Mechanical loading of bioengineered skeletal muscle in vitro recapitulates gene expression signatures of resistance exercise in vivo.

Understanding the role of mechanical loading and exercise in skeletal muscle (SkM) is paramount for delineating the molecular mechanisms that govern changes in muscle mass. However, it is unknown whether loading of bioengineered SkM in vitro adequately recapitulates the molecular responses observed after resistance exercise (RE) in vivo. To address this, the transcriptional and epigenetic (DNA methylation) responses were compared after mechanical loading in bioengineered SkM in vitro and after RE in vivo. Specifically, genes known to be upregulated/hypomethylated after RE in humans were analyzed. Ninety-three percent of these genes demonstrated similar changes in gene expression post-loading in the bioengineered muscle when compared to acute RE in humans. Furthermore, similar differences in gene expression were observed between loaded bioengineered SkM and after programmed RT in rat SkM tissue. Hypomethylation occurred for only one of the genes analysed (GRIK2) post-loading in bioengineered SkM. To further validate these findings, DNA methylation and mRNA expression of known hypomethylated and upregulated genes post-acute RE in humans were also analyzed at 0.5, 3, and 24 h post-loading in bioengineered muscle. The largest changes in gene expression occurred at 3 h, whereby 82% and 91% of genes responded similarly when compared to human and rodent SkM respectively. DNA methylation of only a small proportion of genes analyzed (TRAF1, MSN, and CTTN) significantly increased post-loading in bioengineered SkM alone. Overall, mechanical loading of bioengineered SkM in vitro recapitulates the gene expression profile of human and rodent SkM after RE in vivo. Although some genes demonstrated differential DNA methylation post-loading in bioengineered SkM, such changes across the majority of genes analyzed did not closely mimic the epigenetic response to acute-RE in humans

UBR5 is a Novel E3 Ubiquitin Ligase involved in Skeletal Muscle Hypertrophy and Recovery from Atrophy

We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, was epigenetically altered (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression was positively correlated with increased lean leg mass in humans [1]. This was counterintuitive given the well-defined role of other E3 ligase family members, MuRF1 and MAFbx in muscle atrophy. Therefore, in the present study we aimed to investigate this relatively uncharacterised E3 ubiquitin ligase using multiple in-vivo and in-vitro models of skeletal muscle atrophy, injury, recovery from atrophy as well as anabolism and hypertrophy. We report for the first time, that during atrophy evoked by tetrodotoxin (TTX) nerve silencing in rats, the UBR5 promoter was significantly hypomethylated with a concomitant increase in gene expression early (3 & 7 days) after the induction of atrophy. However, at these timepoints larger increases in MuRF1/MAFbx were observed, and UBR5 expression had returned to baseline levels during later atrophy (14 days) where muscle mass loss was greatest. We confirmed an alternate gene expression profile for UBR5 versus MuRF1/MAFbx in a secondary model of atrophy induced by 7 days continuous low frequency electrical stimulation, where UBR5 demonstrated no significant increase, whereas MuRF1/MAFbx were elevated. Further, after partial (52%) recovery of muscle mass following 7 days TTX-cessation, UBR5 was hypomethylated and increased at the gene expression level, while alternately, reductions in gene expression of MuRF1 and MAFbx were observed. To substantiate these gene expression findings, we observed a significant increase in UBR5 protein abundance after full recovery (14 days) of muscle mass from hindlimb unloading (HU) in rats. Aged rats also demonstrated a similar temporal increase in UBR5 protein abundance after recovery from HU. Further, we confirmed significant increases in UBR5 protein during recovery from nerve crush injury in mice at 28 and 45 days, that related to a full recovery of muscle mass between 45-60 days. During anabolism and hypertrophy, UBR5 gene expression increased following an acute bout of mechanical loading in three-dimensional bioengineered mouse muscle in-vitro, and after chronic electrical stimulation-induced hypertrophy in rats in-vivo, without increases in MuRF1/MAFbx. Additionally, increased UBR5 protein abundance was identified following synergist ablation/functional overload (FO)-induced hypertrophy of the plantaris muscle in mice in-vivo, and finally over a 7-day time-course of regeneration in primary human muscle cells in-vitro. Finally, genetic association studies (> 700,000 SNPs) in human cohorts identified that the A alleles of rs10505025 and rs4734621 SNPs were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power versus endurance/untrained phenotypes. Overall, we suggest that UBR5 is a novel E3 ubiquitin ligase that is alternatively regulated compared to MuRF1/MAFbx, and is elevated during early atrophy (but not later atrophy), recovery, anabolism and hypertrophy in animals in-vivo as well as during human muscle cell regeneration in-vitro. In humans, genetic variations of the UBR5 gene are strongly associated with larger fast-twitch muscle fibres and strength/power performance

The estrogen-injected female mouse: new insight into the etiology of PCOS

<p>Abstract</p> <p>Background</p> <p>Female mice and rats injected with estrogen perinatally become anovulatory and develop follicular cysts. The current consensus is that this adverse response to estrogen involves the hypothalamus and occurs because of an estrogen-induced alteration in the GnRH delivery system. Whether or not this is true has yet to be firmly established. The present study examined an alternate possibility in which anovulation and cyst development occurs through an estrogen-induced disruption in the immune system, achieved through the intermediation of the thymus gland.</p> <p>Methods, Results and Conclusion</p> <p>A putative role for the thymus in estrogen-induced anovulation and follicular cyst formation (a model of PCOS) was examined in female mice by removing the gland prior to estrogen injection. Whereas all intact, female mice injected with 20 ug estrogen at 5–7 days of age had ovaries with follicular cysts, no cysts were observed in animals in which thymectomy at 3 days of age preceded estrogen injection. In fact, after restoring immune function by thymocyte replacement, the majority of thymectomized, estrogen-injected mice had ovaries with corpora lutea. Thus, when estrogen is unable to act on the thymus, ovulation occurs and follicular cysts do not develop. This implicates the thymus in the cysts' genesis and discounts the role of the hypothalamus. Subsequent research established that the disease is transferable by lymphocyte infusion. Transfer took place between 100-day-old estrogen-injected and 15-day-old naïve mice only when recipients were thymectomized at 3 days of age. Thus, a prerequisite for cyst formation is the absence of regulatory T cells. Their absence in donor mice was judged to be the result of an estrogen-induced increase in the thymus' vascular permeability, causing de facto circumvention of the final stages of regulatory T cell development. The human thymus has a similar vulnerability to steroid action during the fetal stage. We propose that in utero exposure to excessive levels of steroids such as estrogen has a long-term effect on the ability of the thymus to produce regulatory T cells. In female offspring this can lead to PCOS.</p

Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival

Population Pharmacokinetics of Telapristone (CDB-4124) and its Active Monodemethylated Metabolite CDB-4453, with a Mixture Model for Total Clearance

Telapristone is a selective progesterone antagonist that is being developed for the long-term treatment of symptoms associated with endometriosis and uterine fibroids. The population pharmacokinetics of telapristone (CDB-4124) and CDB-4453 was investigated using nonlinear mixed-effects modeling. Data from two clinical studies (n = 32) were included in the analysis. A two-compartment (parent) one compartment (metabolite) mixture model (with two populations for apparent clearance) with first-order absorption and elimination adequately described the pharmacokinetics of telapristone and CDB-4453. Telapristone was rapidly absorbed with an absorption rate constant (Ka) of 1.26 h−1. Moderate renal impairment resulted in a 74% decrease in Ka. The population estimates for oral clearance (CL/F) for the two populations were 11.6 and 3.34 L/h, respectively, with 25% of the subjects being allocated to the high-clearance group. Apparent volume of distribution for the central compartment (V2/F) was 37.4 L, apparent inter-compartmental clearance (Q/F) was 21.9 L/h, and apparent peripheral volume of distribution for the parent (V4/F) was 120 L. The ratio of the fraction of telapristone converted to CDB-4453 to the distribution volume of CDB-4453 (Fmetest) was 0.20/L. Apparent volume of distribution of the metabolite compartment (V3/F) was fixed to 1 L and apparent clearance of the metabolite (CLM/F) was 2.43 L/h. A two-compartment parent-metabolite model adequately described the pharmacokinetics of telapristone and CDB-4453. The clearance of telapristone was separated into two populations and could be the result of metabolism via polymorphic CYP3A5

Role of novel targeted therapies in the clinic

The number and variety of novel, molecular-targeted agents offers realistic hope for significant advances in cancer treatment. The potential of these new treatment approaches is unquestionable, but the reality is something that only thorough clinical evaluation and experience can reveal. Clinical experience of targeted therapies is at an early stage but it is likely that we will have an increasing number of treatment options available to us in the near future. This manuscript explores our current understanding of molecular-targeted therapies and considers: What approach should be used? (single vs multitarget agents); When should they be administered? (identifying the optimal point for intervention); How should they be used? (monotherapy or combination therapy regimens); and Who should we be giving them to? (acknowledging the need for patient selection)

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns