Quantifying the improvement of surrogate indices of hepatic insulin resistance using complex measurement techniques

We evaluated the ability of simple and complex surrogate-indices to identify individuals from an overweight/obese cohort with hepatic insulin-resistance (HEP-IR). Five indices, one previously defined and four newly generated through step-wise linear regression, were created against a single-cohort sample of 77 extensively characterised participants with the metabolic syndrome (age 55.6±1.0 years, BMI 31.5±0.4 kg/m2; 30 males). HEP-IR was defined by measuring endogenous-glucose-production (EGP) with [6–62H2] glucose during fasting and euglycemic-hyperinsulinemic clamps and expressed as EGP*fasting plasma insulin. Complex measures were incorporated into the model, including various non-standard biomarkers and the measurement of body-fat distribution and liver-fat, to further improve the predictive capability of the index. Validation was performed against a data set of the same subjects after an isoenergetic dietary intervention (4 arms, diets varying in protein and fiber content versus control). All five indices produced comparable prediction of HEP-IR, explaining 39–56% of the variance, depending on regression variable combination. The validation of the regression equations showed little variation between the different proposed indices (r2 = 27–32%) on a matched dataset. New complex indices encompassing advanced measurement techniques offered an improved correlation (r = 0.75, P<0.001). However, when validated against the alternative dataset all indices performed comparably with the standard homeostasis model assessment for insulin resistance (HOMA-IR) (r = 0.54, P<0.001). Thus, simple estimates of HEP-IR performed comparable to more complex indices and could be an efficient and cost effective approach in large epidemiological investigations

Atrial arrhythmogenesis in wild-type and Scn5a+/Δ murine hearts modelling LQT3 syndrome

Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Δ (KPQ) Langendorff-perfused murine hearts modelling LQT3 (P < 0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (P < 0.001 for both; Fisher’s exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD90), compared to KPQ hearts (8.1 and 3.2 ms, respectively, P < 0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (P < 0.001) and KPQ hearts (P < 0.05). It prolonged the AERP to a larger extent than it did the APD90 in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD90 in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD90 over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD90 reduced such CI values and produced the opposite effect

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Reconstructing terrestrial nutrient cycling using stable nitrogen isotopes in wood

Although recent anthropogenic effects on the global nitrogen (N) cycle have been significant, the consequences of increased anthropogenic N on terrestrial ecosystems are unclear. Studies of the impact of increased reactive N on forest ecosystems—impacts on hydrologic and gaseous loss pathways, retention capacity, and even net primary productivity— have been particularly limited by a lack of long-term baseline biogeochemical data. Stable nitrogen isotope analysis (ratio of ¹⁵N to ¹⁴N, termed δ¹⁵N) of wood chronologies offers the potential to address changes in ecosystem N cycling on millennial timescales and across broad geographic regions. Currently, nearly 50 studies have been published utilizing wood δ¹⁵N records; however, there are significant differences in study design and data interpretation. Here, we identify four categories of wood δ¹⁵N studies, summarize the common themes and primary findings of each category, identify gaps in the spatial and temporal scope of current wood δ¹⁵N chronologies, and synthesize methodological frameworks for future research by presenting eight suggestions for common methodological approaches and enhanced integration across studies. Wood δ¹⁵N records have the potential to provide valuable information for interpreting modern biogeochemical cycling. This review serves to advance the utility of this technique for long-term biogeochemical reconstructions

Linagliptin Improves Insulin Sensitivity and Hepatic Steatosis in Diet-Induced Obesity

Linagliptin (tradjenta™) is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor. DPP-4 inhibition attenuates insulin resistance and improves peripheral glucose utilization in humans. However, the effects of chronic DPP-4 inhibition on insulin sensitivity are not known. The effects of long-term treatment (3–4 weeks) with 3 mg/kg/day or 30 mg/kg/day linagliptin on insulin sensitivity and liver fat content were determined in diet-induced obese C57BL/6 mice. Chow-fed animals served as controls. DPP-4 activity was significantly inhibited (67–89%) by linagliptin (P<0.001). Following an oral glucose tolerance test, blood glucose concentrations (measured as area under the curve) were significantly suppressed after treatment with 3 mg/kg/day (–16.5% to –20.3%; P<0.01) or 30 mg/kg/day (–14.5% to –26.4%; P<0.05) linagliptin (both P<0.01). Liver fat content was significantly reduced by linagliptin in a dose-dependent manner (both doses P<0.001). Diet-induced obese mice treated for 4 weeks with 3 mg/kg/day or 30 mg/kg/day linagliptin had significantly improved glycated hemoglobin compared with vehicle (both P<0.001). Significant dose-dependent improvements in glucose disposal rates were observed during the steady state of the euglycemic–hyperinsulinemic clamp: 27.3 mg/kg/minute and 32.2 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 20.9 mg/kg/minute with vehicle (P<0.001). Hepatic glucose production was significantly suppressed during the clamp: 4.7 mg/kg/minute and 2.1 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 12.5 mg/kg/minute with vehicle (P<0.001). In addition, 30 mg/kg/day linagliptin treatment resulted in a significantly reduced number of macrophages infiltrating adipose tissue (P<0.05). Linagliptin treatment also decreased liver expression of PTP1B, SOCS3, SREBP1c, SCD-1 and FAS (P<0.05). Other tissues like muscle, heart and kidney were not significantly affected by the insulin sensitizing effect of linagliptin. Long-term linagliptin treatment reduced liver fat content in animals with diet-induced hepatic steatosis and insulin resistance, and may account for improved insulin sensitivity

Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis

A quantitative analysis of the effect of cycle length on arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

The clinically established proarrhythmic effect of bradycardia and antiarrhythmic effect of lidocaine (10 μM) were reproduced in hypokalaemic (3.0 mM K+) Langendorff-perfused murine hearts paced over a range (80–180 ms) of baseline cycle lengths (BCLs). Action potential durations (at 90% repolarization, APD90s), transmural conduction times and ventricular effective refractory periods (VERPs) were then determined from monophasic action potential records obtained during a programmed electrical stimulation procedure in which extrasystolic stimuli were interposed following regular stimuli at successively decreasing coupling intervals. A novel graphical analysis of epicardial and endocardial, local and transmural relationships between APD90, corrected for transmural conduction time where appropriate, and VERP yielded predictions in precise agreement with the arrhythmogenic findings obtained over the entire range of BCLs studied. Thus, in normokalaemic (5.2 mM K+) hearts a statistical analysis confirmed that all four relationships were described by straight lines of gradients not significantly (P > 0.05) different from unity that passed through the origin and thus subtended constant critical angles, θ with the abscissa (45.8° ± 0.9°, 46.6° ± 0.5°, 47.6° ± 0.5° and 44.9° ± 0.8°, respectively). Hypokalaemia shifted all points to the left of these reference lines, significantly (P < 0.05) increasing θ at BCLs of 80–120 ms where arrhythmic activity was not observed (∼63°, ∼54°, ∼55° and ∼58°, respectively) and further significantly (P < 0.05) increasing θ at BCLs of 140–180 ms where arrhythmic activity was observed (∼68°, ∼60°, ∼61° and ∼65°, respectively). In contrast, the antiarrhythmic effect of lidocaine treatment was accompanied by a significant (P < 0.05) disruption of this linear relationship and decreases in θ in both normokalaemic (∼40°, ∼33°, ∼39° and ∼41°, respectively) and hypokalaemic (∼40°, ∼44°, ∼50° and ∼48°, respectively) hearts. This extended a previous approach that had correlated alterations in transmural repolarization gradients with arrhythmogenicity in murine models of the congenital long QT syndrome type 3 and hypokalaemia at a single BCL. Thus, the analysis in terms of APD90 and VERP provided a more sensitive indication of the effect of lidocaine than one only considering transmural repolarization gradients and may be particularly applicable in physiological and pharmacological situations in which these parameters diverge

Several Cancer Susceptibility Variants Also Affect Melanoma Risk

<div><p>Background</p><p>Several regions of the genome show pleiotropic associations with multiple cancers. We sought to evaluate whether 181 single-nucleotide polymorphisms previously associated with various cancers in genome-wide association studies were also associated with melanoma risk.</p><p>Methods</p><p>We evaluated 2,131 melanoma cases and 20,353 controls from three studies in the Population Architecture using Genomics and Epidemiology (PAGE) study (EAGLE-BioVU, MEC, WHI) and two collaborating studies (HPFS, NHS). Overall and sex-stratified analyses were performed across studies.</p><p>Results</p><p>We observed statistically significant associations with melanoma for two lung cancer SNPs in the <i>TERT-CLPTM1L</i> locus (Bonferroni-corrected p<2.8x10^-4), replicating known pleiotropic effects at this locus. In sex-stratified analyses, we also observed a potential male-specific association between prostate cancer risk variant rs12418451 and melanoma risk (OR=1.22, p=8.0x10^-4). No other variants in our study were associated with melanoma after multiple comparisons adjustment (p>2.8e^-4).</p><p>Conclusions</p><p>We provide confirmatory evidence of pleiotropic associations with melanoma for two SNPs previously associated with lung cancer, and provide suggestive evidence for a male-specific association with melanoma for prostate cancer variant rs12418451. This SNP is located near <i>TPCN2</i>, an ion transport gene containing SNPs which have been previously associated with hair pigmentation but not melanoma risk. Previous evidence provides biological plausibility for this association, and suggests a complex interplay between ion transport, pigmentation, and melanoma risk that may vary by sex. If confirmed, these pleiotropic relationships may help elucidate shared molecular pathways between cancers and related phenotypes.</p></div

Genome Wide Analysis of Drug-Induced Torsades de Pointes: Lack of Common Variants with Large Effect Sizes

Marked prolongation of the QT interval on the electrocardiogram associated with the polymorphic ventricular tachycardia Torsades de Pointes is a serious adverse event during treatment with antiarrhythmic drugs and other culprit medications, and is a common cause for drug relabeling and withdrawal. Although clinical risk factors have been identified, the syndrome remains unpredictable in an individual patient. Here we used genome-wide association analysis to search for common predisposing genetic variants. Cases of drug-induced Torsades de Pointes (diTdP), treatment tolerant controls, and general population controls were ascertained across multiple sites using common definitions, and genotyped on the Illumina 610k or 1M-Duo BeadChips. Principal Components Analysis was used to select 216 Northwestern European diTdP cases and 771 ancestry-matched controls, including treatment-tolerant and general population subjects. With these sample sizes, there is 80% power to detect a variant at genome-wide significance with minor allele frequency of 10% and conferring an odds ratio of ≥2.7. Tests of association were carried out for each single nucleotide polymorphism (SNP) by logistic regression adjusting for gender and population structure. No SNP reached genome wide-significance; the variant with the lowest P value was rs2276314, a non-synonymous coding variant in C18orf21 (p  =  3×10(-7), odds ratio = 2, 95% confidence intervals: 1.5-2.6). The haplotype formed by rs2276314 and a second SNP, rs767531, was significantly more frequent in controls than cases (p  =  3×10(-9)). Expanding the number of controls and a gene-based analysis did not yield significant associations. This study argues that common genomic variants do not contribute importantly to risk for drug-induced Torsades de Pointes across multiple drugs

Metabolic engineering of Rhizopus oryzae for the production of platform chemicals

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products