expression of Interferon Stimulated Genes (ISGs), we conducted genome-wide cDNA screening in Huh-7 IFIT-1 Luc cells Huh-7 cells stably expressing IFIT1 luciferase reporter were plated into the wells with pre-spotted cDNA clones from MGS collection (http://mgc.nci.nih.gov/) (reverse transfection). The inducibility of luciferase activity upon the introduction of each cDNA clone was then measure to determine the regulator of IFIT1 expression. Two biological replicates were tested and the averages were taken for measurement.
Project description:Four libraries were analyzed to assess the level and effects of inducing the ectopic expression of a luciferase targeting miRNA in mouse embryonic fibroblasts (MEFs). Here, we find low level, leaky expression in the absence of ROSA-rtTA and doxycyclin. When expression is effectively induced, the Luciferase miRNA is expressed as the 8th most abundant species in the cells. Keyword(s): Epigenetics
