Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing® technology

<p>Abstract</p> <p>Background</p> <p>Identification of <it>CYP2A6 </it>alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. <it>CYP2A6*12 </it>is a hybrid allele that results from unequal crossover between <it>CYP2A6 </it>and <it>CYP2A7 </it>genes. The 5' regulatory region and exons 1–2 are derived from <it>CYP2A7</it>, and exons 3–9 are derived from <it>CYP2A6</it>. Conventional methods for detection of <it>CYP2A6*12 </it>consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the <it>CYP2A6*12 </it>allele by Pyrosequencing technology.</p> <p>Methods</p> <p>A single set of PCR primers was designed to specifically amplify both the <it>CYP2A6*1 </it>wild-type allele and the <it>CYP2A6*12 </it>hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. <it>CYP2A6*12 </it>allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow.</p> <p>Results</p> <p>Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the <it>CYP2A6*12 </it>allele in European Caucasians and Hispanics.</p> <p>Conclusion</p> <p>This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.</p

Coxsackievirus-Induced Proteomic Alterations in Primary Human Islets Provide Insights for the Etiology of Diabetes

Enteroviral infections have been associated with the development of type 1 diabetes (T1D), a chronic inflammatory disease characterized by autoimmune destruction of insulin-producing pancreatic beta cells. Cultured human islets, including the insulin-producing beta cells, can be infected with coxsackievirus B4 (CVB4) and thus are useful for understanding cellular responses to infection. We performed quantitative mass spectrometry analysis on cultured primary human islets infected with CVB4 to identify molecules and pathways altered upon infection. Corresponding uninfected controls were included in the study for comparative protein expression analyses. Proteins were significantly and differentially regulated in human islets challenged with virus compared with their uninfected counterparts. Complementary analyses of gene transcripts in CVB4-infected primary islets over a time course validated the induction of RNA transcripts for many of the proteins that were increased in the proteomics studies. Notably, infection with CVB4 results in a considerable decrease in insulin. Genes/proteins modulated during CVB4 infection also include those involved in activation of immune responses, including type I interferon pathways linked to T1D pathogenesis and with antiviral, cell repair, and inflammatory properties. Our study applies proteomics analyses to cultured human islets challenged with virus and identifies target proteins that could be useful in T1D interventions

Star formation in the small magellanic cloud: The youngest star clusters

AbstractWe recently launched a comprehensive ground based (ESO/VLT/NTT) and space (HST & SST) study of the present and past star formation in the Small Magellanic Cloud (SMC), in clusters and in the field, with the goal of understanding how star and cluster formation occur and propagate in an environment of low metallicity, with a gas and dust content that is significantly lower than in the Milky Way. In this paper, we present some preliminary results of the "young cluster" program, where we acquired deep F555W (~V), and F814W (~I) HST/ACS images of the four young and massive SMC star clusters: NGC 346, NGC 602, NGC 299, and NGC 376

Genetic Studies of a Cluster of Acute Lymphoblastic Leukemia Cases in Churchill County, Nevada

OBJECTIVE: In a study to identify exposures associated with 15 cases of childhood leukemia, we found levels of tungsten, arsenic, and dichlorodiphenyldichloroethylene in participants to be higher than mean values reported in the National Report on Human Exposure to Environmental Chemicals. Because case and comparison families had similar levels of these contaminants, we conducted genetic studies to identify gene polymorphisms that might have made case children more susceptible than comparison children to effects of the exposures. DESIGN: We compared case with comparison children to determine whether differences existed in the frequency of polymorphic genes, including genes that code for enzymes in the folate and purine pathways. We also included discovery of polymorphic forms of genes that code for enzymes that are inhibited by tungsten: xanthine dehydrogenase, sulfite oxidase (SUOX gene), and aldehyde oxidase. PARTICIPANTS: Eleven case children were age- and sex-matched with 42 community comparison children for genetic analyses. Twenty parents of case children also contributed to the analyses. RESULTS: One bilalleleic gene locus in SUOX was significantly associated with either case or comparison status, depending on which alleles the child carried (without adjusting for multiple comparisons). CONCLUSIONS: Although genetic studies did not provide evidence that a common agent or genetic susceptibility factor caused the leukemias, the association between a SUOX gene locus and disease status in the presence of high tungsten and arsenic levels warrants further investigation. RELEVANCE: Although analyses of community clusters of cancer have rarely identified causes, these findings have generated hypotheses to be tested in subsequent studies

Spitzer Survey of the Large Magellanic Cloud, Surveying the Agents of a Galaxy's Evolution (SAGE) I: Overview and Initial Results

We are performing a uniform and unbiased, ~7x7 degrees imaging survey of the Large Magellanic Cloud (LMC), using the IRAC and MIPS instruments on board the Spitzer Space Telescope in order to survey the agents of a galaxy's evolution (SAGE), the interstellar medium (ISM) and stars in the LMC. The detection of diffuse ISM with column densities >1.2x10^21 H cm^-2 permits detailed studies of dust processes in the ISM. SAGE's point source sensitivity enables a complete census of newly formed stars with masses >3 solar masses that will determine the current star formation rate in the LMC. SAGE's detection of evolved stars with mass loss rates >1x10^-8 solar masses per year will quantify the rate at which evolved stars inject mass into the ISM of the LMC. The observing strategy includes two epochs in 2005, separated by three months, that both mitigate instrumental artifacts and constrain source variability. The SAGE data are non-proprietary. The data processing includes IRAC and MIPS pipelines and a database for mining the point source catalogs, which will be released to the community in support of Spitzer proposal cycles 4 and 5. We present initial results on the epoch 1 data with a special focus on the N79 and N83 region. The SAGE epoch 1 point source catalog has ~4 million sources. The point source counts are highest for the IRAC 3.6 microns band and decrease dramatically towards longer wavelengths consistent with the fact that stars dominate the point source catalogs and that the dusty objects, e.g. young stellar objects and dusty evolved stars that detected at the longer wavelengths, are rare in comparison. We outline a strategy for identifying foreground MW stars, that may comprise as much as 18% of the source list, and background galaxies, that may comprise ~12% of the source list.Comment: Accepted by the Astronomical Journa

Expert consensus on low-calorie sweeteners:facts, research gaps and suggested actions

A consensus workshop on low-calorie sweeteners (LCS) was held in November 2018 where seventeen experts (the panel) discussed three themes identified as key to the science and policy of LCS: (1) weight management and glucose control; (2) consumption, safety and perception; (3) nutrition policy. The aims were to identify the reliable facts on LCS, suggest research gaps and propose future actions. The panel agreed that the safety of LCS is demonstrated by a substantial body of evidence reviewed by regulatory experts and current levels of consumption, even for high users, are within agreed safety margins. However, better risk communication is needed. More emphasis is required on the role of LCS in helping individuals reduce their sugar and energy intake, which is a public health priority. Based on reviews of clinical evidence to date, the panel concluded that LCS can be beneficial for weight management when they are used to replace sugar in products consumed in the diet (without energy substitution). The available evidence suggests no grounds for concerns about adverse effects of LCS on sweet preference, appetite or glucose control; indeed, LCS may improve diabetic control and dietary compliance. Regarding effects on the human gut microbiota, data are limited and do not provide adequate evidence that LCS affect gut health at doses relevant to human use. The panel identified research priorities, including collation of the totality of evidence on LCS and body weight control, monitoring and modelling of LCS intakes, impacts on sugar reduction and diet quality and developing effective communication strategies to foster informed choice. There is also a need to reconcile policy discrepancies between organisations and reduce regulatory hurdles that impede low-energy product development and reformulation

Spitzer survey of the Large Magellanic Cloud, surveying the agents of a galaxy's evolution (SAGE). IV. Dust properties in the interstellar medium

The goal of this paper is to present the results of a preliminary analysis of the extended infrared (IR) emission by dust in the interstellar medium (ISM) of the Large Magellanic Cloud (LMC). We combine Spitzer Surveying the Agents of Galaxy Evolution (SAGE) and Infrared Astronomical Satellite (IRAS) data and correlate the infrared emission with gas tracers of H I, CO, and Hα. We present a global analysis of the infrared emission as well as detailed modeling of the spectral energy distribution (SED) of a few selected regions. Extended emission by dust associated with the neutral, molecular, and diffuse ionized phases of the ISM is detected at all IR bands from 3.6 μm to 160 μm. The relative abundance of the various dust species appears quite similar to that in the Milky Way (MW) in all the regions we have modeled. We construct maps of the temperature of large dust grains. The temperature map shows variations in the range 12.1-34.7 K, with a systematic gradient from the inner to outer regions, tracing the general distribution of massive stars and individual H II regions as well as showing warmer dust in the stellar bar. This map is used to derive the far-infrared (FIR) optical depth of large dust grains. We find two main departures in the LMC with respect to expectations based on the MW: (1) excess mid-infrared (MIR) emission near 70 μm, referred to as the 70 μm excess, and (2) departures from linear correlation between the FIR optical depth and the gas column density, which we refer to as FIR excess emission. The 70 μm excess increases gradually from the MW to the LMC to the Small Magellanic Cloud (SMC), suggesting evolution with decreasing metallicity. The excess is associated with the neutral and diffuse ionized gas, with the strongest excess region located in a loop structure next to 30 Dor. We show that the 70 μm excess can be explained by a modification of the size distribution of very small grains with respect to that in the MW, and a corresponding mass increase of ≃13% of the total dust mass in selected regions. The most likely explanation is that the 70 μm excess is due to the production of large very small grains (VSG) through erosion of larger grains in the diffuse medium. This FIR excess could be due to intrinsic variations of the dust/gas ratio, which would then vary from 4.6 to 2.3 times lower than the MW values across the LMC, but X_(CO) values derived from the IR emission would then be about three times lower than those derived from the Virial analysis of the CO data. We also investigate the possibility that the FIR excess is associated with an additional gas component undetected in the available gas tracers. Assuming a constant dust abundance in all ISM phases, the additional gas component would have twice the known H I mass. We show that it is plausible that the FIR excess is due to cold atomic gas that is optically thick in the 21 cm line, while the contribution by a pure H_2 phase with no CO emission remains a possible explanation

A Panchromatic View OF NGC 602: Time-Resolved Star Formation with the Hubble and Spitzer Space Telescopes

We present the photometric catalogs for the star-forming cluster NGC 602 in the wing of the Small Magellanic Cloud covering a range of wavelengths from optical HST/ACS (F555W, F814W) and SMARTS/ANDICAM (V, I) to infrared (Spitzer/IRAC 3.6, 4.5, 5.8, and 8 micron and MIPS 24 micron). Combining this with IRSF (InfraRed Survey Facility) near-infrared photometry (J, H, Ks), we compare the young main sequence (MS) and pre-main sequence (PMS) populations prominent in the optical with the current young stellar object (YSO) populations revealed by the infrared (IR). We analyze the MS and PMS population with isochrones in color-magnitude diagrams to derive ages and masses. The optical data reveal ~565 PMS candidates, low mass Stage III YSOs. We characterize ~40 YSOs by fitting their spectral energy distributions (SEDs) to a grid of models (Robitaille et al. 2007) to derive luminosities, masses and evolutionary phase (Stage I-III). The higher resolution HST images reveal that ~70% of the YSO candidates are either multiples or protoclusters. For YSOs and PMS sources found in common, we find a consistency in the masses derived. We use the YSO mass function to derive a present-day star-formation rate of ~0.2-1.0 Msun/yr/kpc^2, similar to the rate derived from the optical star formation history suggesting a constant star formation rate for this region. We demonstrate a progression of star formation from the optical star cluster center to the edge of the star forming dust cloud. We derive lifetimes of a few 10^5 years for the YSO Stages I and II.Comment: 55 pages, 18 Figures; High resolution images available form author Accepted to Ap

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy