A multi-parametric flow cytometric assay to analyze DNA–protein interactions

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA–protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein–DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro

Global and regional brain metabolic scaling and its functional consequences

Background: Information processing in the brain requires large amounts of metabolic energy, the spatial distribution of which is highly heterogeneous reflecting complex activity patterns in the mammalian brain. Results: Here, it is found based on empirical data that, despite this heterogeneity, the volume-specific cerebral glucose metabolic rate of many different brain structures scales with brain volume with almost the same exponent around -0.15. The exception is white matter, the metabolism of which seems to scale with a standard specific exponent -1/4. The scaling exponents for the total oxygen and glucose consumptions in the brain in relation to its volume are identical and equal to $0.86\pm 0.03$, which is significantly larger than the exponents 3/4 and 2/3 suggested for whole body basal metabolism on body mass. Conclusions: These findings show explicitly that in mammals (i) volume-specific scaling exponents of the cerebral energy expenditure in different brain parts are approximately constant (except brain stem structures), and (ii) the total cerebral metabolic exponent against brain volume is greater than the much-cited Kleiber's 3/4 exponent. The neurophysiological factors that might account for the regional uniformity of the exponents and for the excessive scaling of the total brain metabolism are discussed, along with the relationship between brain metabolic scaling and computation.Comment: Brain metabolism scales with its mass well above 3/4 exponen

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Search for the neutral Higgs bosons of the minimal supersymmetric standard model in pp collisions at root s=7 TeV with the ATLAS detector

A search for neutral Higgs bosons of the Minimal Supersymmetric Standard Model (MSSM) is reported. The analysis is based on a sample of proton-proton collisions at a centre-of-mass energy of 7TeV recorded with the ATLAS detector at the Large Hadron Collider. The data were recorded in 2011 and correspond to an integrated luminosity of 4.7 fb-1 to 4.8 fb-1. Higgs boson decays into oppositely-charged muon or τ lepton pairs are considered for final states requiring either the presence or absence of b-jets. No statistically significant excess over the expected background is observed and exclusion limits at the 95% confidence level are derived. The exclusion limits are for the production cross-section of a generic neutral Higgs boson, φ, as a function of the Higgs boson mass and for h/A/H production in the MSSM as a function of the parameters mA and tan β in the mhmax scenario for mA in the range of 90GeV to 500 GeV. Copyright CERN

Stability and change in screen-based sedentary behaviours and associated factors among Norwegian children in the transition between childhood and adolescence

<p>Abstract</p> <p>Background</p> <p>In order to inform interventions to prevent sedentariness, more longitudinal studies are needed focusing on stability and change over time in multiple sedentary behaviours. This paper investigates patterns of stability and change in TV/DVD use, computer/electronic game use and total screen time (TST) and factors associated with these patterns among Norwegian children in the transition between childhood and adolescence.</p> <p>Methods</p> <p>The baseline of this longitudinal study took place in September 2007 and included 975 students from 25 control schools of an intervention study, the HEalth In Adolescents (HEIA) study. The first follow-up took place in May 2008 and the second follow-up in May 2009, with 885 students participating at all time points (average age at baseline = 11.2, standard deviation ± 0.3). Time used for/spent on TV/DVD and computer/electronic games was self-reported, and a TST variable (hours/week) was computed. Tracking analyses based on absolute and rank measures, as well as regression analyses to assess factors associated with change in TST and with tracking high TST were conducted.</p> <p>Results</p> <p>Time spent on all sedentary behaviours investigated increased in both genders. Findings based on absolute and rank measures revealed a fair to moderate level of tracking over the 2 year period. High parental education was inversely related to an increase in TST among females. In males, self-efficacy related to barriers to physical activity and living with married or cohabitating parents were inversely related to an increase in TST. Factors associated with tracking high vs. low TST in the multinomial regression analyses were low self-efficacy and being of an ethnic minority background among females, and low self-efficacy, being overweight/obese and not living with married or cohabitating parents among males.</p> <p>Conclusions</p> <p>Use of TV/DVD and computer/electronic games increased with age and tracked over time in this group of 11-13 year old Norwegian children. Interventions targeting these sedentary behaviours should thus be introduced early. The identified modifiable and non-modifiable factors associated with change in TST and tracking of high TST should be taken into consideration when planning such interventions.</p

Angiotensin II Infusion Induces Marked Diaphragmatic Skeletal Muscle Atrophy

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD

A higher activation threshold of memory CD8+ T cells has a fitness cost that is modified by TCR affinity during Tuberculosis

All relevant data are within the paper and its Supporting Information files except for the primary TCR sequences. The data files for the primary TCR sequences are publicly deposited in the University of Massachusetts Medical School’s institutional repository, eScholarship@UMMS. The permanent link to the data is http://dx.doi.org/10.13028/M2CC70T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.This work was supported by NIH R01 AI106725 as well as fellowship funding to SC from NIH AI T32 007061 and the UMass GSBS Millennium Program. The Small Animal Biocontainment Suite was supported in part by Center for AIDS Research Grant P30 AI 060354. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio