133 research outputs found

    Chapter 23: Food and Drug, Health, and Welfare Law

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    The synergistic effects of elevated temperature and CO2 - induced ocean acidification reduce cardiac performance and increase disease susceptibility in subadult, female American lobsters Homarus americanus H. Milne Edwards, 1837 (Decapoda: Astacidea: Nephropidae) from the Gulf of Maine

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    Increased greenhouse gas emissions have caused rapid ocean warming (OW) and reduced ocean pH via acidification (OA). Both OW and OA will likely impact marine crustaceans, but they are often examined in isolation. We conducted an environmental-stressor experiment to understand how exposure to current summer conditions (16 °C, pH 8.0), OW only (20 °C, pH 8.0), OA only (16 °C, pH 7.6), or both acidification and warming, OAW (20 °C, pH 7.6), differentially influence thermal physiology and immune response of female subadults of the American lobster, Homarus americanus H. Milne Edwards, 1837. Following a 42 d exposure, cardiac performance was assessed during an acute thermal stress, and lobsters were subjected to a subsequent 21 d pathogen challenge with the bacterium Aerococcus viridans var. homari, the causative agent of gaffkemia. Lobsters under OAW had significantly lower (P ≤ 0.02) Arrhenius break temperatures (ABT), an indicator of thermal limits of capacity, compared to lobsters exposed to all other treatments, suggesting these stressors act synergistically to reduce physiological performance. Individuals from the OW and OAW treatments also had significantly lower (P ≤ 0.035) total hemocyte counts (THCs), an indicator of immune response, and showed a reduced median time to death (by up to 5 d sooner) post A. viridans injection compared to lobsters exposed to current summer conditions. Moreover, nearly twice as many lobsters exposed to OAW lost at least one claw during the pathogen challenge compared to all other treatment groups, potentially increasing the risk of mortality due to secondary infection. Together, these results suggest that OAW will impact the physiology and immune response of subadult H. americanus, potentially influencing successful recruitment to the fishery

    Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

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    <p>Abstract</p> <p>Background</p> <p>Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.</p> <p>Findings</p> <p>Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, <it>Schizosaccharomyces pombe</it>. To preserve <it>in vivo </it>molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.</p> <p>Conclusions</p> <p>We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.</p

    Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling

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    Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca2+ regulatory proteins. However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) (by 27 and 41% vs. untreated diabetic rats, respectively, P < 0.05). In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P < 0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P < 0.05), but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.Peer reviewedPhysiological Science

    First radial velocity results from the MINiature Exoplanet Radial Velocity Array (MINERVA)

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    The MINiature Exoplanet Radial Velocity Array (MINERVA) is a dedicated observatory of four 0.7m robotic telescopes fiber-fed to a KiwiSpec spectrograph. The MINERVA mission is to discover super-Earths in the habitable zones of nearby stars. This can be accomplished with MINERVA's unique combination of high precision and high cadence over long time periods. In this work, we detail changes to the MINERVA facility that have occurred since our previous paper. We then describe MINERVA's robotic control software, the process by which we perform 1D spectral extraction, and our forward modeling Doppler pipeline. In the process of improving our forward modeling procedure, we found that our spectrograph's intrinsic instrumental profile is stable for at least nine months. Because of that, we characterized our instrumental profile with a time-independent, cubic spline function based on the profile in the cross dispersion direction, with which we achieved a radial velocity precision similar to using a conventional "sum-of-Gaussians" instrumental profile: 1.8 m s1^{-1} over 1.5 months on the RV standard star HD 122064. Therefore, we conclude that the instrumental profile need not be perfectly accurate as long as it is stable. In addition, we observed 51 Peg and our results are consistent with the literature, confirming our spectrograph and Doppler pipeline are producing accurate and precise radial velocities.Comment: 22 pages, 9 figures, submitted to PASP, Peer-Reviewed and Accepte

    Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

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    Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors
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