The influence of genetic variation on complex diseases is potentially mediated through a range of highly dynamic epigenetic processes exhibiting temporal variation during development and later life. ...Here we present a catalogue of the genetic influences on DNA methylation (methylation quantitative trait loci (mQTL)) at five different life stages in human blood: children at birth, childhood, adolescence and their mothers during pregnancy and middle age.
We show that genetic effects on methylation are highly stable across the life course and that developmental change in the genetic contribution to variation in methylation occurs primarily through increases in environmental or stochastic effects. Though we map a large proportion of the cis-acting genetic variation, a much larger component of genetic effects influencing methylation are acting in trans. However, only 7 % of discovered mQTL are trans-effects, suggesting that the trans component is highly polygenic. Finally, we estimate the contribution of mQTL to variation in complex traits and infer that methylation may have a causal role consistent with an infinitesimal model in which many methylation sites each have a small influence, amounting to a large overall contribution.
DNA methylation contains a significant heritable component that remains consistent across the lifespan. Our results suggest that the genetic component of methylation may have a causal role in complex traits. The database of mQTL presented here provide a rich resource for those interested in investigating the role of methylation in disease.
Abstract Aims Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. ...These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC. Methods and results Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1 , CTGF , c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ. Conclusion Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ–TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention.
DNA methylation is strongly associated with smoking status at multiple sites across the genome. Studies have largely been restricted to European origin individuals yet the greatest increase in ...smoking is occurring in low income countries, such as the Indian subcontinent. We determined whether there are differences between South Asians and Europeans in smoking related loci, and if a smoking score, combining all smoking related DNA methylation scores, could differentiate smokers from non-smokers.
Illumina HM450k BeadChip arrays were performed on 192 samples from the Southall And Brent REvisited (SABRE) cohort. Differential methylation in smokers was identified in 29 individual CpG sites at 18 unique loci. Interaction between smoking status and ethnic group was identified at the AHRR locus. Ethnic differences in DNA methylation were identified in non-smokers at two further loci, 6p21.33 and GNG12. With the exception of GFI1 and MYO1G these differences were largely unaffected by adjustment for cell composition. A smoking score based on methylation profile was constructed. Current smokers were identified with 100% sensitivity and 97% specificity in Europeans and with 80% sensitivity and 95% specificity in South Asians.
Differences in ethnic groups were identified in both single CpG sites and combined smoking score. The smoking score is a valuable tool for identification of true current smoking behaviour. Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.
Abstract Aims Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are ...dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. Methods and results Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. Conclusion cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.
A transient 3-d thermo-fluid model is developed to simulate the heat transfer and weld pool dynamics during the deep penetration laser welding of aluminium alloy AA2024. Numerical modelling and ...non-dimensional analyses are carried out to investigate the effect of driving forces on melt pool dynamics and geometry. The temperature-driven surface tension and its influence on weld pool geometry is analysed quantitatively by varying the input parameters. The normalised dimensions of the weld pool are plotted against dimensionless numbers viz. Peclet, Reynolds, Marangoni numbers to understand the uncertainty and changes in the process. The melt flow in weld pool is found to be laminar for the considered combination of material and process parameters. The conclusions derived in this work could explain the Marangoni convection in materials with a negative gradient of surface tension and provide an insight into the defects formation and their reduction in laser welding of aluminium alloys. The developed model is validated with experimental data and found in good agreement.
•3-D finite element analyses of laser welding of aluminium alloy AA2024.•A solid-liquid model for the molten pool's temperature distribution and fluid flow.•Simulating the weld pool dynamics of deep penetration laser welding.•Assessment of Marangoni convection and buoyancy forces during laser welding.•Investigating the effect of laser welding parameters on weld pool dimensions.
Purpose Aluminium alloys can be used as lightweight and high-strength materials in combination with the technology of laser beam welding, an efficient joining method, in the manufacturing of ...automotive parts. The purposes of this paper are to conduct laser welding experiments with Al2024 in the lap joint configuration, model the laser welding process parameters of Al2024 alloys and use propounded models to optimize the process parameters. Design/methodology/approach Laser welding of Al2024 alloy has been conducted in the lap joint configuration. Then, the influences of explanatory variables (laser peak power, scanning speed and frequency) on outcome variables (weld width WW, throat length TL and breaking load BL) have been investigated with Poisson regression analysis of the data set derived from experimentation. Thereafter, a multi-objective genetic algorithm (MOGA) has been used using MATLAB to find the optimum solutions. The effects of various input process parameters on the responses have also been analysed using response surface plots. Findings The promulgated statistical models, derived with Poisson regression analysis, are evinced to be well-fit ones using the analysis of deviance approach. Pareto fronts have been used to demonstrate the optimization results, and the maximized load-bearing capacity is computed to be 1,263 N, whereas the compromised WW and TL are 714 µm and 760 µm, respectively. Originality/value This work of conducting laser welding of lap joint of Al2024 alloy incorporating the Taguchi method and optimizing the input process parameters with the promulgated statistical models proffers a neoteric perspective that can be useful to the manufacturing industry.
Advanced manufacturing processes have increased industrial productivity and enhanced marketing prospects. Optimization techniques coupled with simulative methods in laser welding can immensely ...contribute to the development of the industries of the future. This work reports the research results emanating from the application of simulative techniques for the prediction of welding parameters of the laser welding process of aluminium alloy 2024. Numerical simulation was conducted in a proprietary software environment and subsequently, artificial neural network (ANN)-based simulation was conducted. The simulatively obtained data were compared with experimentally obtained data and were found to be in good accord. The ANN was trained, validated, and tested with the experimental data. The validity of the ANN-based model was ascertained with linear regression analysis and the correlation coefficients were computed. A comparison was made between the numerical simulation-based model and the ANN-based model and it was observed that the artificial neural network-based model predicts the responses faster hence it could be viewed as a potent tool for quicker and more efficient calculation of the weld dimensions and strength in both laboratory and workshop environments.
To determine the antibacterial activity of newer fluoroquinolones and compare their activity between ciprofloxacin-susceptible and resistant bacterial isolates from patients with keratitis and ...endophthalmitis.
The minimum inhibitory concentration (MIC) of ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin was determined for 123 bacterial isolates, using E test. Among the 123 isolates, 68 were gram-positive (Staphylococcus spp, Streptococcus spp, Corynebacterium spp, Bacillus spp.) and 55 were gram-negative (Pseudomonas aeruginosa). The bacterial isolates were divided into three groups: susceptible/intermediate/resistant to ciprofloxacin. The MIC values for various fluoroquinolones were compared between the three groups and between gram-positive and gram-negative bacteria.
For gram-positive isolates, median MICs of fourth generation fluoroquinolones were lower than second generation. The median MIC was lowest for gatifloxacin and moxifloxacin (0.094 mg/ml) in ciprofloxacin-susceptible isolates of gram-positive bacteria. For ciprofloxacin-susceptible gram-negative bacteria, the median MIC of ciprofloxacin (0.19 mg/ml) was significantly lower than ofloxacin, levofloxacin, gatifloxacin and moxifloxacin (1.5, 0.5, 0.5 and 2 mg/ml respectively). Ciprofloxacin-resistant isolates of gram-positive bacteria showed higher MIC of levofloxacin, moxifloxacin and gatifloxacin though they remained susceptible to them. None of the fluoroquinolones were effective against ciprofloxacin-resistant gram-negative bacteria. Overall, for gram-positive bacteria, median MICs of levofloxacin, moxifloxacin and gatifloxacin were below ciprofloxacin, the MIC of gatifloxacin and moxifloxacin was equal for gram-positive bacteria.
Levofloxacin, gatifloxacin and moxifloxacin are statistically more effective against gram-positive bacteria, the latter two being equally effective. Ciprofloxacin remains the most effective fluoroquinolone against gram-negative bacteria.
Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the ...mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3. Overexpression of CRTC-2 or -3 significantly increased CREB activity and inhibited VSMC proliferation, whereas CRTC-2/3 silencing inhibited CREB activity and reversed the anti-mitogenic effects of adenosine A2B receptor agonists. By contrast, stimulation with serum or PDGF
significantly increased CREB activity, dependent on increased CREB phosphorylation at serine-133 but not on CRTC-2/3 activation. CREB silencing significantly inhibited basal and PDGF induced proliferation. These data demonstrate that cAMP activation of CREB, which is CRTC2/3 dependent and serine-133 independent, is anti-mitogenic. Growth factor activation of CREB, which is serine-133-dependent and CRTC2/3 independent, is pro-mitogenic. Hence, CREB plays a dual role in the regulation of VSMC proliferation with the mode of activation determining its pro- or anti-mitogenic function.
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