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Find video protocols related to scientific articles indexed in Pubmed.
Large-scale gene-centric analysis identifies polymorphisms for resistant hypertension.
J Am Heart Assoc
PUBLISHED: 11-12-2014
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Resistant hypertension (RHTN), defined by lack of blood pressure (BP) control despite treatment with at least 3 antihypertensive drugs, increases cardiovascular risk compared with controlled hypertension. Yet, there are few data on genetic variants associated with RHTN.
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Maternal vitamin D and E intakes during pregnancy are associated with asthma in children.
Eur. Respir. J.
PUBLISHED: 11-01-2014
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Are maternal vitamin D and E intakes during pregnancy associated with asthma in 10-year-old children? In a longitudinal study of 1924 children born to women recruited during pregnancy, maternal vitamin D intake during pregnancy was assessed by the Food Frequency Questionnaire (FFQ) and vitamin E by FFQ and plasma ?-tocopherol; respiratory questionnaires were completed for the 10-year-old children. Their treatment for asthma was also ascertained using administrative data. Longitudinal analyses included data collected at 1, 2, 5 and 10 years. Symptom data were available for 934 (49%) children and use of asthma medication for 1748 (91%). In the children maternal vitamin D intake during pregnancy was negatively associated with doctor-diagnosed asthma at 10 years of age (OR per intake quintile 0.86, 95% CI 0.74-0.99) and over the first 10 years (hazard ratio 0.90, 95% CI 0.81-1.00). Maternal plasma ?-tocopherol at 11 weeks gestation was negatively associated with children receiving asthma treatment (OR per standard deviation increase 0.52, 95% CI 0.31-0.87). Maternal vitamin E intake was negatively associated with doctor-diagnosed asthma (OR 0.89, 95% CI 0.81-0.99) in the first 10 years. Low maternal vitamin D and E intakes during pregnancy are associated with increased risk of children developing asthma in the first 10 years of life. These associations may have significant public health implications.
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Polygenic Overlap Between Kidney Function and Large Artery Atherosclerotic Stroke.
Stroke
PUBLISHED: 10-30-2014
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Epidemiological studies show strong associations between kidney dysfunction and risk of ischemic stroke (IS), the mechanisms of which are incompletely understood. We investigated whether these associations may reflect shared heritability because of a common polygenic basis and whether this differed for IS subtypes.
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Helping Themselves: Optimal Virus-Specific CD4 T Cell Responses Require Help via CD4 T Cell Licensing of Dendritic Cells.
J. Immunol.
PUBLISHED: 10-22-2014
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Although CD4(+) T cell help (Th) is critical for inducing optimal B cell and CD8(+) T cell responses, it remains unclear whether induction of CD4(+) Th responses postinfection are also dependent on CD4(+) T cell help. In this study, we show that activation of adoptively transferred Th cells during primary influenza A virus (IAV) infection enhances both the magnitude and functional breadth of endogenous primary IAV-specific CD4(+) T cell responses. This enhancement was dependent on CD154-CD40-dependent dendritic cell licensing and resulted in a greater recall capacity of IAV-specific CD4(+) and CD8(+) T memory responses after heterologous IAV infection. These data suggest that engaging pre-existing CD4 responses at the time of priming may be a strategy for improving cellular immunity after vaccination.
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Fine particulate matter concentrations in smoking households: just how much secondhand smoke do you breathe in if you live with a smoker who smokes indoors?
Tob Control
PUBLISHED: 10-22-2014
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Using data on fine particulate matter less than 2.5??m (PM2.5) concentrations in smoking and non-smoking homes in Scotland to estimate the mass of PM2.5 inhaled by different age groups.
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Interactions between beta-2 adrenoceptor gene variation, cardiovascular control and dietary sodium in healthy young adults.
J. Physiol. (Lond.)
PUBLISHED: 09-25-2014
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Dietary sodium affects function of the beta-2 adrenoceptor (ADRB2). We tested the hypothesis that haplotype variation in the ADRB2 gene would influence the cardiovascular and regional vasodilator responses to sympathoexcitatory manoeuvres following low, normal and high sodium diets, and ADRB2-mediated forearm vasodilation in the high sodium condition. Seventy-one healthy young adults were grouped by double homozygous haplotypes: Arg16+Gln27 (n = 31), the rare Gly16+Gln27 (n = 10) and Gly16+Glu27 (n = 30). Using a randomized cross-over design, subjects were studied following 5 days of controlled low, normal and high sodium with 1 month or longer between diets (and low hormone phase of the menstrual cycle). All three visits utilized ECG and finger plethysmography for haemodynamic measures, and the high sodium visit included a brachial arterial catheter for forearm vasodilator responses to isoprenaline with plethysmography. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. We found a main effect of haplotype on ADRB2 density (P = 0.03) with the Gly16+Glu27 haplotype having the greatest density (low, normal, high sodium: 12.9 ± 0.9, 13.5 ± 0.9 and 13.6 ± 0.8 fmol mg(-1) protein, respectively) and Arg16+Gln27 having the least (9.3 ± 0.6, 10.1 ± 0.5 and 10.3 ± 0.6  fmol mg(-1) protein, respectively), but there were no sodium or haplotype effects on receptor binding conformation. In the mental stress trial, there was a main effect of haplotype on cardiac output (P = 0.04), as Arg16+Gln27 had the lowest responses. Handgrip and forearm vasodilation yielded no haplotype differences, and no correlations were present for ADRB2 density and haemodynamics. Our findings support cell-based evidence that ADRB2 haplotype influences ADRB2 protein expression independent of dietary sodium, yet the haemodynamic consequences appear modest in healthy humans.
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Reversible Positioning of Single Molecules inside Zero-Mode Waveguides.
Nano Lett.
PUBLISHED: 09-15-2014
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We have developed a hybrid nanopore/zero-mode waveguide device for single-molecule fluorescence and DNA sequencing applications. The device is a freestanding solid-state membrane with sub-5 nm nanopores that reversibly delivers individual biomolecules to the base of 70 nm diameter waveguides for interrogation. Rapid and reversible molecular loading is achieved by controlling the voltage across the device. Using this device we demonstrate protein and DNA loading with efficiency that is orders of magnitude higher than diffusion-based molecular loading.
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Predictors for Glucose Change in Hypertensive Participants Following Short-term Treatment with Atenolol or Hydrochlorothiazide.
Pharmacotherapy
PUBLISHED: 09-09-2014
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To develop and validate a predictive model for glucose change and risk for new-onset impaired fasting glucose in hypertensive participants following treatment with atenolol or hydrochlorothiazide (HCTZ).
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Intermittent montelukast in children aged 10 months to 5 years with wheeze (WAIT trial): a multicentre, randomised, placebo-controlled trial.
Lancet Respir Med
PUBLISHED: 09-08-2014
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The effectiveness of intermittent montelukast for wheeze in young children is unclear. We aimed to assess whether intermittent montelukast is better than placebo for treatment of wheeze in this age group. Because copy numbers of the Sp1-binding motif in the arachidonate 5-lipoxygenase (ALOX5) gene promoter (either 5/5, 5/x, or x/x, where x does not equal 5) modifies response to montelukast in adults, we stratified by this genotype.
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The relationship between diastolic blood pressure and coronary artery calcification is dependent on single nucleotide polymorphisms on chromosome 9p21.3.
BMC Med. Genet.
PUBLISHED: 09-04-2014
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Single nucleotide polymorphisms (SNPs) within the 9p21.3 genomic region have been consistently associated with coronary heart disease (CHD), myocardial infarction, and quantity of coronary artery calcification (CAC), a marker of subclinical atherosclerosis. Prior studies have established an association between blood pressure measures and CAC. To examine mechanisms by which the 9p21.3 genomic region may influence CHD risk, we investigated whether SNPs in 9p21.3 modified associations between blood pressure and CAC quantity.
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T cell mediated immunity to influenza: mechanisms of viral control.
Trends Immunol.
PUBLISHED: 06-20-2014
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Infection with influenza A virus (IAV) is a major cause of worldwide morbidity and mortality. Recent findings indicate that T cell immunity is key to limiting severity of disease arising from IAV infection, particularly in instances where antibody immunity is ineffective. As such, there is a need to understand better the mechanisms that mediate effective IAV-specific cellular immunity, especially given that T cell immunity must form an integral part of any vaccine designed to elicit crossreactive immunity against existing and new strains of influenza virus. Here, we review the current understanding of cellular immunity to IAV, highlighting recent findings that demonstrate important roles for both CD4+ and CD8+ T cell immunity in protection from IAV-mediated disease.
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Fine-mapping IGF1 and prostate cancer risk in African Americans: the multiethnic cohort study.
Cancer Epidemiol. Biomarkers Prev.
PUBLISHED: 06-05-2014
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Genetic variation at insulin-like growth factor 1 (IGF1) has been linked to prostate cancer risk. However, the specific predisposing variants have not been identified. In this study, we fine-mapped the IGF1 locus for prostate cancer risk in African Americans. We conducted targeted Roche GS-Junior 454 resequencing of a 156-kb region of IGF1 in 80 African American aggressive prostate cancer cases. Three hundred and thirty-four IGF1 SNPs were examined for their association with prostate cancer risk in 1,000 African American prostate cancer cases and 991 controls. The top associated SNP in African Americans, rs148371593, was examined in an additional 3,465 prostate cancer cases and 3,425 controls of non-African American ancestry-European Americans, Japanese Americans, Latinos, and Native Hawaiians. The overall association of 334 IGF1 SNPs and prostate cancer risk was assessed using logistic kernel-machine methods. The association between each SNP and prostate cancer risk was evaluated through unconditional logistic regression. A false discovery rate threshold of q < 0.1 was used to determine statistical significance of associations. We identified 8 novel IGF1 SNPs. The cumulative effect of the 334 IGF1 SNPs was not associated with prostate cancer risk (P = 0.13) in African Americans. Twenty SNPs were nominally associated with prostate cancer at P < 0.05. The top associated SNP among African Americans, rs148371593 [minor allele frequency (MAF) = 0.03; P = 0.0014; q > 0.1], did not reach our criterion of statistical significance. This polymorphism was rare in non-African Americans (MAF < 0.003) and was not associated with prostate cancer risk (P = 0.98). Our findings do not support the role of IGF1 variants and prostate cancer risk among African Americans.
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Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.
Nat Commun
PUBLISHED: 04-25-2014
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Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
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The influenza virus-specific CTL immunodominance hierarchy in mice is determined by the relative frequency of high-avidity T cells.
J. Immunol.
PUBLISHED: 04-02-2014
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Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.
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Is diabetes mellitus-linked amino acid signature associated with ?-blocker-induced impaired fasting glucose?
Circ Cardiovasc Genet
PUBLISHED: 03-13-2014
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The 5-amino acid (AA) signature, including isoleucine, leucine, valine, tyrosine, and phenylalanine, has been associated with incident diabetes mellitus and insulin resistance. We investigated whether this same AA signature, single-nucleotide polymorphisms in genes in their catabolic pathway, was associated with development of impaired fasting glucose (IFG) after atenolol treatment.
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Epigenetic plasticity of Cd8a locus during CD8(+) T-cell development and effector differentiation and reprogramming.
Nat Commun
PUBLISHED: 03-04-2014
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Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8(low) effector cells respond to interferon-? by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.
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Heritability of urinary traits that contribute to nephrolithiasis.
Clin J Am Soc Nephrol
PUBLISHED: 02-27-2014
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Kidney stones and their risk factors aggregate in families, yet few studies have systematically estimated heritabilities and genetic correlations of the numerous urinary traits associated with risk of kidney stones.
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Arterial stiffness is associated with increase in blood pressure over time in treated hypertensives.
J Am Soc Hypertens
PUBLISHED: 02-11-2014
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Arterial stiffness is associated with incident hypertension. We hypothesized that measures of arterial stiffness would predict increases in systolic (SBP), mean (MAP), and pulse pressure (PP) over time in treated hypertensives. Blood pressure (BP) was measured a mean of 8.5 ± 0.9 years apart in 414 non-Hispanic white hypertensives (mean age, 60 ± 8 years; 55% women). The average of three supine right brachial BPs was recorded. Measures of arterial stiffness, including carotid-femoral pulse wave velocity (cfPWV), aortic augmentation index (AIx), and central pulse pressure (CPP), were obtained at baseline by applanation tonometry. We performed stepwise multivariable linear regression analyses adjusting for potential confounders to assess the associations of arterial stiffness parameters with BP changes over time. SBP, MAP, and PP increased in 80% of participants. After adjustment for covariates listed, cfPWV (m/s) was associated with increases in SBP (? ± standard error [SE], 0.71 ± 0.31) and PP (? ± SE, 1.09 ± 0.27); AIx (%) was associated with increases in SBP (? ± SE, 0.23 ± 0.10) and MAP (? ± SE, 0.27 ± 0.07); and CPP (mmHg) was associated with increases in SBP (? ± SE, 0.44 ± 0.07), MAP (? ± SE, 0.24 ± 0.05), and PP (? ± SE, 0.42 ± 0.06) over time (P ? .02 for each). In conclusion, arterial stiffness measures were associated with longitudinal increases in SBP, MAP, and PP in treated hypertensives.
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CD154+ CD4+ T-cell dependence for effective memory influenza virus-specific CD8+ T-cell responses.
Immunol. Cell Biol.
PUBLISHED: 02-03-2014
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CD40-CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8(+) cytotoxic T lymphocyte (CTL) responses. This is typically via CD4(+)CD154(+) T-cell-dependent 'licensing' of CD40(+) dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4(+) T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40-CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154(+), but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection.
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Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.
Nat Commun
PUBLISHED: 01-16-2014
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The cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These renin-expressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.
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Glucagon regulates hepatic kisspeptin to impair insulin secretion.
Cell Metab.
PUBLISHED: 01-15-2014
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Early in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic ? cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from ? cells. However, whether hyperglucagonemia is causally linked to ? cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on ? cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Lepr(db/db) mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.
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Reproducible selection of high avidity CD8+ T-cell clones following secondary acute virus infection.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-13-2014
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The recall of memory CD8(+) cytotoxic T lymphocytes (CTLs), elicited by prior virus infection or vaccination, is critical for immune protection. The extent to which this arises as a consequence of stochastic clonal expansion vs. active selection of particular clones remains unclear. Using a parallel adoptive transfer protocol in combination with single cell analysis to define the complementarity determining region (CDR) 3? and CDR3? regions of individual T-cell receptor (TCR) heterodimers, we characterized the antigen-driven recall of the same memory CTL population in three individual recipients. This high-resolution analysis showed reproducible enrichment (or diminution) of particular TCR clonotypes across all challenged animals. These changes in clonal composition were TCR?- and ? chain-dependent and were directly related to the avidity of the TCR for the virus-derived peptide (p) + major histocompatibility complex class I molecule. Despite this shift in clonotype representation indicative of differential selection, there was no evidence of overall repertoire narrowing, suggesting a strategy to optimize CTL responses while safeguarding TCR diversity.
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Reconstructing complex regions of genomes using long-read sequencing technology.
Genome Res.
PUBLISHED: 01-13-2014
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Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.
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The use of a TLR2 agonist-based adjuvant for enhancing effector and memory CD8 T-cell responses.
Immunol. Cell Biol.
PUBLISHED: 01-07-2014
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We have previously shown that the immunogenicity of protein antigens can be significantly enhanced if electrostatically associated with the Toll-like receptor-2 agonist-based lipopeptide R4Pam2Cys. The precise mechanisms and effectiveness of the cytotoxic T-lymphocyte (CTL)-mediated response facilitated by this agonist, however, have not been studied. Here we show that priming by dendritic cells (DCs) in the draining lymph nodes of animals vaccinated with antigen delivered using R4Pam2Cys results in significantly improved T-cell proliferation and induces their differentiation into polyfunctional effector CTLs characterised by granzyme B expression and the ability to secrete interferon-?, interleukin-2 and tumor necrosis factor-? 7 days after vaccination. After 30 days, frequencies of antigen-specific CD62(low)CD127(high) (effector memory), CD62(high)CD127(high) (central memory) and CD43(low)CD27(high) CD8(+) T cells, a phenotype associated with strong recall responses against respiratory infections, are also increased compared with responses obtained with antigens formulated in the adjuvants Alum (alhydrogel) and CFA (complete Freund's adjuvant). The phenotypic changes observed in these mice vaccinated using R4Pam2Cys further correlated with their ability to recall specific T cells into the lung to mediate the reduction of pulmonary viral titres following challenge with a chimeric influenza virus containing the K(b)OVA257-264 epitope compared with animals vaccinated using Alum or CFA. The findings from this study not only demonstrate that better T-cell responses can be elicited using R4Pam2Cys compared with classically utilised adjuvants but also highlight the potential effectiveness of this lipopeptide-based adjuvant particularly against viral infections that require resolution through cell-mediated immunity.
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Linkage analysis incorporating gene-age interactions identifies seven novel lipid loci: the Family Blood Pressure Program.
Atherosclerosis
PUBLISHED: 01-06-2014
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To detect novel loci with age-dependent effects on fasting (? 8 h) levels of total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides using 3600 African Americans, 1283 Asians, 3218 European Americans, and 2026 Mexican Americans from the Family Blood Pressure Program (FBPP).
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Electronic medical records and genomics (eMERGE) network exploration in cataract: Several new potential susceptibility loci.
Mol. Vis.
PUBLISHED: 01-01-2014
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Cataract is the leading cause of blindness in the world, and in the United States accounts for approximately 60% of Medicare costs related to vision. The purpose of this study was to identify genetic markers for age-related cataract through a genome-wide association study (GWAS).
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Validation of high throughput sequencing and microbial forensics applications.
Investig Genet
PUBLISHED: 01-01-2014
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High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.
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Characterization of the nasopharyngeal microbiota in health and during rhinovirus challenge.
Microbiome
PUBLISHED: 01-01-2014
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The bacterial communities of the nasopharynx play an important role in upper respiratory tract infections (URTIs). Our study represents the first survey of the nasopharynx during a known, controlled viral challenge. We aimed to gain a better understanding of the composition and dynamics of the nasopharyngeal microbiome during viral infection.
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High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-30-2013
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Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.
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Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-11-2013
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The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.
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PROX1 Gene Variant is Associated with Fasting Glucose Change After Antihypertensive Treatment.
Pharmacotherapy
PUBLISHED: 10-09-2013
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To assess the relationship of the 33 single nucleotide polymorphisms (SNPs) previously associated with fasting glucose in Caucasians in genome-wide association studies (GWAS) with glucose response to antihypertensive drugs shown to increase risk for hyperglycemia and diabetes.
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Association of Low-Grade Albuminuria with Adverse Cardiac Mechanics: Findings from the HyperGEN Study.
Circulation
PUBLISHED: 09-27-2013
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Albuminuria is a marker of endothelial dysfunction and has been associated with adverse cardiovascular outcomes. The reasons for this association are unclear, but may be due to the relationship between endothelial dysfunction and intrinsic myocardial dysfunction.
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Multiparametric analysis of host response to murine cytomegalovirus in MHC class I-disparate mice reveals primacy of Dk-licensed Ly49G2+ NK cells in viral control.
J. Immunol.
PUBLISHED: 09-25-2013
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MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These data suggested that D(k)-licensed G2(+) NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating D(k) were examined before infection and postinfection, including Ly49(+) NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2(+) NK cell expansion following viral exposure occurred selectively in D(k) offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that D(k)-licensed G2(+) NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.
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Using a new, low-cost air quality sensor to quantify second-hand smoke (SHS) levels in homes.
Tob Control
PUBLISHED: 09-19-2013
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To determine if a low-cost particle counter, the Dylos DC 1700, can be used in homes to quantify second-hand smoke (SHS) concentrations.
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Hypertension in pregnancy is associated with elevated C-reactive protein levels later in life.
J. Hypertens.
PUBLISHED: 09-14-2013
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We assessed whether hypertension in pregnancy is associated with elevated C-reactive protein (CRP) levels in later life, possibly reflecting an increased risk of cardiovascular disease (CVD).
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Common variants in mendelian kidney disease genes and their association with renal function.
Afshin Parsa, Christian Fuchsberger, Anna Köttgen, Conall M O'Seaghdha, Cristian Pattaro, Mariza de Andrade, Daniel I Chasman, Alexander Teumer, Karlhans Endlich, Matthias Olden, Ming-Huei Chen, Adrienne Tin, Young J Kim, Daniel Taliun, Man Li, Mary Feitosa, Mathias Gorski, Qiong Yang, Claudia Hundertmark, Meredith C Foster, Nicole Glazer, Aaron Isaacs, Madhumathi Rao, Albert V Smith, Jeffrey R O'Connell, Maksim Struchalin, Toshiko Tanaka, Guo Li, Shih-Jen Hwang, Elizabeth J Atkinson, Kurt Lohman, Marilyn C Cornelis, Asa Johansson, Anke Tönjes, Abbas Dehghan, Vincent Couraki, Elizabeth G Holliday, Rossella Sorice, Zoltan Kutalik, Terho Lehtimäki, Tonu Esko, Harshal Deshmukh, Sheila Ulivi, Audrey Y Chu, Federico Murgia, Stella Trompet, Medea Imboden, Barbara Kollerits, Giorgio Pistis, Tamara B Harris, Lenore J Launer, Thor Aspelund, Gudny Eiriksdottir, Braxton D Mitchell, Eric Boerwinkle, Helena Schmidt, Edith Hofer, Frank Hu, Ayse Demirkan, Ben A Oostra, Stephen T Turner, Jingzhong Ding, Jeanette S Andrews, Barry I Freedman, Franco Giulianini, Wolfgang Koenig, Thomas Illig, Angela Döring, H-Erich Wichmann, Lina Zgaga, Tatijana Zemunik, Mladen Boban, Cosetta Minelli, Heather E Wheeler, Wilmar Igl, Ghazal Zaboli, Sarah H Wild, Alan F Wright, Harry Campbell, David Ellinghaus, Ute Nöthlings, Gunnar Jacobs, Reiner Biffar, Florian Ernst, Georg Homuth, Heyo K Kroemer, Matthias Nauck, Sylvia Stracke, Uwe Völker, Henry Völzke, Peter Kovacs, Michael Stumvoll, Reedik Mägi, Albert Hofman, André G Uitterlinden, Fernando Rivadeneira, Yurii S Aulchenko, Ozren Polašek, Nick Hastie, Veronique Vitart, Catherine Helmer, Jie Jin Wang, Bénédicte Stengel, Daniela Ruggiero, Sven Bergmann, Mika Kähönen, Jorma Viikari, Tiit Nikopensius, Michael Province, Helen Colhoun, Alex Doney, Antonietta Robino, Bernhard K Krämer, Laura Portas, Ian Ford, Brendan M Buckley, Martin Adam, Gian-Andri Thun, Bernhard Paulweber, Margot Haun, Cinzia Sala, Paul Mitchell, Marina Ciullo, Peter Vollenweider, Olli Raitakari, Andres Metspalu, Colin Palmer, Paolo Gasparini, Mario Pirastu, J Wouter Jukema, Nicole M Probst-Hensch, Florian Kronenberg, Daniela Toniolo, Vilmundur Gudnason, Alan R Shuldiner, Josef Coresh, Reinhold Schmidt, Luigi Ferrucci, Cornelia M van Duijn, Ingrid Borecki, Sharon L R Kardia, Yongmei Liu, Gary C Curhan, Igor Rudan, Ulf Gyllensten, James F Wilson, Andre Franke, Peter P Pramstaller, Rainer Rettig, Inga Prokopenko, Jacqueline Witteman, Caroline Hayward, Paul M Ridker, Murielle Bochud, Iris M Heid, David S Siscovick, Caroline S Fox, W Linda Kao, Carsten A Böger.
J. Am. Soc. Nephrol.
PUBLISHED: 09-12-2013
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Many common genetic variants identified by genome-wide association studies for complex traits map to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2 meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research.
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Predisposed ?? T cell antigen receptor recognition of MHC and MHC-I like molecules?
Curr. Opin. Immunol.
PUBLISHED: 07-25-2013
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The diverse ?? T cell receptor (TCR) repertoire exhibits versatility in its ability to generate antigen (Ag) receptors capable of interacting with polymorphic Major Histocompatibility Complex (MHC) molecules and monomorphic MHC-I like molecules, including the CD1 and MR1 families. Collectively, these evolutionarily related Ag-presenting molecules present peptides, lipids and vitamin B metabolites for T cell surveillance. Interestingly, whilst common TCR gene usage can underpin recognition of these distinct classes of Ags, it is unclear whether the rules that govern ??TCR-Ag MHC interactions are shared. We highlight recent observations in the context of TCR biases towards MHC and MHC-I like molecules.
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Night Blood Pressure Responses to Atenolol and Hydrochlorothiazide in Black and White Patients With Essential Hypertension.
Am. J. Hypertens.
PUBLISHED: 07-25-2013
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Night blood pressure (BP) predicts patient outcomes. Variables associated with night BP response to antihypertensive agents have not been fully evaluated in essential hypertension.
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Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects.
J. Allergy Clin. Immunol.
PUBLISHED: 06-24-2013
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Atopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype.
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Genome-wide methylation profiling demonstrates hypermethylation in maternal leukocyte DNA in preeclamptic compared to normotensive pregnancies.
Hypertens Pregnancy
PUBLISHED: 06-19-2013
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To compare genome-wide methylation profiles in maternal leukocyte DNA between normotensive and preeclamptic pregnant women at delivery.
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Genomic association analysis of common variants influencing antihypertensive response to hydrochlorothiazide.
Hypertension
PUBLISHED: 06-10-2013
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To identify novel genes influencing blood pressure response to thiazide diuretic therapy for hypertension, we conducted genome-wide association meta-analyses of ?1.1 million single-nucleotide polymorphisms in a combined sample of 424 European Americans with primary hypertension treated with hydrochlorothiazide from the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=228) and the Genetic Epidemiology of Responses to Antihypertensive study (n=196). Polymorphisms associated with blood pressure response at P<10(-5) were tested for replication of the associations in independent samples of hydrochlorothiazide-treated European hypertensives. The rs16960228 polymorphism in protein kinase C, ? replicated for same-direction association with diastolic blood pressure response in the Nordic Diltiazem study (n=420) and the Genetics of Drug Responsiveness in Essential Hypertension study (n=206), and the combined 4-study meta-analysis P value achieved genome-wide significance (P=3.3 × 10(-8)). Systolic or diastolic blood pressure responses were consistently greater in carriers of the rs16960228 A allele than in GG homozygotes (>4/4 mm Hg) across study samples. The rs2273359 polymorphism in the GNAS-EDN3 region also replicated for same-direction association with systolic blood pressure response in the Nordic Diltiazem study, and the combined 3-study meta-analysis P value approached genome-wide significance (P=5.5 × 10(-8)). The findings document clinically important effects of genetic variation at novel loci on blood pressure response to a thiazide diuretic, which may be a basis for individualization of antihypertensive drug therapy and identification of new drug targets.
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Acute emergence and reversion of influenza A virus quasispecies within CD8+ T cell antigenic peptides.
Nat Commun
PUBLISHED: 05-22-2013
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Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) provide a degree of cross-strain protection that is potentially subverted by mutation. Here we describe the sequential emergence of such variants within CTL epitopes for a persistently infected, immunocompromised infant. Further analysis in immunodeficient and wild-type mice supports the view that CTL escape variants arise frequently in influenza, accumulate with time and revert in the absence of immune pressure under MHCI-mismatched conditions. Viral fitness, the abundance of endogenous CD8(+) T cell responses and T cell receptor repertoire diversity influence the nature of these de novo mutants. Structural characterization of dominant escape variants shows how the peptide-MHCI interaction is modified to affect variant-MHCI stability. The mechanism of influenza virus escape thus looks comparable to that recognized for chronic RNA viruses like HIV and HCV, suggesting that immunocompromised patients with prolonged viral infection could have an important part in the emergence of influenza quasispecies.
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Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.
J. Immunol.
PUBLISHED: 05-22-2013
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Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-? CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.
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The role of plasma renin activity, age, and race in selecting effective initial drug therapy for hypertension.
Am. J. Hypertens.
PUBLISHED: 04-16-2013
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Strategies for initial drug therapy of hypertension are a thiazide diuretic for all or drug selection based on age/race criteria or on plasma renin activity (PRA). It is uncertain which of these strategies will achieve the highest control rate among patients with stage 1 essential hypertension. We sought to compare control rates among 3 drug selection strategies: (i) thiazide diuretic for all, (ii) thiazide diuretic for all black subjects and white subjects aged ?50 years and a renin-angiotensin system blocker for white subjects aged <50 years, or (iii) thiazide diuretic for PRA < 0.6ng/ml/h (suppressed PRA) and a renin-angiotensin system blocker for PRA ? 0.6ng/ml/h (nonsuppressed PRA).
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Creating smoke-free homes for children.
Nurs Times
PUBLISHED: 04-09-2013
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This article describes a tool that was designed to help nurses deliver health education on the subject of passive smoking. The tool is based on a Scottish study, which encouraged mothers to identify their own strategies for reducing smoking in the home in order to protect their children from being exposed to second-hand smoke. It is designed to give nurses and other health professionals who work with families and children the knowledge, skills and confidence they need to raise the issue of second-hand smoke with parents and help them to find their own solutions to reducing smoking in the home. This article also takes into consideration the implications for nursing practice that relate to raising the issue of exposing children to second-hand smoke with parents and carers.
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Podocyturia predates proteinuria and clinical features of preeclampsia: longitudinal prospective study.
Hypertension
PUBLISHED: 03-25-2013
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Podocyturia, the shedding of live podocytes, is present at delivery in women with preeclampsia. The aim of this study was to test whether podocyturia is present earlier in pregnancy and predicts for preeclampsia. We also aimed to compare test characteristics of podocyturia with those of angiogenic factors previously implicated in the pathogenesis of this disorder. We prospectively enrolled 315 women who provided blood and urine samples at the end of the second trimesters of their pregnancies (median, 27 gestational weeks) and within 24 hours of their deliveries (median, 39.5 gestational weeks). Blood samples were analyzed for angiogenic markers, including placental growth factor, the soluble receptor fms-like tyrosine kinase receptor-1 for vascular endothelial growth factor, and endoglin. The urine sediments were analyzed for podocytes, identified by staining for podocin after culturing the urinary sediments for 24 hours. This analysis included all women who developed preeclampsia (n=15), gestational hypertension (n=15), and a subsample of women who remained normotensive throughout pregnancy (n=44), matched for maternal age and number of previous pregnancies to those who developed preeclampsia. At the second trimester collection, all women who developed preeclampsia had podocyturia, compared with none of those who remained normotensive or were diagnosed with gestational hypertension. Podocyturia in the second trimester had a significantly greater sensitivity and specificity for the subsequent diagnosis of preeclampsia than any single angiogenic marker or a combination thereof. Screening for podocyturia at the end of the second trimester may allow for accurate identification of pregnant women at risk for preeclampsia.
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Preemptive priming readily overcomes structure-based mechanisms of virus escape.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-14-2013
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A reverse-genetics approach has been used to probe the mechanism underlying immune escape for influenza A virus-specific CD8(+) T cells responding to the immunodominant D(b)NP366 epitope. Engineered viruses with a substitution at a critical residue (position 6, P6M) all evaded recognition by WT D(b)NP366-specific CD8(+) T cells, but only the NPM6I and NPM6T mutants altered the topography of a key residue (His155) in the MHC class I binding site. Following infection with the engineered NPM6I and NPM6T influenza viruses, both mutations were associated with a substantial "hole" in the naïve T-cell receptor repertoire, characterized by very limited T-cell receptor diversity and minimal primary responses to the NPM6I and NPM6T epitopes. Surprisingly, following respiratory challenge with a serologically distinct influenza virus carrying the same mutation, preemptive immunization against these escape variants led to the generation of secondary CD8(+) T-cell responses that were comparable in magnitude to those found for the WT NP epitope. Consequently, it might be possible to generate broadly protective T-cell immunity against commonly occurring virus escape mutants. If this is generally true for RNA viruses (like HIV, hepatitis C virus, and influenza) that show high mutation rates, priming against predicted mutants before an initial encounter could function to prevent the emergence of escape variants in infected hosts. That process could be a step toward preserving immune control of particularly persistent RNA viruses and may be worth considering for future vaccine strategies.
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Early-life residential exposure to soil components in rural areas and childhood respiratory health and allergy.
Sci. Total Environ.
PUBLISHED: 03-01-2013
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The increase in asthma and allergies has been attributed to declining exposure to environmental microorganisms. The main source of these is soil, the composition of which varies geographically and which is a major component (40-45%) of household dust. Our hypothesis-generating study aimed to investigate associations between soil components, respiratory health and allergy in a Scottish birth cohort. The cohort was recruited in utero in 1997/8, and followed up at one, two and five years for the development of wheezing, asthma and eczema. Lung function, exhaled nitric oxide and allergic sensitization were measured at age five in a subset. The Scottish Soils Database held at The James Hutton Institute was linked to the birth cohort data by the residential postcode at birth and five years. The soil database contained information on size separates, organic matter concentration, pH and a range of inorganic elements. Soil and clinical outcome data were available for 869, 790 and 727 children at one, two and five years. Three hundred and fifty nine (35%) of children had the same address at birth and five years. No associations were found between childhood outcomes and soil content in the residential area at age five. The soil silt content (2-20 ?m particle size) of the residential area at birth was associated with childhood wheeze (adjusted OR 1.20, 95% CI [1.05; 1.37]), wheeze without a cold (1.41 [1.18; 1.69]), doctor-diagnosed asthma (1.54 [1.04; 2.28]), lung function (FEV1: beta -0.025 [-0.047;-0.001]) and airway inflammation (FENO: beta 0.15 [0.03; 0.27]) at age five, but not with allergic status or eczema. Whilst residual confounding is the most likely explanation for the associations reported, the results of this study lead us to hypothesise that early life exposure to residential soil silt may adversely influence childhood respiratory health, possibly because of the organic components of silt.
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Hypertension in pregnancy is associated with elevated homocysteine levels later in life.
Am. J. Obstet. Gynecol.
PUBLISHED: 02-25-2013
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Hyperhomocysteinemia is associated with an elevated cardiovascular disease risk. We examined whether women with a history of hypertension in pregnancy are more likely to have a high level of serum homocysteine decades after pregnancy.
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Estimating the glomerular filtration rate from serum creatinine is better than from cystatin C for evaluating risk factors associated with chronic kidney disease.
Kidney Int.
PUBLISHED: 02-20-2013
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Chronic kidney disease risk factors may associate with the estimated glomerular filtration rate (eGFR) differently than with the measured GFR. To examine this, we evaluated 1150 patients (mean age 65 years) in two community cohorts for risk factors, measured GFR by iothalamate clearance, and eGFR based on creatinine (Cr), cystatin C (CysC), or both. The interaction between each risk factor and eGFR (relative to measured GFR) identified risk factor associations with eGFR along non-GFR pathways. In a subset of 40 patients with two visits, the mean coefficient of variation was 8.2% for measured GFR, 6.4% for eGFRCr, 8.2% for eGFRCr-CysC, and 10.7% for eGFRCysC. The measured GFR was better correlated with eGFRCr-CysC (r, 0.74) than eGFRCr (r, 0.70) or eGFRCysC (r, 0.68). Lower measured GFR associated with lower 24-hour urine creatinine, albuminuria, hypertension, diabetes, higher triglycerides, and higher uric acid. Lower eGFRCr had these same associations except for an association with higher 24-hour urine creatinine along a non-GFR pathway. Lower eGFRCysC and eGFRCr-CysC also had these same associations but also associated with obesity, albuminuria, hypertension, diabetes, higher triglycerides, higher C-reactive protein, and higher uric acid along non-GFR pathways. Thus, cystatin C improves estimation of GFR over creatinine alone; however, the association between most of the risk factors and GFR was more accurate by eGFR based on creatinine alone. This is explained by the association of these risk factors with the non-GFR determinants of cystatin C.
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Growing up before growing out: secular trends in height, weight and obesity in 5--6-year-old children born between 1970 and 2006.
Arch. Dis. Child.
PUBLISHED: 02-14-2013
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This was a whole-population study of height, weight and obesity prevalence in 5-6-year-old children born between 1970 and 2006 in the Grampian region, north east Scotland.
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Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.
Nat. Methods
PUBLISHED: 01-31-2013
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We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.
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Association of variants in NEDD4L with blood pressure response and adverse cardiovascular outcomes in hypertensive patients treated with thiazide diuretics.
J. Hypertens.
PUBLISHED: 01-29-2013
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Single-nucleotide polymorphisms (SNPs) in NEDD4L may influence the ability of the NEDD4L protein to reduce epithelial sodium channel expression. A variant in NEDD4L, rs4149601, was associated with antihypertensive response and cardiovascular outcomes during treatment with thiazide diuretics and ?-blockers in a Swedish population. We sought to further evaluate associations between NEDD4L polymorphisms, blood pressure response and cardiovascular outcomes with thiazide diuretics and ?-blockers.
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Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.
J Nanobiotechnology
PUBLISHED: 01-25-2013
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Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis.
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Hypertension in pregnancy is a risk factor for microalbuminuria later in life.
J Clin Hypertens (Greenwich)
PUBLISHED: 01-24-2013
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The authors aimed to compare renal function by estimated glomerular filtration rate and albuminuria in 3 groups of women: nulliparous women, women with a history of normotensive pregnancies, and women with a history of at least one hypertensive pregnancy. Women who participated in the second Family Blood Pressure Program Study visit (2000-2004) and had serum creatinine and urine albumin measurements (n=3015) were categorized as having had no pregnancy lasting >6 months (n=341), having had only normotensive pregnancies (n=2199), or having had at least 1 pregnancy with hypertension (n=475) based on a standardized questionnaire. Women who reported having had at least one pregnancy with hypertension were significantly more likely to be hypertensive (75.6% vs 59.4%, P<.001), diabetic (34.2% vs 27.3%, P?.001), and have higher body mass index (32.8 vs 30.5, P<.001) than those who reported normotensive pregnancies. There was a significantly greater risk of microalbuminuria (urine albumin-creatinine ratio >25 mg/g) in those who reported at least one pregnancy with hypertension (odds ratio, 1.37; confidence interval, 1.02-1.85; P=.04) than in those with normotensive pregnancies, after adjusting for risk factors for chronic kidney and cardiovascular disease. Hypertension in pregnancy is associated with an increased risk of future microalbuminuria.
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Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.
BMC Biol.
PUBLISHED: 01-22-2013
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DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.
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Hypertension in pregnancy is a risk factor for peripheral arterial disease decades after pregnancy.
Atherosclerosis
PUBLISHED: 01-17-2013
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An ankle-brachial index (ABI) (the ratio of ankle to brachial artery systolic blood pressure) value ?0.9 identifies patients with peripheral arterial disease (PAD) and elevated cardiovascular event risk. This study examined whether women with a history of hypertension in pregnancy are more likely to have an ABI ?0.9 decades after pregnancy.
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Impact of genetic polymorphisms of SLC2A2, SLC2A5, and KHK on metabolic phenotypes in hypertensive individuals.
PLoS ONE
PUBLISHED: 01-14-2013
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In the past few decades, consumption of added sugars has increased dramatically. Studies have linked high sugar intake with increased risk for a number of diseases. Importantly, fructose, a component of sugar, has been linked with the development of features of metabolic syndrome. This study determined if single nucleotide polymorphisms in genes involved in fructose transport (solute carrier family 2 facilitated glucose transporter, member 2 (SLC2A2) and solute carrier family 2 facilitated glucose/fructose transporter, member 5 (SLC2A5)) and metabolism (ketohexokinase (KHK)) affect inter-individual variability in metabolic phenotypes, such as increased serum uric acid levels.
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Epigenomic association analysis identifies smoking-related DNA methylation sites in African Americans.
Hum. Genet.
PUBLISHED: 01-12-2013
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Cigarette smoking is an environmental risk factor for many chronic diseases, and disease risk can often be managed by smoking control. Smoking can induce cellular and molecular changes, including epigenetic modification, but the short- and long-term epigenetic modifications caused by cigarette smoking at the gene level have not been well understood. Recent studies have identified smoking-related DNA methylation (DNAm) sites in Caucasians. To determine whether the same DNAm sites associate with smoking in African Americans, and to identify novel smoking-related DNAm sites, we conducted a methylome-wide association study of cigarette smoking using a discovery sample of 972 African Americans, and a replication sample of 239 African Americans with two array-based methods. Among 15 DNAm sites significantly associated with smoking after correction for multiple testing in our discovery sample, 5 DNAm sites are replicated in an independent cohort, and 14 sites in the replication sample have effects in the same direction as in the discovery sample. The top two smoking-related DNAm sites in F2RL3 (factor II receptor-like 3) and GPR15 (G-protein-coupled receptor 15) observed in African Americans are consistent with previous findings in Caucasians. The associations between the replicated DNAm sites and smoking remain significant after adjusting for genetic background. Despite the distinct genetic background between African Americans and Caucasians, the DNAm from the two ethnic groups shares common associations with cigarette smoking, which suggests a common molecular mechanism of epigenetic modification influenced by environmental exposure.
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Characterization of birdshot chorioretinopathy using extramacular enhanced depth optical coherence tomography.
JAMA Ophthalmol
PUBLISHED: 01-12-2013
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To combine "extramacular" and "enhanced depth" optical coherence tomographic (OCT) scanning protocols to facilitate enhanced characterization of patients with birdshot chorioretinopathy.
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Variability of inducible expression across the hematopoietic system of tetracycline transactivator transgenic mice.
PLoS ONE
PUBLISHED: 01-11-2013
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The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.
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Matrix gla protein gene polymorphism is associated with increased coronary artery calcification progression.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 01-10-2013
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Matrix gla protein (MGP) inhibits arterial and cartilaginous calcification. A threonine to alanine (Thr83Ala) polymorphism (codon 83) in MGP is associated with myocardial infarction and femoral artery calcification. We examined the association of the MGP Thr83Ala polymorphism with quantity and progression of coronary artery calcification (CAC), a noninvasive measure of subclinical coronary atherosclerosis.
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Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution.
PLoS Genet.
PUBLISHED: 01-03-2013
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In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5-CTAT-3), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5-GAN(7)TAY-3/3-CTN(7)ATR-5). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.
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T cell immunity as a tool for studying epigenetic regulation of cellular differentiation.
Front Genet
PUBLISHED: 01-01-2013
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Cellular differentiation is regulated by the strict spatial and temporal control of gene expression. This is achieved, in part, by regulating changes in histone post-translational modifications (PTMs) and DNA methylation that in turn, impact transcriptional activity. Further, histone PTMs and DNA methylation are often propagated faithfully at cell division (termed epigenetic propagation), and thus contribute to maintaining cellular identity in the absence of signals driving differentiation. Cardinal features of adaptive T cell immunity include the ability to differentiate in response to infection, resulting in acquisition of immune functions required for pathogen clearance; and the ability to maintain this functional capacity in the long-term, allowing more rapid and effective pathogen elimination following re-infection. These characteristics underpin vaccination strategies by effectively establishing a long-lived T cell population that contributes to an immunologically protective state (termed immunological memory). As we discuss in this review, epigenetic mechanisms provide attractive and powerful explanations for key aspects of T cell-mediated immunity - most obviously and notably, immunological memory, because of the capacity of epigenetic circuits to perpetuate cellular identities in the absence of the initial signals that drive differentiation. Indeed, T cell responses to infection are an ideal model system for studying how epigenetic factors shape cellular differentiation and development generally. This review will examine how epigenetic mechanisms regulate T cell function and differentiation, and how these model systems are providing general insights into the epigenetic regulation of gene transcription during cellular differentiation.
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Atenolol Induced HDL-C Change in the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study.
PLoS ONE
PUBLISHED: 01-01-2013
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We sought to identify novel pharmacogenomic markers for HDL-C response to atenolol in participants with mild to moderate hypertension. We genotyped 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study on the Illumina HumanCVD Beadchip. During PEAR, participants were randomized to receive atenolol or hydrochlorothiazide. Blood pressure and cholesterol levels were evaluated at baseline and after treatment. This study focused on participants treated with atenolol monotherapy. Association with atenolol induced HDL-C change was evaluated in 232 whites and 152 African Americans using linear regression. No SNPs achieved a Bonferroni corrected P-value. However, we identified 13 regions with consistent association across whites and African Americans. The most interesting of these regions were seven with prior associations with HDL-C, other metabolic traits, or functional implications in the lipid pathway: GALNT2, FTO, ABCB1, LRP5, STARD3NL, ESR1, and LIPC. Examples are rs2144300 in GALNT2 in whites (P=2.29x10(-4), ?=-1.85 mg/dL) and rs12595985 in FTO in African Americans (P=2.90x10(-4), ?=4.52 mg/dL), both with consistent regional association (P<0.05) in the other race group. Additionally, baseline GALNT2 expression differed by rs2144300 genotype in whites (P=0.0279). In conclusion, we identified multiple gene regions associated with atenolol induced HDL-C change that were consistent across race groups, several with functional implications or prior associations with HDL-C.
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Gene-specific DNA methylation association with serum levels of C-reactive protein in African Americans.
PLoS ONE
PUBLISHED: 01-01-2013
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A more thorough understanding of the differences in DNA methylation (DNAm) profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP), an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.
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Ophthalmic surgical training following modernising medical careers: regional variation in experience across the UK.
BMJ Open
PUBLISHED: 01-01-2013
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To evaluate surgical experience among current doctors appointed into ophthalmology training posts since the introduction of the Modernising Medical Careers programme. Additionally, to identify regional variations in surgical experience and training programme delivery.
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Sex differences in salt sensitivity to nitric oxide dependent vasodilation in healthy young adults.
J. Appl. Physiol.
PUBLISHED: 12-22-2011
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Dietary sodium and blood pressure regulation differs between normotensive men and women, an effect which may involve endothelial production of nitric oxide (NO). Therefore, we tested the hypothesis that differences in the NO component of endothelium-dependent vasodilation between low and high dietary sodium intake depend on sex. For 5 days prior to study, healthy adults consumed a controlled low-sodium diet (10 mmol/day, n = 30, mean age ± SE: 30 ± 1 yr, 16 men) or high-sodium diet (400 mmol/day, n = 36, age 23 ± 1 yr, 13 men). Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of acetylcholine (ACh, 4 ?g·100 ml tissue(-1)·min(-1)) were measured before and after endothelial NO synthase inhibition with N(G)-monomethyl-l-arginine (l-NMMA, 50 mg bolus + 1 mg/min infusion). The NO component of endothelium-dependent dilation was calculated as the response to ACh before and after l-NMMA accounting for changes in baseline FBF: [(FBF ACh - FBF baseline) - (FBF ACh(L-NMMA) - FBF baseline(L-NMMA))]. This value was 5.7 ± 1.3 and 2.5 ± 0.8 ml·100 ml forearm tissue(-1)·min(-1) for the low- and high-sodium diets, respectively (main effect of sodium, P = 0.019). The sodium effect was larger for the men, with values of 7.9 ± 2.0 and 2.2 ± 1.4 for men vs. 3.1 ± 1.3 and 2.7 ± 1.0 ml·100 ml forearm tissue(-1)·min(-1) for the women (P = 0.034, sex-by-sodium interaction). We conclude that the NO component of endothelium-dependent vasodilation is altered by dietary sodium intake based on sex, suggesting that endothelial NO production is sensitive to dietary sodium in healthy young men but not women.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.