190 research outputs found
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Ion-Optics Calculations and Preliminary Precision Estimates of the Gas-Capable Ion Source for the 1-MV LLNL BioAMS Spectrometer
Ion-optics calculations were performed for a new ion source and injection beam line. This source, which can accept both solid and gaseous targets, will be installed onto the 1-MV BioAMS spectrometer at the Center for Accelerator Mass Spectrometry, located at Lawrence Livermore National Laboratory and will augment the current LLNL cesium-sputter solid sample ion source. The ion source and its associated injection beam line were designed to allow direct quantification of {sup 14}C/{sup 12}C and {sup 3}H/{sup 1}H isotope ratios from both solid and gaseous targets without the need for isotope switching. Once installed, this source will enable the direct linking of a nanoflow LC system to the spectrometer to provide for high-throughput LC-AMS quantitation from a continuous flow. Calculations show that, for small samples, the sensitivity of the gas-accepting ion source could be precision limited but zeptomole quantitation should be feasible
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Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae
A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures
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Editorial
The Tenth International Conference on Accelerator Mass Spectrometry (AMS-10) was held from September 5-10 at the University of California, Berkeley campus. The conference attracted 305 attendees from 26 countries who gave 144 platform presentations and presented a total of 170 posters. The conference opened with a special tribute to the late Roy Middleton, which was followed by a companion session on 'ion sourcery'. A plenary talk by Wally Broecker on his '53 years in the Radiocarbon Trenches', provided thought-provoking challenges to commonly accepted paradigms. A workshop on issues in the estimation of isotopic ratios and evaluations of activities from AMS measurements preceded the conference and a workshop on AMS in low-dose bioscience concluded it. Conference attendees had ample opportunity to sample local sights and mid-week excursions to the Napa Valley wine region and the Monterey Bay Aquarium were well attended. The social highlight of the conference was a dinner cruise on San Francisco Bay aboard the San Francisco Belle, which toured the bay on a clear evening and afforded spectacular views of the city front as well as the Bay and Golden Gate bridges. The proceedings of AMS-10 contain 140 peer-reviewed papers that detail recent developments in AMS technology and a broad range of scientific applications. The editors worked to ensure that these contributions represent original research that has not been published elsewhere. We are grateful to the many outside reviewers who provided thoughtful consideration and suggestions in their reviews of these manuscripts. The staff of the Center for Accelerator Mass Spectrometry at the Lawrence Livermore National Laboratory wishes to thank the many members of the international AMS community in allowing us to organize this conference. We are particularly grateful to the University of California's Toxic Substances Research Program, which provided key assistance with conference administration
Biological/Biomedical Accelerator Mass Spectrometry Targets. 1. Optimizing the CO2 Reduction Step Using Zinc Dust
Biological and biomedical applications of accelerator mass spectrometry (AMS) use isotope ratio mass spectrometry to quantify minute amounts of long-lived radioisotopes such as 14C. AMS target preparation involves first the oxidation of carbon (in sample of interest) to CO2 and second the reduction of CO2 to filamentous, fluffy, fuzzy, or firm graphite-like substances that coat a −400-mesh spherical iron powder (−400MSIP) catalyst. Until now, the quality of AMS targets has been variable; consequently, they often failed to produce robust ion currents that are required for reliable, accurate, precise, and high-throughput AMS for biological/biomedical applications. Therefore, we described our optimized method for reduction of CO2 to high-quality uniform AMS targets whose morphology we visualized using scanning electron microscope pictures. Key features of our optimized method were to reduce CO2 (from a sample of interest that provided 1 mg of C) using 100 ± 1.3 mg of Zn dust, 5 ± 0.4 mg of −400MSIP, and a reduction temperature of 500 °C for 3 h. The thermodynamics of our optimized method were more favorable for production of graphite-coated iron powders (GCIP) than those of previous methods. All AMS targets from our optimized method were of 100% GCIP, the graphitization yield exceeded 90%, and δ13C was −17.9 ± 0.3‰. The GCIP reliably produced strong 12C− currents and accurate and precise Fm values. The observed Fm value for oxalic acid II NIST SRM deviated from its accepted Fm value of 1.3407 by only 0.0003 ± 0.0027 (mean ± SE, n = 32), limit of detection of 14C was 0.04 amol, and limit of quantification was 0.07 amol, and a skilled analyst can prepare as many as 270 AMS targets per day. More information on the physical (hardness/color), morphological (SEMs), and structural (FT-IR, Raman, XRD spectra) characteristics of our AMS targets that determine accurate, precise, and high-hroughput AMS measurement are in the companion paper
Quality of Graphite Target for Biological/Biomedical/Environmental Applications of 14C-Accelerator Mass Spectrometry
Catalytic graphitization for 14C-accelerator mass spectrometry (14C-AMS) produced various forms of elemental carbon. Our high-throughput Zn reduction method (C/Fe = 1:5, 500 °C, 3 h) produced the AMS target of graphite-coated iron powder (GCIP), a mix of nongraphitic carbon and Fe3C. Crystallinity of the AMS targets of GCIP (nongraphitic carbon) was increased to turbostratic carbon by raising the C/Fe ratio from 1:5 to 1:1 and the graphitization temperature from 500 to 585 °C. The AMS target of GCIP containing turbostratic carbon had a large isotopic fractionation and a low AMS ion current. The AMS target of GCIP containing turbostratic carbon also yielded less accurate/precise 14C-AMS measurements because of the lower graphitization yield and lower thermal conductivity that were caused by the higher C/Fe ratio of 1:1. On the other hand, the AMS target of GCIP containing nongraphitic carbon had higher graphitization yield and better thermal conductivity over the AMS target of GCIP containing turbostratic carbon due to optimal surface area provided by the iron powder. Finally, graphitization yield and thermal conductivity were stronger determinants (over graphite crystallinity) for accurate/precise/high-throughput biological, biomedical, and environmental14C-AMS applications such as absorption, distribution, metabolism, elimination (ADME), and physiologically based pharmacokinetics (PBPK) of nutrients, drugs, phytochemicals, and environmental chemicals
Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment
Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment
Biological/Biomedical Accelerator Mass Spectrometry Targets. 2. Physical, Morphological, and Structural Characteristics
The number of biological/biomedical applications that require AMS to achieve their goals is increasing, and so is the need for a better understanding of the physical, morphological, and structural traits of high quality of AMS targets. The metrics of quality included color, hardness/texture, and appearance (photo and SEM), along with FT-IR, Raman, and powder X-ray diffraction spectra that correlate positively with reliable and intense ion currents and accuracy, precision, and sensitivity of fraction modern (Fm). Our previous method produced AMS targets of gray-colored iron−carbon materials (ICM) 20% of the time and of graphite-coated iron (GCI) 80% of the time. The ICM was hard, its FT-IR spectra lacked the sp2 bond, its Raman spectra had no detectable G′ band at 2700 cm−1, and it had more iron carbide (Fe3C) crystal than nanocrystalline graphite or graphitizable carbon (g-C). ICM produced low and variable ion current whereas the opposite was true for the graphitic GCI. Our optimized method produced AMS targets of graphite-coated iron powder (GCIP) 100% of the time. The GCIP shared some of the same properties as GCI in that both were black in color, both produced robust ion current consistently, their FT-IR spectra had the sp2 bond, their Raman spectra had matching D, G, G′, D+G, and D′′ bands, and their XRD spectra showed matching crystal size. GCIP was a powder that was easy to tamp into AMS target holders that also facilitated high throughput. We concluded that AMS targets of GCIP were a mix of graphitizable carbon and Fe3C crystal, because none of their spectra, FT-IR, Raman, or XRD, matched exactly those of the graphite standard. Nevertheless, AMS targets of GCIP consistently produced the strong, reliable, and reproducible ion currents for high-throughput AMS analysis (270 targets per skilled analyst/day) along with accurate and precise Fm values
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