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Sample records for efficient isotopic labeling

  1. Labeling Monosaccharides With Stable Isotopes.

    PubMed

    Zhang, Wenhui; Zhao, Shikai; Serianni, Anthony S

    2015-01-01

    Chemical and chemi-enzymic methods are discussed for the preparation of monosaccharide isotopomers that are singly and multiply labeled with (13)C, (2)H, (17/18)O, and (15)N isotopes. The discussion focuses primarily on chemical methods to incorporate stable isotopes into monosaccharides and not on methods to assemble labeled monosaccharides into more complex biomolecules such as oligosaccharides or oligonucleotides. Two primary isotope insertion reactions are considered: cyanohydrin reduction (CR) and molybdate-catalyzed epimerization (MCE). Both methods are described in detail, including discussions of their mechanistic features, and their advantages and limitations. The integration of CR, MCE, and other chemical synthetic processes with enzyme-mediated synthesis is also discussed to illustrate how a wide range of singly and multiply labeled monosaccharides can be prepared for subsequent use in the assembly of more complex isotopically labeled biomolecules. PMID:26577741

  2. Isotope-labeled immunoassays without radiation waste

    E-print Network

    Hammock, Bruce D.

    Isotope-labeled immunoassays without radiation waste Guomin Shan*, Wei Huang*, Shirley J. Gee with radioactive materials, and (iii) short shelf-life of the labeled re- agents. The advantage of isotopic with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay

  3. Glutathione specifically labeled with isotopes

    SciTech Connect

    Murata, K.; Abbott, W.A.; Bridges, R.J.; Meister, A.

    1985-10-01

    A procedure for synthesis of glutathione selectivity labeled with isotopes is described. A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates. The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate. The radiolabeled product is isolated by column chromatography.

  4. Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics

    E-print Network

    Lamond, Angus I.

    Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative, University of Dundee, Dundee, United Kingdom Abstract Stable Isotope Labelling by Amino acids in Cell culture allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine

  5. Simple, rapid method for the preparation of isotopically labeled formaldehyde

    DOEpatents

    Hooker, Jacob Matthew (Port Jefferson, NY); Schonberger, Matthias (Mains, DE); Schieferstein, Hanno (Aabergen, DE); Fowler, Joanna S. (Bellport, NY)

    2011-10-04

    Isotopically labeled formaldehyde (*C.sup..sctn.H.sub.2O) is prepared from labeled methyl iodide (*C.sup..sctn.H.sub.3I) by reaction with an oxygen nucleophile having a pendant leaving group. The mild and efficient reaction conditions result in good yields of *C.sup..sctn.H.sub.2O with little or no *C isotopic dilution. The simple, efficient production of .sup.11CH.sub.2O is described. The use of the .sup.11CH.sub.2O for the formation of positron emission tomography tracer compounds is described. The reaction can be incorporated into automated equipment available to radiochemistry laboratories. The isotopically labeled formaldehyde can be used in a variety of reactions to provide radiotracer compounds for imaging studies as well as for scintillation counting and autoradiography.

  6. Intrinsic isotopic 13C labelling of polyphenols.

    PubMed

    Gleichenhagen, Maike; Zimmermann, Benno F; Herzig, Birgit; Janzik, Ingar; Jahnke, Siegfried; Boner, Markus; Stehle, Peter; Galensa, Rudolf

    2013-12-01

    The intrinsic isotopic labelling of plants with (13)CO2 is an effective method to generate highly labelled compounds using photosynthesis and avoiding labour-intensive complex organic syntheses. In this study, the intrinsic isotopic labelling of polyphenols in parsley, spinach and peppermint is shown for the first time. The plants were grown in an atmosphere where (12)CO2 was replaced by (13)CO2, in order to generate highly labelled compounds. The total content of (13)C as well as the individual polyphenols were analysed by Isotopic Ratio-MS and HPLC-Iontrap-MS(n). After 34 days of plant growth under (13)CO2, degree of labelling was found to be higher than 90 atom% (13)C for most polyphenols, predominantly consisting of highly and fully labelled isotopomers; the total plant material contained more than 88 atom% (13)C. Such highly labelled compounds can be used in future studies to dissect both metabolism and bioavailability of polyphenols in humans. PMID:23870998

  7. Isotope labeling methods for relaxation measurements.

    PubMed

    Lundström, Patrik; Ahlner, Alexandra; Blissing, Annica Theresia

    2012-01-01

    Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly (15)N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on (13)C?, (13)C? or (13)CO or (1)H?. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter. PMID:23076579

  8. Syntheses and NMR studies of isotopically labelled deoxynucleosides and oligodeoxyribonucleotides

    SciTech Connect

    Gao, X.

    1986-01-01

    /sup 2/H and /sup 15/N labelled 2'-deoxyadenosine derivatives were synthesized in sufficient quantity by chemical transformation of nucleosides. Efficient synthetic routes were developed involving trifylation, nucleophilic substitution, and elimination of adenosine derivatives, followed by 1', 2'-ene deuteration to provide 1', 2'(2)-dideutero-2'-deoxyadenosine; and anhydrous diazotization substitution and sulfonylation to convert 2'-deoxyadenosine or 2'-deoxyinosine into appropriate precursors for reaction with /sup 15/N benzylamine. The 6-N-benzyl group was oxidatively removed to afford 6-/sup 15/N-2'-deoxyadenosine. Dimroth rearrangement was employed for the preparation of 1-/sup 15/N-2'-deoxyadenosine. Preliminary studies on the syntheses of oligonucleotides were carried out in order to efficiently incorporate these isotopically labelled deoxyadenosine derivatives into the hexadeoxyribonucleotide d(CGTACG) using the phosphoramidite methodology. Based on the results of these studies, hexamers containing /sup 2/H and /sup 15/N labelled 2'-deoxyadenosine were prepared.

  9. The topology of metabolic isotope labeling networks

    PubMed Central

    Weitzel, Michael; Wiechert, Wolfgang; Nöh, Katharina

    2007-01-01

    Background Metabolic Flux Analysis (MFA) based on isotope labeling experiments (ILEs) is a widely established tool for determining fluxes in metabolic pathways. Isotope labeling networks (ILNs) contain all essential information required to describe the flow of labeled material in an ILE. Whereas recent experimental progress paves the way for high-throughput MFA, large network investigations and exact statistical methods, these developments are still limited by the poor performance of computational routines used for the evaluation and design of ILEs. In this context, the global analysis of ILN topology turns out to be a clue for realizing large speedup factors in all required computational procedures. Results With a strong focus on the speedup of algorithms the topology of ILNs is investigated using graph theoretic concepts and algorithms. A rigorous determination of all cyclic and isomorphic subnetworks, accompanied by the global analysis of ILN connectivity is performed. Particularly, it is proven that ILNs always brake up into a large number of small strongly connected components (SCCs) and, moreover, there are natural isomorphisms between many of these SCCs. All presented techniques are universal, i.e. they do not require special assumptions on the network structure, bidirectionality of fluxes, measurement configuration, or label input. The general results are exemplified with a practically relevant metabolic network which describes the central metabolism of E. coli comprising 10390 isotopomer pools. Conclusion Exploiting the topological features of ILNs leads to a significant speedup of all universal algorithms for ILE evaluation. It is proven in theory and exemplified with the E. coli example that a speedup factor of about 1000 compared to standard algorithms is achieved. This widely opens the door for new high performance algorithms suitable for high throughput applications and large ILNs. Moreover, for the first time the global topological analysis of ILNs allows to comprehensively describe and understand the general patterns of label flow in complex networks. This is an invaluable tool for the structural design of new experiments and the interpretation of measured data. PMID:17727715

  10. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

    PubMed Central

    Basu, Sankha S; Blair, Ian A

    2013-01-01

    Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [13C315N]-pantothenate (vitamin B5), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2–3 weeks. PMID:22157971

  11. Weaving a two-dimensional fishing net from titanoniobate nanosheets embedded with Fe3O4 nanocrystals for highly efficient capture and isotope labeling of phosphopeptides

    NASA Astrophysics Data System (ADS)

    Chen, Xueqin; Li, Siyuan; Zhang, Xiaoxia; Min, Qianhao; Zhu, Jun-Jie

    2015-03-01

    Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests.Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests. Electronic supplementary information (ESI) available: Sequence of phosphopeptides from the digests of ?- and ?-casein percentages of the 4 methylated products from peptide ?1 at different labeling reaction times; sequence of serum phosphopeptides; XPS spectra of Nb 3d and Ti 2p in layered oxides and H+-stacked nanosheets; phosphopeptide enrichment sensitivity of bulk oxides, layered oxides and H+-stacked nanosheets; AFM image of TiNbNS; saturated adsorption isotherm for pNPP adsorbed on bulk oxides, layered oxides and H+-stacked nanosheets; XPS spectra of Fe3O4-TiNbNS nitrogen adsorption-desorption isotherms and pore size distribution curves for the Fe3O4 nanocrystals; phosphopeptide enrichment sensitivity, capacity and selectivity of the Fe3O4-TiNbNS composites; MS/MS spectra of phosphopeptides enriched from serum; linear relationship between the logarithms of peak area ratio and loading volume rat

  12. Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

    SciTech Connect

    Arlinghaus, H.F.; Kwoka, M.N.; Guo, X.Q.; Jacobson, K.B.

    1997-04-15

    A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched tin isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. 34 refs., 5 figs.

  13. Stable isotope labeling strategy based on coding theory.

    PubMed

    Kasai, Takuma; Koshiba, Seizo; Yokoyama, Jun; Kigawa, Takanori

    2015-10-01

    We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as "encoding and decoding" processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells. PMID:26293126

  14. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.

    PubMed

    Snyder, Nathaniel W; Tombline, Gregory; Worth, Andrew J; Parry, Robert C; Silvers, Jacob A; Gillespie, Kevin P; Basu, Sankha S; Millen, Jonathan; Goldfarb, David S; Blair, Ian A

    2015-04-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and ?-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants. PMID:25572876

  15. Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling*S?

    PubMed Central

    Galan, Jacob A.; Guo, Minjie; Sanchez, Elda E.; Cantu, Esteban; Rodriguez-Acosta, Alexis; Perez, John C.; Tao, W. Andy

    2008-01-01

    We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics. PMID:18089550

  16. Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

    PubMed

    Burrell, Richard C; Turley, Wesley A; Bonacorsi, Samuel J

    2015-07-01

    The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8-bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide (13)C5, (15)N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168?mg of (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate. PMID:26158758

  17. Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13-CO2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isotopic labeling of plants provides a unique opportunity for understanding metabolic processes. A significant challenge of isotopic labeling during plant growth is that isotopes must be administered without disrupting plant development and at sufficient levels for mass spectral analysis. We describ...

  18. From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes

    E-print Network

    Maruyama, Shigeo

    From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes Christian Kramberger to this peculiar place? We have used N15 and C13 isotope labeled acetonitrile during the synthesis of single-walled carbon nanotubes to investigate this process. The isotope shifts of phonons and vibrons are observed

  19. Preparation of Uniformly Isotope-labeled DNA Oligonucleotides for NMR Spectroscopy*

    E-print Network

    Clore, G. Marius

    Preparation of Uniformly Isotope-labeled DNA Oligonucleotides for NMR Spectroscopy* (Received and a double-stranded 17-mer DNA uniformly labeled with 15 N and 13 C. Recent advances in NMR spectroscopy have for the large scale preparation of uni- formly isotope-labeled DNA for NMR studies have been developed

  20. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments

    PubMed Central

    Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J.; Macallan, Derek C.; Borghans, José A. M.; Asquith, Becca

    2015-01-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated 2H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  1. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

    PubMed

    Ahmed, Raya; Westera, Liset; Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J; Macallan, Derek C; Borghans, José A M; Asquith, Becca

    2015-10-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  2. Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

    PubMed Central

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M. Francesca

    2014-01-01

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration, and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling. PMID:24457314

  3. Synthesis of Isotopically-Labeled Graphite Films by Cold-Wall Chemical Vapor Deposition and Electronic

    E-print Network

    Synthesis of Isotopically-Labeled Graphite Films by Cold-Wall Chemical Vapor Deposition the synthesis of isotopically-labeled graphite films on nickel substrates by using cold-wall chemical vapor, a nickel foil at high temperature, and a uniform graphite film was segregated from the nickel surface

  4. Simplified Synthesis of Isotopically Labeled 5,5-Dimethyl-pyrroline N-Oxide

    PubMed Central

    Leinisch, Fabian; Jiang, JinJie; Deterding, Leesa J.; Mason, Ronald P.

    2011-01-01

    5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment. PMID:21986521

  5. Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling

    DOE PAGESBeta

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; Want, Elizabeth J.; Smith, Colin; O'Maille, Paul; NordstrÖm, Anders; Morita, Hirotoshi; Qin, Chuan; Uritboonthai, Wilasinee; et al

    2008-01-01

    Comprehensive detection and quantitation of metabolites from a biological source constitute the major challenges of current metabolomics research. Two chemical derivatization methodologies, butylation and amination, were applied to human serum for ionization enhancement of a broad spectrum of metabolite classes, including steroids and amino acids. LC-ESI-MS analysis of the derivatized serum samples provided a significant signal elevation across the total ion chromatogram to over a 100-fold increase in ionization efficiency. It was also demonstrated that derivatization combined with isotopically labeled reagents facilitated the relative quantitation of derivatized metabolites from individual as well as pooled samples.

  6. Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

    DOEpatents

    Brodie, Eoin L; DeSantis, Todd Z; Karaoz, Ulas; Andersen, Gary L

    2014-12-09

    Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

  7. Raman spectroscopic and mass spectrometric investigations of the hydrogen isotopes and isotopically labelled methane

    SciTech Connect

    Jewett, J.R., Fluor Daniel Hanford

    1997-02-24

    Suitable analytical methods must be tested and developed for monitoring the individual process steps within the fuel cycle of a fusion reactor and for tritium accountability. The utility of laser-Raman spectroscopy accompanied by mass spectrometry with an Omegatron was investigated using the analysis of all hydrogen isotopes and isotopically labeled methanes as an example. The Omegatron is useful for analyzing all hydrogen isotopes mixed with the stable helium isotopes. The application of this mass spectrometer were demonstrated by analyzing mixtures of deuterated methanes. In addition, it was employed to study the radiochemical Witzbach exchange reaction between tritium and methanes. A laser-Raman spectrometer was designed for analysis of tritium-containing gases and was built from individual components. A tritium-compatible, metal-sealed Raman cuvette having windows with good optical properties and additional means for measuring the stray light was first used successfully in this work. The Raman spectra of the hydrogen isotopes were acquired in the pure rotation mode and in the rotation-vibration mode and were used for on. The deuterated methanes were measured by Raman spectroscopy, the wavenumbers determined were assigned to the corresponding vibrations, and the wavenumbers for the rotational fine-structure were summarized in tables. The fundamental Vibrations of the deuterated methanes produced Witzbach reactions were detected and assigned. The fundamental vibrations of the molecules were obtained with Raman spectroscopy for the first time in this work. The @-Raman spectrometer assembled is well suited for the analysis of tritium- containing gases and is practical in combination with mass spectrometry using an Omegatron, for studying gases used in fusion.

  8. NMR studies of two spliced leader RNAs using isotope labeling

    SciTech Connect

    Lapham, J.; Crothers, D.M.

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

  9. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells.

    PubMed

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells. PMID:19787297

  10. Soil water utilization by herbaceous species of the southern Great Plains: evidence from isotopically labeled water 

    E-print Network

    Yoder, Carolyn Kay

    1993-01-01

    Understanding spatial and temporal patterns of soil water utilization by plants has broad implications for physiological, ecological, and hydrological processes. Water labeled with the stable isotopes deuterium ('H) or oxygen-18 ("'O) was injected...

  11. Fluorination of isotopically labeled turbostratic and Bernal stacked bilayer graphene.

    PubMed

    Ek Weis, Johan; Costa, Sara D; Frank, Otakar; Bastl, Zdenek; Kalbac, Martin

    2015-01-12

    Fluorination of graphene opens up a bandgap, which creates opportunities for optoelectronics, and also paves the way for the creation of extremely thin insulating layers, which can be important for applications in devices. However, in spite of many interesting features offered by, for example, unequally doped layers in multilayered systems, most of the work has concerned the fluorination of graphene monolayers. Here, the fluorination process of graphene bilayers is investigated through high-resolution Raman mapping followed by analysis of more than 10,000 spectra of bilayer graphene. Isotopically labeled bilayers are used, allowing each individual layer in bilayer graphene to be addressed unambiguously. The fluorinated graphene is prepared through exposure to XeF2. Monolayer graphene is found to be significantly more sensitive to fluorination than bilayer graphene. Through comparison of the D/G area ratio and the position of the G band for turbostratic and Bernal stacked (AB) bilayers, it is found that the fluorination process is more effective for turbostratic than for AB-stacked bilayer graphene. The fluorination changes the electronic structure similarly for the top and bottom layers in turbostratic bilayers. However, the top layer is more sensitive than the bottom layer in AB-stacked bilayers. PMID:25394738

  12. Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

    PubMed Central

    Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

    2014-01-01

    ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

  13. Site-specific isotope labeling of long RNA for structural and mechanistic studies.

    PubMed

    Kawahara, Ikumi; Haruta, Kaichiro; Ashihara, Yuta; Yamanaka, Daichi; Kuriyama, Mituhiro; Toki, Naoko; Kondo, Yoshinori; Teruya, Kenta; Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya; Kojima, Chojiro; Tanaka, Yoshiyuki

    2012-01-01

    A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry. PMID:22080547

  14. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.

    PubMed

    Kerfah, Rime; Plevin, Michael J; Sounier, Remy; Gans, Pierre; Boisbouvier, Jerome

    2015-06-01

    Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins. Here, we describe the evolution of these approaches and discuss their impact on structural and biological studies. The relevant protocols are succinctly reviewed for single and combinatorial isotopic-labeling of methyl-containing residues, and examples of applications on challenging biological systems, including high molecular weight and membrane proteins, are presented. PMID:25881211

  15. Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.

    PubMed

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-03-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  16. Metabolic pathway of inorganic and organic selenocompounds labeled with stable isotope in Japanese quail.

    PubMed

    Anan, Yasumi; Ohbo, Ai; Tani, Yuta; Ogra, Yasumitsu

    2014-12-01

    The distribution and metabolism of an inorganic selenium (Se) compound and a selenoamino acid in quails were evaluated by speciation with inductively coupled plasma mass spectrometry (ICP-MS) and a stable isotope. Quails were orally administered stable isotope [(77)Se]-labeled selenite and selenomethionine (SeMet) at the nutritional dose of 10 ?g Se/bird. Then, the quails were dissected 3, 9, and 24 h after the administration to examine the metabolic pathway and the time-dependent change of Se. The concentrations of exogenous Se in all the organs and tissues of the SeMet-administered group were significantly higher than those of the selenite-administered group 3 h after the administration. This suggested that SeMet was more rapidly and/or efficiently incorporated into the quail body than selenite. A Se-containing protein in the serum was detected only in the SeMet-administered quails, but not in the selenite-administered quails. The major urinary Se metabolite, i.e., Se-methylseleno-N-acetyl-galactosamine (selenosugar), was detected in the quail serum after the administration of both selenite and SeMet. The endogenous amount of Se-methylated selenosugar (MeSeSug) in the serum of quails seemed to be larger than that of the rodents. We conclude that the metabolic pathway of Se in quails was the same as that in rodents, but the metabolic capacity for Se seemed to be larger in quails than in rodents. PMID:25326891

  17. Heavy atom labeled nucleotides for measurement of kinetic isotope effects.

    PubMed

    Weissman, Benjamin P; Li, Nan-Sheng; York, Darrin; Harris, Michael; Piccirilli, Joseph A

    2015-11-01

    Experimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. The implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review, we highlight current approaches to the synthesis of nucleic acids enriched site specifically for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment. PMID:25828952

  18. Turnover of Leaf Waxes in Florida Slash Pine: Results of an Isotopic Labeling Experiment

    NASA Astrophysics Data System (ADS)

    Crumsey, J.; Conte, M. H.; Weber, J. C.; Mortazavi, B.; Smith, M.; Chanton, J.

    2006-12-01

    Isotopic discrimination of terrestrial photosynthesis, atmospheric CO2 concentration, and ?13CO2 are important parameters in global carbon models that are employed to estimate global carbon sources and sinks. Yet, terrestrial isotopic discrimination can be highly variable over space and time, yielding large uncertainties of terrestrial fluxes. The isotopic composition of plant wax aerosols in continental air masses can be used as an indirect measure of the spatial and temporal patterns of photosynthetic discrimination integrated over large (subcontinental) spatial scales. However, the temporal offset between wax biosynthesis and the wax aerosol isotopic signal of photosynthetic discrimination is not well constrained. To further our understanding of this temporal lag, this study sought to determine the turnover time of conifer leaf waxes by performing an isotopic labeling experiment. Four clonal pine saplings were placed in a tent and labeled with enriched 13CO2 for one year, while another four control saplings were grown under ambient CO2. At the end of the year long enrichment, the labeled saplings were removed from the tent and placed in ambient air, such that the wax turnover rate could be determined by analyzing the resultant isotopic and molecular changes. The results of this experiment indicated that after 80 days of sequestering ambient CO2, the wax (and soluble sugar) isotopic composition of the labeled saplings varied minimally. The molecular composition of the waxes, however, did change over time. From these results we concluded that waxes are turning over, but rather than being synthesized de novo from recently fixed carbon precursors they are synthesized using carbon from stored (labeled) carbon pools. Therefore, the ?13C of conifer leaf waxes in aerosols may not reflect recent photosynthetic discrimination, but instead represents photosynthetic discrimination integrated over a longer period of time. The implications of these findings are focused on interpreting the wax aerosol ?13C as an integrative measure of past photosynthetic discrimination in global carbon cycling models, and also provide new insights on internal cycling among plant carbon pools.

  19. X13CMS: global tracking of isotopic labels in untargeted metabolomics.

    PubMed

    Huang, Xiaojing; Chen, Ying-Jr; Cho, Kevin; Nikolskiy, Igor; Crawford, Peter A; Patti, Gary J

    2014-02-01

    Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php . PMID:24397582

  20. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA.

    PubMed Central

    Batey, R T; Inada, M; Kujawinski, E; Puglisi, J D; Williamson, J R

    1992-01-01

    A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR. Images PMID:1383928

  1. Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy

    SciTech Connect

    Unkefer, C.; Hernandez, G.; Springer, P.; Trewhella, J.; Blumenthal, D.; Lidstrom, M.

    1996-04-01

    The project sought to employ stable isotope labeling and NMR spectroscopy to study protein structures and provide insight into important biochemical problems. A methylotrophic bacterial expression system has been developed for uniform deuterium and carbon-13 labeling of proteins for structural studies. These organisms grow using methanol as the sole source of carbon and energy. Because isotopically labeled methanol is relatively inexpensive, the methylotrophs are ideal for expressing proteins labeled uniformly with deuterium and/or carbon-13. This expression system has been employed to prepare deuterated troponin C. NMR spectroscopy measurements have been made on the inhibitory peptide from troponin I (residues 96--115), both as the free peptide and the peptide complexed with deuterated troponin C. Proton-NMR spectroscopy resonance-signal assignments have been made for the free peptide.

  2. Energy-efficient appliance labeling in China: Lessons for successful labeling programs in varied markets

    SciTech Connect

    Lin, Jiang; Townend, Jeanne; Fridley, David; McNeil, Gary; Silva, Tony; Clark, Robin

    2002-08-20

    Appliance ownership and production has increased dramatically in China in the past two decades. From extremely low levels in 1980, China's appliance industry has become one of the largest in the world, with sales topping U.S. $14.4 billion in 2000. In 1981, less than 1 percent of urban Chinese households owned a refrigerator; by 1998, that number had increased to over 75 percent. This dramatic increase in sales and ownership leads to an excellent opportunity to impact energy consumption in China by affecting the energy efficiency of appliances being bought and sold. In general, Chinese consumers value energy efficiency and are knowledgeable about the operating costs of major appliances. However, the Chinese marketplace does not provide information that consumers trust about the energy consumption of specific products. Thus, several interdependent organizations have emerged in China to provide information and market supports for energy efficiency. This paper describes the appliance market in China and the evolution of its standards and labeling programs and the agencies that implement them. It discusses the authors' work with these organizations in developing energy efficiency criteria and supporting an energy efficiency endorsement labeling program in China. It describes how the authors have used their experience with ENERGY STAR{reg_sign} and other programs in the U.S. to work with China to develop a successful program specific to Chinese conditions, with a particular emphasis on refrigerators. It then gives the author's market assessment of the Chinese refrigerator market and recommendations for a successful labeling program and transferable lessons for developing energy efficiency labeling programs in varied markets. This paper is based on the authors' market research, their support in setting energy efficiency criteria in China, interviews with Chinese manufacturers, retailers, and sales staff, and the development and implementation of labeling strategies and promotion in China.

  3. A free-air system for long-term stable carbon isotope labeling of adult forest trees

    EPA Science Inventory

    Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in...

  4. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  5. Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

    PubMed

    Pabst, Martin; Benešová, Iva; Fagerer, Stephan R; Jacobsen, Mathias; Eyer, Klaus; Schmidt, Gregor; Steinhoff, Robert; Krismer, Jasmin; Wahl, Fabian; Preisler, Jan; Zenobi, Renato

    2016-01-01

    We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS. PMID:26573365

  6. Protein N- and C-Termini Identification Using Mass Spectrometry and Isotopic Labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new method for protein N- and C-terminal analysis using mass spectrometry is introduced. A novel stable isotopic labeling scheme has been developed to identify terminal peptides generated from an enzyme digestion for the determination of both N- and C-termini of the protein. This method works dire...

  7. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis™, an isotopic-labeling technique for accurate biochemical profiling

    PubMed Central

    de Jong, Felice A; Beecher, Chris

    2013-01-01

    Metabolomics or biochemical profiling is a fast emerging science; however, there are still many associated bottlenecks to overcome before measurements will be considered robust. Advances in MS resolution and sensitivity, ultra pressure LC MS, ESI, and isotopic approaches such as flux analysis and stable-isotope dilution, have made it easier to quantitate biochemicals. The digitization of mass spectrometers has simplified informatic aspects. However, issues of analytical variability, ion suppression and metabolite identification still plague metabolomics investigators. These hurdles need to be overcome for accurate metabolite quantitation not only for in vitro systems, but for complex matrices such as biofluids and tissues, before it is possible to routinely identify biomarkers that are associated with the early prediction and diagnosis of diseases. In this report, we describe a novel isotopic-labeling method that uses the creation of distinct biochemical signatures to eliminate current bottlenecks and enable accurate metabolic profiling. PMID:23046270

  8. Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.

    PubMed

    Nars, G; Saurel, O; Bordes, F; Saves, I; Remaud-Siméon, M; André, I; Milon, A; Marty, A

    2014-09-01

    Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by X-ray crystallography. These features comprise a mobile domain called the lid that controls access to the catalytic site. Conformational rearrangements of the lid have been suggested to regulate lipase enzymatic activities. We used nuclear magnetic resonance to investigate the dynamics of Lip2 by exploring four expression systems, Escherichia coli, cell-free, Pichia pastoris and Y. lipolytica to produce uniformly labelled enzyme. The expression of Lip2 was assessed by determining its specific activity and measuring (15)N-(1)H HSQC spectra. Y. lipolytica turned out to be the most efficient expression system. Here, we report the first use of Y. lipolytica as an expression host for the production of uniform stable isotopic labelled protein for further structural and dynamics studies using NMR. PMID:24859677

  9. Isotope labeling pattern study of central carbon metabolites using GC/MS.

    PubMed

    Jung, Joon-Young; Oh, Min-Kyu

    2015-01-01

    Determination of fluxes by (13)C tracer experiments depends on monitoring the (13)C labeling pattern of metabolites during isotope experiments. In metabolome-based (13)C metabolic flux analysis, liquid chromatography combined with mass spectrometry or tandem mass spectrometry (LC/MS or LC/MS/MS, respectively) has been mainly used as an analytical platform for isotope pattern studies of central carbon metabolites. However, gas chromatography with mass spectrometry (GC/MS) has several advantages over LC/MS, such as high sensitivity, low cost, ease of operation, and availability of mass spectra databases for comparison. In this study, analysis of isotope pattern for central carbon metabolites using GC/MS was demonstrated. First, a proper set of mass ions for central carbon metabolites was selected based on carbon backbone information and structural isomers of mass fragment ions. A total of 34 mass fragment ions was selected and used for the quantification of 25 central carbon metabolites. Then, to quantify isotope fractions, a natural mass isotopomer library for selected mass fragment ions was constructed and subtracted from isotopomer mass spectra data. The results revealed a surprisingly high abundance of partially labeled (13)C intermediates, such as 56.4% of fructose 6-phosphate and 47.6% of dihydroxyacetone phosphate at isotopic steady state, which were generated in the pentose phosphate pathway. Finally, dynamic changes of isotope fragments of central metabolites were monitored with a U-(13)C glucose stimulus response experiment in Kluyveromyces marxianus. With a comprehensive study of isotope patterns of central carbon metabolites using GC/MS, 25 central carbon metabolites and their isotopic fractions were successfully quantified. Dynamic and precise acquisition of isotope pattern can then be used in combination with proper kinetic models to calculate metabolic fluxes. PMID:25463204

  10. Trypsin is the Primary Mechanism by which the 18O Isotopic Label is Lost in Quantitative Proteomic Studies

    PubMed Central

    Angel, Peggi M.; Orlando, Ron

    2011-01-01

    Labeling with 18O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in 18O water, which incorporates the stable isotope into the C-termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role trypsin plays in isotopic loss, several experiments were performed on the rate of de-labeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme facilitated exchange of 18O in the peptide with 16O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic facilitated exchange to the attention of laboratories using this scheme in order to prevent inaccuracies in quantitative labeling due to loss of the isotopic label. PMID:17046705

  11. Global Potential of Energy Efficiency Standards and Labeling Programs

    SciTech Connect

    McNeil, Michael A; McNeil, Michael A.; Letschert, Virginie; de la Rue du Can, Stephane

    2008-06-15

    This report estimates the global potential reductions in greenhouse gas emissions by 2030 for energy efficiency improvements associated with equipment (appliances, lighting, and HVAC) in buildings by means of energy efficiency standards and labels (EES&L). A consensus has emerged among the world's scientists and many corporate and political leaders regarding the need to address the threat of climate change through emissions mitigation and adaptation. A further consensus has emerged that a central component of these strategies must be focused around energy, which is the primary generator of greenhouse gas emissions. Two important questions result from this consensus: 'what kinds of policies encourage the appropriate transformation to energy efficiency' and 'how much impact can these policies have'? This report aims to contribute to the dialogue surrounding these issues by considering the potential impacts of a single policy type, applied on a global scale. The policy addressed in this report is Energy Efficient Standards and Labeling (EES&L) for energy-consuming equipment, which has now been implemented in over 60 countries. Mandatory energy performance standards are important because they contribute positively to a nation's economy and provide relative certainty about the outcome (both timing and magnitudes). Labels also contribute positively to a nation's economy and importantly increase the awareness of the energy-consuming public. Other policies not analyzed here (utility incentives, tax credits) are complimentary to standards and labels and also contribute in significant ways to reducing greenhouse gas emissions. We believe the analysis reported here to be the first systematic attempt to evaluate the potential of savings from EES&L for all countries and for such a large set of products. The goal of the analysis is to provide an assessment that is sufficiently well-quantified and accurate to allow comparison and integration with other strategies under consideration.

  12. Radiogenic Nd isotope labeling of the northern NE Atlantic during MIS 2

    NASA Astrophysics Data System (ADS)

    Roberts, Natalie L.; Piotrowski, Alexander M.

    2015-08-01

    Paleoceanographic reconstructions rely on chemical proxies which are controlled by physical, chemical, and biological marine parameters. The accurate interpretation of proxy records relies on the integrity of proxy-environmental relationships through time, and under changing conditions. In this study we closely examine paleo controls on authigenic Nd isotope records from five cores in the northern NE Atlantic, approximating a depth-transect, allowing spatial and temporal relationships to be reconstructed. We compare our Nd isotope records with other paleocirculation proxies, and consider the sedimentalogical controls on Nd isotope signals, by comparing ice-rafted detritus lithology and counts, detrital sediment chemistry and redox sensitive element concentrations measured on foraminifera authigenic coatings. With this suite of geochemical and sedimentalogical data we show that Nd isotope records in the northern NE Atlantic were labeled by radiogenic sediments, however this modification did not occur in the pore-waters of each core, but instead likely reflects changes in the Nd isotopic composition of deep-waters caused by the input of ice-rafted sediment during Heinrich events and the last glacial maximum. This study has implications for understanding how localized changes in the Nd isotope signal can set a watermass end-member composition, decoupling chemical proxy-circulation relationships locally, but providing a signal which can be potentially traced along the deep-water flowpath. Such scenarios must be considered in future interpretations of glacial Nd isotope records taken from within the ice-rafted detritus belt and downstream along watermass flowpaths.

  13. Mass-related inversion symmetry breaking and phonon self-energy renormalization in isotopically labeled AB-stacked bilayer graphene

    E-print Network

    Araujo, Paulo Antonio Trinidade

    A mass-related symmetry breaking in isotopically labeled bilayer graphene (2LG) was investigated during in-situ electrochemical charging of AB stacked (AB-2LG) and turbostratic (t-2LG) layers. The overlap of the two ...

  14. 2D-IR Study of a Photoswitchable Isotope-Labeled r-Helix Ellen H. G. Backus,,

    E-print Network

    Caflisch, Amedeo

    2D-IR Study of a Photoswitchable Isotope-Labeled r-Helix Ellen H. G. Backus,, Robbert Bloem, Paul M spectroscopy (2D-IR). Single-isotope labeling with 13 C18 O at various positions in the sequence was employed bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen

  15. Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies.

    PubMed

    Zhang, Tianqi O; Grechko, Maksim; Moran, Sean D; Zanni, Martin T

    2016-01-01

    This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and ?D-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of ?D-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins. PMID:26453203

  16. Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

    PubMed

    Lelyveld, Victor S; Björkbom, Anders; Ransey, Elizabeth M; Sliz, Piotr; Szostak, Jack W

    2015-12-16

    High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex. PMID:26583201

  17. Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria.

    PubMed

    Chang, Shih Chieh; Galea, Charles A; Leung, Eleanor W W; Tajhya, Rajeev B; Beeton, Christine; Pennington, Michael W; Norton, Raymond S

    2012-10-01

    The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T(EM)). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²? iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹?N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K(d) of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T(EM) cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹?N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. PMID:22659540

  18. IMPACT OF DURATION OF INFUSION OF CHOICE ISOTOPE LABEL ON ISOTOPE RECYCLING IN GLUCOSE HOMEOSTASIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purposes of this study were to quantitate the impact of the duration of infusion and choice of stable isotope of glucose on measures of glucose rate of appearance (glucose Ra) and to determine whether the differences observed were due to tracer recycling via the glycogen pool (direct pathway) or...

  19. Site-specific orientation of an ?-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    PubMed

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C?O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  20. Molecular and mass spectroscopic analysis of isotopically labeled organic residues

    NASA Technical Reports Server (NTRS)

    Mendoza-Gomez, Celia X.; Greenberg, J. Mayo; Mccain, P.; Ferris, J. P.; Briggs, R.; Degroot, M. S.; Schutte, Willem A.

    1989-01-01

    Experimental studies aimed at understanding the evolution of complex organic molecules on interstellar grains were performed. The photolysis of frozen gas mixtures of various compositions containing H2O, CO, NH3, and CH4 was studied. These species were chosen because of their astrophysical importance as deducted from observational as well as theoretical studies of ice mantles on interstellar grains. These ultraviolet photolyzed ices were warmed up in order to produce refractory organic molecules like the ones formed in molecular clouds when the icy mantles are being irradiated and warmed up either by a nearby stellar source or impulsive heating. The laboratory studies give estimates of the efficiency of production of such organic material under interstellar conditions. It is shown that the gradual carbonization of organic mantles in the diffuse cloud phase leads to higher and higher visual absorptivity - yellow residues become brown in the laboratory. The obtained results can be applied to explaining the organic components of comets and their relevance to the origin of life.

  1. Selectively Dispersed Isotope Labeling for Protein Structure Determination by Magic Angle Spinning NMR

    PubMed Central

    Eddy, Matthew T.; Belenky, Marina; Sivertsen, Astrid; Griffin, Robert G.; Herzfeld, Judith

    2013-01-01

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-13C] acetate does not label ? carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-13C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

  2. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR.

    PubMed

    Eddy, Matthew T; Belenky, Marina; Sivertsen, Astrid C; Griffin, Robert G; Herzfeld, Judith

    2013-10-01

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new "redox" approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-(13)C] acetate does not label ? carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-(13)C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

  3. Stable isotope labeling strategy for curcumin metabolite study in human liver microsomes by liquid chromatography-tandem mass spectrometry.

    PubMed

    Gao, Dan; Chen, Xiaowu; Yang, Xiaomei; Wu, Qin; Jin, Feng; Wen, Hongliang; Jiang, Yuyang; Liu, Hongxia

    2015-04-01

    The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an (18)O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the (18)O labeled curcumin was successfully synthesized. The non-labeled and (18)O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites. PMID:25592681

  4. Stable Isotope Labeling Strategy for Curcumin Metabolite Study in Human Liver Microsomes by Liquid Chromatography-Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Dan; Chen, Xiaowu; Yang, Xiaomei; Wu, Qin; Jin, Feng; Wen, Hongliang; Jiang, Yuyang; Liu, Hongxia

    2015-04-01

    The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an 18O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the 18O labeled curcumin was successfully synthesized. The non-labeled and 18O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites.

  5. Efficient methods and practical guidelines for simulating isotope effects

    NASA Astrophysics Data System (ADS)

    Ceriotti, Michele; Markland, Thomas E.

    2013-01-01

    The shift in chemical equilibria due to isotope substitution is frequently exploited to obtain insight into a wide variety of chemical and physical processes. It is a purely quantum mechanical effect, which can be computed exactly using simulations based on the path integral formalism. Here we discuss how these techniques can be made dramatically more efficient, and how they ultimately outperform quasi-harmonic approximations to treat quantum liquids not only in terms of accuracy, but also in terms of computational cost. To achieve this goal we introduce path integral quantum mechanics estimators based on free energy perturbation, which enable the evaluation of isotope effects using only a single path integral molecular dynamics trajectory of the naturally abundant isotope. We use as an example the calculation of the free energy change associated with H/D and 16O/18O substitutions in liquid water, and of the fractionation of those isotopes between the liquid and the vapor phase. In doing so, we demonstrate and discuss quantitatively the relative benefits of each approach, thereby providing a set of guidelines that should facilitate the choice of the most appropriate method in different, commonly encountered scenarios. The efficiency of the estimators we introduce and the analysis that we perform should in particular facilitate accurate ab initio calculation of isotope effects in condensed phase systems.

  6. Efficient methods and practical guidelines for simulating isotope effects.

    PubMed

    Ceriotti, Michele; Markland, Thomas E

    2013-01-01

    The shift in chemical equilibria due to isotope substitution is frequently exploited to obtain insight into a wide variety of chemical and physical processes. It is a purely quantum mechanical effect, which can be computed exactly using simulations based on the path integral formalism. Here we discuss how these techniques can be made dramatically more efficient, and how they ultimately outperform quasi-harmonic approximations to treat quantum liquids not only in terms of accuracy, but also in terms of computational cost. To achieve this goal we introduce path integral quantum mechanics estimators based on free energy perturbation, which enable the evaluation of isotope effects using only a single path integral molecular dynamics trajectory of the naturally abundant isotope. We use as an example the calculation of the free energy change associated with H/D and (16)O/(18)O substitutions in liquid water, and of the fractionation of those isotopes between the liquid and the vapor phase. In doing so, we demonstrate and discuss quantitatively the relative benefits of each approach, thereby providing a set of guidelines that should facilitate the choice of the most appropriate method in different, commonly encountered scenarios. The efficiency of the estimators we introduce and the analysis that we perform should in particular facilitate accurate ab initio calculation of isotope effects in condensed phase systems. PMID:23298033

  7. Chemical imaging of biological materials by NanoSIMS using isotopic and elemental labels

    SciTech Connect

    Weber, P K; Fallon, S J; Pett-Ridge, J; Ghosal, S; Hutcheon, I D

    2006-04-10

    The NanoSIMS 50 combines unprecedented spatial resolution (as good as 50 nm) with ultra-high sensitivity (minimum detection limit of {approx}200 atoms). The NanoSIMS 50 incorporates an array of detectors, enabling simultaneous collection of 5 species originating from the same sputtered volume of a sample. The primary ion beam (Cs{sup +} or O{sup -}) can be scanned across the sample to produce quantitative secondary ion images. This capability for multiple isotope imaging with high spatial resolution provides a novel new approach to the study of biological materials. Studies can be made of sub-regions of tissues, mammalian cells, and bacteria. Major, minor and trace element distributions can be mapped on a submicron scale, growth and metabolism can be tracked using stable isotope labels, and biogenic origin can be determined based on composition. We have applied this technique extensively to mammalian and prokaryotic cells and bacterial spores. The NanoSIMS technology enables the researcher to interrogate the fate of molecules of interest within cells and organs through elemental and isotopic labeling. Biological applications at LLNL will be discussed.

  8. Tracing bioavailability of ZnO nanoparticles using stable isotope labeling.

    PubMed

    Larner, Fiona; Dogra, Yuktee; Dybowska, Agnieszka; Fabrega, Julia; Stolpe, Björn; Bridgestock, Luke J; Goodhead, Rhys; Weiss, Dominik J; Moger, Julian; Lead, Jamie R; Valsami-Jones, Eugenia; Tyler, Charles R; Galloway, Tamara S; Rehkämper, Mark

    2012-11-01

    Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator. PMID:23050854

  9. Synthesis of two potent glucocorticoid receptor agonists labeled with carbon-14 and stable isotopes.

    PubMed

    Latli, Bachir; Reeves, Jonathan T; Tan, Zhulin; Hrapchak, Matt; Song, Jinhua J; Busacca, Carl B; Senanayake, Chris H

    2015-09-10

    Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14) C]pyrimidine [(14) C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14) C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14) C]-carbon dioxide. A chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14) C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14) C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14) C]-(18) and finally removal of the chiral auxiliary gave [(14) C]-(2) in 7% overall yield. For stable isotope syntheses, [(13) C6 ]-(1) was obtained in three steps using [(13) C4 ]-(6) and trimethylsilylacetylene-[(13) C2 ] in 26% yield, while [(2) H5 ]-(2) was obtained by first preparing the iodopyridine [(2) H5 ]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2) H5 ]-(2) in 42% overall yield. PMID:26391408

  10. Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC.

    PubMed

    Huang, Sheng-Yu; Tsai, Mei-Ling; Wu, Chin-Jen; Hsu, Jue-Liang; Ho, Shih-Hsin; Chen, Shu-Hui

    2006-03-01

    Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states. PMID:16470654

  11. Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes.

    PubMed

    Su, Xiaoling; Wang, Nan; Chen, Deying; Li, Yunong; Lu, Yingfeng; Huan, Tao; Xu, Wei; Li, Liang; Li, Lanjuan

    2016-01-15

    Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on (12)C-labeling of individual samples and (13)C-labeling of a pooled urine or pooled feces and subsequent analysis of the (13)C-/(12)C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome. PMID:26709303

  12. Isotopically labeled CO{sub 2} from stratosphere: A tracer of carbon biogeochemistry

    SciTech Connect

    Yung, Yuk L.; Thiemens, M.H.

    1993-11-01

    It has been recently discovered that it the stratosphere is a source of isotopically enriched CO{sub 2}: CO{sup 18}O and CO{sub 17}O. The cause of this isotopic enrichment is exchange between heavy O{sub 3} and CO{sub 2} via the excited radical O({sup 1D}). The research effort consists of a coordinated laboratory and model surfaces of isotopomers of CO{sub 2}. The laboratory study yields data on the chemical kinetics of oxygen exchange between CO{sub 2} and O{sub 3}. The modeling study uses the laboratory results as well as atmospheric measurements to model the source and sinks of CO{sub 2} isotopomers in the stratosphere and troposphere. It is expected that this combined study will bring new insights on the exchange of CO{sub 2} between the atmosphere and the biosphere. The goals of this study are to study the kinetic pathways for isotopic exchange between O{sub 2} and CO{sub 2} and to study O{sub 3}: the exchange rate of isotopically labelled CO{sub 2} between the stratosphere and the troposphere.

  13. Tests of isotopic separation efficiency of palladium packed columns

    SciTech Connect

    Heung, L. K.; Staack, G. C.; Klein, J. E.; Jacobs, W. D.

    2008-07-15

    The isotopic effect of palladium has been applied in different ways to separate hydrogen isotopes for many years. At Savannah River Site palladium deposited on kieselguhr (Pd/k) is used in a thermal cycling absorption process (TCAP) to purify tritium for over ten years. The need to design columns for different throughputs and the desire to advance the performance of TCAP created the need to evaluate different column designs and packing materials for their separation efficiency. In this work, columns with variations in length, diameter and metal foam presence were tested using an isotope displacement method. A simple computer model was also developed to calculate the number of theoretical separation stages based on the test results. The effects of column diameter, metal foam presence and gas flow rate were identified. (authors)

  14. Carbon allocation belowground in Pinus pinaster using stable carbon isotope pulse labeling technique

    NASA Astrophysics Data System (ADS)

    Dannoura, M.; Bosc, A.; Chipeaux, C.; Sartore, M.; Lambrot, C.; Trichet, P.; Bakker, M.; Loustau, D.; Epron, D.

    2010-12-01

    Carbon allocation belowground competes with aboveground growth and biomass production. In the other hand, it contributes to resource acquisition such as nutrient, water and carbon sequestration in soil. Thus, a better characterization of carbon flow from plant to soil and its residence time within each compartment is an important issue for understanding and modeling forest ecosystem carbon budget. 13C pulse labeling of whole crown was conducted at 4 seasons to study the fate of assimilated carbon by photosynthesis into the root on 12 year old Pinus pinaster planted in the INRA domain of Pierroton. Maritime pine is the most widely planted species in South-West Europe. Stem, root and soil CO2 effluxes and their isotope composition were measured continuously by tunable diode laser absorption spectroscopy with a trace gas analyzer (TGA 100A; Campbell Scientific) coupled to flow-through chambers. 13CO2 recovery and peak were observed in respiration of each compartment after labeling. It appeared sequentially from top of stem to bottom, and to coarse root. The maximum velocity of carbon transfer was calculated as the difference in time lag of recovery between two positions on the trunk or on the root. It ranged between 0.08-0.2 m h-1 in stem and between 0.04-0.12 m h-1 in coarse root. This velocity was higher in warmer season, and the difference between time lag of recovery and peak increased after first frost. Photosynthates arrived underground 1.5 to 5 days after labeling, at similar time in soil CO2 effluxes and coarse root respiration. 0.08-1.4 g of carbon was respired per tree during first 20 days following labeling. It presented 0.6 -10% of 13C used for labeling and it is strongly related to seasons. The isotope signal was detected in fine root organs and microbial biomass by periodical core sampling. The peak was observed 6 days after labeling in early summer while it was delayed more than 10 days in autumn and winter with less amount of carbon allocated belowground.

  15. Efficiency of background suppression for arterial spin labeling

    E-print Network

    Garcia, Dairon, 1980-

    2005-01-01

    Arterial spin labeling (ASL), a technique developed for the measurement of local tissue perfusion with MRI, is heavily dependent on distinguishing irrelevant static tissue signal from the labeled blood. Background suppression ...

  16. Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.

    PubMed

    Shen, Hongyan; An, Mingrui; Zou, Xiao; Zhao, Xuyang; Wang, Qingsong; Xing, Guowen; Ji, Jianguo

    2015-09-01

    N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis. PMID:25930663

  17. Determination of protein conformation by isotopically labelled cross-linking and dedicated software

    NASA Astrophysics Data System (ADS)

    Nielsen, Tina; Thaysen-Andersen, Morten; Larsen, Nanna; Jørgensen, Flemming S.; Houen, Gunnar; Højrup, Peter

    2007-12-01

    Chemical cross-linking in conjunction with mass spectrometry (MS) can be used for sensitive and rapid investigation of the three-dimensional structure of proteins at low resolution. However, the resulting data are very complex, and on the bioinformatic side, there still exists an urgent need for improving computer software for (semi-) automated cross-linking data analysis. In this study, we have developed dedicated software for rapid and confident identification and validation of cross-linked species using an isotopic labelled cross-linker approach in combination with MS. Deuterated (+4 Da) and non-deuterated (+0 Da) bis(sulfosuccinimidyl)suberate, BS3, was used as homobifunctional cross-linker to tag the cross-linked regions. Peptides generated from proteolysis were separated using high performance liquid chromatography, and peptide mass fingerprinting was obtained for the individual fractions using matrix-assisted laser-desorption ionisation time-of-flight (MALDI TOF) MS. The resulting peptide mass lists were combined and transferred to the program, ProteinXXX, which generated the theoretical mass values of all combinations of cross-linked peptides and dead-end cross-links and compared this to the obtained mass lists. In addition, screening for 4 Da-separated signals aided the identification of potential cross-linked species. Sequence information of these candidates was then obtained using MALDI TOF TOF. The cross-linked peptides could then be validated based on the match of the fragmentation pattern and the theoretical values produced by ProteinXXX. This semi-automated interpretation provided a high analysis speed of cross-linking data, with efficient and confident identification of cross-linked species. Four experiments using different conditions showed a high degree of reproducibility as only 1 and 2 cross-links out of 36 identified was not observed in two experiments. The method was tested using human placenta calreticulin (CRT). Based on the identified cross-links, a few corrections to a model of calreticulin obtained by homology modelling using calnexin as template can be suggested. Furthermore, the cross-links show that the C-terminal of the protein continues along the core region opposite the P-domain for at least 11 residues beyond the known structure. In addition, it was observed that the conformation of CRT does not change significantly in the presence or absence of the divalent ions, Ca2+ and Zn2+.

  18. In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

    PubMed

    Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification. PMID:16396506

  19. Determination of Polychlorinated Biphenyls in Solid Samples by Isotope Dilution Mass Spectrometry Using ³?Cl-Labeled Analogues.

    PubMed

    Somoano-Blanco, Lourdes; Rodriguez-Gonzalez, Pablo; García Fonseca, Sergio; Alonso, J Ignacio Garcia

    2015-08-01

    This work describes the first application of (37)Cl-labeled compounds to isotope dilution mass spectrometry (IDMS). The synthesis of 12 (37)Cl-labeled polychlorinated biphenyls (PCBs) was carried out by the chlorination of biphenyl with isotopically enriched chlorine gas, generated by the direct oxidation of Na(37)Cl with potassium peroxymonosulfate. After an exhaustive purification due to the presence of other congeners, the concentration and the isotopic enrichment of all (37)Cl-labeled PCBs in the mixture was determined. The proposed procedure allows the simultaneous quantification of every isotope diluted PCB congener in a single gas chromatography-tandem mass spectrometry (GC-MS/MS) injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of (37)Cl-labeled analogues provides results in agreement with the certified values of three different Certified Reference Materials (marine sediment SRM 1944, fish tissue 1947, and loamy soil CRM 962-50) and analytical figures of merit comparable to those obtained using regular IDMS procedures based on the use of commercially available (13)C-labeled analogues. PMID:26165349

  20. Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies

    PubMed Central

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains. PMID:9892643

  1. Quantitative Metabolome Analysis Based on Chromatographic Peak Reconstruction in Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry.

    PubMed

    Huan, Tao; Li, Liang

    2015-07-21

    Generating precise and accurate quantitative information on metabolomic changes in comparative samples is important for metabolomics research where technical variations in the metabolomic data should be minimized in order to reveal biological changes. We report a method and software program, IsoMS-Quant, for extracting quantitative information from a metabolomic data set generated by chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). Unlike previous work of relying on mass spectral peak ratio of the highest intensity peak pair to measure relative quantity difference of a differentially labeled metabolite, this new program reconstructs the chromatographic peaks of the light- and heavy-labeled metabolite pair and then calculates the ratio of their peak areas to represent the relative concentration difference in two comparative samples. Using chromatographic peaks to perform relative quantification is shown to be more precise and accurate. IsoMS-Quant is integrated with IsoMS for picking peak pairs and Zero-fill for retrieving missing peak pairs in the initial peak pairs table generated by IsoMS to form a complete tool for processing CIL LC-MS data. This program can be freely downloaded from the www.MyCompoundID.org web site for noncommercial use. PMID:26086729

  2. Measurement of apolipoprotein E and amyloid ? clearance rates in the mouse brain using bolus stable isotope labeling

    PubMed Central

    2012-01-01

    Background Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. Results Cohorts of mice were pulse labeled with 13?C6-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid ? (A?). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and A? clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of A? deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of A?, suggesting that ABCA1 does not regulate A? metabolism in the brain. Conclusions Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents. PMID:22512932

  3. Effect of acetaminophen on the leukocyte-labeling efficiency of indium oxine In 111

    SciTech Connect

    Augustine, S.C.; Schmelter, R.F.; Nelson, K.L.; Petersen, R.J.; Qualfe, M.A.

    1983-11-01

    The effect of acetaminophen on the labeling efficiency of leukocytes with indium oxine In 111 was studied. A blood sample was obtained from eight healthy men before and after they received acetaminophen 650 mg every four hours for 24 hours. After dividing the plasma from each sample into three portions, leukocytes were separated and labeled with indium oxine In 111. In an in vitro study, 200 ml of blood was obtained from one of the men, and the plasma was separated into four portions. Acetaminophen in 95% ethanol was added to three of the plasma fractions to produce acetaminophen concentrations of 4, 20, and 100 micrograms/ml; ethanol was added to the fourth fraction as a control. Each plasma fraction was then subdivided into three aliquots, and leukocytes were labeled as in the in vivo study. Mean leukocyte labeling efficiencies in both studies were calculated from the ratios of leukocyte radioactivity to initial radioactivity in the samples, expressed as percentages. Leukocyte labeling efficiencies before acetaminophen administration ranged from 79 to 85%; after administration, labeling efficiencies ranged from 70 to 87%. No significant differences in mean labeling efficiency before and after acetaminophen administration were noted in any of the subjects. Leukocyte labeling efficiencies in all in vitro plasma fractions were reduced, ranging from 54 to 63%, but no significant differences in labeling efficiency between any of the plasma fractions were found. Using the labeling procedures in this study, exposure of leukocytes from healthy men to acetaminophen in vivo or in vitro does not affect labeling efficiency with indium oxine In 111.

  4. Split-Field Drift Tube/Mass Spectrometry and Isotopic Labeling Techniques for Determination of Single Amino Acid Polymorphisms

    E-print Network

    Clemmer, David E.

    of Single Amino Acid Polymorphisms Stephen J. Valentine, S. Sevugarajan, Ruwan T. Kurulugama, Stormy L/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid

  5. Negative ion ESI-MS analysis of natural yellow dye flavonoids--An isotopic labelling study

    NASA Astrophysics Data System (ADS)

    McNab, Hamish; Ferreira, Ester S. B.; Hulme, Alison N.; Quye, Anita

    2009-07-01

    Flavonoids are amongst the most commonly used natural yellow colourants in paintings, as lakes, and in historical textiles as mordant dyes. In this paper, evidence from isotopically labelled substrates is used to propose negative ion electrospray collision induced decomposition mechanisms of flavones, flavonols and an isoflavone. These mechanisms include a retro-Diels-Alder fragmentation (observed for flavones and flavonols) and an M-122 fragmentation (characteristic of 3',4'-dihydroxyflavonols). In addition, the presence of a m/z 125 fragment ion is shown to be characteristic of 2'-hydroxyflavonols and an ion at m/z 149 is shown to be characteristic of 4'-hydroxyflavones. Applications of these methods are exemplified by the identification of a minor component of Dyer's camomile (Anthemis tinctoria L.) and the identification of the dye source in green threads sampled from an 18th Century Scottish tartan fragment.

  6. Isotopic labeling for the understanding of the alteration of limestone used in built cultural heritage

    NASA Astrophysics Data System (ADS)

    Saheb, Mandana; Chabas, Anne; Mertz, Jean-Didier; Rozenbaum, Olivier; Verney-Carron, Aurélie

    2015-04-01

    This project belongs to a specific work aiming at developing isotopic tools to better understand the alteration of materials used in the built cultural heritage. It is focused on the study of the alteration of limestone used in the facades of historic buildings subject to atmospheric polluted environment. Actually in the elevated parts of the buildings, water as rainfall (runoff or wet deposition) or in vapor form (condensation or dry deposition) is the main agent of alteration. Thus, the rock/water interactions need to be well understood to propose adapted solution to better preserve the buildings. To identify the water transfer within the porous limestone and locate the reaction preferential sites, two isotopic tracers (D and 18O) are used to monitor the alteration solution (D) and locate the zones containing the secondary phases (18O). The Saint-Maximin limestone used in many monuments in the suburbs of Paris (France) as a building and restoration stone has been specifically studied. Pristine materials, stones from monuments (monuments in the Paris area) and samples altered in laboratory constitute the analytical corpus to compare different stages of alteration. In a first step the stones are characterized at different scales to identify the alteration pattern (SEM-EDS, Raman microspectrometry, XRD, rugosimetry) and study the water transfers (X-ray tomography, mercury porosimetry, imbibition kinetics). The samples are then altered in the laboratory by realistic and controlled wet or dry deposition using isotopically labeled solutions to locate the reaction zones by SIMS. The multiscale characterization of the alteration pattern has allowed proposing alteration mechanisms linked to the properties of the stones and their location inside the building. Moreover, the location of the reactive zones inside the materials determined by the isotopic experiments helps examining the role of the evolution of porosity and formation of alteration products within the material, in order to estimate the alteration rate. This innovative methodology will contribute to improve the knowledge of stone alteration processes in order to develop appropriate conservation strategies for the buildings.

  7. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated ?-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  8. Efficient excitation of media for laser isotope separation

    SciTech Connect

    Borisov, S.K.; Kuz`mina, M.A.; Mishin, V.A.

    1995-07-01

    A theoretical investigation is made of the photoionisation of a three-level medium intended for laser isotope separation. The results show that the highest ionisation efficiency can be achieved by dividing the process into two stages: coherent population inversion and photoionisation. A study is made of the possibility of inversion of three-level systems for various detunings of the laser frequencies from the radiative transition frequencies. It is shown that there are detunings for which the population of the third level is maximal for any a priori selected parameters of the medium and field. Such detunings lie near a two-photon resonance and depend on the energy density and duration of the laser pulses, and also on the dipole moments of the transitions. The suitability of this method in the case of inhomogeneously broadend optically dense media is considered. It is shown that the method is most efficient in the case of counterpropagating laser pulses. The efficiency of the method is demonstrated for ytterbium. 6 refs., 5 figs.

  9. Correction: NanoSIMS analysis of an isotopically labelled organometallic ruthenium(ii) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stéphane; Croisier, Marie; Clerc-Rosset, Stéphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-01

    Correction for 'NanoSIMS analysis of an isotopically labelled organometallic ruthenium(ii) drug to probe its distribution and state in vitro' by Ronald F. S. Lee et al., Chem. Commun., 2015, DOI: . PMID:26507472

  10. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    NASA Technical Reports Server (NTRS)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  11. Tracking down sulphate-reducing microorganisms by molecular and isotope-labelling techniques

    NASA Astrophysics Data System (ADS)

    Loy, Alexander

    2010-05-01

    Sulphate-reducing microorganisms (SRM) are of great ecological importance for carbon compound degradation and sulphur cycling in many anoxic ecosystems, including marine sediments, peatlands, and oil reservoirs. However, the activity of SRM can result in oil souring and pipeline corrosion and thus is also an economic burden for the oil industry. Molecular diversity surveys based on rRNA genes and dsrAB, genes that encode major subunits of the dissimilatory sulfite reductase, indicate that our view of the natural diversity of SRM (as we know it from cultivation) is far from being complete. This enormous phylogenetic diversity complicates unbiased identification and quantification of SRM by molecular methods such as fluorescence in situ hybridization, real-time PCR or DNA microarrays. Combining these 16S rRNA and dsrAB-based molecular methods with substrate-mediated isotope labelling techniques is a potential solution for identification and functional characterization of yet uncultivated SRM. Using SRM in peatlands as an example, the problems and opportunities of these techniques for diagnosing and monitoring SRM in the environment will be discussed in this talk.

  12. Investigation of bn-44 peptide fragments using high resolution mass spectrometry and isotope labeling.

    PubMed

    Wang, Bing; Yu, Jiayi; Wang, Huixin; Wei, Zhonglin; Guo, Xinhua; Xiao, Zhaohui; Zeng, Zhoufang; Kong, Wei

    2014-12-01

    An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway. PMID:25280401

  13. Shape-Controlled Synthesis of Isotopic Yttrium-90-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging.

    PubMed

    Paik, Taejong; Chacko, Ann-Marie; Mikitsh, John L; Friedberg, Joseph S; Pryma, Daniel A; Murray, Christopher B

    2015-09-22

    Isotopically labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal-based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we report size- and shape-controlled synthesis of rare earth fluoride nanocrystals doped with the ?-emitting radioisotope yttrium-90 ((90)Y). Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts (e.g., Gd or Y) employed. Radiolabeled nanocrystals are synthesized in high radiochemical yield and purity as well as excellent radiolabel stability in the face of surface modification with different polymeric ligands. We demonstrate the Cerenkov radioluminescence imaging and magnetic resonance imaging capabilities of (90)Y-doped GdF3 nanoplates, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy. PMID:26257288

  14. Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

    NASA Astrophysics Data System (ADS)

    Zhu, Zhikai; Go, Eden P.; Desaire, Heather

    2014-06-01

    N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

  15. Labeled trees and the efficient computation of derivations

    NASA Technical Reports Server (NTRS)

    Grossman, Robert; Larson, Richard G.

    1989-01-01

    The effective parallel symbolic computation of operators under composition is discussed. Examples include differential operators under composition and vector fields under the Lie bracket. Data structures consisting of formal linear combinations of rooted labeled trees are discussed. A multiplication on rooted labeled trees is defined, thereby making the set of these data structures into an associative algebra. An algebra homomorphism is defined from the original algebra of operators into this algebra of trees. An algebra homomorphism from the algebra of trees into the algebra of differential operators is then described. The cancellation which occurs when noncommuting operators are expressed in terms of commuting ones occurs naturally when the operators are represented using this data structure. This leads to an algorithm which, for operators which are derivations, speeds up the computation exponentially in the degree of the operator. It is shown that the algebra of trees leads naturally to a parallel version of the algorithm.

  16. Efficient catalytic synthesis of dendritic polymers having internal fluorescence labels for bioconjugation.

    PubMed

    Chen, Guanghui; Felgner, Philip L; Guan, Zhibin

    2008-07-01

    Here we present an efficient synthesis of functional dendritic polymers carrying internal fluorescence labels for bioconjugation. Specifically, dendritic polymers having pyrene as fluorescence label in the core and N-hydroxysuccinimide (NHS) functional groups at the periphery were synthesized by coupling heterobifunctional PEG to hydroxyl functionalized dendritic polyethylene core. The dendritic polyethylene cores containing one pyrene label per polymer molecule were prepared through a one-step transition-metal-catalyzed polymerization using a pyrene-labeled Pd(II)-alpha-diimine chain walking catalyst. A series of pyrene-labeled dendritic scaffolds were obtained with different molecular weights and sizes. NHS active end groups were introduced to the periphery of the dendritic scaffolds through end-group functionalization. Those NHS-functionalized dendritic scaffolds were successfully used to conjugate a model protein, ovalbumin, to yield protein-polymer conjugates carrying multiple copies of protein attached to each scaffold. PMID:18517245

  17. Effect of different magnetic nanoparticle coatings on the efficiency of stem cell labeling

    NASA Astrophysics Data System (ADS)

    Horák, Daniel; Babi?, Michal; Jendelová, Pavla; Herynek, Vít; Trchová, Miroslava; Likav?anová, Katarina; Kapcalová, Miroslava; Hájek, Milan; Syková, Eva

    2009-05-01

    Maghemite nanoparticles with various coatings were prepared by the coprecipitation method and characterized by transmission electron microscopy, dynamic light scattering and IR in terms of morphology, size, polydispersity and surface coating. The labeling efficiency and the viability of both rat and human mesenchymal stem cells labeled with Endorem ®, poly( L-lysine) (PLL)-modified Endorem ®, uncoated ?-Fe 2O 3, D-mannose-, PLL- or poly( N,N-dimethylacrylamide) (PDMAAm)-coated ?-Fe 2O 3 nanoparticles were compared. Coated ?-Fe 2O 3 nanoparticles labeled cells better than did Endorem ®. High relaxation rates and in vitro magnetic resonance imaging of cells labeled with coated nanoparticles showed clearly visible contrast compared with unlabeled cells or cells labeled with Endorem ®.

  18. Convex Onion Peeling Genetic Algorithm: An Efficient Solution to Map Labeling of Point-Feature

    E-print Network

    Bae, Wan

    Convex Onion Peeling Genetic Algorithm: An Efficient Solution to Map Labeling of Point-Feature Wan-feature and develop a new genetic algorithm to solve this problem. We adopt a data struc- ture called convex onion peeling and utilize it in our pro- posed Convex Onion Peeling Genetic Algorithm (COPGA) to efficiently

  19. Biocompatible Supramolecular Catalytic One-Dimensional Nanofibers for Efficient Labeling of Live Cells.

    PubMed

    Khalily, Mohammad Aref; Gulseren, Gulcihan; Tekinay, Ayse B; Guler, Mustafa O

    2015-12-16

    Understanding complex cellular functions requires study and tracking of biomolecules such as proteins, glycans, and lipids in their natural environment. Herein, we report the first supramolecular nanocatalyst for bioorthogonal click reaction to label live cells. This biocompatible and biodegradable nanocatalyst was formed by self-assembled peptide nanofibers complexed with copper ions. The supramolecular nanocatalyst enhanced azide-alkyne cycloaddition reaction rate under physiological conditions and was shown to be useful for efficient bioorthogonal labeling of live cells. PMID:26457765

  20. Development of a New Extended Motor Product Label for Industrial Energy Efficiency 

    E-print Network

    Rogers, E.; Boteler, R.; Elliot, R. N.

    2014-01-01

    Collection Protocols • Identify and select Data Aggregator Conduct Lab and Field Studies •Collect Data •Report Findings Launch Label(s) •Manufacturers include Standard Product Specifications (Label) in marketing materials Efficiency Programs Determine Scope... Findings •Report Program Launch •Pilot Programs E x te n d e d M o to r P ro d u c t L a b e l In it ia ti v e S c h e d u le Meeting Dec 2013 Meeting Feb 2014 Meeting(s) Mar-Aug 2014 Presentation to Stakeholders Event Oct 2014 Public Launch...

  1. Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants

    PubMed Central

    Warner, Daniel; Ferreira, Luis M. M.; Breuer, Michel J. H.; Dijkstra, Jan; Pellikaan, Wilbert F.

    2013-01-01

    We describe the use of carbon stable isotope (13C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically 13C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha?1) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the ?13C (i.e. the ratio 13C:12C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27–C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K1) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p?0.002) increase with carbon chain length. K1 estimates were comparable to those of the 13C labeled digestible dry matter fraction (3.38%/h; r?=?0.61 to 0.71; p?0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of 13C versus 12C. Our results suggest that 13C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the ?13C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

  2. In Vivo Calibration of Microdialysis Using Infusion of Stable-Isotope Labeled Neurotransmitters

    PubMed Central

    2013-01-01

    In vivo calibration of microdialysis probes is required for interpreting measured concentrations. The most popular method of in vivo calibration is no-net-flux (NNF), which requires infusing several concentrations of neurotransmitters to determine in vivo recoveries (extraction fraction or Ed) and extracellular concentrations. A new method for in vivo calibration of microdialysis of neurotransmitters using glutamate (GLU) and dopamine (DA) as model analytes is reported. 13C6-DA and 13C5-GLU were perfused through microdialysis probes as internal calibrators. Using liquid chromatography with mass spectrometry, it was possible to distinguish the 13C-forms from the endogenous forms of each neurotransmitter. Ed was directly calculated by measuring the loss of the 13C-forms during infusion. The measured endogenous 12C forms of the neurotransmitters could be corrected for Ed to give calibrated extracellular concentrations in vivo. Retrodialysis of stable-isotope-labeled (SIL) neurotransmitters gave Ed and extracellular concentrations of 13C5-GLU and 13C6-DA that matched no-net-flux measurements; however, the values were obtained in a fraction of time because no added measurements were required to obtain the calibration. Ed was reduced during uptake inhibition for GLU and DA when measured by SIL retrodialysis. Because Ed is directly measured at each microdialysis fraction, it was possible to monitor changes in Ed under transient conditions created by systemic injection of uptake inhibitors. The results show that DA and GLU concentrations are underestimated by as much as 50% if not corrected for Ed during uptake inhibition. SIL retrodialysis provides equivalent information to NNF at much reduced time and animal use. PMID:23374073

  3. Development And Evaluation Of Stable Isotope And Fluorescent Labeling And Detection Methodologies For Tracking Injected Bacteria During In Situ Bioremediation

    SciTech Connect

    Mark E. Fuller; Tullis C. Onstott

    2003-12-17

    This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful with regards to the ability to effectively label bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field. However, the development of a novel enumeration method for the 13C-enriched cells, chemical reaction interface/mass spectrometry (CRIMS), was not successful due to difficulties with the proposed instrumentation. Both labeling methodologies were successfully evaluated and validated during laboratory- and field-scale bacterial transport experiments. The methods developed during this research should be useful for future bacterial transport work as well as other microbial ecology research in a variety of environments. A full bibliography of research articles and meeting presentations related to this project is included (including web links to abstracts and full text reprints).

  4. Efficient Inference and Structured Learning for Semantic Role Labeling

    E-print Network

    York oscart@google.com Kuzman Ganchev Google New York kuzman@google.com Dipanjan Das Google New York dipanjand@google.com Abstract We present a dynamic programming algorithm for efficient constrained inference

  5. Measuring supply chain carbon efficiency : a carbon label framework

    E-print Network

    Craig, Anthony (Anthony J.)

    2012-01-01

    In the near term, efficiency improvements represent a key option for reducing the impacts of climate change. The growing awareness of climate change has increased the attention regarding the carbon emissions "embedded" in ...

  6. Subcellular imaging of isotopically labeled carbon compounds in a biological sample by ion microprobe (NanoSIMS).

    PubMed

    Clode, Peta L; Stern, Richard A; Marshall, Alan T

    2007-03-01

    Here we demonstrate the technique of nanoscale secondary ion mass spectrometry, utilizing the Cameca NanoSIMS50 ion microprobe, to detect and image the metabolism of an isotopically labeled compound (NaH(13)CO(3)) in a biological sample. In particular, we have designed and verified protocols for imaging the subcellular distribution and determining the relative abundance of labeled (13)C, within the coral Galaxea fascicularis. Analyses were conducted on 1-mum thick sections of resin-embedded material, using both scanned (mapping) and static (spot analysis) Cs(+) primary ion beam of approximately 100 nm diameter. Using these samples we establish that NanoSIMS has adequate mass resolution to reliably distinguish (13)C from potential isobaric interference by (12)C(1)H and that data extracted from ion maps are comparable to those acquired by spot analyses. Independent of the method of acquisition, ratioing of (13)C to the naturally abundant (12)C is essential if meaningful data, which can be statistically compared to standard and control samples, are to be obtained. These results highlight the potential of NanoSIMS for intracellular tracking of a variety of organic and inorganic compounds labeled with stable isotopes of C, N, O, S, P, and halogens. PMID:17279515

  7. Carbon Allocation of 13CO2-labeled Photoassimilate in Larix gmelinii Saplings - A Physiological Basis for Isotope Dendroclimatology in Eastern Siberia.

    NASA Astrophysics Data System (ADS)

    Kagawa, A.; Sugimoto, A.; Maximov, T. C.

    2006-12-01

    Tree-ring density and widths have been successfully used to reconstruct summer temperatures in high- northern latitudes, although a discrepancy between tree-growth and temperature has been found for recent decades. The so-called "reduced sensitivity" of tree rings to summer temperatures has been observed especially strongly in northern Siberia (Briffa et al., 1998) and drought stress (increased water use efficiency) arose from global warming and/or increasing CO2 are suggested as causes (Barber et al. 2000, Saurer et al. 2004). By using carbon isotope ratio as an indicator of drought stress and ring-width/density as indicators of growth, we can clarify how drought stress caused by recent global warming affects wood formation of Siberian trees. However, isotope dendroclimatology is still in its infancy and our understanding of basic physiological processes of isotope signal transfer from leaves to tree rings is insufficient. In order to understand translocation, storage, and allocation of photoassimilate to different organs of trees, we pulse- labeled ten L. gmelinii growing in a continuous permafrost zone with stable 13CO2. We studied seasonal course of carbon allocation patterns of photoassimilate among needles, branches, stem and roots and also how spring, summer, and autumn photoassimilate is later used for both earlywood and latewood formation. About half of the carbon in new needles was derived from stored material. The starch pool in non- needle parts, which can be used for xylem formation, drew about 43 percent of its carbon from previous year's photoassimilate, suggesting that carbon storage is the key mechanism behind autocorrelation in (isotope) dendroclimatology. Analysis of intra-annual 13C of the tree rings formed after the labeling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it also relied on stored material in some cases. Carbon isotope chronology of recent 100 years shows that the latewood 13C contains stronger climate signal than the earlywood and is significantly correlated to July temperature and July precipitation, corresponding to the timing of carbon incorporation that constitutes latewood. The results suggest the need for separating earlywood and latewood for isotope dendroclimatological study in Siberia. References: 1) Kagawa A., Sugimoto A., & Maximov, T.C. (2006) 13CO2 pulse-labelling of photoassimilates reveals carbon allocation within and between tree rings. Plant, Cell and Environment 29, 1571-1584. 2) Kagawa A., Sugimoto A., & Maximov, T. C. (2006) Seasonal course of translocation, storage, and remobilization of 13C pulse-labeled photoassimilate in naturally growing Larix gmelinii saplings. New Phytologist 171, 793-804. 3) Kagawa A., Naito D., Sugimoto A. & Maximov T. C. (2003) Effects of spatial and temporal variability in soil moisture on widths and 13C values of eastern Siberian tree rings. Journal of Geophysical Research 108 (D16), 4500, doi:10.1029/2002JD003019.

  8. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    PubMed Central

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L.

    2014-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% 13C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation. PMID:25217022

  9. Energy Efficiency Standards and Labels in North America: Opportunities for Harmonization

    SciTech Connect

    Vanwiemcgrory, Laura; Wiel, Stephen; Van Wie McGrory, Laura; Harrington, Lloyd

    2002-05-16

    To support the North American Energy Working Group's Expert Group on Energy Efficiency (NAEWG-EE), USDOE commissioned the Collaborative Labeling and Appliance Standards Program (CLASP) to prepare a resource document comparing current standards, labels, and test procedure regulations in Canada, Mexico, and the United States. The resulting document reached the following conclusions: Out of 24 energy-using products for which at least one of the three countries has energy efficiency regulations, three products -- refrigerators/freezers, split system central air conditioners, and room air conditioners -- have similar or identical minimum energy performance standards (MEPS) in the three countries. These same three products, as well as three-phase motors, have similar or identical test procedures throughout the region. There are 10 products with different MEPS and test procedures, but which have the short-term potential to develop common test procedures, MEPS, and/or labels. Three other noteworthy areas where possible energy efficiency initiatives have potential for harmonization are standby losses, uniform endorsement labels, and a new standard or label on windows. This paper explains these conclusions and presents the underlying comparative data.

  10. Leukocyte labeling in plasma made simple and efficient: Preparation and evaluation

    SciTech Connect

    Thakur, M.L.; McKenney, S.; Seifert, C.; Madsen, M.; Suntherlingam, M.; Desai, A.; Patel, J.; Park, C.H.

    1984-01-01

    Over the past few years the use of In-111 leukocytes (In-111-L) for abscess localization has become increasingly popular. Using less toxic Mercaptopyridine-N-oxide or its Na-salt (Merc, LD/sub 50/ 340 mg/kg b.w.) rather than oxine (LD/sub 50/ 88.8 mg/kg b.w.), In-111-L could be prepared either in salt balance media or preferably by a kit procedure in plasma. In the plasma preparation L concentrated in 0.5 ml plasma are added to 25 ..mu..g dry Merc and subsequently incubated at an ambient temperature for 20 minutes with a weak chelate of In-111 either dry or in solution. Greater than 90/sup 0/ labeling efficiency is achieved which compared favorably to 10 ..mu..g and 50 ..mu..g each of oxine and tropolone. Phagocytic ability of 10 million L exposed to up to 200 ..mu..g Merc remained unaffected. In-111-L and Ga-67 citrate were given to abscess bearing dogs and abscess/blood (24 hr.) ratios of 75 and 12 respectively were achieved. Those were similar to those obtained with In-111-oxine labeled L (75.2 vs. 75.8). Abscess/liver and abscess/spleen ratios obtained with In-111-merc labeled L (14.6 and 4.2 respectively) were more favorable than those obtained with In-111-oxine labeled L (4.2d and 1.8 respectively). These indicated that less quantity of L labeled with In-111-Merc was deposited in these organs. Furthermore, L labeled with In-111-Merc cleared more rapidly from human lungs than L labeled with In-111-oxine. Data indicate that use of Merc permits efficient L labeling in plasma, provides a kit method, and preserves L physiologic function.

  11. Phosphorus use efficiency by cotton measured through 32P isotope technique

    NASA Astrophysics Data System (ADS)

    Marcante, N. C.; Muraoka, T.; Camacho, M. A.; César, F. R. C. F.; Bruno, I. P.

    2012-04-01

    Deficiency of phosphorus (P) is the major limitation to agricultural production in the Brazilian Savannah (Cerrado), which is naturally poor in this nutrient. Most of the P applied by fertilizer in Cerrado soils are converted into low solubility forms and can not be easily absorbed by plants. This occurs for characteristics of adsorption, conditioned by the predominance of low pH and aluminum and iron oxides in the clay fraction. The development of genotypes and cultivars with greater capacity to grow up in soils with low P availability ('phosphorus efficiency') is interesting to improve the agriculture in these areas in a sustainable way. Cotton (Gossypium spp.) is the main product for the fibers used nationally and globally in the textile chain. This study aim was to evaluate the efficiency of absorption and utilization of P by cotton cultivars/genotypes grown in Cerrado soil by the isotopic dilution technique. The soil classified as Ultisols, was labeled with the radioisotope 32P.The experiment was conducted in a greenhouse in a completely randomized design factorial 2 x 17. Factors were considered two levels of P (insufficient = 20 mg kg-1 and sufficient = 120 mg kg-1) and 17 genetic materials of cotton recommended for Cerrado region. Phosphorus levels influenced significantly the shoots dry matter production, the P content and accumulation, the 32P specific activity, the L value and L value less seed cotton P by cultivars and genotypes. The hierarchical clustering analysis used to verify the similarities between the cultivars and genotypes of cotton, classified them into internally homogeneous groups and heterogeneous between different groups. Cultivars FMT 523, FM 910 and CNPA GO 2043 were the most responsive to phosphate fertilizer in sufficient level of P, while the genotype Barbadense 01 and cultivars FM 966LL, IPR Jataí, BRS Aroeira and BRS Buriti were most efficient absorbing P in soils with insufficient level.

  12. An isotope-coded fluorogenic cross-linker for high-performance target identification based on photoaffinity labeling.

    PubMed

    Tomohiro, Takenori; Morimoto, Shota; Shima, Toshiya; Chiba, Junya; Hatanaka, Yasumaru

    2014-12-01

    A photoaffinity labeling (PAL)-based method for the rapid identification of target proteins is presented in which a high-performance chemical tag, an isotope-coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target-selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins. PMID:25382598

  13. Poly(ethylene glycol)-based stable isotope labeling reagents for the quantitative analysis of low molecular weight metabolites by LC-MS.

    PubMed

    Abello, Nicolas; Geurink, Paul P; van der Toorn, Marco; van Oosterhout, Antoon J M; Lugtenburg, Johan; van der Marel, Gijs A; Kerstjens, Huib A M; Postma, Dirkje S; Overkleeft, Hermen S; Bischoff, Rainer

    2008-12-01

    Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of 13C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of 13C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities. PMID:18954088

  14. Energy-efficiency labels and standards: A guidebook for appliances, equipment and lighting

    SciTech Connect

    McMahon, James E.; Wiel, Stephen

    2001-02-16

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and the United Nations Foundation (UNF) recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This guidebook was prepared over the course of the past year with significant contribution from the authors and reviewers mentioned previously. Their diligent participation has made this the international guidance tool it was intended to be. The lead authors would also like to thank the following individuals for their support in the development, production, and distribution of the guidebook: Marcy Beck, Elisa Derby, Diana Dhunke, Ted Gartner, and Julie Osborn of Lawrence Berkeley National Laboratory as well as Anthony Ma of Bevilacqua-Knight, Inc. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards-setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the programs and on the design of the labels and standards themselves. In addition, it directs the reader to references and other resources likely to be useful in conducting the activities described and includes a chapter on energy policies and programs that complement appliance efficiency labels and standards. This guidebook attempts to reflect the essential framework of labeling and standards programs. It is the intent of the authors and sponsors to distribute copies of this book worldwide at no charge for the general public benefit. The guidebook is also available on the web at www.CLASPonline.org and can be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

  15. Efficient isotope separation by single-photon atomic sorting

    SciTech Connect

    Jerkins, M.; Chavez, I.; Raizen, M. G.; Even, U.

    2010-09-15

    We propose a general and scalable approach to isotope separation. The method is based on an irreversible change of the mass-to-magnetic moment ratio of a particular isotope in an atomic beam, followed by a magnetic multipole whose gradients deflect and guide the atoms. The underlying mechanism is a reduction of the entropy of the beam by the information of a single scattered photon for each atom that is separated. We numerically simulate isotope separation for a range of examples, which demonstrate this technique's general applicability to almost the entire periodic table. The practical importance of the proposed method is that large-scale isotope separation should be possible, using ordinary inexpensive magnets and the existing technologies of supersonic beams and lasers.

  16. Optical Label Switching Technology and Energy-Efficient Future Networks S. J. Ben Yoo

    E-print Network

    Yoo, S. J. Ben

    . Future Internet with optical-label switching, wireless, and wireline networking in support of cloud on applications, statistics, and traffic patterns. OLS is possibly the most flexible and complete technologies adapting to various traffic patterns. The industry has taken a greatly simplified Energy Efficiency Index

  17. Status of China's Energy Efficiency Standards and Labels for Appliances and International Collaboration

    SciTech Connect

    Zhou, Nan

    2008-03-01

    China first adopted minimum energy performance standards (MEPS) in 1989. Today, there are standards for a wide range of domestic, commercial and selected industrial equipment. In 1999, China launched a voluntary endorsement label, which has grown to cover over 40 products including water-saving products (See Figure 1). Further, in 2005, China started a mandatory energy information label (also referred to as the 'Energy Label'). Today, the Energy Label is applied to four products including: air conditioners; household refrigerators; clothes washers; and unitary air conditioners (See Figure 2). MEPS and the voluntary endorsement labeling specifications have been updated and revised in order to reflect technology improvements to those products in the market. These programs have had an important impact in reducing energy consumption of appliances in China. Indeed, China has built up a strong infrastructure to develop and implement product standards. Historically, however, the government's primary focus has been on the technical requirements for efficiency performance. Less attention has been paid to monitoring and enforcement with a minimal commitment of resources and little expansion of administrative capacity in this area. Thus, market compliance with both mandatory standards and labeling programs has been questionable and actual energy savings may have been undermined as a result. The establishment of a regularized monitoring system for tracking compliance with the mandatory standard and energy information label in China is a major area for program improvement. Over the years, the Collaborative Labeling and Appliance Standards Program (CLASP) has partnered with several Chinese institutions to promote energy-efficient products in China. CLASP, together with its implementing partner Lawrence Berkeley National Laboratory (LBNL), has assisted China in developing and updating the above-mentioned standards and labeling programs. Because of the increasing need for the development of a monitoring system to track compliance with standards and labeling, CLASP, with support from Japan's Ministry of Economy, Trade and Industry (METI), has expanded its ongoing collaboration with the China National Institute of Standards (CNIS) to include enforcement and monitoring. CNIS has already begun working on the issue of compliance. CNIS has conducted modest sample testing in 2006 for refrigerators, freezers and room air-conditioners, and repeated the same task in 2007 with a similar sample size for three products (refrigerators, freezers, air-conditioners and clothes washers). And, CNIS, with technical support from LBNL, has analyzed the data collected through testing. At the same time, parallel effort has also been paid to look at the potential impact of the label to 2020. In conjunction with CNIS, CLASP technical experts reviewed the standards development timeline of the four products currently subject to the mandatory energy information label. CLASP, with the support of METI/IEEJ, collaborated with CNIS to develop the efficiency grades, providing: technical input to the process; comment and advice on particular technical issues; as well as evaluation of the results. In addition, in order to effectively evaluate the impact of the label on China's market, CLASP further provided assistance to CNIS to collect data on both the efficiency distribution and product volume distribution of refrigerators on the market. This short report summarizes the status of Standards and Labeling program, current enforcement and monitoring mechanism in China, and states the importance of international collaborations.

  18. USE OF OXYGEN-18 ISOTOPE LABELING FOR MEASUREMENT OF OXIDATIVE STRESS

    EPA Science Inventory

    Oxygen-18 (18-O) labeling provides a sensitive means for quantifying oxygen
    binding that occurs during in vivo oxidations. Oxidants (ozone, nitrogen
    oxides, hydrogen peroxide, etc.) are first synthesized using 18-O, then cells
    or tissues are exposed to the labeled ...

  19. CK-LPA: Efficient community detection algorithm based on label propagation with community kernel

    NASA Astrophysics Data System (ADS)

    Lin, Zhen; Zheng, Xiaolin; Xin, Nan; Chen, Deren

    2014-12-01

    With the rapid development of Web 2.0 and the rise of online social networks, finding community structures from user data has become a hot topic in network analysis. Although research achievements are numerous at present, most of these achievements cannot be adopted in large-scale social networks because of heavy computation. Previous studies have shown that label propagation is an efficient means to detect communities in social networks and is easy to implement; however, some drawbacks, such as low accuracy, high randomness, and the formation of a “monster” community, have been found. In this study, we propose an efficient community detection method based on the label propagation algorithm (LPA) with community kernel (CK-LPA). We assign a corresponding weight to each node according to node importance in the whole network and update node labels in sequence based on weight. Then, we discuss the composition of weights, the label updating strategy, the label propagation strategy, and the convergence conditions. Compared with the primitive LPA, existing drawbacks are solved by CK-LPA. Experiments and benchmarks reveal that our proposed method sustains nearly linear time complexity and exhibits significant improvements in the quality aspect of static community detection. Hence, the algorithm can be applied in large-scale social networks.

  20. Isotopic labeling studies of the mechanism of dehydrogenation of methanol to methyl formate over copper-based catalysts

    SciTech Connect

    Cant, N.W.; Tonner, S.P.; Trimm, D.L.; Wainwright, M.S.

    1985-02-01

    Deuterium labeling has been used to study the processes occurring during the conversion of methanol to methyl formate over copper catalysts at 180-210/sup 0/C and pressures of 0.3 to 1 atm. Deuterium substitution has a dramatic effect on rate, which decreases in the ratio 8:4:2:1 in the series CH/sub 3/OH, CH/sub 3/OD, CD/sub 3/OH, CD/sub 3/OD. This can be interpreted as the product of a primary kinetic isotope effect of four for replacement of CH/sub 3/ by CD/sub 3/ and a separate thermodynamic isotope effect of two on the concentration of a surface methoxy intermediate when OH is replaced by OD. The isotope effect provides strong support for a mechanism in which the slow step is the conversion of the methoxy group to formaldehyde. Reversibility of the conversion of methanol to methoxy is reflected by hydroxyl group exchange with D/sub 2/ at a rate much in excess of methyl formate production. H/sub 2//HD/D/sub 2/ equilibration rates are still faster even though methanol coverages are high. The product distribution from CD/sub 3/OD/CH/sub 3/OH mixtures shows that methyl formate formation involves transfer of an H or a D with discrimination isotope effect of two. This rules out coupling by a methoxy plus CHO step but leaves unresolved the possibility that the formate is produced by a hemiacetal intermediate or by formaldehyde dimerization. This lack of resolution results from isotopic scrambling caused by concurrent transesterification reactions as demonstrated using CD/sub 3/OH/CH/sub 3/OCHO mixtures. 24 refs., 6 tabs.

  1. Extrinsic Labeling of Staple Food Crops with Isotopic Iron Does Not Consistently Result in Full Equilibration: Revisiting the Methodology.

    PubMed

    Glahn, Raymond P; Cheng, Zhiqiang; Giri, Shree

    2015-11-01

    Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method assumes that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via in vitro digestion of varieties of maize, white beans, black beans, red beans, and lentils. Prior to digestion, foods were extrinsically labeled with (58)Fe at concentrations of 1, 10, 50, and 100% of the intrinsic (56)Fe. Following an established in vitro digestion protocol, the digest was centrifuged and the Fe solubilities of the extrinsic (58)Fe and the intrinsic (56)Fe were compared as a measure of extrinsic/intrinsic equilibration. In the beans, significantly more of the extrinsic Fe (up to 2-3 times, p < 0.001) partitioned into the supernatant. The effect varied depending upon the seed coat color, the harvest, and the concentration of the extrinsic Fe. For lentils and maize the extrinsic Fe tended to partition into the insoluble fraction and also varied depending on variety and harvest. There was no crop that consistently demonstrated full equilibration of the extrinsic Fe with the intrinsic Fe. These observations challenge the accuracy of Fe absorption studies in which isotopic extrinsic Fe was used to evaluate Fe absorption and bioavailability. PMID:26456842

  2. Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling.

    PubMed

    Vitali, Francesca; Henning, Anke; Oberstrass, Florian C; Hargous, Yann; Auweter, Sigrid D; Erat, Michèle; Allain, Frédéric H-T

    2006-01-11

    The polypyrimidine tract binding protein (PTB) is a 58 kDa protein involved in many aspects of RNA metabolism. In this study, we focused our attention on the structure of the two C-terminal RNA recognition motifs (RRM3 and RRM4) of PTB. In a previous study, it was found that the two RRMs are independent in the free state. We recently determined the structure of the same fragment in complex with RNA and found that the two RRMs interact extensively. This difference made us re-evaluate in detail the free protein structure and in particular the interdomain interface. We used a combination of NMR spectroscopy and segmental isotopic labeling to unambiguously study and characterize the interdomain interactions. An improved segmental isotopic labeling protocol was used, enabling us to unambiguously identify 130 interdomain NOEs between the two RRMs and to calculate a very precise structure. The structure reveals a large interdomain interface, resulting in a very unusual positioning of the two RRM domains relative to one another. PMID:16362043

  3. Untargeted Profiling of Tracer-Derived Metabolites Using Stable Isotopic Labeling and Fast Polarity-Switching LC–ESI-HRMS

    PubMed Central

    2014-01-01

    An untargeted metabolomics workflow for the detection of metabolites derived from endogenous or exogenous tracer substances is presented. To this end, a recently developed stable isotope-assisted LC–HRMS-based metabolomics workflow for the global annotation of biological samples has been further developed and extended. For untargeted detection of metabolites arising from labeled tracer substances, isotope pattern recognition has been adjusted to account for nonlabeled moieties conjugated to the native and labeled tracer molecules. Furthermore, the workflow has been extended by (i) an optional ion intensity ratio check, (ii) the automated combination of positive and negative ionization mode mass spectra derived from fast polarity switching, and (iii) metabolic feature annotation. These extensions enable the automated, unbiased, and global detection of tracer-derived metabolites in complex biological samples. The workflow is demonstrated with the metabolism of 13C9-phenylalanine in wheat cell suspension cultures in the presence of the mycotoxin deoxynivalenol (DON). In total, 341 metabolic features (150 in positive and 191 in negative ionization mode) corresponding to 139 metabolites were detected. The benefit of fast polarity switching was evident, with 32 and 58 of these metabolites having exclusively been detected in the positive and negative modes, respectively. Moreover, for 19 of the remaining 49 phenylalanine-derived metabolites, the assignment of ion species and, thus, molecular weight was possible only by the use of complementary features of the two ion polarity modes. Statistical evaluation showed that treatment with DON increased or decreased the abundances of many detected metabolites. PMID:25372979

  4. Discovery of Histone Modification Crosstalk Networks by Stable Isotope Labeling of Amino Acids in Cell Culture Mass Spectrometry (SILAC MS)*

    PubMed Central

    Guan, Xiaoyan; Rastogi, Neha; Parthun, Mark R.; Freitas, Michael A.

    2013-01-01

    In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation. PMID:23592332

  5. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino sugar 13C analysis showed that oxidizing catabolic pathways and anabolic pathways, i.e. building-up new cellular compounds, occurred in soils simultaneously. This involved an intensive C recycling within the microorganisms that was observed not only for cytosolic compounds but also for cell wall polymers. Fungal metabolism and fluxes were slower than bacterial intracellular C recycling and turnover. Furthermore, position-specific labeling of glutamate and subsequent 13C analysis of microbial phospholipid fatty acids (PLFA) revealed starvation pathways, which were only active in specific microbial groups in soils. These studies revealed that position-specific labeling enables the reconstruction of metabolic pathways of LMWOS within diverse microbial communities in complex media such as soil. Processes occurring simultaneously in soil i.e. 1) within individual, reversible metabolic pathways and 2) in various microbial groups could be traced by position-specific labeling in soils in situ. Tracing these pathways and understanding their regulating factors are crucial for soil C fluxomics, the extremely complex network of transformations towards mineralization versus the formation of microbial biomass compounds. Quantitative models to assess microbial group specific metabolic networks can be generated and parameterized by this approach. The submolecular knowledge of transformation steps and biochemical pathways in soils and their regulating factors is essential for understanding C cycling and long-term C storage in soils.

  6. Kinetic isotope effects significantly influence intracellular metabolite [superscript 13]C labeling patterns and flux determination

    E-print Network

    Stephanopoulos, Gregory

    Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide ...

  7. Automated LC-HRMS(/MS) Approach for the Annotation of Fragment Ions Derived from Stable Isotope Labeling-Assisted Untargeted Metabolomics

    PubMed Central

    2014-01-01

    Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native 12C- and uniformly 13C (U-13C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-13C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research. PMID:24965664

  8. Probing the Metabolic Network in Bloodstream-Form Trypanosoma brucei Using Untargeted Metabolomics with Stable Isotope Labelled Glucose

    PubMed Central

    Creek, Darren J.; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J.; Chokkathukalam, Achuthanunni; Weidt, Stefan K.; Burgess, Karl E. V.; Breitling, Rainer; Watson, David G.; Bringaud, Frédéric; Barrett, Michael P.

    2015-01-01

    Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate. PMID:25775470

  9. Histone H4 Acetylation Dynamics Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry

    PubMed Central

    Su, Xiaodan; Zhang, Liwen; Lucas, David M.; Davis, Melanie E.; Knapp, Amy R.; Green-Church, Kari B.; Marcucci, Guido; Parthun, Mark R.; Byrd, John C.; Freitas, Michael A.

    2007-01-01

    This paper describes an integrated approach that couples Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Acetic acid-Urea Polyacrylamide Gel Electrophoresis (AU-PAGE) and Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular media (Lys-D0) and media in which lysine was substituted with deuterium-labeled lysine (Lys-D4). HDAC activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture media for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Detailed information was obtained for both the change of histone H4 acetylation specific to N-terminus and global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. The current study provides quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli. PMID:17286952

  10. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A. (Santa Fe, NM); Unkefer, Clifford J. (Los Alamos, NM); Alvarez, Marc A. (Santa Fe, NM)

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  11. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

    PubMed Central

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M.; Shoffner, Matthew; Albers, Aaron E.; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  12. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

    PubMed

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  13. Rapid, high-efficiency labeling of leukocytes with In-111 after hemolytic removal of erythrocytes

    SciTech Connect

    Karesh, S.M.; Henkin, R.E.

    1985-05-01

    During the labeling of leukocytes with Indium-111, conventional methodology involves separation and washing to remove red cells. This technique results in the loss of a significant number of leukocytes. Citrated whole blood of ten normal volunteers was studied for an alternate labeling method following sedimentation for 30 to 45 minutes and low speed centrifugation of the leukocyte-rich plasma. The average labeling for these ten volunteers by Indium-111 was 90% versus 60% by the older technique. Viability as measured by the trypan blue exclusion test was greater than 95%, WBC losses were essentially zero, and no WBC clumping was observed. Eighteen patients referred for leukocyte imaging were studied by this method. In this patient population, there was 91% labeling with viability greater than 95% and no evidence of clumping. Less than 5% RBC's were noted in any lot. Indium-111 WBC activity 20 minutes post injection averaged 79% of whole blood activity. This modification results in decreased losses of white cells, reduces preparation time to less than 2 hours, and significantly improves the labeling efficiency of the final product. Liver/spleen ratios and image quality were unchanged from the original method.

  14. Status of the Local Enforcement of Energy Efficiency Standards and Labeling Program in China

    SciTech Connect

    Zhou, Nan; Zheng, Nina; Fino-Chen, Cecilia; Fridley, David; Ning, Cao

    2011-09-26

    As part of its commitment to promoting and improving the local enforcement of appliance energy efficiency standards and labeling, the China National Institute of Standardization (CNIS) launched the National and Local Enforcement of Energy Efficiency Standards and Labeling project on August 14, 2009. The project’s short-term goal is to expand the effort to improve enforcement of standards and labeling requirements to the entire country within three years, with a long-term goal of perfecting overall enforcement. For this project, Jiangsu, Shandong, Sichuan and Shanghai were selected as pilot locations. This report provides information on the local enforcement project’s recent background, activities and results as well as comparison to previous rounds of check-testing in 2006 and 2007. In addition, the report also offers evaluation on the achievement and weaknesses in the local enforcement scheme and recommendations. The results demonstrate both improvement and some backsliding. Enforcement schemes are in place in all target cities and applicable national standards and regulations were followed as the basis for local check testing. Check testing results show in general high labeling compliance across regions with 100% compliance for five products, including full compliance for all three products tested in Jiangsu province and two out of three products tested in Shandong province. Program results also identified key weaknesses in labeling compliance in Sichuan as well as in the efficiency standards compliance levels for small and medium three-phase asynchronous motors and self-ballasted fluorescent lamps. For example, compliance for the same product ranged from as low as 40% to 100% with mixed results for products that had been tested in previous rounds. For refrigerators, in particular, the efficiency standards compliance rate exhibited a wider range of 50% to 100%, and the average rate across all tested models also dropped from 96% in 2007 to 63%, possibly due to the implementation of newly strengthened efficiency standards in 2009. Areas for improvement include: Greater awareness at the local level to ensure that all manufacturers register their products with the label certification project and to minimize their resistance to inspections; improvement of the product sampling methodology to include representative testing of both large and small manufacturers and greater standardization of testing tools and procedures; and continued improvement in local enforcement efforts.

  15. Isotope-Coded and Affinity-Tagged Cross-Linking (ICATXL): An Efficient Strategy to Probe Protein Interaction Surfaces

    E-print Network

    Craik, Charles S.

    Isotope-Coded and Affinity-Tagged Cross-Linking (ICATXL): An Efficient Strategy to Probe Protein, we present a novel cross-linking strategy termed ICATXL (isotope-coded and affinity-tagged cross-linking) that relies on the use of affinity-tagged cross-linkers and isotope coding on the cross

  16. NMR Study of 100 kDa HCV IRES RNA Using Segmental Isotope Labeling Insil Kim, Peter J. Lukavsky, and Joseph D. Puglisi*

    E-print Network

    Puglisi, Joseph

    NMR Study of 100 kDa HCV IRES RNA Using Segmental Isotope Labeling Insil Kim, Peter J. Lukavsky, California 94305-5126 Received April 23, 2002 NMR has proven to be a powerful tool to determine structural and dynamic features of RNA molecules. However, the application of NMR to RNA has been limited to molecules

  17. Evaluation of the impact of matrix effect on quantification of pesticides in foods by gas chromatography-mass spectrometry using isotope-labeled internal standards.

    PubMed

    Yarita, Takashi; Aoyagi, Yoshie; Otake, Takamitsu

    2015-05-29

    The impact of the matrix effect in GC-MS quantification of pesticides in food using the corresponding isotope-labeled internal standards was evaluated. A spike-and-recovery study of nine target pesticides was first conducted using paste samples of corn, green soybean, carrot, and pumpkin. The observed analytical values using isotope-labeled internal standards were more accurate for most target pesticides than that obtained using the external calibration method, but were still biased from the spiked concentrations when a matrix-free calibration solution was used for calibration. The respective calibration curves for each target pesticide were also prepared using matrix-free calibration solutions and matrix-matched calibration solutions with blank soybean extract. The intensity ratio of the peaks of most target pesticides to that of the corresponding isotope-labeled internal standards was influenced by the presence of the matrix in the calibration solution; therefore, the observed slope varied. The ratio was also influenced by the type of injection method (splitless or on-column). These results indicated that matrix-matching of the calibration solution is required for very accurate quantification, even if isotope-labeled internal standards were used for calibration. PMID:25892640

  18. Lewis Acid-Base, Molecular Modeling, and Isotopic Labeling in a Sophomore Inorganic Chemistry Laboratory

    ERIC Educational Resources Information Center

    Nataro, Chip; Ferguson, Michelle A.; Bocage, Katherine M.; Hess, Brian J.; Ross, Vincent J.; Swarr, Daniel T.

    2004-01-01

    An experiment to prepare a deuterium labeled adduct of a Lewis acid and Lewis base, to use computational methods allowing students to visualize the LUMO of Lewis acids, the HOMO of Lewis bases and the molecular orbitals of the adduct that is formed is developed. This allows students to see the interplay between calculated and experimental results.

  19. Analysis of SRC Oncogenic Signaling in Colorectal Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts*

    PubMed Central

    Sirvent, Audrey; Vigy, Oana; Orsetti, Beatrice; Urbach, Serge; Roche, Serge

    2012-01-01

    The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [13C6]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo. PMID:23023324

  20. Insight into the Formation of Molecular Species in Laser-Induced Plasma of Isotopically Labeled Organic Samples.

    PubMed

    Glaus, Reto; Riedel, Jens; Gornushkin, Igor

    2015-10-01

    Recently, the detection of molecular species in laser-induced breakdown spectroscopy (LIBS) has gained increasing interest, particularly for isotopic analysis. In LIBS of organic materials, it is predominantly CN and C2 species that are formed, and multiple mechanisms may contribute to their formation. To gain deeper insight into the formation of these species, laser-induced plasma of (13)C and (15)N labeled organic materials was investigated in a temporally and spatially resolved manner. LIBS on fumaric acid with a (13)C labeled double bond allowed the formation mechanism of C2 to be investigated by analyzing relative signal intensities of (12)C2, (12)C(13)C, and (13)C2 molecules. In the early plasma (<5 ?s), the majority of C2 originates from association of completely atomized target molecules, whereas in the late plasma, the increased concentration of (13)C2 is due to incomplete dissociation of the carbon double bond. The degree of this fragmentation was found to be up to 80% and to depend on the type of the atmospheric gas. Spatial distributions of C2 revealed distinct differences for plasma generated in nitrogen and argon. A study of the interaction of ablated organics with ambient nitrogen showed that the ambient nitrogen contributed mainly to CN formation. The pronounced anisotropy of the C(15)N to C(14)N ratio across the diameter of the plasma was observed in the early plasma, indicating poor initial mixing of the plasma with the ambient gas. Overall, for accurate isotope analysis of organics, LIBS in argon with relatively short integration times (<10 ?s) provides the most robust results. On the other hand, if information about the original molecular structure is of interest, then experiments in nitrogen (or air) with long integration times appear to be the most promising. PMID:26402464

  1. Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.

    PubMed

    Giavalisco, Patrick; Li, Yan; Matthes, Annemarie; Eckhardt, Aenne; Hubberten, Hans-Michael; Hesse, Holger; Segu, Shruthi; Hummel, Jan; Köhl, Karin; Willmitzer, Lothar

    2011-10-01

    The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow. PMID:21699588

  2. Stable carbon isotope labeling reveals different carry-over effects between functional types of tropical trees in an Ethiopian mountain forest.

    PubMed

    Krepkowski, Julia; Gebrekirstos, Aster; Shibistova, Olga; Bräuning, Achim

    2013-07-01

    We present an intra-annual stable carbon isotope (?(13)C) study based on a labeling experiment to illustrate differences in temporal patterns of recent carbon allocation to wood structures of two functional types of trees, Podocarpus falcatus (a late-successional evergreen conifer) and Croton macrostachyus (a deciduous broadleaved pioneer tree), in a tropical mountain forest in Ethiopia. Dendrometer data, wood anatomical thin sections, and intra-annual ?(13)C analyses were applied. Isotope data revealed a clear annual growth pattern in both studied species. For P. falcatus, it was possible to synchronize annual ?(13) C peaks, wood anatomical structures and monthly precipitation patterns. The labeling signature was evident for three consecutive years. For C. macrostachyus, isotope data illustrate a rapid decline of the labeling signal within half a year. Our ?(13)C labeling study indicates a distinct difference in carryover effects between trees of different functional types. A proportion of the labeled ?(13)C is stored in reserves of wood parenchyma for up to 3 yr in P. falcatus. By contrast, C. macrostachyus shows a high turnover of assimilates and a carbon carryover effect is only detectable in the subsequent year. PMID:23586968

  3. geoRge: A Computational Tool To Detect the Presence of Stable Isotope Labeling in LC/MS-Based Untargeted Metabolomics.

    PubMed

    Capellades, Jordi; Navarro, Miriam; Samino, Sara; Garcia-Ramirez, Marta; Hernandez, Cristina; Simo, Rafael; Vinaixa, Maria; Yanes, Oscar

    2016-01-01

    Studying the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell is essential to understanding the alterations in homeostasis that occur in disease. Recently, LC/MS-based untargeted metabolomics and isotopically labeled metabolites have been used to facilitate the unbiased mapping of labeled moieties through metabolic pathways. However, due to the complexity of the resulting experimental data sets few computational tools are available for data analysis. Here we introduce geoRge, a novel computational approach capable of analyzing untargeted LC/MS data from stable isotope-labeling experiments. geoRge is written in the open language R and runs on the output structure of the XCMS package, which is in widespread use. As opposed to the few existing tools, which use labeled samples to track stable isotopes by iterating over all MS signals using the theoretical mass difference between the light and heavy isotopes, geoRge uses unlabeled and labeled biologically equivalent samples to compare isotopic distributions in the mass spectra. Isotopically enriched compounds change their isotopic distribution as compared to unlabeled compounds. This is directly reflected in a number of new m/z peaks and higher intensity peaks in the mass spectra of labeled samples relative to the unlabeled equivalents. The automated untargeted isotope annotation and relative quantification capabilities of geoRge are demonstrated by the analysis of LC/MS data from a human retinal pigment epithelium cell line (ARPE-19) grown on normal and high glucose concentrations mimicking diabetic retinopathy conditions in vitro. In addition, we compared the results of geoRge with the outcome of X(13)CMS, since both approaches rely entirely on XCMS parameters for feature selection, namely m/z and retention time values. To ensure data traceability and reproducibility, and enabling for comparison with other existing and future approaches, raw LC/MS files have been deposited in MetaboLights (MTBLS213) and geoRge is available as an R script at https://github.com/jcapelladesto/geoRge . PMID:26639619

  4. Regional cooperation in energy efficiency standard-setting and labeling in North America

    SciTech Connect

    Wiel, Stephen; Van Wie McGrory, Laura

    2003-08-04

    The North American Energy Working Group (NAEWG) was established in 2001 by the governments of Canada, Mexico, and the United States. The goals of NAEWG are to foster communication and cooperation on energy-related matters of common interest, and to enhance North American energy trade and interconnections consistent with the goal of sustainable development, for the benefit of all three countries. At its outset, NAEWG established teams to address different aspects of the energy sector. One, the Energy Efficiency Expert Group, undertook activity in three areas: (1) analyzing commonalities and differences in the test procedures of Canada, Mexico, and the United States, and identifying specific products for which the three countries might consider harmonization; (2) exploring possibilities for increased mutual recognition of laboratory test results; and (3) looking at possibilities for enhanced cooperation in the Energy Star voluntary endorsement labeling program. To support NAEWG's Expert Group on Energy Efficiency (NAEWG-EE), USDOE commissioned Lawrence Berkeley National Laboratory, representing the Collaborative Labeling and Appliance Standards Program (CLASP), to prepare a resource document comparing current standards, labels, and test procedure regulations in Canada, Mexico, and the United States. The resulting document identified 46 energy-using products for which at least one of the three countries has energy efficiency regulations. Three products--refrigerators/freezers, room air conditioners, and integral horsepower three-phase electric motors--have identical minimum energy performance standards (MEPS) and test procedures in the three countries. Ten other products have different MEPS and test procedures, but have the near-term potential for harmonization. NAEWG-EE is currently working to identify mechanisms for mutual recognition of test results. With consultative support from the United States and Canada through NAEWG-EE, Mexico is exploring possibilities for extending the Energy Star endorsement label to Mexico.

  5. [Effects of stable isotope labeled internal standard on determination of ivabradine and N-demethylivabradine in human plasma].

    PubMed

    Liu, Dong-qin; Yu, Jing-hua; Zhang, Yi-fan; Zhong, Da-fang; He, Ling; Chen, Xiao-yan

    2015-03-01

    This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of ivabradine and N-demethylivabradine in human plasma, and investigate effects of stable isotope labeled (SIL) internal standard (IS) on ivabradine. The analytes and IS were extracted from plasma by protein precipitation with acetonitrile, and chromatographied on a Capcell PAK C18 (100 mm x 4.6 mm, 5 ?m) column using a mobile phase of methanol and 5 mmol x L(-1) ammonium acetate. Multiple reaction monitoring with electrospray ionization (ESI) was used in the positive mode for mass spectrometric detection. The effect of ivabradine isotope peak [M+H+3] + on IS and the effect of SIL IS purity on ivabradine were evaluated. An appropriate concentration of SIL IS was chosen to permit method selectivity and linearity of the assay over the required range. The standard curves were demonstrated to be linear in the range of 0.100 to 60.0 ng x mL(-1) for ivabradine, and 0.050 0 to 20.0 ng x mL(-1) for N-demethylivabradine. The intra and inter day precision and accuracy were within the acceptable limits for all concentrations. Besides, the interaction between IS and ivabradine did not impact the determination of analytes. This method was successfully applied to a pharmacokinetic study of hydrogen sulfate ivabradine sustained release tablets on Chinese healthy volunteers. PMID:26118116

  6. Effect of reversible reactions on isotope label redistribution--analysis of the pentose phosphate pathway.

    PubMed

    Follstad, B D; Stephanopoulos, G

    1998-03-15

    The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon-labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6-13C]hexose 6-phosphate and [4-13C]erythrose 4-phosphate carbon enrichment values resulting from [1-13C]glucose feeding contained more information as compared to those from ribose 5-phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13C- or 14C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14C]glucose or [1-13C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets. PMID:9546650

  7. International Review of the Development and Implementation of Energy Efficiency Standards and Labeling Programs

    SciTech Connect

    Zhou, Nan; Zheng, Nina; Fridley, David

    2012-02-28

    Appliance energy efficiency standards and labeling (S&L) programs have been important policy tools for regulating the efficiency of energy-using products for over 40 years and continue to expand in terms of geographic and product coverage. The most common S&L programs include mandatory minimum energy performance standards (MEPS) that seek to push the market for efficient products, and energy information and endorsement labels that seek to pull the market. This study seeks to review and compare some of the earliest and most well-developed S&L programs in three countries and one region: the U.S. MEPS and ENERGY STAR, Australia MEPS and Energy Label, European Union MEPS and Ecodesign requirements and Energy Label and Japanese Top Runner programs. For each program, key elements of S&L programs are evaluated and comparative analyses across the programs undertaken to identify best practice examples of individual elements as well as cross-cutting factors for success and lessons learned in international S&L program development and implementation. The international review and comparative analysis identified several overarching themes and highlighted some common factors behind successful program elements. First, standard-setting and programmatic implementation can benefit significantly from a legal framework that stipulates a specific timeline or schedule for standard-setting and revision, product coverage and legal sanctions for non-compliance. Second, the different MEPS programs revealed similarities in targeting efficiency gains that are technically feasible and economically justified as the principle for choosing a standard level, in many cases at a level that no product on the current market could reach. Third, detailed survey data such as the U.S. Residential Energy Consumption Survey (RECS) and rigorous analyses provide a strong foundation for standard-setting while incorporating the participation of different groups of stakeholders further strengthen the process. Fourth, sufficient program resources for program implementation and evaluation are critical to the effectiveness of standards and labeling programs and cost-sharing between national and local governments can help ensure adequate resources and uniform implementation. Lastly, check-testing and punitive measures are important forms of enforcement while the cancellation of registration or product sales-based fines have also proven effective in reducing non-compliance. The international comparative analysis also revealed the differing degree to which the level of government decentralization has influenced S&L programs and while no single country has best practices in all elements of standards and labeling development and implementation, national examples of best practices for specific elements do exist. For example, the U.S. has exemplified the use of rigorous analyses for standard-setting and robust data source with the RECS database while Japan?s Top Runner standard-setting principle has motivated manufacturers to exceed targets. In terms of standards implementation and enforcement, Australia has demonstrated success with enforcement given its long history of check-testing and enforcement initiatives while mandatory information-sharing between EU jurisdictions on compliance results is another important enforcement mechanism. These examples show that it is important to evaluate not only the drivers of different paths of standards and labeling development, but also the country-specific context for best practice examples in order to understand how and why certain elements of specific S&L programs have been effective.

  8. ISOTOPES

    E-print Network

    Lederer, C. Michael

    2013-01-01

    nuclear electric power generation involves a number of isotope applications and accounts for most of the separated isotope production.nuclear power industry on a toll basis. According to equations 1.1, 1 .2, and 1.14, the production

  9. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  10. Encoding atlases by randomized classification forests for efficient multi-atlas label propagation.

    PubMed

    Zikic, D; Glocker, B; Criminisi, A

    2014-12-01

    We propose a method for multi-atlas label propagation (MALP) based on encoding the individual atlases by randomized classification forests. Most current approaches perform a non-linear registration between all atlases and the target image, followed by a sophisticated fusion scheme. While these approaches can achieve high accuracy, in general they do so at high computational cost. This might negatively affect the scalability to large databases and experimentation. To tackle this issue, we propose to use a small and deep classification forest to encode each atlas individually in reference to an aligned probabilistic atlas, resulting in an Atlas Forest (AF). Our classifier-based encoding differs from current MALP approaches, which represent each point in the atlas either directly as a single image/label value pair, or by a set of corresponding patches. At test time, each AF produces one probabilistic label estimate, and their fusion is done by averaging. Our scheme performs only one registration per target image, achieves good results with a simple fusion scheme, and allows for efficient experimentation. In contrast to standard forest schemes, in which each tree would be trained on all atlases, our approach retains the advantages of the standard MALP framework. The target-specific selection of atlases remains possible, and incorporation of new scans is straightforward without retraining. The evaluation on four different databases shows accuracy within the range of the state of the art at a significantly lower running time. PMID:25042602

  11. A Low-Storage-Consumption XML Labeling Method for Efficient Structural Information Extraction

    NASA Astrophysics Data System (ADS)

    Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

    Recently, labeling methods to extract and reconstruct the structural information of XML data, which are important for many applications such as XPath query and keyword search, are becoming more attractive. To achieve efficient structural information extraction, in this paper we propose C-DO-VLEI code, a novel update-friendly bit-vector encoding scheme, based on register-length bit operations combining with the properties of Dewey Order numbers, which cannot be implemented in other relevant existing schemes such as ORDPATH. Meanwhile, the proposed method also achieves lower storage consumption because it does not require either prefix schema or any reserved codes for node insertion. We performed experiments to evaluate and compare the performance and storage consumption of the proposed method with those of the ORDPATH method. Experimental results show that the execution times for extracting depth information and parent node labels using the C-DO-VLEI code are about 25% and 15% less, respectively, and the average label size using the C-DO-VLEI code is about 24% smaller, comparing with ORDPATH.

  12. NanoSIMS analysis of an isotopically labelled organometallic ruthenium(ii) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stéphane; Croisier, Marie; Clerc-Rosset, Stéphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-01

    The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(ii)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(ii) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects. PMID:26426486

  13. Separation efficiency of the MASHA facility for short-lived mercury isotopes

    NASA Astrophysics Data System (ADS)

    Rodin, A. M.; Belozerov, A. V.; Chernysheva, E. V.; Dmitriev, S. N.; Gulyaev, A. V.; Gulyaeva, A. V.; Itkis, M. G.; Kliman, J.; Kondratiev, N. A.; Krupa, L.; Novoselov, A. S.; Oganessian, Yu. Ts.; Podshibyakin, A. V.; Salamatin, V. S.; Sivá?ek, I.; Stepantsov, S. V.; Vanin, D. V.; Vedeneev, V. Yu.; Yukhimchuk, S. A.; Granja, C.; Pospisil, S.

    2014-06-01

    The mass-separator MASHA built to identify Super Heavy Elements by their mass-to-charge ratios is described. The results of the off- and on-line measurements of its separation efficiency are presented. In the former case four calibrated leaks of noble gases were used. In the latter the efficiency was measured via 284 MeV Ar beam and with using the hot catcher. The ECR ion source was used in both cases. The -radioactive isotopes of mercury produced in the complete fusion reaction Ar+SmHg+xn were detected at the mass-separator focal plane. The half-lives and the separation efficiency for the short-lived mercury isotopes were measured. Potentialities of the MEDIPIX detector system have been demonstrated for future use at the mass-separator MASHA.

  14. An Efficient Method to Calculate the Aggregated Isotopic Distribution and Exact Center-Masses

    NASA Astrophysics Data System (ADS)

    Claesen, Jürgen; Dittwald, Piotr; Burzykowski, Tomasz; Valkenborg, Dirk

    2012-04-01

    In this article, we present a computation- and memory-efficient method to calculate the probabilities of occurrence and exact center-masses of the aggregated isotopic distribution of a molecule. The method uses fundamental mathematical properties of polynomials given by the Newton-Girard theorem and Viete's formulae. The calculation is based on the atomic composition of the molecule and the natural abundances of the elemental isotopes in normal terrestrial matter. To evaluate the performance of the proposed method, which we named BRAIN, we compare it with the results obtained from five existing software packages ( IsoPro, Mercury, Emass, NeutronCluster, and IsoDalton) for 10 biomolecules. Additionally, we compare the computed mass centers with the results obtained by calculating, and subsequently aggregating, the fine isotopic distribution for two of the exemplary biomolecules. The algorithm will be made available as a Bioconductor package in R, and is also available upon request.

  15. Automated resonance assignment of the 21 kDa stereo-array isotope labeled thioldisulfide oxidoreductase DsbA

    NASA Astrophysics Data System (ADS)

    Schmidt, Elena; Ikeya, Teppei; Takeda, Mitsuhiro; Löhr, Frank; Buchner, Lena; Ito, Yutaka; Kainosho, Masatsune; Güntert, Peter

    2014-12-01

    The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13C/15N-resolved NOESY, CBCA(CO)NH, and 2D [13C/15N,1H]-HSQC were used, and 86.8% if exclusively 3D 13C/15N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa.

  16. A novel method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies

    NASA Astrophysics Data System (ADS)

    Wu, Dianming; Kampf, Christopher; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

    2014-05-01

    We developed a new method (gas-phase stripping-derivatization coupled to LC-MS) to measure the 15N atom percent excess (APE) of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye by the well-known Griess reaction in the Long Path Absorption Photometer (LOPAP). The reaction solutions containing the dye are collected at the outflow of the LOPAP, purified by solid-phase extraction and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The unlabeled azo dye (C18H19O2N5S) with a monoisotopic molecular mass of 369.41 g mol-1 can be detected as its protonated molecular ion ([M+H+], M) by HPLC-MS at a retention time of 2.8 min. Due to the natural isotope distribution M + 0, M + 1, M + 2, and M + 3 ions were considered for the calculation of the 15N APE. The optimal working range was found to be between 20 and 50% for the 15N/14N ratio. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method has been applied for the measurement of HO15NO emissions from soil in a dynamic chamber with and without spiking 15N labeled urea. Our results confirm biogenic HONO emissions from soil as HO15NO was measured after addition of 15N urea.

  17. Stable isotope dimethyl labeling combined with LTQ mass spectrometric detection, a quantitative proteomics technology used in liver cancer research

    PubMed Central

    TANG, BO; LI, YANG; ZHAO, LIANG; YUAN, SHENGGUANG; WANG, ZHENRAN; LI, BO; CHEN, QIAN

    2013-01-01

    Liver cancer is a common malignant disease, with high incidence and mortality rates. The study on the proteomics of liver cancer has attracted particular attention. The quantitative study method of proteomics depends predominantly on two-dimensional (2D) gel electrophoresis. In the present study we reported a rapid and accurate proteomics quantitative study method of high repeatability that includes the use of stable isotope labeling for the extraction of proteins and peptides via enzymolysis to achieve new type 2D capillary liquid chromatography-mass spectrometry separation using the separation mode of cation-exchange chromatography in conjunction with reversed-phase chromatography. LTQ OrbiTrap mass spectrometry detection was also performed. A total of 188 differential proteins were analyzed, including 122 upregulating [deuterium/hydrogen ratio (D/H) >1.5)] and 66 downregulating proteins (D/H<0.67). These proteins may play an important role in the occurrence, drug resistance, metastasis and recurrence of cancer or other pathological processes. Such a proteomics technology may provide biological data as well as a new methodological basis for liver cancer research. PMID:24648984

  18. Stable Isotope Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

    PubMed Central

    Higashi, Richard M.; Fan, Teresa W-M.; Lorkiewicz, Pawel K.; Moseley, Hunter N.B.; Lane, Andrew N.

    2015-01-01

    Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics is poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, Stable Isotope Resolved Metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks, in a wide range of experimental systems, including human subjects. MS offers a wide range of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS affordable by many individual laboratories, to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter will focus on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM. PMID:25270929

  19. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    SciTech Connect

    O'Grady, J; Schwender, J; Shachar-Hill, Y; Morgan, JA

    2012-03-26

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (CO2)-C-13 dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  20. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    SciTech Connect

    O'Grady J.; Schwender J.; Shachar-Hill, Y.; Morgan, J. A.

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on {sup 13}CO{sub 2} dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  1. Stable isotope labeling confirms mixotrophic nature of streamer biofilm communities at alkaline hot springs

    PubMed Central

    Schubotz, Florence; Hays, Lindsay E.; Meyer-Dombard, D'Arcy R.; Gillespie, Aimee; Shock, Everett L.; Summons, Roger E.

    2015-01-01

    Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75–88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and “Bison Pool,” using various 13C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest 13C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. 13C-glucose showed a similar, but a 10–30 times lower uptake across most fatty acids. 13C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at “Bison Pool” and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of 13C-formate occurred only at very low rates at “Bison Pool” and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. 13C-uptake into archaeal lipids occurred predominantly with 13C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth. PMID:25699032

  2. Stable isotope labeling confirms mixotrophic nature of streamer biofilm communities at alkaline hot springs.

    PubMed

    Schubotz, Florence; Hays, Lindsay E; Meyer-Dombard, D'Arcy R; Gillespie, Aimee; Shock, Everett L; Summons, Roger E

    2015-01-01

    Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75-88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and "Bison Pool," using various (13)C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest (13)C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. (13)C-glucose showed a similar, but a 10-30 times lower uptake across most fatty acids. (13)C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at "Bison Pool" and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of (13)C-formate occurred only at very low rates at "Bison Pool" and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. (13)C-uptake into archaeal lipids occurred predominantly with (13)C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth. PMID:25699032

  3. Non-homogeneity of isotopic labelling in 15N gas flux studies: theory, some observations and possible lessons

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Deppe, Marianna; Eschenbach, Wolfram; Gattinger, Andreas; Giesemann, Anette; Krause, Hans-Martin; Lewicka-Szczebak, Dominika

    2015-04-01

    Quantifying dinitrogen (N2) and nitrous oxide (N2O) fluxes from different soil N pools and processes can be accomplished using the 15N tracer technique but this is subject to four different sources of bias (i. - iv.). This approach includes 15N labelling of selected N pools in soil and subsequent isotope analysis of all relevant N pools as well as of gas samples from enclosures, i.e. mixtures of soil-derived and atmospheric N2 and N2O. Depending on the processes of interest, there may be 15N labelling of one or several N pools, were several labelling treatment are needed in the latter case (e.g. Müller et al., 2004). Measuring pool-derived N2 or N2O has been shown to include two calculation problems, (i.) arising from multiple pools (e.g. Arah, 1992) and (ii.) dealing with the non-random distribution of N2 and N2O mole masses (Hauck et al., 1958). Non-randomness can be solved if m/z 28, 29 and 30 are correctly analysed and the 15N enrichment of one (to distinguish two pools, i.e. soil and atmosphere) or two pools (in case of three pools) is known (Spott & Stange, 2008). Moreover (iii.), NO3- pools generating N2 and N2O via denitrification can be identical or different, e.g. if N2O evolved from higher enriched NO3- in deeper soil was more reduced to N2 compared to N2O evolved from N2O from shallow soil with lower enrichment, or vice versa. Apportioning N2O fluxes to NH4+ (nitrification and/or nitrifier denitrification) and NO3- (denitrification) is often conducted by NO3-labeling, measuring ?15N of emitted N2O and applying mixing equations were the measured 15N enrichment of NH4+and NO3-pool is used. However, this assumes that the average 15N enrichment of NH4+and NO3-in the soil is identical to the enrichment in the active soil domain producing N2 and/or N2O. Violation of this precondition must lead to bias in source apportionment (iv.), but to our knowledge this has not been investigated until now. Here we present conceptual models and model calculations addressing cases iii. and iv.. Furthermore we present some experimental data illustrating this. These include two data sets from denitrification experiments exhibiting substantial deviations in 15N enrichment between the N pools producing N2 and N2O. Moreover, results from a lab incubation study to quantify NH4+-derived N2O with increasing NH4+ amendment under conditions favouring nitrification are shown, were non-labelled NH4+ was added together with 15N labelled NO3-. Here we found large deviations between the 15N enrichment of NO3- in extracted soil water and the 15N enrichment of the labelled N pool as calculated from N2O isotopologues (Bergsma et al., 2001). We think that this reflects type iv. bias, probably because enrichment of NO3- in anoxic micro-sites was less diluted by non-labelled NO3- from nitrification compared to NO3- in oxic zones. Our data analysis provides a means to overcome bias iv. and thus to obtain correct source apportionment. References: Arah, J.R.M. (1992): Soil Sci. Soc. Am. J. 56, 795 - 800, 1992. Bergsma, T. et al. (2001): Env. Sci. & Technol. 35(21): 4307-4312. Hauck, R.D., et al.(1958): Soil Science 86, 287 - 291, 1958. Lewicka-Szczebak, D. et al.(2013): Rapid Comm. Mass Spectrom., 27 1548-1558. Müller, C. et al. (2004): Soil Biol. Biochem. 36(4): 619-632. Mulvaney, R.L.(1984):. Soil Sci. Soc. Am. J. 48:690 - 692. Spott, O, et al.. (2006): Rapid Comm. Mass Spectrom., 20: 3267-3274. Spott, O. and C. F. Stange (2007): Rapid Comm. Mass Spectrom., 21: 2398-2406.

  4. Improved quantification of microbial CH4 oxidation efficiency in arctic wetland soils using carbon isotope fractionation

    NASA Astrophysics Data System (ADS)

    Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.

    2013-04-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and ?13CH4 signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (?trans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

  5. Improved quantification of microbial CH4 oxidation efficiency in Arctic wetland soils using carbon isotope fractionation

    NASA Astrophysics Data System (ADS)

    Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.

    2012-12-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the Arctic will cause a deeper permafrost thawing followed by increased carbon mineralization and CH4 formation in water saturated tundra soils which might cause a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River Delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and ?13CH4-signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (e.g. landfill cover soils) have assumed a gas transport dominated by advection (?trans = 1). In tundra soils, however, diffusion is the main gas transport mechanism, aside from ebullition. Hence, diffusive stable isotope fractionation has to be considered. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox, = 1.017 ± 0.009) and needs to be determined individually. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged organic rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost affected ecosystems and their potential strengths in response to global warming.

  6. Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

    SciTech Connect

    Shimizu, K.; Yamaga, N.; Kohara, H.

    1988-03-01

    A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with /sup 2/H/sub 2/O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using (/sup 2/H)diethylene glycol and (/sup 2/H)hydrazine hydrate afforded (11,11,12,12,23,23(-2)H)lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-(11,11,12,12(-2)H)pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-(11,11,12,12(-2)H) progesterone (XIV), consisting of 0.3% /sup 2/H0-, 1.1% /sup 2/H/sub 1/-, 8.6% /sup 2/H/sub 2/-, 37.1% /sup 2/H/sub 3/-, 52.1% /sup 2/H/sub 4/-, and 0.8% /sup 2/H/sub 5/-species.

  7. Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Hao, Yan-Hong; Liu, Ming-Zhou; Yue, Jiang; Ni, Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-09-01

    Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia. PMID:26253834

  8. Energy-Efficiency Labels and Standards: A Guidebook forAppliances, Equipment, and Lighting - 2nd Edition

    SciTech Connect

    Wiel, Stephen; McMahon, James E.

    2005-04-28

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and several other organizations identified on the cover of this guidebook recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This second edition of the guidebook was prepared over the course of the past year, four years after the preparation of the first edition, with a significant contribution from the authors and reviewers mentioned previously. Their diligent participation helps maintain this book as the international guidance tool it has become. The lead authors would like to thank the members of the Communications Office of the Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory for their support in the development, production, and distribution of the guidebook. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the programs and on the design of the labels and standards themselves. In addition, it directs the reader to references and other resources likely to be useful in conducting the activities described and includes a chapter on energy policies and programs that complement appliance efficiency labels and standards. This guidebook attempts to reflect the essential framework of labeling and standards programs. It is the intent of the authors and sponsor to distribute copies of this book worldwide, at no charge, for the general public benefit. The guidebook is also available on the web at www.clasponline.org and may be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

  9. High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

    PubMed Central

    Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

    2008-01-01

    Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

  10. Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme-labeled fluorescence, and turnover times

    NASA Astrophysics Data System (ADS)

    McLaughlin, Karen; Sohm, Jill A.; Cutter, Gregory A.; Lomas, Michael W.; Paytan, Adina

    2013-04-01

    Dissolved inorganic phosphorus (DIP) concentrations in surface water of vast areas of the ocean are extremely low (<10 nM) and phosphorus (P) availability could limit primary productivity in these regions. We explore the use of oxygen isotopic signature of dissolved phosphate (?18OPO4) to investigate biogeochemical cycling of P in the Sargasso Sea, Atlantic Ocean. Additional techniques for studying P dynamics including 33P-based DIP turnover time estimates and percent of cells expressing alkaline phosphatase (AP) activity as measured by enzyme-labeling fluorescence are also used. In surface waters, ?18OPO4 values were lower than equilibrium by 3-6‰, indicative of dissolved organic phosphorous (DOP) remineralization by extracellular enzymes. An isotope mass balance model using a variety of possible combinations of enzymatic pathways and substrates indicates that DOP remineralization in the euphotic zone can account for a large proportion on P utilized by phytoplankton (as much as 82%). Relatively short DIP turnover times (4-8 h) and high expression of AP (38-77% of the cells labeled) are consistent with extensive DOP utilization and low DIP availability in the euphotoc zone. In deep water where DOP utilization rates are lower, ?18OPO4 values approach isotopic equilibrium and DIP turnover times are longer. Our data suggests that in the euphotic zone of the Sargasso Sea, DOP may be appreciably remineralized and utilized by phytoplankton and bacteria to supplement cellular requirements. A substantial fraction of photosynthesis in this region is supported by DOP uptake.

  11. Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

    PubMed

    Brereton, Nicholas J B; Pitre, Frederic E; Shield, Ian; Hanley, Steven J; Ray, Michael J; Murphy, Richard J; Karp, Angela

    2014-11-01

    Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production. PMID:24186940

  12. Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra

    PubMed Central

    Xiao, Kaijie; Yu, Fan; Fang, Houqin; Xue, Bingbing; Liu, Yan; Tian, Zhixin

    2015-01-01

    It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1?+?RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available. PMID:26439836

  13. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

    PubMed

    Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

    2013-01-01

    Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 ?g/kg and a limit of quantitation (LOQ) of 2.89 ?g/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 ?g/kg. PMID:23230907

  14. Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard.

    PubMed

    Zhang, Jingshun; Lai, Shiyun; Cai, Zengxuan; Chen, Qi; Huang, Baifen; Ren, Yiping

    2014-06-01

    A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10-1000 nmol L(-1) showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD<6.5% and RSD<7.1%, respectively. Excellent repeatability (RSD<6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas. PMID:24856400

  15. Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach

    NASA Astrophysics Data System (ADS)

    Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry

    2012-05-01

    In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 ?m diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (??13C) when supplied with diatoms, + 1364‰ (??15N) when supplied with bacteria, and + 24‰ (??13C) and + 135‰ (??15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae and their preys.

  16. Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

    PubMed Central

    Nishiyama, Yusuke; Endo, Yuki; Nemoto, Takahiro; Yamauchi, Kazuo; Asakura, Tetsuo; Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune; Ishii, Yoshitaka

    2015-01-01

    We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems. PMID:25856081

  17. Earthworm Uptake Routes and Rates of Ionic Zn and ZnO Nanoparticles at Realistic Concentrations, Traced Using Stable Isotope Labeling.

    PubMed

    Laycock, Adam; Diez-Ortiz, Maria; Larner, Fiona; Dybowska, Agnieszka; Spurgeon, David; Valsami-Jones, Eugenia; Rehkämper, Mark; Svendsen, Claus

    2016-01-01

    The environmental behavior of ZnO nanoparticles (NPs), their availability to, uptake pathways by, and biokinetics in the earthworm Lumbricus rubellus were investigated using stable isotope labeling. Zinc isotopically enriched to 99.5% in (68)Zn ((68)Zn-E) was used to prepare (68)ZnO NPs and a dissolved phase of (68)Zn for comparison. These materials enabled tracing of environmentally relevant (below background) NP additions to soil of only 5 mg (68)Zn-E kg(-1). Uptake routes were isolated by introducing earthworms with sealed and unsealed mouthparts into test soils for up to 72 h. The Zn isotope compositions of the soils, pore waters and earthworms were then determined using multiple collector inductively coupled plasma mass spectrometry. Detection and quantification of (68)Zn-E in earthworm tissue was possible after only 4 h of dermal exposure, when the uptake of (68)Zn-E had increased the total Zn tissue concentration by 0.03‰. The results demonstrate that at these realistic exposure concentrations there is no distinguishable difference between the uptake of the two forms of Zn by the earthworm L. rubellus, with the dietary pathway accounting for ?95% of total uptake. This stands in contrast to comparable studies where high dosing levels were used and dermal uptake is dominant. PMID:26588002

  18. Isolation, In-111 labeling, and abscess detection efficiency of rabbit polymorphonuclear leukocytes (PMN) from blood and peritoneal fluid

    SciTech Connect

    Bettin, K.M.; Elson, M.K.; Gerding, D.N.; Bamberger, D.M.; Forstrom, L.A.; Shafer, R.B.

    1984-01-01

    In-111 labeled blood and peritoneal exudate PMN were compared for labeling efficiency and ability to migrate to sites of experimental abscesses using both direct sampling and visual imaging techniques. Blood PMN were prepared by combining heparinized blood with 6% Hetastarch for 1 hour and layering the plasma over a double density Ficoll-Hy-paque gradient (S.G. 1.076 over 1.141). The PMN layer (90-99% PMN) at the interface yielded 10/sup 6/-10/sup 7/ PMN from 80-120 ml of blood. Peritoneal PMN were obtained by infusion of 0.1% glycogen, followed by infusion of saline after 4 or 18 hours. The exudate yielded 10/sup 7/-10/sup 8/ PMN (80-99% PMN). PMN suspensions were labeled for 30 minutes by addition of 100 ..mu..Ci of In-111-oxine, then washed twice. Percent cell-associated radioactivity of the labeled blood, 4 hour, and 18 hour peritoneal PMN was 89%, 88%, and 86%. The labeled PMN were injected intravenously into rabbits which had two of three abdominal capsules (table tennis balls drilled with 250 1.5 mm holes) inoculated with Staphylococcus aureus 4 hours earlier. Peak venous recovery of circulating labeled PMN, for blood, 4 hour and 18 hour peritoneal PMN was 60%, 43%, and 19%. Gamma camera images 24 hours after infusion into infected rabbits were superior with 4 hour peritoneal PMN. The peritoneal PMN harvested 4 hours after glycogen stimulation are simple to prepare, are obtainable in greater numbers than blood PMN, and result in better abscess visualization.

  19. Stimulating carbon efficient supply chains : carbon labels and voluntary public private partnerships

    E-print Network

    Tan, Kwan Chong

    2009-01-01

    This thesis looks at the potential of labeling products with life cycle greenhouse gas emission information as a bottom-up, complementary alternative to carbon cap and trade systems. By improving the transparency of product ...

  20. Efficient peak-labeling algorithms for whole-sample mass spectrometry proteomics.

    PubMed

    Pelikan, Richard; Hauskrecht, Milos

    2010-01-01

    Whole-sample mass spectrometry (MS) proteomics allows for a parallel measurement of hundreds of proteins present in a variety of biospecimens. Unfortunately, the association between MS signals and these proteins is not straightforward. The need to interpret mass spectra demands the development of methods for accurate labeling of ion species in such profiles. To aid this process, we have developed a new peak-labeling procedure for associating protein and peptide labels with peaks. This computational method builds upon characteristics of proteins expected to be in the sample, such as the amino sequence, mass weight, and expected concentration within the sample. A new probabilistic score that incorporates this information is proposed. We evaluate and demonstrate our method's ability to label peaks first on simulated MS spectra and then on MS spectra from human serum with a spiked-in calibration mixture. PMID:20150675

  1. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source.

    PubMed

    Ostroumov, P N; Barcikowski, A; Dickerson, C A; Perry, A; Pikin, A I; Sharamentov, S I; Vondrasek, R C; Zinkann, G P

    2015-08-01

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this paper, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz. PMID:26329185

  2. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    NASA Astrophysics Data System (ADS)

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-01

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this paper, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.

  3. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    DOE PAGESBeta

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-28

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstratemore »stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.« less

  4. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    SciTech Connect

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-28

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.

  5. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

  6. Development of isotope labeling LC-MS for human salivary metabolomics and application to profiling metabolome changes associated with mild cognitive impairment.

    PubMed

    Zheng, Jiamin; Dixon, Roger A; Li, Liang

    2012-12-18

    Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C/(12)C isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 ?L of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20 °C freezer, and -80 °C freezer over a period of 1 month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified. PMID:23150892

  7. Nitrogen use efficiency evaluation of aerobic rice under field capacity water potential using 15N isotopic tracer technique

    NASA Astrophysics Data System (ADS)

    Wahid, Ahmad Nazrul Abd; Rahim, Sahibin Abd; Rahim, Khairuddin Abdul; Harun, Abdul Rahim

    2015-09-01

    This study was carried out to evaluate the efficiency use of the nitrogen fertilizer on aerobic rice varieties MR219-4 and MR219-9 which were grown aerobically under field capacity water potential at the controlled environment area or shield house. Direct 15N isotope tracer method was used in this study, whereby the 15N isotope was utilized as a tracer for nitrogen nutrient uptake. 15N isotope presence in the samples is determined by using emission spectrometer analysis and percentage of total nitrogen is determined by using Kjeldahl method. 15N atom access value contained in the sample will be used in determining the effectiveness of the use of nitrogen in fertilizers through the specific calculation formulas. In this work, the data several data of nitrogen derived from fertilizer (Ndff), total nitrogen, nitrogen uptake and nitrogen use efficiency was obtained.

  8. Realistic Fasting Does Not Affect Stable Isotope Levels of a Metabolically Efficient Salamander

    EPA Science Inventory

    Stable isotopes are commonly used to examine various aspects of animal ecology. The use of stable isotopes generally proceeds under the implicit assumption that resource use is the only factor driving variation in stable isotope levels; however, a wealth of studies demonstrate a...

  9. Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

    PubMed

    Onorato, Joelle M; Chu, Ching-Hsuen; Ma, Zhengping; Kopcho, Lisa M; Chao, Hannguang J; Lawrence, R Michael; Cheng, Dong

    2015-03-01

    To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes. PMID:25598079

  10. Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme-labeled

    E-print Network

    Paytan, Adina

    Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition) to investigate biogeochemical cycling of P in the Sargasso Sea, Atlantic Ocean. Additional techniques are longer. Our data suggests that in the euphotic zone of the Sargasso Sea, DOP may be appreciably

  11. Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. OBJECTIVE: Our objectives were to produce heme iron enriched in (58)Fe and to use it to study the absorption of heme iron and the effect of iron and ...

  12. Spatially tracking 13C labeled substrate (bicarbonate) accumulation in microbial communities using laser ablation isotope ratio mass spectrometry

    SciTech Connect

    Moran, James J.; Doll, Charles G.; Bernstein, Hans C.; Renslow, Ryan S.; Cory, Alexandra B.; Hutchison, Janine R.; Lindemann, Stephen R.; Fredrickson, Jim K.

    2014-08-25

    This is a manuscript we would like to submit for publication in Environmental Microbiology Reports. This manuscript contains a description of a laser ablation isotope ratio mass spectrometry methodology developed at PNNL and applied to a microbial system at a PNNL project location – Hot Lake, Washington. I will submit a word document containing the entire manuscript with this Erica input request form.

  13. ISOTOPIC LABELING AND LC-APCI-MS QUANTIFICATION FOR INVESTIGATING ABSORPTION OF CAROTENOIDS AND VITAMIN K1 FROM KALE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability to study bioavailability of nutrients from plant-based foods is an important step in determining the potential health impact of those nutrients. This work describes a new method for studying bioavailability of nutrients from green, leafy vegetables by labeling the nutrients in kale with ...

  14. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards and Labeling Programs for Clothes Washers, Water Dispensers, Vending Machines and CFLs

    SciTech Connect

    Fridley, David; Zheng, Nina; Zhou, Nan

    2010-06-01

    Since the late 1970s, energy labeling programs and mandatory energy performance standards have been used in many different countries to improve the efficiency levels of major residential and commercial equipment. As more countries and regions launch programs covering a greater range of products that are traded worldwide, greater attention has been given to harmonizing the specific efficiency criteria in these programs and the test methods for measurements. For example, an international compact fluorescent light (CFL) harmonization initiative was launched in 2006 to focus on collaboration between Australia, China, Europe and North America. Given the long history of standards and labeling programs, most major energy-consuming residential appliances and commercial equipment are already covered under minimum energy performance standards (MEPS) and/or energy labels. For these products, such as clothes washers and CFLs, harmonization may still be possible when national MEPS or labeling thresholds are revised. Greater opportunity for harmonization exists in newer energy-consuming products that are not commonly regulated but are under consideration for new standards and labeling programs. This may include commercial products such as water dispensers and vending machines, which are only covered by MEPS or energy labels in a few countries or regions. As China continues to expand its appliance standards and labeling programs and revise existing standards and labels, it is important to learn from recent international experiences with efficiency criteria and test procedures for the same products. Specifically, various types of standards and labeling programs already exist in North America, Europe and throughout Asia for products in China's 2010 standards and labeling programs, namely clothes washers, water dispensers, vending machines and CFLs. This report thus examines similarities and critical differences in energy efficiency values, test procedure specifications and other technical performance requirements in existing international programs in order to shed light on where Chinese programs currently stands and considerations for their 2010 programs.

  15. Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W

    PubMed Central

    Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

    2012-01-01

    In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

  16. Efficient approximate and dynamic matching of patterns using a labeling paradigm

    SciTech Connect

    Cenk Sahinalp, S.; Vishkin, U.

    1996-12-31

    A key approach in string processing algorithmics has been the labeling paradigm [KMR72], which is based on assigning labels to some of the substrings of a given string. If these labels are chosen consistently, they can enable fast comparisons of substrings. Until the first optimal parallel algorithm for suffix tree construction was given in [SV94], the labeling paradigm was considered not to be competitive with other approaches. In this paper we show that, this general method is also useful for several central problems in the area of string processing: (1) Approximate String Matching, (2) Dynamic Dictionary Matching, (3) Dynamic Text Indexing. The approximate string matching problem deals with finding all substrings of a text which match a pattern {open_quotes}approximately{close_quotes}, i.e., with at most m differences. The differences can be in the form of inserted, deleted, or replaced characters. The text indexing problem deals with finding all occurrences of a pattern in a text, after the text is preprocessed. In the dynamic text indexing problem, updates to the text in the form of insertions and deletions of substrings are permitted. The dictionary matching problem deals with finding all occurrences of each pattern out of a set of patterns in a text, after the pattern set is preprocessed. In the dynamic dictionary matching problem, insertions and deletions of patterns to the pattern set are permitted.

  17. Luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres as efficient labels in DNA microarrays

    NASA Astrophysics Data System (ADS)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Marinello, F.; Schiavuta, P.

    2009-08-01

    Luminescent nanoparticles are gaining more and more interest in bio-labeling and bio-imaging applications, like for example DNA microarray. This is a high-throughput technology used for detection and quantification of nucleic acid molecules and other ones of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually expressed by PCR and functionalized by a fluorescent dye. These organic labels have well known disadvantages like photobleaching and limited sensitivity. Quantum dots may be used as alternatives, but they present troubles like blinking, toxicity and excitation wavelengths out of the usual range of commercial instruments, lowering their efficiency. Therefore in this work we investigate a different strategy, based on the use of inorganic silica nanospheres incorporating standard luminescent dyes or rare earth doped nanocrystals. In the first case it is possible to obtain a high luminescence emission signal, due to the high number of dye molecules that can be accommodated into each nanoparticle, reduced photobleaching and environmental protection of the dye molecules thanks to the encapsulation in the silica matrix. In the second case, rare earths exhibit narrow emission bands (easy identification), large Stokes shifts (efficient discrimination of excitation and emission) and long luminescence lifetimes (possibility to perform time-delayed analysis) which can be efficiently used for the improvement of signal to noise ratio. The synthesis and characterization of good luminescent silica spheres either by organic dye-doping or by rare-earth-doping are investigated and reported. Moreover, their application in the DNA microarray technology in comparison to the use of standard molecular fluorophores or commercial quantum dots is discussed. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalization and bio-compatibility makes them very promising for present and future applications in bio-labeling and bio-imaging.

  18. A Method Revealing Bacterial Cell-wall Architecture by Time-dependent Isotope Labeling and Quantitative Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Patti, Gary J.; Chen, Jiawei; Gross, Michael L.

    2009-01-01

    The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of post-insertionally modified cell-wall precursors, we find that tripeptides and species without Asx bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. PMID:19281243

  19. Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein-Protein Interactions by Chemical Cross-Linking

    SciTech Connect

    Merkley, Eric D.; Baker, Erin Shammel; Crowell, Kevin L.; Orton, Daniel J.; Taverner, Thomas; Ansong, Charles; Ibrahim, Yehia M.; Burnet, Meagan C.; Cort, John R.; Anderson, Gordon A.; Smith, Richard D.; Adkins, Joshua N.

    2013-02-20

    Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Application of the method to two different homodimeric proteins ? SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis? revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.

  20. Using stable isotopes to reconcile differences in nitrogen uptake efficiency relative to late season fertilization of northern red oak seedlings in Wisconsin bare-root nurseries

    NASA Astrophysics Data System (ADS)

    Fujinuma, R.; Balster, N. J.

    2009-12-01

    Cultural applications (e.g., timing, amount) of nitrogen (N) fertilizer in bareroot tree nurseries have been assessed for some time. However, the use of different metrologies to quantify the efficient use of fertilizer N and its allocation within biomass has confounded comparisons between fertilization regimes. This inconsistency is especially problematic when quantifying N fertilizer uptake efficiency (NFUE) of late season N fertilization in northern red oak (Quercus rubra L.) (NRO) seedlings characterized by episodic flushes in growth and N storage in perennial tissue to support spring growth. The use of isotopic tracers could help elucidate these differences. We therefore hypothesized that: 1) calculations of NFUE using isotopically enriched fertilizer would yield lower, more precise estimates of NFUE relative to traditional methods due to differences in the accounting of mineralized and reabsorbed N, and 2) a significant fraction of leaf N in older leaves (early flushes) would be reabsorbed into root and shoot tissue before abscission relative to leaves produced toward the end of the growing season (late flushes). To test these hypotheses, we conducted an experiment in two-year old NRO seedlings at two bare-root nurseries in Wisconsin. We applied a total of 147 mg N seedling-1 in pulses from early July after the seedlings completed their second leaf flush until late August. The treatments consisted of three replicated plots of 15N enriched (1.000 atom%) ammonium sulfate, three non-enriched plots, and three unfertilized plots (controls) at each nursery. Subsequent changes in plant N uptake and N allocation were quantified from destructively harvested samples taken at 40, 60, and 120 days after the fertilization began. We evaluated three common methods currently used to estimate NFUE (total N without control, total N with control, and isotopic difference). The total N without control method overestimated mean NFUE by 3.2 times relative to the isotope method, because mineralized N uptake and reabsorption of leaf N was unaccounted for. The total N with control method also overestimated mean NFUE, but only by 20% relative to the isotope method; variation associated with the effects of N fertilization on mineralization and immobilization was large enough to preclude significant difference between these methods. The difference of non-labeled N between day 60 and day 120 revealed that the roots and shoots absorbed 95% and 5%, respectively, of initial leaf N. However, isotopic mass balance between day 60 and day 120 indicated that the NRO seedlings did not reabsorb leaf fertilized N from the youngest leaves before abscission. This study shows that using stable isotopes to understand plant-soil interactions in response to fertilization will help elucidate the contribution of additional N fluxes (e.g., N reabsorption) within perennial plants and thus improve fertility management of production systems.

  1. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  2. Exploring metabolic pathways in vivo by a combined approach of mixed stable isotope-labeled Raman microspectroscopy and multivariate curve resolution analysis.

    PubMed

    Noothalapati, Hemanth; Shigeto, Shinsuke

    2014-08-01

    Understanding cellular metabolism is a major challenge in current systems biology and has triggered extensive metabolomics research, which in most cases involves destructive analysis. However, the information obtainable only in a nondestructive manner will be required for accurately mapping the global structure of the organism's metabolic network at a given instant. Here we report that metabolic pathways can be explored in vivo by mixed stable isotope-labeled Raman microspectroscopy in conjunction with multivariate curve resolution analysis. As a model system, we studied ergosterol biosynthesis in single living fission yeast cells grown in mixtures of normal and (13)C-labeled glucose as the sole carbon source. The multivariate spectral data analysis of space-resolved Raman spectra revealed the intrinsic spectra and relative abundances of all isotopomers of ergosterol whose carbon atoms in the 5,7-diene moiety of the sterol skeleton are either partly or fully substituted with (13)C. Our approach is applicable to other metabolites and will earn a place in the toolbox of metabolomic analysis. PMID:24975289

  3. Quantitation of methadone enantiomers in humans using stable isotope-labeled (2H3)-, (2H5)-, and (2H8)Methadone

    SciTech Connect

    Nakamura, K.; Hachey, D.L.; Kreek, M.J.; Irving, C.S.; Klein, P.D.

    1982-01-01

    A new technique for simultaneous stereoselective kinetic studies of methadone enantiomers was developed using three deuterium-labeled forms of methadone and GLC-chemical-ionization mass spectrometry. A racemic mixture (1:1) of (R)-(-)-(2H5)methadone (l-form) and (S)-(R)-(2H3)methadone (d-form) was administered orally in place of a single daily dose of unlabeled (+/-)-(2H0)methadone in long-term maintenance patients. Racemic (+/-)-(2H8)methadone was used as an internal standard for the simultaneous quantitation of (2H0)-, (2H3)-, and (2H5)methadone in plasma and urine. A newly developed extraction procedure, using a short, disposable C18 reversed-phase cartridge and improved chemical-ionization procedures employing ammonia gas, resulted in significant reduction of the background impurities contributing to the ions used for isotopic abundance measurements. These improvements enabled the measurement of labeled plasma methadone levels for 120 hr following a single dose. This methodology was applied to the study of methadone kinetics in two patients; in both patients, the analgesically active l-enantiomer of the drug had a longer plasma elimination half-life and a smaller area under the plasma disappearance curve than did the inactive d-form.

  4. Iron uptake and ferrokinetics in healthy male subjects of an iron-based oral phosphate binder (SBR759) labeled with the stable isotope (58)Fe.

    PubMed

    Gschwind, Hans-Peter; Schmid, Dietmar G; von Blanckenburg, Friedhelm; Oelze, Marcus; van Zuilen, Kirsten; Slade, Alan J; Stitah, Sylvie; Kaufmann, Daniel; Swart, Piet

    2014-11-01

    SBR759 is a novel polynuclear iron(III) oxide-hydroxide starch·sucrose·carbonate complex being developed for oral use in chronic kidney disease (CKD) patients with hyperphosphatemia on hemodialysis. SBR759 binds inorganic phosphate released by food uptake and digestion in the gastro-intestinal tract increasing the fecal excretion of phosphate with concomitant reduction of serum phosphate concentrations. Considering the high content of ?20% w/w covalently bound iron in SBR759 and expected chronic administration to patients, absorption of small amounts of iron released from the drug substance could result in potential iron overload and toxicity. In a mechanistic iron uptake study, 12 healthy male subjects (receiving comparable low phosphorus-containing meal typical for CKD patients: ?1000 mg phosphate per day) were treated with 12 g (divided in 3 × 4 g) of stable (58)Fe isotope-labeled SBR759. The ferrokinetics of [(58)Fe]SBR759-related total iron was followed in blood (over 3 weeks) and in plasma (over 26 hours) by analyzing with high precision the isotope ratios of the natural iron isotopes (58)Fe, (57)Fe, (56)Fe and (54)Fe by multi-collector inductively coupled mass spectrometry (MC-ICP-MS). Three weeks following dosing, the subjects cumulatively absorbed on average 7.8 ± 3.2 mg (3.8-13.9 mg) iron corresponding to 0.30 ± 0.12% (0.15-0.54%) SBR759-related iron which amounts to approx. 5-fold the basal daily iron absorption of 1-2 mg in humans. SBR759 was well-tolerated and there was no serious adverse event and no clinically significant changes in the iron indices hemoglobin, hematocrit, ferritin concentration and transferrin saturation. PMID:25017110

  5. High levels of isotope elimination improve precision and allow individual-based measurements of metabolic rates in animals using the doubly labeled water method.

    PubMed

    Shirai, Masaki; Niizuma, Yasuaki; Yamamoto, Maki; Oda, Emiko; Ebine, Naoyuki; Oka, Nariko; Yoda, Ken

    2015-11-01

    Doubly labeled water (DLW) can be used to measure energy expenditure in free-ranging animals, but questions have been raised about its accuracy in different species or contexts. We investigated whether differences in the extent of isotope elimination affects the precision and accuracy of the DLW method, which can vary according to the experimental design or metabolic rate of the species. Estimated total energy expenditure by the DLW method (TEEdlw) was compared with actual total energy expenditure simultaneously measured via respirometry (TEEresp) in streaked shearwaters Calonectris leucomelas, a pelagic seabird. Subjects were divided into three groups with different experimental conditions: at rest on the ground for 24 h (Group A) or for 48 h (Group B), and at rest on the water for 24 h (Group C). TEEdlw in Group A matched TEEresp, whereas there was an overestimation of TEEdlw in both Groups B and C compared with TEEresp. However, compared with Group A, TEEdlw in Groups B and C had reduced the isotopic analytical variability and thus higher precision. The best regression model (TEEdlw = 1.37 TEEresp - 14.12) showed a high correlation (R(2) = 0.82) between TEEdlw and TEEresp and allows a correction factor for field metabolic rates in streaked shearwaters. Our results demonstrate that the commonly made assumption that the DLW method is not appropriate for individual-based estimates may be incorrect in certain circumstances. Although a correction factor may be necessary when using the DLW method to estimate metabolic rate, greater levels of isotope eliminations provides DLW estimates with high precision, which can adequately represent relative individual estimates. Nevertheless, the DLW method, should be used with caution when characterizing interspecies difference of energy expenditures. PMID:26611463

  6. High levels of isotope elimination improve precision and allow individual-based measurements of metabolic rates in animals using the doubly labeled water method

    PubMed Central

    Shirai, Masaki; Niizuma, Yasuaki; Yamamoto, Maki; Oda, Emiko; Ebine, Naoyuki; Oka, Nariko; Yoda, Ken

    2015-01-01

    Doubly labeled water (DLW) can be used to measure energy expenditure in free-ranging animals, but questions have been raised about its accuracy in different species or contexts. We investigated whether differences in the extent of isotope elimination affects the precision and accuracy of the DLW method, which can vary according to the experimental design or metabolic rate of the species. Estimated total energy expenditure by the DLW method (TEEdlw) was compared with actual total energy expenditure simultaneously measured via respirometry (TEEresp) in streaked shearwaters Calonectris leucomelas, a pelagic seabird. Subjects were divided into three groups with different experimental conditions: at rest on the ground for 24 h (Group A) or for 48 h (Group B), and at rest on the water for 24 h (Group C). TEEdlw in Group A matched TEEresp, whereas there was an overestimation of TEEdlw in both Groups B and C compared with TEEresp. However, compared with Group A, TEEdlw in Groups B and C had reduced the isotopic analytical variability and thus higher precision. The best regression model (TEEdlw = 1.37 TEEresp ? 14.12) showed a high correlation (R2 = 0.82) between TEEdlw and TEEresp and allows a correction factor for field metabolic rates in streaked shearwaters. Our results demonstrate that the commonly made assumption that the DLW method is not appropriate for individual-based estimates may be incorrect in certain circumstances. Although a correction factor may be necessary when using the DLW method to estimate metabolic rate, greater levels of isotope eliminations provides DLW estimates with high precision, which can adequately represent relative individual estimates. Nevertheless, the DLW method, should be used with caution when characterizing interspecies difference of energy expenditures. PMID:26611463

  7. Impact of isotopic effect on density limit and LHCD efficiency in the FT-2 experiments

    NASA Astrophysics Data System (ADS)

    Lashkul, S. I.; Altukhov, A. B.; Gurchenko, A. D.; Gusakov, E. Z.; Dyachenko, V. V.; Esipov, L. A.; Irzak, M. A.; Kantor, M. Yu.; Kouprienko, D. V.; Perevalov, A. A.; Saveliev, A. N.; Shatalin, S. V.; Stepanov, A. Yu.

    2015-07-01

    Current drive by lower hybrid waves (LHCD) is the most effective method to sustain the plasma current, but it is feasible only at the plasma density not exceeding some density limit nDL. In the present work the main attention is paid to the investigation of this effect on the FT-2 (R = 0.55 m, a = 0.08 m, BT ? 3 T, Ipl = 19-40 kA, f0 = 920 MHz) tokamak. The dependence of LHCD efficiency on isotopic plasma content (hydrogen/deuterium) is studied. Characteristic features of such an experiment are a strong influence of the isotope plasma composition on the LH resonance density nLH. For hydrogen plasma nLH ? 3.5 × 1019 m-3, while for deuterium plasma nLH ? 2 × 1020 m-3. The suppression of the LHCD and beginning of the interaction of LH waves with ions are determined by the hydrogen/deuterium plasma density rise. In the hot hydrogen plasma (Te(r = 0 cm) ? 700 eV) the density limit nDL of LHCD is approximately equal to the resonance value nLH ? nLC, where nLC is the point of linear conversion. In the hot deuterium plasma one could expect an increase of nDL because of a much higher value of nLH ? nLC ? 1020 m-3. However it appeared that the observed density limit for LHCD generation nDL ? (3.5-4) × 1019 m-3 is not determined by nLH. The role of parametric instabilities in CD switch-off is considered in both cases. The cooling of the plasma column and density rise could lead to a reduction of the threshold for the parametric decay of f0 and result in early suppression of LHCD. In both cases the LHCD was inversely proportional to the density, which corresponds to the theoretical predictions. In order to analyse the experimentally observed LHCD efficiency the GRILL3D and FRTC codes have been used.

  8. Time-shared experiments for efficient assignment of triple-selectively labeled proteins

    PubMed Central

    Löhr, Frank; Laguerre, Aisha; Bock, Christoph; Reckel, Sina; Connolly, Peter J.; Abdul-Manan, Norzehan; Tumulka, Franz; Abele, Rupert; Moore, Jonathan M.; Dötsch, Volker

    2014-01-01

    Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C’ or 13C? spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns. PMID:25442777

  9. AIL Group, GRE, University of Dundee 2014 SILAC-Stable Isotope Labelling of Amino acids in Culture

    E-print Network

    Lamond, Angus I.

    ) Order no: Sigma, L-Arginine (A8094, 25g), L-lysine (L8662, 25g), L-Methionoine (M5308, 25g) N America, www.isotope.com) for UK see http://www.cgkas.com Amino Acid Symbol Cat. No Pack Size L-arginine-HCL (U-13C6, 98%) R6 CLM-2265 0.5g L-arginine-HCL (U-13C6, 98% : 15N4, 98%) R10 CNLM-539 0.5g Llysine2HCL

  10. Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats

    PubMed Central

    LI, YING-QIN; TANG, YING; FU, RAO; MENG, QIU-HUA; ZHOU, XUE; LING, ZE-MIN; CHENG, XIAO; TIAN, SU-WEI; WANG, GUO-JIE; LIU, XUE-GUO; ZHOU, LI-HUA

    2015-01-01

    Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats. PMID:25816076

  11. Characterization of TATP gas phase product ion chemistry via isotope labeling experiments using ion mobility spectrometry interfaced with a triple quadrupole mass spectrometer.

    PubMed

    Tomlinson-Phillips, Jill; Wooten, Alfred; Kozole, Joseph; Deline, James; Beresford, Pamela; Stairs, Jason

    2014-09-01

    Identification of the fragment ion species associated with the ion reaction mechanism of triacetone triperoxide (TATP), a homemade peroxide-based explosive, is presented. Ion mobility spectrometry (IMS) has proven to be a key analytical technique in the detection of trace explosive material. Unfortunately, IMS alone does not provide chemical identification of the ions detected; therefore, it is unknown what ion species are actually formed and separated by the IMS. In IMS, ions are primarily characterized by their drift time, which is dependent on the ion?s mass and molecular cross-section; thus, IMS as a standalone technique does not provide structural signatures, which is in sharp contrast to the chemical and molecular information that is generally obtained from other customary analytical techniques, such as NMR, Raman and IR spectroscopy and mass spectrometry. To help study the ion chemistry that gives rise to the peaks observed in IMS, the hardware of two different commercial IMS instruments has been directly coupled to triple quadrupole (QQQ) mass spectrometers, in order to ascertain each ion?s corresponding mass/charge (m/z) ratios with different dopants at two temperatures. Isotope labeling was then used to help identify and confirm the molecular identity of the explosive fragment and adduct ions of TATP. The m/z values and isotope labeling experiments were used to help propose probable molecular formulas for the ion fragments. In this report, the fragment and adduct ions m/z 58 and 240 of TATP have been confirmed to be [C3H6NH·H](+) and [TATP·NH4](+), respectively; while the fragment ions m/z 73 and 89 of TATP are identified as having the molecular formulas [C4H9NH2](+) and [C4H9O2](+), respectively. It is anticipated that the work in this area will not only help to facilitate improvements in mobility-based detection (IMS and MS), but also aid in the development and optimization of MS-based detection algorithms for TATP. PMID:24913870

  12. Use of photopatterned porous polymer monoliths as passive micromixers to enhance mixing efficiency of on-chip labeling reactions

    PubMed Central

    Mair, Dieudonne A.; Schwei, Thomas R.; Dinio, Theresa S.; Fréchet, Jean M. J.; Svec, Frantisek

    2009-01-01

    In order to increase the extent of reaction for on-chip fluorescent labeling of proteins, a passive mixer has been prepared by using UV light to photopattern a periodic arrangement of porous polymer monolith structures directly within the channel of a plastic microfluidic chip. By optimizing the composition of the polymerization solution and irradiation time we demonstrated the ability to photopattern monoliths in regularly repeating 100 ?m segments at the tee-junction of the disposable device. To evaluate the efficiency of this dual functional mixer-reactor, fluorescamine and lysine were introduced in separate channels upstream of the tee-junction and the intensity of laser-induced fluorescence resulting from the fluorogenic labeling reaction was monitored. The fluorescence level after passing the photopatterned periodic monolith configuration was better than both an equivalent 1 cm long continuous monolithic segment and an open channel. These results indicate that the periodic arrangement of monoliths, with regularly spaced open areas between 100 ?m plugs, is responsible for enhancing the mixing performance and overall rate of chemical reaction carried out in the system. In addition to facilitating preparation of a dual functional mixer-reactor, the ability to accurately photopattern monoliths in a channel is an enabling technology for seamlessly integrating multiple monoliths into a single microdevice. PMID:19294297

  13. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

    SciTech Connect

    Svensson, Jan; Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm ; Bergman, Ann-Charlotte; Adamson, Ulf; Blombaeck, Margareta; Wallen, Hakan; Joerneskog, Gun

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.

  14. Beta-lactamase-catalyzed aminolysis of depsipeptides: Proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping

    SciTech Connect

    Pazhanisamy, S.; Pratt, R.F. )

    1989-08-22

    The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-(((phenylacetyl)glycyl)oxy)benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.

  15. Analysis of the Membrane Proteome of Ciprofloxacin-Resistant Macrophages by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

    PubMed Central

    Marquez, Béatrice; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.; Devreese, Bart; Van Bambeke, Françoise

    2013-01-01

    Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter. PMID:23505477

  16. Preliminary Quantitative Profile of Differential Expression between Rat L6 Myoblasts and Myotubes by Stable Isotope Labeling by Amino acids in Cell Culture

    PubMed Central

    Cui, Ziyou; Chen, Xiulan; Lu, Bingwen; Park, Sung Kyu; Xu, Tao; Xie, Zhensheng; Xue, Peng; Hou, Junjie; Hang, Haiying; Yates, John R.; Yang, Fuquan

    2010-01-01

    Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muslce myogenesis in vitro. When mononucleate L6 myoblast cells reach confluent in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracelluar matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In sum, our results provide new clues for understanding the mechanism of myogenesis. PMID:19253283

  17. Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture.

    PubMed

    Cui, Ziyou; Chen, Xiulan; Lu, Bingwen; Park, Sung Kyu; Xu, Tao; Xie, Zhensheng; Xue, Peng; Hou, Junjie; Hang, Haiying; Yates, John R; Yang, Fuquan

    2009-03-01

    Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high-accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracellular matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis. PMID:19253283

  18. Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

    PubMed

    Wang, Chunlei; Chen, Sike; Brailsford, John A; Yamniuk, Aaron P; Tymiak, Adrienne A; Zhang, Yingru

    2015-12-24

    Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid. PMID:26674608

  19. Stable isotope-labeled tracers for metabolic pathway elucidation by GC-MS and FT-MS.

    PubMed

    Higashi, Richard M; Fan, Teresa W-M; Lorkiewicz, Pawel K; Moseley, Hunter N B; Lane, Andrew N

    2014-01-01

    Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics are poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, stable isotope-resolved metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks in a wide range of experimental systems, including human subjects. MS offers a wide range of instrumental capabilities involving different levels of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS that is affordable by many individual laboratories to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter focuses on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM. PMID:25270929

  20. Intraoperative beta probe: A device for detecting tissue labeled with positron or electron emitting isotopes during surgery

    SciTech Connect

    Daghighian, F. ); Mazziotta, J.C. ); Hoffman, E.J. ); Shenderov, P.; Eshaghian, B. ); Siegel, S.; Phelps, M.E. )

    1994-01-01

    An intraoperative beta probe was designed, built, and tested for detection of radio-labeled malignant tissues that has the advantage of being selectively sensitive to beta while insensitive to gamma radiation. Since beta radiation (electrons or positrons) has a short range in tissue, this probe is ideal for detecting tracers in tumors at the surface of the surgical field. This probe contains a plastic scintillation detector sensitive to beta rays and to a lesser degree some background gamma rays. A second detector counts spurious gamma rays and allows for their subtraction from the activity measured by the first detector. Sensitivity of the dual probe for I-131 and F-18 was measured to be 108 counts/s/kBq (4000 counts/s/[mu]Ci). The dual-detector probe faithfully measured the 10:1 tumor'' to background ratio of radioactivity concentrations in a simulated environment of a tumor in the presence of intense background 511 keV photons. In another phantom experiment, simulating abdominal tumor deposits with various realistic I-131 radioactive concentrations, the probe was able to accurately identify tumors of approximately 50 mg with a tumor/normal radioactivity concentration of 3/1 in 10 s.

  1. ACCESSING OVERSEAS MARKETS ENERGY EFFICIENCY STANDARDS AND APPLIANCE LABELING IN ASIA AND LATIN AMERICA

    EPA Science Inventory

    The purpose of the project is to reduce pollution and environmental degradation by increasing the efficiency of energy end-uses in the industrial and household sectors of key Asian and Latin American countries. This will be accomplished by encouraging the adoption and harmo...

  2. Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.

    PubMed

    Wu, Dianming; Kampf, Christopher J; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

    2014-07-15

    Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ?60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ?(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ?(15N)=0.2-0.5, the relative standard deviation of ?(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere. PMID:24954648

  3. International Comparison of Energy Labeling and Standards for Energy Efficient and Green Buildings 

    E-print Network

    Hennicke, P.; Shrestha, S.; Schleicher, T.

    2011-01-01

    ., Carraro, C., Hourcade, J.-C., Neuhoff, K. Luderer, C., Flachsland, C., Jakob, M., Popp, A., Steckel, J., Strohschein, J., Bauer, N., Brunner, S., Leimbach, M., Lotze-Campen, H., Bosetti, V., de Cian, E., Tavoni, M., Sassi, O., Waisman, H., Crassous... through higher rent and sales value. It also provides a higher quality of the indoor environment. The approaches to foster energy and resource efficiency in buildings differ among the countries around the world. While the European Union focuses...

  4. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling.

    PubMed

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional (15)N-(1)H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a H?-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for ?-synuclein. PMID:24018100

  5. Alzheimer's disease biomarkers detection in human samples by efficient capturing through porous magnetic microspheres and labelling with electrocatalytic gold nanoparticles.

    PubMed

    de la Escosura-Muñiz, Alfredo; Plichta, Zden?k; Horák, Daniel; Merkoçi, Arben

    2015-05-15

    A nanobiosensor based on the use of porous magnetic microspheres (PMM) as efficient capturing/pre-concentrating platform is presented for detection of Alzheimer's disease (AD) biomarkers. These PMMs prepared by a multistep swelling polymerization combined with iron oxide precipitation afford carboxyl functional groups suitable for immobilization of antibodies on the particle surface allowing an enhanced efficiency in the capturing of AD biomarkers from human serum samples. The AD biomarkers signaling is produced by gold nanoparticle (AuNP) tags monitored through their electrocatalytic effect towards hydrogen evolution reaction (HER). Novel properties of PMMs in terms of high functionality and high active area available for enhanced catalytic activity of the captured AuNPs electrocatalytic tags are exploited for the first time. A thorough characterization by scanning transmission electron microscope in high angle annular dark field mode (STEM-HAADF) demonstrates the enhanced ability of PMMs to capture a higher quantity of analyte and consequently of electrocatalytic label, when compared with commercially available microspheres. The optimized and characterized PMMs are also applied for the first time for the detection of beta amyloid and ApoE at clinical relevant levels in cerebrospinal fluid (CSF), serum and plasma samples of patients suffering from AD. PMID:25153932

  6. The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2.

    PubMed

    Qi, Jenson; Lang, Wensheng; Geisler, John G; Wang, Ping; Petrounia, Ioanna; Mai, Selyna; Smith, Charles; Askari, Hossein; Struble, Geoffrey T; Williams, Robyn; Bhanot, Sanjay; Monia, Brett P; Bayoumy, Shariff; Grant, Eugene; Caldwell, Gary W; Todd, Matthew J; Liang, Yin; Gaul, Micheal D; Demarest, Keith T; Connelly, Margery A

    2012-06-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol. PMID:22493088

  7. The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

    PubMed Central

    Qi, Jenson; Lang, Wensheng; Geisler, John G.; Wang, Ping; Petrounia, Ioanna; Mai, Selyna; Smith, Charles; Askari, Hossein; Struble, Geoffrey T.; Williams, Robyn; Bhanot, Sanjay; Monia, Brett P.; Bayoumy, Shariff; Grant, Eugene; Caldwell, Gary W.; Todd, Matthew J.; Liang, Yin; Gaul, Micheal D.; Demarest, Keith T.; Connelly, Margery A.

    2012-01-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with 13C3-D5-glycerol or 13C18-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that 13C3-D5-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, 13C18-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D5-glycerol to mice and measured plasma levels of D5-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D5-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered 13C18-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol. PMID:22493088

  8. Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

    PubMed

    Zhang, Yang; He, Quanze; Ye, Juanying; Li, Yanhong; Huang, Lin; Li, Qingqing; Huang, Jingnan; Lu, Jianan; Zhang, Xumin

    2015-10-20

    It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 ?g each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409. PMID:26361894

  9. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards & Labeling Programs for Copy Machines, External Power Supplies, LED Displays, Residential Gas Cooktops and Televisions

    SciTech Connect

    Zheng, Nina; Zhou, Nan; Fridley, David

    2012-03-01

    This report presents a technical review of international minimum energy performance standards (MEPS), voluntary and mandatory energy efficiency labels and test procedures for five products being considered for new or revised MEPS in China: copy machines, external power supply, LED displays, residential gas cooktops and flat-screen televisions. For each product, an overview of the scope of existing international standards and labeling programs, energy values and energy performance metrics and description and detailed summary table of criteria and procedures in major test standards are presented.

  10. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  11. The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

    PubMed

    Hong, Sangjin; de Almeida, Wagner B; Taguchi, Alexander T; Samoilova, Rimma I; Gennis, Robert B; O'Malley, Patrick J; Dikanov, Sergei A; Crofts, Antony R

    2014-09-30

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  12. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    NASA Astrophysics Data System (ADS)

    Guo, Bihong; Li, Shaopeng; Song, Lusheng; Yang, Mo; Zhou, Wenfei; Tyagi, Deependra; Zhu, Jinsong

    2015-08-01

    A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein-protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the improvement of the protein bioassay performance, rather than the chemical changes.

  13. Precipitation efficiency derived from isotope ratios in water vapor distinguishes dynamical and microphysical influences on subtropical atmospheric constituents

    NASA Astrophysics Data System (ADS)

    Bailey, A.; Nusbaumer, J.; Noone, D.

    2015-09-01

    With water vapor and clouds expected to effect significant feedbacks on climate, moisture transport through convective processes has important implications for future temperature change. The precipitation efficiency—the ratio of the rates at which precipitation and condensation form (e = P/C)—is useful for characterizing how much boundary layer moisture recycles through precipitation versus mixes into the free troposphere through cloud detrainment. Yet it is a difficult metric to constrain with traditional observational techniques. This analysis characterizes the precipitation efficiency of convection near the Big Island of Hawaii, USA, using a novel tracer: isotope ratios in water vapor. The synoptic circulation patterns associated with high and low precipitation efficiency are identified, and the importance of large-scale dynamics and local convective processes in regulating vertical distributions of atmospheric constituents important for climate is evaluated. The results suggest that high e days are correlated with plume-like transport originating from the relatively clean tropics, while low e days are associated with westerly transport, generated by a branching of the jet stream. Differences in transport pathway clearly modify background concentrations of water vapor and other trace gases measured at Mauna Loa Observatory; however, local convective processes appear to regulate aerosols there. Indeed, differences between observed and simulated diurnal cycles of particle number concentration indicate that precipitation scavenges aerosols and possibly facilitates new particle formation when e is high. As measurements of isotope ratios in water vapor expand across the subtropics, the techniques presented here can further our understanding of how synoptic weather, precipitation processes, and climate feedbacks interrelate.

  14. Identification of subunit-subunit interaction sites in ?A-WT crystallin and mutant ?A-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

    PubMed

    Kannan, Rama; Santhoshkumar, Puttur; Mooney, Brian P; Sharma, K Krishna

    2013-01-01

    Cataract is characterized by progressive protein aggregation and loss of vision. ?-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in ?-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit-subunit interaction sites in cataract-causing mutant ?A-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker-containing peptides. In this study, we cross-linked wild-type (?A-WT) and mutant (?A-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d? and d?) cross-linker-BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88-K99 in the ?5 strand of the mutant ?A-G98R crystallin that is not found in wild-type ?A-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly. PMID:23755258

  15. Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor.

    PubMed

    Buffet, Pierre-Emmanuel; Amiard-Triquet, Claude; Dybowska, Agnieszka; Risso-de Faverney, Christine; Guibbolini, Marielle; Valsami-Jones, Eugénia; Mouneyrac, Catherine

    2012-10-01

    Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone. PMID:22858103

  16. Uptake and Distribution of Soil Applied Zinc by Citrus Trees—Addressing Fertilizer Use Efficiency with 68Zn Labeling

    PubMed Central

    Hippler, Franz Walter Rieger; Boaretto, Rodrigo Marcelli; Quaggio, José Antônio; Boaretto, Antonio Enedi; Abreu-Junior, Cassio Hamilton; Mattos, Dirceu

    2015-01-01

    The zinc (Zn) supply increases the fruit yield of Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO4 enriched with the stable isotope 68Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO4 was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zndff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zndff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays. PMID:25751056

  17. Uptake and distribution of soil applied zinc by citrus trees-addressing fertilizer use efficiency with 68Zn labeling.

    PubMed

    Hippler, Franz Walter Rieger; Boaretto, Rodrigo Marcelli; Quaggio, José Antônio; Boaretto, Antonio Enedi; Abreu-Junior, Cassio Hamilton; Mattos, Dirceu

    2015-01-01

    The zinc (Zn) supply increases the fruit yield of Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO4 enriched with the stable isotope 68Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO4 was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zndff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zndff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays. PMID:25751056

  18. Isotope ratio analysis of actinides, fission products, and geolocators by high-efficiency multi-collector thermal ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Bürger, S.; Riciputi, L. R.; Bostick, D. A.; Turgeon, S.; McBay, E. H.; Lavelle, M.

    2009-09-01

    A ThermoFisher "Triton" multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotope ratio analysis of actinides (uranium, plutonium, and americium), fission products and geolocators (strontium, cesium, and neodymium). Total efficiencies (atoms loaded to ions detected) of up to 0.5-2% for U, Pu, and Am, and 1-30% for Sr, Cs, and Nd can be reported employing resin bead load techniques onto flat ribbon Re filaments or resin beads loaded into a millimeter-sized cavity drilled into a Re rod. This results in detection limits of <0.1 fg (104 atoms to 105 atoms) for 239-242+244Pu, 233+236U, 241-243Am, 89,90Sr, and 134,135,137Cs, and <=1 pg for natural Nd isotopes (limited by the chemical processing blank) using a secondary electron multiplier (SEM) or multiple-ion counters (MICs). Relative standard deviations (RSD) as small as 0.1% and abundance sensitivities of 1 × 106 or better using a SEM are reported here. Precisions of RSD [approximate]0.01-0.001% using a multi-collector Faraday cup array can be achieved at sub-nanogram concentrations for strontium and neodymium and are suitable to gain crucial geolocation information. The analytical protocols reported herein are of particular value for nuclear forensic and nuclear safeguard applications.

  19. Elementary Metabolite Units (EMU): a novel framework for modeling isotopic distributions

    PubMed Central

    Antoniewicz, Maciek R.; Kelleher, Joanne K.; Stephanopoulos, Gregory

    2007-01-01

    Metabolic Flux Analysis (MFA) has emerged as a tool of great significance for metabolic engineering and mammalian physiology. An important limitation of MFA, as carried out via stable isotope labeling and GC/MS and NMR measurements, is the large number of isotopomer or cumomer equations that need to be solved, especially when multiple isotopic tracers are used for the labeling of the system. This restriction reduces the ability of MFA to fully utilize the power of multiple isotopic tracers in elucidating the physiology of realistic situations comprising complex bioreaction networks. Here, we present a novel framework for the modeling of isotopic labeling systems that significantly reduces the number of system variables without any loss of information. The elementary metabolite unit (EMU) framework is based on a highly efficient decomposition method that identifies the minimum amount of information needed to simulate isotopic labeling within a reaction network using the knowledge of atomic transitions occurring in the network reactions. The functional units generated by the decomposition algorithm, called elementary metabolite units, form the new basis for generating system equations that describe the relationship between fluxes and stable isotope measurements. Isotopomer abundances simulated using the EMU framework are identical to those obtained using the isotopomer and cumomer methods, however, require significantly less computation time. For a typical 13C-labeling system the total number of equations that needs to be solved is reduced by one order-of-magnitude (100s EMUs vs. 1000s isotopomers). As such, the EMU framework is most efficient for the analysis of labeling by multiple isotopic tracers. For example, analysis of the gluconeogenesis pathway with 2H, 13C, and 18O tracers requires only 354 EMUs, compared to more than 2 million isotopomers. PMID:17088092

  20. Atmospheric CO2 level affects plants' carbon use efficiency: insights from a 13C labeling experiment on sunflower stands

    NASA Astrophysics Data System (ADS)

    Gong, Xiaoying; Schäufele, Rudi; Schnyder, Hans

    2015-04-01

    The increase of atmospheric CO2 concentration has been shown to stimulate plant photosynthesis and (to a lesser extent) growth, thereby acting as a possible sink for the additional atmospheric CO2. However, this effect is dependent on the efficiency with which plants convert atmospheric carbon into biomass carbon, since a considerable proportion of assimilated carbon is returned to the atmosphere via plant respiration. As a core parameter for carbon cycling, carbon use efficiency of plants (CUE, the ratio of net primary production to gross primary production) quantifies the proportion of assimilated carbon that is incorporated into plant biomass. CUE has rarely been assessed based on measurements of complete carbon balance, due to methodological difficulties in measuring respiration rate of plants in light. Moreover, foliar respiration is known to be inhibited in light, thus foliar respiration rate is generally lower in light than in dark. However, this phenomenon, termed as inhibition of respiration in light (IRL), has rarely been assessed at the stand-scale and been incorporated into the calculation of CUE. Therefore, how CUE responses to atmospheric CO2 levels is still not clear. We studied CUE of sunflower stands grown at sub-ambient CO2 level (200 ?mol mol-1) and elevated CO2 level (1000 ?mol mol-1) using mesocosm-scale gas exchange facilities which enabled continuous measurements of 13CO2/12CO2 exchange. Appling steady-state 13C labeling, fluxes of respiration and photosynthesis in light were separated, and tracer kinetic in respiration was analyzed. This study provides the first data on CUE at a mesocosm-level including respiration in light in different CO2 environments. We found that CUE of sunflower was lower at an elevated CO2 level than at a sub-ambient CO2 level; and the ignorance of IRL lead to erroneous estimations of CUE. Variation in CUE at atmospheric CO2 levels was attributed to several mechanisms. In this study, CO2 enrichment i) affected the size of respiratory substrate pools and the relative contribution of temporary storage pools and current assimilation pools; ii) affected the extent of inhibition of stands' respiration in light, which was related to leaf-level re-fixation of respired CO2; and iii) influenced the ratio of leaf mass to total plant mass. Our study highlights the necessity of integrating measurement of respiration in light in assessing carbon cycling. If the decrease of CUE by CO2 enrichment is a general response of terrestrial ecosystems, the buffering effect of plants C acquisition to the rise of atmospheric CO2 is lower than estimated so far.

  1. Triple-label beta liquid scintillation counting.

    PubMed

    Bukowski, T R; Moffett, T C; Revkin, J H; Ploger, J D; Bassingthwaighte, J B

    1992-07-01

    The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed. PMID:1514684

  2. Top-Down Identification and Quantification of Stable Isotope Labeled Proteins from A. flavus Using Online nano-Flow Reversed Phase Liquid Chromatography Coupled to a LTQ-FT-ICR Mass Spectrometer

    PubMed Central

    Collier, Timothy S.; Hawkridge, Adam M.; Georgianna, D. Ryan; Payne, Gary A.; Muddiman, David C.

    2013-01-01

    Online liquid chromatography – mass spectrometric (LC-MS) analysis of intact proteins (i.e. top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nano-flow reverse phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FT-ICR-MS). Aspergillus flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identificaion and we observed 1,318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail. PMID:18512951

  3. Steps towards High-Efficiency Trapping of Radioactive Isotopes in a Magneto-Optical Trap.*

    NASA Astrophysics Data System (ADS)

    Guckert, R.; Sandberg, V.; Tupa, D.; Vieira, D. J.

    1996-05-01

    As part of an effort to measure parity nonconserving atomic transition rates in a series of Cs radioisotopes(D. J. Vieira, C. E. Wieman et al., LAMPF Proposal 1303 (1992)), we are working on the efficient coupling of an magneto-optical trap (MOT) to a mass separator. We have established a test stand for optical trapping which is used for the off-line development of a high-efficiency MOT. Our trap features a dry-film coated(M. Stephens and C.E. Wieman, Phys. Rev. Let. 72), 24 (1994) cell with large (5 cm) windows to permit the use of large diameter, high power laser beams to increase the efficiency of the trapping process. Studies of the effect of detuning, magnetic field gradient, laser beam intensity and different beam sizes on the number of trapped atoms are presented. *This work is supported by the Department of Energy

  4. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

    PubMed

    Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; de Bolós, Carme; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas

    2015-03-25

    In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (?ZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human ?1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and ?ZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers. PMID:25732693

  5. NTFD--a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data

    E-print Network

    Kelleher, Joanne Keene

    Summary: Most current stable isotope-based methodologies are targeted and focus only on the well-described aspects of metabolic networks. Here, we present NTFD (non-targeted tracer fate detection), a software for the ...

  6. Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation

    NASA Astrophysics Data System (ADS)

    Hendry, Katharine R.; Robinson, Laura F.; McManus, Jerry F.; Hays, James D.

    2014-01-01

    Today’s Sargasso Sea is nutrient starved, except for episodic upwelling events caused by wind-driven winter mixing and eddies. Enhanced diatom opal burial in Sargasso Sea sediments indicates that silicic acid, a limiting nutrient today, may have been more available in subsurface waters during Heinrich Stadials, millennial-scale climate perturbations of the last glacial and deglaciation. Here we use the geochemistry of opal-forming organisms from different water depths to demonstrate changes in silicic acid supply and utilization during the most recent Heinrich Stadial. We suggest that during the early phase (17.5-18?ka), wind-driven upwelling replenished silicic acid to the subsurface, resulting in low Si utilization. By 17?ka, stratification reduced the surface silicic acid supply leading to increased Si utilization efficiency. This abrupt shift in Si cycling would have contributed to high regional carbon export efficiency during the recent Heinrich Stadial, despite being a period of increasing atmospheric CO2.

  7. Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation.

    PubMed

    Hendry, Katharine R; Robinson, Laura F; McManus, Jerry F; Hays, James D

    2014-01-01

    Today's Sargasso Sea is nutrient starved, except for episodic upwelling events caused by wind-driven winter mixing and eddies. Enhanced diatom opal burial in Sargasso Sea sediments indicates that silicic acid, a limiting nutrient today, may have been more available in subsurface waters during Heinrich Stadials, millennial-scale climate perturbations of the last glacial and deglaciation. Here we use the geochemistry of opal-forming organisms from different water depths to demonstrate changes in silicic acid supply and utilization during the most recent Heinrich Stadial. We suggest that during the early phase (17.5-18 ka), wind-driven upwelling replenished silicic acid to the subsurface, resulting in low Si utilization. By 17 ka, stratification reduced the surface silicic acid supply leading to increased Si utilization efficiency. This abrupt shift in Si cycling would have contributed to high regional carbon export efficiency during the recent Heinrich Stadial, despite being a period of increasing atmospheric CO2. PMID:24452197

  8. Nutritional efficiency of alpha-ketoisocaproate relative to leucine, assessed isotopically

    SciTech Connect

    Kang, C.W.; Walser, M.

    1985-10-01

    The efficiency of alpha-ketoisocaproate as a dietary substitute for leucine was assessed in rats by two techniques: first, the minimal dose of alpha-ketoisocaproate required, as a supplement to a leucine-free diet, to achieve a growth rate as great as animals receiving leucine was found to be between 2.2 and 4.4 times larger. Therefore the nutritional efficiency of alpha-ketoisocaproate lies between 0.23 and 0.46. Second, alpha-(1- UC)-ketoisocaproate and (TH)leucine were administered orally and the ratio of UC/TH incorporated into the leucine of whole-body protein and fibrin was measured. This ratio, divided by the ratio UC/TH injected, was the same in fibrin as in whole-body protein and averaged 0.39. Thus both techniques yield the same value, within the error of measurement, for the relative nutritional efficiency of alpha-ketoisocaproate. The authors also found that alpha-ketoisocaproate feeding at varying dosage did not alter this ratio in whole-body protein, suggesting that neither wide variations in growth rate nor exposure for 10 days to alpha-ketoisocaproate alters the relative rates of utilization (or oxidation) of alpha-ketoisocaproate vs. leucine.

  9. Allocation of atmospheric CO2 into labile sub-surface carbon pools: a stable isotope labelling approach in a tundra wetland

    NASA Astrophysics Data System (ADS)

    Rüggen, Norman; Knoblauch, Christian; Pfeiffer, Eva-Maria

    2015-04-01

    Greenhouse gas emissions from permafrost-affected wetlands are intensively studied due to their important role in the global carbon cycle. There are concerns of increasing methane and carbon dioxide fluxes from tundra wetlands due to permafrost degradation and hydrology changes in a warming Arctic. Understanding the sub-surface carbon pool interactions will improve the prediction on how trace gas fluxes from these ecosystems will respond to changing environmental conditions. Partitioning the sources of greenhouse gas fluxes will help to evaluate the quantitative role of recently produced plant photosynthates. Furthermore, partitioning allows separating respiration of long-term stored organic matter and freshly produced plant products. This knowledge is crucial for understanding the response of greenhouse gas fluxes in such wetlands to environmental changes. An in situ 13CO2 pulse-labelling experiment has been conducted in the northeast Siberian tundra (Samoylov island, Lena river delta) in August 2013 to quantify interactions among sub-surface carbon pools (DIC, DOC, CH4) in three depths (6, 16 and 36 cm) of the active layer. The experimental site was a low-centred polygon centre in a polygonal tundra landscape, with a sedge-moss (Carex-Scorpidium) plant association. The water table was at the soils' surface and the permafrost table in a depth of 50 cm. After the system has been 13CO2 pulse labelled, all three studied subsurface carbon pools (CH4, DIC and DOC) were clearly 13C-enriched, which accounts for atmospheric C incorporated into these pools. One day after the labelling, in 6 cm depth 1.5 percent of DIC and 0.1 percent of CH4were replaced by label C, which then steadily declined over a ten days period. The label C content of DOC increased gradually over the same period. In 16 cm depth, the label C increased gradually after labelling in both DIC and CH4. Label C was found in DIC and CH4 even in a depth of 36 cm, although in less pronounced concentrations. Carex material, exposed to the label, also substantially incorporated the label. Deduced from the results, we will present carbon exchange fluxes among sub-surface DIC, DOC and CH4 in a sedge-moss covered polygon-centre.

  10. Fluorescence energy transfer efficiency in labeled yeast cytochrome c: a rapid screen for ion biocompatibility in aqueous ionic liquids

    SciTech Connect

    Baker, Sheila N; Zhao, Hua; Pandey, Siddharth; Heller, William T; Bright, Frank; Baker, Gary A

    2011-01-01

    A fluorescence energy transfer de-quenching assay was implemented to follow the equilibrium unfolding behaviour of site-specific tetramethylrhodamine-labelled yeast cytochrome c in aqueous ionic liquid solutions; additionally, this approach offers the prospect of naked eye screening for biocompatible ion combinations in hydrated ionic liquids.

  11. Leaf oxygen and Carbon Isotopic Signatures Reflect Drought Resistance and Water Use Efficiency in the C4 Grass, Setaria viridis

    NASA Astrophysics Data System (ADS)

    Ellsworth, P.; Cousins, A. B.

    2014-12-01

    Low water availability is a major constraint in crop production, especially as agriculture is pushed to marginal lands. Therefore, improving drought resistance such as increasing water use efficiency (WUE) through plant breeding is needed to expand the range of soil water availability adequate for food production. With the goal of finding the genomic basis for WUE in C4 grasses, Setaria viridis makes an ideal model species because of its small size, short lifespan, and sequenced genome. Also it is part of the panicoid grass clade, which is one of the most important clades for food and biofuel production. In plant breeding programs, large numbers of genotypes must be quickly screened for drought resistance traits, but there is no well-defined method of screening for WUE in C4 grasses. However, bulk leaf oxygen (?18OBL) and carbon (?13C) isotopic signatures have shown potential as recorders of transpiration rate (E) and stomatal conductance (gs), and combined with biomass production potentially serve as a measure of WUE. Values of ?18OBL record differences in transpiration rate because leaf water becomes more enriched as transpiration rate decreases, and leaf tissue records the isotopic composition of leaf water in which it is synthesized. Additionally, in C4 plants ?13C values decrease as gs decreases but the change in ?13C in response to gs may not be adequate to tease apart differences in WUE. In this study, we grew S. viridis plants under well-watered and water-limited conditions to determine if ?18OBL and ?13C could be used as proxies for E and gs, and be used to screen S. viridis for differences in WUE in breeding programs. The ?18OBL and ?13C were significantly different between well-watered and water-limited plants and correlated with each other and with E, gs, and instantaneous water use efficiency (Anet/gs). Therefore, ?18OBL and ?13C can be useful proxies to screen genotypes for drought resistance by recording differences in E, gs, and WUE. Measuring ?18OBL and ?13C are relatively simple and quick, requiring the collection of a single leaf sample from each genotype instead of making laborious gas exchange measurements of E and gs.

  12. The Analysis on Influence of Main Factors on Theoretical Value of Energy Saving Rate for Energy Efficiency Labeling of Civil Buildings

    NASA Astrophysics Data System (ADS)

    Wang, Zhiwei; Wang, Zhenling; Jiang, Bo; Zhang, Fan; Li, Peng; Cao, Wei

    For typical residential buildings, no-large-scale and large-scale public buildings, according to China's Technical Guide for the Energy Efficiency Labeling of Civil Buildings, makes up missing data of the calculation benchmark and determines the boundary conditions for calculating the theoretical values of civil building energy efficiency. Based on equivalent full load hours method, develops a modular program and calculates building energy consumption for the demands of dynamic cooling and heating and lighting etc., finds out the corresponding relationship between star level's theoretical value of energy saving rate and specified-term limiting value in the Guide. With orthogonal experimental design and multiple linear regression, establishes the quantitative function of both the theoretical value of energy saving rate and main factors parameters, analyzes the impact of the control parameter on energy saving rate, and reveals the law of theoretical value of energy saving rate variation with the control parameter. For building energy efficiency labeling upgrade, presents technical measure need to be taken and analyses its feasibility. The results from the study can provide theoretical guidance for energy-saving design or retrofitting of civil buildings.

  13. Technical note: Nitrogen isotopic fractionation can be used to predict nitrogen-use efficiency in dairy cows fed temperate pasture.

    PubMed

    Cheng, L; Sheahan, A J; Gibbs, S J; Rius, A G; Kay, J K; Meier, S; Edwards, G R; Dewhurst, R J; Roche, J R

    2013-12-01

    The objective of this study was to investigate the relationship between nitrogen isotopic fractionation (?(15)N) and nitrogen-use efficiency (milk nitrogen/nitrogen intake; NUE) in pasture-fed dairy cows supplemented with increasing levels of urea to mimic high rumen degradable protein pastures in spring. Fifteen cows were randomly assigned to freshly cut pasture and either supplemented with 0, 250, or 336 g urea/d. Feed, milk, and plasma were analyzed for ?(15)N, milk and plasma for urea nitrogen concentration, and plasma for ammonia concentration. Treatment effects were tested using ANOVA and relationships between variables were established by linear regression. Lower dry matter intake (P = 0.002) and milk yield (P = 0.002) occurred with the highest urea supplementation (336 g urea/d) compared with the other two treatments. There was a strong linear relationship between milk ?(15)N - feed ?(15)N and NUE: [NUE (%) = 58.9 - 10.17 × milk ?(15)N - feed ?(15)N (‰) (r(2) = 0.83, P < 0.001, SE = 1.67)] and between plasma ?(15)N - feed ?(15)N and NUE: [NUE (%) = 52.4 - 8.61 × plasma ?(15)N - feed ?(15)N (‰) (r(2) = 0.85, P < 0.001, SE = 1.56)] . This study confirmed the potential use of ?(15)N to predict NUE in cows consuming different levels of rumen degradable protein. PMID:24085404

  14. Long term changes in Intrinsic Water Use Efficiency, the palaoe record derived from stable carbon isotope measurements from tree rings.

    NASA Astrophysics Data System (ADS)

    Gagen, Mary; McCarroll, Danny; Loader, Neil; Young, Giles; Robertson, Iain

    2015-04-01

    Stable carbon isotope (?13C) measurements from the annual rings of trees are increasingly used to explore long term changes in plant-carbon-water relations, via changes in intrinsic water use efficiency (iWUE); the ratio of photosynthetic rate to stomatal conductance. Many studies report a significant increase in iWEU since industrialisation, which tracks rising global atmospheric CO2. Such changes are logical are trees are known to change their stomatal geometry, number and action in response to rising CO2. However, which increasing iWUE suggests physiological changes which should lead to increased growth increasing iWUE is rarely matched by enhanced tree growth when tree rings are measured, despite increases of up to 30% in iWUE over the recent past (van der Sleen et al 2015). Explanations for the mismatch between iWUE and tree growth records encompass questions over the veracity of ?13C records for recording physiological change (Silva and Howarth 2013), suggestions that moisture stress in warming climates becomes a limit to growth and prevents opportunistic use of rising CO2 by trees (Andreu-Hayles et al 2011) and questions regarding the use of tree ring width, which does not record tree height gain, to record growth. Here we present an extensive range of long term iWUE records, derived broadly from the temperate, high latitude and one tropical forest site to explore the palaeoclimatic perspective on the iWUE-fertilization conundrum in a spatio temporally extensive manner.

  15. The physiological basis for genetic variation in water use efficiency and carbon isotope composition in Arabidopsis thaliana.

    PubMed

    Easlon, Hsien Ming; Nemali, Krishna S; Richards, James H; Hanson, David T; Juenger, Thomas E; McKay, John K

    2014-02-01

    Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (?(13)C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and ?(13)C. We find that stomatal conductance (g s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g m) in variation of WUE. We hypothesize that this is due to an effect through g m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g m than wild-type, consistent with variation in leaf anatomy causing variation in g m and ?(13)C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO2 assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity. PMID:23893317

  16. Use of an automated chromium reduction system for hydrogen isotope ratio analysis of physiological fluids applied to doubly labeled water analysis.

    PubMed

    Schoeller, D A; Colligan, A S; Shriver, T; Avak, H; Bartok-Olson, C

    2000-09-01

    The doubly labeled water method is commonly used to measure total energy expenditure in free-living subjects. The method, however, requires accurate and precise deuterium abundance determinations, which can be laborious. The aim of this study was to evaluate a fully automated, high-throughput, chromium reduction technique for the measurement of deuterium abundances in physiological fluids. The chromium technique was compared with an off-line zinc bomb reduction technique and also subjected to test-retest analysis. Analysis of international water standards demonstrated that the chromium technique was accurate and had a within-day precision of <1 per thousand. Addition of organic matter to water samples demonstrated that the technique was sensitive to interference at levels between 2 and 5 g l(-1). Physiological samples could be analyzed without this interference, plasma by 10000 Da exclusion filtration, saliva by sedimentation and urine by decolorizing with carbon black. Chromium reduction of urine specimens from doubly labeled water studies indicated no bias relative to zinc reduction with a mean difference in calculated energy expenditure of -0.2 +/- 3.9%. Blinded reanalysis of urine specimens from a second doubly labeled water study demonstrated a test-retest coefficient of variation of 4%. The chromium reduction method was found to be a rapid, accurate and precise method for the analysis of urine specimens from doubly labeled water. PMID:11006607

  17. Water use Efficiency in a Blue oak ( Quercus douglasii) Savanna - a Combined Analysis of Stable Isotopes and Eddy Covariance Measurements

    NASA Astrophysics Data System (ADS)

    Mambelli, S.; Tu, K. P.; Knohl, A.; Ma, S.; Baldocchi, D. D.; Dawson, T. E.

    2007-12-01

    Understanding the relationship between carbon assimilation and water consumption by natural vegetation is needed to assess how changes in climate will affect plant carbon and water exchange as well as the energy fluxes of ecosystems. While climate change is expected to cause significant warming, most models also suggest changes in the timing and amount of precipitation received; thus implications of this type of change are particularly acute in Mediterranean regions of the world. Blue oak savannas are already exposed to broad variation in water availability and to severe droughts during the summer months. Our objective was to evaluate the trade-off between carbon gain and water loss (Water Use Efficiency) in this ecosystem at both the leaf and at the ecosystem scales. We monitored the ratio of the partial pressures of CO2 inside the leaf (Ci) and in the outside air (Ca) or Ci/Ca, during the summer months of three subsequent years. This ratio is determined by the balance between photosynthetic capacity and stomatal conductance to water loss. Leaf-level estimates for individual trees were based on the carbon isotope composition (?13C) of bulk leaf tissue and of recently fixed carbohydrates (leaf soluble sugars). These leaf and individual tree based estimates were then compared with canopy-level estimates derived from continuous eddy covariance measurements of fluxes of CO2, water vapor and meteorological variables from two eddy covariance systems, one above (23m) and one below (2m) the tree canopy. We found that savanna Blue oak trees cope with severe drought through coordinated down-regulation of carbon and water fluxes, i.e. the ratio Ci/Ca remained stable over four summer months, despite decreasing soil water content and leaf water potentials. Stable C isotope composition of leaf soluble sugars is the most robust measure of Ci/Ca because it reflects the initial discrimination of photosynthetic products, without the confounding effects ascribed to storage, tissue chemical composition and time of tissue formation. Our findings at the leaf-level were confirmed at the ecosystem-level by using a two tower (above and below canopy) eddy covariance method.

  18. Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

    PubMed Central

    Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

    2014-01-01

    We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

  19. Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling1[W][OPEN

    PubMed Central

    Nelson, Clark J.; Alexova, Ralitza; Jacoby, Richard P.; Millar, A. Harvey

    2014-01-01

    Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis. PMID:25082890

  20. ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.

    SciTech Connect

    BULLOCK,R.M.; BENDER,B.R.

    2000-12-01

    The use of isotope labels has had a fundamentally important role in the determination of mechanisms of homogeneously catalyzed reactions. Mechanistic data is valuable since it can assist in the design and rational improvement of homogeneous catalysts. There are several ways to use isotopes in mechanistic chemistry. Isotopes can be introduced into controlled experiments and followed where they go or don't go; in this way, Libby, Calvin, Taube and others used isotopes to elucidate mechanistic pathways for very different, yet important chemistries. Another important isotope method is the study of kinetic isotope effects (KIEs) and equilibrium isotope effect (EIEs). Here the mere observation of where a label winds up is no longer enough - what matters is how much slower (or faster) a labeled molecule reacts than the unlabeled material. The most careti studies essentially involve the measurement of isotope fractionation between a reference ground state and the transition state. Thus kinetic isotope effects provide unique data unavailable from other methods, since information about the transition state of a reaction is obtained. Because getting an experimental glimpse of transition states is really tantamount to understanding catalysis, kinetic isotope effects are very powerful.

  1. Seasonal variations in photosynthesis, intrinsic water-use efficiency and stable isotope composition of poplar leaves in a short-rotation plantation

    PubMed Central

    Broeckx, L.S.; Fichot, R.; Verlinden, M.S.; Ceulemans, R.

    2014-01-01

    Photosynthetic carbon assimilation and transpirational water loss play an important role in the yield and the carbon sequestration potential of bioenergy-devoted cultures of fast-growing trees. For six poplar (Populus) genotypes in a short-rotation plantation, we observed significant seasonal and genotypic variation in photosynthetic parameters, intrinsic water-use efficiency (WUEi) and leaf stable isotope composition (?13C and ?18O). The poplars maintained high photosynthetic rates (between 17.8 and 26.9??mol?m?2?s?1 depending on genotypes) until late in the season, in line with their fast-growth habit. Seasonal fluctuations were mainly explained by variations in soil water availability and by stomatal limitation upon photosynthesis. Stomatal rather than biochemical limitation was confirmed by the constant intrinsic photosynthetic capacity (Vcmax) during the growing season, closely related to leaf nitrogen (N) content. Intrinsic water-use efficiency scaled negatively with carbon isotope discrimination (?13Cbl) and positively with the ratio between mesophyll diffusion conductance (gm) and stomatal conductance. The WUEi?–??13Cbl relationship was partly influenced by gm. There was a trade-off between WUEi and photosynthetic N-use efficiency, but only when soil water availability was limiting. Our results suggest that seasonal fluctuations in relation to soil water availability should be accounted for in future modelling studies assessing the carbon sequestration potential and the water-use efficiency of woody energy crops. PMID:25074859

  2. An Efficient and Compact Difference-Frequency-Generation Spectrometer and Its Application to 12CH3D/12CH4 Isotope Ratio Measurements

    PubMed Central

    Tsuji, Kiyoshi; Teshima, Hiroaki; Sasada, Hiroyuki; Yoshida, Naohiro

    2010-01-01

    We have developed an efficient and compact 3.4 ?m difference-frequency-generation spectrometer using a 1.55 ?m distributed feedback (DFB) laser diode, a 1.06 ?m DFB laser diode, and a ridge-waveguide periodically poled lithium niobate. It is continuously tunable in the 30 cm?1 span and is applied to 12CH3D/12CH4 isotope ratio measurements. The suitable pair of 12CH3D ?4 pP(7,6) and 12CH4 ? 2+?4 R(6) F1(1) lines enabled us to determine their isotope ratio with a precision repeatability of 0.8‰ using a sample and a working standard of pure methane with an effective signal averaging time of 100 ms. PMID:22163569

  3. Investigation of therapeutic efficiency of bleomycin and bleomycin-glucuronide labeled with (131)I on the cancer cell lines.

    PubMed

    Ediz, Melis; Avc?ba??, U?ur; Unak, Perihan; Müftüler, Fazilet Zümrüt Biber; Medine, Emin ?lker; Yurt K?lçar, Ayfer; Demiro?lu, Hasan; Gümü?er, Fikriye Gül; Sakarya, Serhan

    2013-05-01

    The aim of this study is to determine the incorporations of radiolabeled bleomycin ((131)I-BLM) and bleomycin-glucuronide ((131)I-BLMGLU) on PC-3 (human prostate carcinoma cell line), Caco-2 (human colon adenocarcinoma cell line), Hutu-80 (Human Duodenum adenocarcinoma cell line), and A549 (Human lung adenocarcinoma epithelial cell line) cancerous cell lines. For this purpose, BLM and BLMGLU enyzmatically synthesized were labeled with (131)I, quality control studies were done and the incorporation yields of (131)I-BLM and (131)I-BLMGLU on these cell lines were measured. Quality-control studies showed that the radiolabeling yields were obtained as 95% and 90% for (131)I-BLM and (131)I-BLMGLU, respectively. Also, as a result of the cell culture studies, it was found that (131)I-BLM and (131)I-BLMGLU had higher incorporation on PC-3 cells than that of other cell lines. In addition to this, it was reported that the incorporation yield of (131)I-BLMGLU was higher than that (131)I-BLM. At the end of the study, cytotoxicities of BLM and BLMGLU on PC-3 cancerous cell line were inspected and fluorescent images of BLM and BLMGLU were taken on PC-3 cells by using fluorescein isothiocyanate. In conclusion, cell culture studies demonstrated that the incorporation values of (131)I-BLMGLU on the four cell lines were about five to six times higher than (131)I-BLM. Radiolabeled glucuronide derivatives can be used in cancer therapy and tumor imaging, depending on the properties of radioiodine for the ?-glucuronidase-rich tissues because glucuronidation leads to rapid and higher incorporation on adenocarcinoma cells. PMID:23350895

  4. Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

    SciTech Connect

    Qian, Weijun ); Goshe, Michael B.; Camp, David G.); Yu, Li-Rong ); Tang, Keqi ); Smith, Richard D.)

    2003-10-15

    Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, a sensitive, efficient and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) for isolating and measuring the relative abundance of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported Phosphoprotein Isotope-coded Affinity Tag (PhIAT)approach developed by our laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.

  5. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models. PMID:25561501

  6. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics.

    PubMed

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-09-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  7. Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

    PubMed

    Huang, Ke; Huang, Lingyi; van Breemen, Richard B

    2015-04-01

    Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes. PMID:25774910

  8. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  9. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

    Kim, Jong Seo; Fillmore, Thomas L.; Liu, Tao; Robinson, Errol W.; Hossain, Mahmud; Champion, Boyd L.; Moore, Ronald J.; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2011-10-11

    Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

  10. Soil organic carbon can be up-taken by plant roots and stored in plant biosilica: NanoSIMS and isotopic labeling evidences

    NASA Astrophysics Data System (ADS)

    Alexandre, Anne; Santos, Guaciara M.; balesdent, Jerôme; Basile-Doelsch, Isabelle; Borschneck, Daniel; Cazevieille, Patrick; Chevassus-Rosset, Claire; Doelsch, Emmanuel; Harutyunyan, Araks; Lemee, Laurent; Mazur, Jean-Charles; Reyerson, Paul; Signoret, Patrick

    2015-04-01

    Plant biosilica particles called phytoliths contain occluded organic compounds (phytC). Over the last few years, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle has been the subject of increasing debate[1, 2]. Inconsistencies in phytC quantification were fed by the scarcity of in-situ characterization of phytC in phytoliths and by inadequate extraction methods[3]. Very recently, 14C-AMS analyses of soil organic matter (SOM), amendments, plant tissues, atmospheric CO2 and phytolith samples, evidenced that a small but significant pool of phytC is not photosynthetic but derived from old SOM[4,5]. From there, several investigations were started to go further into the characterization of phytC and the mechanisms in play behind old SOM absorption by plant roots and old SOM occlusion in plant biosilica. Here, we first reconstruct at high spatial resolution the 3-dimentional location of phytC and its C/N signature using 3D X-ray microscopy and Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS). A pool of phytC appears homogeneously distributed in the silica structure and its C:N estimate is in the range of amino acid signatures[6]. Then, we use 13C and 15N-labelled amino acids monitored from an hydroponic solution to grass roots, stems, leaves and phytoliths to evidence that amino acids are absorbed as such by the roots and are concentrated in phytC rather than in the plant tissues. These findings strengthen and complement the 14C evidences. Both of them dissuade attempts to use phytC as a proxy of plant C. Further, they open new avenues of investigation regarding the processes which drive SOM mobilization by plant uptake, for a better understanding of soil/plant interactions involved in the terrestrial C cycle. [1] Santos et al. 2010. Radiocarbon 52:113 [2] Santos et al. 2012. Biogeosci. 9:1873 [3] Corbineau et al. 2013 R. Paleobot. Palyn. 197: 179 [4] Reyerson et al. 2013 AGU Fall meeting 2013 (1803125) [5] Santos et al. 2014 AGU Fall meeting 2014 (B51A-0011) [6] Alexandre, et al., 2014. Biogeosci. Discuss. 11, 14699 :14727.

  11. Radioactively labelled porphyrin derivatives

    NASA Astrophysics Data System (ADS)

    Koní?ová, R.; Ernestová, M.; Jedináková-K?ížová, V.; Král, V.

    2003-01-01

    Radioactive labelling of guanidine-bearing tetraphenylporphyrin and Dy—texaphyrin with selected radionuclides (166Ho and 90Y) is described. A basic characterisation of studied porphyrin and texaphyrin, including their behaviour in a wide range of pH values and data on holmium and yttrium complexation with these compounds was probed using UV-VIS absorption spectrometry. The labelling yield of these macrocyclic molecules depends on the pH of the reaction mixture, metal: ligand ratio and time of incubation. Optimal reaction conditions for formation of porphyrin and texaphyrin radioactive complexes were determined by thin layer chromatography with the detection of ?- activity. The ability of porphyrin derivatives to bind anions was examined as well. Our experiments were focused on perrhenate ion (ReO4 -) because radiopharmaceuticals labelled with isotopes 186Re and 188Re play an important role in therapy of numerous tumour diseases. The possibility of applying ReO4 - anion directly for labelling purposes, without the necessity of its reduction to lower oxidation state, was not proved.

  12. Combining Computational Prediction of Cis-Regulatory Elements with a New Enhancer Assay to Efficiently Label Neuronal Structures in the Medaka Fish

    PubMed Central

    Bourrat, Franck; Gruhl, Franziska; Dewar, Ken; Blanchette, Mathieu; Wittbrodt, Joachim; Ettwiller, Laurence

    2011-01-01

    The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates. PMID:21637758

  13. Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

    PubMed

    Yeh, Chao-Chi; Hsu, Chih-Hung; Shao, Yu-Yun; Ho, Wen-Ching; Tsai, Mong-Hsun; Feng, Wen-Chi; Chow, Lu-Ping

    2015-06-01

    Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1? signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy. PMID:25850433

  14. High-resolution twin-ion metabolite extraction (HiTIME) mass spectrometry: nontargeted detection of unknown drug metabolites by isotope labeling, liquid chromatography mass spectrometry, and automated high-performance computing.

    PubMed

    Leeming, Michael G; Isaac, Andrew P; Pope, Bernard J; Cranswick, Noel; Wright, Christine E; Ziogas, James; O'Hair, Richard A J; Donald, William A

    2015-04-21

    The metabolic fate of a compound can often determine the success of a new drug lead. Thus, significant effort is directed toward identifying the metabolites formed from a given molecule. Here, an automated and nontargeted procedure is introduced for detecting drug metabolites without authentic metabolite standards via the use of stable isotope labeling, liquid chromatography mass spectrometry (LC/MS), and high-performance computing. LC/MS of blood plasma extracts from rats that were administered a 1:1 mixture of acetaminophen (APAP) and (13)C6-APAP resulted in mass spectra that contained "twin" ions for drug metabolites that were not detected in control spectra (i.e., no APAP administered). Because of the development of a program (high-resolution twin-ion metabolite extraction; HiTIME) that can identify twin-ions in high-resolution mass spectra without centroiding (i.e., reduction of mass spectral peaks to single data points), 9 doublets corresponding to APAP metabolites were identified. This is nearly twice that obtained by use of existing programs that make use of centroiding to reduce computational cost under these conditions with a quadrupole time-of-flight mass spectrometer. By a manual search for all reported APAP metabolite ions, no additional twin-ion signals were assigned. These data indicate that all the major metabolites of APAP and multiple low-abundance metabolites (e.g., acetaminophen hydroxy- and methoxysulfate) that are rarely reported were detected. This methodology can be used to detect drug metabolites without prior knowledge of their identity. HiTIME is freely available from https://github.com/bjpop/HiTIME . PMID:25818563

  15. Tree-Ring Stable Isotopes Reveal Twentieth-Century Increases in Water-Use Efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean Mountains

    PubMed Central

    Tognetti, Roberto; Lombardi, Fabio; Lasserre, Bruno; Cherubini, Paolo; Marchetti, Marco

    2014-01-01

    Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in ?13C, ?13C, ?18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, ?13C and ?18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and ?13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations. PMID:25398040

  16. Elevated CO2 increases tree-level intrinsic water use efficiency: insights from carbon and oxygen isotope analyses in tree rings across three forest FACE sites

    SciTech Connect

    Battipaglia, Giovanna; Saurer, Matthias; Cherubini, Paulo; Califapietra, Carlo; McCarthy, Heather R; Norby, Richard J; Cotrufo, M. Francesca

    2013-01-01

    Elevated CO2 increases intrinsic water use efficiency (WUEi) of forests, but the magnitude of this effect and its interaction with climate is still poorly understood. We combined tree ring analysis with isotope measurements at three Free Air CO2 Enrichment (FACE, POP-EUROFACE, in Italy; Duke FACE in North Carolina and ORNL in Tennessee, USA) sites, to cover the entire life of the trees. We used 13C to assess carbon isotope discrimination ( 13C ci/ca) and changes in WUEi, while direct CO2 effects on stomatal conductance were explored using 18O as a proxy. Across all the sites, elevated CO2 increased 13C-derived WUEi on average by 73% for Liquidambar styraciflua, 77% for Pinus taeda and 75% for Populus sp., but through different ecophysiological mechanisms. Our findings provide a robust means of predicting WUEi responses from a variety of tree species exposed to variable environmental conditions over time, and species-specific relationships that can help modeling elevated CO2 and climate impacts on forest productivity, carbon and water balances.

  17. Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation

    PubMed Central

    Beer, Lynn A.; Tang, Hsin-Yao; Barnhart, Kurt T.; Speicher, David W.

    2011-01-01

    In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers. PMID:21468938

  18. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  19. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  20. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  1. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  2. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  3. Isotopic Biogeochemistry

    NASA Technical Reports Server (NTRS)

    Hayes, J. M.

    1985-01-01

    An overview is provided of the biogeochemical research. The funding, productivity, personnel and facilities are reviewed. Some of the technical areas covered are: carbon isotopic records; isotopic studies of banded iron formations; isotope effects in microbial systems; studies of organic compounds in ancient sediments; and development in isotopic geochemistry and analysis.

  4. A new procedure for leukocytes labeling

    SciTech Connect

    Kurkowski, C.; Biddle, B.; Ahern, M.; Grisanti, K.; Gilani, S.S.H.

    1985-05-01

    Abscess localization using leukocytes labeled with In-oxine-111 is an established procedure. However, In-111 offers one half life (67 hr.) to the clinician to work with and is an expensive isotope. As such, a new approach offering versatility to the clinician in the manipulation of isotopic half life by substituting In-111 with isotopes of iodine I-123 (13 hr.) and I-131 (8 days) is studied. Oxine having an activated aromatic moiety was radioiodinated via Hunter-Greenwood method using I-125, yield being 90%. Therefore, judging from the in vitro tests, leukocytes stay viable after being labeled with radioiodinated oxine. Since isotopes by definition have similar chemical properties, for in vivo studies, I-125 (30-35 kev) could be substituted with I-123 (159 kev) and I-131 (364 kev).

  5. Rare-isotope and kinetic studies of Pt/SnO2 catalysts

    NASA Technical Reports Server (NTRS)

    Upchurch, Billy T.; Wood, George M.; Schryer, David R.; Hess, Robert V.; Miller, Irvin M.; Kielin, Erik J.

    1990-01-01

    Closed-cycle pulsed CO2 laser operation requires the use of an efficient CO-O2 recombination catalyst for these dissociation products which otherwise would degrade the laser operation. The catalyst must not only operate at low temperatures but also must operate efficiently for long periods. In the case of the Laser Atmospheric Wind Sounder (LAWS) laser, an operational lifetime of 3 years is required. Additionally, in order to minimize atmospheric absorption and enhance aerosol scatter of laser radiation, the LAWS system will operate at 9.1 micrometers with an oxygen-18 isotope CO2 lasing medium. Consequently, the catalyst must not only operate at low temperatures but must also preserve the isotopic integrity of the rare-isotope composition in the recombination mode. Several years ago an investigation of commercially available and newly synthesized recombination catalysts for use in closed-cycle pulsed common and rare-isotope CO2 lasers was implemented at the NASA Langley Research Center. Since that time, mechanistic efforts utilizing both common and rare oxygen isotopes have been implemented and continue. Rare-isotope studies utilizing commercially available platinum-tin oxide catalyst have demonstrated that the catalyst contributes oxygen-16 to the product carbon dioxide thus rendering it unusable for rare-isotope applications. A technique has been developed for modification of the surface of the common-isotope catalyst to render it usable. Results of kinetic and isotope label studies using plug flow, recycle plug flow, and closed internal recycle plug flow reactor configuration modes are discussed.

  6. INCORPORATING CONCENTRATION DEPENDENCE IN STABLE ISOTOPE MIXING MODELS

    EPA Science Inventory

    Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source li...

  7. Correlated optical and isotopic nanoscopy

    NASA Astrophysics Data System (ADS)

    Saka, Sinem K.; Vogts, Angela; Kröhnert, Katharina; Hillion, François; Rizzoli, Silvio O.; Wessels, Johannes T.

    2014-04-01

    The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100?nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

  8. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

  9. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, Richard A. (Contra Costa County, CA); Huang, Xiaohua C. (Mt. View, CA); Quesada, Mark A. (San Francisco, CA)

    1995-01-01

    A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

  10. GEO label: The General Framework for Labeling and Certification

    NASA Astrophysics Data System (ADS)

    Bye, B. L.; McCallum, I.; Maso, J.

    2012-04-01

    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data providers; it will create trustworthiness; and it will stimulate increased production and sharing of data and services. The survey of labeling and analysis with respect to certification, user ratings and user requirements management constitutes a useful input to the general discussion on data distribution and management of data.

  11. Physicochemical isotope anomalies

    SciTech Connect

    Esat, T.M.

    1988-06-01

    Isotopic composition of refractory elements can be modified, by physical processes such as distillation and sputtering, in unexpected patterns. Distillation enriches the heavy isotopes in the residue and the light isotopes in the vapor. However, current models appear to be inadequate to describe the detailed mass dependence, in particular for large fractionations. Coarse- and fine-grained inclusions from the Allende meteorite exhibit correlated isotope effects in Mg both as mass-dependent fractionation and residual anomalies. This isotope pattern can be duplicated by high temperature distillation in the laboratory. A ubiquitous property of meteoritic inclusions for Mg as well as for most of the other elements, where measurements exist, is mass-dependent fractionation. In contrast, terrestrial materials such as microtektites, tektite buttons as well as lunar orange and green glass spheres have normal Mg isotopic composition. A subset of interplanetary dust particles labelled as chondritic aggregates exhibit excesses in {sup 26}Mg and deuterium anomalies. Sputtering is expected to be a dominant mechanism in the destruction of grains within interstellar dust clouds. An active proto-sun as well as the present solar-wind and solar-flare flux are of sufficient intensity to sputter significant amounts of material. Laboratory experiments in Mg show widespread isotope effects including residual {sup 26}Mg excesses and mass dependent fractionation. It is possible that the {sup 26}Mg excesses in interplanetary dust is related to sputtering by energetic solar-wind particles. The implication if the laboratory distillation and sputtering effects are discussed and contrasted with the anomalies in meteoritic inclusions the other extraterrestrial materials the authors have access to.

  12. Stable isotope, site-specific mass tagging for protein identification

    DOEpatents

    Chen, Xian

    2006-10-24

    Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

  13. Comparison of water-use efficiency estimates based on tree-ring carbon isotopes with simulations of a dynamic vegetation model

    NASA Astrophysics Data System (ADS)

    Saurer, Matthias; Renato, Spahni; Fortunat, Joos; David, Frank; Kerstin, Treydte; Rolf, Siegwolf

    2015-04-01

    Tree-ring d13C-based estimates of intrinsic water-use efficiency (iWUE, reflecting the ratio of assimilation A to stomatal conductance gs) generally show a strong increase during the industrial period, likely associated with the increase in atmospheric CO2. However, it is not clear, first, if tree-ring d13C-derived iWUE-values indeed reflect actual plant and ecosystem-scale variability in fluxes and, second, what physiological changes were the drivers of the observed iWUE increase, changes in A or gs or both. To address these questions, we used a complex dynamic vegetation model (LPX) that combines process-based vegetation dynamics with land-atmosphere carbon and water exchange. The analysis was conducted for three functional types, representing conifers, oaks, larch, and various sites in Europe, where tree-ring isotope data are available. The increase in iWUE over the 20th century was comparable in LPX-simulations as in tree-ring-estimates, strengthening confidence in these results. Furthermore, the results from the LPX model suggest that the cause of the iWUE increase was reduced stomatal conductance during recent decades rather than increased assimilation. High-frequency variation reflects the influence of climate, like for example the 1976 summer drought, resulting in strongly reduced A and g in the model, particularly for oak.

  14. Comparison of seasonal variations in water-use efficiency calculated from the carbon isotope composition of tree rings and flux data in a temperate forest.

    PubMed

    Michelot, Alice; Eglin, Thomas; Dufrêne, Eric; Lelarge-Trouverie, Caroline; Damesin, Claire

    2011-02-01

    Tree-ring ?(13) C is often interpreted in terms of intrinsic water-use efficiency (WUE) using a carbon isotope discrimination model established at the leaf level. We examined whether intra-ring ?(13) C could be used to assess variations in intrinsic WUE (W(g), the ratio of carbon assimilation and stomatal conductance to water) and variations in ecosystem WUE (W(t) , the ratio of C assimilation and transpiration) at a seasonal scale. Intra-ring ?(13) C was measured in 30- to 60-µm-thick slices in eight oak trees (Quercus petraea). Canopy W(g) was simulated using a physiologically process-based model. High between-tree variability was observed in the seasonal variations of intra-ring ?(13) C. Six trees showed significant positive correlations between W(g) calculated from intra-ring ?(13) C and canopy W(g) averaged over several days during latewood formation. These results suggest that latewood is a seasonal recorder of W(g) trends, with a temporal lag corresponding to the mixing time of sugars in the phloem. These six trees also showed significant negative correlations between photosynthetic discrimination ? calculated from intra-ring ?(13) C, and ecosystem W(t), during latewood formation. Despite the observed between-tree variability, these results indicate that intra-ring ?(13) C can be used to access seasonal variations in past W(t). PMID:20955221

  15. A roadmap for interpreting 13C metabolite labeling patterns from cells

    PubMed Central

    Buescher, Joerg M.; Antoniewicz, Maciek R.; Boros, Laszlo G.; Burgess, Shawn C.; Brunengraber, Henri; Clish, Clary B.; DeBerardinis, Ralph J.; Feron, Olivier; Frezza, Christian; Ghesquiere, Bart; Gottlieb, Eyal; Hiller, Karsten; Jones, Russell G.; Kamphorst, Jurre J.; Kibbey, Richard G.; Kimmelman, Alec C.; Locasale, Jason W.; Lunt, Sophia Y.; Maddocks, Oliver D. K.; Malloy, Craig; Metallo, Christian M.; Meuillet, Emmanuelle J.; Munger, Joshua; Nöh, Katharina; Rabinowitz, Joshua D.; Ralser, Markus; Sauer, Uwe; Stephanopoulos, Gregory; St-Pierre, Julie; Tennant, Daniel A.; Wittmann, Christoph; Vander Heiden, Matthew G.; Vazquez, Alexei; Vousden, Karen; Young, Jamey D.; Zamboni, Nicola; Fendt, Sarah-Maria

    2015-01-01

    Measuring intracellular metabolism has increasingly led to important insights in biomedical research. 13C tracer analysis, although less information-rich than quantitative 13C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting 13C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. PMID:25731751

  16. A roadmap for interpreting (13)C metabolite labeling patterns from cells.

    PubMed

    Buescher, Joerg M; Antoniewicz, Maciek R; Boros, Laszlo G; Burgess, Shawn C; Brunengraber, Henri; Clish, Clary B; DeBerardinis, Ralph J; Feron, Olivier; Frezza, Christian; Ghesquiere, Bart; Gottlieb, Eyal; Hiller, Karsten; Jones, Russell G; Kamphorst, Jurre J; Kibbey, Richard G; Kimmelman, Alec C; Locasale, Jason W; Lunt, Sophia Y; Maddocks, Oliver D K; Malloy, Craig; Metallo, Christian M; Meuillet, Emmanuelle J; Munger, Joshua; Nöh, Katharina; Rabinowitz, Joshua D; Ralser, Markus; Sauer, Uwe; Stephanopoulos, Gregory; St-Pierre, Julie; Tennant, Daniel A; Wittmann, Christoph; Vander Heiden, Matthew G; Vazquez, Alexei; Vousden, Karen; Young, Jamey D; Zamboni, Nicola; Fendt, Sarah-Maria

    2015-08-01

    Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. PMID:25731751

  17. Multiple linear regression for isotopic measurements

    NASA Astrophysics Data System (ADS)

    Garcia Alonso, J. I.

    2012-04-01

    There are two typical applications of isotopic measurements: the detection of natural variations in isotopic systems and the detection man-made variations using enriched isotopes as indicators. For both type of measurements accurate and precise isotope ratio measurements are required. For the so-called non-traditional stable isotopes, multicollector ICP-MS instruments are usually applied. In many cases, chemical separation procedures are required before accurate isotope measurements can be performed. The off-line separation of Rb and Sr or Nd and Sm is the classical procedure employed to eliminate isobaric interferences before multicollector ICP-MS measurement of Sr and Nd isotope ratios. Also, this procedure allows matrix separation for precise and accurate Sr and Nd isotope ratios to be obtained. In our laboratory we have evaluated the separation of Rb-Sr and Nd-Sm isobars by liquid chromatography and on-line multicollector ICP-MS detection. The combination of this chromatographic procedure with multiple linear regression of the raw chromatographic data resulted in Sr and Nd isotope ratios with precisions and accuracies typical of off-line sample preparation procedures. On the other hand, methods for the labelling of individual organisms (such as a given plant, fish or animal) are required for population studies. We have developed a dual isotope labelling procedure which can be unique for a given individual, can be inherited in living organisms and it is stable. The detection of the isotopic signature is based also on multiple linear regression. The labelling of fish and its detection in otoliths by Laser Ablation ICP-MS will be discussed using trout and salmon as examples. As a conclusion, isotope measurement procedures based on multiple linear regression can be a viable alternative in multicollector ICP-MS measurements.

  18. 1. Isotope Definitions and terms a) Isotopes and isotope ratios.

    E-print Network

    Saleska, Scott

    3/24/2011 1 Outline 1. Isotope Definitions and terms a) Isotopes and isotope ratios. Isotopes fractionation c) Simple illustration with the water cycle 2. CO2 isotopes in photosynthesis a) Photosynthetic discrimination in C3 plants b) C3 vs C4 photosynthesis and the distinction in isotopes c) Measuring isotopic

  19. Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics

    SciTech Connect

    Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.

    2011-07-01

    Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

  20. Simultaneous tracing of {sup 76}Se-selenite and {sup 77}Se-selenomethionine by absolute labeling and speciation

    SciTech Connect

    Suzuki, Kazuo T. . E-mail: ktsuzuki@p.chiba-u.ac.jp; Somekawa, Layla; Kurasaki, Kazuki; Suzuki, Noriyuki

    2006-11-15

    Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ({sup 82}Se-selenite) and then administered {sup 76}Se-selenite and {sup 77}Se-selenomethionine ({sup 77}Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled {sup 76}Se and {sup 77}Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys.

  1. An Efficient Labelling Approach to Harness Backbone and Side-Chain Protons in 1H-Detected Solid-State NMR Spectroscopy

    PubMed Central

    Mance, Deni; Sinnige, Tessa; Kaplan, Mohammed; Narasimhan, Siddarth; Daniëls, Mark; Houben, Klaartje; Baldus, Marc; Weingarth, Markus

    2015-01-01

    1H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of 1H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for 1H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent 1H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR. PMID:26555653

  2. Efficient detection of Escherichia coli O157:H7 using a reusable microfluidic chip embedded with antimicrobial peptide-labeled beads.

    PubMed

    Chang, Mi-Sook; Yoo, Jeong Ha; Woo, Deok Ha; Chun, Myung-Suk

    2015-12-01

    The ability of antimicrobial peptides (AMPs) for effective binding to multiple target microbes has drawn lots of attention as an alternative to antibodies for detecting whole bacteria. We investigated pathogenic Escherichia coli (E. coli) detection by applying a microfluidic based biosensing device embedded with AMP-labeled beads. According to a new channel design, our device is reusable by the repeated operation of detection and regeneration modes, and the binding rate is more enhanced due to even distribution of the bacterial suspension inside the chamber by implementing influx side channels. We observed higher binding affinity of pathogenic E. coli O157:H7 for AMP-labeled beads than nonpathogenic E. coli DH5?, and the fluorescence intensity of pathogenic E. coli was about 3.4 times higher than the nonpathogenic one. The flow rate of bacterial suspension should be applied above a certain level for stronger binding and rapid detection by attaining a saturation level of detection within a short time of less than 20 min. A possible improvement in the limit of detection in the level of 10 cells per mL for E. coli O157:H7 implies that the AMP-labeled beads have high potential for the sensitive detection of pathogenic E. coli at an appropriate flow rate. PMID:26524182

  3. Relative Quantification of Carboxylic Acid Metabolites by Liquid-Chromatography Mass-Spectrometry Using Isotopic Variants of Cholamine

    PubMed Central

    Lamos, Shane M.; Shortreed, Michael R.; Frey, Brian L.; Belshaw, Peter J.; Smith, Lloyd M.

    2008-01-01

    Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC/MS conditions, regardless of mobile phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reverse-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for ten observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements. PMID:17563114

  4. Failure to label baboon milk intrinsically with iron

    SciTech Connect

    Figueroa-Colon, R.; Elwell, J.H.; Jackson, E.; Osborne, J.W.; Fomon, S.J. )

    1989-11-01

    The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically.

  5. Labelling technique of biomolecules for target radiotherapy

    E-print Network

    Hong Sheng Bai; Fan Hong Qiang; Jia Bing; Jin Xiao Hai; Lu Wei Wei

    1998-01-01

    Labelling techniques were developed for the preparation of biomolecules (DOTA-IgG, DOTA-lanreotide, anti-hepatoma antibody fragment, lanreotide) with radionuclides such as sup 9 sup 0 Y, sup 1 sup 5 sup 3 Sm and sup 1 sup 8 sup 8 Re. The labelling yield and radiochemical purity of these labelling biomolecules were determined by PC, ITLC and Sep-Pak C18 cartridge. The stability in vitro and bio-behaviour in normal rats were also evaluated. The experimental results showed that labelling efficiency of biomolecules (DOTA-IgG and DOTA-lanreotide) with sup 9 sup 0 Y and sup 1 sup 5 sup 3 Sm is more than 95% and had good stability in vitro, but the labelling efficiency of biomolecules (anti-hepatoma antibody fragment and lanroetide) with sup 1 sup 8 sup 8 Re via directly labelling technique is at range of 88%approx 95% and stability in vitro was less.

  6. Genetic control of water use efficiency and leaf carbon isotope discrimination in sunflower (Helianthus annuus L.) subjected to two drought scenarios.

    PubMed

    Adiredjo, Afifuddin Latif; Navaud, Olivier; Muños, Stephane; Langlade, Nicolas B; Lamaze, Thierry; Grieu, Philippe

    2014-01-01

    High water use efficiency (WUE) can be achieved by coordination of biomass accumulation and water consumption. WUE is physiologically and genetically linked to carbon isotope discrimination (CID) in leaves of plants. A population of 148 recombinant inbred lines (RILs) of sunflower derived from a cross between XRQ and PSC8 lines was studied to identify quantitative trait loci (QTL) controlling WUE and CID, and to compare QTL associated with these traits in different drought scenarios. We conducted greenhouse experiments in 2011 and 2012 by using 100 balances which provided a daily measurement of water transpired, and we determined WUE, CID, biomass and cumulative water transpired by plants. Wide phenotypic variability, significant genotypic effects, and significant negative correlations between WUE and CID were observed in both experiments. A total of nine QTL controlling WUE and eight controlling CID were identified across the two experiments. A QTL for phenotypic response controlling WUE and CID was also significantly identified. The QTL for WUE were specific to the drought scenarios, whereas the QTL for CID were independent of the drought scenarios and could be found in all the experiments. Our results showed that the stable genomic regions controlling CID were located on the linkage groups 06 and 13 (LG06 and LG13). Three QTL for CID were co-localized with the QTL for WUE, biomass and cumulative water transpired. We found that CID and WUE are highly correlated and have common genetic control. Interestingly, the genetic control of these traits showed an interaction with the environment (between the two drought scenarios and control conditions). Our results open a way for breeding higher WUE by using CID and marker-assisted approaches and therefore help to maintain the stability of sunflower crop production. PMID:24992022

  7. Genetic Control of Water Use Efficiency and Leaf Carbon Isotope Discrimination in Sunflower (Helianthus annuus L.) Subjected to Two Drought Scenarios

    PubMed Central

    Adiredjo, Afifuddin Latif; Navaud, Olivier; Muños, Stephane; Langlade, Nicolas B.; Lamaze, Thierry; Grieu, Philippe

    2014-01-01

    High water use efficiency (WUE) can be achieved by coordination of biomass accumulation and water consumption. WUE is physiologically and genetically linked to carbon isotope discrimination (CID) in leaves of plants. A population of 148 recombinant inbred lines (RILs) of sunflower derived from a cross between XRQ and PSC8 lines was studied to identify quantitative trait loci (QTL) controlling WUE and CID, and to compare QTL associated with these traits in different drought scenarios. We conducted greenhouse experiments in 2011 and 2012 by using 100 balances which provided a daily measurement of water transpired, and we determined WUE, CID, biomass and cumulative water transpired by plants. Wide phenotypic variability, significant genotypic effects, and significant negative correlations between WUE and CID were observed in both experiments. A total of nine QTL controlling WUE and eight controlling CID were identified across the two experiments. A QTL for phenotypic response controlling WUE and CID was also significantly identified. The QTL for WUE were specific to the drought scenarios, whereas the QTL for CID were independent of the drought scenarios and could be found in all the experiments. Our results showed that the stable genomic regions controlling CID were located on the linkage groups 06 and 13 (LG06 and LG13). Three QTL for CID were co-localized with the QTL for WUE, biomass and cumulative water transpired. We found that CID and WUE are highly correlated and have common genetic control. Interestingly, the genetic control of these traits showed an interaction with the environment (between the two drought scenarios and control conditions). Our results open a way for breeding higher WUE by using CID and marker-assisted approaches and therefore help to maintain the stability of sunflower crop production. PMID:24992022

  8. The anatomy of a laser label

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an efficient alternative to adhesive tags. The advantages of this system are numerous. In general the label consists of alphanumerical characters formed by laser generated pinhole depressions that penetrate the produce’s surface creating visible markings. H...

  9. 76 FR 20233 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-12

    ...The Commission extends the effective date for its new light bulb labeling requirements to January 1, 2012, to provide manufacturers with additional compliance time. In addition, the Commission exempts from the new label requirements incandescent bulbs that will not be produced after January 1, 2013, due to Federal efficiency...

  10. Bcl-2-functionalized ultrasmall superparamagnetic iron oxide nanoparticles coated with amphiphilic polymer enhance the labeling efficiency of islets for detection by magnetic resonance imaging

    PubMed Central

    Yang, Bin; Cai, Haolei; Qin, Wenjie; Zhang, Bo; Zhai, Chuanxin; Jiang, Biao; Wu, Yulian

    2013-01-01

    Based on their versatile, biocompatible properties, superparamagnetic iron oxide (SPIO) or ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are utilized for detecting and tracing cells or tumors in vivo. Here, we developed an innoxious and concise synthesis approach for a novel B-cell lymphoma (Bcl)-2 monoclonal antibody-functionalized USPIO nanoparticle coated with an amphiphilic polymer (carboxylated polyethylene glycol monooleyl ether [OE-PEG-COOH]). These nanoparticles can be effectively internalized by beta cells and label primary islet cells, at relatively low iron concentration. The biocompatibility and cytotoxicity of these products were investigated by comparison with the commercial USPIO product, FeraSpin™ S. We also assessed the safe dosage range of the product. Although some cases showed a hypointensity change at the site of transplant, a strong magnetic resonance imaging (MRI) was detectable by a clinical MRI scanner, at field strength of 3.0 Tesla, in vivo, and the iron deposition/attached in islets was confirmed by Prussian blue and immunohistochemistry staining. It is noteworthy that based on our synthesis approach, in future, we could exchange the Bcl-2 with other probes that would be more specific for the targeted cells and that would have better labeling specificity in vivo. The combined results point to the promising potential of the novel Bcl-2-functionalized PEG-USPIO as a molecular imaging agent for in vivo monitoring of islet cells or other cells. PMID:24204136

  11. Quantitative Microbial Ecology through Stable Isotope Probing.

    PubMed

    Hungate, Bruce A; Mau, Rebecca L; Schwartz, Egbert; Caporaso, J Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J; Liu, Cindy M; McHugh, Theresa A; Marks, Jane C; Morrissey, Ember M; Price, Lance B

    2015-11-01

    Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in (18)O and (13)C composition after exposure to [(18)O]water or [(13)C]glucose. The addition of glucose increased the assimilation of (18)O into DNA from [(18)O]water. However, the increase in (18)O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing. PMID:26296731

  12. Understanding Food Labels

    MedlinePLUS

    ... Healthy eating for girls Understanding food labels Understanding food labels There is lots of info on food ... need to avoid because of food allergies. Other food label terms top In addition to the Nutrition ...

  13. Aircraft profile measurements of 18O/16O and D/H isotope ratios of cloud condensate and water vapor constrain precipitation efficiency and entrainment rates in tropical clouds

    NASA Astrophysics Data System (ADS)

    Noone, D. C.; Raudzens Bailey, A.; Toohey, D. W.; Twohy, C. H.; Heymsfield, A.; Rella, C.; Van Pelt, A. D.

    2011-12-01

    Convective clouds play a significant role in the moisture and heat balance of the tropics. The dynamics of organized and isolated convection are a function of the background thermodynamic profile and wind shear, buoyancy sources near the surface and the latent heating inside convective updrafts. The stable oxygen and hydrogen isotope ratios in water vapor and condensate can be used to identify dominant moisture exchanges and aspects of the cloud microphysics that are otherwise difficult to observe. Both the precipitation efficiency and the dilution of cloud updrafts by entrainment can be estimated since the isotopic composition outside the plume is distinct from inside. Measurements of the 18O/16O and D/H isotope ratios were made in July 2011 on 13 research flights of the NCAR C130 aircraft during the ICE-T (Ice in Clouds Experiment - Tropical) field campaign near St Croix. Measurements were made using an instrument based on the Picarro Wave-Length Scanning Cavity Ring Down platform that includes a number of optical, hardware and software modifications to allow measurements to be made at 5 Hz for deployment on aircraft. The measurement system was optimized to make precise measurements of the isotope ratio of liquid and ice cloud condensate by coupling the gas analyzer to the NCAR Counter flow Virtual Impactor inlet. The inlet system provides a particle enhancement while rejecting vapor. Sample air is vigorously heated before flowing into the gas phase analyzer. We present statistics that demonstrate the performance and calibration of the instrument. Measured profiles show that environmental air exhibits significant layering showing controls from boundary layer processes, large scale horizontal advection and regional subsidence. Condensate in clouds is consistent with generally low precipitation efficiency, although there is significant variability in the isotope ratios suggesting heterogeneity within plumes and the stochastic nature of detrainment processes. Entrainment of air into the plume is seen as evaporation of condensate. In the plume between about -7 and -12C, the ice condensate fraction increases with height, and the isotope ratios are used to discern ice formation from deposition from ice formed from in situ freezing of cloud liquid. The observed profiles demonstrate a new capacity for cloud process studies and provide new insight into the water budget of clouds.

  14. Impacts of China's Current Appliance Standards and Labeling Program to 2020

    E-print Network

    Fridley, David; Aden, Nathaniel; Zhou, Nan; Lin, Jiang

    2007-01-01

    energy conservation target values, energy con- sumption test methods,energy conservation (voluntary energy efficiency label specifications) energy consumption test methods;energy conservation evaluation values (voluntary energy efficiency labeling specifications); energy consumption test methods;

  15. Check-Testing of Manufacturer Self Reported Labeling Data & Compliance with MEPS

    E-print Network

    Zhou, Nan

    2008-01-01

    energy conservation (voluntary energy efficiency label specifications) energy consumption test methods;energy conservation evaluation values (voluntary energy efficiency labeling speci- fications); maximum allowable values for water consumption; energy consumption test methods;

  16. Monitoring CO[subscript 2] Fixation Using GC-MS Detection of a [superscript 13]C-Label

    ERIC Educational Resources Information Center

    Hammond, Daniel G.; Bridgham, April; Reichert, Kara; Magers, Martin

    2010-01-01

    Much of our understanding of metabolic pathways has resulted from the use of chemical and isotopic labels. In this experiment, a heavy isotope of carbon, [superscript 13]C, is used to label the product of the well-known RuBisCO enzymatic reaction. This is a key reaction in photosynthesis that converts inorganic carbon to organic carbon; a process…

  17. A fully enzymatic method for site-directed spin labeling of long RNA

    PubMed Central

    Lebars, Isabelle; Vileno, Bertrand; Bourbigot, Sarah; Turek, Philippe; Wolff, Philippe; Kieffer, Bruno

    2014-01-01

    Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5?-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies. PMID:24981512

  18. 10 CFR 431.30 - Applicability of labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Applicability of labeling requirements. 431.30 Section 431.30 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability of labeling requirements....

  19. 10 CFR 431.30 - Applicability of labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Applicability of labeling requirements. 431.30 Section 431.30 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability of labeling requirements....

  20. 10 CFR 431.30 - Applicability of labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Applicability of labeling requirements. 431.30 Section 431.30 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability of labeling requirements....

  1. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A. (Santa Fe, NM); Silks, III, Louis A. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Atcher, Robert (White Rock, NM)

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  2. DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells

    SciTech Connect

    Schwartz, J.L. |; Mustafi, R.; Hughes, A.; DeSombre, E.R.

    1996-08-01

    The effects of the Auger electron-emitting isotope {sup 123}I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with {sup 60}Co {gamma} rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-labeled estrogen. The {sup 123}I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 {+-} 1.7, which is similar to that seen with high-LET radiations. Our results suggest that {sup 123}I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by {sup 123}I bound to estrogen is very like damage induced by high-LET radiation, and the {sup 123}I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA. 40 refs., 7 figs.

  3. Efficiency of monolaurin in mitigating ruminal methanogenesis and modifying C-isotope fractionation when incubating diets composed of either C3 or C4

    E-print Network

    Gilli, Adrian

    when incubating diets composed of either C3 or C4 plants in a rumen simulation technique (Rusitec in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal metha-isotope preferences. Using the rumen simulation technique, four basal diets, characterised either by the C3 plants

  4. Structural Changes of Cephalopod Rhodopsin and -Arrestin Measured by FTIR Difference Spectroscopy and Isotope Editing

    E-print Network

    Xie, Xiaoliang Sunney

    and Isotope Editing Joel M Kralj1, Erica Raber1, Jose Sarmiento2, David Shumate2, Christie Stanzel2, Carrie is perturbing at least one carboxyl group in s-Rh. In order to assign these changes, total 15N isotope labeling total isotope results in a shift of difference bands indicating that the arrestin is undergoing

  5. Assessment of vitamin A status in rats by isotope dilution: A simplified model

    SciTech Connect

    Furr, H.C.; Cooper, D.A.; Olson, J.A. )

    1990-02-26

    Isotope-dilution analysis of vitamin A status requires giving a known quantity of labeled vitamin A to the subject and measuring the ratio of labeled to unlabeled retinol in the blood after a period for equilibration. To calculate total body stores from the isotopic ratio of plasma retinol, several assumptions must be made. In considering new ways of better calculating liver vitamin A stores from isotope-dilution data, the authors used the data of Green et al. to estimate loss of vitamin A tracer as a function of time and of vitamin A status. This correction markedly improves the correlation between calculated and analyzed liver vitamin A stores and also quantitively explains the hyperbolic relationship between fraction of tracer dose recovered in liver and mass of liver vitamin A stores. Agreement of this model with experimental data suggests that efficiency of absorption and storage of vitamin A is not affected by vitamin A status. This model can be used to estimate both the amount of tracer needed for a given lower limit of detection and an optimum sampling time.

  6. Transuranium isotopes

    SciTech Connect

    Hoffman, D.C.

    1985-12-01

    The needs of the research community for the production of transuranium isotopes, the quantities required, the continuity of production desired, and what a new steady state neutron source would have to provide to satisfy these needs are discussed. Examples of past frontier research which need these isotopes as well as an outline of the proposed Large Einsteinium Activation Program, LEAP, which requires roughly ten times the current production of /sup 254/Es are given. 15 refs., 5 figs., 4 tabs.

  7. Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.

    PubMed

    Wang, Shanquan; He, Jianzhong

    2011-09-01

    Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

  8. Extrinsic labeling method may not accurately measure Fe absorption from cooked pinto beans (Phaseolus vulgaris): comparison of extrinsic and intrinsic labeling of beans.

    PubMed

    Jin, Fuxia; Cheng, Zhiqiang; Rutzke, Michael A; Welch, Ross M; Glahn, Raymond P

    2008-08-27

    Isotopic labeling of food has been widely used for the measurement of Fe absorption in determining requirements and evaluating the factors involved in Fe bioavailability. An extrinsic labeling technique will not accurately predict the total Fe absorption from foods unless complete isotopic exchange takes place between an extrinsically added isotope label and the intrinsic Fe of the food. We examined isotopic exchange in the case of both white beans and colored beans (Phaseolus vulgaris) with an in vitro digestion model. There are significant differences in (58)Fe/(56)Fe ratios between the sample digest supernatant and the pellet of extrinsically labeled pinto bean. The white bean digest shows significantly better equilibration of the extrinsic (58)Fe with the intrinsic (56)Fe. In contrast to the extrinsically labeled samples, both white and red beans labeled intrinsically with (58)Fe demonstrated consistent ratios of (58)Fe/(56)Fe in the bean meal, digest, supernatant, and pellet. It is possible that the polyphenolics in the bean seed coat may bind Fe and thus interfere with extrinsic labeling of the bean meals. These observations raise questions on the accuracy of studies that used extrinsic tags to measure Fe absorption from beans. Intrinsic labeling appears necessary to accurately measure Fe bioavailability from beans. PMID:18620404

  9. Effects of soil strength on the relation of water-use efficiency and growth to carbon isotope discrimination in wheat seedlings.

    PubMed

    Masle, J; Farquhar, G D

    1988-01-01

    The ratio of carbon accumulation to transpiration, W, of wheat (Triticum aestivum L.) seedlings increased with increasing soil strength, measured as soil penetrometer resistance, and this was already apparent at the two leaf stage. The ratio was negatively correlated with carbon isotope discrimination, in accord with theory. This means that decrease in intercellular partial pressure of CO(2) accounted for an important part of the increase in W with increasing soil strength. Despite a lower CO(2) concentration in the leaves at high soil strength, assimilation rate per unit leaf area was enhanced. Greater ribulose 1,5-bisphosphate carboxylase activity confirmed that photosynthetic capacity was actually increased. This pattern of opposite variation of assimilation rate and of stomatal conductance is unusual. The ratio of plant carbon mass to leaf area increased markedly with increasing soil strength, mainly because of a greater investment of carbon into roots than into shoots. A strong negative correlation was found between this ratio and carbon isotope discrimination. For a given increase in discrimination, decrease in carbon mass per leaf area was proportionally larger than decrease in assimilation rate, so that relative growth rate was positively correlated to carbon isotope discrimination. PMID:16665888

  10. Labeling strategies for 13C-detected aligned-sample solid-state NMR of proteins

    NASA Astrophysics Data System (ADS)

    Filipp, Fabian V.; Sinha, Neeraj; Jairam, Lena; Bradley, Joel; Opella, Stanley J.

    2009-12-01

    13C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13C sites so that dipole-dipole couplings among the 13C are minimized, thus eliminating the need for homonuclear 13C- 13C decoupling in either indirect or direct dimensions of one- or multi-dimensional NMR experiments that employ 13C detection. The optimal percentage for random fractional 13C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on [2- 13C]-glucose-containing media was found to be most effective.

  11. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  12. Leukemic cell labeling with indium-111-oxine

    SciTech Connect

    Uchida, T.; Takagi, Y.; Matsuda, S.; Yui, T.; Ishibashi, T.; Kimura, H.; Kariyone, S.

    1984-01-01

    Leukemic cells were labeled with In-111-oxine in patients with acute leukemia. In vitro labeling studies revealed that labeling efficiency reached maximum 80.8 +- 3.6% (mean +- 1SD) by 2 times washes after 20 minutes incubation time. Cell viability was assessed by trypan blue exclusion test and in vitro culture of leukemic cells, which showed no cellular damage during labeling procedure. Elution of In-111 from the labeled cells was 10.0 +- 1.2% at 12 hours after labeling. For in vivo leukemic cell kinetic studies, more than 10/sup 8/ leukemic cells separated from Ficoll-Hypacque sedimentation were labeled by 30 minutes of In-111-oxine incubation and two times washes at 37/sup 0/C. In vivo studies were performed in 7 patients with acute myeloblastic, lymphoblastic leukemia and blastic crisis of chronic myelocytic leukemia. Labeled leukemic cells disappeared in single exponential fashion with half life of 9.6 to 31.8 hours. Total leukemic cell pool in peripheral circulation was calculated, which correlated well with peripheral leukemic cell counts (r=0.99). No relationship was observed between total leukemic cell pool and leukemic cell turnover rate. Migration patterns of labeled leukemic cells showed that pulmonary uptake was evident within 15 minutes after the infusion and returned to base-line. Splenic and hepatic uptake showed gradual increase up to 24 hours. Bone marrow accumulation was shown only in 2 cases. Presently, there are no suitable radionuclides for leukemic cell labeling. In-111-oxine labeled leukemic cells would overcome this difficulty.

  13. ISOTOP Camera 

    E-print Network

    Unknown

    2011-08-17

    Our group has recently described a new form of kinetic isotope effect that arises from dynamic selectivity in the bifurcation of a reaction pathway on the slope of an energy surface. Since the selection between products does not occur at a potential...

  14. Investigations into agents for improving cell labeling with positron- and gamma-emitting radionuclides

    SciTech Connect

    Zoghbi, S.S.; Thakur, M.L.; Gottschalk, A.; Pande, S.; Srivastava, S.C.; Richards, P.

    1982-01-01

    It was possible to label leukocytes with Co-oxine, but a large proportion of the radioactivity was eluted from the cells upon washing. Ruthenium oxine labeled platelets efficiently in plasma while negligible proportion of radioactivity was eluted from the cells. Three factors influence the labeling efficiency of the cells: duration of the incubation periods; cell concentration; and ACD concentration.

  15. An oligonucleotide microarray for microRNA expression analysis based on labeling RNA

    E-print Network

    Tian, Weidong

    terminus with biotin and hybridized with com- plementary oligo-DNA probes immobilized on glass slides membranes and detected the miRNAs by labeling filtered low molecular weight RNA with radioactive isotopes (9). To avoid the need of large amounts of total RNA and using radioactive isotopes as in the above methods, Liu

  16. Mass Spectrometry-Based Label-Free Quantitative Proteomics

    PubMed Central

    Zhu, Wenhong; Smith, Jeffrey W.; Huang, Chun-Ming

    2010-01-01

    In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies. PMID:19911078

  17. A new [2H]-labelled ?-trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates.

    PubMed

    Heinkele, Georg; Geditz, Mirjam C K; Ganchev, Boian; Kerb, Reinhold; Hofmann, Ute; Mürdter, Thomas E

    2014-10-01

    A new reaction pathway for the synthesis of a [(2)H]-labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope-labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis. PMID:25339577

  18. BIOAVAILABILITY OF LUTEIN IN HUMANS FROM INTRINSICALLY LABELED VEGETABLES DETERMINED BY LC-APCI-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of the investigation was to assess a stable isotope method for determining the relative bioavailability of food-derived lutein in humans. Subjects were administered a single dose of deuterium-labeled carotenoids from intrinsically labeled spinach or collard green; 10 mL blood samples were d...

  19. Assimilation efficiency for sediment-sorbed benzo(a)pyrene by Diporeia spp.

    USGS Publications Warehouse

    Lydy, M.J.; Landrum, P.F.

    1993-01-01

    Two methods are currently available for determining contaminant assimilation efficiencies (AE) from ingested material in benthic invertebrates. These methods were compared using the Great Lakes amphipod Diporeia spp. and [14C]benzo(a)pyrene (BaP) sorbed to Florissant sediment (< 63 ??m). The first approach, the direct measurement method, uses total organic carbon as a tracer and yielded AE values ranging from 45.9-60.4%. The second approach, the dual-labeled method, uses 51Cr as a non-assimilated tracer and did not yield AE values for our data. The inability of the dual- labeled approach to estimate AEs was due, in part, to the selective feeding by Diporeia resulting in a failure of the non-assimilated tracer (51Cr) to track with the assimilated tracer ([14C]BaP). The failure of the dual- labeled approach was not a result of an uneven distribution of the labels among particle size classes, but more likely resulted from differential sorption of the two isotopically labeled materials to particles of differing composition. The [14C]BaP apparently sorbs to organic particles that are selectively ingested, while the 51Cr apparently sorbs to particles which are selectively excluded by Diporeia. The dual-labeled approach would be a viable and easier experimental approach for determining AE values if the characteristics that govern selective feeding can be determined.

  20. Metabolic flux analysis using 13C peptide label measurements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...

  1. DNA Hybridization: Nonradioactive Labeling Now Available for the Laboratory.

    ERIC Educational Resources Information Center

    Freeman, Lenore Gardner

    1984-01-01

    The advantages of DNA hybridization procedures for classroom and clinical use can now be realized by the recent development of nonradioactive DNA labeling/detection procedures. These methods (which are described) can replace the use of isotopes in standard DNA hybridization procedures. (JN)

  2. Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short-strong hydrogen bonded cluster

    E-print Network

    Iyengar, Srinivasan S.

    Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short/deuterium isotope effects, in a fundamental organic hydrogen bonded system using multiple experimental infrared the isotopically labeled systems arises from an analysis of the simulated cluster spectroscopy and leads

  3. Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short-strong hydrogen bonded cluster

    E-print Network

    Iyengar, Srinivasan S.

    Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short, Bloomington, IN 47405 Abstract We investigate and analyze the vibrational properties, including H/D isotope between simulated cluster spectroscopy of the isotopically labeled systems were analyzed from a system

  4. Isotope separation apparatus and method

    DOEpatents

    Feldman, Barry J. (Los Alamos, NM)

    1985-01-01

    The invention relates to an improved method and apparatus for laser isotope separation by photodeflection. A molecular beam comprising at least two isotopes to be separated intersects, preferably substantially perpendicular to one broad side of the molecular beam, with a laser beam traveling in a first direction. The laser beam is reflected back through the molecular beam, preferably in a second direction essentially opposite to the first direction. Because the molecules in the beam occupy various degenerate energy levels, if the laser beam comprises chirped pulses comprising selected wavelengths, the laser beam will very efficiently excite substantially all unexcited molecules and will cause stimulated emission of substantially all excited molecules of a selected one of the isotopes in the beam which such pulses encounter. Excitation caused by first direction chirped pulses moves molecules of the isotope excited thereby in the first direction. Stimulated emission of excited molecules of the isotope is brought about by returning chirped pulses traveling in the second direction. Stimulated emission moves emitting molecules in a direction opposite to the photon emitted. Because emitted photons travel in the second direction, emitting molecules move in the first direction. Substantial molecular movement of essentially all the molecules containing the one isotope is accomplished by a large number of chirped pulse-molecule interactions. A beam corer collects the molecules in the resulting enriched divergent portions of the beam.

  5. Dynamic Visualization of Graphs with Extended Labels

    SciTech Connect

    Wong, Pak C.; Mackey, Patrick S.; Perrine, Kenneth A.; Eagan, James R.; Foote, Harlan P.; Thomas, Jim

    2005-10-23

    The paper describes a novel technique to visualize graphs with extended node and link labels. The lengths of these labels range from a short phrase to a full sentence to an entire paragraph and beyond. Our solution is different from all the existing approaches that almost always rely on intensive computational effort to optimize the label placement problem. Instead, we share the visualization resources with the graph and present the label information in static, interactive, and dynamic modes without the requirement for tackling the intractability issues. This allows us to reallocate the computational resources for dynamic presentation of real-time information. The paper includes a user study to evaluate the effectiveness and efficiency of the visualization technique.

  6. Ideal regularization for learning kernels from labels.

    PubMed

    Pan, Binbin; Lai, Jianhuang; Shen, Lixin

    2014-08-01

    In this paper, we propose a new form of regularization that is able to utilize the label information of a data set for learning kernels. The proposed regularization, referred to as ideal regularization, is a linear function of the kernel matrix to be learned. The ideal regularization allows us to develop efficient algorithms to exploit labels. Three applications of the ideal regularization are considered. Firstly, we use the ideal regularization to incorporate the labels into a standard kernel, making the resulting kernel more appropriate for learning tasks. Next, we employ the ideal regularization to learn a data-dependent kernel matrix from an initial kernel matrix (which contains prior similarity information, geometric structures, and labels of the data). Finally, we incorporate the ideal regularization to some state-of-the-art kernel learning problems. With this regularization, these learning problems can be formulated as simpler ones which permit more efficient solvers. Empirical results show that the ideal regularization exploits the labels effectively and efficiently. PMID:24824969

  7. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  8. Comprehensive analysis of metabolic pathways through the combined use of multiple isotopic tracers

    E-print Network

    Antoniewicz, Maciek Robert

    2006-01-01

    Metabolic Flux Analysis (MFA) has emerged as a tool of great significance for metabolic engineering and the analysis of human metabolic diseases. An important limitation of MFA, as carried out via stable isotope labeling ...

  9. Combining solvent isotope effects with substrate isotope effects in mechanistic studies of alcohol and amine oxidation by enzymes.

    PubMed

    Fitzpatrick, Paul F

    2015-11-01

    Oxidation of alcohols and amines is catalyzed by multiple families of flavin- and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. PMID:25448013

  10. Monitoring electron donor metabolism under variable electron acceptor conditions using 13C-labeled lactate

    NASA Astrophysics Data System (ADS)

    Bill, M.; Conrad, M. E.; Yang, L.; Beller, H. R.; Brodie, E. L.

    2010-12-01

    Three sets of flow-through columns constructed with aquifer sediment from Hanford (WA) were used to study reduction of Cr(VI) to poorly soluble Cr(III) under denitrifying, sulfate-reducing/fermentative, and iron-reducing conditions with lactate as the electron donor. In order to understand the relationship between electron donors and biomarkers, and to determine the differences in carbon isotope fractionation resulting from different microbial metabolic processes, we monitored the variation in carbon isotopes in dissolved inorganic carbon (DIC), in total organic carbon (TOC), and in lactate, acetate and propionate. The greatest enrichment in 13C in columns was observed under denitrifying conditions. The ?13C of DIC increased by ~1750 to ~2000‰ fifteen days after supplementation of natural abundance lactate with a 13C-labeled lactate tracer (for an influent ?13C of ~2250‰ for the lactate) indicating almost complete oxidation of the electron donor. The denitrifying columns were among the most active columns and had the highest cell counts and the denitrification rate was highly correlated with Cr(VI) reduction rate. ?13C values of DIC ranged from ~540 to ~1170‰ for iron-reducing conditions. The lower enrichment in iron columns was related to the lower biological activity observed with lower yields of RNA and cell numbers in the column effluents. The carbon isotope shift in the sulfate-reducing ~198 to ~1960‰ for sulfate-reducing conditions reflecting the lower levels of the lactate in these columns. Additionally, in two of the sulfate columns, almost complete fermentation of the lactate occurred, producing acetate and propionate with the labeled carbon signature, but relatively smaller amounts of inorganic carbon. For all electron-accepting conditions, TOC yielded similar ?13C values as lactate stock solutions. Differences in C use efficiency, metabolic rate or metabolic pathway contributed to the differing TOC ?13C to DIC ?13C ratios between treatments. Carbon isotope signatures of DIC can be useful for monitoring the efficiency of 13C-enriched electron donor consumption associated with bioactivity under reducing conditions.

  11. Utraviolet-B effects on stomatal density, water use efficiency, and stable carbon isotope discrimination in four glasshouse-grown soybean cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interactions between UV-B radiation and drought stress have been observed but the underlying mechanisms have not been thoroughly investigated. We hypothesized that UV-B radiation would improve water use efficiency by its effects on epidermal development, specifically stomatal density and leaf gas e...

  12. Tritium labeling of detonation nanodiamonds.

    PubMed

    Girard, Hugues A; El-Kharbachi, Abdelouahab; Garcia-Argote, Sébastien; Petit, Tristan; Bergonzo, Philippe; Rousseau, Bernard; Arnault, Jean-Charles

    2014-03-18

    For the first time, the radioactive labeling of detonation nanodiamonds was efficiently achieved using a tritium microwave plasma. According to our measurements, the total radioactivity reaches 9120 ± 120 ?Ci mg(-1), with 93% of (3)H atoms tightly bonded to the surface and up to 7% embedded into the diamond core. Such (3)H doping will ensure highly stable radiolabeled nanodiamonds, on which surface functionalization is still allowed. This breakthrough opens the way to biodistribution and pharmacokinetics studies of nanodiamonds, while this approach can be scalable to easily treat bulk quantities of nanodiamonds at low cost. PMID:24492594

  13. Introduction Balanced Group Labeled Graphs

    E-print Network

    Diwan, Ajit A

    Introduction Results Summary Balanced Group Labeled Graphs M. Joglekar N. Shah A.A. Diwan.A.Diwan Balanced Group Labeled Graphs #12;Introduction Results Summary Outline 1 Introduction Group Labeled Graphs Balanced Labellings Characterization 2 Results Counting Number of Balanced labellings Proof Markable Graphs

  14. Labeling and Identification of Direct Kinase Substrates

    PubMed Central

    Carlson, Scott M.; White, Forest M.

    2013-01-01

    Identifying kinase substrates is an important step in mapping signal transduction pathways, but remains a difficult and time-consuming process. Analog-sensitive kinases (AS-kinases) have been used to selectively tag and identify direct kinase substrates in lysates from whole cells. In this approach a gamma-thiol ATP-analog and AS-kinase are used to selectively thiophosphorylate target proteins. Thiophosphate is used as a chemical handle to purify peptides from a tryptic digest, and target proteins are identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we describe an updated strategy for labeling AS-kinase substrates, solid-phase capture of thiophosphorylated peptides, incorporation of stable-isotopic labeling in cell culture (SILAC) for filtering nonspecific background peptides, enrichment of phosphorylated target peptides to identify low-abundance targets, and analysis by LC-MS/MS. PMID:22669844

  15. Nanovehicles based Bioassay Labels

    SciTech Connect

    Liu, Guodong; Wang, Jun; Wu, Hong; Lin, Ying-Ying; Lin, Yuehe

    2007-04-01

    In this article, we review recent advances of our group in nanoparticle labels based bioassay. Apoferritin and silica nanoparticles have been used as nanovehicles to load large amount of markers for highly sensitive bioassay. Markers loaded apoferritin, apoferritin-templated metallic phosphate nanoparticles, and poly [guanine] coated silica nanoparticles have been prepared, characterized and used as labels for highly sensitive bioassay of protein and DNA. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin nanovehicle provides a simple and convenient route to prepare versatile nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. Additionally, the use of apoferritin nanovehicle as template for synthesis of metallic phosphate nanoparticle labels offers fast route to prepare uniform-size metallic nanoparticle labels for electrochemical bioassay and avoids the traditional harsh dissolution conditions to dissolve metallic nanoparticle tags (that is, the strong-acid dissolution of quantum dots and gold nanoparticles) during the stripping analysis step. Silica nanoparticle has also been used as nanovehicle to carry thousands of poly [guanine] tracers, which was used to enhance the oxidation current of Ru(bpy)32+, resulting in enhanced sensitivity of electrochemical immunoassay. The new nanovehicle-based labels have been used for highly sensitive electrochemical detection of DNA and protein biomarkers, such as tumor necrosis factor-alpha (TNF-a). The high sensitivity and selectivity make these labels a useful addition to the armory of nanoparticle-based bioassay. The new nanovehicles based labels hold great promise for multiplex protein and DNA detection and for enhancing the sensitivity of other bioassays.

  16. How to read food labels

    MedlinePLUS

    Food labels tell you the nutrition facts about the foods you buy. Use the food labels to help you choose healthier foods. ... also pay attention to trans fats on any food label. These fats raise "bad" cholesterol and lower your " ...

  17. How to Read Drug Labels

    MedlinePLUS

    ... and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to Read Drug ... read drug labels How to read a prescription drug label View a text version of this picture. ...

  18. Nutrition Facts: Reading the Label

    MedlinePLUS

    ... My Go4Life Get Free Stuff Be a Partner Nutrition Facts: Reading the Label Reading labels can help ... of information on their labels or packaging about nutrition and food safety. Product dates . You might see ...

  19. Bioavailability of xenobiotics in unsaturated soils – implications for nucleic acid based stable isotope probing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of stable isotopes to label phylogenetically informative biomolecules (phospholipid fatty acids, DNA, or RNA), typically referred to as stable isotope probing (SIP) has the potential of providing definitive evidence that a detected population is active in a specific process, if that process ...

  20. The Doubly Labeled Water Method for Measuring Human Energy Expenditure: Adaptations for Spaceflight

    NASA Technical Reports Server (NTRS)

    Schulz, Leslie O.

    1991-01-01

    It is essential to determine human energy requirements in space, and the doubly labeled water method has been identified as the most appropriate means of indirect calorimetry to meet this need. The method employs naturally occurring, stable isotopes of hydrogen (H-2, deuterium) and oxygen (O-18) which, after dosing, mix with body water. The deuterium is lost from the body as water while the O-18 is eliminated as both water and CO2. The difference between the two isotope elimination rates is therefore a measure of CO2 production and hence energy expenditure. Spaceflight will present a unique challenge to the application of the doubly labeled water method. Specifically, interpretation of doubly labeled water results assumes that the natural abundance or 'background' levels of the isotopes remain constant during the measurement interval. To address this issue, an equilibration model will be developed in an ongoing ground-based study. As energy requirements of women matched to counterparts in the Astronauts Corps are being determined by doubly labeled water, the baseline isotope concentration will be changed by consumption of 'simulated Shuttle water' which is artificially enriched. One group of subjects will be equilibrated on simulated Shuttle water prior to energy determinations by doubly labeled water while the others will consume simulated Shuttle water after dosing. This process will allow us to derive a prediction equation to mathematically model the effect of changing background isotope concentrations.

  1. Stable isotope studies

    SciTech Connect

    Ishida, T.

    1992-01-01

    The research has been in four general areas: (1) correlation of isotope effects with molecular forces and molecular structures, (2) correlation of zero-point energy and its isotope effects with molecular structure and molecular forces, (3) vapor pressure isotope effects, and (4) fractionation of stable isotopes. 73 refs, 38 figs, 29 tabs.

  2. Embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenetic mechanisms

    SciTech Connect

    Spielmann, H.; Nau, H.

    1986-07-01

    Experiments on teratogenic effects of stable isotopes from our own and other laboratories are evaluated. In the first series of investigations, the enrichment of the stable isotope /sup 13/C derived from U-/sup 13/C-glucose was studied in mouse embryos at various stages of development, including limb buds in organ culture. Preimplantation mouse embryos incubated in vitro in /sup 13/C-enriched medium for 48 hours showed normal development during subsequent differentiation in vitro and also in vivo after embryo transfer to faster mothers. These embryos were 15% to 20% enriched in /sup 13/C. Administration of U-13-C-glucose to pregnant mice during organogenesis led to an increase of the absolute /sup 13/C content of the embryo for several days after the end of isotope administration, whereas the enrichment in maternal tissue decreased. No alterations of embryonic development were detected due to stable isotope enrichment. Development of cultured mouse limb buds was unaffected by incubation with 82 mol% U-/sup 13/C-glucose as judged from morphologic and biochemical criteria. The second part of the article describes the value of deuterium-labeled drugs as probes into the mechanism of activation of teratogenic metabolites. A comparison of the pharmacokinetics as well as the teratogenicity between cyclophosphamide and some specific deuterium-labeled analogues showed that the isotope effect observed can be related to a particular metabolic pathway crucial for teratogenic activation by this drug.

  3. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  4. Indium--111 tropolone, a new high-affinity platelet label: preparation and evaluation of labeling parameters

    SciTech Connect

    Dewanjee, M.K.; Rao, S.A.; Didisheim, P.

    1981-11-01

    Platelets were labeled with a new neutral, lipid-soluble metal complex of indium-111 and tropolone. Unlike oxine, which must be dissolved in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with In-111 tropolone can be performed in both acid-citrate-dextrose (ACD)-plasma and ACD-saline media within two hours' time. Labeling efficiency has been 80-90% in ACD-saline and 60-70% in the ACD-plasma medium. Optimum concentrations for the labeling of platelets with In-111 tropolone were 5 ..mu..g/ml in ACD-saline and 10 ..mu..g/ml in ACD-plasma, using a 15-min incubation at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. Seven parameters of platelet labeling were studied: concentration of tropolone, citrate, plasma proteins, and calcium ions; also platelet density, temperature, and pH of incubation medium. Their effects on the mechanism of platelet labeling with lipid-soluble tracers are discussed.

  5. CARBON ISOTOPE DISCRIMINATION AND GROWTH RESPONSE TO STAND DENSITY REDUCTIONS IN OLD PINUS PONDEROSA TREES

    EPA Science Inventory

    Carbon isotope ratios ( 13C) of tree rings are commonly used for paleoclimatic reconstruction and for inferring canopy water-use efficiency (WUE). However, the responsiveness of carbon isotope discrimination ( ) to site disturbance and resource availability has only rarely been ...

  6. Probes labelled with energy transfer coupled dyes

    DOEpatents

    Mathies, R.A.; Glazer, A.; Ju, J.

    1997-11-18

    Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids. 7 figs.

  7. Covalent Protein Labeling by Enzymatic Phosphocholination.

    PubMed

    Heller, Katharina; Ochtrop, Philipp; Albers, Michael F; Zauner, Florian B; Itzen, Aymelt; Hedberg, Christian

    2015-08-24

    We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo. PMID:26147231

  8. Labeling Organic Products

    MedlinePLUS

    ... gov/NOPOrganicLabeling . 100 PERCENT ORGANIC Raw or processed agricultural products in the “100 percent organic” category must ... asterisk or other mark. ORGANIC Raw or processed agricultural products in the “organic” category must meet these ...

  9. Theoretical study on isotope separation of an ytterbium atomic beam by laser deflection

    NASA Astrophysics Data System (ADS)

    Zhou, Min; Xu, Xin-Ye

    2014-01-01

    Isotope separation by laser deflecting an atomic beam is analyzed theoretically. Interacting with a tilted one-dimensional optical molasses, an ytterbium atomic beam is split into multi-beams with different isotopes like 172Yb,173Yb, and 174Yb. By using the numerical calculation, the dependences of the splitting angle on the molasses laser intensity and detuning are studied, and the optimal parameters for the isotope separation are also investigated. Furthermore, the isotope separation efficiency and purity are estimated. Finally a new scheme for the efficient isotope separation is proposed. These findings will give a guideline for simply obtaining pure isotopes of various elements.

  10. China Refrigerator Information Label: Specification Development and Potential Impact

    SciTech Connect

    Fridley, David; Fridley, David; Zheng, Nina; Zhou, Nan; Aden, Nathaniel; Lin, Jiang; Jianhong, Cheng; Sakamoto, Tomoyuki

    2008-02-01

    In the last five years, China's refrigerator market has grown rapidly, and now urban markets are showing signs of saturation, with ownership rates in urban households reaching 92%. Rural markets continue to grow from a much lower base. As a result of this growth, the Chinese government in 2006 decided to revise the refrigerator standards and its associated efficiency grades for the mandatory energy information label. In the Chinese standards process, the efficiency grades for the information label are tied to the minimum standards. Work on the minimum standards revision began in 2006 and continued through the first half of 2007, when the draft standard was completed under the direction of the China National Institute of Standardization (CNIS). Development of the information label grades required consideration of stakeholder input, continuity with the previous grade classification, ease of implementation, and potential impacts on the market. In this process, CLASP, with the support of METI/IEEJ, collaborated with CNIS to develop the efficiency grades, providing technical input to the process, comment and advice on particular technical issues, and evaluation of the results. After three months of effort and three drafts of the final grade specifications, this work was completed. In addition, in order to effectively evaluate the impact of the label on China's market, CLASP further provided assistance to CNIS to collect data on both the efficiency distribution and product volume distribution of refrigerators on the market. The new information label thresholds to be implemented in 2008 maintain the approach first adopted in 2005 of establishing efficiency levels relative to the minimum standard, but increased the related required efficiency levels by 20% over those established in 2003 and implemented in 2005. The focus of improvement was on the standard refrigerator/freezer (class 5), which constitutes the bulk of the Chinese market. Indeed, the new requirements to achieve grade 1 on the label are now virtually as stringent as those for US Energy Star-qualified or EU A-grade refrigerators. When the energy information label went into effect in March 2005, refrigerator manufacturers were required to display their declared level of efficiency on the label and report it to the China Energy Label Center (CELC), a newly established unit of CNIS responsible for label program management. Because of the visible nature of the label, it was found, through a METI/IEEJ-supported study, that MEPS non-compliance dropped from 4% to zero after the label became mandatory, and that the percentage of higher-grade refrigerators increased. This suggests that the label itself does have potential for shifting the market to higher-efficiency models (Lin 2007). One challenge, however, of assessing this potential impact is the lack of a comprehensive baseline of market efficiency and a program to evaluate the market impact on a yearly basis. As a result, the impact evaluation in this study draws upon the market transformation experience of the related EU energy information label, for which quantitative assessments of its market impact exist. By assuming a parallel process unfolding in China, it is possible to look at the potential impact of the label to 2020. The results of the analysis demonstrates that a robust market transformation program in China focused on the energy information label could save substantial amounts of electricity by 2020, totaling 16.4 TWh annually by that year, compared to a case in which the efficiency distribution of refrigerators was frozen at the 2007 level. Remarkably, the impact of a successful market transformation program with the label would essentially flatten the consumption of electricity for refrigerator use throughout most of the next decade, despite the expectations of continued growth in total stock by nearly 190 million units. At the end of this period, total consumption begins to rise again, as the least efficient of the units have been mostly removed from the market. Such a level of savings would reduce CO{sub

  11. On Gracefully Labeling Trees

    E-print Network

    Dhananjay P. Mehendale

    2013-10-25

    A method to obtain all possible graceful spanning trees in a complete graph is proposed. An algorithm to generate all the labeled spanning trees in a complete graph is developed and modified to generate all graceful spanning trees. The count of all possible graceful graphs in a complete graph is obtained. An upper bound on the count of gracefully labeled trees in a complete graph is obtained. We settle Graceful Tree Conjecture in the affirmative in two ways: 1) We show that all trees can be gracefully labeled by assigning the lowest label 1 to the so called special vertices of trees, i.e. prependant vertices or pendant vertices adjacent to prependant vertices. 2) We establish the existence of graceful labeling for all trees by associating distinct lattice paths with trees and by showing the existence of a lattice path for a tree of each isomorphism type by showing how to construct a lattice path recursively by starting from the lattice path for its pendant vertex deleted subtree, which is assumed to exists by induction, and carrying out appropriate modification of this lattice path. 3) We propose a crisp algorithm to gracefully label tree of every isomorphism type with the help of novel representation for any tree in terms of juxtaposition of paths. Lastly, we discuss an algorithm to find arbitrarily degree constrained graceful spanning tree and propose some problems for further investigation.

  12. Re-188 labelled antibodies.

    PubMed

    Rhodes, B A; Lambert, C R; Marek, M J; Knapp, F F; Harvey, E B

    1996-01-01

    Monoclonal antibodies can be directly labelled with 188Re using a simple one-step radiolabelling kit. Using B72.3 as a model antibody, the formulation was optimized and kits were made and tested and compared to data previously reported for the same antibody labelled with other radioisotopes. Labelling with Re-188 was carried out with the eluate of a W-188/Re-188 generator from Oak Ridge National Laboratory. Fresh generator eluate was added to the pre-reduced lyophilized antibody and the mixture allowed to incubate overnight at room temperature. The radiochemical purity, immunoreactive fraction, and biodistribution in normal and LS174T tumor bearing nude mice was determined. The radiochemical purity was 88 +/- 7%, the immunoreactive fraction was 68.46 +/- 3.8%. The immunoreactive fraction was higher than any previously reported for this antibody when labelled with other radioisotopes. At 48 h, 7.9 +/- 2.4% of the injected dose per gram was found in the tumor. The biodistribution and tumor uptake of Re-188 labelled B72.3 was similar to that previously reported for Re-186 and In-111 labelled B72.3. PMID:8589673

  13. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

    PubMed Central

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian

    2015-01-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13C/15N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13C/15N-labeled amino acids. The most cost-effective production of 13C/15N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% 13C-glycerol and 0.5% 15N-ammonium sulfate, supplemented with only 0.025% of 13C/15N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  14. Off-Label Drug Use

    MedlinePLUS

    ... Your Local Offices Close + - Text Size Off-label Drug Use What is off-label drug use? In the United States new drugs are ... unapproved use of a drug. Is off-label drug use legal? The off-label use of FDA- ...

  15. Isotope Tales: Remaining Problems, Unsolvable Questions, and Gentle Successes

    NASA Astrophysics Data System (ADS)

    fogel, marilyn; bradley, christina; newsome, seth; filipp, fabian

    2014-05-01

    Earth's biomes function and adapt today as climate changes and ecosystems and the organisms within them adapt. Stable isotope biogeochemistry has had a major influence in understanding climate perturbations and continues to be an active area of research on many fronts. Banking on the success of compound specific stable isotope analyses of amino acids, nitrogen, carbon, and hydrogen isotopes continue to reveal subtle shifts in oceanic food webs and metabolic changes in microbes, plants, and animals. A biochemical understanding of exactly how organisms process and partition stable isotopes during metabolism remains unsolved, but is required if this field is to move beyond description to quantitation. Although the patterns of carbon and nitrogen isotopes are fairly well established in the common amino acids, we need to consider specifics: How do shifting metabolic pathways (metabolomics) influence the outcome of stable isotope partitioning? What influence does the gut microflora in animals have on isotopic labeling? What are the intramolecular isotope patterns of common amino acids and what do they tell us? What can be learned with other isotope systems, such as hydrogen? Results and ideas of how to move forward in this field will be presented starting at the molecular level and ending with ecosystems.

  16. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ...for Container Labels and Carton Labeling Design To Minimize Medication Errors; Availability...for Container Labels and Carton Labeling Design to Minimize Medication Errors.'' The...the container label and carton labeling design for prescription drug and biological...

  17. Direct photoaffinity labeling of tubulin with colchicine

    SciTech Connect

    Wolff, J.; Knipling, L.; Cahnmann, H.J.; Palumbo, G. )

    1991-04-01

    Ultraviolet irradiation of the ({sup 3}H)colchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to greater than 90%) of the labeling occurs on beta-tubulin and appears early after irradiation, whereas {alpha}-tubulin is labeled later. The labeling ratio of {beta}-tubulin to {alpha}-tubulin ({beta}/{alpha} ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the {beta}/{alpha} ratio. Limited data with ({sup 3}H)podophyllotoxin show that it covalently bound with a similar {beta}/{alpha} distribution. Vinblastine, on the other hand, exhibits preferential attachment to {alpha}-tubulin. The possibilities that colchicine binds at the interface between {alpha}-tubulin and {beta}-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.

  18. Manifold Adaptive Label Propagation for Face Clustering.

    PubMed

    Pei, Xiaobing; Lyu, Zehua; Chen, Changqing; Chen, Chuanbo

    2015-08-01

    In this paper, a novel label propagation (LP) method is presented, called the manifold adaptive label propagation (MALP) method, which is to extend original LP by integrating sparse representation constraint into regularization framework of LP method. Similar to most LP, first of all, MALP also finds graph edges from given data and gives weights to the graph edges. Our goal is to find graph weights matrix adaptively. The key advantage of our approach is that MALP simultaneously finds graph weights matrix and predicts the label of unlabeled data. This paper also derives efficient algorithm to solve the proposed problem. Extensions of our MALP in kernel space and robust version are presented. The proposed method has been applied to the problem of semi-supervised face clustering using the well-known ORL, Yale, extended YaleB, and PIE datasets. Our experimental evaluations show the effectiveness of our method. PMID:25291812

  19. Reuse or Never Reuse the Deleted Labels in XML Query Processing Based on Labeling Schemes

    E-print Network

    Ling, Tok Wang

    , yet it can efficiently determine the ancestor-descendant (A-D) and parent-child (P-C) relationships have been proposed. Based on the labeling schemes, the ancestor- descendant and parent-child relationships in XML queries can be quickly de- termined without accessing the original XML file. Recently, more

  20. Application of Screening Experimental Designs to Assess Chromatographic Isotope Effect upon Isotope-Coded Derivatization for Quantitative Liquid Chromatography–Mass Spectrometry

    PubMed Central

    2015-01-01

    Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography–mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, 13C6-, 15N2-, or 15N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett–Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of 15N or 13C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus 15N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

  1. Differential protein labeling based on electrochemically generated reactive intermediates.

    PubMed

    Büter, Lars; Faber, Helene; Wigger, Tina; Vogel, Martin; Karst, Uwe

    2015-10-01

    A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as ?-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments. PMID:26327615

  2. Synthetic procedures for the preparation of deuterium-labeled analogs of naturally occurring steroids

    SciTech Connect

    Wudy, S.A. )

    1990-10-01

    The object of this article is to review the procedures that have been published concerning the preparation of deuterium-labeled analogs of naturally occurring steroid hormones. In combination with mass spectrometric methods, these stable isotope-labeled compounds should be applicable for human metabolism studies or as internal standards. Deuteration techniques for the elucidation of stereochemical problems, procedures for the preparation of monodeuterated steroids, and synthesis of deuterated analogs of nonbiologic steroids have therefore not been included in this review. 41 refs.

  3. Base-sequence dependence of emission lifetimes for DNA oligomers and duplexes covalently labeled with pyrene: Relative electron-transfer quenching efficiencies of A, G, C, and T nucleosides toward pyrene

    SciTech Connect

    Manoharan, M.; Tivel, K.L.; Zhao, M.; Nafisi, K.; Netzel, T.L.

    1995-11-30

    This paper reports both continuous and time-resolved spectroscopic studies of the emission properties of photoexcited pyrene labels covalently attached to uridine nucleosides and oligonucleotides. For all nucleic acid systems, uridine is substituted with pyrene at the 2`-oxygen position, 2`-O-[hexyl-N-(1-pyrenepropylcarbonyl)amino]uridine, U(12){sup *} . Three types of nucleic nucleic acid systems are investigated: the 5`-OH (1) and the 5`-ODMT (2) substituted U(12){sup *}-nucleosides; four pentameric oligonucleotides, X{sub 2}U(12){sup *}X{sub 2}, where X is 2`-deoxyadenosine (A), 2`-deoxyguanosine (G), 2`-deoxythymidine (T), or 2`-deoxycytidine (C); and four duplexes with 18 base pairs each containing one strand with a central U(12){sup *} label. The longest {pi},{pi}{sup *} emission lifetimes of U(12){sup *}-labeled DNA duplexes exceed those of the corresponding parameters. A measure of duplex-induced restricted access pyrene{sup *} to base-paired nucleosides in double-strand (ds) versus single-strand (ss) DNA can be obtained. 111 refs., 6 figs., 6 tabs.

  4. Cosmic ray isotopes

    NASA Technical Reports Server (NTRS)

    Stone, E. C.

    1973-01-01

    The isotopic composition of cosmic rays is studied in order to develop the relationship between cosmic rays and stellar processes. Cross section and model calculations are reported on isotopes of H, He, Be, Al and Fe. Satellite instrument measuring techniques separate only the isotopes of the lighter elements.

  5. Production of (15)N-labeled ?-amanitin in Galerina marginata.

    PubMed

    Luo, Hong; DuBois, Brandon; Sgambelluri, R Michael; Angelos, Evan R; Li, Xuan; Holmes, Daniel; Walton, Jonathan D

    2015-09-01

    ?-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but ?-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces ?-amanitin in culture, we describe a method for producing (15)N-labeled ?-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, ?-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR. PMID:26100667

  6. Labeling and Functionalizing Amphipols for Biological Applications

    PubMed Central

    Bon, Christel Le; Popot, Jean-Luc; Giusti, Fabrice

    2014-01-01

    Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes. PMID:24696186

  7. 40 CFR 156.10 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS LABELING REQUIREMENTS FOR PESTICIDES AND DEVICES General Provisions § 156... —(1) Contents of the label. Every pesticide product shall bear a label...

  8. Preparation of (68)Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    PubMed

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies. PMID:26492321

  9. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... Label Approval System: Generic Label Approval AGENCY: Food Safety and Inspection Service, USDA. ACTION... the Agency (76 FR 75809). FSIS also proposed to combine the regulations that provide for the approval... ingredients actually used in the food product that is to bear the label--the only way to determine...

  10. Pesticide Labeling: Miscellaneous Label Parts1 Frederick M. Fishel2

    E-print Network

    Hill, Jeffrey E.

    PI-109 Pesticide Labeling: Miscellaneous Label Parts1 Frederick M. Fishel2 1. This document is PI, professor, Agronomy Department, and director, Pesticide Information Office; UF/IFAS Extension, Gainesville pesticides safely. Read and follow directions on the manufacturer's label. The Institute of Food

  11. Pesticide Labeling: Labeling Claims1 Frederick M. Fishel2

    E-print Network

    Hill, Jeffrey E.

    PI-105 Pesticide Labeling: Labeling Claims1 Frederick M. Fishel2 1. This document is PI-105, one, Agronomy Department, and Director, Pesticide Information Office, UF/IFAS Extension, Gainesville, FL 32611. Use pesticides safely. Read and follow directions on the manufacturer's label. The Institute of Food

  12. 99m tc labeled liposomes

    SciTech Connect

    Phillips, W.T.; Klipper, R.W.; Timmons, J.H.; Rudolph, A.S.

    1992-10-27

    This patent describes a method of preparing stable gamma-emitting radionuclide-labeled alkyleneamine oxime, the incubating being for a period of time sufficient to form labeled liposome-encapsulated protein.

  13. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  14. Isotope Dilution Mass Spectrometry for the Quantification of Sulfane Sulfurs

    PubMed Central

    Liu, Chunrong; Zhang, Faya; Munske, Gerhard; Zhang, Hui

    2014-01-01

    Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that can attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H2S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfurs concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulphide product PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a 13C3-labelled phosphine sulfide PS1 as the internal standard. The specificity and efficiency of the method were proved by model reactions. It was also applied in the measurement of sulfane sulfurs in mice tissues including brain, kidney, lung, liver, heart, spleen, and blood. PMID:25152234

  15. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label

  16. Method for labeling phagocytic cells

    SciTech Connect

    Gamble, R. C.; Tin, G. W.; Williams, L. E.

    1985-02-05

    Described herein is a process for labeling leukocytes and other phagocytic cells with labeled micellular particles involving incubating the cells with the micellular particles. Also described is a process for detecting the locus of an infection by administering to a subject leukocytes radiolabeled by incubation with labeled micellular particles followed by scanning the subject to detect the locus of radiation emitted by the particles.

  17. Food Labels Tell the Story!

    MedlinePLUS

    ... page Share From the Label to the Table! Food Labels Tell the Story! What is in food? Food provides your body with all of the ... your food choices. Nutrition Facts—the Labels on Food Products Beginning in 1994, the US government began ...

  18. 13C? CEST experiment on uniformly 13C-labeled proteins.

    PubMed

    Zhou, Yang; Yang, Daiwen

    2015-02-01

    A new HSQC-based (13)C? CEST pulse scheme is proposed, which is suitable for uniformly (13)C- or (13)C, (15)N-labeled samples in either water or heavy water. Except for Thr and Ser residues, the sensitivity of this scheme for uniformly labeled samples is similar to that of the previous scheme for selectively (13)C?-labeled samples with 100% isotope enrichment. The experiment is demonstrated on an acyl carrier protein domain. Our (13)C? CEST data reveal that the minor state of the acyl carrier protein has high helical propensity. The new scheme will facilitate structural characterization of invisible minor states. PMID:25465387

  19. ROUTING IN TIME-DEPENDENT AND LABELED NETWORKS

    SciTech Connect

    Barrett, C. L.; Bisset, K. R.; Jacob, R.; Konjevod, G.; Marathe, M. V.

    2001-01-01

    We study routing problems in time-dependent and edge and/or vertex-labeled transportation networks. Labels allow one to express a number of discrete properties of the edges and nodes. The main focus is a unified algorithm that efficiently solves a number of seemingly unrelated problems in transportation science. Experimental data gained from modeling practical situations suggest that the formalism allows interesting compromises between the conflicting goals of generality and efficiency. 1. We use edge/vertex labels in the framework of Formal Language Constrained Path Problems to handle discrete choice constraints. The label set is usually small and does not depend on the graph. Edge labels induct! path labels, which allows us to impose feasibility constraints on the set of paths considered as shortest path candidates. Second, we propose monotonic piecewise-linear traversal functions to represent the time-dependent aspect of link delays. The applications that can be modeled include scheduled transit and time-windows. 3. Third, we combine the above models and capture a variety of natural problems in transportatiou science such as time-window constrained trip-chaining. The results demonstrate the robustness of the proposed formalisms. As evidence for our claims of practical efficiency in a realistic setting, we report preliminary computational experience from TRANSIMS case studies of Portland, Oregon.

  20. Impact of nutritional labelling on 10-d energy intake, appetite perceptions and attitudes towards food.

    PubMed

    Carbonneau, Elise; Perron, Julie; Drapeau, Vicky; Lamarche, Benoît; Doucet, Éric; Pomerleau, Sonia; Provencher, Véronique

    2015-12-01

    The purpose of this study was to investigate the impact of nutritional labelling on energy intake, appetite perceptions and attitudes towards food. During a 10-d period, seventy normal-weight (BMI<25 kg/m2) and seventy-one obese women (BMI?30 kg/m2) were given three meals per d under ad libitum conditions. Participants were randomly assigned to one of three experimental labelling groups in which the only difference was the label posted on lunch meal entrée: (1) low-fat label, (2) energy label (energy content of the entrée and average daily needs) and (3) no label (control). Average energy intake was calculated by weighing all foods before v. after daily consumption. Hunger and fullness perceptions were rated on visual analogue scales immediately before and after each meal. Satiety efficiency was assessed through the calculation of the satiety quotient (SQ). The appreciation and perceived healthiness of the lunch entrées were rated on eight-point Likert scales. There was no difference in energy intake, SQ and attitudes towards food between the three labelling groups. Fasting hunger perception was higher in the low-fat label group compared with the two others groups (P=0·0037). No interactions between labelling groups and BMI categories were observed. In conclusion, although labelling does not seem to influence energy intake, a low-fat label may increase women's fasting hunger perceptions compared with an energy label or no label. PMID:26439975

  1. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are...you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these...

  2. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are...you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these...

  3. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are...you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these...

  4. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are...you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these...

  5. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are...you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these...

  6. 21 CFR 225.80 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...correct labeling is employed for the medicated feed. (2) Labels and labeling, including placards, upon receipt from the printer shall be proofread against the Master Record File to verify their suitability and accuracy. The proofread label shall...

  7. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Alternative Fuels Label Specifications § 309.17 Labels. All labels must meet the following specifications: (a) Layout: (1) Non-liquid alternative vehicle fuel (other than electricity) labels with disclosure of principal component...

  8. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... All labels must meet the following specifications: (a) Layout —(1) For gasoline labels. The label is 3? (7.62...of this rule are prototype labels that demonstrate the proper layout. “Helvetica Black” type is used throughout except...

  9. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

  10. Microfluidic Radiometal Labeling Systems for Biomolecules

    SciTech Connect

    Reichert, D E; Kenis, P J. A.

    2011-12-29

    In a typical labeling procedure with radiometals, such as Cu-64 and Ga-68; a very large (~ 100-fold) excess of the non-radioactive reactant (precursor) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. In order to achieve high specific activities, careful control of reaction conditions and extensive chromatographic purifications are required in order to separate the labeled compounds from the cold precursors. Here we propose a microfluidic approach to overcome these problems, and achieve high specific activities in a more convenient, semi-automated fashion and faster time frame. Microfluidic reactors, consisting of a network of micron-sized channels (typical dimensions in the range 10 â�� 300 �¼m), filters, separation columns, electrodes and reaction loops/chambers etched onto a solid substrate, are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes. The ability to manipulate, process and analyze reagent concentrations and reaction interfaces in both space and time within the channel network of a microreactor provides the fine level of reaction control that is desirable in PET radiochemistry practice. These factors can bring radiometal labeling, specifically the preparation of radio-labeled biomolecules such as antibodies, much closer to their theoretical maximum specific activities.

  11. Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density? †

    PubMed Central

    Buckley, Daniel H.; Huangyutitham, Varisa; Hsu, Shi-Fang; Nelson, Tyrrell A.

    2007-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml?1 for 15N-labeled DNA, relative to 0.036 g ml?1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml?1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications. PMID:17369331

  12. Site-Specific Carbon Isotopes in Organics

    NASA Astrophysics Data System (ADS)

    Piasecki, A.; Eiler, J. M.

    2012-12-01

    Natural organic molecules exhibit a wide range of internal site-specific isotope variation (i.e., molecules with same isotopic substitution type but different site). Such variations are generally unconstrained by bulk isotopic measurements. If known, site-specific variations might constrain temperatures of equilibrium, mechanisms of formation or consumption reactions, and possibly other details. For example, lipids can exhibit carbon isotope differences of up to 30‰ between adjacent carbon sites as a result of fractionations arising during decarboxylation of pyruvate and other steps in lipid biosynthesis(1). We present a method for site-specific carbon isotope analysis of propane, based on high-resolution, multi-collector gas source mass spectrometry, using a novel prototype instrument - the Thermo MAT 253 Ultra. This machine has an inlet system and electron bombardment ion source resembling those in conventional stable isotope gas source mass spectrometers, and the energy filter, magnet, and detector array resembling those in multi-collector ICPMS and TIMS. The detector array has 7 detector positions, 6 of which are movable, and each of which can collect ions with either a faraday cup (read through amplifiers ranging from 107-1012 ohms) or an SEM. High mass resolving power (up to 27,000, MRP = M/dM definition) is achieved through a narrow entrance slit, adjustable from 250 to 5 ?m. Such resolution can cleanly separate isobaric interferences between isotopologues of organic molecules having the same cardinal mass (e.g., 13CH3 and 12CH2D). We use this technology to analyze the isotopologues and fragments of propane, and use such data to solve for the site-specific carbon isotope fractionation. By measuring isotopologues of both the one-carbon (13CH3) and the two-carbon (13C12CH4) fragment ion, we can solve for both bulk ?13C and the difference in ?13C between the terminal and central carbon position. We tested this method by analyzing mixtures between natural propane and labeled propane (13CH3-12CH2-12CH3). Results are consistent with the expected relative fractionations between the two fragments, indicating limited 'scrambling' of carbon positions of less than 2% in the source. The limits of precision of this method are currently ~0.5 ‰, sufficient to resolve known or suspected position-specific isotope effects in propane. We have explored the expected temperature-dependent equilibrium isotopic distributions of propane using density functional theory and quantum mechanical models of vibrational isotope effects. These models predict the homogeneous isotope exchange equilibria among the various isotopologues of propane, which include several of a wide range of effects that should be measurable by our methods. At 300 K we predict that the central carbon site is 15‰ higher in ?13C and 95 ‰ higher in ?D than the terminal carbon site; similarly the molecule containing both a 13C and D in the central site is enriched by ~120 ‰ relative to a random isotopic distribution at 300 K. These predictions present targets for future experimental and empirical studies of the temperature dependence of isotopic ordering in propane. More generally, the methods we are developing for the study of intramolecular isotopic distributions in propane will serve as a model for future study of similar effects in other organic compounds. [1]DeNiro, Epstein (1977) Science Volume 197, 261-263.

  13. Review of nutrition labeling formats.

    PubMed

    Geiger, C J; Wyse, B W; Parent, C R; Hansen, R G

    1991-07-01

    This article examines nutrition labeling history as well as the findings of nine research studies of nutrition labeling formats. Nutrition labeling regulations were announced in 1973 and have been periodically amended since then. In response to requests from consumers and health care professionals for revision of the labeling system, the Food and Drug Administration initiated a three-phase plan for reform of nutrition labeling in 1990. President Bush signed the Nutrition Labeling and Education Act in November 1990. Literature analysis revealed that only nine studies with an experimental design have focused on nutrition labeling since 1971. Four were conducted before 1975, which was the year that nutrition labeling was officially implemented, two were conducted in 1980, and three were conducted after 1986. Only two of the nine studies supported the traditional label format mandated by the Code of Federal Regulations, and one study partially supported it. Four of the nine studies that evaluated graphic presentations of nutrition information found that consumer comprehension of nutrition information was improved with a graphic format for nutrition labeling: three studies supported the use of bar graphs and one study supported the use of a pie chart. Full disclosure (ie, complete nutrient and ingredient labeling) was preferred by consumers in two of the three studies that examined this variable. The third study supported three types of information disclosure dependent upon socioeconomic class. In those studies that tested graphics, a bar graph format was significantly preferred and showed better consumer comprehension than the traditional format. PMID:2071796

  14. Map labeling and its generalizations

    SciTech Connect

    Doddi, S. |; Marathe, M.V.; Mirzaian, A.; Moret, B.M.E.; Zhu, B. |

    1997-01-01

    Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

  15. 16 CFR 305.17 - Television labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... in paragraph (i), the labels must be affixed to the product in the form of either an adhesive label, cling label, or alternative label as follows: (1) Adhesive label. All adhesive labels shall be applied... applied with an adhesive with an adhesion capacity sufficient to prevent their dislodgment during...

  16. In vivo gallbladder absorption: a new dual-isotope technique

    SciTech Connect

    Conter, R.L.; Porter-Fink, V.; Denbesten, L.; Roslyn, J.J.

    1986-10-01

    Available methods for measuring in vivo gallbladder absorption preclude the use of animals in which hepatic bile enters the gallbladder via accessory or aberrant channels. However, accessory bile ducts are present in many of the animal models currently used in gallstone research. The aim of this study, therefore, was to evaluate a new dual-isotope technique that corrects for accessory bile flow and to compare data on electrolyte and water absorption with those derived from the standard, single-isotope technique. Prairie dogs underwent gallbladder exclusion by cystic duct ligation and common bile duct cannulation. Carbon 14-polyethylene glycol-labeled lactated Ringer's solution was instilled into the gallbladder while tritiated cholic acid was administered intravenously to label the bile acid pool. There is no correlation between water or electrolyte absorption and time, nor between water and electrolyte absorption, when these parameters are calculated by the standard, single-isotope technique. In contrast, use of the dual-isotope technique quantifies accessory bile duct flow and yields a linear increase in water and electrolyte absorption, both of which are time dependent. These data suggest that the dual-isotope technique provides a means to accurately measure in vivo gallbladder absorption in animals with or without accessory bile ducts.

  17. Synthesis and applications of RNAs with position-selective labelling and mosaic composition.

    PubMed

    Liu, Yu; Holmstrom, Erik; Zhang, Jinwei; Yu, Ping; Wang, Jinbu; Dyba, Marzena A; Chen, De; Ying, Jinfa; Lockett, Stephen; Nesbitt, David J; Ferré-D'Amaré, Adrian R; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

    2015-06-18

    Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics. PMID:25938715

  18. Hybrid isotope separation scheme

    DOEpatents

    Maya, Jakob (Brookline, MA)

    1991-01-01

    A method of yielding selectively a desired enrichment in a specific isotope including the steps of inputting into a spinning chamber a gas from which a scavenger, radiating the gas with a wave length or frequency characteristic of the absorption of a particular isotope of the atomic or molecular gas, thereby inducing a photochemical reaction between the scavenger, and collecting the specific isotope-containing chemical by using a recombination surface or by a scooping apparatus.

  19. Hybrid isotope separation scheme

    DOEpatents

    Maya, J.

    1991-06-18

    A method is described for yielding selectively a desired enrichment in a specific isotope including the steps of inputting into a spinning chamber a gas from which a scavenger, radiating the gas with a wave length or frequency characteristic of the absorption of a particular isotope of the atomic or molecular gas, thereby inducing a photochemical reaction between the scavenger, and collecting the specific isotope-containing chemical by using a recombination surface or by a scooping apparatus. 2 figures.

  20. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2015-09-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

  1. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies.

    PubMed

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E; Gross, Michael L

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. Graphical Abstract ?. PMID:26384685

  2. Gastric ulcer localization: Potential use of in vivo labeling

    SciTech Connect

    Pera, A.; Rose, H.; Seavers, R.; Bekerman, C.; Pinsky, S.

    1984-01-01

    A previous work suggests that sucralfate labeled by binding to Tc-99m HSA permits the visualization of gastric ulcers. Potential problems with this technique are: 1) decreased binding of sucralfate to ulcer sites due to the labeling method of binding to exogenous protein (HSA); 2) overlying activity that may obscure identification of the ulcer. Because of these problems we have examined the possibility of direct in vivo Tc-99m labeling of sucralfate after it has already bound to the ulcer. In vitro studies were done to determine the binding of Tc-99m pertechnetate to sucralfate in the presence of tin in HCl solution at pHs comparable to those found in the stomach. Rapid and efficient labeling was achieved with 75-95% of the label bound to sucralfate at 30 minutes. In vivo studies were performed in rabbits with aspirin induced ulcers and in ulcer free human volunteers. The animal studies confirm that orally administered Tc-99m pertechnetate will bind to previously ingested sucralfate and that the labeled material will bind to the ulcers. Tc-99m pertechnetate was also shown to bind well to previously ingested sucralfate in humans. The results suggest that it is possible to label sucralfate in vivo. This method would offer the following advantages: 1) a simpler labeling procedure; 2) the potential of increased sensitivity by delaying the labeling until much of the sucralfate not bound to ulcer has passed, and thus decreasing the activity that remains in the stomach; and also by leaving the protein binding sites of the sucralfate free to interact with the ulcer since no exogenous protein is involved in labeling.

  3. Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization.

    PubMed

    Wang, Xiang; Wei, Fang; Xu, Ji-Qu; Lv, Xin; Dong, Xu-Yan; Han, Xianlin; Quek, Siew-Young; Huang, Feng-Hong; Chen, Hong

    2016-01-01

    Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d0-acetone) and deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased. PMID:26703264

  4. Discovery of the Calcium Isotopes

    E-print Network

    J. L. Gross; M. Thoennessen

    2010-09-08

    Twenty four calcium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  5. Discovery of the Vanadium Isotopes

    E-print Network

    Shore, A; Heim, M; Schuh, A; Thoennessen, M

    2009-01-01

    Twenty-four vanadium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  6. Discovery of the Vanadium Isotopes

    E-print Network

    A. Shore; A. Fritsch; M. Heim; A. Schuh; M. Thoennessen

    2009-07-11

    Twenty-four vanadium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  7. Discovery of the Einsteinium Isotopes

    E-print Network

    Bury, A; Ginepro, J Q; Heim, M; Schuh, A; Shore, A; Thoennessen, M

    2009-01-01

    Seventeen einsteinium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  8. Discovery of the Scandium Isotopes

    E-print Network

    Meierfrankenfeld, D

    2010-01-01

    Twenty-three scandium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  9. Discovery of the Mercury Isotopes

    E-print Network

    D. Meierfrankenfeld; M. Thoennessen

    2010-09-08

    Forty mercury isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  10. Discovery of the Einsteinium Isotopes

    E-print Network

    A. Bury; A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; A. Shore; M. Thoennessen

    2010-09-08

    Seventeen einsteinium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  11. Discovery of the Titanium Isotopes

    E-print Network

    D. Meierfrankenfeld; M. Thoennessen

    2010-09-08

    Twentyfive titanium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  12. Discovery of the Arsenic Isotopes

    E-print Network

    Shore, A; Heim, M; Schuh, A; Thoennessen, M

    2009-01-01

    Twenty-nine arsenic isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  13. Discovery of the Arsenic Isotopes

    E-print Network

    A. Shore; A. Fritsch; M. Heim; A. Schuh; M. Thoennessen

    2009-02-25

    Twenty-nine arsenic isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  14. Discovery of the Cobalt Isotopes

    E-print Network

    T. Szymanski; M. Thoennessen

    2009-09-04

    Twenty-six cobalt isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  15. Discovery of the Barium Isotopes

    E-print Network

    A. Shore; A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; M. Thoennessen

    2009-08-13

    Thirty-eight barium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  16. Discovery of the barium isotopes

    SciTech Connect

    Shore, A.; Fritsch, A.; Ginepro, J.Q.; Heim, M.; Schuh, A.; Thoennessen, M.

    2010-11-15

    Thirty-eight barium isotopes have been observed so far and the discovery of these isotopes is discussed here. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  17. Discovery of the Platinum Isotopes

    E-print Network

    Gross, J L

    2010-01-01

    Thirty-nine platinum isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  18. Discovery of the Krypton Isotopes

    E-print Network

    M. Heim; A. Fritsch; A. Schuh; A. Shore; M. Thoennessen

    2009-04-15

    Thirty-two krypton isotopes have been observed so far; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  19. Discovery of the krypton isotopes

    SciTech Connect

    Heim, M.; Fritsch, A.; Schuh, A.; Shore, A.; Thoennessen, M.

    2010-07-15

    Thirty-two krypton isotopes have been observed so far and the discovery of these isotopes is discussed here. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  20. Discovery of the Gold Isotopes

    E-print Network

    A. Schuh; A. Fritsch; J. Q. Ginepro; M. Heim; A. Shore; M. Thoennessen

    2009-03-10

    Thirty-six gold isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  1. Discovery of the Indium Isotopes

    E-print Network

    S. Amos; M. Thoennessen

    2010-09-08

    Thirty-eight indium isotopes (A = 98-135) have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  2. Discovery of the Silver Isotopes

    E-print Network

    A. Schuh; A. Fritsch; J. Q. Ginepro; M. Heim; A. Shore; M. Thoennessen

    2009-07-09

    Thirty-eight silver isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  3. Discovery of the Tin Isotopes

    E-print Network

    S. Amos; M. Thoennessen

    2010-09-08

    Thirty-eight tin isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  4. Discovery of the Cadmium Isotopes

    E-print Network

    S. Amos; M. Thoennessen

    2009-10-22

    Thirty-seven cadmium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  5. Discovery of the Iron Isotopes

    E-print Network

    A. Schuh; A. Fritsch; M. Heim; A. Shore; M. Thoennessen

    2009-09-01

    Twenty-eight iron isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  6. Discovery of the Tungsten Isotopes

    E-print Network

    A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; A. Shore; M. Thoennessen

    2009-03-25

    Thirty-five tungsten isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

  7. Leukocyte labeling with technetium-99m tin colloids

    SciTech Connect

    Mock, B.H.; English, D.

    1987-09-01

    Triple density gradients of metrizamide in plasma (MP) were used to characterize label distribution in human leukocyte preparations incubated with /sup 99m/Tc tin colloids. Less than 50% of the cell-associated radioactivity was specifically bound to leukocytes when heparinized blood was rotated with stannous fluoride colloid ((Tc)SFC). Labeling efficiency in leukocyte rich plasma (LRP) averaged 44%, of which greater than 90% was specifically bound to leukocytes. MP-gradient analysis also revealed that leukocyte labeling did not occur with stannous chloride colloid, nor when citrate was present during rotation with (Tc)SFC. When citrate was added after labeling to solubilize unbound (Tc)SFC, radiocolloid was removed from the leukocytes, indicating that the mechanism of (Tc)SFC labeling is adherence rather than phagocytosis. Technetium-labeled neutrophils exhibited normal in vitro chemotaxis and no lung uptake in vivo. Technetium-labeled mononuclear leukocytes, on the other hand, exhibited prolonged lung transit in vivo. Neither (Tc)SFC cell preparation showed signs of in vivo reoxidation to pertechnetate.

  8. Label Scrambling During CID of Covalently Labeled Peptide Ions

    NASA Astrophysics Data System (ADS)

    Borotto, Nicholas B.; Degraan-Weber, Nicholas; Zhou, Yuping; Vachet, Richard W.

    2014-10-01

    Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification.

  9. Intracellular Cadmium Isotope Fractionation

    NASA Astrophysics Data System (ADS)

    Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.

    2011-12-01

    Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.

  10. Label propagation with ?-degree neighborhood impact for network community detection.

    PubMed

    Sun, Heli; Huang, Jianbin; Zhong, Xiang; Liu, Ke; Zou, Jianhua; Song, Qinbao

    2014-01-01

    Community detection is an important task for mining the structure and function of complex networks. In this paper, a novel label propagation approach with ?-degree neighborhood impact is proposed for efficiently and effectively detecting communities in networks. Firstly, we calculate the neighborhood impact of each node in a network within the scope of its ?-degree neighborhood network by using an iterative approach. To mitigate the problems of visiting order correlation and convergence difficulty when updating the node labels asynchronously, our method updates the labels in an ascending order on the ?-degree neighborhood impact of all the nodes. The ?-degree neighborhood impact is also taken as the updating weight value, where the parameter impact scope ? can be set to a positive integer. Experimental results from several real-world and synthetic networks show that our method can reveal the community structure in networks rapidly and accurately. The performance of our method is better than other label propagation based methods. PMID:25525425

  11. Holographic labeling for automated identification

    NASA Astrophysics Data System (ADS)

    McOwan, Peter W.; Powell, Andrew K.; Burge, Ronald E.

    1991-02-01

    The optical reading of labels encoding data in the form of a one dimensional barcode is a well developed technology. This paper presents a novel approach to the optical retrieval of a high volume of data contained in a new type of label. The labelling method uses the techniques of computer generated holography to encode the required two dimensional label data in the form of a digitally synthesised wavefront. This wavefront is optimally encoded using models based on optical holography and the calculated structure mechanically plotted into a new polymer based reflective substrate to form the label. The label is laser illuminated and the reflected wavefront optically reconstructed and decoded to remove the desired information.

  12. Optimizing connected component labeling algorithms

    SciTech Connect

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-01-16

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 {approx} 100 in our tests on random binary images.

  13. Rare isotope studies involving catalytic oxidation of CO over platinum-tin oxide

    NASA Technical Reports Server (NTRS)

    Upchurch, Billy T.; Wood, George M., Jr.; Hess, Robert V.; Hoyt, Ronald F.

    1987-01-01

    Results of studies utilizing normal and rare oxygen isotopes in the catalytic oxidation of carbon monoxide over a platinum-tin oxide catalyst substrate are presented. Chemisorption of labeled carbon monoxide on the catalyst followed by thermal desorption yielded a carbon dioxide product with an oxygen-18 composition consistent with the formation of a carbonate-like intermediate in the chemisorption process. The efficacy of a method developed for the oxygen-18 labeling of the platinum-tin oxide catalyst surface for use in closed cycle pulsed care isotope carbon dioxide lasers is demonstrated for the equivalent of 10 to the 6th power pulses at 10 pulses per second.

  14. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism.

    PubMed

    Steinhauser, Matthew L; Bailey, Andrew P; Senyo, Samuel E; Guillermier, Christelle; Perlstein, Todd S; Gould, Alex P; Lee, Richard T; Lechene, Claude P

    2012-01-26

    Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research. PMID:22246326

  15. (Carbon isotope fractionation inplants)

    SciTech Connect

    O'Leary, M.H.

    1990-01-01

    The objectives of this research are: To develop a theoretical and experimental framework for understanding isotope fractionations in plants; and to develop methods for using this isotope fractionation for understanding the dynamics of CO{sub 2} fixation in plants. Progress is described.

  16. Detecting isotopic ratio outliers

    SciTech Connect

    Bayne, C.K.; Smith, D.H.

    1985-01-01

    An alternative method is proposed for improving isotopic ratio estimates. This method mathematically models pulse-count data and uses iterative reweighted Poisson regression to estimate model parameters to calculate the isotopic ratios. This computer-oriented approach provides theoretically better methods than conventional techniques to establish error limits and to identify outliers. 6 refs., 3 figs., 3 tabs.

  17. Isotope - based Quantum Information

    E-print Network

    Vladimir G. Plekhanov

    2010-07-22

    This paper is brief review of three aspects of the isotope - based quantum information: computation, teleportation and cryptography. Our results demonstrate not only that entanglement exists in elementary excitation of isotope - mixed solids but also it can be used for quantum information processing.

  18. Instant magnetic labeling of tumor cells by ultrasound in vitro

    NASA Astrophysics Data System (ADS)

    Mo, Runyang; Yang, Jian; Wu, Ed X.; Lin, Shuyu

    2011-09-01

    Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 ?g/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.

  19. Photochemical isotope separation

    DOEpatents

    Robinson, C.P.; Jensen, R.J.; Cotter, T.P.; Greiner, N.R.; Boyer, K.

    1987-04-28

    A process is described for separating isotopes by selective excitation of isotopic species of a volatile compound by tuned laser light. A highly cooled gas of the volatile compound is produced in which the isotopic shift is sharpened and defined. Before substantial condensation occurs, the cooled gas is irradiated with laser light precisely tuned to a desired wavelength to selectively excite a particular isotopic species in the cooled gas. The laser light may impart sufficient energy to the excited species to cause it to undergo photochemical reaction or even to photoionize. Alternatively, a two-photon irradiation may be applied to the cooled gas to induce photochemical reaction or photoionization. The process is particularly applicable to the separation of isotopes of uranium and plutonium. 8 figs.

  20. Energy expenditure by doubly labeled water: validation in humans and proposed calculation

    SciTech Connect

    Schoeller, D.A.; Ravussin, E.; Schutz, Y.; Acheson, K.J.; Baertschi, P.; Jequier, E.

    1986-05-01

    To further validate the doubly labeled water method for measurement of CO/sub 2/ production and energy expenditure in humans, we compared it with near-continuous respiratory gas exchange in nine healthy young adult males. Subjects were housed in a respiratory chamber for 4 days. Each received /sup 2/H/sub 2/(18)O at either a low (n = 6) or a moderate (n = 3) isotope dose. Low and moderate doses produced initial /sup 2/H enrichments of 5 and 10 X 10(-3) atom percent excess, respectively, and initial 18O enrichments of 2 and 2.5 X 10(-2) atom percent excess, respectively. Total body water was calculated from isotope dilution in saliva collected at 4 and 5 h after the dose. CO/sub 2/ production was calculated by the two-point method using the isotopic enrichments of urines collected just before each subject entered and left the chamber. Isotope enrichments relative to predose samples were measured by isotope ratio mass spectrometry. At low isotope dose, doubly labeled water overestimated average daily energy expenditure by 8 +/- 9% (SD) (range -7 to 22%). At moderate dose the difference was reduced to +4 +/- 5% (range 0-9%). The isotope elimination curves for /sup 2/H and 18O from serial urines collected from one of the subjects showed expected diurnal variations but were otherwise quite smooth. The overestimate may be due to approximations in the corrections for isotope fractionation and isotope dilution. An alternative approach to the corrections is presented that reduces the overestimate to 1%.

  1. Simulating electron spin resonance spectra of macromolecules labeled with two dipolar-coupled nitroxide spin labels from trajectories.

    PubMed

    Sezer, Deniz; Sigurdsson, Snorri Th

    2011-07-28

    An efficient method for simulating continuous-wave electron spin resonance spectra (ESR) of molecules labeled with two dipolar-coupled nitroxides from trajectories of the molecular motion is presented. Two approximate treatments of the dipolar spin evolution, resulting in significantly shorter simulation times, are examined in order to determine their range of applicability. The approach is illustrated in the context of a double-helical B-DNA. ESR spectra for DNA undergoing anisotropic global diffusion and internal stretching dynamics are calculated for three different labeling geometries with the spin labels bracketing, respectively, three, two and one base pairs. While multifrequency spectra of all three labeling schemes are very sensitive to DNA tumbling, the last one is found to be most informative about the local DNA dynamics. PMID:21691643

  2. Exploring the isotopes

    SciTech Connect

    Firestone, R.B.; Chu, S.Y.F.; Nordberg, H.

    1997-12-31

    The explosion in computer technology has led to a revolution in the way nuclear structure and decay data is being disseminated. For nearly sixty years the Table of Isotopes and later the Nuclear Data Sheets were primary sources of information for nuclear researchers. In 1996, the 8th edition of the Table of Isotopes (John Wiley and Sons, Inc.) was published. This two-volume, 3100 page book signaled the end of the era of publishing nuclear data comprehensively on paper. The CD-ROM version that comes with the 8th edition provides the same information spread over 14,000 {open_quotes}pages{close_quotes} for better readability on the computer screen. With Adobe Acrobat technology to view the data, over 100,000 hypertext links to navigate through it, and nearly unlimited expansion possibilities, the CD-ROM becomes the format of choice for {open_quotes}hard copy{close_quotes} publishing of nuclear data. Even a CD-ROM cannot be updated continuously to provide the most current data. We are therefore developing an Internet version of the Table of Isotopes (http://isotopes lbl.gov/isotopes/toi.html) to supplement the CD-ROM. The website provides home pages focusing on nuclear structure data, radioactive decay data, nuclear astrophysics data, capture gamma data, and atomic masses. The nuclear structure (ENSDF) and reference (NSR) databases are available on both the CD-ROM and Internet. We have developed the Isotopes Explorer application (previously known as VuENSDF) to view these databases and display level schemes, tables, plots, references, and nuclear charts. The databases can be searched and derived quantities can be calculated. The Isotopes Explorer is a 32-bit Windows application that can download information directly from the CD-ROM, Internet, or user generated files. Information about the Isotopes Explorer can be found at http://isotopes.lbl.gov/isotopes/vuensdf.html.

  3. Multilabel image classification via high-order label correlation driven active learning.

    PubMed

    Zhang, Bang; Wang, Yang; Chen, Fang

    2014-03-01

    Supervised machine learning techniques have been applied to multilabel image classification problems with tremendous success. Despite disparate learning mechanisms, their performances heavily rely on the quality of training images. However, the acquisition of training images requires significant efforts from human annotators. This hinders the applications of supervised learning techniques to large scale problems. In this paper, we propose a high-order label correlation driven active learning (HoAL) approach that allows the iterative learning algorithm itself to select the informative example-label pairs from which it learns so as to learn an accurate classifier with less annotation efforts. Four crucial issues are considered by the proposed HoAL: 1) unlike binary cases, the selection granularity for multilabel active learning need to be fined from example to example-label pair; 2) different labels are seldom independent, and label correlations provide critical information for efficient learning; 3) in addition to pair-wise label correlations, high-order label correlations are also informative for multilabel active learning; and 4) since the number of label combinations increases exponentially with respect to the number of labels, an efficient mining method is required to discover informative label correlations. The proposed approach is tested on public data sets, and the empirical results demonstrate its effectiveness. PMID:24723538

  4. Isotope production target design for LANSCE

    SciTech Connect

    Cappiello, M.; Poston, D.; Pitcher, E.; Barber, R.

    2000-07-01

    A conceptual design has been completed for an isotope production facility at the high energy end of the Los Alamos Neutron Science Center (LANSCE) accelerator. The authors propose a system that provides a unique irradiation environment that makes use of the 1-mA, 800-MeV proton beam. By placing the isotope production targets within a tungsten neutron source, they obtain a neutron flux of similar magnitude to the proton flux. In many cases, this mixed environment significantly increases the isotope production rate above that of using protons alone. A location for the facility is proposed near the existing hot cell that can be efficiently used for loading and unloading targets. A rabbit system provides easy access without replacing the tungsten neutron source. The design consists of a vacuum vessel, a spallation target, rabbits, reflector, shielding, heat removal systems, coolant purification systems, and beam transport and control systems.

  5. Stable isotope methodology in the pharmacokinetic studies of androgenic steroids in humans

    SciTech Connect

    Shinohara, Y.; Baba, S. )

    1990-04-01

    The use of stable isotopically labeled steroids combined with gas chromatography/mass spectrometry (GC/MS) has found a broad application in pharmacologic studies. Initially, stable isotopically labeled steroids served as the ideal analytic internal standard for GC/MS analysis; however, their in vivo use has expanded and has proven to be a powerful pharmacokinetic tool. We have successfully used stable isotope methodology to study the pharmacokinetic/bioavailability of androgens. The primary advantage of the technique is that endogenous and exogenous steroids with the same basic structure can be differentiated by using stable isotopically labeled analogs. The method was used to examine the pharmacokinetics of testosterone and testosterone propionate, and to clarify the influence of endogenous testosterone. Another advantage of the isotope methods is that steroidal drugs can be administered concomitantly in two formulations (e.g., solution and solid dosage). A single set of blood samples serves to describe the time course of the formulations being compared. This stable isotope coadministration technique was used to estimate the relative bioavailability of 17 alpha-methyltestosterone. 35 references.

  6. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  7. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ...The Food Safety and Inspection Service (FSIS) is proposing to amend the meat and poultry products inspection regulations to expand the circumstances in which FSIS will generically approve the labels of meat and poultry products. The Agency also is proposing to combine the regulations that provide for the approval of labels for meat products and poultry products into a new CFR...

  8. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ...The Food Safety and Inspection Service (FSIS) is amending the meat and poultry products inspection regulations to expand the circumstances in which FSIS will generically approve the labels of meat and poultry products. The Agency also is consolidating the regulations that provide for the approval of labels for meat products and poultry products into a new Code of Federal Regulations (CFR)...

  9. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... limited types of labels (e.g., labels for raw, single ingredient meat and poultry products) (48 FR 11410... products will take effect January 1, 2012 (75 FR 82148, Dec. 29, 2010). These mandatory features must be... Agency. On March 25, 1992, FSIS published an Advance Notice of Proposed Rulemaking (ANPRM) (57 FR...

  10. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  11. Pesticide Labeling: Unique Product Labeling1 Frederick M. Fishel2

    E-print Network

    Hill, Jeffrey E.

    PI-110 Pesticide Labeling: Unique Product Labeling1 Frederick M. Fishel2 1. This document is PI-110, professor, Agronomy Department, and Director, Pesticide Information Office; Florida Cooperative Extension not signify our approval to the exclusion of other products of suitable composition. Use pesticides safely

  12. Theoretical Basis for Dynamic Label Propagation in Stationary Metabolic Networks under Step and Periodic Inputs

    PubMed Central

    Sokol, Serguei; Portais, Jean-Charles

    2015-01-01

    The dynamics of label propagation in a stationary metabolic network during an isotope labeling experiment can provide highly valuable information on the network topology, metabolic fluxes, and on the size of metabolite pools. However, major issues, both in the experimental set-up and in the accompanying numerical methods currently limit the application of this approach. Here, we propose a method to apply novel types of label inputs, sinusoidal or more generally periodic label inputs, to address both the practical and numerical challenges of dynamic labeling experiments. By considering a simple metabolic system, i.e. a linear, non-reversible pathway of arbitrary length, we develop mathematical descriptions of label propagation for both classical and novel label inputs. Theoretical developments and computer simulations show that the application of rectangular periodic pulses has both numerical and practical advantages over other approaches. We applied the strategy to estimate fluxes in a simulated experiment performed on a complex metabolic network (the central carbon metabolism of Escherichia coli), to further demonstrate its value in conditions which are close to those in real experiments. This study provides a theoretical basis for the rational interpretation of label propagation curves in real experiments, and will help identify the strengths, pitfalls and limitations of such experiments. The cases described here can also be used as test cases for more general numerical methods aimed at identifying network topology, analyzing metabolic fluxes or measuring concentrations of metabolites. PMID:26641860

  13. Photosynthetic carbon metabolism in seagrasses C-labeling evidence for the c(3) pathway.

    PubMed

    Andrews, T J; Abel, K M

    1979-04-01

    The delta(13)C values of several seagrasses were considerably less negative than those of terrestrial C(3) plants and tended toward those of terrestrial C(4) plants. However, for Thalassia hemprichii (Ehrenb.) Aschers and Halophila spinulosa (R. Br.) Aschers, phosphoglycerate and other C(3) cycle intermediates predominated among the early labeled products of photosynthesis in (14)C-labeled seawater (more than 90% at the earliest times) and the labeling pattern at longer times was brought about by the operation of the C(3) pathway. Malate and aspartate together accounted for only a minor fraction of the total fixed label at all times and the kinetic data of this labeling were not at all consistent with these compounds being early intermediates in seagrass photosynthesis. Pulse-chase (14)C-labeling studies further substantiated these conclusions. Significant labeling of photorespiratory intermediates was observed in all experiments. The kinetics of total fixation of label during some steady-state and pulse-chase experiments suggested that there may be an intermediate pool of inorganic carbon of variable size closely associated with the leaves, either externally or internally. Such a pool may be one cause for the C(4)-like carbon isotope ratios of seagrasses. PMID:16660784

  14. Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans

    PubMed Central

    Szewc, Mark A.; Garrett, Timothy; Menger, Robert F.; Yost, Richard A.; Beecher, Chris; Edison, Arthur S.

    2014-01-01

    We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass spectrometry-based technique called Isotopic Ratio Outlier Analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95% and 5% 13C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: 1) compounds arising from biosynthesis are easily distinguished from artifacts, 2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, 3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulae, and 4) relative concentrations of all metabolites are easily determined. A heat shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway, which we use to demonstrate the approach. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline. PMID:24274725

  15. Stable isotope method to measure drug release from nanomedicines.

    PubMed

    Skoczen, Sarah; McNeil, Scott E; Stern, Stephan T

    2015-12-28

    Existing methods to measure nanomedicine drug release in biological matrices are inadequate. A novel drug release method utilizing a stable isotope tracer has been developed. Stable isotope-labeled drug is spiked into plasma containing nanomedicine. The labeled drug equilibrates with plasma components identical to the normoisotopic drug released from the nanomedicine formulation. Therefore, the ultrafilterable fraction of the isotope-labeled drug represents a reliable measure of free normoisotopic drug fraction in plasma, and can be used to calculate nanomedicine encapsulated and unencapsulated drug fractions. To demonstrate the utility of this method, we performed a plasma drug release study with both a fast releasing commercial docetaxel formulation, Taxotere®, and a delayed releasing nanomicellar formulation of a docetaxel prodrug, Procet 8. The instability of the unencapsulated prodrug in plasma allowed us to compare our calculated prodrug release and docetaxel conversion with the actual docetaxel concentration measured directly without fractionation. Drug release estimates for the fast releasing Taxotere formulation demonstrated accuracy deviation and precision (%CV) of <15%. For the controlled release Procet 8 formulation, we calculated a slow release and conversion of the prodrug in rat plasma that was highly correlated with the direct docetaxel measurement (R(2)=0.98). We believe that this method will have tremendous utility in the development and regulatory evaluation of nanomedicines, and aid in determination of generic bioequivalence. PMID:26596375

  16. Electrochemical Isotope Effect and Lithium Isotope Separation Jay R. Black,

    E-print Network

    Mcdonough, William F.

    Electrochemical Isotope Effect and Lithium Isotope Separation Jay R. Black, Grant Umeda, Bruce Dunn May 19, 2009; E-mail: akavner@ucla.edu The electrochemical separation of lithium isotopes is of growing interest due to the need for pure 6 Li and 7 Li isotopes in the nuclear industry.1 Here we present

  17. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  18. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  19. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  20. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  1. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  2. 16 CFR 305.17 - Television labeling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... to the product in the form of either an adhesive label, cling label, or alternative label as follows: (1) Adhesive label. All adhesive labels shall be applied so they can be easily removed without the use of tools or liquids, other than water, but shall be applied with an adhesive with an...

  3. 16 CFR 305.17 - Television labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... to the product in the form of either an adhesive label, cling label, or alternative label as follows: (1) Adhesive label. All adhesive labels shall be applied so they can be easily removed without the use of tools or liquids, other than water, but shall be applied with an adhesive with an...

  4. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  5. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  6. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  7. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  8. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  9. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  10. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  11. Phage Selection Assisted by Sfp Phosphopantetheinyl Transferase Catalyzed Site-Specific Protein Labeling

    PubMed Central

    Zhao, Bo; Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Zhou, Han; Zhang, Mengnan; Yin, Jun

    2015-01-01

    Summary Phosphopantetheinyl transferases (PPTase) Sfp and AcpS catalyze a highly efficient reaction that conjugates chemical probes of diverse structures to proteins. PPTases have been widely used for site-specific protein labeling and live cell imaging of the target proteins. Here we describe the use of PPTase catalyzed protein labeling in protein engineering by facilitating high throughput phage selection. PMID:25560074

  12. Phage selection assisted by Sfp phosphopantetheinyl transferase-catalyzed site-specific protein labeling.

    PubMed

    Zhao, Bo; Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Zhou, Han; Zhang, Mengnan; Yin, Jun

    2015-01-01

    Phosphopantetheinyl transferases (PPTase) Sfp and AcpS catalyze a highly efficient reaction that conjugates chemical probes of diverse structures to proteins. PPTases have been widely used for site-specific protein labeling and live cell imaging of the target proteins. Here we describe the use of PPTase-catalyzed protein labeling in protein engineering by facilitating high-throughput phage selection. PMID:25560074

  13. Looks Aren't Everything: 24-Month-Olds' Willingness to Accept Unexpected Labels

    ERIC Educational Resources Information Center

    Jaswal, Vikram K.; Markman, Ellen M.

    2007-01-01

    A label can efficiently convey nonobvious information about category membership, but this information can sometimes conflict with one's own expectations. Two studies explored whether 24-month-olds (N = 56) would be willing to accept a category label indicating that an animal (Study 1) or artifact (Study 2) that looked like a member of one familiar…

  14. Stable isotopes in plant physiology: using water isotopes to study water fluxes in a temperate forest

    NASA Astrophysics Data System (ADS)

    Gerlein, C.; Wolf, A.; Caylor, K. K.

    2013-12-01

    Drought has profound consequences on vegetation, including decreases in instantaneous carbon uptake; damage that limits future uptake for the life of the plant; mortality that can lead to large sources of carbon to the atmosphere; and shifts in biogeography that alter future potential for carbon uptake and capacitance. These processes are largely absent from global models, for lack of understanding in how co-occurring plants compete for water, weak understanding of how plant hydraulics is coordinated to minimize risk of drought, and few empirical data to constrain superior models of these processes. Here we present the results of a large-scale field experiment at Silas Little Experimental Forest (NJ), where rainwater was diverted from a 10m^2 area around selected trees from two different species (either oak or pine trees) and either re-injected (control plots), discarded (drought plots) or replaced by isotopically labeled water (isotope plots). We sampled heavily the drought plots and collected valuable information on tree hydraulics under drought conditions, such as water potentials of soil, leaf and stem, photosynthetic rate or sap flow. At the isotope plots, we followed the injected water within the injection trees and the surrounding ones. In particular, using an innovative setup for in-situ measurement paired with a laser spectrometer, we studied the isotopes effects within the tree xylem, which gave us a better understanding of water uptake by the roots and its transport to the leaves. By tracking the labeled water in the surrounding trees, we were also able to quantify the importance of plant competition for water availability below ground. We show here the importance of understanding all the phases of the water transport in the biosphere to help constraining climate models.

  15. Sparse labeling of proteins: Structural characterization from long range constraints

    NASA Astrophysics Data System (ADS)

    Prestegard, James H.; Agard, David A.; Moremen, Kelley W.; Lavery, Laura A.; Morris, Laura C.; Pederson, Kari

    2014-04-01

    Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1). This is not intended as a comprehensive review, but as a discussion of future prospects that promise impact on important questions in the structural biology area.

  16. Stable-label intravenous glucose tolerance test minimal model

    SciTech Connect

    Avogaro, A.; Bristow, J.D.; Bier, D.M.; Cobelli, C.; Toffolo, G. )

    1989-08-01

    The minimal model approach to estimating insulin sensitivity (Sl) and glucose effectiveness in promoting its own disposition at basal insulin (SG) is a powerful tool that has been underutilized given its potential applications. In part, this has been due to its inability to separate insulin and glucose effects on peripheral uptake from their effects on hepatic glucose inflow. Prior enhancements, with radiotracer labeling of the dosage, permit this separation but are unsuitable for use in pregnancy and childhood. In this study, we labeled the intravenous glucose tolerance test (IVGTT) dosage with (6,6-{sup 2}H{sub 2})glucose, (2-{sup 2}H)glucose, or both stable isotopically labeled glucose tracers and modeled glucose kinetics in six postabsorptive, nonobese adults. As previously found with the radiotracer model, the tracer-estimated S*l derived from the stable-label IVGTT was greater than Sl in each case except one, and the tracer-estimated SG* was less than SG in each instance. More importantly, however, the stable-label IVGTT estimated each parameter with an average precision of +/- 5% (range 3-9%) compared to average precisions of +/- 74% (range 7-309%) for SG and +/- 22% (range 3-72%) for Sl. In addition, because of the different metabolic fates of the two deuterated tracers, there were minor differences in basal insulin-derived measures of glucose effectiveness, but these differences were negligible for parameters describing insulin-stimulated processes. In conclusion, the stable-label IVGTT is a simple, highly precise means of assessing insulin sensitivity and glucose effectiveness at basal insulin that can be used to measure these parameters in individuals of all ages, including children and pregnant women.

  17. Tin isotope fractionation in terrestrial cassiterites

    SciTech Connect

    McNaughton, N.J. ); Rosman, K.J.R. )

    1991-02-01

    The isotopic composition of tin has been measured in a range of cassiterites and pure reagents to assess the extent to which this element is isotopically fractionated in natural processes. Only two samples showed evidence of isotopic fractionation, and it is concluded that natural Sn isotope fractionation is small and uncommon. This feature reflects the world dominance of Sn-oxide ores Sn-sulfide ores, and the highly efficient processes of Sn dissolution and precipitation which negate equilibrium and kinetic fractionation of Sn isotopes, respectively. The two samples which show slight fractionation are a highly purified and cassiterite from the Archaean Greenbushes pegmatite, Western Australia. The latter Sn is 0.15{per thousand} per mass unit heavier than the authors laboratory standard, whereas the former is 0.12{per thousand} per mass unit lighter. Although the cassiterite fractionation is considered to result from natural geological processes, the fractionation of purified Sn may be either natural or relate to the purification process, the fractionation of this magnitude has a negligible effect on the current best estimate of the atomic weight of Sn, but it does place a lower limit on its associated accuracy.

  18. Development of radioactively labelled cancer seeking biomolecules for targeted therapy

    E-print Network

    Pillai, M R A; Bapat, K; Chakraborty, S; Das, T; Dash, A K; Korde, A; Kothari, K; Mukherjee, A; Pandey, U; Samuel, G; Sharma, H D; Unni, P R; Venkatesh, M

    1998-01-01

    Several radioisotopes such as sup 9 sup 0 Y, sup 1 sup 8 sup 6 Re, sup 1 sup 6 sup 6 Ho, sup 1 sup 7 sup 7 Lu, were produced and processed in our laboratory for evaluation as potential isotopes for therapy. sup 9 sup 0 Y was sourced from sup 9 sup 0 Sr in the form of a generator based on a supported liquid membrane separating technique as well as by solvent extraction /adsorption procedure. The other isotopes were obtained by irradiation of the natural/enriched targets. Among the various isotopes studies for possible therapeutic use, sup 1 sup 7 sup 7 Lu was identified as an ideal candidate for labelling of peptides owing to its high specific activity. Bifunctional chelating agents for complexing with these isotopes were synthesized and characterized. Radiolabelling procedures were standardized to obtain maximum complexation. Standard quality control techniques such as PC, TLC, HPLC have been utilized to characterize the radiolabelled species. Lanreotide, a somatostatin analogue was coupled to DOTA in a three...

  19. Lead in petrol. The isotopic lead experiment

    SciTech Connect

    Facchetti, S. )

    1989-10-01

    Many studies were dedicated to the evaluation of the impact of automotive lead on the environment and to the assessment of its absorption in the human population. They can be subdivided into two groups, those based on changes of air and blood lead concentrations and those based on changes of air and blood lead isotopic compositions. According to various authors, 50-66% of the lead added to petrol is mobilized in the atmosphere, while most of the remainder adheres to the walls of the exhaust system from which it is expelled by mechanical and thermal shocks in the forms of easily sedimented particles. The fraction directly emitted by engine exhaust fumes is found in the form of fine particles, which can be transferred a long way from the emitting sources. However important the contribution of petrol lead to the total airborne lead may be, our knowledge does not permit a straightforward calculation of the percentage of petrol lead in total blood lead, which of course can also originate from other sources (e.g., industrial, natural). To evaluate this percentage in 1973, the idea of the Isotopic Lead Experiment (ILE project) was conceived to label, on a regional scale, petrol with a nonradioactive lead of an isotopic composition sufficiently different from that of background lead and sufficiently stable in time. This Account summarizes the main results obtained by the ILE project.

  20. Stable isotope tracing: a powerful tool for selenium speciation and metabolic studies in non-hyperaccumulator plants (ryegrass Lolium perenne L.).

    PubMed

    Di Tullo, Pamela; Versini, Antoine; Bueno, Maïté; Le Hécho, Isabelle; Thiry, Yves; Biron, Philippe; Castrec-Rouelle, Maryse; Pannier, Florence

    2015-12-01

    Selenium is both essential and toxic for mammals; the range between the two roles is narrow and not only dose-dependent but also related to the chemical species present in foodstuff. Unraveling the metabolism of Se in plants as a function of Se source may thus lead to ways to increase efficiency of fertilization procedures in selenium deficient regions. In this study, stable-isotope tracing was applied for the first time in plants to simultaneously monitor the bio-incorporation of two inorganic Se species commonly used as foodstuff enrichment sources. Occurrence and speciation of Se coming from different Se sources were investigated in root and leaf extracts of ryegrass (Lolium perenne L.), which had been co-exposed to two labeled Se species ((77)SeIV and (82)SeVI). Although the plant absorbed similar amounts of Se when supplied in the form of selenite or selenate, the results evidenced marked differences in speciation and tissues allocation. Selenite was converted into organic forms incorporated mostly into high molecular weight compounds with limited translocation to leaves, whereas selenate was highly mobile being little assimilated into organic forms. Double-spike isotopic tracer methodology makes it possible to compare the metabolism of two species-specific Se sources simultaneously in a single experiment and to analyze Se behavior in not-hyperaccumulator plants, the ICP-MS sensitivity being improved by the use of enriched isotopes. PMID:26427506