An Ac/Ds-mediated gene trap system for functional genomics in barley

<p>Abstract</p> <p>Background</p> <p>Gene trapping is a powerful tool for gene discovery and functional genomics in both animals and plants. Upon insertion of the gene trap construct into an expressed gene, splice donor and acceptor sites facilitate the generation of transcriptional fusions between the flanking sequence and the reporter. Consequently, detection of reporter gene expression allows the identification of genes based on their expression pattern. Up to now rice is the only cereal crop for which gene trap approaches exist. In this study we describe a gene trap system in barley (<it>Hordeum vulgare </it>L.) based on the maize transposable elements <it>Ac/Ds</it>.</p> <p>Results</p> <p>We generated gene trap barley lines by crossing <it>Ac </it>transposase expressing plants with multiple independent transformants carrying the <it>Ds </it>based gene trap construct GT<it>Ds</it>B. Upstream of the β-Glucuronidase start codon GT<it>Ds</it>B carries splice donor and acceptor sites optimized for monocotyledonous plants. DNA blot analysis revealed GT<it>Ds</it>B transposition frequencies of 11% and 26% in the F₁and F₂generation of gene trap lines and perpetuation of transposition activity in later generations. Furthermore, analysis of sequences flanking transposed GT<it>Ds</it>B elements evidenced preferential insertion into expressed regions of the barley genome. We screened leaves, nodes, immature florets, pollinated florets, immature grains and seedlings of F₂plants and detected GUS expression in 51% (72/141) of the plants. Thus, reporter gene expression was found in 24 of the 28 F₁lines tested and in progeny of all GT<it>Ds</it>B parental lines.</p> <p>Conclusion</p> <p>Due to the frequent transposition of GT<it>Ds</it>B and the efficient expression of the GUS reporter gene, we conclude that this <it>Ac/Ds</it>-based gene trap system is an applicable approach for gene discovery in barley. The successful introduction of a gene trap construct optimized for monocots in barley contributes a novel functional genomics tool for this cereal crop.</p

Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation

Summary: Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM

Discovery of a novel series of tankyrase inhibitors by a hybridization approach

Abstract A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC₅₀ values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor <b>16</b> displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC₅₀ values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor <b>16</b> displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC₅₀ values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker