Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer

Multi-Way Multi-Group Segregation and Diversity Indices

Background: How can we compute a segregation or diversity index from a three-way or multi-way contingency table, where each variable can take on an arbitrary finite number of values and where the index takes values between zero and one? Previous methods only exist for two-way contingency tables or dichotomous variables. A prototypical three-way case is the segregation index of a set of industries or departments given multiple explanatory variables of both sex and race. This can be further extended to other variables, such as disability, number of years of education, and former military service. Methodology/Principal Findings: We extend existing segregation indices based on Euclidean distance (square of coefficient of variation) and Boltzmann/Shannon/Theil index from two-way to multi-way contingency tables by including multiple summations. We provide several biological applications, such as indices for age polyethism and linkage disequilibrium. We also provide a new heuristic conceptualization of entropy-based indices. Higher order association measures are often independent of lower order ones, hence an overall segregation or diversity index should be the arithmetic mean of the normalized association measures at all orders. These methods are applicable when individuals selfidentify as multiple races or even multiple sexes and when individuals work part-time in multiple industries. Conclusions/Significance: The policy implications of this work are enormous, allowing people to rigorously test whethe

Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Modulates Oscillations of Pancreatic Islet Metabolism

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca2+ pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca2+ oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis

Postprandial differences in the plasma metabolome of healthy Finnish subjects after intake of a sourdough fermented endosperm rye bread versus white wheat bread

<p>Abstract</p> <p>Background</p> <p>The mechanism behind the lowered postprandial insulin demand observed after rye bread intake compared to wheat bread is unknown. The aim of this study was to use the metabolomics approach to identify potential metabolites related to amino acid metabolism involved in this mechanism.</p> <p>Methods</p> <p>A sourdough fermented endosperm rye bread (RB) and a standard white wheat bread (WB) as a reference were served in random order to 16 healthy subjects. Test bread portions contained 50 g available carbohydrate. <it>In vitro </it>hydrolysis of starch and protein were performed for both test breads. Blood samples for measuring glucose and insulin concentrations were drawn over 4 h and gastric emptying rate (GER) was measured. Changes in the plasma metabolome were investigated by applying a comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry metabolomics platform (GC×GC-TOF-MS).</p> <p>Results</p> <p>Plasma insulin response to RB was lower than to WB at 30 min (P = 0.004), 45 min (P = 0.002) and 60 min (P < 0.001) after bread intake, and plasma glucose response was significantly higher at time point 90 min after RB than WB intake (P = 0.045). The starch hydrolysis rate was higher for RB than WB, contrary to the <it>in vitro </it>protein digestibility. There were no differences in GER between breads. From 255 metabolites identified by the metabolomics platform, 26 showed significant postprandial relative changes after 30 minutes of bread intake (p and q values < 0.05). Among them, there were changes in essential amino acids (phenylalanine, methionine, tyrosine and glutamic acid), metabolites involved in the tricarboxylic acid cycle (alpha-ketoglutaric, pyruvic acid and citric acid) and several organic acids. Interestingly, the levels of two compounds involved in the tryptophan metabolism (picolinic acid, ribitol) significantly changed depending on the different bread intake.</p> <p>Conclusions</p> <p>A single meal of a low fibre sourdough rye bread producing low postprandial insulin response brings in several changes in plasma amino acids and their metabolites and some of these might have properties beneficial for health.</p

Glucocorticoid Effects on the Programming of AT1b Angiotensin Receptor Gene Methylation and Expression in the Rat

Adverse events in pregnancy may ‘programme’ offspring for the later development of cardiovascular disease and hypertension. Previously, using a rodent model of programmed hypertension we have demonstrated the role of the renin-angiotensin system in this process. More recently we showed that a maternal low protein diet resulted in undermethylation of the At1b angiotensin receptor promoter and the early overexpression of this gene in the adrenal of offspring. Here, we investigate the hypothesis that maternal glucocorticoid modulates this effect on fetal DNA methylation and gene expression. We investigated whether treatment of rat dams with the 11β-hydroxylase inhibitor metyrapone, could prevent the epigenetic and gene expression changes we observed. Offspring of mothers subjected to a low protein diet in pregnancy showed reduced adrenal Agtr1b methylation and increased adrenal gene expression as we observed previously. Treatment of mothers with metyrapone for the first 14 days of pregnancy reversed these changes and prevented the appearance of hypertension in the offspring at 4 weeks of age. As a control for non-specific effects of programmed hypertension we studied offspring of mothers treated with dexamethasone from day 15 of pregnancy and showed that, whilst they had raised blood pressure, they failed to show any evidence of Agtr1b methylation or increase in gene expression. We conclude that maternal glucocorticoid in early pregnancy may induce changes in methylation and expression of the Agtr1b gene as these are clearly reversed by an 11 beta-hydroxylase inhibitor. However in later pregnancy a converse effect with dexamethasone could not be demonstrated and this may reflect either an alternative mechanism of this glucocorticoid or a stage-specific influence

Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta'), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases

Sry delivery to the adrenal medulla increases blood pressure and adrenal medullary tyrosine hydroxylase of normotensive WKY rats

BACKGROUND: Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in the SHR rat and in WKY rats that had the SHR Y chromosome locus crossed into their genome (SHR/y rat). A potential candidate for this Y chromosome hypertension locus is Sry, a gene that encodes a transcription factor that is responsible for testes development and the Sry protein may affect other target genes. METHODS: The following study examined if exogenous Sry would elevate adrenal Th, adrenal catecholamines, plasma catecholamines and blood pressure. We delivered 10 μg of either the expression construct, Sry1/pcDNA 3.1, or control vector into the adrenal medulla of WKY rats by electroporation. Blood pressure was measured by the tail cuff technique and Th and catecholamines by HPLC with electrochemical detection. RESULTS: In the animals receiving Sry there were significant increases after 3 weeks in resting plasma NE (57%) and adrenal Th content (49%) compared to vector controls. BP was 30 mmHg higher in Sry injected animals (160 mmHg, p < .05) compared to vector controls (130 mmHg) after 2–3 weeks. Histological analysis showed that the electroporation procedure did not produce morphological damage. CONCLUSION: These results provide continued support that Sry is a candidate gene for hypertension. Also, these results are consistent with a role for Sry in increasing BP by directly or indirectly activating sympathetic nervous system activity

Seasonal Climate Effects Anemotaxis in Newly Emerged Adult Anopheles gambiae Giles in Mali, West Africa

The direction and magnitude of movement by the malaria vector Anopheles gambiae Giles has been of great interest to medical entomologists for over 70 years. This direction of movement is likely to be affected by many factors, from environmental conditions and stage of life history of the mosquito to the existence of attractants in the vicinity. We report here the direction of movement of newly emerged An. gambiae in nature, around the village of Donéguébougou, Mali. We assessed the direction of movement for individual mosquitoes by placing them in a novel enclosure with exit traps oriented in the direction of the cardinal and intermediate points of the compass. We consistently found predominantly Southward directions of movement during 2009 and 2010, with an additional Eastward component during the dry season and a Westward one during the wet season. Our data indicate that wind has an important effect on the direction of movement, but that this effect varied by season: Average directions of movement were downwind during the dry season and upwind during the wet season. A switch in anemotactic response suggests that the direction of movement of An. gambiae relative to the wind immediately after emergence under varying conditions of humidity should be further investigated under controlled conditions

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

Coronary collaterals and risk for restenosis after percutaneous coronary interventions: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>The benefit of the coronary collateral circulation (natural bypass network) on survival is well established. However, data derived from smaller studies indicates that coronary collaterals may increase the risk for restenosis after percutaneous coronary interventions. The purpose of this systematic review and meta-analysis of observational studies was to explore the impact of the collateral circulation on the risk for restenosis.</p> <p>Methods</p> <p>We searched the MEDLINE, EMBASE and ISI Web of Science databases (2001 to 15 July 2011). Random effects models were used to calculate summary risk ratios (RR) for restenosis. The primary endpoint was angiographic restenosis > 50%.</p> <p>Results</p> <p>A total of 7 studies enrolling 1,425 subjects were integrated in this analysis. On average across studies, the presence of a good collateralization was predictive for restenosis (risk ratio (RR) 1.40 (95% CI 1.09 to 1.80); <it>P </it>= 0.009). This risk ratio was consistent in the subgroup analyses where collateralization was assessed with intracoronary pressure measurements (RR 1.37 (95% CI 1.03 to 1.83); <it>P </it>= 0.038) versus visual assessment (RR 1.41 (95% CI 1.00 to 1.99); <it>P </it>= 0.049). For the subgroup of patients with stable coronary artery disease (CAD), the RR for restenosis with 'good collaterals' was 1.64 (95% CI 1.14 to 2.35) compared to 'poor collaterals' (<it>P </it>= 0.008). For patients with acute myocardial infarction, however, the RR for restenosis with 'good collateralization' was only 1.23 (95% CI 0.89 to 1.69); <it>P </it>= 0.212.</p> <p>Conclusions</p> <p>The risk of restenosis after percutaneous coronary intervention (PCI) is increased in patients with good coronary collateralization. Assessment of the coronary collateral circulation before PCI may be useful for risk stratification and for the choice of antiproliferative measures (drug-eluting stent instead bare-metal stent, cilostazol).</p