Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2

International audienceEukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eu-karyotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In ar-chaea, orthologs of eIF5 are not found and aIF2 GT-Pase activity is thought to be non-assisted. However , no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2␥, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2␥. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

International audienceHeterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2-GDPNP-tRNA complex. © The Author(s) 2012. Published by Oxford University Press

Theoretical modelling of the three-dimensional wake of vertical axis turbines

Arrays of Vertical Axis Wind Turbines (VAWTs) can achieve larger power generation per land area than horizontal axis turbines farms, due to the positive synergy between VATs in close proximity. Theoretical wake models enable the reliable design of the array layout that maximises the energy output, which need to depict the driving wake dynamics. VAWTs generate a highly complex wake that evolves according to two governing length-scales, namely the turbine rotor's diameter and height which define a rectangular shape of the wake cross-section, and feature distinct wake expansion rates. This paper presents analytical VAWT wake models that account for an asymmetric distribution of such wake expansion adopting a top-hat and Gaussian velocity deficit distribution. Our proposed analytical Gaussian model leads to an enhanced initial wake expansion prediction with the wake width ($\varepsilon$) behind the rotor equal to $(\beta/4 \pi)^{1/2}$ with $\beta$ being the ratio of initial wake area to the VAWT's frontal area, which addresses the limitations of previous models that under-predicted the wake onset area. Velocity deficit predictions are calculated in a series of numerical benchmarks consisting of a single and an array of four in-line vertical axis wind turbines. In comparisons with field data and large-eddy simulations, our models provide a good accuracy to represent the mean wake distribution, maximum velocity deficit, and momentum thickness, with the Gaussian model attaining the best predictions.These models will aid to drive the design of VAT arrays and accelerate this technology.Comment: 22 pages, 8 figure

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERalpha expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy

Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

10.1371/journal.pone.0057015PLoS ONE83

Optimum positioning for AP pelvis radiography : a literature review

Aim Pelvic radiography is used for the identification of hip joint changes, including pathologies such as osteoarthritis. Several studies have recommended that the position for this radiological procedure should be standing, not supine, to reflect the functional appearances of the hip joint. The aim of this review was to evaluate pelvis radiography positioning with respect to the image appearances and information provided for clinical decision-making. Aside from this, potential recommendations to the radiographic technique for an erect pelvis projection will be considered. Method A literature search was performed using databases/abstract systems (ScienceDirect, Web of Science, PubMed, and MEDLINE). Only articles written in English were included. Results Twenty-five articles were identified. Findings from the review describe the effect of repositioning from supine to erect on a series of specific hip measurements. These include pelvic tilt, joint space width, and the acetabular component. Conclusion Evidence within the literature illustrates that in several studies, there were differences when repositioning from supine to standing for a number of pelvic metrics. Standing positioning is promoted by some authors since this may facilitate the early diagnosis of hip joint pathology and assist in the planning of surgical interventions. Literature is very limited on how to optimally perform erect pelvis radiography, and this should be an area for future research