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Sample records for 3hthymidine incorporation assay

  1. Rapid aquatic toxicity assay utilizing labeled thymidine incorporation in sea urchin embryos

    SciTech Connect

    Jackim, E.; Nacci, D.

    1984-01-01

    Aquatic toxicity was evaluated in the sea urchin embryo (Arbacea punctulata) by the inhibition of tritiated thymidine incorporation after a brief exposure to toxic chemicals. Arbacia is a useful surrogate species for assay: well-studied, easily cultured and fertile virtually year round. The simplicity and speed of this test system lends itself to screening large numbers of compounds, mixtures or water samples.

  2. Clinical correlations with chemosensitivities measured in a rapid thymidine incorporation assay

    SciTech Connect

    Sondak, V.K.; Bertelsen, C.A.; Tanigawa, N.; Hildebrand-Zanki, S.U.; Morton, D.L.; Korn, E.L.; Kern, D.H.

    1984-04-01

    A rapid assay for in vitro chemosensitivity testing measuring (3H)thymidine incorporation has been developed. Results of this assay correlate highly with chemosensitivities determined by the soft-agar clonogenic assay. A correlative study was carried out on 219 solid tumor specimens to assess the ability of the rapid assay to predict clinical response to antineoplastic therapy. One hundred forty-two of 219 tumors (65%) yielded chemosensitivity data. Of these, 33 were evaluable for in vitro-in vivo correlations. In vitro sensitivity (greater than or equal to 80% inhibition of thymidine uptake) was associated with clinical response in 6 of 13 patients. In vitro resistance was associated with progressive disease in 20 of 20 patients. The rapid assay offers several advantages over the soft-agar clonogenic assay, including higher success rate, avoidance of clumping artifact, shorter time course (5 days), and very low false-negative rate. Further refinement may be necessary, but the rapid assay appears to have potential for individualizing solid tumor chemotherapy.

  3. Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots.

    PubMed

    Cimaglia, Fabio; Liandris, Emmanouil; Gazouli, Maria; Sechi, Leonardo; Chiesa, Maurizio; De Lorenzis, Enrico; Andreadou, Margarita; Taka, Styliani; Mataragka, Antonia; Ikonomopoulos, John

    2015-12-01

    Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure.

  4. Performance of a MALDI-TOF MS-based imipenem hydrolysis assay incorporating zinc sulfate.

    PubMed

    Knox, James; Palombo, Enzo

    2017-03-01

    A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.

  5. Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.

    PubMed

    Lorand, L; Urayama, T; De Kiewiet, J W; Nossel, H L

    1969-06-01

    Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-(5-aminopentyl)-5-dimethylamino-1-naphthalene-sulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase). In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.The amine incorporation test was applied to a clinical case of hereditary total fibrin-stabilizing factor deficiency. The effect of transfusion therapy was studied, and some of the patient's relatives were examined. Whereas a paternal aunt and uncle gave values well within the normal range, a brother and the mother proved to be partially deficient and could be considered as heterozygous carriers. The father appeared to have a reduced level of fibrin

  6. Measurement of suppressor activity of T CD4⁺CD25⁺ T reg cells using bromodeoxyuridine incorporation assay.

    PubMed

    Avalos-Martínez, Claudia E; Rodríguez-Alba, Juan Carlos; Berrón-Ruiz, Laura; Romero-Ramírez, Hector; Santos-Argumedo, Leopoldo; Jiménez-Zamudio, Luis Antonio; Domínguez-López, Maria Lilia; Vega-López, Armando; García-Latorre, Ethel

    2013-01-01

    The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4⁺CD25⁺ cells were also FOXP3⁺ therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials.

  7. A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

    PubMed

    Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

    2016-11-15

    Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening.

  8. Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

    PubMed

    Åström, Johan; Pettersson, Thomas J R; Reischer, Georg H; Norberg, Tommy; Hermansson, Malte

    2015-02-03

    Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

  9. Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

    PubMed Central

    Hawkins, M E; Pfleiderer, W; Mazumder, A; Pommier, Y G; Balis, F M

    1995-01-01

    We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions. PMID:7659509

  10. Assay of anti-cancer drugs in tissue culture: conditions affecting their ability to incorporate 3H-leucine after drug treatment.

    PubMed

    Freshney, R I; Paul, J; Kane, I M

    1975-01-01

    An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid the ingerent variability and heterogeneity of primary cultures from human tumours, and an assay has been constructed using microtitration trays to provide large numbers of replicate cultures without the requirement of a large number cells. An increase in the duration of the exposure to drug increased sensitivity in nearly all cases examined. Similarly, an increase in the period of culture following drug removal produced increased sensitivity to alkylating agents but allowed recovery from exposure to certain cycle-dependent drugs. Some of the drugs used were shown to be unstable under culture conditions and vinblastine was actively metabolized, although this instability was not necessarily reflected in the time course of the drug's effect. Mustine sensitivity was shown to be reduced by an increase in cell density at a level where density limitation of 3H-thymidine incorporation becomes apparent. These variations and possible methods of minimizing their effects are discussed.

  11. Assay of anti-cancer drugs in tissue culture: conditions affecting their ability to incorporate 3H-leucine after drug treatment.

    PubMed Central

    Freshney, R. I.; Paul, J.; Kane, I. M.

    1975-01-01

    An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid the ingerent variability and heterogeneity of primary cultures from human tumours, and an assay has been constructed using microtitration trays to provide large numbers of replicate cultures without the requirement of a large number cells. An increase in the duration of the exposure to drug increased sensitivity in nearly all cases examined. Similarly, an increase in the period of culture following drug removal produced increased sensitivity to alkylating agents but allowed recovery from exposure to certain cycle-dependent drugs. Some of the drugs used were shown to be unstable under culture conditions and vinblastine was actively metabolized, although this instability was not necessarily reflected in the time course of the drug's effect. Mustine sensitivity was shown to be reduced by an increase in cell density at a level where density limitation of 3H-thymidine incorporation becomes apparent. These variations and possible methods of minimizing their effects are discussed. PMID:1156513

  12. Relative Impact of Incorporating Pharmacokinetics on Predicting In Vivo Hazard and Mode of Action from High-Throughput In Vitro Toxicity Assays

    EPA Science Inventory

    The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo e...

  13. Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

    PubMed

    Pessina, Augusto; Bonomi, Arianna; Baglio, Carolina; Cavicchini, Loredana; Gribaldo, Laura

    2009-09-01

    The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

  14. Hereditary orotic aciduria, Lesch-Nyhan syndrome, and xeroderma pigmentosum probed by herpes simplex virus: /sup 125/I-iododeoxycytidine incorporation as an assay for viral growth. [Human fibroblasts

    SciTech Connect

    Campisi, J.; Hafner, J.; Boorstein, R.; Pardee, A.B.

    1983-01-01

    /sup 125/I-Iododeoxycytidine (/sup 125/IdC) incorporation into acid-insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV-1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV-1 was used to probe the metabolic capabilities of three mutant human fibroblast strains. /sup 125/IdC incorporation quantitatively measured the ability of the virus to grow in these cells. Viral /sup 125/IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8- to 10-fold greater resistance to TG in fibroblasts derived from patients with Lesch-Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV-1 in normal fibroblasts was 5-fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral /sup 125/IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2-fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. HSV-1 infected cells could be individually identified by their incorporated /sup 125/IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. (JMT)

  15. Comparative evaluation of the antiproliferative effect of cyclosporin A and gamma-interferon on normal and HPV-transformed keratinocytes by cell counting, MTT assay and tritiated thymidine incorporation.

    PubMed

    Marionnet, A V; Lizard, G; Chardonnet, Y; Schmitt, D

    1997-02-01

    We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-gamma (IFN-gamma) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papilomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 micrograms/ml) and IFN-gamma (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-gamma, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages, of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigations shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-gamma treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.

  16. Rapid aquatic toxicity assay using incorporation of tritiated-thymidine into sea urchin, Arbacia punctulata, embryo: evaluation of toxicant exposure procedures

    SciTech Connect

    Nacci, D.E.; Jackim, E.

    1985-01-01

    Toxicity of substances in seawater was measured using growth inhibition of embryonic sea urchins during a short period after fertilization. Growth of Arbacia punctulata embryos was monitored by incorporation of tritium-labeled thymidine. The paper presents a comparison of toxicant exposure procedures using the Arbacia embryo thymidine incorporation test. Toxicant exposure began before, at the time of, or after fertilization and continued for 4 h following fertilization. In addition to the eight organic chemicals tested for comparison to acute toxicity values for other species, several chemicals with embryotoxic potentials (tumor promoters and teratogens) were tested to determine differential sensitivities of exposed life-stages: unfertilized egg, fertilization, and early embryo. EC50 values for any one substance were not significantly changed by exposure modification. Toxicity values for exposures that included fertilization as well as early embryo growth were at least as sensitive as post-fertilization exposure values for all compounds tested except one. Because of technical ease and potential sensitivity, toxicant exposure that includes fertilization as well as early embryo growth (but not unfertilized egg exposure) is recommended for future testing.

  17. TumorNext: A comprehensive tumor profiling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy

    PubMed Central

    Gray, Phillip N.; Vuong, Huy; Tsai, Pei; Lu, Hsaio-Mei; Mu, Wenbo; Hsuan, Vickie; Hoo, Jayne; Shah, Swati; Uyeda, Lisa; Fox, Susanne; Patel, Harshil; Janicek, Mike; Brown, Sandra; Dobrea, Lavinia; Wagman, Lawrence; Plimack, Elizabeth; Mehra, Ranee; Golemis, Erica A.; Bilusic, Marijo; Zibelman, Matthew; Elliott, Aaron

    2016-01-01

    The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the “second hit” in hereditary cancer patients. PMID:27626691

  18. Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films.

    PubMed

    Iturriaga, L; Olabarrieta, I; de Marañón, I Martínez

    2012-08-01

    The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30 °C or 37 °C) and refrigeration temperature (4 °C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43±3% relative humidity and 24±3 °C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation.

  19. Evaluation of a Test Article in the Salmonella typhimurium/Escherichia coli Plate Incorporation Mutation Assay in the Presence and Absence of Induced Rat Liver S-9. Test Article: N,N,N’,N’-tetramethyl ethanediamine (TMEDA)

    DTIC Science & Technology

    2008-06-12

    Chemical Company St. Louis, MO 63178 Storage Conditions: Room Temperature Lot No.: 46081638 Expiration Dates: February 9,2012 November 20, 2012 Lot...1983 STORAGE: At or below -70°C INDUCING AGENT(s): Aroclor 1254 ( Monsanto KL615), 500 mglkg i.p. BIOCHEMISTRY: -PROTEIN 38.9 mglm! Assayed

  20. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  1. Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids.

    PubMed

    da Silva, Marcelo Santos; Muñoz, Paula Andrea Marin; Armelin, Hugo Aguirre; Elias, Maria Carolina

    2017-03-04

    Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: Indirect Immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2+M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring. This article is protected by copyright. All rights reserved.

  2. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  3. Measuring ribonucleotide incorporation into DNA in vitro and in vivo.

    PubMed

    Clausen, Anders R; Williams, Jessica S; Kunkel, Thomas A

    2015-01-01

    Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome.

  4. Measuring Ribonucleotide Incorporation into DNA In Vitro and In Vivo

    PubMed Central

    Clausen, Anders R.; Williams, Jessica S.; Kunkel, Thomas A.

    2017-01-01

    Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome. PMID:25916710

  5. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  6. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  7. Permanent hair dye-incorporated hyaluronic acid nanoparticles.

    PubMed

    Lee, Hye-Young; Jeong, Young-Il; Kim, Da-Hye; Choi, Ki-Choon

    2013-01-01

    We prepared p-phenylenediamine (PDA)-incorporated nanoparticles using hyaluronic acid (HA). PDA-incorporated HA nanoparticles have spherical shapes and sizes were less than 300 nm. The results of FT-IR spectra indicated that PDA-incorporated HA nanoparticles were formed by ion-complex formation between amine group of PDA and carboxyl group of HA. Furthermore, powder-X-ray diffractogram (XRD) measurement showed that intrinsic crystalline peak of PDA disappeared by formation of nanoparticle with HA at XRD measurement. These results indicated that PDA-incorporated HA nanoparticles were formed by ion-complex formation. At drug release study, the higher PDA contents induced faster release rate from nanoparticles. PDA-incorporated nanoparticles showed reduced intrinsic toxicity against HaCaT human keratinocyte cells at MTT assay and apoptosis assay. We suggest that PDA-incorporated HA nanoparticles are promising candidates for novel permanent hair dye.

  8. Bearings Incorporating Deadband Rollers

    NASA Technical Reports Server (NTRS)

    Gualtieri, Guy V.

    1996-01-01

    Bearings in high-pressure turbopump redesigned to incorporate rollers allowing limited axial motion within small deadband. Does not permit radial deadband motion. Axial deadband motion used for rotor-thrust-balance control. Design eliminates some nonlinearities in dynamics of pump rotor and assists in suppressing vibrations at harmonics of frequency of rotation.

  9. Zinc Incorporation Into Hydroxylapatite

    SciTech Connect

    Tang, Y.; Chappell, H; Dove, M; Reeder, R; Lee, Y

    2009-01-01

    By theoretical modeling and X-ray absorption spectroscopy, the local coordination structure of Zn incorporated into hydroxylapatite was examined. Density function theory (DFT) calculations show that Zn favors the Ca2 site over the Ca1 site, and favors tetrahedral coordination. X-ray absorption near edge structure (XANES) spectroscopy results suggest one dominant coordination environment for the incorporated Zn, and no evidence was observed for other Zn-containing phases. Extended X-ray absorption fine structure (EXAFS) fitting of the synthetic samples confirms that Zn occurs in tetrahedral coordination, with two P shells at 2.85-3.07 {angstrom}, and two higher Ca shells at 3.71-4.02 {angstrom}. These fit results are consistent with the most favored DFT model for Zn substitution in the Ca2 site.

  10. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  11. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  12. A lateral electrophoretic flow diagnostic assay.

    PubMed

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  13. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  14. DISTRIBUTED AMPLIFIER INCORPORATING FEEDBACK

    DOEpatents

    Bell, P.R. Jr.

    1958-10-21

    An improved distributed amplifier system employing feedback for stabilization is presented. In accordance with the disclosed invention, a signal to be amplified is applled to one end of a suitable terminated grid transmission line. At intervals along the transmission line, the signal is fed to stable, resistance-capacitance coupled amplifiers incorporating feedback loops therein. The output current from each amplifier is passed through an additional tube to minimize the electrostatic capacitance between the tube elements of the last stage of the amplifier, and fed to appropriate points on an output transmission line, similar to the grid line, but terminated at the opposite (input) end. The output taken from the unterminated end of the plate transmission line is proportional to the input voltage impressed upon the grid line.

  15. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  16. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  17. Nepal CRS project incorporates.

    PubMed

    1983-01-01

    The Nepal Contraceptive Retail Sales (CRS) Project, 5 years after lauching product sales in June 1978, incorporated as a private, nonprofit company under Nepalese management. The transition was finalized in August 1983. The Company will work through a cooperative agreement with USAID/Kathmandu to complement the national family planning goals as the program continues to provide comtraceptives through retail channels at subsidized prices. Company objectives include: increase contraceptive sales by at least 15% per year; make CRS cost effective and move towards self sufficiency; and explore the possibility of marketing noncontraceptive health products to improve primary health care. After only5 years the program can point to some impressive successes. The number of retial shops selling family planning products increased from 100 in 1978 to over 8000, extending CRS product availability to 66 of the country's 75 districts. Retail sales have climbed dramatically in the 5-year period, from Rs 46,817 in 1978 to Rs 271,039 in 1982. Sales in terms of couple year protection CYP) have grown to 24,451 CYP(1982), a 36% increase over 1980 CYP. Since the beginning of the CRS marketing program, total distribution of contraceptives--through both CRS and the Family Planning Maternal and Child Haelth (FP/MCH) Project--has been increasing. While the FP/MCH program remains the largest distributor,contribution of CRS Products is increasing, indicating that CRS is creating new product acceptors. CRS market share in 1982 was 43% for condoms and 16% for oral contraceptives (OCs). CRS markets 5 products which are subsidized in order to be affordable to consumers as well as attractive to sellers. The initial products launched in June 1978 were Gulaf standard dose OCs and Dhaal lubricated colored condoms. A less expensive lubricates, plain Suki-Dhaal condom was introduced in June 1980 in an attempt to reach poorer rural populations, but rural distribution costs are excessive and Suki

  18. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  19. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  20. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  1. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  2. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  3. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  4. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  5. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  6. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  7. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  8. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  9. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  10. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  11. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  12. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  13. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R; Benett, William J; Coleman, Matthew A; Pearson, Francesca S; Nasarabadi, Shanavaz L

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  14. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  15. Cascade enzyme-linked immunosorbent assay (CELISA).

    PubMed

    Lee, Young-mi; Jeong, Yujin; Kang, Hyo Jin; Chung, Sang J; Chung, Bong Hyun

    2009-10-15

    Immunoassays are representative biochemical detection methods. Among them, sandwich-type immunoassays, typified by sandwich ELISA, have used in disease diagnosis or biochemical detection with high target selectivity. Horseradish peroxidase and alkaline phosphatase have been typically used for signal amplification in ELISA. Recently developed sandwich-type immunoassays such as biobarcode immunoassays, immuno-PCR, and immuno-RCA have improved sensitivity by changing mainly the signal amplification method. To develop a novel amplification method in ELISA, an enzyme-cascading system was incorporated into an ELISA, and the new assay is termed a cascading enzyme-linked immunosorbent assay (CELISA). This CELISA includes a trypsinogen-enterokinase combination as the cascading enzyme system, and was used to detect alpha-fetoprotein (AFP), which is a liver cancer marker, and prostate-specific antigen (PSA). Using a colorimetric reagent for signal generation, CELISA had 0.1-10pM limits-of-detection for AFP and PSA in whole human serum and assay buffers, depending on the platform, well plate, or microbead type used. This study represents the first example that incorporated an enzyme cascading step in an ELISA system, resulting in successful signal amplification with sensitive detection of pathogenic antigens in serum.

  16. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  17. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  18. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  19. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  20. Overview of Plant Incorporated Protectants

    EPA Pesticide Factsheets

    When assessing the potential risks of genetically engineered plant-incorporated protectants, EPA requires extensive studies examining numerous factors. Learn more about the history and process for regulating PIPs.

  1. Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase.

    PubMed

    Takagi, Toshimitsu; Shum, David; Parisi, Monika; Santos, Ruth E; Radu, Constantin; Calder, Paul; Rizvi, Zahra; Frattini, Mark G; Djaballah, Hakim

    2011-09-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

  2. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  3. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  4. Automated filtration capture immunoelectrochemical assay of bacteria

    NASA Astrophysics Data System (ADS)

    Brewster, Jeffrey D.

    1999-01-01

    An automated system for filtration capture and immunoelectrochemical detection of bactria in liquid samples is described. The detector incorporates a porous electrode in contact with the filter, rather than the solid electrode used previously, to allow sample and reagent solutions to be delivered in a flowing stream. This eliminated the need for manual assembly of the electrode and filter for each assay and allowed repetitive assays on a single filter/electrode. The electrochemical response of the novel gold grid electrode under static and flow conditions was found to be consistent with theory for a planar electrode operating in laminar flow conditions. A computer-controlled fluid handling system was coupled to the detector for delivery of samples and reagents at controlled flow rates and times. The combination of flow detector and fluid handling system allows for automation of the previous assay protocol as well as providing new operating modes with enhanced background rejection and improved sensitivity. The use of these operating modes is demonstrated by a simple assay for Escherichia coli O157:H7 with virtually no background current.

  5. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  6. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges.

    PubMed

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-10-20

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group.

  7. ABCs of DNA aptamer and related assay development.

    PubMed

    Sharma, Tarun Kumar; Bruno, John G; Dhiman, Abhijeet

    This review is intended to guide the novice in aptamer research and development to understand virtually all of the aptamer development options and currently available assay modalities. Aptamer development topics range from discussions of basic and advanced versions of Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and SELEX variations involving incorporation of exotic unnatural nucleotides to expand library diversity for even greater aptamer affinity and specificity to improved next generation methods of DNA sequencing, screening and tracking aptamer development throughout the SELEX process and characterization of lead aptamer candidates. Aptamer assay development topics include descriptions of various colorimetric and fluorescent assays in microplates or on membranes including homogeneous beacon and multiplexed Fluorescence Resonance Energy Transfer (FRET) assays. Finally, a discussion of the potential for marketing successful aptamer-based assays or test kits is included.

  8. Host thin films incorporating nanoparticles

    NASA Astrophysics Data System (ADS)

    Qureshi, Uzma

    The focus of this research project was the investigation of the functional properties of thin films that incorporate a secondary nanoparticulate phase. In particular to assess if the secondary nanoparticulate material enhanced a functional property of the coating on glass. In order to achieve this, new thin film deposition methods were developed, namely use of nanopowder precursors, an aerosol assisted transport technique and an aerosol into atmospheric pressure chemical vapour deposition system. Aerosol assisted chemical vapour deposition (AACVD) was used to deposit 8 series of thin films on glass. Five different nanoparticles silver, gold, ceria, tungsten oxide and zinc oxide were tested and shown to successfully deposit thin films incorporating nanoparticles within a host matrix. Silver nanoparticles were synthesised and doped within a titania film by AACVD. This improved solar control properties. A unique aerosol assisted chemical vapour deposition (AACVD) into atmospheric pressure chemical vapour deposition (APCVD) system was used to deposit films of Au nanoparticles and thin films of gold nanoparticles incorporated within a host titania matrix. Incorporation of high refractive index contrast metal oxide particles within a host film altered the film colour. The key goal was to test the potential of nanopowder forms and transfer the suspended nanopowder via an aerosol to a substrate in order to deposit a thin film. Discrete tungsten oxide nanoparticles or ceria nanoparticles within a titanium dioxide thin film enhanced the self-cleaning and photo-induced super-hydrophilicity. The nanopowder precursor study was extended by deposition of zinc oxide thin films incorporating Au nanoparticles and also ZnO films deposited from a ZnO nanopowder precursor. Incorporation of Au nanoparticles within a VO: host matrix improved the thermochromic response, optical and colour properties. Composite VC/TiC and Au nanoparticle/V02/Ti02 thin films displayed three useful

  9. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  10. 17 CFR 260.7a-30 - Identification of material incorporated; form of incorporation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... incorporated; form of incorporation. 260.7a-30 Section 260.7a-30 Commodity and Securities Exchanges SECURITIES... Incorporation by Reference § 260.7a-30 Identification of material incorporated; form of incorporation. In each case of incorporation by reference, the matter incorporated shall be clearly identified in...

  11. Influence of grape pomace extract incorporation on chitosan films properties.

    PubMed

    Ferreira, Andreia S; Nunes, Cláudia; Castro, Alichandra; Ferreira, Paula; Coimbra, Manuel A

    2014-11-26

    Chitosan has been studied as a renewable polymer to form edible films allowing the incorporation of functional compounds. The aim of this work was to evaluate the effects in the chitosan films properties of the incorporation of grape pomace extracts: 0.15% of hot water extract (mainly polysaccharides), 0.15 and 0.3% of chloroform extract (wax), and 0.3 and 0.75% of n-hexane extract (oil). The evaluation of the surface morphology revealed that the films with the aqueous extract had the most homogeneous and smoother topography. The incorporation of higher proportion of wax and oil led to changes in mechanical properties of the films, namely lower resistance and stiffness. The chitosan-based films with 0.75% oil demonstrated a 75% decrease of solubility in water, due to their hydrophobicity, as confirmed by the contact angle and surface free energy measurements. The hydrophobic films showed higher antioxidant capacity in organic medium (ABTS and DPPH assays) whereas the most hydrophilic films showed an improvement in FRAP and reducing power assays. Therefore, all the chitosan-based films prepared by incorporation of these grape pomace extracts are promising for food shelf life extension.

  12. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  13. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  14. Incorporating Argumentation through Forensic Science

    ERIC Educational Resources Information Center

    Wheeler, Lindsay B.; Maeng, Jennifer L.; Smetana, Lara K.

    2014-01-01

    This article outlines how to incorporate argumentation into a forensic science unit using a mock trial. Practical details of the mock trial include: (1) a method of scaffolding students' development of their argument for the trial, (2) a clearly outlined set of expectations for students during the planning and implementation of the mock…

  15. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  16. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  17. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  18. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  19. Selenium incorporation using recombinant techniques.

    PubMed

    Walden, Helen

    2010-04-01

    Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  20. Survival assays using Caenorhabditis elegans

    PubMed Central

    Park, Hae-Eun H.; Jung, Yoonji; Lee, Seung-Jae V.

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

  1. A fluorescent microsphere-based method for assay of multiple analytes in plasma.

    PubMed

    Bernhard, Oliver K; Mathias, Rommel A; Barnes, Thomas W; Simpson, Richard J

    2011-01-01

    Measurement of multiple analytes can provide increased sensitivity and specificity for the detection and management of disease. The enzyme-linked immunosorbent assay (ELISA) is currently the "gold standard" for protein quantification; however, individual assays for each analyte must be performed, placing demand on sample volume. On the contrary, multiplex assays using microsphere-based technologies allow for multiple analytes to be simultaneously assayed within a single sample. Here, we present a protocol for the preparation and development of a multiple-analyte assay in human plasma using the BioPlex 200 platform (Bio-Rad), which incorporates xMAP technology (Luminex).

  2. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  3. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  4. Incorporating Spirituality in Primary Care.

    PubMed

    Isaac, Kathleen S; Hay, Jennifer L; Lubetkin, Erica I

    2016-06-01

    Addressing cultural competency in health care involves recognizing the diverse characteristics of the patient population and understanding how they impact patient care. Spirituality is an aspect of cultural identity that has become increasingly recognized for its potential to impact health behaviors and healthcare decision-making. We consider the complex relationship between spirituality and health, exploring the role of spirituality in primary care, and consider the inclusion of spirituality in existing models of health promotion. We discuss the feasibility of incorporating spirituality into clinical practice, offering suggestions for physicians.

  5. Incorporation of additives into polymers

    DOEpatents

    McCleskey, T. Mark; Yates, Matthew Z.

    2003-07-29

    There has been invented a method for incorporating additives into polymers comprising: (a) forming an aqueous or alcohol-based colloidal system of the polymer; (b) emulsifying the colloidal system with a compressed fluid; and (c) contacting the colloidal polymer with the additive in the presence of the compressed fluid. The colloidal polymer can be contacted with the additive by having the additive in the compressed fluid used for emulsification or by adding the additive to the colloidal system before or after emulsification with the compressed fluid. The invention process can be carried out either as a batch process or as a continuous on-line process.

  6. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  7. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  8. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  9. Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay

    DTIC Science & Technology

    2010-09-01

    provide data relating to the test substance’s health effects, environmental effects, or environmental fate testing regulated by the U.S. Environmental...procedures will be consistent with the Office of Prevention, Pesticides and Toxic Substances (OPPTS), Health Effects Test Guidelines, 870.5100 (U.S...Mutat. Res. 455: 29-60, 2000. U.S. Environmental Protection Agency (EPA). Office of Prevention, Pesticides and Toxic Substances (OPPTS), Health

  10. Enzymatic incorporation and utilization of an emissive 6-azauridine.

    PubMed

    Hopkins, Patrycja A; McCoy, Lisa S; Tor, Yitzhak

    2017-01-18

    To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.

  11. Incorporation of heparin into biomaterials.

    PubMed

    Sakiyama-Elbert, Shelly E

    2014-04-01

    This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas.

  12. Click-iT assay with improved DNA distribution histograms.

    PubMed

    Hamelik, Ronald M; Krishan, Awtar

    2009-10-01

    The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

  13. Click-iT proliferation assay with improved DNA histograms.

    PubMed

    Krishan, Awtar; Hamelik, Ronald M

    2010-04-01

    The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.

  14. Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA▿

    PubMed Central

    Ginocchio, C. C.; Barth, D.; Zhang, F.

    2008-01-01

    This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay). PMID:18367578

  15. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  16. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.180 Section 572.180... Test Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part...

  17. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  18. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  19. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  20. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  1. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.30 Section 572.30....30 Incorporated materials. (a) The drawings and specifications referred to in this regulation that... Federal Register has approved the materials incorporated by reference. For materials subject to...

  2. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.190 Section 572.190... Test Dummy, Small Adult Female § 572.190 Incorporated materials. (a) The following materials are hereby... Register approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR...

  3. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  4. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  5. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  6. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  7. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  8. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 587.5 Section 587.5... Barrier § 587.5 Incorporated materials. (a) The drawings and specifications referred to in this regulation that are not set forth in full are hereby incorporated in this part by reference. These materials...

  9. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.180 Section 572.180... Test Dummy, 50th Percentile Adult Male § 572.180 Incorporated materials. (a) The following materials... approved the materials incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part...

  10. 49 CFR 572.80 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.80 Section 572.80... Incorporated materials. The drawings and specifications referred to in § 572.81(a) that are not set forth in full are hereby incorporated in this part by reference. These materials are thereby made part of...

  11. Numeral Incorporation in Japanese Sign Language

    ERIC Educational Resources Information Center

    Ktejik, Mish

    2013-01-01

    This article explores the morphological process of numeral incorporation in Japanese Sign Language. Numeral incorporation is defined and the available research on numeral incorporation in signed language is discussed. The numeral signs in Japanese Sign Language are then introduced and followed by an explanation of the numeral morphemes which are…

  12. Nondestructive boxed transuranic (TRU) waste assay systems

    NASA Astrophysics Data System (ADS)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.

    1999-01-01

    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  13. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  14. Face Recognition Incorporating Ancillary Information

    NASA Astrophysics Data System (ADS)

    Kim, Sang-Ki; Toh, Kar-Ann; Lee, Sangyoun

    2007-12-01

    Due to vast variations of extrinsic and intrinsic imaging conditions, face recognition remained to be a challenging computer vision problem even today. This is particularly true when the passive imaging approach is considered for robust applications. To advance existing recognition systems for face, numerous techniques and methods have been proposed to overcome the almost inevitable performance degradation due to external factors such as pose, expression, occlusion, and illumination. In particular, the recent part-based method has provided noticeable room for verification performance improvement based on the localized features which have good tolerance to variation of external conditions. The part-based method, however, does not really stretch the performance without incorporation of global information from the holistic method. In view of the need to fuse the local information and the global information in an adaptive manner for reliable recognition, in this paper we investigate whether such external factors can be explicitly estimated and be used to boost the verification performance during fusion of the holistic and part-based methods. Our empirical evaluations show noticeable performance improvement adopting the proposed method.

  15. Incorporating transgenerational testing and epigenetic ...

    EPA Pesticide Factsheets

    A number of environmental chemicals have been shown to alter markers of epigenetic change. Some published multi-generation rodent studies have identified effects on F2 and greater generations after chemical exposures solely to F0 dams, but were not focused on chemical safety. We were interested in how outcomes related to epigenetic changes could be identified and incorporated into chemical testing and risk assessment. To address this question, we conducted a systematic literature review to identify transgenerational (TG) epigenetic studies in rodents. These were analyzed to characterize the methods and observed outcomes, and to evaluate strengths, limitations, and biases. Our analysis found that test substances were administered to pregnant F0 dams; endpoints assessed in F1 to F4 generation offspring included growth, puberty timing, steroid hormone levels, abdominal adiposity, organ weights, histopathology, and epigenetic biomarkers. Biases were minimized through, e.g., randomization procedures, avoiding sibling or cousin matings, and independent multiple reviews of histopathology data. However, the numbers of litters assigned to control and test groups were not always transparently reported, nested statistical analyses of data was not always utilized to address litter effects, and “blind” testing was seldom performed. Many of these studies identified chemicals or combinations of chemicals that produced TG effects and/or adult-onset diseases, but there is a

  16. Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

    EPA Science Inventory

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

  17. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  18. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  19. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  20. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  1. A microassay for colony-stimulating factor based on thymidine incorporation.

    PubMed Central

    Prystowsky, M. B.; Naujokas, M. F.; Ihle, J. N.; Goldwasser, E.; Fitch, F. W.

    1984-01-01

    A variety of growth factors and lectins were tested; only colony-stimulating factors CSF-1, Interleukin 3, and a T-lymphocyte GM CSF induced colony formation in semisolid medium and stimulated thymidine incorporation in liquid culture. All other growth factors and lectins were inactive in both assays. Factor-stimulated thymidine incorporation was detectable 24 hours after stimulation and reached maximal levels 4-6 days after stimulation. A convenient microassay for measuring CSF activity has been developed, enabling a large number of samples to be screened qualitatively in 2 days and permitting CSF activity to be measured quantitatively in 4-5 days. This microassay can supplement the clonal-cell assay method and be especially useful as an initial screening assay for CSF activity. PMID:6606982

  2. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  3. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  4. Use of the spiral Salmonella assay to detect the mutagenicity of complex environmental mixtures

    SciTech Connect

    Houk, V.S.; Early, G.; Claxton, L.D. )

    1991-01-01

    The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose-response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: (1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. (2) Samples were more mutagenic on rev/microgram basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. (3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. (4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay.

  5. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  6. Incorporating Sociology into Community Service Classes

    ERIC Educational Resources Information Center

    Hochschild, Thomas R., Jr.; Farley, Matthew; Chee, Vanessa

    2014-01-01

    Sociologists and instructors who teach about community service share an affinity for understanding and addressing social problems. While many studies have demonstrated the benefits of incorporating community service into sociology courses, we examine the benefits of incorporating sociological content into community service classes. The authors…

  7. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  8. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  9. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  10. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Incorporated materials. 572.40 Section 572.40... Percentile Male § 572.40 Incorporated materials. (a) The drawings, specifications, manual, and computer... by reference. These materials are thereby made part of this regulation. The Director of the...

  11. Fenugreek Incorporated Silk Fibroin Nanofibers-A Potential Antioxidant Scaffold for Enhanced Wound Healing.

    PubMed

    Selvaraj, Sowmya; Fathima, Nishter Nishad

    2017-02-22

    Free radicals are generated by various biochemical pathways in the living system, causing severe oxidative damage to the biomolecules leading to adverse disease conditions. Hence, there is an increasing interest in antioxidant studies for preventing the effects of these free radicals. Herein, we propose a novel electrospun scaffold with antioxidant properties that can be used as wound healing material. Fenugreek, a natural antioxidant incorporated silk fibroin nanofiber, was prepared in four different ratios by the co-electrospinning method. The biocompatibility of the nanofibers and its antioxidant activity were evaluated through 3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay, respectively. The experimental observations indicate that the incorporation of fenugreek increases the thermal and mechanical properties of silk fibroin nanofibers. DPPH assay proves that the antioxidant property is enhanced with increasing concentration of fenugreek in nanofiber mats, and the Swiss albino 3T6 fibroblasts show better proliferation on the nanofibrous scaffolds. Further, the wound healing efficiency of fenugreek incorporated silk fibroin nanofibrous scaffolds was evaluated using full thickness excisional wounds in rat model. Wound healing was accelerated in silk fibroin-fenugreek nanofibers treated wounds with complete re-epithelialization and enhanced collagen deposition. The present study validates the use of fenugreek incorporated silk fibroin nanofiber mats as antioxidant scaffolds in wound healing applications.

  12. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  13. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  14. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  15. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  16. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  17. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  18. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  19. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  20. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  1. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  2. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  3. Toxin activity assays, devices, methods and systems therefor

    DOEpatents

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2016-04-05

    Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  4. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  5. Important Norwegian crude assays updated

    SciTech Connect

    Corbett, R.A

    1990-03-12

    New assays on two important Norwegian North Sea crude oils, Statfjord and Gullfaks, are presented. Both are high-quality, low-sulfur crudes that will yield a full range of good-quality products. All assay data came from industry-standard test procedures. The Statfjord field is the largest in the North Sea. Production started in 1979. Statfjord is a typical North Sea crude, produced from three separate platforms and three separate loading buoys with interconnecting lines. Current production is about 700,000 b/d. Gullfaks is produced from a large field in Block 34/10 of the Norwegian sector of the North Sea production area. Gullfaks crude oil is more biodegraded than other crudes from the region. Biodegradation has removed most of the waxy normal paraffins, resulting in a heavier, more naphthenic and aromatic crude.

  6. Comet Assay in Cancer Chemoprevention.

    PubMed

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  7. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  8. [Visible spectrophotometric assay of ranitidine].

    PubMed

    Apostu, M; Dorneanu, V; Bibire, Nela

    2003-01-01

    Ranitidine, belonging to H2-antagonist group, is a compound containing a furanic moiety and is used in peptic ulcer therapy. This paper debates the possibility of developing a new visible spectrophotometric assessment by using the reaction between ranitidine and eosine. We carried out our determinations at 505 nm, where the absorbency of ranitidine-eosine complex is maximal, and we have established the optimal reaction conditions. This method was successfully applied for ranitidine assay from pharmaceutical dosage forms.

  9. Two offshore Australian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-09

    Two light, sweet crudes from offshore Australia have been assayed. Gippsland crude, also called Bass Strait, is produced off the coast of Victoria, in southeastern Australia. The 47 API, 0.09% sulfur crude was analyzed in mid-1993. Skua, a 42 API, 0.06 wt % sulfur crude, is produced in the Timor Sea. Data are given on the whole crude and fractions for both deposits. Both chemical and physical properties are listed.

  10. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  11. Optimization of a UDP-glucuronosyltransferase assay for trout ...

    EPA Pesticide Factsheets

    An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate

  12. Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins.

    PubMed

    Bekedam, E Koen; De Laat, Marieke P F C; Schols, Henk A; Van Boekel, Martinus A J S; Smit, Gerrit

    2007-02-07

    The charge properties of melanoidins in high molecular weight (HMw) coffee brew fractions, isolated by diafiltration and membrane dialysis, were studied. Ion exchange chromatography experiments with the HMw fractions showed that coffee brew melanoidins were negatively charged whereas these molecules did not expose any positive charge at the pH of coffee brew. Fractions with different ionic charges were isolated and subsequently characterized by means of the specific extinction coefficient (K(mix 405nm)), sugar composition, phenolic group content, nitrogen content, and the arabinogalactan protein (AGP) specific Yariv gel-diffusion assay. The isolated fractions were different in composition and AGP was found to be present in one of the HMw fractions. The AGP accounted for 6% of the coffee brew dry matter and had a moderate negative charge, probably caused by the presence of uronic acids. As the fraction that precipitated with Yariv was brown (K(mix 405nm) = 1.2), compared to a white color in the green bean, it was concluded that these AGPs had undergone Maillard reaction resulting in an AGP-melanoidin complex. The presence of mannose (presumably from galactomannan) indicates the incorporation of galactomannans in the AGP-melanoidin complex. As the uronic acid content in the more negatively charged melanoidin-rich, AGP-poor HMw fractions decreased, it was hypothesized that acidic groups are formed or incorporated during melanoidin formation.

  13. Effects of MMP inhibitors incorporated within dental adhesives.

    PubMed

    Almahdy, A; Koller, G; Sauro, S; Bartsch, J W; Sherriff, M; Watson, T F; Banerjee, A

    2012-06-01

    Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the "hybrid layer" and to the "adhesive", respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability.

  14. Effects of MMP Inhibitors Incorporated within Dental Adhesives

    PubMed Central

    Almahdy, A.; Koller, G.; Sauro, S.; Bartsch, J.W.; Sherriff, M.; Watson, T.F.; Banerjee, A.

    2012-01-01

    Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the “hybrid layer” and to the “adhesive”, respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability. PMID:22518030

  15. The Rabbit Corneal Pocket Assay.

    PubMed

    Morbidelli, Lucia; Ziche, Marina

    2016-01-01

    The rabbit corneal micropocket angiogenesis assay uses the avascular cornea as a substrate canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 1-2 weeks and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor and a synthetic polymer to allow slow release. A micropocket is surgically created in the rabbit cornea under anesthesia and a pellet implanted. On the days later, the angiogenic response is measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay are used to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter, the experimental details of the rabbit cornea assay and technical implementations to the original protocol are described.

  16. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  17. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

  18. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    PubMed

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  19. Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

    PubMed

    Yi, Lian; Wang, Xue; Bethge, Lucas; Klussmann, Sven; Roper, Michael G

    2016-03-21

    The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

  20. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  1. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  2. Radioenzymatic assay for quinolinic acid

    SciTech Connect

    Foster, A.C.; Okuno, E.; Brougher, D.S.; Schwarcz, R.

    1986-10-01

    A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing (/sup 3/H)ATP, further to (/sup 3/H) deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

  3. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor.

  4. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  5. Assay of potentially contaminated propellant

    SciTech Connect

    Koster, J.E.; Williams, H.E. III; Scott, W.S.

    1995-02-01

    One of the decontamination and decommissioning projects within DOD is demilitarization of an aging stockpile of munitions. A large portion of the stockpile contains depleted uranium (DU) as an armor piercing core and so these munitions must be assayed for the presence of uranium in other components. The assay method must be fast and preferably easy to implement. Presence of DU is indicated by its alpha decay. The alpha particles in turn produce ions in the ambient air. If a significant fraction of these ions can escape the quantity of propellant, the ions can be detected instead of the alpha particles. As a test of the feasibility of detecting alpha emissions from DU somewhere within a cartridge of propellant, the transmission of ions through layers of real propellant was measured. The propellant is in the form of graphite-coated cylindrical pellets. A 105nun cartridge was modified for use as a pellet chamber. A check source served as an ion source. The ion detector consisted of a grid held at 300V coupled to an ammeter. Results confirm that this is a promising technique for testing the propellant for the presence of DU quickly yet with sensitivity.

  6. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  7. Rotor assembly and assay method

    DOEpatents

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  8. Therapy for incorporated radionuclides: scope and need

    SciTech Connect

    Smith, V.H.

    1981-03-01

    In the United States the recent termination of funding for research on therapy for incorporated radionuclides has virtually halted progress on improved or new agents and procedures for removing radioactivity from the body. Research was eliminated, but is still needed on new removal agents, improved delivery system, in vitro test systems, and the toxicology of treatments. For many radionuclides, no adequate therapy exists. The relationship between radionuclide removal and reduction in cancer risk is still unanswered. Without proper research support, needed improvements in the treatment for incorporated radionuclides in the US are uncertain.

  9. Erythropoietin assay: present status of methods, pitfalls, and results in polycythemic disorders.

    PubMed

    Popovic, W J; Adamson, J W

    1978-01-01

    Mammalian erythropoiesis is regulated primarily by the hormone erythropoietin (ESP). Studies of ESF have provided information about its biochemistry and its role in regulating hemoglobin synthesis. Such studies rely on assays for erythropoietic activity in biological fluid. The assay which has proven most valuable and is used most widely is based upon the incorporation of radioactive iron into newly-formed red cells of polycythemic mice. While this assay has gained wide acceptance, it is expensive, cumbersome, imprecise, and insensitive, capable of reliably detecting no less than 50 milliunits of erythropoietin. Improvements in assay techniques will require new methodology relying primarily on immunologic recognition for the determination of hormone activity. Currently under development and in experimental use are radioimmunoassays and a hemagglutination inhibition assay. While work has progressed in these areas, these assays are not of proven value at present and meaningful physiological correlations have not emerged from their use. Alternatively, assays for hormone activity using suspensions of hematopoietic cells and the measurement of incorporation of radioactive isotopes into hemoglobin have provided both improvement in sensitivity and precision. The disadvantage of these types of assays is that they are sensitive to factors other than ESF and may give misleading information, depending on whether the factors present stimulate or inhibit cellular proliferation and hemoglobin synthesis. While such techniques may provide a temporary solution to some problems associated with assaying ESF for purification or physiological studies, they are not the best answer to the overall problem of hormone detection and characterization. The most important contribution to this field will be the availability of large amounts of highly purified and well-characterized ESF.

  10. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  11. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  12. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  13. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  14. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  15. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  16. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  17. Eimeria tenella: parasite-specific incorporation of /sup 3/H-uracil as a quantitative measure of intracellular development

    SciTech Connect

    Schmatz, D.M.; Crane, M.S.; Murray, P.K.

    1986-02-01

    An assay has been developed using parasite-specific incorporation of /sup 3/H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, /sup 3/H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional /sup 3/H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of /sup 3/H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.

  18. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  19. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

    PubMed

    Yang, T T; Sinai, P; Kain, S R

    1996-10-01

    We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

  20. Forensically relevant SNaPshot(®) assays for human DNA SNP analysis: a review.

    PubMed

    Mehta, Bhavik; Daniel, Runa; Phillips, Chris; McNevin, Dennis

    2017-01-01

    Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot(®) technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot(®) assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot(®) assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot(®) assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.

  1. Incorporating the National Standards in Performance Classes.

    ERIC Educational Resources Information Center

    Kerchner, Jody L.

    2001-01-01

    Addresses how the National Standards can be incorporated into band rehearsals using the music piece, "Balladair" (Frank Erickson), as an example when teaching techniques and musical concepts. Provides ideas such as using material from "Balladair" to create warm-ups or relating the piece to other arts. (CMK)

  2. Incorporating Engineering Design Challenges into STEM Courses

    ERIC Educational Resources Information Center

    Householder, Daniel L., Ed.; Hailey, Christine E., Ed.

    2012-01-01

    Successful strategies for incorporating engineering design challenges into science, technology, engineering, and mathematics (STEM) courses in American high schools are presented in this paper. The developers have taken the position that engineering design experiences should be an important component of the high school education of all American…

  3. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE Recommended...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal... 20879, (301) 670-0090. (2) The SAE materials referred to in paragraphs (a)(4) and (a)(5) of this...

  4. 49 CFR 572.190 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Impact Crash Test Dummy, July 1, 2008,” incorporated by reference in § 572.191; (4) SAE Recommended...) SAE J1733 of 1994-12, “Sign Convention for Vehicle Crash Testing.” (b) The Director of the Federal... 20879, (301) 670-0090. (2) The SAE materials referred to in paragraphs (a)(4) and (a)(5) of this...

  5. 49 CFR 572.40 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.40 Section 572.40 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Side Impact Dummy...

  6. Combined cycle power plant incorporating coal gasification

    DOEpatents

    Liljedahl, Gregory N.; Moffat, Bruce K.

    1981-01-01

    A combined cycle power plant incorporating a coal gasifier as the energy source. The gases leaving the coal gasifier pass through a liquid couplant heat exchanger before being used to drive a gas turbine. The exhaust gases of the gas turbine are used to generate both high pressure and low pressure steam for driving a steam turbine, before being exhausted to the atmosphere.

  7. The Incorporation and Abjection of Official Knowledge

    ERIC Educational Resources Information Center

    Kearl, Benjamin Kelsey

    2012-01-01

    In this essay, the author analyzes two theoretical perspectives--incorporation and abjection--that inform official knowledge generally and high school American history textbooks specifically. While contemporary textbooks increasingly depict the experiences of historically marginalized groups such as women, African Americans, Latinos, American…

  8. 77 FR 16761 - Incorporation by Reference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-22

    ...; ] OFFICE OF THE FEDERAL REGISTER 1 CFR Part 51 Incorporation by Reference AGENCY: Office of the Federal... Register (OFR or we) received a petition to revise our regulations at 1 CFR part 51 on February 13, 2012... 11414. You can view the petition and its suggested revisions to the regulations 1 CFR part 51 on...

  9. 49 CFR 587.5 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 587.5 Section 587.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) DEFORMABLE BARRIERS Side Impact Moving...

  10. Incorporating Technology into a Hawaiian Language Curriculum.

    ERIC Educational Resources Information Center

    Ka'awa, Makalapua; Hawkins, Emily

    This paper describes Hawaiian language courses that incorporate computer technology at the University of Hawaii at Manoa. In the past decade, enrollments in all types of Hawaiian language programs have increased rapidly. The University of Hawaii is committed to extending Hawaiian language education, especially the full development of Hawaiian…

  11. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...,” incorporated by reference in §§ 572.180(a)(2), and 572.181(a); (4) Society of Automotive Engineers (SAE) Recommended Practice J211, Rev. Mar 95 “Instrumentation for Impact Tests—Part 1—Electronic Instrumentation... the National Archives and Records Administration (NARA), and in electronic format through...

  12. Semiconducting compounds and devices incorporating same

    DOEpatents

    Marks, Tobin J.; Facchetti, Antonio; Boudreault, Pierre-Luc; Miyauchi, Hiroyuki

    2016-01-19

    Disclosed are molecular and polymeric compounds having desirable properties as semiconducting materials. Such compounds can exhibit desirable electronic properties and possess processing advantages including solution-processability and/or good stability. Organic transistor and photovoltaic devices incorporating the present compounds as the active layer exhibit good device performance.

  13. Semiconducting compounds and devices incorporating same

    DOEpatents

    Marks, Tobin J; Facchetti, Antonio; Boudreault, Pierre-Luc; Miyauchi, Hiroyuki

    2014-06-17

    Disclosed are molecular and polymeric compounds having desirable properties as semiconducting materials. Such compounds can exhibit desirable electronic properties and possess processing advantages including solution-processability and/or good stability. Organic transistor and photovoltaic devices incorporating the present compounds as the active layer exhibit good device performance.

  14. Design of Schools to Incorporate Fallout Protection.

    ERIC Educational Resources Information Center

    Folley, Milo D.

    Means are suggested by which a school district may incorporate low-cost fallout protection in a school construction program, through construction of an underground shelter beneath the concrete slab foundation. Ways of controlling distribution and filtering air are discussed. The author also suggests consideration of a completely underground…

  15. Incorporating Mobile Learning into Athletic Training Education

    ERIC Educational Resources Information Center

    Davie, Emily

    2009-01-01

    Objective: To introduce and present techniques for incorporating mobile learning into athletic training education. Background: The matriculation of digital natives into college has stimulated the identification and development of new teaching and learning strategies. Electronic learning (e-learning), including the use of learning management…

  16. Incorporating the Aesthetic Dimension into Pedagogy

    ERIC Educational Resources Information Center

    Webster, R. Scott; Wolfe, Melissa

    2013-01-01

    This paper reports on a case study that was undertaken to discover not only the belief and intent behind the everyday opportunities that four exemplary teachers offered their high performing students but what activities they incorporated into their everyday lessons in an attempt to make sense of how aesthetic experiences may enhance learning. The…

  17. Incorporation of labeled small molecules into rubratoxin.

    PubMed

    Emeh, C O; Marth, E H

    1978-07-01

    A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28 degrees C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1(14)C]acetate, [1,5(14)C]citrate, [2(14)C]malonate, [1(14)C]glucose, [U14C]glucose or [1(14)C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1(14)C]acetate and [2(14)C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.

  18. 49 CFR 572.30 - Incorporated materials.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Incorporated materials. 572.30 Section 572.30 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES Hybrid III Test Dummy §...

  19. Incorporating Social Media in the Classroom

    ERIC Educational Resources Information Center

    McMeans, April

    2015-01-01

    Incorporating social media into the classroom will provide a positive, upbeat learning environment that students are engaged in on a regular basis. In doing this, educators will be ensuring discussion, collaboration, critical thinking, and creativity amongst their students. Social media is a knowledgeable topic for our students, and it is an…

  20. Identification, Introjection, Incorporation, Internalization: A Bibliography.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    Identification, introjection, incorporation, and internalization dealt with the same basic process according to S. Freud. The process has been discussed and researched in many and varied personal-social contexts. There have been two general areas of concern: identification for positive reasons (often called developmental) and identification for…

  1. Incorporating Feminism into Rehabilitation Counselor Education

    ERIC Educational Resources Information Center

    Jeon, Mookyong

    2015-01-01

    Purpose: The author describes how rehabilitation counselor educators can incorporate the feminist perspective in teaching rehabilitation counselors-in-training by exploring history, core values, and training methods of feminism. Method: Based on a literature review, the author compares philosophy and concepts of rehabilitation counseling and…

  2. Steroid assays in paediatric endocrinology.

    PubMed

    Honour, John W

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  3. Steroid Assays in Paediatric Endocrinology

    PubMed Central

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation. Conflict of interest:None declared. PMID:21274330

  4. Optimized microturbidimetric assay for fibrinogen.

    PubMed

    Macart, M; Koffi, A; Henocque, G; Mathieu, J F; Guilbaud, J C

    1989-02-01

    In this assay we measure the turbidity produced by precipitation of plasma fibrinogen with a reagent composed of ammonium sulfate, EDTA, and guanidine hydrochloride. The two-step reagent addition, and use of fixed reaction times, eliminates interference from bilirubin, hemoglobin, and chylomicrons. We checked 135 monoclonal proteins for interference, finding the probability of encountering major interference in samples from adults to be very low, P = 0.0002. The method is calibrated with purified fibrinogen and the response is linear over the range 0-10 g/L. Within-run precision (CV) is less than 2% from 1 to 10 g/L. Correlations with the immunoturbidimetric (r = 0.99), chronometric (r = 0.99), and clotting (r = 0.97) methods were extremely high.

  5. Human Arterial Ring Angiogenesis Assay.

    PubMed

    Seano, Giorgio; Primo, Luca

    2016-01-01

    In this chapter we describe a model of human angiogenesis where artery explants from umbilical cords are embedded in gel matrices and subsequently produce capillary-like structures. The human arterial ring (hAR) assay is an innovative system that enables three-dimensional (3D) and live studies of human angiogenesis. This ex vivo model has the advantage of recapitulating several steps of angiogenesis, including endothelial sprouting, migration, and differentiation into capillaries. Furthermore, it can be exploited for (1) identification of new genes regulating sprouting angiogenesis, (2) screening for pro- or anti-angiogenic drugs, (3) identification of biomarkers to monitor the efficacy of anti-angiogenic regimens, and (4) dynamic analysis of tumor microenvironmental effects on vessel formation.

  6. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  7. Fluorogenic assays for immediate confirmation of Escherichia coli.

    PubMed Central

    Feng, P C; Hartman, P A

    1982-01-01

    Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains). Images PMID:7049088

  8. Predictive assay for cancer targets

    NASA Astrophysics Data System (ADS)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  9. Predictive Assay For Cancer Targets

    SciTech Connect

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  10. Microdroplet chain array for cell migration assays.

    PubMed

    Ma, Yan; Pan, Jian-Zhang; Zhao, Shi-Ping; Lou, Qi; Zhu, Ying; Fang, Qun

    2016-11-29

    Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

  11. Development of forensic assay signatures for ebolaviruses.

    PubMed

    Song, Jian; Doggett, Norman; Wren, Melinda; Burr, Tom; Fenimore, P W; Hatcher, Eneida L; Bruno, William J; Li, Po-E; Stubben, Chris; Wolinsky, Murray

    2015-03-01

    Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

  12. Evaluating 6 ricin field detection assays.

    PubMed

    Slotved, Hans-Christian; Sparding, Nadja; Tanassi, Julia Tanas; Steenhard, Nina R; Heegaard, Niels H H

    2014-01-01

    This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.

  13. 46 CFR 105.01-3 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... COMMERCIAL FISHING VESSELS DISPENSING PETROLEUM PRODUCTS Administration § 105.01-3 Incorporation by reference..., Standard Test Method for Vapor Pressure of Petroleum Products (Reid Method), incorporation by...

  14. Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation

    PubMed Central

    Herceg, Zdenko

    2013-01-01

    Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the ‘normal’ epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing. PMID:23749751

  15. Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation.

    PubMed

    Herceg, Zdenko; Lambert, Marie-Pierre; van Veldhoven, Karin; Demetriou, Christiana; Vineis, Paolo; Smith, Martyn T; Straif, Kurt; Wild, Christopher P

    2013-09-01

    Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the 'normal' epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing.

  16. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  17. Rocking adhesion assay system to study adhesion and transendothelial migration of cancer cells.

    PubMed

    Bapu, Deepashree; Khadim, Munira; Brooks, Susan A

    2014-01-01

    Adhesion of metastatic cancer cells to the vascular endothelium of the target organs and their subsequent transendothelial migration is one of the critical, yet poorly understood, steps of the metastatic cascade. Conventionally, the mechanisms of this complex process have been studied using static adhesion systems or flow assay systems. Static assay systems are easy to set up and perform but do not mimic the physiological conditions of blood flow. Flow assays closely mimic physiological conditions of flow but are time consuming and require specialist equipment. In this chapter we describe the rocking adhesion system which incorporates the key advantages of both the static and flow assay systems and not only is easy to set up and perform but also mimics conditions of blood flow.

  18. Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

    PubMed

    Sun, Yanli; Sun, Yu; Lin, Guigao; Zhang, Rui; Zhang, Kuo; Xie, Jiehong; Wang, Lunan; Li, Jinming

    2012-10-01

    EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [(3) H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU(+) cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10-50 μM, the optimal incubation time 8-12 h and the proper volume of Click volume 100 μl for labeling 1 × 10(6) lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU(+) cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, -80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

  19. Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts

    SciTech Connect

    Snyder, R.D.; Matheson, D.W.

    1985-01-01

    An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

  20. Emissive sensors and devices incorporating these sensors

    SciTech Connect

    Swager, Timothy M; Zhang, Shi-Wei

    2013-02-05

    The present invention generally relates to luminescent and/or optically absorbing compositions and/or precursors to those compositions, including solid films incorporating these compositions/precursors, exhibiting increased luminescent lifetimes, quantum yields, enhanced stabilities and/or amplified emissions. The present invention also relates to sensors and methods for sensing analytes through luminescent and/or optically absorbing properties of these compositions and/or precursors. Examples of analytes detectable by the invention include electrophiles, alkylating agents, thionyl halides, and phosphate ester groups including phosphoryl halides, cyanides and thioates such as those found in certain chemical warfare agents. The present invention additionally relates to devices and methods for amplifying emissions, such as those produced using the above-described compositions and/or precursors, by incorporating the composition and/or precursor within a polymer having an energy migration pathway. In some cases, the compositions and/or precursors thereof include a compound capable of undergoing a cyclization reaction.

  1. Toxicity of sediment-incorporated drilling fluids

    SciTech Connect

    Clark, J.R.; Patrick, J.M.

    1987-01-01

    The 24, 96, or 168-h LC50s of four used drilling fluids or barite incorporated into sediment were determined in toxicity tests with lancelets (Branchiostoma caribaeum), a benthic chordate. The number of lancelets that did not burrow into contaminated sediments was used to calculate EC50s at the same times that LC50s were determined. Observations of the burrowing behavior allowed quantitation of effects after 24-h exposures to each of the drilling fluids whereas lancelet mortality was sufficient to calculate 24-h LC50s for only one drilling fluid. Drilling fluids were less toxic to lancelets when incorporated into sediments than to mysids (Mysidopsis bahia) or benthic invertebrate communities in water-column exposures.

  2. 49 CFR 572.180 - Incorporated materials.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...,” incorporated by reference in §§ 572.180(a)(2), and 572.181(a); (4) Society of Automotive Engineers (SAE...”; and, (5) SAE J1733 of 1994-12 “Sign Convention for Vehicle Crash Testing.” (b) The Director of the... Woodfield Road, Gaithersburg, MD 20879, telephone (301) 670-0090. (2) The SAE materials referred to...

  3. Improving techniques for clonogenic assays.

    PubMed

    Eliason, J F; Fekete, A; Odartchenko, N

    1984-01-01

    A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

  4. Benefits of oxygen incorporation in atomic laminates

    NASA Astrophysics Data System (ADS)

    Dahlqvist, Martin

    2016-04-01

    Atomic laminates such as MAX phases benefit from the addition of oxygen in many ways, from the formation of a protective oxide surface layer with self-healing capabilities when cracks form to the tuning of anisotropic conductivity. In this paper oxygen incorporation and vacancy formation in M 2AlC (M  =  Ti, V, Cr) MAX phases have been studied using first-principles calculations where the focus is on phase stability and electronic structure for different oxygen and/or vacancy configurations. Oxygen prefers different lattice sites depending on M-element and this can be correlated to the number of available non-bonding M d-electrons. In Ti2AlC, oxygen substitutes carbon while in Cr2AlC it is located interstitially within the Al-layer. I predict that oxygen incorporation in Ti2AlC stabilizes the material, which explains the experimentally observed 12.5 at% oxygen (x  =  0.5) in Ti2Al(C1-x O x ). In addition, it is also possible to use oxygen to stabilize the hypothetical Zr2AlC and Hf2AlC. Hence, oxygen incorporation may be beneficial in many ways. Not only can it make a material more stable, but it also can act as a reservoir for internal self-healing with shorter diffusion paths.

  5. Structural incorporation of As5+ into hematite.

    PubMed

    Bolanz, Ralph M; Wierzbicka-Wieczorek, Mária; Čaplovičová, Mária; Uhlík, Peter; Göttlicher, Jörg; Steininger, Ralph; Majzlan, Juraj

    2013-08-20

    Hematite (α-Fe2O3) is one of the most common iron oxides and a sink for the toxic metalloid arsenic. Arsenic can be immobilized by adsorption to the hematite surface; however, the incorporation of As in hematite was never seriously considered. In our study we present evidence that, besides adsorption, the incorporation of As into the hematite crystals can be of great relevance for As immobilization. With the coupling of nanoresolution techniques and X-ray absorption spectroscopy the presence of As (up to 1.9 wt %) within the hematite crystals could be demonstrated. The incorporated As(5+) displays a short-range order similar to angelellite-like clusters, epitaxially intergrown with hematite. Angelellite (Fe4As2O11), a triclinic iron arsenate with structural relations to hematite, can epitaxially intergrow along the (210) plane with the (0001) plane of hematite. This structural composite of hematite and angelellite-like clusters represents a new immobilization mechanism and potentially long-lasting storage facility for As(5+) by iron oxides.

  6. Use of luminescent Leptospira interrogans for enumeration in biological assays.

    PubMed

    Murray, Gerald L; King, Amy M; Srikram, Amporn; Sermswan, Rasana W; Adler, Ben

    2010-06-01

    Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10(4) leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R(2) = 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R(2) = 0.916, complement killing assay R(2) = 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.

  7. Assay development status report for total cyanide

    SciTech Connect

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN{sup {minus}} ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation.

  8. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    SciTech Connect

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  9. Sensitive field assays for water analysis

    SciTech Connect

    Douglas, W.L.

    1984-08-01

    The goal of the project is to develop a rapid, simple, and inexpensive dry-film assay device for detection of environmental contaminants using the compound geosmin as a model. Phase I activities centered upon the immunochemical reagents necessary for the assay, development of an enzyme-cycling system that makes possible detection of substances in the parts per billion (PPB) range or lower, and demonstration of how the Immuno-Replacement-Assay can be used to detect geosmin.

  10. Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

    PubMed

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

    2016-01-01

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  11. Magnetoresistive Sensors in Biological Assays

    NASA Astrophysics Data System (ADS)

    Tondra, Mark

    2010-03-01

    Magnetic beads or nanoparticles can be used as ``labels'' in biochemical assays by attaching the beads to the biospecies of interest using a bio-specific attachment. Once the labels are attached, they can be used to manipulate, capture, and detect the species to be analyzed. Magnetoresistive (MR) sensors may be used to detect and count these labels, and thus make an inference about the concentration of the species of interest. MR technology is especially promising for biosensor applications where making the detector small and integrated with related sample handling tools to form a ``lab-on-a-chip'' miniaturized system. The function of the MR sensors is to detect stray magnetic fields from the beads while they are exposed to a magnetic excitation field. Generally, the stray fields from beads and clusters of beads are complicated functions of geometry, so some care is required to relate the detected magnetic signal to the number and location of the bead labels. This presentation will begin with a broad overview of results from many groups working in this area. For convenience, the applications are divided into three categories, detection of: flowing magnetic beads, immobilized beads, and scanned samples. Next will be some discussion of how the choice of spintronic sensor technology might affect detection capabilities (AMR, GMR, TMR, Hall effect, etc). Then, challenges relating to integration of MR sensors into microfluidic products will be discussed. This is the focus of the presenter's current day-to-day work on developing and producing MR-based biosensors. And finally, a description of possible future avenues of study and development will be presented.

  12. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    PubMed

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  13. Manganese incorporation into ferroelectric lead titanate

    NASA Astrophysics Data System (ADS)

    Stoupin, Stanislav

    Substitution with 3d magnetic transition elements in ABO 3 ferroelectric perovskite host media is widely utilized to produce relaxor ferroelectrics. Many resulting solid solutions exhibit magnetoelectric properties affected by concentration levels of the introduced magnetic ions. For conventional material preparation techniques such as firing of mechanically mixed oxides, incorporation is often limited by 5 mol% concentration level. Doping at higher concentrations requires introduction of other substituents to compensate charge within the unit cell to promote formation of the perovskite phase. In contrast, molecular mixing of precursor materials at the initial phase of preparation procedure offers an advantage of achieving higher incorporation levels of the 3d elements without additional charge-compensating ions. Presented in this thesis is a new sol-gel procedure utilized for high level incorporation of 3d magnetic ions into ferroelectric lead titanate. The technique was applied to produce PbTi1-xMnxO 3 solid solution, a perovskite system promising for high degree of magnetoelectric coupling. Concentration dependent studies were performed to characterize structural, thermal, ferroelectric and magnetic properties of the material. The solubility limit of Mn has been found to be 20 mol% and the material remains tetragonally distorted. X-ray Absorption Spectroscopy confirms that local structural environment of Mn, Ti, and Pb is consistent with tetragonal symmetry of the unit cell. Increase in Mn concentration leads to reduction in melting point, broadening of the ferroelectric transition, reduction of the transition temperature and increase in dielectric constant of the material. At the solubility limit the system was found to be ferromagnetic below 50 K.

  14. Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

    PubMed Central

    Hansenová Maňásková, Silvie; Nazmi, Kamran; van ‘t Hof, Wim; van Belkum, Alex; Martin, Nathaniel I.; Bikker, Floris J.; van Wamel, Willem J. B.; Veerman, Enno C. I.

    2016-01-01

    The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall. PMID:26799839

  15. Parameters influencing zeolite incorporation in PDMS membranes

    SciTech Connect

    Vankelecom, I.F.J.; Scheppers, E.; Heus, R.; Uytterhoeven, J.B. )

    1994-11-24

    The incorporation of several types of zeolite in PDMS membranes is studied, by measuring the tensile strength, xylene sorption, and density of the membranes. The zeolite is shown to be involved in the cross-linking of the membrane. The interaction between the PDMS matrix and the zeolites results in reinforced membranes in the case of zeolite Y. The parameters influencing the dispersion of the zeolite in the membrane are investigated, as well as several aspects of the preparation method. Finally, the idea of cross-linking is applied to explain the results of water/ethanol pervaporation. 25 refs., 9 figs., 4 tabs.

  16. Incorporating social concerns in environmental impact assessments

    SciTech Connect

    Wolfe, A.K.

    1990-03-01

    Social impact assessments most often focus on the population-driven impacts of projects. Such impacts may be insignificant when compared with social structural impacts of complex, controversial projects. This set of impacts includes social disruption, social group formation, and stigma effects. The National Environmental Policy Act does not explicitly call for assessment of, and assessors often are reluctant to address, these complex issues. This paper discusses why such impacts are critical to assess and gives examples of how they have been incorporated into environmental assessment documents. 6 refs.

  17. Incorporation of noble metals into aerogels

    DOEpatents

    Hair, L.M.; Sanner, R.D.; Coronado, P.R.

    1998-12-22

    Aerogels or xerogels containing atomically dispersed noble metals for applications such as environmental remediation are disclosed. New noble metal precursors, such as Pt--Si or Pd(Si--P){sub 2}, have been created to bridge the incompatibility between noble metals and oxygen, followed by their incorporation into the aerogel or xerogel through sol-gel chemistry and processing. Applications include oxidation of hydrocarbons and reduction of nitrogen oxide species, complete oxidation of volatile organic carbon species, oxidative membranes for photocatalysis and partial oxidation for synthetic applications.

  18. Incorporation of noble metals into aerogels

    DOEpatents

    Hair, Lucy M.; Sanner, Robert D.; Coronado, Paul R.

    1998-01-01

    Aerogels or xerogels containing atomically dispersed noble metals for applications such environmental remediation. New noble metal precursors, such as Pt--Si or Pd(Si--P).sub.2, have been created to bridge the incompatibility between noble metals and oxygen, followed by their incorporation into the aerogel or xerogel through sol-gel chemistry and processing. Applications include oxidation of hydrocarbons and reduction of nitrogen oxide species, complete oxidation of volatile organic carbon species, oxidative membranes for photocatalysis and partial oxidation for synthetic applications.

  19. Radiation collimator and systems incorporating same

    DOEpatents

    Norman, Daren R.; Yoon, Woo Y.; Jones, James L.; Haskell, Kevin J.; Bennett, Brion D.; Tschaggeny, Charles W.; Jones, Warren F.

    2011-09-13

    A collimator including a housing having disposed therein a shield element surrounding a converter core in which a photon beam is generated from electrons emanating from a linear accelerator. A beam channeler longitudinally adjacent the shield element has a beam aperture therethrough coaxially aligned with, and of the same diameter as, an exit bore of the converter core. A larger entry bore in the converter core is coaxial with, and longitudinally separated from, the exit bore thereof. Systems incorporating the collimator are also disclosed.

  20. Incorporation of Nanosensors into Aerospace Vehicles

    NASA Technical Reports Server (NTRS)

    Medelius, Pedro J.

    2007-01-01

    Traditional sensors are too big and heavy for installation in space vehicles, including the Space Shuttle Orbiter as well as future manned and unmanned vehicles currently in the early design phase. Advances in nanotechnology have led to the availability of smaller and more accurate sensors. Multiple and redundant nanosensors can be used to conduct more accurate and comprehensive measurements in a space vehicle. Early planning can lead to the relatively easy incorporation of miniature sensors sharing power and communication lines, thus reducing the requirement for large amount of electrical and/or optical cabling.

  1. Incorporating opponent models into adversary search

    SciTech Connect

    Carmel, D.; Markovitch, S.

    1996-12-31

    This work presents a generalized theoretical framework that allows incorporation of opponent models into adversary search. We present the M* algorithm, a generalization of minimax that uses an arbitrary opponent model to simulate the opponent`s search. The opponent model is a recursive structure consisting of the opponent`s evaluation function and its model of the player. We demonstrate experimentally the potential benefit of using an opponent model. Pruning in M* is impossible in the general case. We prove a sufficient condition for pruning and present the {alpha}{beta}* algorithm which returns the M* value of a tree while searching only necessary branches.

  2. Valve system incorporating single failure protection logic

    DOEpatents

    Ryan, Rodger; Timmerman, Walter J. H.

    1980-01-01

    A valve system incorporating single failure protective logic. The system consists of a valve combination or composite valve which allows actuation or de-actuation of a device such as a hydraulic cylinder or other mechanism, integral with or separate from the valve assembly, by means of three independent input signals combined in a function commonly known as two-out-of-three logic. Using the input signals as independent and redundant actuation/de-actuation signals, a single signal failure, or failure of the corresponding valve or valve set, will neither prevent the desired action, nor cause the undesired action of the mechanism.

  3. A homogeneous fluorometric assay platform based on novel synthetic proteins

    SciTech Connect

    Vardar-Schara, Goenuel; Krab, Ivo M.; Yi, Guohua; Su, Wei Wen . E-mail: wsu@hawaii.edu

    2007-09-14

    Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step 'mix and read' noncompetitive homogeneous assay process, based on modulating the Foerster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications.

  4. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  5. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  6. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  7. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  8. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  9. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  10. Statistical inference for serial dilution assay data.

    PubMed

    Lee, M L; Whitmore, G A

    1999-12-01

    Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.

  11. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  12. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  13. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  14. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  15. Incorporated of tritiated water in fish

    SciTech Connect

    Sprous, D.G.; Fox, J.E.; Jackson, B.A.

    1989-01-01

    Tritiated water {sup 3}H{sub 2}O is routinely discharged into the environment near nuclear power plants and reactors. The radioactive water is rapidly equilibriated with cell water in the aquatic life forms. The purpose of this study was to determine the uptake of the radioactive hydrogen into the various lipid classes. Oak Ridge National Laboratory has been discharging tritiated water into White Oak Lake. Blue gill and mosquito fish from White Oak Lake were analyzed. An experimental fish tank with water having a specific activity of 1.2 {times} 10{sup 6} dpm of {sup 3}H/mL was set up. Mosquito fish were exposed to this level of radioactivity for thirty days. After this time the fish were lypolyzed an the lipids were extracted. The phospholipid fraction incorporated the greatest percentage of the radioactivity. Significant incorporation of activity was also seen in the triglyceride and cholesterol fractions. Phospholipids and cholesterol are important structural components of the cell, insuring persistence of the radioactivity in the organism. The long term effects are not known.

  16. Optoelectronic devices incorporating fluoropolymer compositions for protection

    DOEpatents

    Chen, Xuming; Chum, Pak-Wing S.; Howard, Kevin E.; Lopez, Leonardo C.; Sumner, William C.; Wu, Shaofu

    2015-12-22

    The fluoropolymer compositions of the present invention generally incorporate ingredients comprising one or more fluoropolymers, an ultraviolet light protection component (hereinafter UV protection component), and optionally one or more additional ingredients if desired. The UV protection component includes a combination of at least one hindered tertiary amine (HTA) compound having a certain structure and a weight average molecular weight of at least 1000. This tertiary amine is used in combination with at least one organic, UV light absorbing compound (UVLA compound) having a weight average molecular weight greater than 500. When the HTA compound and the UVLA compound are selected according to principles of the present invention, the UV protection component provides fluoropolymer compositions with significantly improved weatherability characteristics for protecting underlying materials, features, structures, components, and/or the like. In particular, fluoropolymer compositions incorporating the UV protection component of the present invention have unexpectedly improved ability to resist blackening, coloration, or other de gradation that may be caused by UV exposure. As a consequence, devices protected by these compositions would be expected to have dramatically improved service life. The compositions have a wide range of uses but are particularly useful for forming protective layers in optoelectronic devices.

  17. Incorporating Geospatial Technology into Teacher Professional Development

    NASA Astrophysics Data System (ADS)

    Sproles, E. A.; Songer, L.

    2009-12-01

    The need for students to think spatially and use geospatial technologies is becoming more critical as these tools and concepts are increasingly incorporated into a broad range of occupations and academic disciplines. Geospatial Teaching Across the Curriculum (Geo-STAC) is a collaborative program that provides high school teachers with mentored professional development workshops in geospatial thought and technology. The seminars, led by community college faculty, give high school teachers the ability to incorporate geospatial technology into coursework across the curriculum — in Science, Technology, Engineering, and Math (STEM) and non-STEM disciplines. Students participating in the hands-on lessons gain experience in web-based and desktop Geographic Information Systems (GIS). The goals of the workshop are for teachers to: (1) understand the importance of geospatial thinking; (2) learn how to employ geospatial thinking in each discipline; (3) learn about geospatial technologies; (4) develop a Web-based GIS lesson; and, (5) implement a Web-based GIS lesson. Additionally, Geo-STAC works with high school students so that they: (1) understand the importance of geospatial technologies and careers in future job markets; (2) learn how to use Web-based GIS to solve problems; and, (3) visit the community college GIS lab and experience using desktop GIS. Geo-STAC actively disseminates this collaborative model to colleges to community colleges and high schools across the country.

  18. Incorporating intelligence into structured radiology reports

    NASA Astrophysics Data System (ADS)

    Kahn, Charles E.

    2014-03-01

    The new standard for radiology reporting templates being developed through the Integrating the Healthcare Enterprise (IHE) and DICOM organizations defines the storage and exchange of reporting templates as Hypertext Markup Language version 5 (HTML5) documents. The use of HTML5 enables the incorporation of "dynamic HTML," in which documents can be altered in response to their content. HTML5 documents can employ JavaScript, the HTML Document Object Model (DOM), and external web services to create intelligent reporting templates. Several reporting templates were created to demonstrate the use of scripts to perform in-template calculations and decision support. For example, a template for adrenal CT was created to compute contrast washout percentage from input values of precontrast, dynamic postcontrast, and delayed adrenal nodule attenuation values; the washout value can used to classify an adrenal nodule as a benign cortical adenoma. Dynamic templates were developed to compute volumes and apply diagnostic criteria, such as those for determination of internal carotid artery stenosis. Although reporting systems need not use a web browser to render the templates or their contents, the use of JavaScript creates innumerable opportunities to construct highly sophisticated HTML5 reporting templates. This report demonstrates the ability to incorporate dynamic content to enhance the use of radiology reporting templates.

  19. Self-Incorporation of Coenzymes by Ribozymes

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

  20. Incorporating Experience Curves in Appliance Standards Analysis

    SciTech Connect

    Garbesi, Karina; Chan, Peter; Greenblatt, Jeffery; Kantner, Colleen; Lekov, Alex; Meyers, Stephen; Rosenquist, Gregory; Buskirk, Robert Van; Yang, Hung-Chia; Desroches, Louis-Benoit

    2011-10-31

    The technical analyses in support of U.S. energy conservation standards for residential appliances and commercial equipment have typically assumed that manufacturing costs and retail prices remain constant during the projected 30-year analysis period. There is, however, considerable evidence that this assumption does not reflect real market prices. Costs and prices generally fall in relation to cumulative production, a phenomenon known as experience and modeled by a fairly robust empirical experience curve. Using price data from the Bureau of Labor Statistics, and shipment data obtained as part of the standards analysis process, we present U.S. experience curves for room air conditioners, clothes dryers, central air conditioners, furnaces, and refrigerators and freezers. These allow us to develop more representative appliance price projections than the assumption-based approach of constant prices. These experience curves were incorporated into recent energy conservation standards for these products. The impact on the national modeling can be significant, often increasing the net present value of potential standard levels in the analysis. In some cases a previously cost-negative potential standard level demonstrates a benefit when incorporating experience. These results imply that past energy conservation standards analyses may have undervalued the economic benefits of potential standard levels.

  1. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  2. Paper disk assay for glycosaminoglycan sulfotransferases

    SciTech Connect

    Sugahara, K.; Ishii, T.; Yamashina, I.

    1987-11-01

    A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the (/sup 35/S)sulfate-labeled products are precipitated and then separated from a sulfate donor ((/sup 35/S)PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.

  3. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  4. Rapid fluorescence-based assay for radiosensitivity and chemosensitivity testing in mammalian cells in vitro

    SciTech Connect

    Begg, A.C.; Mooren, E.

    1989-02-01

    An efficient and rapid cytotoxicity assay has been developed, particularly for radiobiological studies, utilizing 96-well microtiter plates. Several days after treatment, cell numbers per well were measured by fluorescent intensity using an automatic reader after staining with the DNA specific dye Hoechst 33258. For radiobiological applications, a microtiter plate irradiation box was designed and built which allowed a variable number of wells (minimum 4, maximum 16) to be irradiated at one time. In this manner, complete dose-response curves could be obtained from one plate. The assay depends on the growth of surviving and untreated cells, and by appropriate choice of conditions (cell numbers plated, time of assay), cell survival curves for this quick fluorescence assay were in reasonable agreement with those from a clonogenic assay for cisplatin and X-ray-induced cell killing. The assay can span 1.5-2 decades of cell survival and is suitable for any cell line which grows as a monolayer. Radiobiological applications were tested using agents or conditions which modified radiation damage. Firstly, sublethal damage repair could be demonstrated in RIF1 mouse tumor cells by comparing the survival curve for a single X-ray dose with that for two fractions separated by 4 h. Secondly, incorporation of 5-iodo-2'-deoxyuridine into cellular DNA was shown to radiosensitize Chinese Hamster cells, with similar enhancement ratios obtained from the fluorescence and clonogenic assays. Thirdly, radiosensitization by cisplatin and radioprotection by cysteamine could be readily measured using the quick fluorescence assay. The ability to have multiple dose groups per plate makes it an efficient assay for both radiosensitivity and chemosensitivity testing.

  5. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity

    SciTech Connect

    Carmichael, J.; DeGraff, W.G.; Gazdar, A.F.; Minna, J.D.; Mitchell, J.B.

    1987-02-15

    Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D0 and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D0S were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (less than 4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

  6. Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.

    PubMed

    Rösslein, Matthias; Elliott, John T; Salit, Marc; Petersen, Elijah J; Hirsch, Cordula; Krug, Harald F; Wick, Peter

    2015-01-20

    An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.

  7. Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes.

    PubMed

    Lammers, Christoph; Hahn, Liljan E; Neumann, Heinz

    2014-08-18

    Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.

  8. Laboratory Assays for Epstein-Barr Virus-Related Disease

    PubMed Central

    Gulley, Margaret L.; Tang, Weihua

    2008-01-01

    Epstein-Barr virus (EBV) infects various cell types in a wide spectrum of benign and malignant diseases. Laboratory tests for EBV have improved and are increasingly used in diagnosis, prognosis, prediction, and prevention of diseases ranging from infectious mononucleosis to selected subtypes of lymphoma, sarcoma, and carcinoma. Indeed, the presence of EBV is among the most effective tumor markers supporting clinical management of cancer patients. In biopsies, localization of EBER transcripts by in situ hybridization remains the gold standard for identifying latent infection. Other RNA- and protein-based assays detect lytic viral replication and can distinguish carcinoma-derived from lymphocyte-derived EBV in saliva or nasopharyngeal brushings. Analysis of blood using EBV viral load and serology reflects disease status and risk of progression. This review summarizes prior research in the context of basic virologic principles to provide a rational strategy for applying and interpreting EBV tests in various clinical settings. Such assays have been incorporated into standard clinical practice in selected settings such as diagnosis of primary infection and management of patients with immune dysfunction or nasopharyngeal carcinoma. As novel therapies are developed that target virus-infected cells or overcome the adverse effects of infection, laboratory testing becomes even more critical for determining when intervention is appropriate and the extent to which it has succeeded. PMID:18556771

  9. INCORPORATING DYNAMIC 3D SIMULATION INTO PRA

    SciTech Connect

    Steven R Prescott; Curtis Smith

    2011-07-01

    Through continued advancement in computational resources, development that was previously done by trial and error production is now performed through computer simulation. These virtual physical representations have the potential to provide accurate and valid modeling results and are being used in many different technical fields. Risk assessment now has the opportunity to use 3D simulation to improve analysis results and insights, especially for external event analysis. By using simulations, the modeler only has to determine the likelihood of an event without having to also predict the results of that event. The 3D simulation automatically determines not only the outcome of the event, but when those failures occur. How can we effectively incorporate 3D simulation into traditional PRA? Most PRA plant modeling is made up of components with different failure modes, probabilities, and rates. Typically, these components are grouped into various systems and then are modeled together (in different combinations) as a “system” with logic structures to form fault trees. Applicable fault trees are combined through scenarios, typically represented by event tree models. Though this method gives us failure results for a given model, it has limitations when it comes to time-based dependencies or dependencies that are coupled to physical processes which may themselves be space- or time-dependent. Since, failures from a 3D simulation are naturally time related, they should be used in that manner. In our simulation approach, traditional static models are converted into an equivalent state diagram representation with start states, probabilistic driven movements between states and terminal states. As the state model is run repeatedly, it converges to the same results as the PRA model in cases where time-related factors are not important. In cases where timing considerations are important (e.g., when events are dependent upon each other), then the simulation approach will typically

  10. Development and Utilization of an Ex Vivo Bromodeoxyuridine Local Lymph Node Assay (LLNA) Protocol for Assessing Potential Chemical Sensitizers

    EPA Science Inventory

    The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]m...

  11. Choline incorporation by Schistosoma mansoni: distribution of choline metabolites during development and after sexual differentiation

    SciTech Connect

    Ancelin, M.L.; Torpier, G.; Vial, H.J.; Capron, A.

    1987-06-01

    Choline metabolism was investigated in Schistosoma mansoni during the main phases of its development, namely, schistosomula, 11- and 15-day-old worms, and adults. At the physiological choline concentration used in the assay (20 microM), betaine was, along with phosphatidylcholine, one of the most abundant choline metabolites, revealing considerable choline oxidation activity. Very little radioactivity was associated with CDP-choline, whereas a sustained incorporation into phosphocholine occurred. These results provide good evidence that CTP:phosphocholine cytidylyltransferase plays a regulatory role in the de novo pathway of phosphatidylcholine biosynthesis. During development, the incorporation of choline into its various metabolites was maximal in 11-day-old worms. At this stage, the oxidative pathway predominated over the Kennedy pathway, whereas at all other stages the de novo phosphatidylcholine biosynthesis was predominant. Furthermore, choline incorporation into betaine was much more important in the adult female worm than in the male, indicating a major difference in choline incorporation and distribution between the 2 sexes of the adult worms.

  12. Assessment of phospholipid deacylation-reacylation cycles by H/sub 2/ /sup 18/O incorporation

    SciTech Connect

    Kuwae, T.; Schmid, P.C.; Johnson, S.B.; Schmid, H.H.O.

    1987-05-01

    Quantitative estimation of metabolic turnover involving hydrolysis is possible with the use of H/sub 2/ /sup 18/O which readily equilibrates throughout subcellular compartments and thus obviates many problems associated with the use of exogenous metabolites. Incubation of cells in H/sub 2/ /sup 18/O results in the time-dependent incorporation of /sup 18/O into ester carbonyls of glycerophospholipids. Preparation of methyl esters by alkaline methanolysis preserves the carbonyl oxygen and hydrogenation prior to analysis by gas-chromatography/mass-spectrometry simplifies determination of /sup 18/O//sup 16/O ratios. Mouse peritoneal exudate cells incorporate /sup 18/O from H/sub 2/ /sup 18/O into acyl groups of phosphatidylcholine (PC) and phosphatidylinositol (PI), and smaller amounts into phosphatidylethanolamine (PE) and phosphatidylserine (PS). Acyl groups at the sn-1 position of PC and PE show about the same rate of turnover as those at the sn-2 position. Considering the route of /sup 18/O incorporation via free fatty acid derived through ester hydrolysis in 40% H/sub 2/ /sup 18/O, acyl turnover may exceed 20% per hour. Turnover of 2-O-acyl groups in the plasmalogen analog of PE, as assayed by /sup 18/O incorporation, is drastically lower than in diacylPE. This indicates either a much lower rate of hydrolysis, or reacylation of lysoplasmalogen by direct acyl transfer not involving free fatty acid.

  13. A preclinical assay for chemosensitivity in multiple myeloma

    PubMed Central

    Khin, Zayar P.; Ribeiro, Maria L. C.; Jacobson, Timothy; Hazlehurst, Lori; Perez, Lia; Baz, Rachid; Shain, Kenneth; Silva, Ariosto S.

    2013-01-01

    Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma (MM), an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating 3D extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used convert experimental data into dose-exposure-response "surfaces" which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical MM treatments, in 3 MM cell lines and 7 patient-derived primary MM cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology. Major Findings By designing an experimental platform with the specific intent of generating experimental parameters for a computational clinical model of personalized therapy in multiple myeloma, while taking in consideration the limitations of working with patient primary cells, and the need to incorporate elements of the tumor microenvironment, we have generated patient-individualized estimations of initial response and time to relapse

  14. Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide*

    PubMed Central

    Aquilini, Eleonora; Azevedo, Joana; Merino, Susana; Jimenez, Natalia; Tomás, Juan M.; Regué, Miguel

    2010-01-01

    The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to l-glycero-d-manno-heptopyranose II (l-glycero-α-d-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is d-glucosamine, whereas in other P. mirabilis strains, it corresponds to d-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of d-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase. PMID:20959463

  15. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  16. Incorporating LCA tools in integrated simulation environments

    SciTech Connect

    Pal, Vineeta; Papamichael, Konstantinos; Bourassa, Norman; Loffeld, John J.

    2001-02-01

    In this paper we address the issue of building data schema evolution in integrated simulation environments, as seen from the perspective of incorporating LCA tools within these environments. First we describe the key features of an integrated simulation environment designed for expandability, focusing on (a) the mechanism for the expansion of the integrated environment, and (b) its overall system architecture that allows processes and data to be added to the system without modifications or restructuring of existing code. We then focus on how the data schema allows the inclusion and maintenance of specialized construction objects bearing LCA data. Finally, we discuss various integration issues that arise from modeling capabilities and idiosyncrasies of individual simulation and analysis tools.

  17. Active containment systems incorporating modified pillared clays

    SciTech Connect

    Lundie, P. |; McLeod, N.

    1997-12-31

    The application of treatment technologies in active containment systems provides a more advanced and effective method for the remediation of contaminated sites. These treatment technologies can be applied in permeable reactive walls and/or funnel and gate systems. The application of modified pillared clays in active containment systems provides a mechanism for producing permeable reactive walls with versatile properties. These pillared clays are suitably modified to incorporate reactive intercalatants capable of reacting with both a broad range of organic pollutants of varying molecular size, polarity and reactivity. Heavy metals can be removed from contaminated water by conventional ion-exchange and other reactive processes within the clay structure. Complex contamination problems can be addressed by the application of more than one modified clay on a site specific basis. This paper briefly describes the active containment system and the structure/chemistry of the modified pillared clay technology, illustrating potential applications of the in-situ treatment process for contaminated site remediation.

  18. Incorporating Pharmacy Scholarship to Management Responsibilities

    PubMed Central

    Hertig, John B.; Weber, Robert J.

    2015-01-01

    Practice advancement demands innovation. Amidst professional change, pharmacy leaders have the opportunity to collaborate with colleagues to develop transformational ideas, implement these solutions, and share those successes with professionals around the state, country, and world. Scholarship, defined as contributing to the literature through publications, presentations, and other writings, is an ideal way to advance innovation within the profession. It is critical for pharmacy leaders to build scholarship into their professional workflow. Ensuring that successful projects are published or presented may translate into shared best practices. Many pharmacy leaders may find it difficult to participate in scholarship activities because of their busy schedules. This column serves to outline recommendations on how to effectively incorporate writing for publications, making presentations, and other scholarly work into the role of pharmacy leaders and managers. To reduce the barriers to scholarship, pharmacy leaders role can apply project management principles to their work and identify projects that otherwise would not be published and support their development. PMID:26823623

  19. Incorporating immigrant flows into microsimulation models.

    PubMed

    Duleep, Harriet Orcutt; Dowhan, Daniel J

    2008-01-01

    Building on the research on immigrant earnings reviewed in the first article of this series, "Research on Immigrant Earnings," the preceding article, "Adding Immigrants to Microsimulation Models," linked research results to various issues essential for incorporating immigrant earnings into microsimulation models. The discussions of that article were in terms of a closed system. That is, it examined a system in which immigrant earnings and emigration are forecast for a given population represented in the base sample in the microsimulation model. This article, the last in the series, addresses immigrant earnings projections for open systems--microsimulation models that include projections of future immigration. The article suggests a simple method to project future immigrants and their earnings. Including the future flow of immigrants in microsimulation models can dramatically affect the projected Social Security benefits of some groups.

  20. Radiation shielding materials and containers incorporating same

    DOEpatents

    Mirsky, Steven M.; Krill, Stephen J.; Murray, Alexander P.

    2005-11-01

    An improved radiation shielding material and storage systems for radioactive materials incorporating the same. The PYRolytic Uranium Compound ("PYRUC") shielding material is preferably formed by heat and/or pressure treatment of a precursor material comprising microspheres of a uranium compound, such as uranium dioxide or uranium carbide, and a suitable binder. The PYRUC shielding material provides improved radiation shielding, thermal characteristic, cost and ease of use in comparison with other shielding materials. The shielding material can be used to form containment systems, container vessels, shielding structures, and containment storage areas, all of which can be used to house radioactive waste. The preferred shielding system is in the form of a container for storage, transportation, and disposal of radioactive waste. In addition, improved methods for preparing uranium dioxide and uranium carbide microspheres for use in the radiation shielding materials are also provided.

  1. Radiation Shielding Materials and Containers Incorporating Same

    DOEpatents

    Mirsky, Steven M.; Krill, Stephen J.; and Murray, Alexander P.

    2005-11-01

    An improved radiation shielding material and storage systems for radioactive materials incorporating the same. The PYRolytic Uranium Compound (''PYRUC'') shielding material is preferably formed by heat and/or pressure treatment of a precursor material comprising microspheres of a uranium compound, such as uranium dioxide or uranium carbide, and a suitable binder. The PYRUC shielding material provides improved radiation shielding, thermal characteristic, cost and ease of use in comparison with other shielding materials. The shielding material can be used to form containment systems, container vessels, shielding structures, and containment storage areas, all of which can be used to house radioactive waste. The preferred shielding system is in the form of a container for storage, transportation, and disposal of radioactive waste. In addition, improved methods for preparing uranium dioxide and uranium carbide microspheres for use in the radiation shielding materials are also provided.

  2. [Effectiveness of incorporating a quality management system].

    PubMed

    Seki, Akira; Hankins, Raleigh W; Miya, Tetsumasa

    2010-01-01

    In 2003, the ISO 15189 international standardization program on the quality and competence of the clinical reference laboratory was introduced. To date, 46 facilities have committed themselves to providing a higher level of medical service by incorporating a quality management system (QMS) and acquiring accreditation. QMS is defined as "setting up a policy and goals pertaining to quality, and adopting an appropriate system," and is a scheme that includes all managerial and technical factors that can affect test results. Regarding the Health Sciences Research Institute Group, 4 facilities have previously received the accreditation described above, but in the process of implementing the QMS, a number of problems have been identified. Here, we report on the effectiveness of adopting such a QMS based on the results of employee questionnaires, internal audits, customer complaint analyses, and external audits by the Japan Accreditation Board for Conformity Assessment (JAB), the official inspection body for accreditation.

  3. Solar cells incorporating light harvesting arrays

    DOEpatents

    Lindsey, Jonathan S.; Meyer, Gerald J.

    2003-07-22

    A solar cell incorporates a light harvesting array that comprises: (a) a first substrate comprising a first electrode; and (b) a layer of light harvesting rods electrically coupled to the first electrode, each of the light harvesting rods comprising a polymer of Formula I: ##EQU1## wherein m is at least 1, and may be from two, three or four to 20 or more; X.sup.1 is a charge separation group (and preferably a porphyrinic macrocycle, which may be one ligand of a double-decker sandwich compound) having an excited-state of energy equal to or lower than that of X.sup.2 ; and X.sup.2 through X.sup.m+1 are chromophores (and again are preferably porphyrinic macrocycles).

  4. Solar cells incorporating light harvesting arrays

    DOEpatents

    Lindsey, Jonathan S.; Meyer, Gerald J.

    2002-01-01

    A solar cell incorporates a light harvesting array that comprises: (a) a first substrate comprising a first electrode; and (b) a layer of light harvesting rods electrically coupled to the first electrode, each of the light harvesting rods comprising a polymer of Formula I: X.sup.1.paren open-st.X.sup.m+1).sub.m (I) wherein m is at least 1, and may be from two, three or four to 20 or more; X.sup.1 is a charge separation group (and preferably a porphyrinic macrocycle, which may be one ligand of a double-decker sandwich compound) having an excited-state of energy equal to or lower than that of X.sup.2 ; and X.sup.2 through X.sup.m+1 are chromophores (and again are preferably porphyrinic macrocycles).

  5. Jay Carter Enterprises, Incorporated steam engine

    NASA Technical Reports Server (NTRS)

    1981-01-01

    The Small Community Solar Thermal Power Experiment (SCSE) selected an organic rankine cycle (ORC) engine driving a high speed permanent magnet alternator (PMA) as the baseline power conversion subsystem (PCS) design. The back-up conceptual PCS design is a steam engine driving an induction alternator delivering power directly to the grid. The development of the automotive reciprocating simple rankine cycle steam engine and how an engine of similar design might be incorporated into the SCSE is discussed. A description of the third generation automotive engine is included along with some preliminary test data. Tests were conducted with the third generation engine driving an induction alternator delivering power directly to the grid. The purpose of these tests is to further verify the effects of expander inlet temperature, input thermal power level, expansion ratio, and other parameters affecting engine performance to aid in the development of an SCSE PCS.

  6. Upgrading carbon dioxide by incorporation into heterocycles.

    PubMed

    Yu, Bing; He, Liang-Nian

    2015-01-01

    Carbon dioxide is commonly regarded as the primary greenhouse gas, but from a synthetic standpoint can be utilized as an alternative and sustainable C1 synthon in organic synthesis rather than a waste. This results in the production of organic carbonates, carboxylic acids, and derivatives. Recently, CO2 has emerged as an appealing tool for heterocycle synthesis under mild conditions without using stoichiometric amounts of organometallic reducing reagents. This Minireview summarizes recent advances on methodologies for CO2 incorporation into N-, O-, and C-nucleophiles to provide various heterocycles, including cyclic carbamates, benzoxazine-2-one, 4-hydroxyquinolin-2-one, quinazoline-2,4(1 H,3 H)-diones, benzimidazolones, α-alkylidene cyclic carbonate.

  7. Incorporating global components into ethics education.

    PubMed

    Wang, George; Thompson, Russell G

    2013-03-01

    Ethics is central to science and engineering. Young engineers need to be grounded in how corporate social responsibility principles can be applied to engineering organizations to better serve the broader community. This is crucial in times of climate change and ecological challenges where the vulnerable can be impacted by engineering activities. Taking a global perspective in ethics education will help ensure that scientists and engineers can make a more substantial contribution to development throughout the world. This paper presents the importance of incorporating the global and cross culture components in the ethic education. The authors bring up a question to educators on ethics education in science and engineering in the globalized world, and its importance, necessity, and impendency. The paper presents several methods for discussion that can be used to identify the differences in ethics standards and practices in different countries; enhance the student's knowledge of ethics in a global arena.

  8. SkinSensDB: a curated database for skin sensitization assays.

    PubMed

    Wang, Chia-Chi; Lin, Ying-Chi; Wang, Shan-Shan; Shih, Chieh; Lin, Yi-Hui; Tung, Chun-Wei

    2017-01-01

    Skin sensitization is an important toxicological endpoint for chemical hazard determination and safety assessment. Prediction of chemical skin sensitizer had traditionally relied on data from rodent models. The development of the adverse outcome pathway (AOP) and associated alternative in vitro assays have reshaped the assessment of skin sensitizers. The integration of multiple assays as key events in the AOP has been shown to have improved prediction performance. Current computational models to predict skin sensitization mainly based on in vivo assays without incorporating alternative in vitro assays. However, there are few freely available databases integrating both the in vivo and the in vitro skin sensitization assays for development of AOP-based skin sensitization prediction models. To facilitate the development of AOP-based prediction models, a skin sensitization database named SkinSensDB has been constructed by curating data from published AOP-related assays. In addition to providing datasets for developing computational models, SkinSensDB is equipped with browsing and search tools which enable the assessment of new compounds for their skin sensitization potentials based on data from structurally similar compounds. SkinSensDB is publicly available at http://cwtung.kmu.edu.tw/skinsensdb.

  9. The evolution of chemotaxis assays from static models to physiologically relevant platforms.

    PubMed

    Toetsch, Stephanie; Olwell, Peter; Prina-Mello, Adriele; Volkov, Yuri

    2009-02-01

    The role of chemotactic gradients in the immunological response is an area which elicits a lot of attention due to its impact on the outcome of the inflammatory process. Consequently there are numerous standard in vitro designs which attempt to mimic chemotactic gradients, albeit in static conditions, and with no control over the concentration of the chemokine gradient. In recent times the design of the standard chemotaxis assay has incorporated modern microfluidic platforms, computer controlled flow devices and cell tracking software. Assays under fluid flow which use biochips have provided data which highlight the importance of shear stress on cell attachment and migration towards a chemokine gradient. However, the in vivo environment is far more complex in comparison to conventional cell assay chambers. The designs of biochips are therefore in constant flux as advances in technology permit ever greater imitations of in vivo conditions. Researchers are focused on designing a generation of new biochips and enhancing the physiological relevance of the current assays. The challenge is to combine a shear flow with a 3D scaffold containing the endothelial layer and permitting a natural diffusion of chemokines through a tissue-like basal matrix. Here we review the latest range of chemotaxis assays and assess the innovative features of their designs which enable them to better imitate the in vivo environment. We also present some alternative designs that were initially employed in tissue engineering which could potentially be used in the establishment of novel chemotaxis assays.

  10. Incorporation of RAM techniques into simulation modeling

    SciTech Connect

    Nelson, S.C. Jr.; Haire, M.J.; Schryver, J.C.

    1995-07-01

    This work concludes that reliability, availability, and maintainability (RAM) analytical techniques can be incorporated into computer network simulation modeling to yield an important new analytical tool. This paper describes the incorporation of failure and repair information into network simulation to build a stochastic computer model represents the RAM Performance of two vehicles being developed for the US Army: The Advanced Field Artillery System (AFAS) and the Future Armored Resupply Vehicle (FARV). The AFAS is the US Army`s next generation self-propelled cannon artillery system. The FARV is a resupply vehicle for the AFAS. Both vehicles utilize automation technologies to improve the operational performance of the vehicles and reduce manpower. The network simulation model used in this work is task based. The model programmed in this application requirements a typical battle mission and the failures and repairs that occur during that battle. Each task that the FARV performs--upload, travel to the AFAS, refuel, perform tactical/survivability moves, return to logistic resupply, etc.--is modeled. Such a model reproduces a model reproduces operational phenomena (e.g., failures and repairs) that are likely to occur in actual performance. Simulation tasks are modeled as discrete chronological steps; after the completion of each task decisions are programmed that determine the next path to be followed. The result is a complex logic diagram or network. The network simulation model is developed within a hierarchy of vehicle systems, subsystems, and equipment and includes failure management subnetworks. RAM information and other performance measures are collected which have impact on design requirements. Design changes are evaluated through ``what if`` questions, sensitivity studies, and battle scenario changes.

  11. Methods for threshold determination in multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  12. Nondestructive assay confirmatory assessment experiments: mixed oxide

    SciTech Connect

    Lemming, J.F.

    1980-04-30

    The confirmatory assessment experiments demonstrate traceable nondestructive assay (NDA) measurements of plutonium in mixed oxide powder using commercially available spontaneous-fission assay systems. The experiments illustrate two major concepts: the production of calibration materials using calorimetric assay, and the use of paired measurements for measurement assurance. Two batches of well-characterized mixed oxide powder were used to establish the random and systematic error components. The major components of an NDA measurement assurance technique to establish and maintain traceability are identified and their functions are demonstrated. 20 refs., 10 figs., 10 tabs.

  13. Assuring reliable performance of antibiotic assay media.

    PubMed

    Freeman, K A; Johnson, D P; Garth, M A

    1977-11-01

    The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.

  14. Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria.

    PubMed

    Anastassopoulos, Elias

    2006-10-01

    An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.

  15. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  16. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  17. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  18. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  19. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  20. 27 CFR 21.6 - Incorporations by reference.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Analytical Chemists (13th Edition 1980)” (AOAC) is incorporated by reference in this part. This incorporation... the Association of Official Analytical Chemists, 11 North 19th Street, Suite 210, Arlington,...

  1. Incorporation of pollution prevention principles in environmental methods

    SciTech Connect

    Erickson, M.D.; Alvarado, J.S.; Lu, C.-S.; Peterson, D.P.; Silzer, J.

    1996-07-01

    The principles of pollution prevention (P2) have not been sufficiently incorporated into analytical methods. In this paper, we focus on the needs for and the benefits of incorporating the principles of P2 into environmental analytical methods.

  2. Cooperative cell invasion: matrix metalloproteinase–mediated incorporation between cells

    PubMed Central

    Mitchell, Camilla B.; O’Neill, Geraldine M.

    2016-01-01

    Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion. PMID:27605703

  3. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  4. FACS binding assay for analysing GDNF interactions.

    PubMed

    Quintino, Luís; Baudet, Aurélie; Larsson, Jonas; Lundberg, Cecilia

    2013-08-15

    Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

  5. Linearization of the bradford protein assay.

    PubMed

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  6. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  7. BIOMARKER ASSAYS IN NIPPLE APIRATE FLUID

    EPA Science Inventory

    ABSTRACT

    The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA and performing protein electrophoresis on nipple aspirate fluid was explored. ...

  8. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  9. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  10. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Matter incorporated by reference. 385.4 Section 385.4 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by...

  11. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  12. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Matter incorporated by reference. 385.4 Section 385.4 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by...

  13. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Matter incorporated by reference. 385.4 Section... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by reference. Part 385 includes references to certain matter or materials, as listed in paragraph (b) of this...

  14. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  15. 49 CFR 384.107 - Matter incorporated by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Matter incorporated by reference. 384.107 Section... COMPLIANCE WITH COMMERCIAL DRIVER'S LICENSE PROGRAM General § 384.107 Matter incorporated by reference. (a) Incorporation by reference. This part includes references to certain matter or materials. The text of...

  16. 49 CFR 385.4 - Matter incorporated by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Matter incorporated by reference. 385.4 Section... FITNESS PROCEDURES General § 385.4 Matter incorporated by reference. (a) Incorporation by reference. Part 385 includes references to certain matter or materials, as listed in paragraph (b) of this...

  17. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  18. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  19. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  20. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  1. 14 CFR 25.5 - Incorporations by reference.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES General § 25.5 Incorporations by reference. (a) The materials listed in this section are incorporated by reference in the corresponding sections noted. These...(a) and 1 CFR part 51. These materials are incorporated as they exist on the date of the...

  2. 46 CFR 107.115 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Incorporation by reference. 107.115 Section 107.115 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS INSPECTION AND CERTIFICATION General § 107.115 Incorporation by reference. (a) The standards referred to in this subchapter are incorporated by...

  3. 40 CFR 282.2 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    .... (a) Material listed as incorporated by reference in part 282 was approved for incorporation by.... Material is incorporated as it exists on the date of the approval, and notice of any change in the material... Courtland St., NE, Atlanta, GA 30365. (5) Region 5 (Illinois, Indiana, Michigan, Minnesota, Ohio,...

  4. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  5. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  6. 43 CFR 34.7 - Incorporation by operation of law.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 1 2014-10-01 2014-10-01 false Incorporation by operation of law. 34.7... OPPORTUNITY DURING CONSTRUCTION AND OPERATION OF THE ALASKA NATURAL GAS TRANSPORTATION SYSTEM § 34.7 Incorporation by operation of law. (a) The Equal Opportunity Clause shall be deemed incorporated into...

  7. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  8. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  9. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  10. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  11. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  12. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  13. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  14. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  15. 46 CFR 160.077-5 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS... Incorporation by reference. (a) Certain materials are incorporated by reference into this subpart with the..., “Material Approved for Incorporation by Reference,” which appears in the Finding Aids section of this...

  16. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  17. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  18. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  19. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  20. 46 CFR 109.105 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Incorporation by reference. 109.105 Section 109.105 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS OPERATIONS General § 109.105 Incorporation by reference. (a) Certain material is incorporated by...

  1. 46 CFR 38.01-3 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Incorporation by reference. 38.01-3 Section 38.01-3....01-3 Incorporation by reference. (a) Certain standards and specifications are incorporated by... ASME Boiler and Pressure Vessel Code Section V, Nondestructive Examination (1986)...

  2. 48 CFR 1952.102-2 - Incorporation in full text.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Incorporation in full text... Clauses 1952.102-2 Incorporation in full text. All IAAR provisions and clauses shall be incorporated in solicitations and/or contracts in full text....

  3. 48 CFR 2852.102-270 - Incorporation in full text.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Incorporation in full text... 2852.102-270 Incorporation in full text. JAR provisions or clauses shall be incorporated in solicitations and contracts in full text....

  4. 75 FR 17737 - Industrial Economics, Incorporated; Transfer of Data

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-07

    ... AGENCY Industrial Economics, Incorporated; Transfer of Data AGENCY: Environmental Protection Agency (EPA... Economics, Incorporated in accordance with 40 CFR 2.307(h)(3) and 2.308(i)(2). Industrial Economics... enable Industrial Economics, Incorporated to fulfill the obligations of the contract. DATES:...

  5. Identification of an amphotericin B resistant strain of Candida albicans using a rapid 3H-glucose incorporation microassay.

    PubMed

    Sweeney, J F; Greene, J N; Hiemenz, J W; Wei, S; Rosemurgy, A S; Djeu, J Y

    1996-11-01

    Using a 3H-glucose incorporation assay, antifungal sensitivity testing undertaken on an isolate of Candida albicans cultured from the blood of a bone marrow transplant patient documented resistance to amphotericin B but sensitivity to fluconazole and itraconazole. Information obtained from in vitro antifungal sensitivity testing can be used to direct in vivo antifungal therapy. Widespread application of standardized in vitro antifungal sensitivity testing is needed.

  6. Incorporation of OSI-7836 into DNA of Calu-6 and H460 xenograft tumors.

    PubMed

    Richardson, Frank; Black, Chris; Richardson, Katherine; Franks, April; Wells, Edward; Karimi, Susan; Sennello, Gina; Hart, Karen; Meyer, Denny; Emerson, David; Brown, Eric; LeRay, Jeremy; Nilsson, Christy; Tomkinson, Blake; Bendele, Raymond

    2005-03-01

    OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during

  7. Electrochemical Assay of Gold-Plating Solutions

    NASA Technical Reports Server (NTRS)

    Chiodo, R.

    1982-01-01

    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  8. Optical assay for biotechnology and clinical diagnosis.

    PubMed

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  9. Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth

    PubMed Central

    Caballero, Iván; Colmenarejo, Gonzalo; Sanz, Laura M.; Álvarez-Ruiz, Emilio; Gamo, Francisco-Javier; Cid, Concepción

    2016-01-01

    The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [3H]hypoxanthine incorporation assay is considered the “gold standard” assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum. However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [3H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [3H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method. PMID:27458216

  10. Incorporating Student Activities into Climate Change Education

    NASA Astrophysics Data System (ADS)

    Steele, H.; Kelly, K.; Klein, D.; Cadavid, A. C.

    2013-12-01

    Under a NASA grant, Mathematical and Geospatial Pathways to Climate Change Education, students at California State University, Northridge integrated Geographic Information Systems (GIS), remote sensing, satellite data technologies, and climate modelling into the study of global climate change under a Pathway for studying the Mathematics of Climate Change (PMCC). The PMCC, which is an interdisciplinary option within the BS in Applied Mathematical Sciences, consists of courses offered by the departments of Mathematics, Physics, and Geography and is designed to prepare students for careers and Ph.D. programs in technical fields relevant to global climate change. Under this option students are exposed to the science, mathematics, and applications of climate change science through a variety of methods including hands-on experience with computer modeling and image processing software. In the Geography component of the program, ESRI's ArcGIS and ERDAS Imagine mapping, spatial analysis and image processing software were used to explore NASA satellite data to examine the earth's atmosphere, hydrosphere and biosphere in areas that are affected by climate change or affect climate. These technology tools were incorporated into climate change and remote sensing courses to enhance students' knowledge and understanding of climate change through hands-on application of image processing techniques to NASA data. Several sets of exercises were developed with specific learning objectives in mind. These were (1) to increase student understanding of climate change and climate change processes; (2) to develop student skills in understanding, downloading and processing satellite data; (3) to teach remote sensing technology and GIS through applications to climate change; (4) to expose students to climate data and methods they can apply to solve real world problems and incorporate in future research projects. In the Math and Physics components of the course, students learned about

  11. Protein Crystal Growth Dynamics and Impurity Incorporation

    NASA Technical Reports Server (NTRS)

    Chernov, Alex A.; Thomas, Bill

    2000-01-01

    The general concepts and theories of crystal growth are proven to work for biomolecular crystallization. This allowed us to extract basic parameters controlling growth kinetics - free surface energy, alpha, and kinetic coefficient, beta, for steps. Surface energy per molecular site in thermal units, alpha(omega)(sup 2/3)/kT approx. = 1, is close to the one for inorganic crystals in solution (omega is the specific molecular volume, T is the temperature). Entropic restrictions on incorporation of biomolecules into the lattice reduce the incorporation rate, beta, by a factor of 10(exp 2) - 10(exp 3) relative to inorganic crystals. A dehydration barrier of approx. 18kcal/mol may explain approx. 10(exp -6) times difference between frequencies of adding a molecule to the lattice and Brownian attempts to do so. The latter was obtained from AFM measurements of step and kink growth rates on orthorhombic lysozyme. Protein and many inorganic crystals typically do not belong to the Kossel type, thus requiring a theory to account for inequivalent molecular positions within its unit cell. Orthorhombic lysozyme will serve as an example of how to develop such a theory. Factors deteriorating crystal quality - stress and strain, mosaicity, molecular disorder - will be reviewed with emphasis on impurities. Dimers in ferritin and lysozyme and acetylated lysozyme, are microheterogeneous i.e. nearly isomorphic impurities that are shown to be preferentially trapped by tetragonal lysozyme and ferritin crystals, respectively. The distribution coefficient, K defined as a ratio of the (impurity/protein) ratios in crystal and in solution is a measure of trapping. For acetylated lysoyzme, K = 2.15 or, 3.42 for differently acetylated forms, is independent of both the impurity and the crystallizing protein concentration. The reason is that impurity flux to the surface is constant while the growth rate rises with supersaturation. About 3 times lower dimer concentration in space grown ferritin and

  12. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  13. In vitro cell migration and invasion assays.

    PubMed

    Kramer, Nina; Walzl, Angelika; Unger, Christine; Rosner, Margit; Krupitza, Georg; Hengstschläger, Markus; Dolznig, Helmut

    2013-01-01

    Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

  14. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  15. Operational and Regulatory Performance of Waste Crate Assay Systems at RFETS

    SciTech Connect

    Clapham, M. J.; Franco, J.; Simpson, A.; Santo, J.; Menlove, H. O.; Durel, F. M.

    2003-02-27

    As Rocky Flats Environmental Technology Site (RFETS) approaches its closure target of 2006 emphasis for Non-Destructive Assay (NDA) has shifted from small waste package assay systems towards larger systems that are designed to accommodate Standard Waste Boxes (SWB) and larger Low Level Waste (LLW) containers. To this end, Kaiser Hill, with the support of BNFL Instruments, Inc. (BII) and Los Alamos National Laboratory (LANL), has recently deployed two new crate assay systems. These systems provide the capacity to meet the assay requirements associated with the Deactivation and Decommissioning (D&D) at RFETS. The Super High Efficiency Neutron Coincidence Counting System (SuperHENC) was designed and fabricated as a collaborative effort between RFETS, LANL and BII. The purpose of this counter is to provide a WIPP certified assay capability for SWBs with a sensitivity that allows for TRU/LLW sorting. The SuperHENC has been in operation since early 2001. The BII Multi-Purpose Crate Counter (MPCC) is based on the Imaging Passive Active Neutron (IPAN) technology. This counter was designed to provide diverse capacity for WIPP certified assay of SWBs and to provide assay capability for larger LLW crates that are generated at RFETS. The MPCC has been in operation since early 2002. In order to meet the requirement for measurement of the WIPP tracked radionuclides, both systems incorporate a BII Gamma Energy Analysis sub-system. The unique Energy Times Attenuation (ETA) method is used to provide isotopic mass fractions for diverse waste streams. These systems were the first, and at this time the only, waste crate assay systems that have achieved WIPP certification. This represents a significant achievement given that the performance criteria applied to the measurements of large crates is identical to the criteria for 55-gallon (208 liter) drums. They are now both fully operational at RFETS and continue to successfully support the site closure mission.

  16. Operational and regulatory performance of waste crate assay systems at RFETS.

    SciTech Connect

    Clapham, M.; Franco, J. B.; Simpson, A.; Santo, J.; Menlove, Howard O.; Durel, F. M.

    2003-01-01

    As Rocky Flats Environmental Technology Site (RFETS) approaches its closure target of 2006 emphasis for Non-Destructive Assay (NDA) has shifted from small waste package assay systems towards larger systems that are designed to accommodate Standard Waste Boxes (SWB) and larger Low Level Waste (LLW) containers. To this end, Kaiser Hill, with the support of BNFL Instruments, Inc . (BIn) and Los Alamos National Laboratory (LANL), has recently deployed two new crate assay systems . These systems provide the capacity to meet the assay requirements associated with the Deactivation and Decommissioning (D&D) at RFETS . The Super High Efficiency Neutron Coincidence Counting System (SuperHENC) was designed and fabricated as a collaborative effort between RFETS, LANL and BII. The purpose of this counter is to provide a WIPP certified assay capability for SWBs with a sensitivity that allows for TRU/LLW sorting. The SuperHENC has been in operation since early 2001 . The BII Mu1ti-Purpose Crate Counter (MPCC) is based on the Imaging Passive Active Neutron (IPANTM) technology. This counter was designed to provide diverse capacity for WIPP certified assay of SWBs and to provide assay capability for larger LLW crates that are generated at RFETS. The MPCC h as been in operation since early 2002 . In order to meet the requirement for measurement of the WIPP tracked radionuclides, both systems incorporate a BII Gamma Energy Analysis sub-system . The unique Energy Times . Attenuation (ETA) method is used to provide isotopic mass fractions for diverse wastes treams: These systems were the first, and at this time the only, waste crate assay systems that have achieved WIPP certification. This represents a significant achievement given that the performance criteria applied to the measurements of large crates is identical to the criteria for 55-gallon (208 liter) drums . They are now both fully operational at RFETS and continue to successfully support the site closure mission .

  17. Utilization of fluorogenic assay for rapid detection of Escherichia coli in acidic fruit juice.

    PubMed

    Pao, Steven; Davis, Craig L; Friedrich, Loretta M; Parish, Mickey E

    2002-12-01

    This study was undertaken to investigate interference by acids commonly found in fruit juice in Escherichia coli assays involving the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) as a fluorogenic substrate for enzyme reaction. Fluorescence intensity was negatively correlated (P < 0.001) with the volume of fresh citrus juice tested by the lauryl tryptose broth (LST)-MUG assay, and the permissible sample sizes were limited to 0.3 and 0.5 ml for fresh citrus juices with pHs of 3.3 and 3.9, respectively. In addition, false-negative results were visually observed under UV light when the E*Colite assay was used to test large volumes (5 to 10 ml per test) of fresh citrus juice or when the test broth used for the LST-MUG assay was supplemented with citric, malic, or tartaric acid at 2 to 4 g/liter. These results suggest that the size and pH of acidic samples should be controlled in MUG-based fluorogenic assays. The inhibitory effect on fluorescence was due to high acidity, which reduces fluorescence from 4-methylumbelliferone. Buffering improved the assays. When sodium bicarbonate was incorporated in the enrichment broth at 10 g/liter, the permissible sample sizes for fresh grapefruit juice (pH 3.1) increased from 0.3 to 1 ml for the LST-MUG (with 9.9 ml of broth) assay and from 3 to 10 ml for the E*Colite (with 99 ml of broth) assay.

  18. Incorporation of salinity in Water Availability Modeling

    NASA Astrophysics Data System (ADS)

    Wurbs, Ralph A.; Lee, Chihun

    2011-10-01

    SummaryNatural salt pollution from geologic formations in the upper watersheds of several large river basins in the Southwestern United States severely constrains the use of otherwise available major water supply sources. The Water Rights Analysis Package modeling system has been routinely applied in Texas since the late 1990s in regional and statewide planning studies and administration of the state's water rights permit system, but without consideration of water quality. The modeling system was recently expanded to incorporate salinity considerations in assessments of river/reservoir system capabilities for supplying water for environmental, municipal, agricultural, and industrial needs. Salinity loads and concentrations are tracked through systems of river reaches and reservoirs to develop concentration frequency statistics that augment flow frequency and water supply reliability metrics at pertinent locations for alternative water management strategies. Flexible generalized capabilities are developed for using limited observed salinity data to model highly variable concentrations imposed upon complex river regulation infrastructure and institutional water allocation/management practices.

  19. Incorporating routing into reservoir planning optimization models

    NASA Astrophysics Data System (ADS)

    Zmijewski, Nicholas; Wörman, Anders; Bottacin-Busolin, Andrea

    2015-04-01

    To achieve the best overall operation result in a reservoir network, optimization models are used. For larger reservoir networks the computational cost increases, making simplification of the hydrodynamic description necessary. In-accuracy in flow prediction can be related to an incurred sub-optimality in production planning. Flow behavior in a management optimization model is often described using a constant time-lag model. A simplified hydraulic model was used, describing the stream flow in a reservoir network for short term production planning of a case-study reservoir network (Dalälven River). In this study, the importance of incorporating hydrodynamic wave diffusion for optimized hydropower production planning in a regulated water system was examined, comparing the kinematic-wave model to the constant time-lag. The receding horizon optimization procedure was applied, emulating the data-assimilation procedure present in modern operations. Power production was shown to deviate from the planned production while considering a single time-lag, as a function of the stream description. The simplification of using a constant time-lag could be considered acceptable for streams characterized by high Peclet number. Examining the effect of the effect of the length of the decision time-step demonstrated the importance of high frequency data assimilation for streams characterized by low Peclet numbers. Further, it was shown that the variability in flow becomes more ordered as a result of management and that the Peclet number contributes to that goal.

  20. Incorporating Ecosystem Services into Community-level ...

    EPA Pesticide Factsheets

    EPA’s Office of Research and Development’s Sustainable and Healthy Communities Research Program is developing tools and approaches to incorporate ecosystem goods and services concepts into community-level decision-making. The San Juan Community Study is one of a series of coordinated community studies, which also include Mobile Bay, AL, Great Lakes Areas of Concern, and the Pacific Northwest. Common elements across the community studies include a focus on watershed management and national estuary programs (National Estuary Program, National Estuarine Research Reserve System). San Juan, Puerto Rico, is unique from the other community studies in that it is located in a highly urbanized watershed integrated with a number of freshwater and coastal ecosystems. The San Juan Community Study will explore linkages between watershed management decisions (e.g., dredging canals, restoration of mangrove buffers, sewage discharge interventions, climate adaptive strategies) targeting priority stressors (e.g., nutrients, contaminants, and pathogens; aquatic debris; habitat loss; modified hydrology and water circulation; sea level rise; storm intensity and frequency) effecting the condition of ecosystems (e.g., estuarine habitats and fish, as well as the connected terrestrial and coastal ecosystems), associated ecosystem goods and services (e.g., tourism and recreation, fishing, nutrient & sediment retention, contaminant processing, carbon sequestration, flood protection),

  1. Incorporating flow into practice and performance.

    PubMed

    Kirchner, Joann Marie

    2011-01-01

    In the music profession, individuals often work under stress filled conditions. This is especially true for individuals making their living as performing musicians. Musical performance anxiety has been well documented in both students and professionals. For some, the experience may lead to a termination of what might otherwise remain a successful performing career. Humans are susceptible to anxiety and so the phenomenon of musical performance anxiety is not likely to disappear. Learning how to effectively deal with musical performance anxiety is paramount for those in the performing arts. Entering a state of flow, in which there is total absorption in an activity, allows for the possiblity of any ensuing anxiety to become facilitative, rather than debilitative. This article will discuss several characteristics of flow, as defined by Mihalyi Csikszentmihalyi, and provide practical applications for musical practice and performance in an attempt to counterbalance musical performance anxiety. Musicians will benefit from a closer examination of the elements of flow and means of incorporating these elements into practice and performance.

  2. Incorporating Hypnosis into Pediatric Clinical Encounters

    PubMed Central

    Pendergrast, Robert A.

    2017-01-01

    Increasing numbers of licensed health professionals who care for children have been trained in clinical hypnosis. The evidence base for the safety and efficacy of this therapeutic approach in a wide variety of conditions is also growing. Pediatricians and other health professionals who have received training may wish to apply these skills in appropriate clinical scenarios but still may be unsure of the practical matters of how to incorporate this skill-set into day to day practice. Moreover, the practical application of such skills will take very different forms depending on the practice setting, types of acute or chronic conditions, patient and family preferences, and the developmental stages of the child or teen. This article reviews the application of pediatric clinical hypnosis skills by describing the use of hypnotic language outside of formal trance induction, by describing natural trance states that occur in children and teens in healthcare settings, and by describing the process of planning a clinical hypnosis encounter. It is assumed that this article does not constitute training in hypnosis or qualify its readers for the application of such skills; rather, it may serve as a practical guide for those professionals who have been so trained, and may serve to inform other professionals what to expect when referring a patient for hypnotherapy. The reader is referred to specific training opportunities and organizations. PMID:28300761

  3. Incorporating climate change into systematic conservation planning

    USGS Publications Warehouse

    Groves, Craig R.; Game, Edward T.; Anderson, Mark G.; Cross, Molly; Enquist, Carolyn; Ferdana, Zach; Girvetz, Evan; Gondor, Anne; Hall, Kimberly R.; Higgins, Jonathan; Marshall, Rob; Popper, Ken; Schill, Steve; Shafer, Sarah L.

    2012-01-01

    The principles of systematic conservation planning are now widely used by governments and non-government organizations alike to develop biodiversity conservation plans for countries, states, regions, and ecoregions. Many of the species and ecosystems these plans were designed to conserve are now being affected by climate change, and there is a critical need to incorporate new and complementary approaches into these plans that will aid species and ecosystems in adjusting to potential climate change impacts. We propose five approaches to climate change adaptation that can be integrated into existing or new biodiversity conservation plans: (1) conserving the geophysical stage, (2) protecting climatic refugia, (3) enhancing regional connectivity, (4) sustaining ecosystem process and function, and (5) capitalizing on opportunities emerging in response to climate change. We discuss both key assumptions behind each approach and the trade-offs involved in using the approach for conservation planning. We also summarize additional data beyond those typically used in systematic conservation plans required to implement these approaches. A major strength of these approaches is that they are largely robust to the uncertainty in how climate impacts may manifest in any given region.

  4. Incorporating infiltration modelling in urban flood management

    NASA Astrophysics Data System (ADS)

    Jumadar, A. S.; Pathirana, A.; Gersonius, B.; Zevenbergen, C.

    2008-06-01

    Increasing frequency and intensity of flood events in urban areas can be linked to increase in impervious area due to urbanization, exacerbated by climate change. The established approach of conveying storm water by conventional drainage systems has contributed to magnification of runoff volume and peak flows beyond those of undeveloped catchments. Furthermore, the continuous upgrading of such conventional systems is costly and unsustainable in the long term. Sustainable drainage systems aim at addressing the adverse effects associated with conventional systems, by mimicking the natural drainage processes, encouraging infiltration and storage of storm water. In this study we model one of the key components of SuDS, the infiltration basins, in order to assert the benefits of the approach. Infiltration modelling was incorporated in the detention storage unit within the one-dimensional urban storm water management model, EPA-SWMM 5.0. By introduction of infiltration modelling in the storage, the flow attenuation performance of the unit was considerably improved. The study also examines the catchment scale impact of both source and regional control storage/infiltration systems. Based on the findings of two case study areas modelled with the proposed options, it was observed that source control systems have a greater and much more natural impact at a catchment level, with respect to flow attenuation, compared to regional control systems of which capacity is equivalent to the sum of source control capacity at the catchment.

  5. Antitumor activity of sorafenib-incorporated nanoparticles of dextran/poly(dl-lactide- co-glycolide) block copolymer

    NASA Astrophysics Data System (ADS)

    Kim, Do Hyung; Kim, Min-Dae; Choi, Cheol-Woong; Chung, Chung-Wook; Ha, Seung Hee; Kim, Cy Hyun; Shim, Yong-Ho; Jeong, Young-Il; Kang, Dae Hwan

    2012-01-01

    Sorafenib-incoporated nanoparticles were prepared using a block copolymer that is composed of dextran and poly( DL-lactide- co-glycolide) [Dex bLG] for antitumor drug delivery. Sorafenib-incorporated nanoparticles were prepared by a nanoprecipitation-dialysis method. Sorafenib-incorporated Dex bLG nanoparticles were uniformly distributed in an aqueous solution regardless of the content of sorafenib. Transmission electron microscopy of the sorafenib-incorporated Dex bLG nanoparticles revealed a spherical shape with a diameter < 300 nm. Sorafenib-incorporated Dex bLG nanoparticles at a polymer/drug weight ratio of 40:5 showed a relatively uniform size and morphology. Higher initial drug feeding was associated with increased drug content in nanoparticles and in nanoparticle size. A drug release study revealed a decreased drug release rate with increasing drug content. In an in vitro anti-proliferation assay using human cholangiocarcinoma cells, sorafenib-incorporated Dex bLG nanoparticles showed a similar antitumor activity as sorafenib. Sorafenib-incorporated Dex bLG nanoparticles are promising candidates as vehicles for antitumor drug targeting.

  6. Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents.

    PubMed

    Dezi, Manuela; Di Cicco, Aurelie; Bassereau, Patricia; Lévy, Daniel

    2013-04-30

    Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.

  7. Investigation of tritium incorporation of tritium incorporation by means of excreted metabolites.

    PubMed

    Biró, T; Szilágyi, M

    1978-01-01

    The commonly accepted urine analysis by liquid scintillation method was applied for whole body dose estimating. After the separation of metabolite fractions the organically bound tritium in urine could be measured. Urine samples from workers repeatedly exposed to tritium incorporation during the chemical processing of various labeled compounds have been collected and analyzed. The time dependence of tritium activity in certain metabolites was found to be characteristic, significantly differing from the 3H concentration curve of the native or treated urine sample.

  8. Breads enriched with guava flour as a tool for studying the incorporation of phenolic compounds in bread melanoidins.

    PubMed

    Alves, Genilton; Perrone, Daniel

    2015-10-15

    In the present study we aimed at studying, for the first time, the incorporation of phenolic compounds into bread melanoidins. Fermentation significantly affected the phenolics profile of bread doughs. Melanoidins contents continuously increased from 24.1 mg/g to 71.9 mg/g during baking, but their molecular weight decreased at the beginning of the process and increased thereafter. Enrichment of white wheat bread with guava flour increased the incorporation of phenolic compounds up to 2.4-fold. Most phenolic compounds showed higher incorporation than release rates during baking, leading to increases from 3.3- to 13.3-fold in total melanoidin-bound phenolics. Incorporation patterns suggested that phenolic hydroxyls, but not glycosidic bonds of melanoidin-bound phenolics are cleaved during thermal processing. Antioxidant capacity of bread melanoidins increased due to enrichment with guava flour and increasing baking periods and was partially attributed to bound phenolics. Moreover, FRAP assay was more sensitive to measure this parameter than TEAC assay.

  9. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  10. A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

    PubMed

    Limsirichaikul, Siripan; Niimi, Atsuko; Fawcett, Heather; Lehmann, Alan; Yamashita, Shunichi; Ogi, Tomoo

    2009-03-01

    Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

  11. Evaluation of estrogenic activity of parabens and their chlorinated derivatives by using the yeast two-hybrid assay and the enzyme-linked immunosorbent assay.

    PubMed

    Terasaki, Masanori; Kamata, Ryo; Shiraishi, Fujio; Makino, Masakazu

    2009-01-01

    We assessed the estrogen agonist activities of 21 parabens and their chlorinated derivatives by using yeast two-hybrid assays incorporating either the human or medaka (Oryzias latipes) estrogen receptor alpha (hERalpha and medERalpha, respectively), and by using hERalpha competitive enzyme-linked immunosorbent assay (ER-ELISA). In the two-hybrid assay with hERalpha, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17beta-estradiol [E2] = 1) ranged from 2.0 x 10(-5) to 2.0 x 10(-4), with the highest activity observed in i-butylparaben. In the medERalpha assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 x 10(-5) to 3.5 x 10(-3), with the highest activity observed in benzylparaben, its monochlorinated derivative, i-butylparaben, and n-butylparaben. Although medERalpha demonstrated an activity to E2 that was three times lower than that demonstrated by hERalpha, medERalpha has a higher sensitivity to parabens than hERa (1.3-8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ERa in the ER-ELISA; of the parabens, i-butylparaben exhibited the strongest binding affinity. The yeast two-hybrid assay and the ER-ELISA also revealed that many of the assayed chlorinated parabens were much weaker than the parent compound. In addition, the results mainly showed that parabens with a bulk substituent (e.g., i-butyl and benzyl groups) had a higher activity than those with a sterically small substituent. It is considered that derivatization masks the apparent estrogenic activity of parabens, but the resulting chlorinated compounds may represent a potential hazard and therefore other toxicity tests should be performed to determine the toxicity of the chlorinated derivatives.

  12. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  13. Incorporating neurophysiological concepts in mathematical thermoregulation models

    NASA Astrophysics Data System (ADS)

    Kingma, Boris R. M.; Vosselman, M. J.; Frijns, A. J. H.; van Steenhoven, A. A.; van Marken Lichtenbelt, W. D.

    2014-01-01

    Skin blood flow (SBF) is a key player in human thermoregulation during mild thermal challenges. Various numerical models of SBF regulation exist. However, none explicitly incorporates the neurophysiology of thermal reception. This study tested a new SBF model that is in line with experimental data on thermal reception and the neurophysiological pathways involved in thermoregulatory SBF control. Additionally, a numerical thermoregulation model was used as a platform to test the function of the neurophysiological SBF model for skin temperature simulation. The prediction-error of the SBF-model was quantified by root-mean-squared-residual (RMSR) between simulations and experimental measurement data. Measurement data consisted of SBF (abdomen, forearm, hand), core and skin temperature recordings of young males during three transient thermal challenges (1 development and 2 validation). Additionally, ThermoSEM, a thermoregulation model, was used to simulate body temperatures using the new neurophysiological SBF-model. The RMSR between simulated and measured mean skin temperature was used to validate the model. The neurophysiological model predicted SBF with an accuracy of RMSR < 0.27. Tskin simulation results were within 0.37 °C of the measured mean skin temperature. This study shows that (1) thermal reception and neurophysiological pathways involved in thermoregulatory SBF control can be captured in a mathematical model, and (2) human thermoregulation models can be equipped with SBF control functions that are based on neurophysiology without loss of performance. The neurophysiological approach in modelling thermoregulation is favourable over engineering approaches because it is more in line with the underlying physiology.

  14. Coral barium incorporation: implications for proxy applications

    NASA Astrophysics Data System (ADS)

    Gonneea, M. E.; Cohen, A. L.; Charette, M. A.

    2012-12-01

    Coral Ba/Ca ratios have been proposed as proxies for various environmental variables including sediment loading, upwelling and groundwater input. Two assumptions that underpin the application of Ba/Ca ratios as an environmental proxy is 2) that corals take up Ba/Ca in concentrations proportional to seawater concentrations and 1) that the specified forcing mechanism influences seawater [Ba]. Here we present data from laboratory experiments that demonstrates corals reared in a range of seawater [Ba] linearly incorporate this signature in their skeletal Ba/Ca ratios. Observed coral Ba/Ca perturbations above baseline typically range from 5-15 μmol/mol which is ~100-500% increase over baseline. Other factors known to influence coral Ba/Ca include the temperature dependence on the partition coefficient and mass fraction aragonite precipitated by the coral which may be linked to calcification rate. In our experiments, calcification rate increased with temperature, thus the observed coral partition coefficient is the net effect of temperature (Ba/Ca increase at lower temperature) and calcification rate (Ba/Ca increase at higher temperature). We observed that the partition coefficient for reared coral Ba/Ca increased 20% 27.7 to 22.5 C, much less than observed Ba/Ca perturbations. Thus we predict that seawater [Ba] drives coral Ba/Ca signals in many locations. We present a model framework to calculate the expected contribution from sediment input, upwelling and groundwater discharge that is needed to produce this signature in corals growing in receiving waters. Finally we apply this model to a coral record from the Yucatan Peninsula, Mexico, recording groundwater discharge to the coastal ocean.

  15. Incorporating Web Tracking Into EOSDIS Metrics Collection

    NASA Astrophysics Data System (ADS)

    Murphy, K. J.; Boquist, C. L.; Hines-Watts, T. M.; Moses, J. F.; Sofinowski, E. J.

    2007-12-01

    The Earth Observing System Data and Information System (EOSDIS) and related data centers have been collecting and analyzing information on the archiving, processing and distribution of Earth science data for more than 10 years. Long-standing approaches for evaluating the performance of systems managed by EOSDIS have provided insights into how system engineering requirements are being met, how user communities are being served and the levels of interest across the science data products. However, data and services are increasingly being made available to users via web-based applications that cannot be evaluated with current methodologies. New tools are required to record and analyze how users find, navigate and use the continuously evolving landscape of web-based resources supported by EOSDIS. Without such tools a significant portion of the data and services EOSDIS provides would not be captured. EOSDIS will be able to incorporate metrics collection on new web-based applications across geographically distributed and differently configured systems while not interfering in those systems' operations. A consistent process has been devised for collection and reporting of information on web data usage and the characterization of the community of web service users. To provide a more complete picture of system utilization, knowledge of the web resource usage is coupled with improvements to the current approaches for collection of processing, archival and distribution metrics of Earth science products. This combined information improves evaluation of engineering requirements and understanding of how the new web capabilities are impacting usage and distribution of science data and services provided by EOSDIS. This paper describes the new, non-intrusive methodology EOSDIS is using to capture and analyze information on the usage of web services across all related systems. The paper will discuss initial results demonstrating the benefits of the reengineered system.

  16. Incorporating psychological influences in probabilistic cost analysis

    SciTech Connect

    Kujawski, Edouard; Alvaro, Mariana; Edwards, William

    2004-01-08

    Today's typical probabilistic cost analysis assumes an ''ideal'' project that is devoid of the human and organizational considerations that heavily influence the success and cost of real-world projects. In the real world ''Money Allocated Is Money Spent'' (MAIMS principle); cost underruns are rarely available to protect against cost overruns while task overruns are passed on to the total project cost. Realistic cost estimates therefore require a modified probabilistic cost analysis that simultaneously models the cost management strategy including budget allocation. Psychological influences such as overconfidence in assessing uncertainties and dependencies among cost elements and risks are other important considerations that are generally not addressed. It should then be no surprise that actual project costs often exceed the initial estimates and are delivered late and/or with a reduced scope. This paper presents a practical probabilistic cost analysis model that incorporates recent findings in human behavior and judgment under uncertainty, dependencies among cost elements, the MAIMS principle, and project management practices. Uncertain cost elements are elicited from experts using the direct fractile assessment method and fitted with three-parameter Weibull distributions. The full correlation matrix is specified in terms of two parameters that characterize correlations among cost elements in the same and in different subsystems. The analysis is readily implemented using standard Monte Carlo simulation tools such as {at}Risk and Crystal Ball{reg_sign}. The analysis of a representative design and engineering project substantiates that today's typical probabilistic cost analysis is likely to severely underestimate project cost for probability of success values of importance to contractors and procuring activities. The proposed approach provides a framework for developing a viable cost management strategy for allocating baseline budgets and contingencies. Given the

  17. Research highlights: digital assays on chip.

    PubMed

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino

    2015-01-07

    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  18. Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus.

    PubMed

    Pulford, David; Meyer, Hermann; Brightwell, Gale; Damon, Inger; Kline, Richard; Ulaeto, David

    2004-04-01

    PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay's specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

  19. Evaluation of adenosine deaminase assay for analyzing T-lymphocyte density in vitro.

    PubMed

    Kainthla, Rani Poonam; Kashyap, Rajpal Singh; Prasad, Sweta; Purohit, Hemant J; Taori, Giridhar M; Daginawala, Hatim F

    2006-01-01

    The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.

  20. Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

    PubMed Central

    Yu, Chang-Ping; Ahuja, Rajiv; Sayler, Gary; Chu, Kung-Hui

    2005-01-01

    A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats. PMID:15746346

  1. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders.

    PubMed

    Mumford, Andrew D; Frelinger, Andrew L; Gachet, Christian; Gresele, Paolo; Noris, Patrizia; Harrison, Paul; Mezzano, Diego

    2015-07-01

    Measurement of platelet granule release to detect inherited platelet secretion disorders (IPSDs) is essential for the evaluation of patients with abnormal bleeding and is necessary to distinguish which granule sub-types are affected and whether there is abnormal granule bio-synthesis or secretion. The radioactive serotonin incorporation and release assay, described before 1970, is still considered the "gold standard" test to assess platelet δ-granule release, although is unsuitable for clinical diagnostic laboratories. Luciferin-based assays, such as lumiaggregometry, are the most widely performed alternatives, although these methods do not distinguish defects in δ-granule biosynthesis from defects in secretion. Platelet α-granule release is commonly evaluated using flow cytometry by measuring surface exposure of P-selectin after platelet activation. However, this assay has poor sensitivity for some α-granule disorders. Only few studies have been published with more recently developed assays and no critical reviews on these methods are available. In this review, we describe the rationale for developing robust and accurate laboratory tests of platelet granule release and describe the characteristics of the currently available tests. We identify an unmet need for further systematic evaluation of new assays and for standardisation of methodologies for clinical diagnostic laboratories.

  2. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    PubMed

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  3. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

    PubMed Central

    Dozier, Jonathan K; Distefano, Mark D

    2012-01-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays use either radiolabeled substrates and are discontinuous, or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format and that it can reproduce IC50 values for several previously reported FDPS inhibitors. This new method offers a simple, safe and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target. PMID:22085443

  4. A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

    PubMed

    Vainshtein, Inna; Silveria, Scott; Kaul, Poonam; Rouhani, Riaz; Eglen, Richard M; Wang, John

    2002-12-01

    A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta

  5. Incorporating Scientific Publishing into an Undergraduate Neuroscience Course: A Case Study Using IMPULSE.

    PubMed

    Jones, Leslie Sargent; Allen, Laura; Cronise, Kim; Juneja, Natasha; Kohn, Rebecca; McClellan, Katherine; Miller, Ashley; Nazir, Azka; Patel, Andy; Sweitzer, Sarah M; Vickery, Erin; Walton, Anna; Young, Robert

    2011-01-01

    The journal IMPULSE offers undergraduates worldwide the opportunity to publish research and serve as peer reviewers for the submissions of others. Undergraduate faculty have recognized the journal's value in engaging students working in their labs in the publication process. However, integration of scientific publication into an undergraduate laboratory classroom setting has been lacking. We report here on a course at Ursinus College where 20 students taking Molecular Neurobiology were required to submit manuscripts to IMPULSE. The syllabus allowed for the laboratory research to coincide with the background research and writing of the manuscript. Students completed their projects on the impact of drugs on the Daphnia magna nervous system while producing manuscripts ready for submission by week 7 of the course. Findings from a survey completed by the students and perceptions of the faculty member teaching the course indicated that students spent much more time writing, were more focused on completing the assays, completed the assays with larger data sets, were more engaged in learning the scientific concepts and were more thorough with their revisions of the paper knowing that it might be published. Further, the professor found she was more thorough in critiquing students' papers knowing they would be externally reviewed. Incorporating journal submission into the course stimulated an in depth writing experience and allowed for a deeper exploration of the topic than students would have experienced otherwise. This case study provides evidence that IMPULSE can be successfully used as a means of incorporating scientific publication into an undergraduate laboratory science course. This approach to teaching undergraduate neuroscience allows for a larger number of students to have hands-on research and scientific publishing experience than would be possible with the current model of a few students in a faculty member's laboratory. This report illustrates that IMPULSE can

  6. Incorporating Scientific Publishing into an Undergraduate Neuroscience Course: A Case Study Using IMPULSE

    PubMed Central

    Jones, Leslie Sargent; Allen, Laura; Cronise, Kim; Juneja, Natasha; Kohn, Rebecca; McClellan, Katherine; Miller, Ashley; Nazir, Azka; Patel, Andy; Sweitzer, Sarah M.; Vickery, Erin; Walton, Anna; Young, Robert

    2011-01-01

    The journal IMPULSE offers undergraduates worldwide the opportunity to publish research and serve as peer reviewers for the submissions of others. Undergraduate faculty have recognized the journal’s value in engaging students working in their labs in the publication process. However, integration of scientific publication into an undergraduate laboratory classroom setting has been lacking. We report here on a course at Ursinus College where 20 students taking Molecular Neurobiology were required to submit manuscripts to IMPULSE. The syllabus allowed for the laboratory research to coincide with the background research and writing of the manuscript. Students completed their projects on the impact of drugs on the Daphnia magna nervous system while producing manuscripts ready for submission by week 7 of the course. Findings from a survey completed by the students and perceptions of the faculty member teaching the course indicated that students spent much more time writing, were more focused on completing the assays, completed the assays with larger data sets, were more engaged in learning the scientific concepts and were more thorough with their revisions of the paper knowing that it might be published. Further, the professor found she was more thorough in critiquing students’ papers knowing they would be externally reviewed. Incorporating journal submission into the course stimulated an in depth writing experience and allowed for a deeper exploration of the topic than students would have experienced otherwise. This case study provides evidence that IMPULSE can be successfully used as a means of incorporating scientific publication into an undergraduate laboratory science course. This approach to teaching undergraduate neuroscience allows for a larger number of students to have hands-on research and scientific publishing experience than would be possible with the current model of a few students in a faculty member’s laboratory. This report illustrates that IMPULSE

  7. Incorporating population variability and susceptible subpopulations into dosimetry for high-throughput toxicity testing.

    PubMed

    Wetmore, Barbara A; Allen, Brittany; Clewell, Harvey J; Parker, Timothy; Wambaugh, John F; Almond, Lisa M; Sochaski, Mark A; Thomas, Russell S

    2014-11-01

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework.

  8. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian J.; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-29

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  9. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  10. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  11. A high-throughput radiometric kinase assay

    PubMed Central

    Duong-Ly, Krisna C.; Peterson, Jeffrey R.

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  12. Robot speeds assays and enhances safeguards

    SciTech Connect

    Phelan, P.F.; Powell, W.D.; Blankenship, R.W.

    1990-01-01

    At the Los Alamos National Laboratory Plutonium Facility, a robotics system utilizing a gantry robot and an automated inventory system operates five calorimeters and two gamma isotopic assay instruments. This system has significantly improved safeguards, because the opportunity for diversion has been greatly reduced. Not only is the accountability much more timely because throughput has doubled but the special nuclear material has been made physically more secure in several ways. First, items awaiting assay are kept in the inventory system, whose doors remain locked whenever the robot is unattended. An alarm sounds if the doors are unlocked without authorization. Second, light curtains surround the robot's work envelope and pressure-sensitive pads cover the floor to detect entry into the assay area. Third, the robot weighs each item whenever it is moved, and the result is compared with the weight that was measured when the item was first put into inventory. 2 refs., 3 figs.

  13. EDTA interference in electrochemiluminescence ACTH assay.

    PubMed

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer

    2016-11-01

    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  14. Bacillus Spore Inactivation Methods Affect Detection Assays

    PubMed Central

    Dang, Jessica L.; Heroux, Karen; Kearney, John; Arasteh, Ameneh; Gostomski, Mark; Emanuel, Peter A.

    2001-01-01

    Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment. PMID:11472945

  15. Development of an objective comedogenicity assay.

    PubMed

    Tucker, S B; Flannigan, S A; Dunbar, M; Drotman, R B

    1986-06-01

    The rabbit ear comedogenicity assay is useful as a screening procedure for evaluating agents that come in contact with human skin. Controversy exists regarding the reliability of this assay because of differences in results from various laboratories. The subjective nature of the standard method of grading may also contribute to this variation. We use a more objective comedogenicity assay that utilizes increasing follicular orifice size on the rabbit ear as a measure of comedogenic activity. A generally linear increase in the degree of follicular orifice area was noted with several agents evaluated over a four-week application period. Further, a noninvasive Silastic elastomer mold was used to allow measurement of the same follicular orifice areas over time.

  16. Enzyme immunometric assay for leukotriene C4.

    PubMed

    Volland, H; Vulliez Le Normand, B; Mamas, S; Grassi, J; Créminon, C; Ezan, E; Pradelles, P

    1994-09-30

    An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

  17. Safety performance functions incorporating design consistency variables.

    PubMed

    Montella, Alfonso; Imbriani, Lella Liana

    2015-01-01

    Highway design which ensures that successive elements are coordinated in such a way as to produce harmonious and homogeneous driver performances along the road is considered consistent and safe. On the other hand, an alignment which requires drivers to handle high speed gradients and does not meet drivers' expectancy is considered inconsistent and produces higher crash frequency. To increase the usefulness and the reliability of existing safety performance functions and contribute to solve inconsistencies of existing highways as well as inconsistencies arising in the design phase, we developed safety performance functions for rural motorways that incorporate design consistency measures. Since the design consistency variables were used only for curves, two different sets of models were fitted for tangents and curves. Models for the following crash characteristics were fitted: total, single-vehicle run-off-the-road, other single vehicle, multi vehicle, daytime, nighttime, non-rainy weather, rainy weather, dry pavement, wet pavement, property damage only, slight injury, and severe injury (including fatal). The design consistency parameters in this study are based on operating speed models developed through an instrumented vehicle equipped with a GPS continuous speed tracking from a field experiment conducted on the same motorway where the safety performance functions were fitted (motorway A16 in Italy). Study results show that geometric design consistency has a significant effect on safety of rural motorways. Previous studies on the relationship between geometric design consistency and crash frequency focused on two-lane rural highways since these highways have the higher crash rates and are generally characterized by considerable inconsistencies. Our study clearly highlights that the achievement of proper geometric design consistency is a key design element also on motorways because of the safety consequences of design inconsistencies. The design consistency measures

  18. Kennedy Space Center's Partnership with Graftel Incorporated

    NASA Technical Reports Server (NTRS)

    Dunn, Carol Anne

    2010-01-01

    NASA Kennedy Space Center (KSC) has recently partnered with Graftel Incorporated under an exclusive license agreement for the manufacture and sale of the Smart Current Signature Sensor. The Smart Current Signature Sensor and software were designed and developed to be utilized on any application using solenoid valves. The system monitors the electrical and mechanical health of solenoids by comparing the electrical current profile of each solenoid actuation to a typical current profile and reporting deviation from its learned behavior. The objective of this partnership with Graftel is for them to develop the technology into a hand-held testing device for their customer base in the Nuclear Power Industry. The device will be used to perform diagnostic testing on electromechanical valves used in Nuclear Power plants. Initially, Graftel plans to have working units within the first year of license in order to show customers and allow them to put purchase requests into their next year's budget. The subject technology under discussion was commercialized by the Kennedy Space Center Technology Programs and Partnerships Office, which patented the technology and licensed it to Graftel, Inc., a company providing support, instrumentation, and calibration services to the nuclear community and private sector for over 10 years. For the nuclear power industry, Graftel designs, manufacturers, and calibrates a full line of testing instrumentation. Grafters smart sensors have been in use in the United States since 1993 and have proved to decrease set-up time and test durations. The project was funded by Non-Destructive Engineering, and it is felt that this technology will have more emphasis on future vehicles. Graftel plans to market the Current Signature Sensor to the Electric Utility industry. Graftel currently supplies product and services to the Nuclear Power Industry in the United States as well as internationally. Product and services sold are used in non-destructive testing for

  19. Incorporating scale into digital terrain analysis

    NASA Astrophysics Data System (ADS)

    Dragut, L. D.; Eisank, C.; Strasser, T.

    2009-04-01

    Digital Elevation Models (DEMs) and their derived terrain attributes are commonly used in soil-landscape modeling. Process-based terrain attributes meaningful to the soil properties of interest are sought to be produced through digital terrain analysis. Typically, the standard 3 X 3 window-based algorithms are used for this purpose, thus tying the scale of resulting layers to the spatial resolution of the available DEM. But this is likely to induce mismatches between scale domains of terrain information and soil properties of interest, which further propagate biases in soil-landscape modeling. We have started developing a procedure to incorporate scale into digital terrain analysis for terrain-based environmental modeling (Drăguţ et al., in press). The workflow was exemplified on crop yield data. Terrain information was generalized into successive scale levels with focal statistics on increasing neighborhood size. The degree of association between each terrain derivative and crop yield values was established iteratively for all scale levels through correlation analysis. The first peak of correlation indicated the scale level to be further retained. While in a standard 3 X 3 window-based analysis mean curvature was one of the poorest correlated terrain attribute, after generalization it turned into the best correlated variable. To illustrate the importance of scale, we compared the regression results of unfiltered and filtered mean curvature vs. crop yield. The comparison shows an improvement of R squared from a value of 0.01 when the curvature was not filtered, to 0.16 when the curvature was filtered within 55 X 55 m neighborhood size. This indicates the optimum size of curvature information (scale) that influences soil fertility. We further used these results in an object-based image analysis environment to create terrain objects containing aggregated values of both terrain derivatives and crop yield. Hence, we introduce terrain segmentation as an alternative

  20. Uranyl carboxyphosphonates that incorporate Cd(II)

    SciTech Connect

    Alsobrook, Andrea N.; Alekseev, Evgeny V.; Depmeier, Wulf; Albrecht-Schmitt, Thomas E.

    2011-05-15

    The hydrothermal treatment of UO{sub 3}, Cd(CH{sub 3}CO{sub 2}){sub 2}.2H{sub 2}O, and triethyl phosphonoacetate results in the formation of Cd{sub 2}[(UO{sub 2}){sub 6}(PO{sub 3}CH{sub 2}CO{sub 2}){sub 3}O{sub 3}(OH)(H{sub 2}O){sub 2}].16H{sub 2}O (CdUPAA-1), [Cd{sub 3}(UO{sub 2}){sub 6}(PO{sub 3}CH{sub 2}CO{sub 2}){sub 6}(H{sub 2}O){sub 13}].6H{sub 2}O (CdUPAA-2), and Cd(H{sub 2}O){sub 2}[(UO{sub 2})(PO{sub 3}CH{sub 2}CO{sub 2})(H{sub 2}O)]{sub 2} (CdUPAA-3). CdUPAA-1 adopts a cubic three-dimensional structure constructed from planar uranyl oxide clusters containing both UO{sub 7} pentagonal bipyramids and UO{sub 8} hexagonal bipyramids that are linked by Cd(II) cations and phosphonoacetate to yield large cavities approximately 16 A across that are filled with disordered water molecules. CdUPAA-2 forms a rhombohedral three-dimensional channel structure that is assembled from UO{sub 7} pentagonal bipyramids that are bridged by phosphonoacetate. CdUPAA-3 is layered with the hydrated Cd(II) cations incorporated directly into the layers linking one-dimensional uranyl phosphonate substructures together. In this structure, there are complex networks of hydrogen bonds that exist within the sheets, and also stitch the sheets together. -- Graphical abstract: A view of part of the cubic structure of Cd{sub 2}[(UO{sub 2}){sub 6}(PO{sub 3}CH{sub 2}CO{sub 2}){sub 3}O{sub 3}(OH)(H{sub 2}O){sub 2}].16H{sub 2}O. Display Omitted highlights: > High symmetry uranyl compounds. > Three-dimensional structures. > Porous materials. > Heterobimetallic compounds.

  1. Preparation and Characterization of Lanthanum-Incorporated Hydroxyapatite Coatings on Titanium Substrates

    PubMed Central

    Lou, Weiwei; Dong, Yiwen; Zhang, Hualin; Jin, Yifan; Hu, Xiaohui; Ma, Jianfeng; Liu, Jinsong; Wu, Gang

    2015-01-01

    Titanium (Ti) has been widely used in clinical applications for its excellent biocompatibility and mechanical properties. However, the bioinertness of the surface of Ti has motivated researchers to improve the physicochemical and biological properties of the implants through various surface modifications, such as coatings. For this purpose, we prepared a novel bioactive material, a lanthanum-incorporated hydroxyapatite (La-HA) coating, using a dip-coating technique with a La-HA sol along with post-heat treatment. The XRD, FTIR and EDX results presented in this paper confirmed that lanthanum was successfully incorporated into the structure of HA. The La-HA coating was composed of rod-like particles which densely compacted together without microcracks. The results of the interfacial shear strength test indicated that the incorporation of lanthanum increased the bonding strength of the HA coating. The mass loss ratios under acidic conditions (pH = 5.5) suggested that the La-HA coatings have better acid resistance. The cytocompatibility of the La-HA coating was also revealed by the relative activity of alkaline phosphatase, cellular morphology and cell proliferation assay in vitro. The present study suggested that La-HA coated on Ti has promising potential for applications in the development of a new type of bioactive coating for metal implants. PMID:26404255

  2. Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin.

    PubMed

    Pan, C J; Tang, J J; Shao, Z Y; Wang, J; Huang, N

    2007-09-01

    This paper dealt with improving the blood compatibility of the rapamycin-eluting stent by incorporating curcumin. The rapamycin- and rapamycin/curcumin-loaded PLGA (poly(d,l-lactic acid-co-glycolic acid)) coatings were fabricated onto the surface of the stainless steel stents using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier transform infrared spectroscopy (FTIR). The optical microscopy and scanning electron microscopy (SEM) images of the drug-eluting stents indicated that the surface of all drug-eluting stents was very smooth and uniform, and there were not webbings and "bridges" between struts. There were not any cracks and delaminations on stent surface after expanded by the angioplasty balloon. The in vitro platelet adhesion and activation were investigated by static platelet adhesion test and GMP140 (P-selection), respectively. The clotting time was examined by activated partially prothromplastin time (APTT) test. The fibrinogen adsorption on the drug-loaded PLGA films was evaluated by enzyme-linked immunosorbent assay (ELISA). All obtained data showed that incorporating curcumin in rapamycin-loaded PLGA coating can significantly decrease platelet adhesion and activation, prolong APTT clotting time as well as decrease the fibrinogen adsorption. All results indicated that incorporating curcumin in rapamycin-eluting coating obviously improve the blood compatibility of rapamycin-eluting stents. It was suggested that it may be possible to develop a drug-eluting stent which had the characteristics of not only good anti-proliferation but also improved anticoagulation.

  3. Assessment of DNA interstrand crosslinks using the modified alkaline comet assay.

    PubMed

    Wu, Jian Hong; Jones, Nigel J

    2012-01-01

    The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.

  4. Sensitive radioenzymatic assay for catechol drugs

    SciTech Connect

    Durrett, L.R.; Ziegler, M.G.

    1980-01-01

    This assay measures picogram quantities of catechol drugs and endogenous catecholamines in body tissues and fluids. The catechols are converted to their 3H-O-methyl metabolites during incubation with 3H-S-adenosylmethionine then separated by solvent extraction and thin-layer chromatography. Most drugs containing the catechol structure can be radiolabeled and separated from norepinephrine and epinephrine by this technique to provide simultaneous measurement of endogenous and exogenously administered catechols. The disposition of isoproterenol in tissues and fluids of man and experimental animals is measured to illustrate the utility of this assay. The reactivity of several commonly administered catechol drugs with COMT is described and the possible implications discussed.

  5. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C; Bourne, Mark M; Crooks, William J; Evans, Louise; Mayo, Douglas R; Miko, David K; Salazar, William R; Stange, Sy; Valdez, Jose I; Vigil, Georgiana M

    2012-07-13

    Waste will be removed from confinement vessels remaining from 1970s-era experiments. Los Alamos has 9+ spherical confinement vessels remaining from experiments. Each vessel contains {approx} 500 lbs of radioactive debris such as actinide metals and oxides, metals, powdered silica, graphite, and wires and hardware. In order to dispose of the vessels, debris and contamination must be removed. Neutron assay system was designed to assay vessels before and after cleanout. System requirements are: (1) Modular and moveable; (2) Capable of detecting {approx}100g {sup 239}Pu equivalent in a 2-inch thick steel sphere with 6 foot diameter; and (3) Capable of safeguards-quality assays. Initial design parameters arethe use of 4-atm {sup 3}He tubes with length of 6 feet, and {sup 3}He tubes embedded in polyethelene for moderation. This paper describes the calibration of the Confinement Vessel Assay System (CVAS) and quantification of its uncertainties. Assay uncertainty depends on five factors: (1) Statistical uncertainty in the assay measurement; (2) Statistical uncertainty in the background measurement; (3) Statistical uncertainty in the isotopics determination - This should be much smaller than the other uncertainties; (4) Systematic uncertainty due to position bias; and (5) Systematic uncertainty due to fluctuations in cosmic ray spallation. This one can be virtually eliminated by performing the background measurement with an empty vessel - but that may not be possible. We used modeling and experiments to quantify the systematic uncertainties. The calibration assumes a uniform distribution of material, but reality will be different. MCNPX modeling was used to quantify the positional bias. The model was benchmarked to build confidence in its results. Material at top of vessel is 44% greater than amount assayed, according to singles. Material near 19-tube detector is 38% less than amount assayed, according to singles. Cosmic ray spallation contributes significantly to the

  6. Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases.

    PubMed

    Egan, Alexander J F; Vollmer, Waldemar

    2016-01-01

    Bacterial cell wall peptidoglycan is synthesized from its precursor lipid II by two enzymatic reactions. First, glycosyltransferases polymerize the glycan strands and second, DD-transpeptidases form cross-links between peptides of neighboring strands. Most bacteria possess bifunctional peptidoglycan synthesis enzymes capable of catalyzing both reactions. Here, we describe a continuous fluorescence glycosyltransferase assay using Dansyl-labeled lipid II as substrate. Progression of the reaction is monitored by the reduction in fluorescence over time. The assay is suitable to investigate the effect of protein interaction partners on the glycan strand synthesis activity of peptidoglycan polymerases.

  7. Bicinchoninic acid (BCA) assay in low volume.

    PubMed

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.

  8. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  9. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    Cancer.gov

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page: https://assays.cancer.gov/support/

  10. SWEPP Assay System Version 2.0 software design description

    SciTech Connect

    East, L.V.; Marwil, E.S.

    1996-08-01

    The Idaho National Engineering Laboratory (INEL) Stored Waste Examination Pilot Plant (SWEPP) operations staff use nondestructive analysis methods to characterize the radiological contents of contact-handled radioactive waste containers. Containers of waste from Rocky Flats Environmental Technology Site and other Department of Energy (DOE) sites are currently stored at SWEPP. Before these containers can be shipped to the Waste Isolation Pilot Plant (WIPP), SWEPP must verify compliance with storage, shipping, and disposal requirements. This program has been in operation since 1985 at the INEL Radioactive Waste Management Complex (RWMC). One part of the SWEPP program measures neutron emissions from the containers and estimates the mass of plutonium and other transuranic (TRU) isotopes present. A Passive/Active Neutron (PAN) assay system developed at the Los Alamos National Laboratory is used to perform these measurements. A computer program named NEUT2 was originally used to perform the data acquisition and reduction functions for the neutron measurements. This program was originally developed at Los Alamos and extensively modified by a commercial vendor of PAN systems and by personnel at the INEL. NEUT2 uses the analysis methodology outlined, but no formal documentation exists on the program itself. The SWEPP Assay System (SAS) computer program replaced the NEUT2 program in early 1994. The SAS software was developed using an `object model` approach and is documented in accordance with American National Standards Institute (ANSI) and Institute of Electrical and Electronic Engineers (IEEE) standards. The new program incorporates the basic analysis algorithms found in NEUT2. Additional functionality and improvements include a graphical user interface, the ability to change analysis parameters without program code modification, an `object model` design approach and other features for improved flexibility and maintainability.

  11. Deuterium incorporation in biomass cell wall components by NMR analysis

    SciTech Connect

    Foston, Marcus B; McGaughey, Joseph; O'Neill, Hugh Michael; Evans, Barbara R; Ragauskas, Arthur J

    2012-01-01

    A commercially available deuterated kale sample was analyzed for deuterium incorporation by ionic liquid solution 2H and 1H nuclear magnetic resonance (NMR). This protocol was found to effectively measure the percent deuterium incorporation at 33%, comparable to the 31% value determined by combustion. The solution NMR technique also suggested by a qualitative analysis that deuterium is preferentially incorporated into the carbohydrate components of the kale sample.

  12. 46 CFR 54.01-1 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Code”), 54.01-2; 54.01-5; 54.01-15; 54.01-18; 54.01-25; 54.01-30; 54.01-35; 54.03-1; 54.05-1; 54.10-1... 46 Shipping 2 2010-10-01 2010-10-01 false Incorporation by reference. 54.01-1 Section 54.01-1... General Requirements § 54.01-1 Incorporation by reference. (a) Certain material is incorporated...

  13. 46 CFR 54.01-1 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Code”), 54.01-2; 54.01-5; 54.01-15; 54.01-18; 54.01-25; 54.01-30; 54.01-35; 54.03-1; 54.05-1; 54.10-1... 46 Shipping 2 2011-10-01 2011-10-01 false Incorporation by reference. 54.01-1 Section 54.01-1... General Requirements § 54.01-1 Incorporation by reference. (a) Certain material is incorporated...

  14. Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

    PubMed

    Lin, H J; Wu, P C; Lai, C L; Chak, W

    1984-04-01

    A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

  15. In vitro Sirius Red collagen assay measures the pattern shift from soluble to deposited collagen.

    PubMed

    Chen, Chun; Yang, Shanmin; Zhang, Mei; Zhang, Zhenhuan; Zhang, Bingrong; Han, Deping; Ma, Jun; Wang, Xiaohui; Hong, Jingshen; Guo, Yansong; Okunieff, Paul; Zhang, Lurong

    2013-01-01

    In this study, we compared two in vitro collagen production assays ([(3)H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-β), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [(3)H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agent's capacity for collagen production and (b) Sirius Red is more useful than [(3)H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.

  16. 46 CFR 147.7 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... certain standards and specifications are incorporated by reference as the governing requirements for.... (ASHRAE), Publication Sales Department, 1791 Tullie Circle, NE, Atlanta, GA 30329 ANSI/ASHRAE...

  17. Training Paraprofessionals in Programming: An Experimental Course Incorporating Developmental Theory.

    ERIC Educational Resources Information Center

    Croteau, James M.; Tinsley, Diane J.

    1984-01-01

    Describes a training program designed to train undergraduate paraprofessionals to plan, facilitate, and evaluate developmental workshops for college students. The program incorporated developmental theory and concepts. (JAC)

  18. Energy Efficient Electrochromic Windows Incorporating Ionic Liquids

    SciTech Connect

    Cheri Boykin; James Finley; Donald Anthony; Julianna Knowles; Richard Markovic; Michael Buchanan; Mary Ann Fuhry; Lisa Perrine

    2008-11-30

    One approach to increasing the energy efficiency of windows is to control the amount of solar radiation transmitted through a window by using electrochromic technology. What is unique about this project is that the electrochromic is based on the reduction/oxidation reactions of cathodic and anodic organic semi-conducting polymers using room temperature ionic liquids as ion transport electrolytes. It is believed that these types of coatings would be a lower cost alternative to traditional all inorganic thin film based electrochromic technologies. Although there are patents1 based on the proposed technology, it has never been reduced to practice and thoroughly evaluated (i.e. durability and performance) in a window application. We demonstrate that by using organic semi-conductive polymers, specific bands of the solar spectrum (specifically visible and near infrared) can be targeted for electrochemical variable transmittance responsiveness. In addition, when the technology is incorporated into an insulating glass unit, the energy parameters such as the solar heat gain coefficient and the light to solar gain ratio are improved over that of a typical insulating glass unit comprised of glass with a low emissivity coating. A minimum of {approx}0.02 quads of energy savings per year with a reduction of carbon emissions for electricity of {approx}320 MKg/yr benefit is achieved over that of a typical insulating glass unit including a double silver low-E coating. Note that these values include a penalty in the heating season. If this penalty is removed (i.e. in southern climates or commercial structures where cooling is predominate year-round) a maximum energy savings of {approx}0.05 quad per year and {approx}801 MKg/yr can be achieved over that of a typical insulating glass unit including a double silver low-E coating. In its current state, the technology is not durable enough for an exterior window application. The primary downfall is that the redox chemistry fails to

  19. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  20. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  1. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  2. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and anemia. (b) Classification. Class II. The special control for this device is FDA's “Document for...

  3. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  4. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  5. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  6. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  7. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  8. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  9. Three-dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichert, Anke

    2001-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flue virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  10. A Rapid and Quantitative Recombinase Activity Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  11. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Product Quality Assurance §...

  12. Implementation of a radioreceptor assay for dexmedetomidine.

    PubMed

    Salonen, M; Maze, M

    1993-11-01

    We have implemented a radioreceptor assay for dexmedetomidine, a novel alpha 2-adrenoceptor agonist. Receptor-bearing membranes were prepared from rat cerebral cortex and 3H-clonidine, 4 nM, was used as the labeled ligand. Dexmedetomidine displaced 3H-clonidine in a linear fashion over a concentration of 2 x 10(-10) to 2 x 10(-8)M. The detection limit of dexmedetomidine (i.e. 10% of radiolabeled ligand displaced) in this assay was 50 pg.ml-1 which is comparable to that seen with the reference method which utilizes gas chromotography with mass spectrometer (GC/MS) in series (Vuorilehto et al. 1989). Endogenous catecholamines, which can displace the radiolabeled ligand from its binding site, could easily be eliminated with a one-step extraction procedure. A comparison was made with the reference method (GC/MS) in 47 human plasma samples; the correlation coefficient (r2) was 0.61 (P < 0.001). The radioreceptor assay was also successfully applied for determining dexmedetomidine concentration in rabbit samples. These data indicate that the radioreceptor assay can be utilized for characterizing the pharmacokinetics of novel alpha 2 agonists which are now being introduced into the clinical practice of anaesthesia.

  13. Assay for Angiotensin-Converting Enzyme.

    ERIC Educational Resources Information Center

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  14. Advanced analysis techniques for uranium assay

    SciTech Connect

    Geist, W. H.; Ensslin, Norbert; Carrillo, L. A.; Beard, C. A.

    2001-01-01

    Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples count rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.

  15. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  16. Sensitive radioenzymatic assay for epinephrine forming enzymes

    SciTech Connect

    Ziegler, M.G.; Kennedy, B.; Elayan, H.

    1988-01-01

    Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces /sup 3/H-E from norepinephrine (NE) and /sup 3/H-S-adenosylmethionine. The /sup 3/H-E is isolated on alumina, /sup 3/H-S-adenosylmethionine is precipitated and the /sup 3/H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.

  17. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  18. Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

    PubMed Central

    Kehl, K S; Havens, P; Behnke, C E; Acheson, D W

    1997-01-01

    An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children. PMID:9230380

  19. Bioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites.

    PubMed

    Leippe, Donna; Sobol, Mary; Vidugiris, Gediminas; Cali, James J; Vidugiriene, Jolanta

    2017-04-01

    Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1-5 pmol/sample), broad linear range (0.1-100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z' = 0.6-0.9) and could be multiplexed with a real-time viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

  20. Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

    PubMed

    Miller, Michelle M; Alyea, Rebecca A; LeSommer, Caroline; Doheny, Daniel L; Rowley, Sean M; Childs, Kristin M; Balbuena, Pergentino; Ross, Susan M; Dong, Jian; Sun, Bin; Andersen, Melvin A; Clewell, Rebecca A

    2016-11-01

    A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERβ, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments.

  1. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    PubMed

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

  2. Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation.

    PubMed

    Vasquez, Marie Z

    2010-03-01

    Despite regulatory directives requiring the reduction of animal use in safety testing, recent modifications to genotoxicity testing guidelines now propose the use of two in vivo genotoxicity assays as a follow-up to an in vitro positive (International Conference on Harmonization Consensus Draft Guidance S2[R1] released March, 2008). To address both goals, the in vivo comet and micronucleus (MN) assays can be successfully combined into one informative study. Combining these two assays with such differences in sensitivity, endpoints measured and the type of data generated significantly improves upon the current standard capabilities for detecting genotoxicity without requiring additional animals. But to take full advantage of the benefits of incorporating the comet assay in safety testing, these same differences must be recognized and considered. Developed from over 15 years experience using the in vivo comet and MN assays in genotoxicity testing of chemicals and pharmaceuticals, this paper presents guidelines for the appropriate experimental design, dose selection and data interpretation for combined in vivo comet/MN assay studies. To illustrate the approach, data from combined assay studies are presented and discussed.

  3. Toxicity of fungicides to natural bacterial communities in wetland water and sediment measured using leucine incorporation and potential denitrification.

    PubMed

    Milenkovski, Susann; Bååth, Erland; Lindgren, Per-Eric; Berglund, Olof

    2010-02-01

    We assessed potential toxicity of fungicides to natural bacterial communities from a constructed wetland, located in southern Sweden, and compared the sensitivity of two endpoints indicating bacterial activity, leucine incorporation, and potential denitrification, in detecting toxicity. The effects of eight fungicides (benomyl, carbendazim, carboxin, captan, cycloheximide, fenpropimorph, propiconazole, and thiram), two bactericides (bronopol and chlortetracycline) as controls, and one reference compound (3,5-dichlorophenol), were tested in a water-sediment microcosm set-up. Leucine incorporation was measured in both the water and sediment column, while potential denitrification was measured for the entire microcosm. The bactericides and the reference compound gave sigmoid concentration-response curves for both endpoints in all but one case. The fungicides thiram, captan, and benomyl, and to a lesser extent fenpropimorph and propiconazole had quantifiable toxic effects on leucine incorporation, with EC(50) values ranging from 3 to 70 mg l(-1), while carbendazim, carboxin, and cycloheximide had little effect at the investigated concentrations. Only thiram and captan inhibited potential denitrification; the other fungicides showed no quantifiable effect. A greater toxic effect on leucine incorporation was recorded for bacterial communities associated with the water column, compared to the sediment column, for all tested compounds. Leucine incorporation was the more sensitive method for toxicity assessment of bacterial communities, and also allowed for a rapid and simple way of comparing exposure in the sediment and water column, making it an attractive standard method for community based toxicological assays in aquatic environments.

  4. Paraprotein interference with turbidimetric gentamicin assay

    PubMed Central

    Bassett, Kendra; Brown, Nigel

    2015-01-01

    Introduction Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays. Materials and methods A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser. Results The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery. Conclusion IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol. PMID:25672475

  5. Assays for mammalian tyrosinase: a comparative study

    SciTech Connect

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.

  6. Production and assay of forskolin antibodies

    SciTech Connect

    Ho, L.T.; Ho, R.J.

    1986-05-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  7. Effects of tumour necrosis factor-α on BrdU incorporation in cultured human enterocytes

    PubMed Central

    McDevitt, J.; Feighery, C.; O'Farrelly, C.; Martin, G.; Weir, D. G.

    1995-01-01

    Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFα in organ culture. TNFα caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G0, the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFα. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFα on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFα. Exposure to TNFα alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFα there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFα and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFα may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel. PMID:18475613

  8. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    NASA Astrophysics Data System (ADS)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  9. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  10. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  11. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  12. Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

    PubMed

    Kondas, Ashley V; Olson, Victoria A; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard; Damon, Inger K

    2015-04-01

    A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

  13. 21 CFR 866.3305 - Herpes simplex virus serological assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices... herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus...

  14. Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

    PubMed

    Stornetta, Alessia; Angelov, Todor; Guengerich, F Peter; Sturla, Shana J

    2013-09-02

    O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.

  15. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  16. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  17. 49 CFR 393.7 - Matter incorporated by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., WSTDA-T1, 1998, incorporation by reference approved for § 393.104(e). (21) Wire Rope Users Manual, 2nd Edition, Wire Rope Technical Board November 1985, incorporation by reference approved for § 393.104(e... Association, Inc., 5024-R Campbell Boulevard, Baltimore, Maryland 21236-5974. (5) Manuals of the Wire...

  18. 49 CFR 393.7 - Matter incorporated by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Association, WSTDA-T1, 1998, incorporation by reference approved for § 393.104(e). (21) Wire Rope Users Manual, 2nd Edition, Wire Rope Technical Board November 1985, incorporation by reference approved for § 393... Association, Inc., 5024-R Campbell Boulevard, Baltimore, Maryland 21236-5974. (5) Manuals of the Wire...

  19. 49 CFR 393.7 - Matter incorporated by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Association, WSTDA-T1, 1998, incorporation by reference approved for § 393.104(e). (21) Wire Rope Users Manual, 2nd Edition, Wire Rope Technical Board November 1985, incorporation by reference approved for § 393... Association, Inc., 5024-R Campbell Boulevard, Baltimore, Maryland 21236-5974. (5) Manuals of the Wire...

  20. 49 CFR 393.7 - Matter incorporated by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., WSTDA-T1, 1998, incorporation by reference approved for § 393.104(e). (21) Wire Rope Users Manual, 2nd Edition, Wire Rope Technical Board November 1985, incorporation by reference approved for § 393.104(e... Association, Inc., 5024-R Campbell Boulevard, Baltimore, Maryland 21236-5974. (5) Manuals of the Wire...

  1. 49 CFR 393.7 - Matter incorporated by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Association, WSTDA-T1, 1998, incorporation by reference approved for § 393.104(e). (21) Wire Rope Users Manual, 2nd Edition, Wire Rope Technical Board November 1985, incorporation by reference approved for § 393... Association, Inc., 5024-R Campbell Boulevard, Baltimore, Maryland 21236-5974. (5) Manuals of the Wire...

  2. 28 CFR 10.5 - Incorporation of papers previously filed.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Incorporation of papers previously filed... CARRYING ON ACTIVITIES WITHIN THE UNITED STATES Registration Statement § 10.5 Incorporation of papers previously filed. Papers and documents already filed with the Attorney General pursuant to the said act...

  3. 24 CFR 3280.4 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...—American National Standards Institute, 1430 Broadway, New York, New York 10018 APA—American Plywood... URBAN DEVELOPMENT MANUFACTURED HOME CONSTRUCTION AND SAFETY STANDARDS General § 3280.4 Incorporation by reference. (a) The specifications, standards and codes of the following organizations are incorporated...

  4. 46 CFR 160.171-3 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Incorporation by reference. 160.171-3 Section 160.171-3...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Immersion Suits § 160.171-3 Incorporation by reference. (a... Practice for Operating Salt Spray (Fog) Apparatus—160.171-17 ASTM C 177-85 (1993), Standard Test Method...

  5. Incorporating interprofessional communication skills (ISBARR) into an undergraduate nursing curriculum.

    PubMed

    Enlow, Michele; Shanks, Linda; Guhde, Jacqueline; Perkins, Michelle

    2010-01-01

    The AACN, in their 2008 Essentials of Baccalaureate Education for Professional Nursing Practice, recommends that colleges of nursing faculty incorporate competencies into their baccalaureate curriculum that focus on the development of professional communication skills. The authors provide a plan to incorporate a standardized communication tool (ISBARR) throughout all levels of an undergraduate curriculum.

  6. 28 CFR 10.5 - Incorporation of papers previously filed.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Incorporation of papers previously filed... CARRYING ON ACTIVITIES WITHIN THE UNITED STATES Registration Statement § 10.5 Incorporation of papers previously filed. Papers and documents already filed with the Attorney General pursuant to the said act...

  7. Incorporating Twitter, Instagram, and Facebook in Economics Classrooms

    ERIC Educational Resources Information Center

    Al-Bahrani, Abdullah; Patel, Darshak

    2015-01-01

    Social media is one of the most current and dynamic developments in education. In general, the field of economics has lagged behind other disciplines in incorporating technologies in the classroom. In this article, the authors provide a guide for economics educators on how to incorporate Twitter, Instagram, and Facebook inside and outside of the…

  8. 27 CFR 31.122 - Incorporation of business.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Incorporation of business... Requiring Registration As A New Business § 31.122 Incorporation of business. Where an individual or a firm engaged in business requiring registration under this part forms a corporation to take over and...

  9. 49 CFR 572.140 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Version) September 2001,” incorporated by reference in § 572.141; (3) SAE Recommended Practice J211/1, Rev... in § 572.146; (4) SAE J1733 1994-12 “Sign Convention for Vehicle Crash Testing” incorporated by..., (301) 419-5070. (2) The SAE materials referred to in paragraphs (a)(3) and (a)(4) of this section...

  10. 49 CFR 572.140 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Version) September 2001,” incorporated by reference in § 572.141; (3) SAE Recommended Practice J211/1, Rev... in § 572.146; (4) SAE J1733 1994-12 “Sign Convention for Vehicle Crash Testing” incorporated by..., (301) 419-5070. (2) The SAE materials referred to in paragraphs (a)(3) and (a)(4) of this section...

  11. 40 CFR 72.13 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 17 2013-07-01 2013-07-01 false Incorporation by reference. 72.13 Section 72.13 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Program General Provisions § 72.13 Incorporation by reference....

  12. 40 CFR 72.13 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Incorporation by reference. 72.13 Section 72.13 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Program General Provisions § 72.13 Incorporation by reference....

  13. 40 CFR 76.4 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 17 2014-07-01 2014-07-01 false Incorporation by reference. 76.4 Section 76.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) ACID RAIN NITROGEN OXIDES EMISSION REDUCTION PROGRAM § 76.4 Incorporation by reference. (a)...

  14. 40 CFR 72.13 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 16 2011-07-01 2011-07-01 false Incorporation by reference. 72.13 Section 72.13 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Program General Provisions § 72.13 Incorporation by reference....

  15. 40 CFR 76.4 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 16 2011-07-01 2011-07-01 false Incorporation by reference. 76.4 Section 76.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) ACID RAIN NITROGEN OXIDES EMISSION REDUCTION PROGRAM § 76.4 Incorporation by reference. (a)...

  16. 40 CFR 76.4 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 17 2013-07-01 2013-07-01 false Incorporation by reference. 76.4 Section 76.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) ACID RAIN NITROGEN OXIDES EMISSION REDUCTION PROGRAM § 76.4 Incorporation by reference. (a)...

  17. 40 CFR 76.4 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 17 2012-07-01 2012-07-01 false Incorporation by reference. 76.4 Section 76.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) ACID RAIN NITROGEN OXIDES EMISSION REDUCTION PROGRAM § 76.4 Incorporation by reference. (a)...

  18. 40 CFR 72.13 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 17 2014-07-01 2014-07-01 false Incorporation by reference. 72.13 Section 72.13 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Program General Provisions § 72.13 Incorporation by reference....

  19. 40 CFR 72.13 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 17 2012-07-01 2012-07-01 false Incorporation by reference. 72.13 Section 72.13 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Program General Provisions § 72.13 Incorporation by reference....

  20. 42 CFR 489.74 - Incorporation into existing provider agreements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 5 2010-10-01 2010-10-01 false Incorporation into existing provider agreements... HUMAN SERVICES (CONTINUED) STANDARDS AND CERTIFICATION PROVIDER AGREEMENTS AND SUPPLIER APPROVAL Surety Bond Requirements for HHAs § 489.74 Incorporation into existing provider agreements. The...

  1. 46 CFR 197.510 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Incorporation by reference. 197.510 Section 197.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Benzene § 197.510 Incorporation by reference. (a) Certain materials...

  2. 46 CFR 197.510 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Incorporation by reference. 197.510 Section 197.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Benzene § 197.510 Incorporation by reference. (a) Certain materials...

  3. 46 CFR 197.510 - Incorporation by reference.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Incorporation by reference. 197.510 Section 197.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Benzene § 197.510 Incorporation by reference. (a) Certain materials...

  4. 46 CFR 197.510 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Incorporation by reference. 197.510 Section 197.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Benzene § 197.510 Incorporation by reference. (a) Certain materials...

  5. 46 CFR 197.510 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Incorporation by reference. 197.510 Section 197.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Benzene § 197.510 Incorporation by reference. (a) Certain materials...

  6. 14 CFR 253.5 - Notice of incorporated terms.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... information concerning: (1) Limits on the air carrier's liability for personal injury or death of passengers... instrument given to a passenger, that embodies the contract of carriage and incorporates terms by reference... contract, passengers may inspect the full text of each term incorporated by reference at the...

  7. 14 CFR 253.5 - Notice of incorporated terms.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... information concerning: (1) Limits on the air carrier's liability for personal injury or death of passengers... instrument given to a passenger, that embodies the contract of carriage and incorporates terms by reference... contract, passengers may inspect the full text of each term incorporated by reference at the...

  8. 14 CFR 253.5 - Notice of incorporated terms.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... information concerning: (1) Limits on the air carrier's liability for personal injury or death of passengers... instrument given to a passenger, that embodies the contract of carriage and incorporates terms by reference... contract, passengers may inspect the full text of each term incorporated by reference at the...

  9. 14 CFR 253.5 - Notice of incorporated terms.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... information concerning: (1) Limits on the air carrier's liability for personal injury or death of passengers... instrument given to a passenger, that embodies the contract of carriage and incorporates terms by reference... contract, passengers may inspect the full text of each term incorporated by reference at the...

  10. 14 CFR 253.5 - Notice of incorporated terms.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... information concerning: (1) Limits on the air carrier's liability for personal injury or death of passengers... instrument given to a passenger, that embodies the contract of carriage and incorporates terms by reference... contract, passengers may inspect the full text of each term incorporated by reference at the...

  11. 49 CFR 572.160 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., Upper Torso Assembly, incorporated by reference in §§ 572.161, 572.164, and 572.165 as part of a...-3000, Lower Torso Assembly, incorporated by reference in §§ 572.161, and 572.165 as part of a...

  12. 49 CFR 572.160 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., Upper Torso Assembly, incorporated by reference in §§ 572.161, 572.164, and 572.165 as part of a...-3000, Lower Torso Assembly, incorporated by reference in §§ 572.161, and 572.165 as part of a...

  13. 49 CFR 572.160 - Incorporation by reference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... No. 167-2000, Upper Torso Assembly, incorporated by reference in §§ 572.161, 572.164, and 572.165 as...; (iv) Drawing No. 167-3000, Lower Torso Assembly, incorporated by reference in §§ 572.161, and...

  14. 49 CFR 572.160 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., Upper Torso Assembly, incorporated by reference in §§ 572.161, 572.164, and 572.165 as part of a...-3000, Lower Torso Assembly, incorporated by reference in §§ 572.161, and 572.165 as part of a...

  15. 49 CFR 572.120 - Incorporation by reference.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... § 572.123, (iii) Drawing No. 127-2000, Upper Torso Assembly, incorporated by reference in § 572.124, (iv) Drawing No. 127-3000, Lower Torso Assembly, incorporated by reference in § 572.125, (v) Drawing No....

  16. 49 CFR 572.120 - Incorporation by reference.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... § 572.123, (iii) Drawing No. 127-2000, Upper Torso Assembly, incorporated by reference in § 572.124, (iv) Drawing No. 127-3000, Lower Torso Assembly, incorporated by reference in § 572.125, (v) Drawing No....

  17. 28 CFR 10.5 - Incorporation of papers previously filed.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Incorporation of papers previously filed... CARRYING ON ACTIVITIES WITHIN THE UNITED STATES Registration Statement § 10.5 Incorporation of papers previously filed. Papers and documents already filed with the Attorney General pursuant to the said act...

  18. 28 CFR 10.5 - Incorporation of papers previously filed.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Incorporation of papers previously filed... CARRYING ON ACTIVITIES WITHIN THE UNITED STATES Registration Statement § 10.5 Incorporation of papers previously filed. Papers and documents already filed with the Attorney General pursuant to the said act...

  19. 28 CFR 10.5 - Incorporation of papers previously filed.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Incorporation of papers previously filed... CARRYING ON ACTIVITIES WITHIN THE UNITED STATES Registration Statement § 10.5 Incorporation of papers previously filed. Papers and documents already filed with the Attorney General pursuant to the said act...

  20. 49 CFR 537.10 - Incorporation by reference.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 6 2011-10-01 2011-10-01 false Incorporation by reference. 537.10 Section 537.10 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AUTOMOTIVE FUEL ECONOMY REPORTS § 537.10 Incorporation by...