407-3 Glutathione Peroxidase Prevents the Inactivation of Nitric Oxide and Restores the Inhibition of Platelet Function by S-Nitrosothiols

The interaction of nitric oxide (NO) with reactive oxygen species in the vasculature can inactivate NO leading to potentially adverse vascular consequences. Glutathione peroxidases (GSH-Px), a family of antioxidant enzymes present at reduced concentrations in plasma and platelets of patients with coronary artery disease, catalyze the reduction of hydrogen peroxide and lipid hydroperoxides (LOOH) by glutathione. Given the role of LOOH in platelet eicosanoid metabolism and their presence in atherosclerotic plaque, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring NO donor, S-nitroso-glutathione (SNO-Glu). Subthreshold inhibitory concentrations of SNO-Glu were added to platelet-rich plasma, and aggregation was induced by arachidonate. The addition of GSHPx (0.2–20U/ml) to this system led to a dose-dependent inhibition of platelet aggregation with an IC50= 0.6U/ml GSH-Px (p < 0.05 by ANOVA). Superoxide dismutase (0.1–200U/ml), catalase (0.1–200U/ml), or GSH-Px without SNO-Glu did not alter platelet aggregation responses. The addi ion of GSH-Px to a subthreshold inhibitory concentration of sodium nitroprusside also did not affect platelet aggregation responses. LOOH increased platelet aggregation in the presence of SNO-Glu, an effect reversed by GSH-Px. Levels of cGMP were measured after platelets were incubated with SNO-Glu, exogenous LOOH, and GSH-Px. SNO-Glu alone increased cGMP levels, and this effect was attenuated by LOOH but restored by the addition of GSH-Px. GSH-Px activity was equivalent with either SNO-Glu or glutathione as cosubstrate. Incubation of SNO-Glu with GSH-Px led to a 48.5% decrease in the concentration of SNO-Glu as determined by HPLC-electrochemical detection. Incubation of SNO-Glu with albumin in the presence of GSH-Px led to increased formation of S-nitroso-albumin, a prevalent reservoir of EDRF in plasma. These results show that GSH-Px, at physiologically relevant concentrations, has a potent effect on NO-induced inhibition of platelet aggregation and that this enzyme may have two functions: (i) metabolism of LOOH, thereby preventing its inactivation of NO; and (ii) metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions These findings suggest that GSH-Px, in addition to its antioxidant functions, regulates the availability of NO in the vasculature and possibly alters plateletdependent thrombotic events

Integrated genome-wide analysis of expression quantitative trait loci aids interpretation of genomic association studies

BACKGROUND: Identification of single nucleotide polymorphisms (SNPs) associated with gene expression levels, known as expression quantitative trait loci (eQTLs), may improve understanding of the functional role of phenotype-associated SNPs in genome-wide association studies (GWAS). The small sample sizes of some previous eQTL studies have limited their statistical power. We conducted an eQTL investigation of microarray-based gene and exon expression levels in whole blood in a cohort of 5257 individuals, exceeding the single cohort size of previous studies by more than a factor of 2. RESULTS: We detected over 19,000 independent lead cis-eQTLs and over 6000 independent lead trans-eQTLs, targeting over 10,000 gene targets (eGenes), with a false discovery rate (FDR) \u3c 5%. Of previously published significant GWAS SNPs, 48% are identified to be significant eQTLs in our study. Some trans-eQTLs point toward novel mechanistic explanations for the association of the SNP with the GWAS-related phenotype. We also identify 59 distinct blocks or clusters of trans-eQTLs, each targeting the expression of sets of six to 229 distinct trans-eGenes. Ten of these sets of target genes are significantly enriched for microRNA targets (FDR \u3c 5%). Many of these clusters are associated in GWAS with multiple phenotypes. CONCLUSIONS: These findings provide insights into the molecular regulatory patterns involved in human physiology and pathophysiology. We illustrate the value of our eQTL database in the context of a recent GWAS meta-analysis of coronary artery disease and provide a list of targeted eGenes for 21 of 58 GWAS loci

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories [preprint]

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols

The Carnegie Hubble Program: The Distance and Structure of the SMC as Revealed by Mid-infrared Observations of Cepheids

Using Spitzer observations of classical Cepheids we have measured the true average distance modulus of the SMC to be $18.96 \pm 0.01_{stat} \pm 0.03_{sys}$ mag (corresponding to $62 \pm 0.3$ kpc), which is $0.48 \pm 0.01$ mag more distant than the LMC. This is in agreement with previous results from Cepheid observations, as well as with measurements from other indicators such as RR Lyrae stars and the tip of the red giant branch. Utilizing the properties of the mid--infrared Leavitt Law we measured precise distances to individual Cepheids in the SMC, and have confirmed that the galaxy is tilted and elongated such that its eastern side is up to 20 kpc closer than its western side. This is in agreement with the results from red clump stars and dynamical simulations of the Magellanic Clouds and Stream.Comment: Accepted for publication in ApJ. 38 Pages, 11 figures. Figure 9 is interactive. Spitzer photometry for all Cepheids available as online tabl

The Carnegie Hubble Program

We present an overview of and preliminary results from an ongoing comprehensive program that has a goal of determining the Hubble constant to a systematic accuracy of 2%. As part of this program, we are currently obtaining 3.6 micron data using the Infrared Array Camera (IRAC) on Spitzer, and the program is designed to include JWST in the future. We demonstrate that the mid-infrared period-luminosity relation for Cepheids at 3.6 microns is the most accurate means of measuring Cepheid distances to date. At 3.6 microns, it is possible to minimize the known remaining systematic uncertainties in the Cepheid extragalactic distance scale. We discuss the advantages of 3.6 micron observations in minimizing systematic effects in the Cepheid calibration of the Hubble constant including the absolute zero point, extinction corrections, and the effects of metallicity on the colors and magnitudes of Cepheids. We are undertaking three independent tests of the sensitivity of the mid-IR Cepheid Leavitt Law to metallicity, which when combined will allow a robust constraint on the effect. Finally, we are providing a new mid-IR Tully-Fisher relation for spiral galaxies

The Cepheid Period-Luminosity Relation at Mid-Infrared Wavelengths: II. Second-Epoch LMC Data

We present revised and improved mid-infrared Period-Luminosity (PL) relations for Large Magellanic Cloud (LMC) Cepheids based on double-epoch data of 70 Cepheids observed by Spitzer at 3.6, 4.5, 5.8 and 8.0um. The observed scatter at all wavelengths is found to decrease from +/-0.17 mag to +/-0.14 mag, which is fully consistent with the prediction that the total scatter is made up of roughly equal contributions from random sampling of the light curve and nearly-uniform samplings of stars across the instability strip. It is calculated that the Cepheids in this sample have a full amplitude of about 0.4 mag and that their fully-sampled, time-averaged magnitudes should eventually reveal mid-infrared PL relations that each have intrinsic scatter at most at the +/-0.12 mag level, and as low as +/-0.08 mag after correcting for the tilt of the LMC.Comment: 7 pages, 6 figures, 1 data tabl

The Cepheid Period-Luminosity Relation (The Leavitt Law) at Mid-Infrared Wavelengths: III. Cepheids in NGC 6822

We present the first application of mid-infrared Period-Luminosity relations to the determination of a Cepheid distance beyond the Magellanic Clouds. Using archival IRAC imaging data on NGC 6822 from Spitzer we were able to measure single-epoch magnitudes for sixteen long-period (10 to 100-day) Cepheids at 3.6um, fourteen at 4.5um, ten at 5.8um and four at 8.0um. The measured slopes and the observed scatter both conform to the relations previously measured for the Large Magellanic Cloud Cepheids, and fitting to those relations gives apparent distance moduli of mod{3.6} = 23.57 +/- 0.06, mod{4.5} = 23.55 +/- 0.07, mod{5.8} = 23.60 +/- 0.09 and mod{8.0} = 23.51 +/-0.08 mag. A multi-wavelength fit to the new IRAC moduli, and previously published BVRIJHK moduli, allows for a final correction for interstellar reddening and gives a true distance modulus of 23.49 +/- 0.03 mag with E(B-V) = 0.26 mag, corresponding to a metric distance of 500 +/-8 kpc.Comment: Accepted to ApJ, 2 figure

The Cepheid Period-Luminosity Relation at Mid-Infrared Wavelengths: I. First-Epoch LMC Data

We present the first mid-infrared Period-Luminosity (PL) relations for Large Magellanic Cloud (LMC) Cepheids. Single-epoch observations of 70 Cepheids were extracted from Spitzer IRAC observations at 3.6, 4.5, 5.8 and 8.0 microns, serendipitously obtained during the SAGE (Surveying the Agents of a Galaxy's Evolution) imaging survey of the LMC. All four mid-infrared PL relations have nearly identical slopes over the period range 6 - 88 days, with a small scatter of only +/-0.16 mag independent of period for all four of these wavelengths. We emphasize that differential reddening is not contributing significantly to the observed scatter, given the nearly two orders of magnitude reduced sensitivity of the mid-IR to extinction compared to the optical. Future observations, filling in the light curves for these Cepheids, should noticeably reduce the residual scatter. These attributes alone suggest that mid-infrared PL relations will provide a practical means of significantly improving the accuracy of Cepheid distances to nearby galaxies.Comment: 19 pages, 4 figures, 1 table, Accepted for publication in the Astrophysical Journa

Diverse human extracellular RNAs are widely detected in human plasma

There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population