Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus

<p>Abstract</p> <p>Background</p> <p>Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to <it>Evc </it>and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.</p> <p>Results</p> <p>Bioinformatic analysis reveals that the <it>Evc </it>and <it>Evc2 </it>genes arose through a duplication event early in metazoan evolution and were subsequently lost in arthropods and nematodes. Here we demonstrate that Evc2 is essential for Hh pathway activation in response to the Smo agonist purmorphamine. A yeast two-hybrid screen using Evc as bait identified Evc2 as an Evc binding partner and we confirmed the interaction by immunoprecipitation. We developed anti-Evc2 antibodies and show that Evc2 and Evc co-localize at the basal body and also on primary cilia. In transfected cells, basal body and cilia localization is observed when Evc and Evc2 constructs are co-transfected but not when either construct is transfected individually. We show that Evc and Evc2 are cilia transmembrane proteins, the C-terminus for both being intracellular and Evc2, but not Evc, having an extracellular portion. Furthermore, Evc is absent at the basal body in Evc2 null cells. Using Western blots of cytoplasmic and nuclear protein, we also demonstrate that full length Evc2 but not Evc, is located in the nucleus.</p> <p>Conclusions</p> <p>We demonstrate for the first time that Evc2 is a positive regulator of the Hh signalling pathway and that it is located at the basal body of primary cilia. We show that the presence of Evc and Evc2 at the basal body and cilia membrane is co-dependent. In addition, Evc2, but not Evc, is present in the cell nucleus suggesting movement of Evc2 between the cilium and nucleus.</p

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

OPTIMA: A prospective randomized trial to validate the predictive utility and cost-effectiveness of gene expression test-directed chemotherapy decisions in early breast cancer

Background: Multi-parameter gene expression assays (MPAs) are widely used to estimate individual patient residual risk in hormone-sensitive HER2-negative node-negative early breast cancer, allowing patients with low risk to safely avoid chemotherapy. Evidence for MPA use in node-positive breast cancer is limited. OPTIMA (Optimal Personalised Treatment of early breast cancer usIng Multi-parameter Analysis) aims to validate MPA’s as predictors of chemotherapy sensitivity in a largely node-positive breast cancer population

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Glycosylation of immunoglobulin G is regulated by a large network of genes pleiotropic with inflammatory diseases

Effector functions of immunoglobulin G (IgG) are regulated by the composition of a glycan moiety, thus affecting activity of the immune system. Aberrant glycosylation of IgG has been observed in many diseases, but little is understood about the underlying mechanisms. We performed a genome-wide association study of IgG N-glycosylation (N = 8090) and, using a data-driven network approach, suggested how associated loci form a functional network. We confirmed in vitro that knockdown of IKZF1 decreases the expression of fucosyltransferase FUT8, resulting in increased levels of fucosylated glycans, and suggest that RUNX1 and RUNX3, together with SMARCB1, regulate expression of glycosyltransferase MGAT3. We also show that variants affecting the expression of genes involved in the regulation of glycoenzymes colocalize with variants affecting risk for inflammatory diseases. This study provides new evidence that variation in key transcription factors coupled with regulatory variation in glycogenes modifies IgG glycosylation and has influence on inflammatory diseases

The life history of 21 breast cancers.

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis

Mutational processes molding the genomes of 21 breast cancers

All cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed “kataegis,” was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed