Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates

Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4′,6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by reduction. Trapped complexes impeded the association of DAPI in a manner dependent on the enzyme used and the location of the DAPI-binding site in relation to the lesion. Of the sequences tested, one was shown to give an accurate measure of the fraction of active molecules for each enzyme tested from both the Fpg/Nei family and HhH-GPD Nth superfamily of DNA glycosylases. The validity of the approach was demonstrated by direct comparison with current gel-based methods. Additionally, the results are supported by in silico modeling based on available crystal structures

DOE EPSCoR Initiative in Structural and computational Biology/Bioinformatics

The overall goal of the DOE EPSCoR Initiative in Structural and Computational Biology was to enhance the competiveness of Vermont research in these scientific areas. To develop self-sustaining infrastructure, we increased the critical mass of faculty, developed shared resources that made junior researchers more competitive for federal research grants, implemented programs to train graduate and undergraduate students who participated in these research areas and provided seed money for research projects. During the time period funded by this DOE initiative: (1) four new faculty were recruited to the University of Vermont using DOE resources, three in Computational Biology and one in Structural Biology; (2) technical support was provided for the Computational and Structural Biology facilities; (3) twenty-two graduate students were directly funded by fellowships; (4) fifteen undergraduate students were supported during the summer; and (5) twenty-eight pilot projects were supported. Taken together these dollars resulted in a plethora of published papers, many in high profile journals in the fields and directly impacted competitive extramural funding based on structural or computational biology resulting in 49 million dollars awarded in grants (Appendix I), a 600% return on investment by DOE, the State and University

Structure/Function Analysis of DNA-glycosylases That Repair Oxidized Purines and Pyrimidines and the Influence of Surrounding DNA Sequence on Their Interactions

The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner

Peripheral Blood Mitochondrial DNA as a Biomarker of Cerebral Mitochondrial Dysfunction Following Traumatic Brain Injury in a Porcine Model

Background Traumatic brain injury (TBI) has been shown to activate the peripheral innate immune system and systemic inflammatory response, possibly through the central release of damage associated molecular patterns (DAMPs). Our main purpose was to gain an initial understanding of the peripheral mitochondrial response following TBI, and how this response could be utilized to determine cerebral mitochondrial bioenergetics. We hypothesized that TBI would increase peripheral whole blood relative mtDNA copy number, and that these alterations would be associated with cerebral mitochondrial bioenergetics triggered by TBI. Methodology Blood samples were obtained before, 6 h after, and 25 h after focal (controlled cortical impact injury: CCI) and diffuse (rapid non-impact rotational injury: RNR) TBI. PCR primers, unique to mtDNA, were identified by aligning segments of nuclear DNA (nDNA) to mtDNA, normalizing values to nuclear 16S rRNA, for a relative mtDNA copy number. Three unique mtDNA regions were selected, and PCR primers were designed within those regions, limited to 25-30 base pairs to further ensure sequence specificity, and measured utilizing qRT-PCR. Results Mean relative mtDNA copy numbers increased significantly at 6 and 25 hrs after following both focal and diffuse traumatic brain injury. Specifically, the mean relative mtDNA copy number from three mitochondrial-specific regions pre-injury was 0.84 ± 0.05. At 6 and 25 h after diffuse non-impact TBI, mean mtDNA copy number was significantly higher: 2.07 ± 0.19 (P \u3c 0.0001) and 2.37 ± 0.42 (P \u3c 0.001), respectively. Following focal impact TBI, relative mtDNA copy number was also significantly higher, 1.35 ± 0.12 (P \u3c 0.0001) at 25 hours. Alterations in mitochondrial respiration in the hippocampus and cortex post-TBI correlated with changes in the relative mtDNA copy number measured in peripheral blood. Conclusions Alterations in peripheral blood relative mtDNA copy numbers may be a novel biosignature of cerebral mitochondrial bioenergetics with exciting translational potential for non-invasive diagnostic and interventional studies

Increased rates of large-magnitude explosive eruptions in Japan in the late Neogene and Quaternary

Tephra layers in marine sediment cores from scientific ocean drilling largely record high-magnitude silicic explosive eruptions in the Japan arc for up to the last 20 million years. Analysis of the thickness variation with distance of 180 tephra layers from a global dataset suggests that the majority of the visible tephra layers used in this study are the products of caldera-forming eruptions with magnitude (M) >6, considering their distances at the respective drilling sites to their likely volcanic sources. Frequency of visible tephra layers in cores indicates a marked increase in rates of large magnitude explosive eruptions at ~8 Ma, 6–4 Ma and further increase after ~2 Ma. These changes are attributed to major changes in tectonic plate interactions. Lower rates of large magnitude explosive volcanism in the Miocene are related to a strike-slip dominated boundary (and temporary cessation or deceleration of subduction) between the Philippine Sea Plate and southwest Japan, combined with the possibility that much of the arc in northern Japan was submerged beneath sea level partly due to previous tectonic extension of Northern Honshu related to formation of the Sea of Japan. Changes in plate motions and subduction dynamics during the ~8 Ma to present period led to (1) increased arc-normal subduction in southwest Japan (and resumption of arc volcanism) and (2) shift from extension to compression of the upper plate in northeast Japan, leading to uplift, crustal thickening and favourable conditions for accumulation of the large volumes of silicic magma needed for explosive caldera-forming eruptions

Plasma Phospholipid Saturated Fatty Acids and Incident Atrial Fibrillation: The Cardiovascular Health Study

Background: Prior studies suggest that circulating fatty acids may influence the risk of atrial fibrillation (AF), but little is known about the associations of circulating saturated fatty acids with risk of AF. Methods and Results: The study population included 2899 participants from the Cardiovascular Health Study, a community‐based longitudinal cohort of adults aged 65 years or older in the United States who were free of prevalent coronary heart disease and AF in 1992. Cox regression was used to assess the association of all the long‐chain saturated fatty acids—palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0)—with incident AF. During a median of 11.2 years of follow‐up, 707 cases of incident AF occurred. After adjustment for other AF risk factors, higher levels of circulating 16:0 were associated with a higher risk of AF (hazard ratio comparing highest and lowest quartiles: 1.48; 95% CI: 1.18, 1.86). In contrast, higher levels of circulating 18:0, 20:0, 22:0, and 24:0 were each associated with a lower risk of AF. The hazard ratios (95% CI) for AF in the top and bottom quartiles were 0.76 (95% CI: 0.61, 0.95) for 18:0; 0.78 (95% CI: 0.63, 0.97) for 20:0; 0.62 (95% CI: 0.50, 0.78) for 22:0; and 0.68 (95% CI: 0.55, 0.85) for 24:0. Conclusions: Results from this prospective cohort study of older adults demonstrate divergent associations of circulating 16:0 versus longer‐chain saturated fatty acids with incident AF, highlighting the need to investigate both determinants of these levels and potential pathways of the observed differential risk

The Revised TESS Input Catalog and Candidate Target List

We describe the catalogs assembled and the algorithms used to populate the revised TESS Input Catalog (TIC), based on the incorporation of the Gaia second data release. We also describe a revised ranking system for prioritizing stars for 2-minute cadence observations, and assemble a revised Candidate Target List (CTL) using that ranking. The TIC is available on the Mikulski Archive for Space Telescopes (MAST) server, and an enhanced CTL is available through the Filtergraph data visualization portal system at the URL http://filtergraph.vanderbilt.edu/tess_ctl.Comment: 30 pages, 16 figures, submitted to AAS Journals; provided to the community in advance of publication in conjunction with public release of the TIC/CTL on 28 May 201

The 1999 international emergency humanitarian evacuation of the Kosovars to Canada: A qualitative study of service providers' perspectives at the international, national and local levels

BACKGROUND: In response to the Kosovo crisis, Canada received 5,500 Albanian Kosovar refugees in 1999 as part of the emergency humanitarian evacuation and settlement effort. This study attempts to describe the experiences of service providers at the international, national, and local levels, involved in the organization and delivery of health and settlement services in Canada for the Kosovar refugees. METHODS: A qualitative case study design using key informant interviews was used. Nominated sampling was used to identify 17 individuals involved in the organization and delivery of health and settlement. Key themes were identified and recommendations made to provide a framework for the development of policy to guide response to future humanitarian emergencies. RESULTS: Six themes emerged: (1) A sense of being overwhelmed, (2) A multitude of health issues, (3) critical challenges in providing health care, (4) access to health and settlement services, (5) overall successes and (6) need for a coordinated approach to migration health. CONCLUSIONS: For those involved, the experience was overwhelming but rewarding. Interviewees' major concerns were the need for a more comprehensive and coordinated approach to the flow of medical information and handling of specific health problems