Chemogenomic profiling predicts antifungal synergies

Chemotherapies, HIV infections, and treatments to block organ transplant rejection are creating a population of immunocompromised individuals at serious risk of systemic fungal infections. Since single-agent therapies are susceptible to failure due to either inherent or acquired resistance, alternative therapeutic approaches such as multi-agent therapies are needed. We have developed a bioinformatics-driven approach that efficiently predicts compound synergy for such combinatorial therapies. The approach uses chemogenomic profiles in order to identify compound profiles that have a statistically significant degree of similarity to a fluconazole profile. The compounds identified were then experimentally verified to be synergistic with fluconazole and with each other, in both Saccharomyces cerevisiae and the fungal pathogen Candida albicans. Our method is therefore capable of accurately predicting compound synergy to aid the development of combinatorial antifungal therapies

Gap junctions in early human placental development

grantor: University of TorontoGap junctional intercellular communication (GJIC) is known to mediate cell proliferation, differentiation, and invasion. We hypothesize that GJIC plays a role in early human placental development. By RTPCR, we identified transcripts for Cx32, Cx37, Cx40, Cx43, and Cx45 in the first trimester human placenta. Of these, Cx40 mRNA and protein were prominent in anchoring extra-villous trophoblast (EVT) columns. Inhibition of GJIC in first trimester placental villous explant cultures by treatment with carbenoxolone and heptanol induced a switch from a proliferative EVT phenotype to an early invasive phenotype, as evident from: (a) a loss of the proliferation marker Ki67, and (b) a decrease in matrix-metalloproteinase-2 activity (which is normally present in proliferative EVT columns). Furthermore, the morphology of EVT outgrowths changed dramatically upon GJIC-blockade from compact, organized outgrowths to a scattered group of individual EVT cells, reminiscent of an early invasive phenotype. Since Cx40 is predominant in anchoring columns 'in vivo ', we treated placental villous explants with antisense oligonucleotides to Cx40. This resulted in identical changes in EVT outgrowth morphology as the non-specific GJIC inhibitors. Together, our data suggest that the presence of functional GJIC, particularly through Cx40 channels, is necessary for the establishment of proliferative EVT cell columns in the early human placenta, and that a loss of GJIC may contribute to the switch to the invasive trophoblast phenotype.M.Sc

Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs

Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs

The eIF4E-Binding Protein 4E-T Is a Component of the mRNA Decay Machinery that Bridges the 5′ and 3′ Termini of Target mRNAs

Eukaryotic mRNA degradation often initiates with the recruitment of the CCR4-NOT deadenylase complex and decay factors to the mRNA 3′ terminus. How the 3′-proximal decay machinery interacts with the 5′-terminal cap structure in order to engender mRNA decapping and 5′–3′ degradation is unclear. Human 4E-T is an eIF4E-binding protein that has been reported to promote mRNA decay, albeit via an unknown mechanism. Here, we show that 4E-T is a component of the mRNA decay machinery and interacts with factors including DDX6, LSM14, and the LSM1-7-PAT1 complex. We also provide evidence that 4E-T associates with, and enhances the decay of, mRNAs targeted by the CCR4-NOT deadenylase complex, including microRNA targets. Importantly, we demonstrate that 4E-T must interact with eIF4E to engender mRNA decay. Taken together, our data support a model where 4E-T promotes mRNA turnover by physically linking the 3′-terminal mRNA decay machinery to the 5′ cap via its interaction with eIF4E