A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

PubMed

Liu, Bing-Hsien; Lin, Yu-Chen; Ho, Chia-Shin; Yang, Che-Chuan; Chang, Yun-Tsui; Chang, Jui-Feng; Li, Chun-Yuan; Cheng, Cheng-Shun; Huang, Jiun-Yan; Lee, Yen-Fu; Hsu, Ming-Hung; Lin, Feng-Chun; Wang, Hao-Ching; Lo, Chu-Fang; Yang, Shieh-Yueh; Wang, Han-Ching

2015-01-01

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Reduction in a commercial potato irrigation schedule during tuber bulking in Florida: Physiological, Yield, and Quality effects

USDA-ARS?s Scientific Manuscript database

Proper irrigation scheduling in potato (Solanum tuberosum L.) can lead to higher returns and more sustainable production practices when compared to systems that do not take plant water demand into account. In an attempt to reduce irrigation applications while minimizing yield reduction, we evaluated...

Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication.

PubMed

Wieten, Rosanne W; Jonker, Emile F F; Pieren, Daan K J; Hodiamont, Caspar J; van Thiel, Pieter P A M; van Gorp, Eric C M; de Visser, Adriëtte W; Grobusch, Martin P; Visser, Leo G; Goorhuis, Abraham

2016-03-04

The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and the Plaque Reduction Neutralization Test) in a group of Dutch immune-compromised travellers with a median of 33 days (IQR [28-49]) after primary YF vaccination. We collected samples of 15 immune-compromised vaccinees vaccinated with the 17D yellow fever vaccine between 2004 and 2012. All samples measured in the plaque reduction neutralization test yielded positive results (>80% virus neutralization with a 1:10 serum dilution). Immune Fluorescence Assay sensitivity was 28% (95% CI [0.12-0.49]). No adverse events were reported. All immune-compromised patients mounted an adequate response with protective levels of virus neutralizing antibodies to the 17-D YF vaccine. No adverse effects were reported. Compared to the plaque reduction neutralization test, the sensitivity of the Immune Fluorescence Assay test was low. Further research is needed to ascertain that 17D vaccination in immune-compromised patients is safe. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-reductive fabrication of carbon nanodots with high quantum yield for bioimaging of bacteria

PubMed Central

Wang, Jiajun; Liu, Xia; Milcovich, Gesmi; Chen, Tzu-Yu; Durack, Edel; Mallen, Sarah; Ruan, Yongming

2018-01-01

A simple and straightforward synthetic approach for carbon nanodots (C-dots) is proposed. The strategy is based on a one-step hydrothermal chemical reduction with thiourea and urea, leading to high quantum yield C-dots. The obtained C-dots are well-dispersed with a uniform size and a graphite-like structure. A synergistic reduction mechanism was investigated using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The findings show that using both thiourea and urea during the one-pot synthesis enhances the luminescence of the generated C-dots. Moreover, the prepared C-dots have a high distribution of functional groups on their surface. In this work, C-dots proved to be a suitable nanomaterial for imaging of bacteria and exhibit potential for application in bioimaging thanks to their low cytotoxicity. PMID:29441259

Evaluation of cytotoxicity, genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay, comet assay and zebrafish embryo test.

PubMed

Yang, Fan; Zhang, Qianqian; Guo, Huarong; Zhang, Shicui

2010-10-01

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC>ZQ>OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause. Copyright © 2010 Elsevier Ltd. All rights reserved.

Nitrate concentration effects on NO3-N uptake and reduction, growth, and fruit yield in strawberry

NASA Technical Reports Server (NTRS)

Darnell, R. L.; Stutte, G. W.; Sager, J. C. (Principal Investigator)

2001-01-01

Strawberries (Fragaria xananassa Duch. 'Osogrande') were grown hydroponically with three NO3-N concentrations (3.75, 7.5, or 15.0 mM) to determine effects of varying concentration on NO3-N uptake and reduction rates, and to relate these processes to growth and fruit yield. Plants were grown for 32 weeks, and NO3-N uptake and nitrate reductase (NR) activities in roots and shoots were measured during vegetative and reproductive growth. In general, NO3-N uptake rates increased as NO3-N concentration in the hydroponics system increased. Tissue NO3- concentration also increased as external NO3-N concentration increased, reflecting the differences in uptake rates. There was no effect of external NO3-N concentration on NR activities in leaves or roots during either stage of development. Leaf NR activity averaged approximately 360 nmol NO2 formed/g fresh weight (FW)/h over both developmental stages, while NR activity in roots was much lower, averaging approximately 115 nmol NO2 formed/g FW/h. Vegetative organ FW, dry weight (DW), and total fruit yield were unaffected by NO3-N concentration. These data suggest that the inability of strawberry to increase growth and fruit yield in response to increasing NO3-N concentrations is not due to limitations in NO3-N uptake rates, but rather to limitations in NO3- reduction and/or assimilation in both roots and leaves.

Yield Stress Reduction of DWPF Melter Feed Slurries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stone, M.E.; Smith, M.E.

2007-07-01

a flame, then quenched with a water spray. Approximately 90% of the frit was converted to beads by this process. Yield stress reduction was measured by preparing melter feed slurries (using nonradioactive HLW simulants) that contain beads and comparing the yield stress with melter feed containing frit. A second set of tests was performed with beads of various diameters to determine if a decrease in diameter affected the results. Smaller particle size was shown to increase yield stress when frit is utilized. The settling rate of the beads was required to match the settling rate of the frit, therefore a decrease in particle size was anticipated. Settling tests were conducted in water, xanthan gum solutions, and in non-radioactive simulants of the HLW. The tests used time-lapse video-graphy as well as solids sampling to evaluate the settling characteristics of beads compared to frit of the same particle size. A preliminary melt rate evaluation was performed using a dry-fed Melt Rate Furnace (MRF) developed by SRNL. Preliminary evaluation of the impact of beading the frit on the frit addition system were completed by conducting flow loop testing. A recirculation loop was built with a total length of about 30 feet. Pump power, flow rate, outlet pressure, and observations of the flow in the horizontal upper section of the loop were noted. The recirculation flow was then gradually reduced and the above items recorded until settling was noted in the recirculation line. Overall, the data shows that the line pressure increased as the solids were increased for the same flow rate. In addition, the line pressure was higher for Frit 320 than the beads at the same solids level and flow. With the observations, a determination of minimum velocity to prevent settling could be done, but a graph of the line pressures versus velocity for the various tests was deemed to more objective. The graph shows that the inflection point in pressure drop is about the same for the beads and Frit 320

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Photocatalytic reduction of CO2 to CO over copper decorated g-C3N4 nanosheets with enhanced yield and selectivity

NASA Astrophysics Data System (ADS)

Shi, Guodong; Yang, Lin; Liu, Zhuowen; Chen, Xiao; Zhou, Jianqing; Yu, Ying

2018-01-01

Photocatalytic reduction of CO2 to fuel has attracted considerable attention due to the consumption of fossil fuels and serious environmental problems. Although there are many photocatalysts reported for CO2 reduction, the improvement of activity and selectivity is still in great need of. In this work, a series of Cu nanoparticle decorated g-C3N4 nanosheets with different Cu loadings were fabricated by a facile secondary calcination and subsequent microwave hydrothermal method. The designed catalysts shown good photocatalytic activity and selectivity for CO2 reduction to CO. The optimal sample exhibited a 3-fold augmentation of the CO yield in comparison with pristine g-C3N4 under visible light. It is revealed that with the loading of Cu nanoparticles, the resulting photocatalyst possessed an improved charge carrier transfer and separation efficiency as well as increased surface reactive sites, resulting in a significant enhancement of CO yield. It is anticipated that the designed Cu/C3N4 photocatalyst may provide new insights for two dimensional layer materials and non-noble particles applied to CO2 reduction.

Stimulation and Inhibition of Anaerobic Digestion by Nickel and Cobalt: A Rapid Assessment Using the Resazurin Reduction Assay.

PubMed

Chen, Jian Lin; Steele, Terry W J; Stuckey, David C

2016-10-03

Stimulation of anaerobic digestion by essential trace metals is beneficial from a practical point of view to enhance the biodegradability and degradation rate of wastes. Hence, a quick method to determine which metal species, and at what concentration, can optimize anaerobic digestion is of great interest to both researchers and operators. In this present study, we investigated the effect of nickel(II), cobalt(II), and their mixture, on the anaerobic digestion of synthetic municipal wastewater. Using a volumetric method, that is, measuring methane production over time, revealed that anaerobic digestion was stimulated by the addition of 5 mg L -1 nickel(II), and cobalt(II), and their mixture in day(s). However, using a novel resazurin reduction assay, and based on its change in rate over time, we evaluated both inhibition at 250 mg L -1 nickel(II) and cobalt(II), and also the stimulatory effect of 5 mg L -1 nickel(II), and cobalt(II), and their mixture, in just 6 h. By investigating the dynamic distribution of these metals in the liquid phase of the anaerobic system and kinetics of resazurin reduction by nickel spiked anaerobic sludge, the concentration of nickel(II) on anaerobic digestion performance was profiled. Three critical concentrations were determined; stimulation starting (around 1 mg L -1 ), stimulation ending (around 100 mg L -1 ) and stimulation maximizing (around 10 mg L -1 ). Hence, we propose that the resazurin reduction assay is a novel and quick protocol for studying the stimulation of anaerobic bioprocesses by bioavailable essential trace metals.

Post-anaerobic digestion thermal hydrolysis of sewage sludge and food waste: Effect on methane yields, dewaterability and solids reduction.

PubMed

Svensson, Kine; Kjørlaug, Oda; Higgins, Matthew J; Linjordet, Roar; Horn, Svein J

2018-04-01

Post-anaerobic digestion (PAD) treatment technologies have been suggested for anaerobic digestion (AD) to improve process efficiency and assure hygenization of organic waste. Because AD reduces the amount of organic waste, PAD can be applied to a much smaller volume of waste compared to pre-digestion treatment, thereby improving efficiency. In this study, dewatered digestate cakes from two different AD plants were thermally hydrolyzed and dewatered, and the liquid fraction was recirculated to a semi-continuous AD reactor. The thermal hydrolysis was more efficient in relation to methane yields and extent of dewaterability for the cake from a plant treating waste activated sludge, than the cake from a plant treating source separated food waste (SSFW). Temperatures above 165 °C yielded the best results. Post-treatment improved volumetric methane yields by 7% and the COD-reduction increased from 68% to 74% in a mesophilic (37 °C) semi-continuous system despite lowering the solid retention time (from 17 to 14 days) compared to a conventional system with pre-treatment of feed substrates at 70 °C. Results from thermogravimetric analysis showed an expected increase in maximum TS content of dewatered digestate cake from 34% up to 46% for the SSFW digestate cake, and from 17% up to 43% in the sludge digestate cake, after the PAD thermal hydrolysis process (PAD-THP). The increased dewatering alone accounts for a reduction in wet mass of cake leaving the plant of 60% in the case of sludge digestate cake. Additionaly, the increased VS-reduction will contribute to further reduce the mass of wet cake. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of 4th-Generation HIV Antigen/Antibody Combination Assay With 3rd-Generation HIV Antibody Assays for the Occurrence of False-Positive and False-Negative Results.

PubMed

Muthukumar, Alagarraju; Alatoom, Adnan; Burns, Susan; Ashmore, Jerry; Kim, Anne; Emerson, Brian; Bannister, Edward; Ansari, M Qasim

2015-01-01

To assess the false-positive and false-negative rates of a 4th-generation human immunodeficiency virus (HIV) assay, the Abbott ARCHITECT, vs 2 HIV 3rd-generation assays, the Siemens Centaur and the Ortho-Clinical Diagnostics Vitros. We examined 123 patient specimens. In the first phase of the study, we compared 99 specimens that had a positive screening result via the 3rd-generation Vitros assay (10 positive, 82 negative, and 7 indeterminate via confirmatory immunofluorescent assay [IFA]/Western blot [WB] testing). In the second phase, we assessed 24 HIV-1 RNA-positive (positive result via the nuclear acid amplification test [NAAT] and negative/indeterminate results via the WB test) specimens harboring acute HIV infection. The 4th-generation ARCHITECT assay yielded fewer false-positive results (n = 2) than the 3rd-generation Centaur (n = 9; P = .02) and Vitros (n = 82; P <.001) assays. One confirmed positive case had a false-negative result via the Centaur assay. When specimens from the 24 patients with acute HIV-1 infection were tested, the ARCHITECT assay yielded fewer false-negative results (n = 5) than the Centaur (n = 10) (P = .13) and the other 3rd-generation tests (n = 16) (P = .002). This study indicates that the 4th-generation ARCHITECT HIV assay yields fewer false-positive and false-negative results than the 3rd-generation HIV assays we tested. Copyright© by the American Society for Clinical Pathology (ASCP).

Results of the Abbott RealTime HIV-1 assay for specimens yielding "target not detected" results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test.

PubMed

Babady, N Esther; Germer, Jeffrey J; Yao, Joseph D C

2010-03-01

No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

Tolerance assays: measuring the unknown.

PubMed

Newell, Kenneth A; Larsen, Christian P

2006-06-15

Distinguishing transplant recipients who will benefit from a reduction in, or even the withdrawal of, immunosuppression from those who require intensive, lifelong immunosuppression will be dependent on developing strategies for immune monitoring. Currently, no assays have been shown to accurately predict the development or presence of donor-specific tolerance in humans after transplantation. In this overview we describe and discuss those assays that we believe may be useful for identifying tolerant transplant recipients. Validation of "tolerance" assays will be critical for the safe development of tolerance regimens in humans.

A dose-response relationship for marketable yield reduction of two lettuce (Lactuca sativa L.) cultivars exposed to tropospheric ozone in Southern Europe.

PubMed

Marzuoli, Riccardo; Finco, Angelo; Chiesa, Maria; Gerosa, Giacomo

2017-12-01

The present study investigated the response to ozone (O 3 ) of two cultivars (cv.'Romana' and cv. 'Canasta') of irrigated lettuce grown in an open-top chamber (OTC) experiment in Mediterranean conditions. Two different levels of O 3 were applied, ambient O 3 in non-filtered OTCs (NF-OTCs) and -40% of ambient O 3 in charcoal-filtered OTCs (CF-OTCs), during four consecutive growing cycles. At the end of each growing cycle, the marketable yield (fresh biomass) was assessed while during the growing periods, measurements of the stomatal conductance at leaf level were performed and used to define a stomatal conductance model for calculation of the phytotoxic ozone dose (POD) absorbed by the plants.Results showed that O 3 caused statistically significant yield reductions in the first and in the last growing cycle. In general, the marketable yield of the NF-OTC plants was always lower than the CF-OTC plants for both cultivars, with mean reductions of -18.5 and -14.5% for 'Romana' and 'Canasta', respectively. On the contrary, there was no statistically significant difference in marketable yield due to the cultivar factor or to the interaction between O 3 and cultivar in any of the growing cycle performed.Dose-response relationships for the marketable relative yield based on the POD values were calculated according to different flux threshold values (Y). The best regression fit was obtained using an instantaneous flux threshold of 6 nmol O 3 m -2  s -1 (POD 6 ); the same value was obtained also for other crops. According to the generic lettuce dose-response relationship, an O 3 critical level of 1 mmol O 3 m -2 of POD 6 for a 15% of marketable yield loss was found.

A two-fold reduction in measurement time for neutron assay: Initial tests of a prototype dual-gated shift register

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, J.E.; Bourret, S.C.; Krick, M.S.

1996-09-01

Neutron coincidence counting (NCC) is used routinely around the world for nondestructive mass assay of uranium and plutonium in many forms, including waste. Compared with other methods, NCC is generally the most flexible, economic, and rapid. Many applications of NCC would benefit from a reduction in counting time required for a fixed random error. We have developed and tested the first prototype of a dual- gated, shift-register-based electronics unit that offers the potential of decreased measurement time for all passive and active NCC applications.

A two-fold reduction in measurement time for neutron assay: Initial tests of a prototype dual-gated shift register

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, J.E.; Bourret, S.C.; Krick, M.S.

1996-12-31

Neutron coincidence counting (NCC) is used routinely around the world for nondestructive mass assay of uranium and plutonium in many forms, including waste. Compared with other methods, NCC is generally the most flexible, economic, and rapid. Many applications of NCC would benefit from a reduction in counting time required for a fixed random error. The authors have developed and tested the first prototype of a dual-gated, shift-register-based electronics unit that offers the potential of decreased measurement time for all passive and active NCC applications.

Yields of damage to C4' deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation.

PubMed

Peoples, Anita R; Lee, Jane; Weinfeld, Michael; Milligan, Jamie R; Bernhard, William A

2012-07-01

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the (32)P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) (32)P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091-1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ ≈ 2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4' radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ ≈ 2.5 was found to be 30.2 ± 1.0 nmol/J (RT) and 12.9 ± 0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.

Comparison of Five Assays for Detection of Clostridium difficile Toxin

PubMed Central

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

Specific detection of membrane-toxic substances with a conductivity assay.

PubMed

Eich, J; Dürholt, H; Steger-Hartmann, T; Wagner, E

2000-03-01

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds. Copyright 2000 Academic Press.

Yield: it's now an entitlement

NASA Astrophysics Data System (ADS)

George, Bill

1994-09-01

Only a few years ago, the primary method of cost reduction and productivity improvement in the semiconductor industry was increasing manufacturing yields throughout the process. Many of the remarkable reliability improvements realized over the past decade have come about as a result of actions that were originally taken primarily to improve device yields. Obviously, the practice of productivity improvement through yield enhancement is limited to the attainment of 100% yield, at which point some other mechanism must be employed. Traditionally, new products have been introduced to manufacturing at a point of relative immaturity, and semiconductor producers have relied on the traditional `learning curve' method of yield improvement to attain profitable levels of manufacturing yield. Recently, results of a survey of several fabs by a group of University of California at Berkeley researchers in the Competitive Semiconductor Manufacturing Program indicate that most factories learn at about the same rate after startup, in terms of both line yield and defectivity. If this is indeed generally true, then the most competitive factor is the one that starts with the highest yield, and it is difficult to displace a leader once his lead has been established. The two observations made above carry enormous implications for the semiconductor development or manufacturing professional. First, one must achieve very high yields in order to even play the game. Second, the achievement of competitive yields over time in the life of a factory is determined even before the factory is opened, in the planning and development phase. Third, and perhaps most uncomfortable for those of us who have relied on yield improvement as a cost driver, the winners of the nineties will find new levers to drive costs down, having already gotten the benefit of very high yield. This paper looks at the question of how the winners will achieve the critical measures of success, high initial yield and utilization

Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle.

PubMed

Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M

2015-11-30

This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P < 0.0001), however, praziquantel did not record any reduction of Fasciola egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P < 0.01). In albendazole, the hatching varied according to the drug concentration. At the highest two concentrations; 0.2 and 2.0 μg/ml, the hatching percentages were 7.4 ± 1.6 and 5.6 ± 1.5 (P < 0.01) respectively. On the contrary, there were no significant differences in egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect. Copyright © 2015 Elsevier B.V. All rights

LINE-1 Cultured Cell Retrotransposition Assay

PubMed Central

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

2016-01-01

Summary 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. PMID:26895052

Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

PubMed

Naidoo, N; Ghai, M; Moodley, K; Mkize, L; Martin, L; McFarlane, S; Rutherford, S

2017-12-01

Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U μl -1 ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities. This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis. © 2017

Mouse assay for determination of arsenic bioavailability in contaminated soils.

PubMed

Bradham, Karen D; Diamond, Gary L; Scheckel, Kirk G; Hughes, Michael F; Casteel, Stan W; Miller, Bradley W; Klotzbach, Julie M; Thayer, William C; Thomas, David J

2013-01-01

A mouse assay for measuring the relative bioavailability (RBA) of arsenic (As) in soil was developed. In this study, results are presented of RBA assays of 16 soils, including multiple assays of the same soils, which provide a quantitative assessment of reproducibility of mouse assay results, as well as a comparison of results from the mouse assay with results from a swine and monkey assay applied to the same test soils. The mouse assay is highly reproducible; three repeated assays on the same soils yielded RBA estimates that ranged from 1 to 3% of the group mean. The mouse, monkey, and swine models yielded similar results for some, but not all, test materials. RBA estimates for identical soils (nine test soils and three standard reference materials [SRM]) assayed in mice and swine were significantly correlated (r = 0.70). Swine RBA estimates for 6 of the 12 test materials were higher than those from the mouse assay. RBA estimates for three standard reference materials (SRM) were not statistically different (mouse/swine ratio ranged from 0.86-1). When four test soils from the same orchard were assessed in the mouse, monkey, and swine assays, the mean soil As RBA were not statistically different. Mouse and swine models predicted similar steady state urinary excretion fractions (UEF) for As of 62 and 74%, respectively, during repeated ingestion doses of sodium arsenate, the water-soluble As form used as the reference in the calculation of RBA. In the mouse assay, the UEF for water soluble As(V) (sodium arsenate) and As(III) (sodium [meta] arsenite) were 62% and 66%, respectively, suggesting similar absolute bioavailabilities for the two As species. The mouse assay can serve as a highly cost-effective alternative or supplement to monkey and swine assays for improving As risk assessments by providing site-specific assessments of RBA of As in soils.

Comparison of five assays for detection of Clostridium difficile toxin.

PubMed

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

PubMed

Vaidya, Sunil R; Kumbhar, Neelakshi S; Bhide, Vandana S

2014-12-01

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

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.

YIELD STRESS REDUCTION OF DWPF MELTER FEED SLURRIES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stone, M; Michael02 Smith, M

2006-12-28

The Defense Waste Processing Facility (DWPF) at the Savannah River Site vitrifies High Level Waste for repository internment. The process consists of three major steps: waste pretreatment, vitrification, and canister decontamination/sealing. The HLW consists of insoluble metal hydroxides (primarily iron, aluminum, magnesium, manganese, and uranium) and soluble sodium salts (carbonate, hydroxide, nitrite, nitrate, sulfate). The pretreatment process acidifies the sludge with nitric and formic acids, adds the glass formers as glass frit, then concentrates the resulting slurry to approximately 50 weight percent (wt%) total solids. This slurry is fed to the joule-heated melter where the remaining water is evaporated followedmore » by calcination of the solids and conversion to glass. The Savannah River National Laboratory (SRNL) is currently assisting DWPF efforts to increase throughput of the melter. As part of this effort, SRNL has investigated methods to increase the solids content of the melter feed to reduce the heat load required to complete the evaporation of water and allow more of the energy available to calcine and vitrify the waste. The process equipment in the facility is fixed and cannot process materials with high yield stresses, therefore increasing the solids content will require that the yield stress of the melter feed slurries be reduced. Changing the glass former added during pretreatment from an irregularly shaped glass frit to nearly spherical beads was evaluated. The evaluation required a systems approach which included evaluations of the effectiveness of beads in reducing the melter feed yield stress as well as evaluations of the processing impacts of changing the frit morphology. Processing impacts of beads include changing the settling rate of the glass former (which effects mixing and sampling of the melter feed slurry and the frit addition equipment) as well as impacts on the melt behavior due to decreased surface area of the beads versus

Abbott ARCHITECT iPhenytoin assay versus similar assays for measuring free phenytoin concentrations.

PubMed

Tacker, Danyel Hermes; Robinson, Randy; Perrotta, Peter L

2014-01-01

To measure free phenytoin (FP) concentrations in filtered specimens using the Abbott ARCHITECT iPhenytoin assay and to compare results from this method with results from the Abbott TDx/FLx assays. We verified accuracy, analytic measurement range, and precision for FP measurements. For correlation and therapeutic interval studies, we used filtered calibrators, controls, proficiency-testing materials, and surplus clinical samples. After implementation, we determined proficiency testing results. The analytic measurement range was 2.0 to 25.0 micromol/L. Quality control materials (6.1, 12.6, and 20.1 micromol/L) provided mean (SD) recoveries of 96.1 (5.0%), 99.2 (5.0%), and 99.3 (5.7%), respectively, and coefficients of variation of 5.2%, 5.0%, and 5.8%, respectively. Clinical specimens produced mean (SD) FP recovery levels of 103.7 (10.6%) (bias, 0.1 [0.3] micromol/L). Altering the FP therapeutic range (4.0-8.0 micromol/L) was unnecessary. Proficiency testing yielded consistently acceptable results. Our accuracy, precision, and correlation results were similar for the TDx/FLx and ARCHITECT assays, which demonstrates that the ARCHITECT iPhenytoin assay is acceptable for clinical FP measurements.

Balancing Immunity and Yield in Crop Plants.

PubMed

Ning, Yuese; Liu, Wende; Wang, Guo-Liang

2017-12-01

Crop diseases cause enormous yield losses and threaten global food[ED1] security. The use of highly resistant cultivars can effectively control plant diseases, but in crops, genetic immunity to disease often comes with an unintended reduction in growth and yield. Here, we review recent advances in understanding how nucleotide-binding domain, leucine-rich repeat (NLR) receptors and cell wall-associated kinase (WAK) proteins function in balancing immunity and yield. We also discuss the role of plant hormones and transcription factors in regulating the trade-offs between plant growth and immunity. Finally, we describe how a novel mechanism of translational control of defense proteins can enhance immunity without the reduction in fitness. Copyright © 2017 Elsevier Ltd. All rights reserved.

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

PubMed

Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

2011-01-01

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

Reduction of tobacco smoke components yield in commercial cigarette brands by addition of HUSY, NaY and Al-MCM-41 to the cigarette rod.

PubMed

Marcilla, A; Gómez-Siurana, A; Berenguer, D; Martínez-Castellanos, I; Beltrán, M I

2015-01-01

The effect of two zeolites, HUSY, NaY and a mesoporous synthesized Al-MCM-41 material on the smoke composition of ten commercial cigarettes brands has been studied. Cigarettes were prepared by mixing the tobacco with the three powdered materials, and the smoke obtained under the ISO conditions was analyzed. Up to 32 compounds were identified and quantified in the gas fraction and 80 in the total particulate matter (TPM) condensed in the cigarettes filters and in the traps located after the mouth end of the cigarettes. Al-MCM-41 is by far the best additive, providing the highest reductions of the yield for most compounds and brands analyzed. A positive correlation was observed among the TPM and nicotine yields with the reduction obtained in nicotine, CO, and most compounds with the three additives. The amount of ashes in additive free basis increases due to the coke deposited on the solids, especially with Al-MCM-41. Nicotine is reduced with Al-MCM-41 by an average of 34.4% for the brands studied (49.5% for the brand where the major reduction was obtained and 18.5 for the brand behaving the worst). CO is reduced by an average of 18.6% (ranging from 10.3 to 35.2% in the different brands).

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

PubMed

Tsukatani, Tadayuki; Suenaga, Hikaru; Ishiyama, Munetaka; Ezoe, Takatoshi; Matsumoto, Kiyoshi

2011-07-15

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. Copyright © 2011 Elsevier Ltd. All rights reserved.

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

PubMed Central

Dilworth, M J; Eady, R R; Eldridge, M E

1988-01-01

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed. PMID:3162672

Impacts of aerosol pollutant mitigation on lowland rice yields in China

NASA Astrophysics Data System (ADS)

Zhang, Tianyi; Li, Tao; Yue, Xu; Yang, Xiaoguang

2017-10-01

Aerosol pollution in China is significantly altering radiative transfer processes and is thereby potentially affecting rice photosynthesis and yields. However, the response of rice photosynthesis to aerosol-induced radiative perturbations is still not well understood. Here, we employ a process-based modelling approach to simulate changes in incoming radiation (RAD) and the diffuse radiation fraction (DF) with aerosol mitigation in China and their associated impacts on rice yields. Aerosol reduction has the positive effect of increasing RAD and the negative effect of decreasing DF on rice photosynthesis and yields. In rice production areas where the average RAD during the growing season is lower than 250 W m-2, aerosol reduction is beneficial for higher rice yields, whereas in areas with RAD>250 W m-2, aerosol mitigation causes yield declines due to the associated reduction in the DF, which decreases the light use efficiency. As a net effect, rice yields were estimated to significantly increase by 0.8%-2.6% with aerosol concentrations reductions from 20 to 100%, which is lower than the estimates obtained in earlier studies that only considered the effects of RAD. This finding suggests that both RAD and DF are important processes influencing rice yields and should be incorporated into future assessments of agricultural responses to variations in aerosol-induced radiation under climate change.

Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water

PubMed Central

Shin, Gwy-Am; Sobsey, Mark D.

2003-01-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone. PMID:12839770

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water.

PubMed

Shin, Gwy-Am; Sobsey, Mark D

2003-07-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.

Study of water stress effects in different growth stages on yield and yield components of different rice (Oryza sativa L.) cultivars.

PubMed

Sarvestani, Zinolabedin Tahmasebi; Pirdashti, Hemmatollah; Sanavy, Seyed Ali Mohammad Modarres; Balouchi, Hamidreza

2008-05-15

A field experiment was conducted during 2001-2003 to evaluate the effect of water stress on the yield and yield components of four rice cultivars commonly grown in Mazandaran province, Iran. In northern Iran irrigated lowland rice usually experiences water deficit during the growing season include of land preparation time, planting, tillering stage, flowering and grain filing period. Recently drought affected 20 of 28 provinces in Iran; with the southeastern, central and eastern parts of the country being most severely affected. The local and improved cultivars used were Tarom, Khazar, Fajr and Nemat. The different water stress conditions were water stress during vegetative, flowering and grain filling stages and well watered was the control. Water stress at vegetative stage significantly reduced plant height of all cultivars. Water stress at flowering stage had a greater grain yield reduction than water stress at other times. The reduction of grain yield largely resulted from the reduction in fertile panicle and filled grain percentage. Water deficit during vegetative, flowering and grain filling stages reduced mean grain yield by 21, 50 and 21% on average in comparison to control respectively. The yield advantage of two semidwarf varieties, Fajr and Nemat, were not maintained under drought stress. Total biomass, harvest index, plant height, filled grain, unfilled grain and 1000 grain weight were reduced under water stress in all cultivars. Water stress at vegetative stage effectively reduced total biomass due to decrease of photosynthesis rate and dry matter accumulation.

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.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Nitric oxide alleviates wheat yield reduction by protecting photosynthetic system from oxidation of ozone pollution.

PubMed

Li, Caihong; Song, Yanjie; Guo, Liyue; Gu, Xian; Muminov, Mahmud A; Wang, Tianzuo

2018-05-01

Accelerated industrialization has been increasing releases of chemical precursors of ozone. Ozone concentration has risen nowadays, and it's predicted that this trend will continue in the next few decades. The yield of many ozone-sensitive crops suffers seriously from ozone pollution, and there are abundant reports exploring the damage mechanisms of ozone to these crops, such as winter wheat. However, little is known on how to alleviate these negative impacts to increase grain production under elevated ozone. Nitric oxide, as a bioactive gaseous, mediates a variety of physiological processes and plays a central role in response to biotic and abiotic stresses. In the present study, the accumulation of endogenous nitric oxide in wheat leaves was found to increase in response to ozone. To study the functions of nitric oxide, its precursor sodium nitroprusside was spayed to wheat leaves under ozone pollution. Wheat leaves spayed with sodium nitroprusside accumulated less hydrogen peroxide, malondialdehyde and electrolyte leakage under ozone pollution, which can be accounted for by the higher activities of superoxide dismutase and peroxidase than in leaves treated without sodium nitroprusside. Consequently, net photosynthetic rate of wheat treated using sodium nitroprusside was much higher, and yield reduction was alleviated under ozone fumigation. These findings are important for our understanding of the potential roles of nitric oxide in responses of crops in general and wheat in particular to ozone pollution, and provide a viable method to mitigate the detrimental effects on crop production induced by ozone pollution, which is valuable for keeping food security worldwide. Copyright © 2018 Elsevier Ltd. All rights reserved.

The effect of biomass densification on structural sugar release and yield in biofuel feedstock and feedstock blends

DOE PAGES

Wolfrum, Edward J.; Nagle, Nicholas J.; Ness, Ryan M.; ...

2017-01-13

In this work, we examined the behavior of feedstock blends and the effect of a specific feedstock densification strategy (pelleting) on the release and yield of structural carbohydrates in a laboratory-scale dilute acid pretreatment (PT) and enzymatic hydrolysis (EH) assay. We report overall carbohydrate release and yield from the two-stage PT-EH assay for five single feedstocks (two corn stovers, miscanthus, switchgrass, and hybrid poplar) and three feedstock blends (corn stover-switchgrass, corn stover-switchgrass-miscanthus, and corn stover-switchgrass-hybrid poplar). We first examined the experimental results over time to establish the robustness of the PT-EH assay, which limits the precision of the experimental results.more » The use of two different control samples in the assay enabled us to identify (and correct for) a small bias in the EH portion of the combined assay for some runs. We then examined the effect of variable pretreatment reaction conditions (residence time, acid loading, and reactor temperature) on the conversion of a single feedstock (single-pass corn stover, CS-SP) in order to establish the range of pretreatment reaction conditions likely to provide optimal conversion data. Finally, we applied the assay to the 16 materials (8 feedstocks in 2 formats, loose and pelleted) over a more limited range of pretreatment experimental conditions. The four herbaceous feedstocks behaved similarly, while the hybrid poplar feedstock required higher pretreatment temperatures for optimal results. As expected, the yield data for three blended feedstocks were the average of the yield data for the individual feedstocks. As a result, the pelleting process appears to provide a slightly positive effect on overall total sugar yield.« less

The effect of biomass densification on structural sugar release and yield in biofuel feedstock and feedstock blends

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolfrum, Edward J.; Nagle, Nicholas J.; Ness, Ryan M.

In this work, we examined the behavior of feedstock blends and the effect of a specific feedstock densification strategy (pelleting) on the release and yield of structural carbohydrates in a laboratory-scale dilute acid pretreatment (PT) and enzymatic hydrolysis (EH) assay. We report overall carbohydrate release and yield from the two-stage PT-EH assay for five single feedstocks (two corn stovers, miscanthus, switchgrass, and hybrid poplar) and three feedstock blends (corn stover-switchgrass, corn stover-switchgrass-miscanthus, and corn stover-switchgrass-hybrid poplar). We first examined the experimental results over time to establish the robustness of the PT-EH assay, which limits the precision of the experimental results.more » The use of two different control samples in the assay enabled us to identify (and correct for) a small bias in the EH portion of the combined assay for some runs. We then examined the effect of variable pretreatment reaction conditions (residence time, acid loading, and reactor temperature) on the conversion of a single feedstock (single-pass corn stover, CS-SP) in order to establish the range of pretreatment reaction conditions likely to provide optimal conversion data. Finally, we applied the assay to the 16 materials (8 feedstocks in 2 formats, loose and pelleted) over a more limited range of pretreatment experimental conditions. The four herbaceous feedstocks behaved similarly, while the hybrid poplar feedstock required higher pretreatment temperatures for optimal results. As expected, the yield data for three blended feedstocks were the average of the yield data for the individual feedstocks. As a result, the pelleting process appears to provide a slightly positive effect on overall total sugar yield.« less

Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

PubMed

Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

2015-08-01

Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

Proline catalyzed sequential α-aminooxylation or -amination/reductive cyclization of o-nitrohydrocinnamaldehydes: a high yield synthesis of chiral 3-substituted tetrahydroquinolines.

PubMed

Rawat, Varun; Kumar, B Senthil; Sudalai, Arumugam

2013-06-14

A new sequential organocatalytic method for the synthesis of chiral 3-substituted (X = OH, NH2) tetrahydroquinoline derivatives (THQs) [ee up to 99%, yield up to 87%] based on α-aminooxylation or -amination followed by reductive cyclization of o-nitrohydrocinnamaldehydes has been described. This methodology has been efficiently demonstrated in the synthesis of two important bioactive molecules namely (-)-sumanirole (96% ee) and 1-[(S)-3-(dimethylamino)-3,4-dihydro-6,7-dimethoxy-quinolin-1(2H)-yl]propanone (92% ee).

Water yield and hydrology

Treesearch

Pamela J. Edwards; Charles A. Troendle

2012-01-01

Investigations of hydrologic responses resulting from reducing vegetation density are fairly common throughout the Eastern United States. Although most studies have focused on the potential for increasing water yields or documenting effects from intensive practices that far exceed what would be done for fuel-reduction objectives, data from some less-intensive...

Fluorescence lifetime assays: current advances and applications in drug discovery.

PubMed

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

PubMed Central

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-01-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

PubMed

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-03-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

Studies of multi-ion-fluid yield anomaly in shock-driven implosions

NASA Astrophysics Data System (ADS)

Rinderknecht, H. G.; Rosenberg, M. J.; Li, C. K.; Zylstra, A. B.; Sio, H.; Gatu Johnson, M.; Frenje, J. A.; Séguin, F. H.; Petrasso, R. D.; Amendt, P. A.; Bellei, C.; Wilks, S. C.; Zimmerman, G.; Hoffman, N. M.; Kagan, G.; Molvig, K.; Glebov, V. Yu.; Stoeckl, C.; Marshall, F. J.; Seka, W.; Delettrez, J. A.; Sangster, T. C.; Betti, R.; Goncharov, V. N.; Meyerhofer, D. D.

2014-10-01

A. NIKROO, GA - Anomalously reduced yields relative to hydrodynamically calculated values have been observed for mixtures of D:3He compared to pure D2 gas-filled implosions in a series of shock-driven implosions at OMEGA. An extensive suite of measurements including temporal and spatial measurements of both the DD- and D3He-fusion reactions were obtained to identify the origin and physics behind this anomalous yield reduction. Measured spectral linewidths of fusion products suggest that the D ions are not thermalized to 3He during the burn, contributing to the reduced yield. The hypothesis that ion-species separation due to diffusive processes contributes to the observed yield reduction is explored using hydrodynamic simulations incorporating ion diffusion. Recent observations by Rosenberg et al. of a yield reduction with increased ion-ion mean free path do not explain the observed anomalous yield trend. Future work that will directly probe species separation with high-precision relative fusion reaction rate measurements between DD-neutrons and D3He-protons using the DualPTD instrument is discussed. This work was supported in part by the U.S. DOE, NLUF, LLE, and LLNL.

Rapid assay for microbially reducible ferric iron in aquatic sediments

USGS Publications Warehouse

Lovely, Derek R.; Philips , Elizabeth J.P.

1987-01-01

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.

PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS

EPA Science Inventory

A bactericidal assay developed to assess the ability of oyster (Crassostrea virginica) hemocytes to kill the human pathogen Vibrio parahaemolyticus was optimized to estimate killing of the oyster parasite Perkinsus marinus. Assay variables, temperature, hemocyte:parasite ratio, i...

The prognostic value of histidine-rich glycoprotein RNA in breast tissue using unmodified gold nanoparticles assay.

PubMed

Eissa, Sanaa; Azzazy, Hassan M E; Matboli, Marwa; Shawky, Sherif M; Said, Hebatallah; Anous, Fatin A

2014-09-01

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis.

Quality control of estrogen receptor assays.

PubMed

Godolphin, W; Jacobson, B

1980-01-01

Four types of material have been used for the quality control of routine assays of estrogen receptors in human breast tumors. Pieces of hormone-dependent Nb rat mammary tumors gave a precision about 40%. Rat uteri and rat tumors pulverized at liquid nitrogen temperature and stored as powder yielded precision about 30%. Powdered and lyophilised human tumors appear the best with precision as good as 17%.

Increase of chromium yield by slag reduction during production of chromium steels

NASA Astrophysics Data System (ADS)

Bažan, J.; Socha, L.; Kurka, V.; Jonšta, P.; Sušovský, M.

2017-02-01

The paper is focused on the evaluation of the course of Cr2O3 reduction from slag to alloyed steel under laboratory conditions. The experiments were aimed at the evaluation of increase in the chromium content in the melt together with the studying the behaviour of Cr2O3 and the mechanism of reduction by means of three reducing agents. Anthracite, ferrosilicon and mixtures of anthracite and ferrosilicon belong among the selected reducing agents. The experimental melts were focused on the proposal of a theoretical calculation of the consumption of selected reducing agents, study of reduction under laboratory conditions at application of alloyed steel with content of chromium of 12.16 wt. %, temperatures of 1600 °C and 1650 °C, together with the change of amount of reducing agents and reduction time. The results indicated in the paper constitute basic information on the possibilities of Cr2O3 reduction from slag; they will be used for verification of results in the pilot plant and operation experiments which will simulate operating conditions in the electric arc furnace.

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method.

PubMed

Bektaşoğlu, Burcu; Esin Celik, Saliha; Ozyürek, Mustafa; Güçlü, Kubilay; Apak, Reşat

2006-07-07

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more

The Importance of Juvenile Root Traits for Crop Yields

NASA Astrophysics Data System (ADS)

White, Philip; Adu, Michael; Broadley, Martin; Brown, Lawrie; Dupuy, Lionel; George, Timothy; Graham, Neil; Hammond, John; Hayden, Rory; Neugebauer, Konrad; Nightingale, Mark; Ramsay, Gavin; Thomas, Catherine; Thompson, Jacqueline; Wishart, Jane; Wright, Gladys

2014-05-01

Genetic variation in root system architecture (RSA) is an under-exploited breeding resource. This is partly a consequence of difficulties in the rapid and accurate assessment of subterranean root systems. However, although the characterisation of root systems of large plants in the field are both time-consuming and labour-intensive, high-throughput (HTP) screens of root systems of juvenile plants can be performed in the field, glasshouse or laboratory. It is hypothesised that improving the root systems of juvenile plants can accelerate access to water and essential mineral elements, leading to rapid crop establishment and, consequently, greater yields. This presentation will illustrate how aspects of the juvenile root systems of potato (Solanum tuberosum L.) and oilseed rape (OSR; Brassica napus L.) correlate with crop yields and examine the reasons for such correlations. It will first describe the significant positive relationships between early root system development, phosphorus acquisition, canopy establishment and eventual yield among potato genotypes. It will report the development of a glasshouse assay for root system architecture (RSA) of juvenile potato plants, the correlations between root system architectures measured in the glasshouse and field, and the relationships between aspects of the juvenile root system and crop yields under drought conditions. It will then describe the development of HTP systems for assaying RSA of OSR seedlings, the identification of genetic loci affecting RSA in OSR, the development of mathematical models describing resource acquisition by OSR, and the correlations between root traits recorded in the HTP systems and yields of OSR in the field.

Evidence of ATP assay as an appropriate alternative of MTT assay for cytotoxicity of secondary effluents from WWTPs.

PubMed

Yang, Yang; Lu, Yun; Wu, Qian-Yuan; Hu, Hong-Ying; Chen, Ying-Hua; Liu, Wan-Li

2015-12-01

Biological tests are effective and comprehensive methods to assess toxicity of environmental pollutants to ensure the safety of reclaimed water. In this study, the canonical MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cytotoxicity of dissolved organic matters (DOMs) of secondary effluents from wastewater treatment plants (WWTPs). It was surprising that most concentrated DOMs treated HepG2 cells yielded much higher signal compared with vehicle control regardless of difference of treatment technologies and seasons. However, there was actually no obvious enhancement of the cell proliferation by microscopy. In order to find out potential reason for the discrepancy, another three assays were performed. The results of ATP assay and flow cytometry showed expected toxicity, which was consistent with microscopy and previous studies, while DNA assay did not exhibit apparent change in treated cells. The possible mechanisms of abnormal MTT signal could be that some materials in secondary effluents isolated by solid extraction with HLB resin directly reacted with MTT and/or enhanced the activity of mitochondrial dehydrogenase. Therefore, the MTT assay is not suitable to assess cytotoxicity of complex mixtures such as secondary effluents, while ATP assay is an optional sensitive method. This study also suggests the importance of choosing both suitable extraction methods and detection assays for toxicity evaluation of component-unknown environmental samples. Copyright © 2015 Elsevier Inc. All rights reserved.

A single secreted luciferase-based gene reporter assay.

PubMed

Barriscale, Kathy A; O'Sullivan, Sharon A; McCarthy, Tommie V

2014-05-15

Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary. Copyright © 2014 Elsevier Inc. All rights reserved.

Hot spots of wheat yield decline with rising temperatures.

PubMed

Asseng, Senthold; Cammarano, Davide; Basso, Bruno; Chung, Uran; Alderman, Phillip D; Sonder, Kai; Reynolds, Matthew; Lobell, David B

2017-06-01

Many of the irrigated spring wheat regions in the world are also regions with high poverty. The impacts of temperature increase on wheat yield in regions of high poverty are uncertain. A grain yield-temperature response function combined with a quantification of model uncertainty was constructed using a multimodel ensemble from two key irrigated spring wheat areas (India and Sudan) and applied to all irrigated spring wheat regions in the world. Southern Indian and southern Pakistani wheat-growing regions with large yield reductions from increasing temperatures coincided with high poverty headcounts, indicating these areas as future food security 'hot spots'. The multimodel simulations produced a linear absolute decline of yields with increasing temperature, with uncertainty varying with reference temperature at a location. As a consequence of the linear absolute yield decline, the relative yield reductions are larger in low-yielding environments (e.g., high reference temperature areas in southern India, southern Pakistan and all Sudan wheat-growing regions) and farmers in these regions will be hit hardest by increasing temperatures. However, as absolute yield declines are about the same in low- and high-yielding regions, the contributed deficit to national production caused by increasing temperatures is higher in high-yielding environments (e.g., northern India) because these environments contribute more to national wheat production. Although Sudan could potentially grow more wheat if irrigation is available, grain yields would be low due to high reference temperatures, with future increases in temperature further limiting production. © 2016 John Wiley & Sons Ltd.

DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing.

PubMed

Austin, Melissa C; Smith, Christina; Pritchard, Colin C; Tait, Jonathan F

2016-02-01

Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. All samples with a volume greater than 8 mm(3) yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm(3) of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.

2011-09-10

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenousmore » nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.« less

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.

Strategy for continuous improvement in IC manufacturability, yield, and reliability

NASA Astrophysics Data System (ADS)

Dreier, Dean J.; Berry, Mark; Schani, Phil; Phillips, Michael; Steinberg, Joe; DePinto, Gary

1993-01-01

Continual improvements in yield, reliability and manufacturability measure a fab and ultimately result in Total Customer Satisfaction. A new organizational and technical methodology for continuous defect reduction has been established in a formal feedback loop, which relies on yield and reliability, failed bit map analysis, analytical tools, inline monitoring, cross functional teams and a defect engineering group. The strategy requires the fastest detection, identification and implementation of possible corrective actions. Feedback cycle time is minimized at all points to improve yield and reliability and reduce costs, essential for competitiveness in the memory business. Payoff was a 9.4X reduction in defectivity and a 6.2X improvement in reliability of 256 K fast SRAMs over 20 months.

Reductions in India's crop yield due to ozone

NASA Astrophysics Data System (ADS)

Ghude, Sachin D.; Jena, Chinmay; Chate, D. M.; Beig, G.; Pfister, G. G.; Kumar, Rajesh; Ramanathan, V.

2014-08-01

This bottom-up modeling study, supported by emission inventories and crop production, simulates ozone on local to regional scales. It quantifies, for the first time, potential impact of ozone on district-wise cotton, soybeans, rice, and wheat crops in India for the first decade of the 21st century. Wheat is the most impacted crop with losses of 3.5 ± 0.8 million tons (Mt), followed by rice at 2.1 ± 0.8 Mt, with the losses concentrated in central and north India. On the national scale, this loss is about 9.2% of the cereals required every year (61.2 Mt) under the provision of the recently implemented National Food Security Bill (in 2013) by the Government of India. The nationally aggregated yield loss is sufficient to feed about 94 million people living below poverty line in India.

Multi-site analytical evaluation of the Abbott ARCHITECT tacrolimus assay.

PubMed

Wallemacq, Pierre; Goffinet, Jean-Sebastien; O'Morchoe, Susan; Rosiere, Thomas; Maine, Gregory T; Labalette, Myriam; Aimo, Giuseppe; Dickson, Diana; Schmidt, Ed; Schwinzer, Reinhard; Schmid, Rainer W

2009-04-01

The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Tacrolimus immunoassay. Proficiency panels and specimens from a population of organ transplant recipients were analyzed in 6 clinical laboratories in Europe and the United States, and the results were compared with other methods. The ARCHITECT assay requires a whole blood specimen pretreatment step with methanol/zinc sulfate to precipitate protein and extract the drug, followed by a 30-minute immunoassay using anti-tacrolimus antibody-coated paramagnetic microparticles and an acridinium-tacrolimus tracer. The assay was free from hematocrit interference in the range 25%-55% and from interference by extremes of cholesterol, triglycerides, bilirubin, total protein, and uric acid. The total percent of coefficient of variations of the assay were 4.9%-7.6% at 3 ng/mL, 2.9%-4.6% at 8.6 ng/mL, and 3.1%-8.2% at 15.5 ng/mL. Limit of detection was < or =0.5 ng/mL and limit of quantification (LOQ) ranged from 0.69 to 1.07 ng/mL across the 6 sites (based on the upper 95% confidence interval concentrations). The 2007 European Consensus Conference on Tacrolimus Optimization recommended the use of assay methods with an LOQ around 1 ng/mL, based upon the need to measure trough tacrolimus blood concentrations precisely down to 3 ng/mL during low-dose tacrolimus regimens. Tacrolimus International Proficiency Testing Scheme samples were measured by the ARCHITECT immunoassay at 5 sites and showed an average bias of -0.28 to +0.85 ng/mL versus IMx Tacrolimus II immunoassay historical values and -0.21 to +0.68 ng/mL versus liquid chromatography/tandem mass spectrometry (LC-MSMS) Tacrolimus historical values. Method comparison studies were performed with the ARCHITECT Tacrolimus immunoassay on patient specimens with the following results: ARCHITECT Tacrolimus assay versus the Abbott IMx Tacrolimus II immunoassay (4 sites) yielded average biases between -0.94 and +0.26 ng/mL; ARCHITECT assay

Heterophilic interference in specimens yielding false-reactive results on the Abbott 4th generation ARCHITECT HIV Ag/Ab Combo assay.

PubMed

Lavoie, S; Caswell, D; Gill, M J; Kadkhoda, K; Charlton, C L; Levett, P N; Hatchette, T; Garceau, R; Maregmen, J; Mazzulli, T; Needle, R; Kadivar, K; Kim, J

2018-07-01

False-reactivity in HIV-negative specimens has been detected in HIV fourth-generation antigen/antibody or 'combo' assays which are able to detect both anti-HIV-1/HIV-2 antibodies and HIV-1 antigen. We sought to characterize these specimens and determine the effect of heterophilic interference. Specimens previously testing as false-reactive on the Abbott ARCHITECT HIV Ag/Ab combo assay and re-tested on a different (Siemens ADVIA Centaur HIV Ag/Ab) assay. A subset of these specimens were also pre-treated with heterophilic blocking agents and re-tested on the Abbott assay. Here we report that 95% (252/264) of clinical specimens that were repeatedly reactive on the Abbott ARCHITECT HIV Ag/Ab combo assay (S/Co range, 0.94-678) were negative when re-tested on a different fourth generation HIV combo assay (Siemens ADVIA Centaur HIV Ag/Ab). All 264 samples were subsequently confirmed to be HIV negative. On a small subset (57) of specimens with available volume, pre-treatment with two different reagents (HBT; Heterophilic Blocking Tube, NABT; Non-Specific Blocking Tube) designed to block heterophilic antibody interference either eliminated (HBT) or reduced (NABT) the false reactivity when re-tested on the ARCHITECT HIV Ag/Ab combo assay. Our results suggest that the Abbott ARCHITECT HIV Ag/Ab combo assay can be prone to heterophilic antibody interference. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

A new method for the simultaneous enhancement of methane yield and reduction of hydrogen sulfide production in the anaerobic digestion of waste activated sludge.

PubMed

Dai, Xiaohu; Hu, Chongliang; Zhang, Dong; Chen, Yinguang

2017-11-01

The biogas generated from anaerobic digestion (AD) also includes undesirable by-product such as hydrogen sulfide (H 2 S), which must be removed before the biogas can be used as a clean energy source. Therefore, it is necessary to find an appropriate strategy to simultaneously enhance the methane yield and reduce H 2 S production. An efficient strategy-pretreating sludge at pH 10 for 8d and adjusting the system at neutral pH to produce methane for 20d-is reported for the synchronous enhancement of methane production and reduction of H 2 S production during AD. The experimental results showed that the cumulative methane yield was 861.2±6.1mL/g volatile solids (VS) of sludge pretreated at pH 10 in semi-continuous stirred anaerobic reactors for 84d, an increase of 49.6% over the yield in the control. Meanwhile, the cumulative production of H 2 S was 144.1×10 -4 mL/g VS, 54.2% lower than that in the control. Copyright © 2017 Elsevier Ltd. All rights reserved.

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results. PMID:3032390

Comparison of the Chiron Quantiplex branched DNA (bDNA) assay and the Abbott Genostics solution hybridization assay for quantification of hepatitis B viral DNA.

PubMed

Kapke, G E; Watson, G; Sheffler, S; Hunt, D; Frederick, C

1997-01-01

Several assays for quantification of DNA have been developed and are currently used in research and clinical laboratories. However, comparison of assay results has been difficult owing to the use of different standards and units of measurements as well as differences between assays in dynamic range and quantification limits. Although a few studies have compared results generated by different assays, there has been no consensus on conversion factors and thorough analysis has been precluded by small sample size and limited dynamic range studied. In this study, we have compared the Chiron branched DNA (bDNA) and Abbott liquid hybridization assays for quantification of hepatitis B virus (HBV) DNA in clinical specimens and have derived conversion factors to facilitate comparison of assay results. Additivity and variance stabilizing (AVAS) regression, a form of non-linear regression analysis, was performed on assay results for specimens from HBV clinical trials. Our results show that there is a strong linear relationship (R2 = 0.96) between log Chiron and log Abbott assay results. Conversion factors derived from regression analyses were found to be non-constant and ranged from 6-40. Analysis of paired assay results below and above each assay's limit of quantification (LOQ) indicated that a significantly (P < 0.01) larger proportion of observations were below the Abbott assay LOQ but above the Chiron assay LOQ, indicating that the Chiron assay is significantly more sensitive than the Abbott assay. Testing of replicate specimens showed that the Chiron assay consistently yielded lower per cent coefficients of variance (% CVs) than the Abbott assay, indicating that the Chiron assay provides superior precision.

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay

PubMed Central

Logue, James; Tuznik, Kaylie; Follmann, Dean; Grandits, Greg; Marchand, Jonathan; Reilly, Cavan; Sarro, Yeya dit Sadio; Pettitt, James; Stavale, Eric J.; Fallah, Mosoka; Olinger, Gene G.; Bolay, Fatorma K.; Hensley, Lisa E.

2018-01-01

As part of the scientific community’s development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I–III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity. PMID:29481881

Reductive methods for isotopic labeling of antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Champney, W.S.

1989-08-15

Methods for the reductive methylation of the amino groups of eight different antibiotics using {sup 3}HCOH or H{sup 14}COH are presented. The reductive labeling of an additional seven antibiotics by NaB{sub 3}H{sub 4} is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Establishing Assay Cutoffs for HLA Antibody Screening of Apheresis Donors

PubMed Central

Carrick, Danielle M.; Norris, Philip J.; Endres, Robert O.; Pandey, Suchitra; Kleinman, Steven H.; Wright, David; Sun, Yu; Busch, Michael P.

2011-01-01

BACKGROUND TRALI is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability. STUDY DESIGN Pregnancy history and HLA antibody screening and single antigen bead (SAB) data from blood donors in the REDS-II Leukocyte Antibody Prevalence Study (LAPS) were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody reactive donations and loss of donors and donations. RESULTS We provide evidence that higher HLA Ab screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending upon the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%. CONCLUSION This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis platelets that is consistent with how much donation loss the blood center can tolerate. PMID:21332726

Comparison of the Second-Generation Digene Hybrid Capture Assay with the Branched-DNA Assay for Measurement of Hepatitis B Virus DNA in Serum

PubMed Central

Ho, Stephen K. N.; Chan, Tak Mao; Cheng, Ignatius K. P.; Lai, Kar Neng

1999-01-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 ± 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 × [HBV DNA by bDNA (megaequivalents per milliliter)]0.866. The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants. PMID:10405385

Comparison of the second-generation digene hybrid capture assay with the branched-DNA assay for measurement of hepatitis B virus DNA in serum.

PubMed

Ho, S K; Chan, T M; Cheng, I K; Lai, K N

1999-08-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 +/- 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 x [HBV DNA by bDNA (megaequivalents per milliliter)](0.866). The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants.

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

USGS Publications Warehouse

Stephen J. Amish,; Paul A. Hohenlohe,; Sally Painter,; Robb F. Leary,; Muhlfeld, Clint C.; Fred W. Allendorf,; Luikart, Gordon

2012-01-01

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

Green synthesis of gold and silver nanoparticles using gallic acid: catalytic activity and conversion yield toward the 4-nitrophenol reduction reaction

NASA Astrophysics Data System (ADS)

Park, Jisu; Cha, Song-Hyun; Cho, Seonho; Park, Youmie

2016-06-01

In the present report, gallic acid was used as both a reducing and stabilizing agent to synthesize gold and silver nanoparticles. The synthesized gold and silver nanoparticles exhibited characteristic surface plasmon resonance bands at 536 and 392 nm, respectively. Nanoparticles that were approximately spherical in shape were observed in high-resolution transmission electron microscopy and atomic force microscopy images. The hydrodynamic radius was determined to be 54.4 nm for gold nanoparticles and 33.7 nm for silver nanoparticles in aqueous medium. X-ray diffraction analyses confirmed that the synthesized nanoparticles possessed a face-centered cubic structure. FT-IR spectra demonstrated that the carboxylic acid functional groups of gallic acid contributed to the electrostatic binding onto the surface of the nanoparticles. Zeta potential values of -41.98 mV for the gold nanoparticles and -53.47 mV for the silver nanoparticles indicated that the synthesized nanoparticles possess excellent stability. On-the-shelf stability for 4 weeks also confirmed that the synthesized nanoparticles were quite stable without significant changes in their UV-visible spectra. The synthesized nanoparticles exhibited catalytic activity toward the reduction reaction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride. The rate constant of the silver nanoparticles was higher than that of the gold nanoparticles in the catalytic reaction. Furthermore, the conversion yield (%) of 4-nitrophenol to 4-aminophenol was determined using reversed-phase high-performance liquid chromatography with UV detection at 254 nm. The silver nanoparticles exhibited an excellent conversion yield (96.7-99.9 %), suggesting that the synthesized silver nanoparticles are highly efficient catalysts for the 4-nitrophenol reduction reaction.

[Predicting the impact of climate change in the next 40 years on the yield of maize in China].

PubMed

Ma, Yu-ping; Sun, Lin-li; E, You-hao; Wu, Wei

2015-01-01

Climate change will significantly affect agricultural production in China. The combination of the integral regression model and the latest climate projection may well assess the impact of future climate change on crop yield. In this paper, the correlation model of maize yield and meteorological factors was firstly established for different provinces in China by using the integral regression method, then the impact of climate change in the next 40 years on China's maize production was evaluated combined the latest climate prediction with the reason be ing analyzed. The results showed that if the current speeds of maize variety improvement and science and technology development were constant, maize yield in China would be mainly in an increasing trend of reduction with time in the next 40 years in a range generally within 5%. Under A2 climate change scenario, the region with the most reduction of maize yield would be the Northeast except during 2021-2030, and the reduction would be generally in the range of 2.3%-4.2%. Maize yield reduction would be also high in the Northwest, Southwest and middle and lower reaches of Yangtze River after 2031. Under B2 scenario, the reduction of 5.3% in the Northeast in 2031-2040 would be the greatest across all regions. Other regions with considerable maize yield reduction would be mainly in the Northwest and the Southwest. Reduction in maize yield in North China would be small, generally within 2%, under any scenarios, and that in South China would be almost unchanged. The reduction of maize yield in most regions would be greater under A2 scenario than under B2 scenario except for the period of 2021-2030. The effect of the ten day precipitation on maize yield in northern China would be almost positive. However, the effect of ten day average temperature on yield of maize in all regions would be generally negative. The main reason of maize yield reduction was temperature increase in most provinces but precipitation decrease in a few

The decrease in yield strength in NiAl due to hydrostatic pressure

NASA Technical Reports Server (NTRS)

Margevicius, R. W.; Lewandowski, J. J.; Locci, I.

1992-01-01

The decrease in yield strength in NiAl due to hydrostatic pressure is examined via a comparison of the tensile flow behavior in the low strain regime at 0.1 MPa for NiAl which was cast, extruded, and annealed for 2 hr at 827 C in argon and very slowly cooled to room temperature. Pressurization to 1.4 GPa produces a subsequent reduction at 0.1 MP in proportional limit by 40 percent as well as a 25-percent reduction in the 0.2-percent offset yield strength, while pressurization with lower pressures produces a similar reduction, although smaller in magnitude.

Interaction of nucleic acids with Coomassie Blue G-250 in the Bradford assay.

PubMed

Wenrich, Broc R; Trumbo, Toni A

2012-09-15

The Bradford assay has been used reliably for decades to quantify protein in solution. The analyte is incubated in acidic solution of Coomassie Blue G-250 dye, during which reversible ionic and nonionic binding interactions form. Bradford assay color yields were determined for salmon, bovine, shrimp, and kiwi fruit genomic DNA; baker's yeast RNA; bovine serum albumin (BSA); and hen egg lysozyme. Pure DNA and RNA bound the dye, with color yields of 0.0017 mg⁻¹ cm⁻¹ and 0.0018 mg⁻¹ cm⁻¹, respectively. The nucleic acid-Coomassie Blue response was significant, at roughly 9% of that for BSA and 18% of that for lysozyme. Copyright © 2012 Elsevier Inc. All rights reserved.

Manganese catalyzed reductive amination of aldehydes using hydrogen as a reductant.

PubMed

Wei, Duo; Bruneau-Voisine, Antoine; Valyaev, Dmitry A; Lugan, Noël; Sortais, Jean-Baptiste

2018-04-24

A one-pot two-step procedure was developed for the alkylation of amines via reductive amination of aldehydes using molecular dihydrogen as a reductant in the presence of a manganese pyridinyl-phosphine complex as a pre-catalyst. After the initial condensation step, the reduction of imines formed in situ is performed under mild conditions (50-100 °C) with 2 mol% of catalyst and 5 mol% of tBuOK under 50 bar of hydrogen. Excellent yields (>90%) were obtained for a large combination of aldehydes and amines (40 examples), including aliphatic aldehydes and amino-alcohols.

Incremental Yield of Including Determine-TB LAM Assay in Diagnostic Algorithms for Hospitalized and Ambulatory HIV-Positive Patients in Kenya.

PubMed

Huerga, Helena; Ferlazzo, Gabriella; Bevilacqua, Paolo; Kirubi, Beatrice; Ardizzoni, Elisa; Wanjala, Stephen; Sitienei, Joseph; Bonnet, Maryline

2017-01-01

Determine-TB LAM assay is a urine point-of-care test useful for TB diagnosis in HIV-positive patients. We assessed the incremental diagnostic yield of adding LAM to algorithms based on clinical signs, sputum smear-microscopy, chest X-ray and Xpert MTB/RIF in HIV-positive patients with symptoms of pulmonary TB (PTB). Prospective observational cohort of ambulatory (either severely ill or CD4<200cells/μl or with Body Mass Index<17Kg/m2) and hospitalized symptomatic HIV-positive adults in Kenya. Incremental diagnostic yield of adding LAM was the difference in the proportion of confirmed TB patients (positive Xpert or MTB culture) diagnosed by the algorithm with LAM compared to the algorithm without LAM. The multivariable mortality model was adjusted for age, sex, clinical severity, BMI, CD4, ART initiation, LAM result and TB confirmation. Among 474 patients included, 44.1% were severely ill, 69.6% had CD4<200cells/μl, 59.9% had initiated ART, 23.2% could not produce sputum. LAM, smear-microscopy, Xpert and culture in sputum were positive in 39.0% (185/474), 21.6% (76/352), 29.1% (102/350) and 39.7% (92/232) of the patients tested, respectively. Of 156 patients with confirmed TB, 65.4% were LAM positive. Of those classified as non-TB, 84.0% were LAM negative. Adding LAM increased the diagnostic yield of the algorithms by 36.6%, from 47.4% (95%CI:39.4-55.6) to 84.0% (95%CI:77.3-89.4%), when using clinical signs and X-ray; by 19.9%, from 62.2% (95%CI:54.1-69.8) to 82.1% (95%CI:75.1-87.7), when using clinical signs and microscopy; and by 13.4%, from 74.4% (95%CI:66.8-81.0) to 87.8% (95%CI:81.6-92.5), when using clinical signs and Xpert. LAM positive patients had an increased risk of 2-months mortality (aOR:2.7; 95%CI:1.5-4.9). LAM should be included in TB diagnostic algorithms in parallel to microscopy or Xpert request for HIV-positive patients either ambulatory (severely ill or CD4<200cells/μl) or hospitalized. LAM allows same day treatment initiation in patients at

Nitrogen rate strategies for reducing yield-scaled nitrous oxide emissions in maize

NASA Astrophysics Data System (ADS)

Zhao, Xu; Nafziger, Emerson D.; Pittelkow, Cameron M.

2017-12-01

Mitigating nitrogen (N) losses from agriculture without negatively impacting crop productivity is a pressing environmental and economic challenge. Reductions in N fertilizer rate are often highlighted as a solution, yet the degree to which crop yields and economic returns may be impacted at the field-level remains unclear, in part due to limited data availability. Farmers are risk averse and potential yield losses may limit the success of voluntary N loss mitigation protocols, thus understanding field-level yield tradeoffs is critical to inform policy development. Using a case study of soil N2O mitigation in the US Midwest, we conducted an ex-post assessment of two economic and two environmental N rate reduction strategies to identify promising practices for maintaining maize yields and economic returns while reducing N2O emissions per unit yield (i.e. yield-scaled emissions) compared to an assumed baseline N input level. Maize yield response data from 201 on-farm N rate experiments were combined with an empirical equation predicting N2O emissions as a function of N rate. Results indicate that the economic strategy aimed at maximizing returns to N (MRTN) led to moderate but consistent reductions in yield-scaled N2O emissions with small negative impacts on yield and slight increases in median returns. The economic optimum N rate strategy reduced yield-scaled N2O emissions in 75% of cases but increased them otherwise, challenging the assumption that this strategy will automatically reduce environmental impacts per unit production. Both environmental strategies, one designed to increase N recovery efficiency and one to balance N inputs with grain N removal, further reduced yield-scaled N2O emissions but were also associated with negative yield penalties and decreased returns. These results highlight the inherent tension between achieving agronomic and economic goals while reducing environmental impacts which is often overlooked in policy discussions. To enable the

The impact exploration of agricultural drought on winter wheat yield in the North China Plain

NASA Astrophysics Data System (ADS)

Yang, Jianhua; Wu, Jianjun; Han, Xinyi; Zhou, Hongkui

2017-04-01

Drought is one of the most serious agro-climatic disasters in the North China Plain, which has a great influence on winter wheat yield. Global warming exacerbates the drought trend of this region, so it is important to study the effect of drought on winter wheat yield. In order to assess the drought-induced winter wheat yield losses, SPEI (standardized precipitation evapotranspiration index), the widely used drought index, was selected to quantify the drought from 1981 to 2013. Additionally, the EPIC (Environmental Policy Integrated Climate) crop model was used to simulate winter wheat yield at 47 stations in this region from 1981 to 2013. We analyzed the relationship between winter wheat yield and the SPEI at different time scales in each month during the growing season. The trends of the SPEI and the trends of winter wheat yield at 47 stations over the past 32 years were compared with each other. To further quantify the effect of drought on winter wheat yield, we defined the year that SPEI varied from -0.5 to 0.5 as the normal year, and calculated the average winter wheat yield of the normal years as a reference yield, then calculated the reduction ratios of winter wheat based on the yields mentioned above in severe drought years. As a reference, we compared the results with the reduction ratios calculated from the statistical yield data. The results showed that the 9 to 12-month scales' SPEI in April, May and June had a high correlation with winter wheat yield. The trends of the SPEI and the trends of winter wheat yield over the past 32 years showed a positive correlation (p<0.01) and have similar spatial distributions. The proportion of the stations with the same change trend between the SPEI and winter wheat yield was 70%, indicating that drought was the main factor leading to a decline in winter wheat yield in this region. The reduction ratios based on the simulated yield and the reduction ratios calculated from the statistical yield data have a high positive

Toward an Economic Definition of Sustainable Yield for Coastal Aquifers

NASA Astrophysics Data System (ADS)

Jenson, J. W.; Habana, N. C.; Lander, M.

2016-12-01

The concept of aquifer sustainable yield has long been criticized, debated, and even disparaged among groundwater hydrologists, but policy-makers and professional water resource managers inevitably ask them for unequivocal answers to such questions as "What is the absolute maximum volume of water that could be sustainably withdrawn from this aquifer?" We submit that it is therefore incumbent upon hydrologists to develop and offer valid practical definitions of sustainable yield that can be usefully applied to given conditions and types of aquifers. In coastal aquifers, water quality—in terms of salinity—is affected by changes in the natural water budget and the volume rate of artificial extraction. In principle, one can identify a family of assay curves for a given aquifer, showing the specific relationships between the quantity and quality of the water extracted under given conditions of recharge. The concept of the assay curve, borrowed from the literature of natural-resource extraction economics, has to our knowledge not yet found its way into the literature of applied hydrology. The relationships between recharge, extraction, and water quality that define the assay curve can be determined empirically from sufficient observations of groundwater response to recharge and extraction and can be estimated from models that have been reliably history-matched ("calibrated") to such data. We thus propose a working definition of sustainable yield for coastal aquifers in terms of the capacity that ultimately could be achieved by an ideal production system, given what is known or can be assumed about the natural limiting conditions. Accordingly, we also offer an approach for defining an ideal production system for a given aquifer, and demonstrate how observational data and/or modeling results can be used to develop assay curves of quality vs. quantity extracted, which can serve as reliable predictive tools for engineers, managers, regulators, and policy

[Comparison of the clinical performance of the ECLusys HBsAg II assay with the Lumipulse f and HISCL 2000-i HBsAg screening assays].

PubMed

Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

2012-02-01

We compared the ECLusys HBsAgII (ECL HBsAg) assay to the Lumipulse Forte (LPf HBsAg) and HISCL (HIS HBsAg) assays. Measurement of dilution panels for which the WHO HBsAg international reference panel was the parent specimen revealed that the ECL and HIS assays enabled detection to a theoretical level of 0.04 IU/mL, whereas the LPf assay enabled detection to a level of 0.08 IU/mL. In a specificity test using high RF positive specimens (n = 33), pregnancy specimens (n = 35), cytomegalovirus antibody positive specimens (n = 36), and high M protein positive specimens (n = 21) that were confirmed negative for HBsAg by the LPf assay, negative results were obtained for all specimens on the HIS assay, but the ECL assay yielded a positive result for one of the high RF positive specimens. This individual was suggested on further testing to be an HBV carrier who was strongly positive for HBc antibody. In HBsAg mutants detection test, the detection rate was 92.3% with the ECL assay and 69.2% with the HIS assay. In a correlation test using routinely collected clinical specimens (n = 155), including positive stock specimens, aside from the one case where the LPf assay gave a negative result but both the ECL and HIS assays gave positive results, all of the results were consistent for all specimens. The above results confirmed that the ECL assay is both highly sensitive and specific, and also enables a high rate of HBsAg mutant detection.

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

PubMed Central

Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

2015-01-01

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox. PMID:26426117

Multicenter evaluation of the Bayer Immuno I CA 15-3 assay.

PubMed

Cheli, C D; Morris, D L; Kish, L; Goldblatt, J; Neaman, I; Allard, W J; Yeung, K K; Wu, A H; Moore, R; Chan, D W; Fritsche, H A; Schwartz, M K; Very, D L

1998-04-01

We conducted a multicenter evaluation of the analytical and clinical features of the automated Bayer Immuno 1 CA 15-3 assay and compared assay performance to two manual tests. Results of the 10-day imprecision study of the Bayer Immuno 1 assay pooled across four evaluation sites and three lots of reagent produced total CV < or = 4%. Lot-to-lot reproducibility for 26 different lots of reagents and calibrators manufactured over a 2-year period was demonstrated (CV, 1.1%). Results for the Bayer Immuno 1 assay correlated well with the Biomira TRUQUANT BR 27.29 and Centocor CA 15-3 RIAs (r > or = 0.94). The upper limit of the reference interval for the Bayer Immuno 1 assay was 35.9 kilounits/L (35.9 units/mL); values were similar for all methods. Longitudinal monitoring of healthy women yielded assay values with an average CV of 11% and 21% for the Bayer Immuno 1 and Biomira assays, respectively. The Bayer Immuno 1 assay demonstrated the analytical features, intermethod correlation, and long-term performance characteristics that are essential for longitudinal monitoring of breast cancer patients.

Ochratoxin A reduction in meat sausages using processing methods practiced in households.

PubMed

Pleadin, Jelka; Perši, Nina; Kovačević, Dragan; Vulić, Ana; Frece, Jadranka; Markov, Ksenija

2014-01-01

The aim of this study was to investigate the possibilities of ochratoxin A (OTA) reduction in home-made meat products. Meat sausages (n = 50) produced from raw materials coming from pigs exposed to OTA-contaminated feed, were subject to common heat processes practiced in households (cooking, frying and baking). Concentrations of OTA in pre- and post-processed products were quantified using a validated immunoassay method, enzyme-linked immunosorbent assay, and confirmed using a high-performance liquid chromatography with fluorescence detection. In line with the differences in recipes used and the degree of OTA accumulation in raw materials, OTA concentrations established in Mediterranean and roast sausages were lower than those found in liver and blood sausages. Baking of contaminated sausages at the temperatures of 190-220°C (for 60 min) resulted in significant reduction of OTA levels (75.8%), while 30-min cooking (at 100°C) and frying (at 170°C) proved to be significantly less effective (e.g. yielding OTA reductions of 7.4% and 12.6%, respectively). The results pointed out that despite high OTA stability, heat processes are capable of reducing its concentration in home-made meat products, depending on the processing modality used.

In vitro versus in vivo concordance: a case study of the replacement of an animal potency test with an immunochemical assay.

PubMed

Schofield, T

2002-01-01

Early in its development, the potency of Merck's recombinant hepatitis B vaccine, RECOMBIVAX HB, was monitored using an assay performed in mice. A specification was determined to be the lowest potency which induced acceptable response in clinical trials. As a post-licensing commitment, Merck was asked to replace its mouse potency assay with an in vitro procedure for product release in the US market. Early studies with a commercial enzyme immunoassay (EIA) yielded highly variable results. That assay, combined with a sample pretreatment step, proved more dependable and predictive of potency in the mouse assay. Based on measurements made on manufactured materials, combined with experiments contrived to yield a wide range of reactivity in the two assays, concordance was established between the EIA and the mouse potency assay. This concordance was used to calibrate a specification for the in vitro assay that is predictive of a satisfactory response in vivo. Data from clinical trials established a correspondence between human immunogenicity and these potency markers.

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay.

PubMed

Logue, James; Tuznik, Kaylie; Follmann, Dean; Grandits, Greg; Marchand, Jonathan; Reilly, Cavan; Sarro, Yeya Dit Sadio; Pettitt, James; Stavale, Eric J; Fallah, Mosoka; Olinger, Gene G; Bolay, Fatorma K; Hensley, Lisa E

2018-05-01

As part of the scientific community's development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I-III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Anomalous yield reduction in direct-drive DT implosions due to 3He addition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herrmann, Hans W; Langenbrunner, James R; Mack, Joseph M

2008-01-01

Glass capsules were imploded in direct drive on the OMEGA laser [T. R. Boehly et aI., Opt. Commun. 133, 495, 1997] to look for anomalous degradation in deuterium/tritium (DT) yield (i.e., beyond what is predicted) and changes in reaction history with {sup 3}He addition. Such anomalies have previously been reported for D/{sup 3}He plasmas, but had not yet been investigated for DT/{sup 3}He. Anomalies such as these provide fertile ground for furthering our physics understanding of ICF implosions and capsule performance. A relatively short laser pulse (600 ps) was used to provide some degree of temporal separation between shock andmore » compression yield components for analysis. Anomalous degradation in the compression component of yield was observed, consistent with the 'factor of two' degradation previously reported by MIT at a 50% {sup 3}He atom fraction in D{sub 2} using plastic capsules [Rygg et aI., Phys. Plasmas 13, 052702 (2006)]. However, clean calculations (i.e., no fuel-shell mixing) predict the shock component of yield quite well, contrary to the result reported by MIT, but consistent with LANL results in D{sub 2}/{sup 3}He [Wilson, et aI., lml Phys: Conf Series 112, 022015 (2008)]. X-ray imaging suggests less-than-predicted compression ofcapsules containing {sup 3}He. Leading candidate explanations are poorly understood Equation-of-State (EOS) for gas mixtures, and unanticipated particle pressure variation with increasing {sup 3}He addition.« less

The Effect of Carbonaceous Reductant Selection on Chromite Pre-reduction

NASA Astrophysics Data System (ADS)

Kleynhans, E. L. J.; Beukes, J. P.; Van Zyl, P. G.; Bunt, J. R.; Nkosi, N. S. B.; Venter, M.

2017-04-01

Ferrochrome (FeCr) production is an energy-intensive process. Currently, the pelletized chromite pre-reduction process, also referred to as solid-state reduction of chromite, is most likely the FeCr production process with the lowest specific electricity consumption, i.e., MWh/t FeCr produced. In this study, the effects of carbonaceous reductant selection on chromite pre-reduction and cured pellet strength were investigated. Multiple linear regression analysis was employed to evaluate the effect of reductant characteristics on the aforementioned two parameters. This yielded mathematical solutions that can be used by FeCr producers to select reductants more optimally in future. Additionally, the results indicated that hydrogen (H)- (24 pct) and volatile content (45.8 pct) were the most significant contributors for predicting variance in pre-reduction and compressive strength, respectively. The role of H within this context is postulated to be linked to the ability of a reductant to release H that can induce reduction. Therefore, contrary to the current operational selection criteria, the authors believe that thermally untreated reductants ( e.g., anthracite, as opposed to coke or char), with volatile contents close to the currently applied specification (to ensure pellet strength), would be optimal, since it would maximize H content that would enhance pre-reduction.

Gold nanoparticles mediated colorimetric assay for HIV-Tat protein detection

NASA Astrophysics Data System (ADS)

Hashwan, Saeed S. Ba; Ruslinda, A. Rahim; Fatin, M. F.; Gopinath, Subash C. B.; Thivina, V.; Tony, V. C. S.; Arshad, M. K. Md.; Hashim, U.

2016-07-01

Gold-nanoparticle (AuNP) based colorimetric assays have been formulated for different biomolecular interactions. With this assay the probe such as antibody immobilized on the Au surface and in the presence of appropriate binding partner (antigen), will interact with each other on the Au surface. By following this strategy, herein we formulated a detection system with two anti-HIV-Tat antibodies, Mono (McAb) - and polyclonal (PcAb) by immobilizing them independently with different AuNPs. Under this condition, these two antibodies are under dispersed condition, and in the presence of HIV-Tat antigen, these molecules will be connected and forms the aggregation of AuNPs. This strategy yield rapid results, can be monitored by the spectral changes in UV-Vis spectrophotometry. Experiments were performed with two different methods using two anti-HIV-Tats monoclonal and one Polyclonal antibody against the antigen HIV-Tat. Between these methods conjugation of HIV-Tat and McAb on the AuNP followed by addition of PcAb yielded better results.

Comparison of bioluminescent kinase assays using substrate depletion and product formation.

PubMed

Tanega, Cordelle; Shen, Min; Mott, Bryan T; Thomas, Craig J; MacArthur, Ryan; Inglese, James; Auld, Douglas S

2009-12-01

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

Comparison of amino acid digestibility of feedstuffs determined with the precision-fed cecectomized rooster assay and the standardized ileal amino acid digestibility assay.

PubMed

Kim, E J; Utterback, P L; Applegate, T J; Parsons, C M

2011-11-01

The objective of this study was to evaluate and compare amino acid digestibility of several feedstuffs using 2 commonly accepted methods: the precision-fed cecectomized rooster assay (PFR) and the standardized ileal amino acid assay (SIAAD). Six corn, 6 corn distillers dried grains with or without solubles (DDGS/DDG), one wet distillers grains, one condensed solubles, 2 meat and bone meal (MBM) and a poultry byproduct meal were evaluated. Due to insufficient amounts, the wet distillers grains and condensed solubles were only evaluated in roosters. Standardized amino acid digestibility varied among the feed ingredients and among samples of the same ingredient for both methods. For corn, there were generally no differences in amino acid digestibility between the 2 methods. When differences did occur, there was no consistent pattern among the individual amino acids and methods. Standardized amino acid digestibility was not different between the 2 methods for 4 of the DDG samples; however, the PFR yielded higher digestibility values for a high protein DDG and a conventionally processed DDGS. The PFR yielded higher amino acid digestibility values than the SIAAD for several amino acids in 1 MBM and the poultry byproduct meal, but it yielded lower digestibility values for the other MBM. Overall, there were no consistent differences between methods for amino acid digestibility values. In conclusion, the PFR and SIAAD methods are acceptable for determining amino acid digestibility. However, these procedures do not always yield similar results for all feedstuffs evaluated. Thus, further studies are needed to understand the underlying causes in this variability.

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bavari, Sina; Kaplan, Gerardo

2018-04-01

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials. Published by Elsevier B.V.

Nongenotoxic effects and a reduction of the DXR-induced genotoxic effects of Helianthus annuus Linné (sunflower) seeds revealed by micronucleus assays in mouse bone marrow.

PubMed

Boriollo, Marcelo Fabiano Gomes; Souza, Luiz Silva; Resende, Marielly Reis; Silva, Thaísla Andrielle da; Oliveira, Nelma de Mello Silva; Resck, Maria Cristina Costa; Dias, Carlos Tadeu dos Santos; Fiorini, João Evangelista

2014-04-02

This research evaluated the genotoxicity of oil and tincture of H. annuus L. seeds using the micronucleus assay in bone marrow of mice. The interaction between these preparations and the genotoxic effects of doxorubicin (DXR) was also analysed (antigenotoxicity test). Experimental groups were evaluated at 24-48 h post treatment with N-Nitroso-N-ethylurea (positive control - NEU), DXR (chemotherapeutic), NaCl (negative control), a sunflower tincture (THALS) and two sources of sunflower oils (POHALS and FOHALS). Antigenotoxic assays were carried out using the sunflower tincture and oils separately and in combination with NUE or DXR. For THALS, analysis of the MNPCEs showed no significant differences between treatment doses (250-2,000 mg.Kg-1) and NaCl. A significant reduction in MNPCE was observed when THALS (2,000 mg.Kg-1) was administered in combination with DXR (5 mg.Kg-1). For POHALS or FOHALS, analysis of the MNPCEs also showed no significant differences between treatment doses (250-2,000 mg.Kg-1) and NaCl. However, the combination DXR + POHALS (2,000 mg.Kg-1) or DXR + FOHALS (2,000 mg.Kg-1) not contributed to the MNPCEs reduction. This research suggests absence of genotoxicity of THALS, dose-, time- and sex-independent, and its combination with DXR can reduce the genotoxic effects of DXR. POHALS and FOHALS also showed absence of genotoxicity, but their association with DXR showed no antigenotoxic effects.

A photometric method for the estimation of the oil yield of oil shale

USGS Publications Warehouse

Cuttitta, Frank

1951-01-01

A method is presented for the distillation and photometric estimation of the oil yield of oil-bearing shales. The oil shale is distilled in a closed test tube and the oil extracted with toluene. The optical density of the toluene extract is used in the estimation of oil content and is converted to percentage of oil by reference to a standard curve. This curve is obtained by relating the oil yields determined by the Fischer assay method to the optical density of the toluene extract of the oil evolved by the new procedure. The new method gives results similar to those obtained by the Fischer assay method in a much shorter time. The applicability of the new method to oil-bearing shale and phosphatic shale has been tested.

A batch assay to measure microbial hydrogen sulfide production from sulfur-containing solid wastes.

PubMed

Sun, Mei; Sun, Wenjie; Barlaz, Morton A

2016-05-01

Large volumes of sulfur-containing wastes enter municipal solid waste landfills each year. Under the anaerobic conditions that prevail in landfills, oxidized forms of sulfur, primarily sulfate, are converted to sulfide. Hydrogen sulfide (H2S) is corrosive to landfill gas collection and treatment systems, and its presence in landfill gas often necessitates the installation of expensive removal systems. For landfill operators to understand the cost of managing sulfur-containing wastes, an estimate of the H2S production potential is needed. The objective of this study was to develop and demonstrate a biochemical sulfide potential (BSP) test to measure the amount of H2S produced by different types of sulfur-containing wastes in a relatively fast (30days) and inexpensive (125mL serum bottles) batch assay. This study confirmed the toxic effect of H2S on both sulfate reduction and methane production in batch systems, and demonstrated that removing accumulated H2S by base adsorption was effective for mitigating inhibition. H2S production potentials of coal combustion fly ash, flue gas desulfurization residual, municipal solid waste combustion ash, and construction and demolition waste were determined in BSP assays. After 30days of incubation, most of the sulfate in the wastes was converted to gaseous or aqueous phase sulfide, with BSPs ranging from 0.8 to 58.8mLH2S/g waste, depending on the chemical composition of the samples. Selected samples contained solid phase sulfide which contributed to the measured H2S yield. A 60day incubation in selected samples resulted in 39-86% additional sulfide production. H2S production measured in BSP assays was compared with that measured in simulated landfill reactors and that calculated from chemical analyses. H2S production in BSP assays and in reactors was lower than the stoichiometric values calculated from chemical composition for all wastes tested, demonstrating the importance of assays to estimate the microbial sulfide production

Platelet aggregation inhibitors from Philippine marine invertebrate samples screened in a new microplate assay.

PubMed

Pimentel, Sheila Marie V; Bojo, Zenaida P; Roberto, Amy V D; Lazaro, Jose Enrico H; Mangalindan, Gina C; Florentino, Leila M; Lim-Navarro, Pilar; Tasdemir, Deniz; Ireland, Chris M; Concepcion, Gisela P

2003-01-01

A new microplate assay for Ca(2+)-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 micro g/ml, and epinephrine-induced aggregation by 78% at 20 micro g/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 micro g/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity.

Development and evaluation of a rapid recombinase polymerase amplification assay for detection of coxsackievirus A6.

PubMed

Wang, Kaifeng; Wu, Yue; Yin, Dan; Tang, Shixing; Hu, Guifang; He, Yaqing

2017-01-01

Coxsackievirus A6 (CV-A6) is an important pathogen causing hand, foot and mouth disease (HFMD). The aim of this study was to develop and evaluate a rapid real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of CV-A6. The sensitivity of this assay was 202 copies/reaction, with 100 % specificity. Furthermore, this assay yielded consistent results comparable with a commercial qRT-PCR diagnostic kit. This assay is therefore potentially useful for surveillance of CV-A6 infections and outbreak control.

Diffusive tunneling for alleviating Knudsen-layer reactivity reduction under hydrodynamic mix

NASA Astrophysics Data System (ADS)

Tang, Xianzhu; McDevitt, Chris; Guo, Zehua

2017-10-01

Hydrodynamic mix will produce small features for intermixed deuterium-tritium fuel and inert pusher materials. The geometrical characteristics of the mix feature have a large impact on Knudsen layer yield reduction. We considered two features. One is planar structure, and the other is fuel cells segmented by inert pusher material which can be represented by a spherical DT bubble enclosed by a pusher shell. The truly 3D fuel feature, the spherical bubble, has the largest degree of yield reduction, due to fast ions being lost in all directions. The planar fuel structure, which can be regarded as 1D features, has modest amount of potential for yield degradation. While the increasing yield reduction with increasing Knudsen number of the fuel region is straightforwardly anticipated, we also show, by a combination of direct simulation and simple model, that once the pusher materials is stretched sufficiently thin by hydrodynamic mix, the fast fuel ions diffusively tunnel through them with minimal energy loss, so the Knudsen layer yield reduction becomes alleviated. This yield recovery can occur in a chunk-mixed plasma, way before the far more stringent, asymptotic limit of an atomically homogenized fuel and pusher assembly. Work supported by LANL LDRD program.

MTBDRplus and MTBDRsl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

PubMed Central

Georghiou, Sophia B.; Catanzaro, Donald; Rodrigues, Camilla; Crudu, Valeriu; Victor, Thomas C.; Garfein, Richard S.; Catanzaro, Antonino; Rodwell, Timothy C.

2016-01-01

Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplus and MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results. PMID:26763971

A highly sensitive and versatile virus titration assay in the 96-well microplate format.

PubMed

Borisevich, V; Nistler, R; Hudman, D; Yamshchikov, G; Seregin, A; Yamshchikov, V

2008-02-01

This report describes a fast, reproducible, inexpensive and convenient assay system for virus titration in the 96-well format. The micromethod substantially increases assay throughput and improves the data reproducibility. A highly simplified variant of virus quantification is based on immunohistochemical detection of virus amplification foci obtained without use of agarose or semisolid overlays. It can be incorporated into several types of routine virological assays successfully replacing the laborious and time-consuming conventional methods based on plaque formation under semisolid overlays. The method does not depend on the development of CPE and can be accommodated to assay viruses with substantial differences in growth properties. The use of enhanced immunohistochemical detection enabled a five- to six-fold reduction of the total assay time. The micromethod was specifically developed to take advantage of multichannel pipettor use to simplify handling of a large number of samples. The method performs well with an inexpensive low-power binocular, thus offering a routine assay system usable outside of specialized laboratory setting, such as for testing of clinical or field samples. When used in focus reduction-neutralization tests (FRNT), the method accommodates very small volumes of immune serum, which is often a decisive factor in experiments involving small rodent models.

Reduction of ethanol yield from switchgrass infected with rust caused by Puccinia emaculata

DOE PAGES

Sykes, Virginia R.; Allen, Fred L.; Mielenz, Jonathan R.; ...

2015-10-16

Switchgrass ( Panicum virgatum) is an important biofuel crop candidate thought to have low disease susceptibility. As switchgrass production becomes more prevalent, monoculture and production fields in close proximity to one another may increase the spread and severity of diseases such as switchgrass rust caused by the pathogen Puccinia emaculata. The objective of this research was to examine the impact of rust on ethanol yield in switchgrass. In 2010 and 2012, naturally infected leaves from field-grown Alamo and Kanlow in Knoxville, TN (2010, 2012) and Crossville, TN (2012) were visually categorized as exhibiting low, medium, or high disease based onmore » the degree of chlorosis and sporulation. P. emaculata was isolated from each disease range to confirm infection. Samples from 2010 were acid/heat pretreated and subjected to two runs of simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae D 5A to measure ethanol yield. Near-infrared spectroscopy (NIRS) was used to estimate ethanol yield for 2012 samples. SSF and NIRS data were analyzed separately using ANOVA. Disease level effects were significant within both models (P < 0.05) and both models explained a large amount of variation in ETOH (SSF: R 2 = 0.99, NIRS: R 2 = 0.99). In the SSF dataset, ethanol was reduced by 35 % in samples exhibiting medium disease symptoms and by 55 % in samples exhibiting high disease symptoms. In the NIRS dataset, estimated ethanol was reduced by 10 % in samples exhibiting medium disease symptoms and by 21 % in samples exhibiting high disease symptoms. Lastly, results indicate that switchgrass rust will likely have a negative impact on ethanol yield in switchgrass grown as a biofuel crop.« less

Reduction of ethanol yield from switchgrass infected with rust caused by Puccinia emaculata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sykes, Virginia R.; Allen, Fred L.; Mielenz, Jonathan R.

Switchgrass ( Panicum virgatum) is an important biofuel crop candidate thought to have low disease susceptibility. As switchgrass production becomes more prevalent, monoculture and production fields in close proximity to one another may increase the spread and severity of diseases such as switchgrass rust caused by the pathogen Puccinia emaculata. The objective of this research was to examine the impact of rust on ethanol yield in switchgrass. In 2010 and 2012, naturally infected leaves from field-grown Alamo and Kanlow in Knoxville, TN (2010, 2012) and Crossville, TN (2012) were visually categorized as exhibiting low, medium, or high disease based onmore » the degree of chlorosis and sporulation. P. emaculata was isolated from each disease range to confirm infection. Samples from 2010 were acid/heat pretreated and subjected to two runs of simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae D 5A to measure ethanol yield. Near-infrared spectroscopy (NIRS) was used to estimate ethanol yield for 2012 samples. SSF and NIRS data were analyzed separately using ANOVA. Disease level effects were significant within both models (P < 0.05) and both models explained a large amount of variation in ETOH (SSF: R 2 = 0.99, NIRS: R 2 = 0.99). In the SSF dataset, ethanol was reduced by 35 % in samples exhibiting medium disease symptoms and by 55 % in samples exhibiting high disease symptoms. In the NIRS dataset, estimated ethanol was reduced by 10 % in samples exhibiting medium disease symptoms and by 21 % in samples exhibiting high disease symptoms. Lastly, results indicate that switchgrass rust will likely have a negative impact on ethanol yield in switchgrass grown as a biofuel crop.« less

Effects of vegetation cover of natural grassland on runoff and sediment yield in loess hilly region of China.

PubMed

Zhao, Xining; Chen, Xiaoli; Huang, Jun; Wu, Pute; Helmers, Matthew J

2014-02-01

The effects of vegetation cover (VC) on runoff and sediment yield were investigated from rainfall simulation experiments in the Loess Plateau of China. Five VCs from 0% to 80% and three different rainfall intensities (I₂.₀, ₁.₅, ₀.₇₅) were implemented. The results indicated that runoff and sediment yields in slopes were significantly affected by I and VC, and when the VC amounted to 40% there occurred obvious benefits of runoff and sediment reductions and then amplitude decreased with the increase of VC. The runoff reduction benefits at I₁.₅ and I₀.₇₅ were much greater than that at I₂.₀, while the sediment reduction benefits had no significant difference among different rainfall intensities. At I₂.₀, the natural grassland slopes with high VC exhibited the characteristics of high runoff but low sediment production. There existed a power function relationship between cumulative runoff and sediment yield. The increase in cumulative sediment yield was less than the increase in cumulative runoff with increasing VC, and the sediment reduction benefit was greater than runoff reduction on natural grassland slopes. The ratio of runoff reduction to sediment reduction can be used as a comprehensive index for assessing the benefits of runoff and sediment reduction in natural grassland. © 2013 Society of Chemical Industry.

Turbulent drag reduction for external flows

NASA Technical Reports Server (NTRS)

Bushnell, D. M.

1985-01-01

A summary of turbulent drag reduction approaches applicable to external flows is given. Because relatively recent and exhaustive reviews exist for laminar flow control and polymer (hydrodynamic) drag reduction, the focus here is upon the emerging areas of nonplanar geometry and large-eddy alteration. Turbulent control techniques for air generally result in modest (but technologically significant) drag reductions (order of 20 percent or less), whereas hydrodynamic approaches can yield drag reductions the order of 70 percent. Suggestions are included for alternative concepts and optimization of existing approaches.

CPTAC Assay Portal: a repository of targeted proteomic assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

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 investigatorsmore » 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.« less

Measurement of Fission Product Yields from Fast-Neutron Fission

NASA Astrophysics Data System (ADS)

Arnold, C. W.; Bond, E. M.; Bredeweg, T. A.; Fowler, M. M.; Moody, W. A.; Rusev, G.; Vieira, D. J.; Wilhelmy, J. B.; Becker, J. A.; Henderson, R.; Kenneally, J.; Macri, R.; McNabb, D.; Ryan, C.; Sheets, S.; Stoyer, M. A.; Tonchev, A. P.; Bhatia, C.; Bhike, M.; Fallin, B.; Gooden, M. E.; Howell, C. R.; Kelley, J. H.; Tornow, W.

2014-09-01

One of the aims of the Stockpile Stewardship Program is a reduction of the uncertainties on fission data used for analyzing nuclear test data [1,2]. Fission products such as 147Nd are convenient for determining fission yields because of their relatively high yield per fission (about 2%) and long half-life (10.98 days). A scientific program for measuring fission product yields from 235U,238U and 239Pu targets as a function of bombarding neutron energy (0.1 to 15 MeV) is currently underway using monoenergetic neutron beams produced at the 10 MV Tandem Accelerator at TUNL. Dual-fission chambers are used to determine the rate of fission in targets during activation. Activated targets are counted in highly shielded HPGe detectors over a period of several weeks to identify decaying fission products. To date, data have been collected at neutron bombarding energies 4.6, 9.0, 14.5 and 14.8 MeV. Experimental methods and data reduction techniques are discussed, and some preliminary results are presented.

Nongenotoxic effects and a reduction of the DXR-induced genotoxic effects of Helianthus annuus Linné (sunflower) seeds revealed by micronucleus assays in mouse bone marrow

PubMed Central

2014-01-01

Background This research evaluated the genotoxicity of oil and tincture of H. annuus L. seeds using the micronucleus assay in bone marrow of mice. The interaction between these preparations and the genotoxic effects of doxorubicin (DXR) was also analysed (antigenotoxicity test). Methods Experimental groups were evaluated at 24-48 h post treatment with N-Nitroso-N-ethylurea (positive control – NEU), DXR (chemotherapeutic), NaCl (negative control), a sunflower tincture (THALS) and two sources of sunflower oils (POHALS and FOHALS). Antigenotoxic assays were carried out using the sunflower tincture and oils separately and in combination with NUE or DXR. Results For THALS, analysis of the MNPCEs showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. A significant reduction in MNPCE was observed when THALS (2,000 mg.Kg-1) was administered in combination with DXR (5 mg.Kg-1). For POHALS or FOHALS, analysis of the MNPCEs also showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. However, the combination DXR + POHALS (2,000 mg.Kg-1) or DXR + FOHALS (2,000 mg.Kg-1) not contributed to the MNPCEs reduction. Conclusions This research suggests absence of genotoxicity of THALS, dose-, time- and sex-independent, and its combination with DXR can reduce the genotoxic effects of DXR. POHALS and FOHALS also showed absence of genotoxicity, but their association with DXR showed no antigenotoxic effects. PMID:24694203

Microbiological assay for the determination of meropenem in pharmaceutical dosage form.

PubMed

Mendez, Andreas S L; Weisheimer, Vanessa; Oppe, Tércio P; Steppe, Martin; Schapoval, Elfrides E S

2005-04-01

Meropenem is a highly active carbapenem antibiotic used in the treatment of a wide range of serious infections. The present work reports a microbiological assay, applying the cylinder-plate method, for the determination of meropenem in powder for injection. The validation method yielded good results and included linearity, precision, accuracy and specificity. The assay is based on the inhibitory effect of meropenem upon the strain of Micrococcus luteus ATCC 9341 used as the test microorganism. The results of assay were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9999) in the range of 1.5-6.0 microg ml(-1), precise (intra-assay: R.S.D.=0.29; inter-assay: R.S.D.=0.94) and accurate. A preliminary stability study of meropenem was performed to show that the microbiological assay is specific for the determination of meropenem in the presence of its degradation products. The degraded samples were also analysed by the HPLC method. The proposed method allows the quantitation of meropenem in pharmaceutical dosage form and can be used for the drug analysis in routine quality control.

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

Turbulent drag reduction for external flows

NASA Technical Reports Server (NTRS)

Bushnell, D. M.

1983-01-01

Paper presents a review and summary of turbulent drag reduction approaches applicable to external flows. Because relatively recent and exhaustive reviews exist for laminar flow control and polymer (hydrodynamic) drag reduction, the paper focuses upon the emerging areas of nonplanar geometry and large eddy alteration. Turbulent control techniques for air generally result in modest (but technologically significant) drag reductions (order of 20 percent or less) whereas hydrodynamic approaches can yield drag reductions the order of 70 percent. Paper also includes suggestions for alternative concepts and optimization of existing approaches.

Effect of microalgae storage conditions on methane yields.

PubMed

Barreiro-Vescovo, Santiago; de Godos, Ignacio; Tomás-Pejó, Elia; Ballesteros, Mercedes; González-Fernández, Cristina

2018-05-01

During the last decade, a lot of research has been focused on identifying the methane yields achievable when using microalgae biomass (fresh and pretreated) as a substrate in anaerobic digestion. Encountered differences are frequently attributed to the different microalgae strains (cell walls and macromolecular profiles) or the different metabolic activities of anaerobic sludge used as inoculum. Nevertheless, under the hypothesis that the state of microalgae upon biomass storage may also play a significant role, this study was designed to evaluate the effect of biomass processing and storage on methane yields and hydrolysis kinetics in batch mode assays. Slight changes in the macromolecular profile distribution of the different tested biomass were observed. Regardless of the time that the biomass was stored, results revealed that frozen biomass doubled the hydrolysis constant and enhanced methane yield by 1.56-fold compared to fresh microalgae biomass (82.4 mL CH 4  g COD in -1 ). Similar enhancement was obtained with the freeze-dried biomass, and slightly lower values were obtained (1.34-fold) for the biomass kept at 4 °C longer than a week. Likewise, the semi-continuously operated reactor fed with microalgae biomass stored for 28 days at 4 °C did not show any effect in terms of methane production, although nitrogen mineralization was higher than expected. Remarkably, the initial stage of the biomass should be carefully considered for comparison purposes with the available literature on batch mode assays. This study highlights the importance of considering how the biomass is stored before the anaerobic digestion process to avoid misleading conclusions.

The Z {yields} cc-bar {yields} {gamma}{gamma}*, Z {yields} bb-bar {yields} {gamma}{gamma}* triangle diagrams and the Z {yields} {gamma}{psi}, Z {yields} {gamma}Y decays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achasov, N. N., E-mail: achasov@math.nsc.ru

2011-03-15

The approach to the Z {yields} {gamma}{psi} and Z {yields} {gamma}Y decay study is presented in detail, based on the sum rules for the Z {yields} cc-bar {yields} {gamma}{gamma}* and Z {yields} bb-bar {yields} {gamma}{gamma}* amplitudes and their derivatives. The branching ratios of the Z {yields} {gamma}{psi} and Z {yields} {gamma}Y decays are calculated for different hypotheses on saturation of the sum rules. The lower bounds of {Sigma}{sub {psi}} BR(Z {yields} {gamma}{psi}) = 1.95 Multiplication-Sign 10{sup -7} and {Sigma}{sub {upsilon}} BR(Z {yields} {gamma}Y) = 7.23 Multiplication-Sign 10{sup -7} are found. Deviations from the lower bounds are discussed, including the possibilitymore » of BR(Z {yields} {gamma}J/{psi}(1S)) {approx} BR(Z {yields} {gamma}Y(1S)) {approx} 10{sup -6}, that could be probably measured in LHC. The angular distributions in the Z {yields} {gamma}{psi} and Z {yields} {gamma}Y decays are also calculated.« less

What Do Chaotrope-Based Avidity Assays for Antibodies to HIV-1 Envelope Glycoproteins Measure?

PubMed Central

Alexander, Marina R.; Ringe, Rajesh; Sanders, Rogier W.; Voss, James E.; Moore, John P.

2015-01-01

ABSTRACT When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an added chaotrope (such as thiocyanate). Based on that assay, an avidity index was devised for assessing the affinity maturation of antibodies of unknown concentration in polyclonal sera. Since a high avidity index was linked to protection in animal models of HIV-1 infection, it has become a criterion for evaluating antibody responses to vaccine candidates. But what does the assay measure and what does an avidity index mean? Here, we have used a panel of monoclonal antibodies to well-defined epitopes on Env (gp120, gp41, and SOSIP.664 trimers) to explore how the chaotrope acts. We conclude that the chaotrope sensitivity of antibody binding to Env depends on several properties of the epitopes (continuity versus tertiary- and quaternary-structural dependence) and that the avidity index has no simple relationship to antibody affinity for functional Env spikes on virions. We show that the binding of broadly neutralizing antibodies against quaternary-structural epitopes is particularly sensitive to chaotrope treatment, whereas antibody binding to epitopes in variable loops and to nonneutralization epitopes in gp41 is generally resistant. As a result of such biases, the avidity index may at best be a mere surrogate for undefined antibody or other immune responses that correlate weakly with protection. IMPORTANCE An effective HIV-1 vaccine is an important goal. Such a vaccine will probably need to induce antibodies that neutralize typically transmitted variants of HIV-1, preventing them from infecting target cells. Vaccine candidates have so far failed to induce such antibody responses, although some do protect weakly against infection in animals and, possibly, humans

Modified batch anaerobic digestion assay for testing efficiencies of trace metal additives to enhance methane production of energy crops.

PubMed

Brulé, Mathieu; Bolduan, Rainer; Seidelt, Stephan; Schlagermann, Pascal; Bott, Armin

2013-01-01

Batch biochemical methane potential (BMP) assays to evaluate the methane yield of biogas substrates such as energy crops are usually carried out with undiluted inoculum. A BMP assay was performed on two energy crops (green cuttings and grass silage). Anaerobic digestion was performed both with and without supplementation of three commercial additives containing trace metals in liquid, solid or adsorbed form (on clay particles). In order to reveal positive effects of trace metal supplementation on the methane yield, besides undiluted inoculum, 3-fold and 10-fold dilutions of the inoculum were applied for substrate digestion. Diluted inoculum variants were supplemented with both mineral nutrients and pH-buffering substances to prevent a collapse of the digestion process. As expected, commercial additives had no effect on the digestion process performed with undiluted inoculum, while significant increases of methane production through trace element supplementation could be observed on the diluted variants. The effect of inoculum dilution may be twofold: (1) decrease in trace metal supplementation from the inoculum and (2) reduction in the initial number of bacterial cells. Bacteria require higher growth rates for substrate degradation and hence have higher trace element consumption. According to common knowledge of the biogas process, periods with volatile fatty acids accumulation and decreased pH may have occurred in the course ofanaerobic digestion. These effects may have led to inhibition, not only ofmethanogenes and acetogenes involved in the final phases of methane production, but also offibre-degrading bacterial strains involved in polymer hydrolysis. Further research is required to confirm this hypothesis.

Biomass production of 12 winter cereal cover crop cultivars and their effect on subsequent no-till corn yield

USDA-ARS?s Scientific Manuscript database

Cover crops can improve the sustainability and resilience of corn and soybean production systems. However, there have been isolated reports of corn yield reductions following winter rye cover crops. Although there are many possible causes of corn yield reductions following winter cereal cover crops,...

Photofission product yields of 238U and 239Pu with 22-MeV bremsstrahlung

NASA Astrophysics Data System (ADS)

Wen, Xianfei; Yang, Haori

2016-06-01

In homeland security and nuclear safeguards applications, non-destructive techniques to identify and quantify special nuclear materials are in great demand. Although nuclear materials naturally emit characteristic radiation (e.g. neutrons, γ-rays), their intensity and energy are normally low. Furthermore, such radiation could be intentionally shielded with ease or buried in high-level background. Active interrogation techniques based on photofission have been identified as effective assay approaches to address this issue. In designing such assay systems, nuclear data, like photofission product yields, plays a crucial role. Although fission yields for neutron-induced reactions have been well studied and readily available in various nuclear databases, data on photofission product yields is rather scarce. This poses a great challenge to the application of photofission techniques. In this work, short-lived high-energy delayed γ-rays from photofission of 238U were measured in between linac pulses. In addition, a list-mode system was developed to measure relatively long-lived delayed γ-rays from photofission of 238U and 239Pu after the irradiation. Time and energy information of each γ-ray event were simultaneously recorded by this system. Cumulative photofission product yields were then determined using the measured delayed γ-ray spectra.

Effects of different nitrogen reduction modes on yield of spring maize and nitrate - N residue in soils of the southern Loess Plateau

NASA Astrophysics Data System (ADS)

Dang, Tinghui; Dong, Qiang

2017-04-01

【Objectives】Excessive fertilization in farmland caused nitrate leaching and accumulation in soil, which not only reduced the fertilizer utilization rate, but also had potential environmental problems. The effects of different nitrogen reduction modes on yield of spring maize and soil NO3-N in the southern Loess Plateau were discussed, and scientific fertilization suggestions were put forward, which were of great significance to instruct local maize fertilization and protect environment safety. 【Methods】A field experiment was conducted in the south of the Loess Plateau for three years. Spring maize was planted with half plastic film mulching in one crop per annum．The experiment consisted of 5 N fertilization treatments: control treatment (CK),conventional N fertilization rate (Con), optimal N fertilizationⅠ(OptⅠ), optimal N fertilizationⅡ (Opt Ⅱ) and optimal N fertilization Ⅲ(Opt Ⅲ), The changes of yield of maize, nitrogen uptake and soil NO3-N were measured.【Results】The results showed that, compared with the conventional fertilization, the grain yield and N uptake of maize had no significant change under the three optimal N fertilization application models, the rate of the grain yield's change is 100 300kg/hm2. Compared with the conventional fertilization, agronomic efficiency of fertilizer-nitrogen and N partial fertilizer productivity were increased by 20.2% 23.2% and 21.9% 23.7%, respectively. The accumulation of nitrate nitrogen in profile (0－200 cm) decreased by 90.7 kg / hm2, 97.3 kg / hm2, 100.7 kg / hm2 ,respectively, with the decreases of 44.7%, 47.9% and 49.6% respectively.【Conclusions】The optimum nitrogen fertilization pattern did not affect spring maize yield and N uptake, and could improve agronomic efficiency of fertilizer-nitrogen and N partial fertilizer productivity. Under the same nitrogen application rate, the effects of adding nitrification inhibitor or slow-release fertilizer on nitrate-N residue were not obvious

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

Cupric ion reducing antioxidant capacity assay for antioxidants in human serum and for hydroxyl radical scavengers.

PubMed

Apak, Reşat; Güçlü, Kubilay; Ozyürek, Mustafa; Bektaşoğlu, Burcu; Bener, Mustafa

2010-01-01

, for which the FRAP (ferric reducing antioxidant potency) test is basically nonresponsive. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test do not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance, and that a total antioxidant capacity (TAC) assay of serum is possible. As a distinct advantage over other electron-transfer based assays (e.g., Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favorable redox potential, accessibility and stability of reagents, and applicability to lipophilic antioxidants as well as hydrophilic ones. The CUPRAC procedure can also assay hydroxyl radicals, being the most reactive oxygen species (ROS). As a more convenient, efficient, and less costly alternative to HPLC/electrochemical detection techniques and to the nonspecific, low-yield TBARS test, we use p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of [Fe(II)+EDTA] with hydrogen peroxide. The produced hydroxyl radicals attack both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2 h. The CUPRAC absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreases in the presence of (.)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

PubMed

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Effects of two sediment types on the fluorescence yield of two Hawaiian scleractinian corals

USGS Publications Warehouse

Piniak, G.A.

2007-01-01

This study used non-invasive pulse-amplitude modulated (PAM) fluorometry to measure the maximum fluorescence yield (Fv/Fm) of two Hawaiian scleractinian coral species exposed to short-term sedimentation stress. Beach sand or harbor mud was applied to coral fragments in a flow-through aquarium system for 0-45 h, and changes in Fv/Fm were measured as a function of sediment type and length of exposure. Corals were monitored for up to 90 h to document recovery after sediment removal. Sediment deposition significantly decreased Fv/Fm in both species and was a function of sediment type and time. Corals that received sediment for 30 h or more had the greatest reduction in yield and exhibited little recovery over the course of the experiment. Harbor mud caused a greater reduction in Porites lobata yield than beach sand, whereas both sediment types had equally deleterious effects on Montipora capitata. Colony morphology and sediment type were important factors in determining yield reduction-P. lobata minimized damage from coarse sand grains by passive sediment rejection or accumulation in depressions in the skeleton, and fluorescence yield decreased most in corals exposed to sticky harbor mud or in colonies with flattened morphologies. Species-specific differences could not be tested due to differences in colony morphology and surface area. ?? 2007.

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

PubMed

Stellrecht, K A; Espino, A A; Maceira, V P; Nattanmai, S M; Butt, S A; Wroblewski, D; Hannett, G E; Musser, K A

2014-05-01

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Effects of physical agitation on yield of greenhouse-grown soybean

NASA Technical Reports Server (NTRS)

Jones, R. S.; Mitchell, C. A.

1992-01-01

Agronomic and horticultural crop species experience reductions in growth and harvestable yield after exposure to physical agitation (also known as mechanical stress), as by wind or rain. A greenhouse study was conducted to test the influence of mechanical stress on soybean yield and to determine if exposure to mechanical stress during discrete growth periods has differential effects on seed yield. A modified rotatory shaker was used to apply seismic (i.e., shaking) stress. Brief, periodic episodes of seismic stress reduced stem length, total seed dry weight, and seed number of soybean [Glycine max (L.) Merr.]. Lodging resistance was greater for plants stressed during vegetative growth or throughout vegetative and reproductive growth than during reproductive growth only. Seed dry weight yield was reduced regardless of the timing or duration of stress application, but was lowest when applied during reproductive development. Seismic stress applied during reproductive growth stages R1 to R2 (Days 3 to 4) was as detrimental to seed dry weight accumulation as was stress applied during growth stages R1 to R6 (Days 39 to 42). Seed dry weight per plant was highly correlated with seed number per plant, and seed number was correlated with the seed number of two- and three-seeded pods. Dry weight per 100 seeds was unaffected by seismic-stress treatment. Growth and yield reductions resulting from treatments applied only during the vegetative stage imply that long-term mechanical effects were induced, from which the plants did not fully recover. It is unclear which yield-controlling physiological processes were affected by mechanical stress. Both transient and long-term effects on yield-controlling processes remain to be elucidated.

On the Yield Strength of Oceanic Lithosphere

NASA Astrophysics Data System (ADS)

Jain, C.; Korenaga, J.; Karato, S. I.

2017-12-01

The origin of plate tectonic convection on Earth is intrinsically linked to the reduction in the strength of oceanic lithosphere at plate boundaries. A few mechanisms, such as deep thermal cracking [Korenaga, 2007] and strain localization due to grain-size reduction [e.g., Ricard and Bercovici, 2009], have been proposed to explain this reduction in lithospheric strength, but the significance of these mechanisms can be assessed only if we have accurate estimates on the strength of the undamaged oceanic lithosphere. The Peierls mechanism is likely to govern the rheology of old oceanic lithosphere [Kohlstedt et al., 1995], but the flow-law parameters for the Peierls mechanism suggested by previous studies do not agree with each other. We thus reanalyze the relevant experimental deformation data of olivine aggregates using Markov chain Monte Carlo inversion, which can handle the highly nonlinear constitutive equation of the Peierls mechanism [Korenaga and Karato, 2008; Mullet et al., 2015]. Our inversion results indicate nontrivial nonuniqueness in every flow-law parameter for the Peierls mechanism. Moreover, the resultant flow laws, all of which are consistent with the same experimental data, predict substantially different yield stresses under lithospheric conditions and could therefore have different implications for the origin of plate tectonics. We discuss some future directions to improve our constraints on lithospheric yield strength.

Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting Density of Functional Groups for DNA Assays.

PubMed

Švorc, Ĺubomír; Jambrec, Daliborka; Vojs, Marian; Barwe, Stefan; Clausmeyer, Jan; Michniak, Pavol; Marton, Marián; Schuhmann, Wolfgang

2015-09-02

The impact of different doping levels of boron-doped diamond on the surface functionalization was investigated by means of electrochemical reduction of aryldiazonium salts. The grafting efficiency of 4-nitrophenyl groups increased with the boron levels (B/C ratio from 0 to 20,000 ppm). Controlled grafting of nitrophenyldiazonium was used to adjust the amount of immobilized single-stranded DNA strands at the surface and further on the hybridization yield in dependence on the boron doping level. The grafted nitro functions were electrochemically reduced to the amine moieties. Subsequent functionalization with a succinic acid introduced carboxyl groups for subsequent binding of an amino-terminated DNA probe. DNA hybridization significantly depends on the probe density which is in turn dependent on the boron doping level. The proposed approach opens new insights for the design and control of doped diamond surface functionalization for the construction of DNA hybridization assays.

Simulating crop yield losses in Switzerland for historical and present Tambora climate scenarios

NASA Astrophysics Data System (ADS)

Flückiger, Simon; Brönnimann, Stefan; Holzkämper, Annelie; Fuhrer, Jürg; Krämer, Daniel; Pfister, Christian; Rohr, Christian

2017-07-01

Severe climatic anomalies in summer 1816, partly due to the eruption of Tambora in April 1815, contributed to delayed growth and poor harvests of important crops in Central Europe. Coinciding with adverse socio-economic conditions, this event triggered the last subsistence crisis in the western World. Here, we model reductions in potential crop yields for 1816 and 1817 and address the question, what impact a similar climatic anomaly would have today. We reconstructed daily weather for Switzerland for 1816/17 on a 2 km grid using historical observations and an analogue resampling method. These data were used to simulate potential crop yields for potato, grain maize, and winter barley using the CropSyst model calibrated for current crop cultivars. We also simulated yields for the same weather anomalies, but referenced to a present-day baseline temperature. Results show that reduced temperature delayed growth and harvest considerably, and in combination with reduced solar irradiance led to a substantial reduction (20%-50%) in the potential yield of potato in 1816. Effects on winter barley were smaller. Significant reductions were also modelled for 1817 and were mainly due to a cold late spring. Relative reductions for the present-day scenario for the two crops were almost indistinguishable from the historical ones. An even stronger response was found for maize, which was not yet common in 1816/17. Waterlogging, which we assessed using a stress-day approach, likely added to the simulated reductions. The documented, strong east-west gradient in malnutrition across Switzerland in 1817/18 could not be explained by biophysical yield limitations (though excess-water limitation might have contributed), but rather by economic, political and social factors. This highlights the importance of these factors for a societies’ ability to cope with extreme climate events. While the adaptive capacity of today’s society in Switzerland is much greater than in the early 19th century

Cost of gentamicin assays carried out by microbiology laboratories.

PubMed Central

Vacani, P F; Malek, M M; Davey, P G

1993-01-01

AIMS--To assess the current range of prices charged for gentamicin assays in United Kingdom laboratories; and to examine the laboratories' likely response to increases or decreases in the demand for the service. METHODS--A postal survey of the 420 members of the Association of Medical Microbiologists was used to establish the range of prices charged for aminoglycoside assays. Additionally, eight private institutions were contacted to determine what the private sector was charging for aminoglycoside assays. Reagent costs in the NHS laboratories were calculated by dividing the total cost of all aminoglycoside assay kits by the number of samples analysed. RESULTS--The NHS and the private institutions both showed a wide price variation. Prices charged to an in-hospital requester for a peak and trough assay ranged from 5.00 pounds to 68.20 pounds (n = 44), and to an external private hospital, under a bulk service contract, from 5.00 pounds to 96.00 pounds (n = 47). Prices charged by private laboratories ranged from 49.00 pounds to 84.00 pounds (n = 8). There was a log linear correlation in the NHS laboratories between the reagent costs per assay and the number of assays performed per year, and most laboratories thought that their price per assay would be sensitive to increases or decreases in demand. Laboratories which had purchased their assay machines had lower reagent costs per assay but higher repair and maintenance costs. Overall, number of assays performed and method of payment for assay machinery only accounted for 44.8% of the observed variation in assay kit costs. CONCLUSIONS--The price range for gentamicin assays in the United Kingdom is wide and is only partially explained by the number of assays performed. Most laboratories believe that they would experience a reduction in unit cost as output increases. The currently offered range of prices is, in part, due to variation in the laboratories' approach to costing the service provided and some laboratories

Forest age-induced changes in evapotranspiration and water yield in a eucalypt forest

NASA Astrophysics Data System (ADS)

Cornish, P. M.; Vertessy, R. A.

2001-02-01

Water yields in a regrowth eucalypt forest were found to increase initially and then to decline below pre-treatment levels during the 16-year period which followed the logging of a moist old-growth eucalypt forest in Eastern Australia. Both regrowth and old-growth stands were dominated by Sydney Blue Gum ( Eucalyptus saligna Smith) and Silvertop Stringybark ( Eucalyptus laevopinea R. Baker). Using a paired-catchment approach we observed significant reductions in five of six gauged catchments, and were able to associate their magnitude with forest growth rate, canopy cover and soil depth. Regular yield declines were interrupted for a period in some catchments, possibly due to foliar insect attack. Yield reductions of up to a maximum 600 mm per year in logged and regenerated areas were in accord with water yield reductions observed in Mountain Ash ( Eucalyptus regnans F.J. Muell.) regeneration in Victoria. This study therefore represents the first confirmation of these Maroondah Mountain Ash results in another forest type that has also undergone eucalypt-to-eucalypt succession. Baseflow analysis indicated that baseflow and stormflow both increased after logging, with stormflow increases dominant in catchments with shallower soils. The lower runoff observed when the regenerating forest was aged 13-16 years was principally a consequence of lower baseflow.

Reduction of FeO contents in sinter under high bed operation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujii, K.; Hazama, K.; Hoshikuma, Y.

1996-12-31

High-bed operation (bed height more than 700 mm) is currently being carried out at the Kure No. 1 sintering plant. Before initiating this high-bed operation, the authors conducted sinter pot tests at various bed heights to investigate the effect of bed height on sintering. The following results were obtained from these pot tests: Heightening of the sinter bed increased yield at the upper layer, but at the lower layer, the yield reached a maximum value at a certain bed height. From observation of the sinter cakes, the reduction in yield is attributed to uneven burn caused by surplus heat atmore » the lower layers. Therefore, when high-bed operation is carried out, reduction of the burning energy (reduction of the FeO content in the sinter) is required. This high-bed operation with lower FeO content has enabled the company to reduce fuel consumption and SiO{sub 2} content, while maintaining high yield and high sinter quality.« less

DNA Meter: Energy Tunable, Quantitative Hybridization Assay

PubMed Central

Braunlin, William; Völker, Jens; Plum, G. Eric; Breslauer, Kenneth J.

2015-01-01

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial “pre-binding” of a probe strand modulates its effective “availability” for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. PMID:23529692

Impacts of variability in cellulosic biomass yields on energy security.

PubMed

Mullins, Kimberley A; Matthews, H Scott; Griffin, W Michael; Anex, Robert

2014-07-01

The practice of modeling biomass yields on the basis of deterministic point values aggregated over space and time obscures important risks associated with large-scale biofuel use, particularly risks related to drought-induced yield reductions that may become increasingly frequent under a changing climate. Using switchgrass as a case study, this work quantifies the variability in expected yields over time and space through switchgrass growth modeling under historical and simulated future weather. The predicted switchgrass yields across the United States range from about 12 to 19 Mg/ha, and the 80% confidence intervals range from 20 to 60% of the mean. Average yields are predicted to decrease with increased temperatures and weather variability induced by climate change. Feedstock yield variability needs to be a central part of modeling to ensure that policy makers acknowledge risks to energy supplies and develop strategies or contingency plans that mitigate those risks.

Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.

PubMed

Cowan, Lauren S; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

HVM die yield improvement as a function of DRSEM ADC

NASA Astrophysics Data System (ADS)

Maheshwary, Sonu; Haas, Terry; McGarvey, Steve

2010-03-01

Given the current manufacturing technology roadmap and the competitiveness of the global semiconductor manufacturing environment in conjunction with the semiconductor manufacturing market dynamics, the market place continues to demand a reduced die manufacturing cost. This continuous pressure on lowering die cost in turn drives an aggressive yield learning curve, a key component of which is defect reduction of manufacturing induced anomalies. In order to meet and even exceed line and die yield targets there is a need to revamp defect classification strategies and place a greater emphasize on increasing the accuracy and purity of the Defect Review Scanning Electron Microscope (DRSEM) Automated Defect Classification (ADC) results while placing less emphasis on the ADC results of patterned/un-patterned wafer inspection systems. The increased emphasis on DRSEM ADC results allows for a high degree of automation and consistency in the classification data and eliminates variance induced by the manufacturing staff. This paper examines the use of SEM based Auto Defect Classification in a high volume manufacturing environment as a key driver in the reduction of defect limited yields.

Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

PubMed

Kraynak, A R; Barnum, J E; Cunningham, C L; Ng, A; Ykoruk, B A; Bennet, B; Stoffregen, D; Merschman, M; Freeland, E; Galloway, S M

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery. Copyright © 2015 Elsevier B.V. All rights reserved.

Fischer Assays of Oil-Shale Drill Cores and Rotary Cuttings from the Greater Green River Basin, Southwestern Wyoming

USGS Publications Warehouse

,

2008-01-01

Chapter 1 of this CD-ROM is a database of digitized Fischer (shale-oil) assays of cores and cuttings from boreholes drilled in the Eocene Green River oil shale deposits in southwestern Wyoming. Assays of samples from some surface sections are also included. Most of the Fischer assay analyses were made by the former U.S. Bureau of Mines (USBM) at its laboratory in Laramie, Wyoming. Other assays, made by institutional or private laboratories, were donated to the U.S. Geological Survey (USGS) and are included in this database as well as Adobe PDF-scanned images of some of the original laboratory assay reports and lithologic logs prepared by USBM geologists. The size of this database is 75.2 megabytes and includes information on 971 core holes and rotary-drilled boreholes and numerous surface sections. Most of these data were released previously by the USBM and the USGS through the National Technical Information Service but are no longer available from that agency. Fischer assays for boreholes in northeastern Utah and northwestern Colorado have been published by the USGS. Additional data include geophysical logs, groundwater data, chemical and X-ray diffraction analyses, and other data. These materials are available for inspection in the office of the USGS Central Energy Resources Team in Lakewood, Colorado. The digitized assays were checked with the original laboratory reports, but some errors likely remain. Other information, such as locations and elevations of core holes and oil and gas tests, were not thoroughly checked. However, owing to the current interest in oil-shale development, it was considered in the public interest to make this preliminary database available at this time. Chapter 2 of this CD-ROM presents oil-yield histograms of samples of cores and cuttings from exploration drill holes in the Eocene Green River Formation in the Great Divide, Green River, and Washakie Basins of southwestern Wyoming. A database was compiled that includes about 47

The buffer value of groundwater when well yield is limited

NASA Astrophysics Data System (ADS)

Foster, T.; Brozović, N.; Speir, C.

2017-04-01

A large proportion of the total value of groundwater in conjunctive use systems is associated with the ability to smooth out shortfalls in surface water supply during droughts. Previous research has argued that aquifer depletion in these regions will impact farmers negatively by reducing the available stock of groundwater to buffer production in future periods, and also by increasing the costs of groundwater extraction. However, existing studies have not considered how depletion may impact the productivity of groundwater stocks in conjunctive use systems through reductions in well yields. In this work, we develop a hydro-economic modeling framework to quantify the effects of changes in well yields on the buffer value of groundwater, and apply this model to an illustrative case study of tomato production in California's Central Valley. Our findings demonstrate that farmers with low well yields are forced to forgo significant production and profits because instantaneous groundwater supply is insufficient to buffer surface water shortfalls in drought years. Negative economic impacts of low well yields are an increasing function of surface water variability, and are also greatest for farmers operating less efficient irrigation systems. These results indicate that impacts of well yield reductions on the productivity of groundwater are an important economic impact of aquifer depletion, and that failure to consider this feedback may lead to significant errors in estimates of the value of groundwater management in conjunctive use systems.

Biogeochemical N signatures from rate-yield trade-offs during in vitro chemosynthetic NO3- reduction by deep-sea vent ε-Proteobacteria and Aquificae growing at different temperatures

NASA Astrophysics Data System (ADS)

Pérez-Rodríguez, Ileana; Sievert, Stefan M.; Fogel, Marilyn L.; Foustoukos, Dionysis I.

2017-08-01

NO3- reduction is a metabolism that is widespread among ε-Proteobacteria and Aquificae, two abundant classes of microorganisms found at deep-sea vents. In this study, we used Sulfurovum lithotrophicum, Caminibacter mediatlanticus and Thermovibrio ammonificans as representatives of these groups to study ecophysiological, metabolic and biogeochemical parameters associated with chemolithoautotrophic NO3- reduction under different temperature regimes. We observed that while S. lithotrophicum and C. mediatlanticus achieved higher cell densities than T. ammonificans, the overall NO3- consumption by the latter was on average ∼9 and ∼5 times faster on a per cell basis, respectively. Comparison with previously published data from other cultured vent ε-Proteobacteria and Aquificae suggests that the rate-yield trade-offs observed in our experiments are generally conserved between these two groups in line with their ecophysiologies. Kinetic isotope effects of N from NO3- reduction were 9.6 ± 2.7‰ for S. lithotrophicum, 6.4 ± 0.7‰ for C. mediatlanticus and 8.8 ± 0.6‰ for T. ammonificans. Our results help evaluate how metabolic partitioning between growth efficiency and reaction kinetics during chemolithoautotrophic NO3- reduction affect the concentration and isotope composition of N compounds at deep-sea hydrothermal vents.

Transfer of a Mycobacterium tuberculosis Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System

PubMed Central

Cowan, Lauren S.; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T.

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay. PMID:14715809

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

PubMed

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

A universally calibrated microplate ferric reducing antioxidant power (FRAP) assay for foods and applications to Manuka honey.

PubMed

Bolanos de la Torre, Amparo Angelica S; Henderson, Terence; Nigam, Poonam Singh; Owusu-Apenten, Richard K

2015-05-01

The ferric reducing antioxidant power (FRAP) assay was recently adapted to a microplate format. However, microplate-based FRAP (mFRAP) assays are affected by sample volume and composition. This work describes a calibration process for mFRAP assays which yields data free of volume effects. From the results, the molar absorptivity (ε) for the mFRAP assay was 141,698 M(-1) cm(-1) for gallic acid, 49,328 M(-1) cm(-1) for ascorbic acid, and 21,606 M(-1) cm(-1) for ammonium ferrous sulphate. The significance of ε (M(-1) cm(-1)) is discussed in relation to mFRAP assay sensitivity, minimum detectable concentration, and the dimensionless FRAP-value. Gallic acid showed 6.6 mol of Fe(2+) equivalents compared to 2.3 mol of Fe(+2) equivalents for ascorbic acid. Application of the mFRAP assay to Manuka honey samples (rated 5+, 10+, 15+, and 18+ Unique Manuka Factor; UMF) showed that FRAP values (0.54-0.76 mmol Fe(2+) per 100g honey) were strongly correlated with UMF ratings (R(2)=0.977) and total phenols content (R(2) = 0.982)whilst the UMF rating was correlated with the total phenols (R(2) = 0.999). In conclusion, mFRAP assay results were successfully standardised to yield data corresponding to 1-cm spectrophotometer which is useful for quality assurance purposes. The antioxidant capacity of Manuka honey was found to be directly related to the UMF rating. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nitrite reduction mechanism on a Pd surface.

PubMed

Shin, Hyeyoung; Jung, Sungyoon; Bae, Sungjun; Lee, Woojin; Kim, Hyungjun

2014-11-04

Nitrate (NO3-) is one of the most harmful contaminants in the groundwater, and it causes various health problems. Bimetallic catalysts, usually palladium (Pd) coupled with secondary metallic catalyst, are found to properly treat nitrate-containing wastewaters; however, the selectivity toward N2 production over ammonia (NH3) production still requires further improvement. Because the N2 selectivity is determined at the nitrite (NO2-) reduction step on the Pd surface, which occurs after NO3- is decomposed into NO2- on the secondary metallic catalyst, we here performed density functional theory (DFT) calculations and experiments to investigate the NO2- reduction pathway on the Pd surface activated by hydrogen. Based on extensive DFT calculations on the relative energetics among ∼100 possible intermediates, we found that NO2- is easily reduced to NO* on the Pd surface, followed by either sequential hydrogenation steps to yield NH3 or a decomposition step to N* and O* (an adsorbate on Pd is denoted using an asterisk). Based on the calculated high migration barrier of N*, we further discussed that the direct combination of two N* to yield N2 is kinetically less favorable than the combination of a highly mobile H* with N* to yield NH3. Instead, the reduction of NO2- in the vicinity of the N* can yield N2O* that can be preferentially transformed into N2 via diverse reaction pathways. Our DFT results suggest that enhancing the likelihood of N* encountering NO2- in the solution phase before combination with surface H* is important for maximizing the N2 selectivity. This is further supported by our experiments on NO2- reduction by Pd/TiO2, showing that both a decreased H2 flow rate and an increased NO2- concentration increased the N2 selectivity (78.6-93.6% and 57.8-90.9%, respectively).

Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase

PubMed Central

Yano, Takahiro; Kassovska-Bratinova, Sacha; Teh, J. Shin; Winkler, Jeffrey; Sullivan, Kevin; Isaacs, Andre; Schechter, Norman M.; Rubin, Harvey

2011-01-01

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species. PMID:21193400

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

PubMed

Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for detection of beet necrotic yellow vein virus.

PubMed

Almasi, Mohammad Amin; Almasi, Galavizh

2017-02-01

Sugar beet can be infected by many different viruses that can reduce yield; beet necrotic yellow vein virus (BNYVV) is one of the most economically important viruses of this crop plant. This report describes a new reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for identification of BNYVV. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection (without RNA extraction) of BNYVV was developed here and compared with DAS-ELISA and RT-LAMP assays. Our results show that the IC-RT-LAMP assay is a highly reliable alternative assay for identification of BNYVV.

Factors affecting a cyanogen bromide-based assay of thiamin.

PubMed

Wyatt, D T; Lee, M; Hillman, R E

1989-11-01

We analyzed extensively a modified thiochrome method for thiamin analysis. Acid phosphatase (EC 3.1.3.2) from potato was superior to either alpha-amylase or acid phosphatase from wheat germ as a dephosphorylating agent. Timing of cyanogen bromide exposure was important, but the assay had good precision and accuracy. The standard curve was linear from 10 to 3000 nmol/L. The within-run and between-run coefficients of variation for total thiamin in whole blood were 3.6% and 7.4%, respectively. Analytical recoveries for low, intermediate, and high additions of thiamin to whole blood were 93-109%. Sample yield was increased by 41% (+/- 29% SD) with pre-assay freezing. Samples were stable for two days at room temperature, for seven days when refrigerated, and for two years when frozen. Previously unreported interference was seen with penicillin derivatives, and with several commonly used diuretic and antiepileptic medications. This assay may be suitable for population screening; 200 samples could be analyzed weekly at a cost of +0.20 per sample.

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

PubMed

Huber, Alexander; France, Richard M; Riccalton-Banks, Lisa; McLaren, Jane; Cox, Helen; Quirk, Robin A; Shakesheff, Kevin M; Thompson, David; Panjwani, Naveed; Shipley, Sarah; Pickett, Andy

2008-05-01

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

Anomalous yield reduction in direct-drive deuterium/tritium implosions due to 3He addition

NASA Astrophysics Data System (ADS)

Herrmann, H. W.; Langenbrunner, J. R.; Mack, J. M.; Cooley, J. H.; Wilson, D. C.; Evans, S. C.; Sedillo, T. J.; Kyrala, G. A.; Caldwell, S. E.; Young, C. S.; Nobile, A.; Wermer, J.; Paglieri, S.; McEvoy, A. M.; Kim, Y.; Batha, S. H.; Horsfield, C. J.; Drew, D.; Garbett, W.; Rubery, M.; Glebov, V. Yu.; Roberts, S.; Frenje, J. A.

2009-05-01

Glass capsules were imploded in direct drive on the OMEGA laser [Boehly et al., Opt. Commun. 133, 495 (1997)] to look for anomalous degradation in deuterium/tritium (DT) yield and changes in reaction history with H3e addition. Such anomalies have previously been reported for D/H3e plasmas but had not yet been investigated for DT/H3e. Anomalies such as these provide fertile ground for furthering our physics understanding of inertial confinement fusion implosions and capsule performance. Anomalous degradation in the compression component of yield was observed, consistent with the "factor of 2" degradation previously reported by Massachusetts Institute of Technology (MIT) at a 50% H3e atom fraction in D2 using plastic capsules [Rygg, Phys. Plasmas 13, 052702 (2006)]. However, clean calculations (i.e., no fuel-shell mixing) predict the shock component of yield quite well, contrary to the result reported by MIT but consistent with Los Alamos National Laboratory results in D2/H3e [Wilson et al., J. Phys.: Conf. Ser. 112, 022015 (2008)]. X-ray imaging suggests less-than-predicted compression of capsules containing H3e. Leading candidate explanations are poorly understood equation of state for gas mixtures and unanticipated particle pressure variation with increasing H3e addition.

Heat-induced phytohormone changes are associated with disrupted early reproductive development and reduced yield in rice

PubMed Central

Wu, Chao; Cui, Kehui; Wang, Wencheng; Li, Qian; Fahad, Shah; Hu, Qiuqian; Huang, Jianliang; Nie, Lixiao; Peng, Shaobing

2016-01-01

Heat stress causes morphological and physiological changes and reduces crop yield in rice (Oryza sativa). To investigate changes in phytohormones and their relationships with yield and other attributes under heat stress, four rice varieties (Nagina22, Huanghuazhan, Liangyoupeijiu, and Shanyou 63) were grown in pots and subjected to three high temperature treatments plus control in temperature-controlled greenhouses for 15 d during the early reproductive phase. Yield reductions in Nagina22, Huanghuazhan, and Liangyoupeijiu were attributed to reductions in spikelet fertility, spikelets per panicle, and grain weight. The adverse effects of high temperature were alleviated by application of exogenous 6-benzylaminopurine (6-BA) in the heat-susceptible Liangyoupeijiu. High temperature stress reduced active cytokinins, gibberellin A1 (GA1), and indole-3-acetic acid (IAA), but increased abscisic acid (ABA) and bound cytokinins in young panicles. Correlation analyses and application of exogenous 6-BA revealed that high temperature-induced cytokinin changes may regulate yield components by modulating the differentiation and degradation of branches and spikelets, panicle exsertion, pollen vigor, anther dehiscence, and grain size. Heat-tolerant Shanyou 63 displayed minor changes in phytohormones, panicle formation, and grain yield under high temperature compared with those of the other three varieties. These results suggest that phytohormone changes are closely associated with yield formation, and a small reduction or stability in phytohormone content is required to avoid large yield losses under heat stress. PMID:27713528

The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays.

PubMed Central

Rice, L.; Urano, M.; Suit, H. D.

1980-01-01

The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells. PMID:6932931

Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng

Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor,more » we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.« less

Fusion yield rate recovery by escaping hot-spot fast ions in the neighboring fuel layer

NASA Astrophysics Data System (ADS)

Tang, Xian-Zhu; McDevitt, C. J.; Guo, Zehua; Berk, H. L.

2014-02-01

Free-streaming loss by fast ions can deplete the tail population in the hot spot of an inertial confinement fusion (ICF) target. Escaping fast ions in the neighboring fuel layer of a cryogenic target can produce a surplus of fast ions locally. In contrast to the Knudsen layer effect that reduces hot-spot fusion reactivity due to tail ion depletion, the inverse Knudsen layer effect increases fusion reactivity in the neighboring fuel layer. In the case of a burning ICF target in the presence of significant hydrodynamic mix which aggravates the Knudsen layer effect, the yield recovery largely compensates for the yield reduction. For mix-dominated sub-ignition targets, the yield reduction is the dominant process.

Effects of Management Practices on Meloidogyne incognita and Snap Bean Yield.

PubMed

Smittle, D A; Johnson, A W

1982-01-01

Phenamiphos applied at 6.7 kg ai/ha through a solid set or a center pivot irrigation system with 28 mm of water effectively controlled root-knot nematodes, Meloidogyne incognita, and resulted in greater snap bean growth and yields irrespective of growing season, tillage method, or cover crop system. The percentage yield increases attributed to this method of M. incognita control over nontreated controls were 45% in the spring crop, and 90% and 409% in the fall crops following winter rye and fallow, respectively. Root galling was not affected by tillage systems or cover crop, but disk tillage resulted in over 50% reduction in bean yield compared with yields from the subsoil-bed tillage system.

Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

PubMed Central

Phillips, Richard Odame; Badziklou, Kossi; Piten, Ebekalisai; Maman, Issaka; Sarfo, Fred Stephen; Huber, Kristina Lydia; Rhomberg, Agata; Symank, Dominik; Wagner, Magdalena; Wiedemann, Franz; Nitschke, Jörg; Banla Kere, Abiba; Herbinger, Karl-Heinz; Adjei, Ohene; Löscher, Thomas; Bretzel, Gisela

2014-01-01

This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence. PMID:24478404

Root Traits Enhancing Rice Grain Yield under Alternate Wetting and Drying Condition

PubMed Central

Sandhu, Nitika; Subedi, Sushil R.; Yadaw, Ram B.; Chaudhary, Bedanand; Prasai, Hari; Iftekharuddaula, Khandakar; Thanak, Tho; Thun, Vathany; Battan, Khushi R.; Ram, Mangat; Venkateshwarlu, Challa; Lopena, Vitaliano; Pablico, Paquito; Maturan, Paul C.; Cruz, Ma. Teresa Sta.; Raman, K. Anitha; Collard, Bertrand; Kumar, Arvind

2017-01-01

Reducing water requirements and lowering environmental footprints require attention to minimize risks to food security. The present study was conducted with the aim to identify appropriate root traits enhancing rice grain yield under alternate wetting and drying conditions (AWD) and identify stable, high-yielding genotypes better suited to the AWD across variable ecosystems. Advanced breeding lines, popular rice varieties and drought-tolerant lines were evaluated in a series of 23 experiments conducted in the Philippines, India, Bangladesh, Nepal and Cambodia in 2015 and 2016. A large variation in grain yield under AWD conditions enabled the selection of high-yielding and stable genotypes across locations, seasons and years. Water savings of 5.7–23.4% were achieved without significant yield penalty across different ecosystems. The mean grain yield of genotypes across locations ranged from 3.5 to 5.6 t/ha and the mean environment grain yields ranged from 3.7 (Cambodia) to 6.6 (India) t/ha. The best-fitting Finlay-Wilkinson regression model identified eight stable genotypes with mean grain yield of more than 5.0 t/ha across locations. Multidimensional preference analysis represented the strong association of root traits (nodal root number, root dry weight at 22 and 30 days after transplanting) with grain yield. The genotype IR14L253 outperformed in terms of root traits and high mean grain yield across seasons and six locations. The 1.0 t/ha yield advantage of IR14L253 over the popular cultivar IR64 under AWD shall encourage farmers to cultivate IR14L253 and also adopt AWD. The results suggest an important role of root architectural traits in term of more number of nodal roots and root dry weight at 10–20 cm depth on 22–30 days after transplanting (DAT) in providing yield stability and preventing yield reduction under AWD compared to continuous flooded conditions. Genotypes possessing increased number of nodal roots provided higher yield over IR64 as well as no

[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

PubMed

Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

2015-12-01

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

Spectrophotometric total reducing sugars assay based on cupric reduction.

PubMed

Başkan, Kevser Sözgen; Tütem, Esma; Akyüz, Esin; Özen, Seda; Apak, Reşat

2016-01-15

As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method. Copyright © 2015 Elsevier B.V. All rights reserved.

Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

PubMed

Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

2017-10-15

Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

PubMed

Barber, L E; Tjepkema, J D; Russell, S A; Evans, H J

1976-07-01

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.

Profiling Sugars in Tall Fescue and Kentucky Bluegrass Extracts Assayed for Total Water- and Ethanol-Soluble Carbohydrates: Relationship of Chromatographic to Spectrophotometric Data

USDA-ARS?s Scientific Manuscript database

Water-soluble carbohydrates (WSC) and ethanol-soluble carbohydrates (ESC) of herbage are often quantified by spectrophotometric assays. To determine if quantifying individual sugars from chromatograms could yield results comparable to those obtained by the assays, WSC and ESC were extracted from fr...

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

Performance evaluation of the Aptima® HCV Quant Dx assay for hepatitis C virus (HCV) RNA detection and quantification in comparison to the Abbott RealTime HCV assay.

PubMed

Garbuglia, Anna Rosa; Bibbò, Angela; Sciamanna, Roberta; Pisciotta, Marina; Capobianchi, Maria Rosaria

2017-07-01

The Aptima HCV Quant Dx assay (Aptima) is a real-time transcription-mediated amplification assay CE-approved for the diagnosis and monitoring of hepatitis C virus (HCV) infection. Aptima's analytical performance was compared to the Abbott RealTime HCV assay (RealTime) in a clinical routine setting. Overall 295 clinical plasma samples (117 prospective/fresh; 178 retrospective/frozen) from HCV-infected patients were tested in Aptima and RealTime to determine concordance on qualitative and quantitative results. Linearity and precision at low viral loads (VLs; 0.8-3.3LogIU/mL) was tested using dilutions of the 5th WHO standard, in 10 and 20 replicates in the two assays, respectively. The ability to measure different HCV genotypes and accuracy were analyzed using the Seracare EQA panel. Inter-assay agreement for qualitative results (prospective samples) was 88% (kappa=0.78). For the 127 samples with quantitative results in both assays, Aptima yielded on average slightly higher values (by 0.24LogIU/mL; Bland-Altman method) than RealTime. Concordance between assay results was excellent (R=0.98). At low VLs (0.8-3.3LogIU/mL), Aptima demonstrated good linearity and precision, similar to RealTime. Aptima detected and accurately quantified all main HCV genotypes. Aptima demonstrated excellent precision, linearity, and accuracy in all genotypes tested. Good concordance was observed between Aptima and RealTime assays in clinical samples. The performance of the Aptima assay, on the fully automated Panther platform, makes it an excellent candidate for the detection and monitoring of HCV RNA in plasma and serum samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays.

PubMed

Saleh, Lanja; Mueller, Daniel; von Eckardstein, Arnold

2016-04-01

We evaluated the analytical and clinical performance of the new Lumipulse® G 25-OH vitamin D assay from Fujirebio, and compared it to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and three other commercial automated assays. Total 25 hydroxy vitamin D (25(OH)D) levels were measured in 100 selected serum samples from our routine analysis with Fujirebio 25(OH)D assay. The results were compared with those obtained with LC-MS/MS and three other automated 25(OH)D assays (Abbott, Beckman, and Roche). The accuracy of each assay tested was evaluated against a Labquality reference serum panel for 25(OH)D (Ref!25OHD; University of Ghent). Intra- and inter-day imprecision of the Fujirebio 25(OH)D assay was <5%. Fujirebio 25(OH)D assay showed the highest correlation among the assays tested with the LC-MS/MS method (R=0.986). The mean relative bias obtained was -15.6% (Fujirebio), -12.7% (Beckman), -2.1% (Abbott) and 9.7% (Roche) as compared to LC-MS/MS. Comparison with the Labquality certified reference serum panel yielded a mean bias of -11.8% (Fujirebio), -14.1% (Beckman), 4.4% (Abbott) and 3.2% (Roche), respectively. Compared to LC-MS/MS, the sensitivity of different methods in detecting vitamin D insufficiency (<50 nmol/L) varied from 100% for the Fujirebio assay to 72.7% for Roche, and specificity ranged from 94.4% for Roche to 87.6% for Beckman. The Lumipulse G 25-OH vitamin D assay from Fujirebio demonstrated a good correlation with LC-MS/MS and some immunoassays. The performance of the assay is well-suited for routine 25(OH)D measurement in clinical serum samples. A correction for the observed negative bias vs. LC-MS/MS could be considered.

Effect of Damping and Yielding on the Seismic Response of 3D Steel Buildings with PMRF

PubMed Central

Haldar, Achintya; Rodelo-López, Ramon Eduardo; Bojórquez, Eden

2014-01-01

The effect of viscous damping and yielding, on the reduction of the seismic responses of steel buildings modeled as three-dimensional (3D) complex multidegree of freedom (MDOF) systems, is studied. The reduction produced by damping may be larger or smaller than that of yielding. This reduction can significantly vary from one structural idealization to another and is smaller for global than for local response parameters, which in turn depends on the particular local response parameter. The uncertainty in the estimation is significantly larger for local response parameter and decreases as damping increases. The results show the limitations of the commonly used static equivalent lateral force procedure where local and global response parameters are reduced in the same proportion. It is concluded that estimating the effect of damping and yielding on the seismic response of steel buildings by using simplified models may be a very crude approximation. Moreover, the effect of yielding should be explicitly calculated by using complex 3D MDOF models instead of estimating it in terms of equivalent viscous damping. The findings of this paper are for the particular models used in the study. Much more research is needed to reach more general conclusions. PMID:25097892

Effect of damping and yielding on the seismic response of 3D steel buildings with PMRF.

PubMed

Reyes-Salazar, Alfredo; Haldar, Achintya; Rodelo-López, Ramon Eduardo; Bojórquez, Eden

2014-01-01

The effect of viscous damping and yielding, on the reduction of the seismic responses of steel buildings modeled as three-dimensional (3D) complex multidegree of freedom (MDOF) systems, is studied. The reduction produced by damping may be larger or smaller than that of yielding. This reduction can significantly vary from one structural idealization to another and is smaller for global than for local response parameters, which in turn depends on the particular local response parameter. The uncertainty in the estimation is significantly larger for local response parameter and decreases as damping increases. The results show the limitations of the commonly used static equivalent lateral force procedure where local and global response parameters are reduced in the same proportion. It is concluded that estimating the effect of damping and yielding on the seismic response of steel buildings by using simplified models may be a very crude approximation. Moreover, the effect of yielding should be explicitly calculated by using complex 3D MDOF models instead of estimating it in terms of equivalent viscous damping. The findings of this paper are for the particular models used in the study. Much more research is needed to reach more general conclusions.

Outer-layer manipulators for turbulent drag reduction

NASA Technical Reports Server (NTRS)

Anders, J. B., Jr.

1990-01-01

The last ten years have yielded intriguing research results on aerodynamic boundary outer-layer manipulators as local skin friction reduction devices at low Reynolds numbers; net drag reduction device systems for entire aerodynamic configurations are nevertheless noted to remain elusive. Evidence has emerged for dramatic alterations of the structure of a turbulent boundary layer which persist for long distances downstream and reduce wall shear as a results of any one of several theoretically possible mechanisms. Reduced effectiveness at high Reynolds numbers may, however, limit the applicability of outer-layer manipulators to practical aircraft drag reduction.

Two continuous coupled assays for ornithine-δ-aminotransferase.

PubMed

Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

2013-09-15

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

Multi-center validation of the Response Biomedical Corporation RAMP NT-proBNP assay with comparison to the Roche Diagnostics GmbH Elecsys proBNP assay.

PubMed

Lee-Lewandrowski, Elizabeth; Januzzi, James L; Green, Sandy M; Tannous, Bakhos; Wu, Alan H B; Smith, Andrew; Wong, Alicia; Murakami, MaryAnn M; Kaczmarek, Jason; Apple, Fred S; Miller, Wayne L; Hartman, Karen; Jaffe, Allan S

2007-01-01

NT-proBNP measurements aid in the evaluation of patients with suspected heart failure (HF) and may facilitate risk stratification in patients with HF and acute coronary syndrome (ACS). Point-of-care (POC) assays may provide more timely results and potentially improve patient outcomes. We evaluated the analytical performance of the Response Biomedical Corporation whole blood RAMP amino-terminal pro-B type natriuretic peptide (NT-proBNP) POC assay compared to the Roche Elecsys proBNP (NT-proBNP) assay. Intra-day and total imprecision (% CV) ranged from 5.5% to 10.3% at 140, 449 and 1675 ng/L. The lowest concentration that yields a 20% CV was 57 ng/L. The lower limit of detection was 18 ng/L. The upper limit of linearity was validated to 23,428 ng/L with an average recovery of 95%. Correlation by Passing and Bablok regression yielded RAMP=1.01 Elecsys+14.6, r=0.98 (n=540; range of Elecsys values <5 to >35,000). Concordance of RAMP versus Elecsys using cut-offs of 125 ng/L for subjects <75 years and 450 ng/L for subjects > or =75 was 92% (95% CI 89-94%) for a group consisting of 127 apparently healthy individuals and 208 non-healthy subjects without HF, and 99% (95% CI 97-100%) for patients with HF, using the New York Heart Association (NYHA) functional classification. Overall, 80%, 87%, 97% and 100% of the RAMP results and 77%, 85%, 96% and 100% of the Elecsys results were greater than the age appropriate cut-off for NYHA I, II, III or IV groups. For both the RAMP and Elecsys results, the median NT-proBNP value was statistically correlated (increasing) with NYHA I, II, III or IV groups, respectively (p<0.0001), with no significant difference between the two methods. The POC Response Biomedical RAMP NT-proBNP assay provides comparable results that measured on the FDA cleared Roche Elecsys central laboratory platform.

Seed yield, development, and variation in diverse poa pratensis accessions

USDA-ARS?s Scientific Manuscript database

Post harvest residue removal is critical for continued high seed production of Kentucky bluegrass (Poa pratensis L.). Previous work showed some accessions have little or no yield reduction with mechanical residue removal compared with the controversial practice of open field burning. Using 10 of t...

Multi-tiered Approach to Development of Increased Throughput Assay Models to Assess Endocrine-Disrupting Activity of Chemicals

EPA Science Inventory

Screening for endocrine-disrupting chemicals (EDCs) requires sensitive, scalable assays. Current high-throughput screening (HTPS) approaches for estrogenic and androgenic activity yield rapid results, but many are not sensitive to physiological hormone concentrations, suggesting ...

Blackleg (Leptosphaeria maculans) Severity and Yield Loss in Canola in Alberta, Canada

PubMed Central

Hwang, Sheau-Fang; Strelkov, Stephen E.; Peng, Gary; Ahmed, Hafiz; Zhou, Qixing; Turnbull, George

2016-01-01

Blackleg, caused by Leptosphaeria maculans, is an important disease of oilseed rape (Brassica napus L.) in Canada and throughout the world. Severe epidemics of blackleg can result in significant yield losses. Understanding disease-yield relationships is a prerequisite for measuring the agronomic efficacy and economic benefits of control methods. Field experiments were conducted in 2013, 2014, and 2015 to determine the relationship between blackleg disease severity and yield in a susceptible cultivar and in moderately resistant to resistant canola hybrids. Disease severity was lower, and seed yield was 120%–128% greater, in the moderately resistant to resistant hybrids compared with the susceptible cultivar. Regression analysis showed that pod number and seed yield declined linearly as blackleg severity increased. Seed yield per plant decreased by 1.8 g for each unit increase in disease severity, corresponding to a decline in yield of 17.2% for each unit increase in disease severity. Pyraclostrobin fungicide reduced disease severity in all site-years and increased yield. These results show that the reduction of blackleg in canola crops substantially improves yields. PMID:27447676

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays.

PubMed

Mistry, Bhaven A; D'Orsogna, Maria R; Chou, Tom

2018-06-19

Many biological assays are employed in virology to quantify parameters of interest. Two such classes of assays, virus quantification assays (VQAs) and infectivity assays (IAs), aim to estimate the number of viruses present in a solution and the ability of a viral strain to successfully infect a host cell, respectively. VQAs operate at extremely dilute concentrations, and results can be subject to stochastic variability in virus-cell interactions. At the other extreme, high viral-particle concentrations are used in IAs, resulting in large numbers of viruses infecting each cell, enough for measurable change in total transcription activity. Furthermore, host cells can be infected at any concentration regime by multiple particles, resulting in a statistical multiplicity of infection and yielding potentially significant variability in the assay signal and parameter estimates. We develop probabilistic models for statistical multiplicity of infection at low and high viral-particle-concentration limits and apply them to the plaque (VQA), endpoint dilution (VQA), and luciferase reporter (IA) assays. A web-based tool implementing our models and analysis is also developed and presented. We test our proposed new methods for inferring experimental parameters from data using numerical simulations and show improvement on existing procedures in all limits. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Modeling systematic errors: polychromatic sources of Beer-Lambert deviations in HPLC/UV and nonchromatographic spectrophotometric assays.

PubMed

Galli, C

2001-07-01

It is well established that the use of polychromatic radiation in spectrophotometric assays leads to excursions from the Beer-Lambert limit. This Note models the resulting systematic error as a function of assay spectral width, slope of molecular extinction coefficient, and analyte concentration. The theoretical calculations are compared with recent experimental results; a parameter is introduced which can be used to estimate the magnitude of the systematic error in both chromatographic and nonchromatographic spectrophotometric assays. It is important to realize that the polychromatic radiation employed in common laboratory equipment can yield assay errors up to approximately 4%, even at absorption levels generally considered 'safe' (i.e. absorption <1). Thus careful consideration of instrumental spectral width, analyte concentration, and slope of molecular extinction coefficient is required to ensure robust analytical methods.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate

PubMed Central

Levoye, Angélique; Zwier, Jurriaan M.; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization. PMID:26617570

One-week 96-well soft agar growth assay for cancer target validation.

PubMed

Ke, Ning; Albers, Aaron; Claassen, Gisela; Yu, De-hua; Chatterton, Jon E; Hu, Xiuyuan; Meyhack, Bernd; Wong-Staal, Flossie; Li, Qi-Xiang

2004-05-01

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

High performance of a cobalt–nitrogen complex for the reduction and reductive coupling of nitro compounds into amines and their derivatives

PubMed Central

Zhou, Peng; Jiang, Liang; Wang, Fan; Deng, Kejian; Lv, Kangle; Zhang, Zehui

2017-01-01

Replacement of precious noble metal catalysts with low-cost, non-noble heterogeneous catalysts for chemoselective reduction and reductive coupling of nitro compounds holds tremendous promise for the clean synthesis of nitrogen-containing chemicals. We report a robust cobalt–nitrogen/carbon (Co–Nx/C-800-AT) catalyst for the reduction and reductive coupling of nitro compounds into amines and their derivates. The Co–Nx/C-800-AT catalyst was prepared by the pyrolysis of cobalt phthalocyanine–silica colloid composites and the subsequent removal of silica template and cobalt nanoparticles. The Co–Nx/C-800-AT catalyst showed extremely high activity, chemoselectivity, and stability toward the reduction of nitro compounds with H2, affording full conversion and >97% selectivity in water after 1.5 hours at 110°C and under a H2 pressure of 3.5 bar for all cases. The hydrogenation of nitrobenzene over the Co–Nx/C-800-AT catalyst can even be smoothly performed under very mild conditions (40°C and a H2 pressure of 1 bar) with an aniline yield of 98.7%. Moreover, the Co–Nx/C-800-AT catalyst has high activity toward the transfer hydrogenation of nitrobenzene into aniline and the reductive coupling of nitrobenzene into other derivates with high yields. These processes were carried out in an environmentally friendly manner without base and ligands. PMID:28232954

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bandhuvula, Padmavathi; Li Zaiguo; Bittman, Robert

2009-03-06

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an {omega}-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K{sub m} ofmore » 35 {mu}M for BODIPY-sphingosine 1-phosphate.« less

Magnetic Resonance Imaging Assays for Dimethyl Sulfoxide Effect on Cancer Vasculature

PubMed Central

Cyran, Clemens C.; Sennino, Barbara; Chaopathomkul, Bundit; Fu, Yanjun; Rogut, Victor; Shames, David M.; Wendland, Michael F.; McDonald, Donald M.; Brasch, Robert C.

2015-01-01

Objectives To evaluate the potential of quantitative assays of vascular characteristics based on dynamic contrast-enhanced magnetic resonance imaging (MRI) using a macromolecular contrast medium (MMCM) to search for and measure effects of dimethyl sulfoxide (DMSO) on cancer vasculature with microscopic correlations. Material and Methods Saline-treated control (n = 8) and DMSO-treated (n = 7) human breast cancer xenografts (MDA-MB-435) in rats were imaged dynamically by MMCM-enhanced MRI using albumin-(Gd-DTPA)27-(biotin)11 (molecular weight approximately 90 kDa), before and after a 1-week, 3-dose treatment course. After the posttreatment MRI examinations, tumors were perfused with lectin and fixative and subsequently stained with RECA-1 and streptavidin for quantitative fluorescent microscopy. Quantitative MRI estimates of cancer microvessel permeability (KPS; µL/ min·100 cm3) and fractional plasma volume (fPV; %) were based on a 2-compartment kinetic model. Fluorescent microscopy yielded estimates of MMCM extravasation and vascular density that were compared to the MRI results. Results DMSO decreased cancer vascular endothelial permeability significantly (P < 0.05) from tumor KPSday0 = 19.3 ± 8.8 µL/min·100 cm3 to KPSday7 = 0 µL/min·100 cm3). KPS values in the saline-treated tumors did not change significantly. The amount of extravasated albumin-Gd-(DTPA)27-(biotin)11, as assayed by a fluorescently labeled streptavidin stain that strongly binds to the biotin tag on the MMCM, was significantly (P < 0.05) lower in the DMSO-treated cancers than in the control cancers (57.7% ± 5.5% vs. 34.2% ± 4.9%). Tumor vascular richness as reflected by the MRI-assayed fPV and by the RECA-1 and lectin-stained microscopy did not change significantly with DMSO or saline treatment. Conclusion Reductions in cancer microvascular leakiness induced by a 7-day course of DMSO could be detected and measured by dynamic MMCM-enhanced MRI and were confirmed by microscopic measurements

A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

PubMed

First, Eric A

2015-08-15

A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

A protein chip membrane-capture assay for botulinum neurotoxin activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marconi, Severine; Universite de la Mediterranee-Aix Marseille 2, Faculte de medecine secteur nord, Bd P. Dramard, Marseille F-13916; Ferracci, Geraldine

2008-12-15

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capturemore » providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.« less

A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

PubMed

Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

1984-10-01

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

2013-12-01

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of surfactant assisted sonic pretreatment on liquefaction of fruits and vegetable residue: Characterization, acidogenesis, biomethane yield and energy ratio.

PubMed

Shanthi, M; Rajesh Banu, J; Sivashanmugam, P

2018-05-15

The present study explored the disintegration potential of fruits and vegetable residue through sodium dodecyl sulphate (SDS) assisted sonic pretreatment (SSP). In SSP method, initially the biomass barrier (lignin) was removed using SDS at different dosage, subsequently it was sonically disintegrated. The effect of SSP were assessed based on dissolved organic release (DOR) of fruits and vegetable waste and specific energy input. SSP method achieved higher DOR rate and suspended solids reduction (26% and 16%) at optimum SDS dosage of 0.035 g/g SS with least specific energy input of 5400 kJ/kg TS compared to ultrasonic pretreatment (UP) (16% and 10%). The impact of fermentation and biomethane potential assay revealed highest production of volatile fatty acid and methane yield in SSP (1950 mg/L, 0.6 g/g COD) than UP. The energy ratio obtained was 0.9 for SSP, indicating proposed method is energetically efficient. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adapting the CROPGRO cotton model to simulate cotton biomass and yield under southern root-knot nematode parasitism

USDA-ARS?s Scientific Manuscript database

Cotton (Gossypium hirsutum L.) yield losses by southern root-knot nematode [Meloidogyne incognita (Kofoid & White) Chitwood] (RKN) are usually estimated after significant damage has been caused. However, estimation of potential yield reduction before planting is possible by using crop simulation mod...

Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

PubMed Central

Anderson, Courtney M.; Zhang, Bingqing; Miller, Melanie; Butko, Emerald; Wu, Xingyong; Laver, Thomas; Kernag, Casey; Kim, Jeffrey; Luo, Yuling; Lamparski, Henry; Park, Emily; Su, Nan

2016-01-01

ABSTRACT Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27191821

Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

PubMed Central

Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

PubMed

Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

The unbearable lightness of "light" cigarettes: a comparison of smoke yields in six varieties of Canadian "light" cigarettes.

PubMed

Gendreau, Paul L; Vitaro, Frank

2005-01-01

Labelling cigarettes as "light" or "mild" is claimed to be one of the biggest marketing scams in Canadian history. Arguably, such labelling implies that these varieties of cigarettes are less harmful than "regular" cigarettes. In Canada, a food product can be labelled "light" if there is a 25% reduction from the "reference food" and if the constituent being reduced is clearly identified (e.g., light in fat). Cigarette labelling does not comply with these regulations, however. To examine whether or not some tobacco constituents meet the 25% reduction criterion, we compared yields of 41 toxic and/or carcinogenic smoke constituents in six varieties of "light" cigarettes to the yields of "regular" cigarettes. We selected cigarettes from the two most popular Canadian brands, Du Maurier and Players. Using a set of data provided by Imperial Tobacco Canada and made available to the public by the Government of British Columbia, we compared yields measured under a laboratory protocol (modified ISO) that was designed to provide a more rigorous evaluation of the differences between varieties of cigarettes and a more accurate assessment of smokers' potential smoke intake than the traditional protocol (standard ISO). For all six varieties of "light" cigarettes, the yields of nicotine were higher by an average of 5% (range: 1% to 13%). The 25% reduction criterion was not met for any variety of "light" cigarettes concerning yields of tar. For all cigarettes tested, yields of tar were reduced on average by only 16% (range: 5% to 22%). For carbon monoxide (CO), only Player's Smooth Light had an over 25% reduction (30%) compared with Player's Regular. Conversely, yield of CO was 24% higher for Du Maurier Lights compared with Du Maurier Regular. As for the other smoke constituents, the majority (75%) were not reduced by 25% or more in "light" cigarettes, and a sizeable proportion of yields (e.g., acrylonitrile, benzene, chromium, m+p cresol, mercury, nickel, toluene) were larger in

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays.

PubMed

Cwach, Kevin T; Sandbulte, Heather R; Klonoski, Joshua M; Huber, Victor C

2012-03-01

Prior to detection of an antibody response toward influenza viruses using the hemagglutination inhibition assay (HAI), sera are routinely treated to inactivate innate inhibitors using both heat inactivation (56°C) and recombinant neuraminidase [receptor-destroying enzyme (RDE)]. We revisited the contributions of innate serum inhibitors toward interference with influenza viruses in immune assays, using murine sera, with emphasis on the interactions with influenza A viruses of the H3N2 subtype. We used individual serum treatments: 56°C alone, RDE alone, or RDE + 56°C, to treat sera prior to evaluation within HAI, microneutralization, and macrophage uptake assays. Our data demonstrate that inhibitors present within untreated murine sera interfere with the HAI assay in a manner that is different from that seen for the microneutralization assay. Specifically, the γ class inhibitor α(2) -Macroglobulin (A2-M) can inhibit H3N2 viruses within the HAI assay, but not in the microneutralization assay. Based on these findings, we used a macrophage uptake assay to demonstrate that these inhibitors can increase uptake by macrophages when the influenza viruses express an HA from a 1968 H3N2 virus isolate, but not a 1997 H3N2 isolate. The practice of treating sera to inactivate innate inhibitors of influenza viruses prior to evaluation within immune assays has allowed us to effectively detect influenza virus-specific antibodies for decades. However, this practice has yielded an under-appreciation for the contribution of innate serum inhibitors toward host immune responses against these viruses, including contributions toward neutralization and macrophage uptake. © 2011 Blackwell Publishing Ltd.

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae.

PubMed

Pagliardini, Julien; Hubmann, Georg; Alfenore, Sandrine; Nevoigt, Elke; Bideaux, Carine; Guillouet, Stephane E

2013-03-28

Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved

Brazilian Soybean Yields and Yield Gaps Vary with Farm Size

NASA Astrophysics Data System (ADS)

Jeffries, G. R.; Cohn, A.; Griffin, T. S.; Bragança, A.

2017-12-01

Understanding the farm size-specific characteristics of crop yields and yield gaps may help to improve yields by enabling better targeting of technical assistance and agricultural development programs. Linking remote sensing-based yield estimates with property boundaries provides a novel view of the relationship between farm size and yield structure (yield magnitude, gaps, and stability over time). A growing literature documents variations in yield gaps, but largely ignores the role of farm size as a factor shaping yield structure. Research on the inverse farm size-productivity relationship (IR) theory - that small farms are more productive than large ones all else equal - has documented that yield magnitude may vary by farm size, but has not considered other yield structure characteristics. We examined farm size - yield structure relationships for soybeans in Brazil for years 2001-2015. Using out-of-sample soybean yield predictions from a statistical model, we documented 1) gaps between the 95th percentile of attained yields and mean yields within counties and individual fields, and 2) yield stability defined as the standard deviation of time-detrended yields at given locations. We found a direct relationship between soy yields and farm size at the national level, while the strength and the sign of the relationship varied by region. Soybean yield gaps were found to be inversely related to farm size metrics, even when yields were only compared to farms of similar size. The relationship between farm size and yield stability was nonlinear, with mid-sized farms having the most stable yields. The work suggests that farm size is an important factor in understanding yield structure and that opportunities for improving soy yields in Brazil are greatest among smaller farms.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays

PubMed Central

Cwach, Kevin T.; Sandbulte, Heather R.; Klonoski, Joshua M.; Huber, Victor C.

2011-01-01

Please cite this paper as: Cwach et al. (2011) Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2011.00283.x. Background  Prior to detection of an antibody response toward influenza viruses using the hemagglutination inhibition assay (HAI), sera are routinely treated to inactivate innate inhibitors using both heat inactivation (56°C) and recombinant neuraminidase [receptor‐destroying enzyme (RDE)]. Objectives  We revisited the contributions of innate serum inhibitors toward interference with influenza viruses in immune assays, using murine sera, with emphasis on the interactions with influenza A viruses of the H3N2 subtype. Methods  We used individual serum treatments: 56°C alone, RDE alone, or RDE + 56°C, to treat sera prior to evaluation within HAI, microneutralization, and macrophage uptake assays. Results  Our data demonstrate that inhibitors present within untreated murine sera interfere with the HAI assay in a manner that is different from that seen for the microneutralization assay. Specifically, the γ class inhibitor α2‐Macroglobulin (A2‐M) can inhibit H3N2 viruses within the HAI assay, but not in the microneutralization assay. Based on these findings, we used a macrophage uptake assay to demonstrate that these inhibitors can increase uptake by macrophages when the influenza viruses express an HA from a 1968 H3N2 virus isolate, but not a 1997 H3N2 isolate. Conclusions  The practice of treating sera to inactivate innate inhibitors of influenza viruses prior to evaluation within immune assays has allowed us to effectively detect influenza virus‐specific antibodies for decades. However, this practice has yielded an under‐appreciation for the contribution of innate serum inhibitors toward host immune responses against these viruses, including contributions toward neutralization and macrophage uptake. PMID

Evaluating the synchronicity in yield variations of staple crops at global scale

NASA Astrophysics Data System (ADS)

Yokozawa, M.

2014-12-01

Reflecting the recent globalization trend in world commodity market, several major production countries are producing large amount of staple crops, especially, maize and soybean. Thus, simultaneous crop failure (abrupt reduction in crop yield, lean year) due to extreme weather and/or climate change could lead to unstable food supply. This study try to examine the synchronicity in yield variations of staple crops at global scale. We use a gridded crop yields database, which includes the historical year-to-year changes in staple crop yields with a spatial resolution of 1.125 degree in latitude/longitude during a period of 1982-2006 (Iizumi et al. 2013). It has been constructed based on the agriculture statistics issued by local administrative bureaus in each country. For the regions being lack of data, an interpolation was conducted to obtain the values referring to the NPP estimates from satellite data as well as FAO country yield. For each time series of the target crop yield, we firstly applied a local kernel regression to represent the long-term trend component. Next, the deviations of yearly yield from the long-term trend component were defined as ΔY(i, y) in year y at grid i. Then, the correlation of deviation between grids i and j in year y is defined as Cij(y) = ΔY(i, y) ΔY(j, y). In addition, Pij = <ΔY(i, y) ΔY(j, y)> represents the time-averaged correlation of deviation between grids i and j. Bracket <...> means the time average operation over 25 years (1982-2006). As the results, figures show the time changes in the number of grid pairs, in which both the deviation are negative. It represent the time changes in ratio of the grid pairs where both crop yields synchronically decreased to the total grid pairs. The years denoted by arrows in the figures indicate the case that all the ratios of three country pairs (i.e. China-USA, USA-Brazil and Brazil-China) are relatively larger (>0.6 for soybean and >0.5 for maize). This suggests that the reductions in

Revised estimates for ozone reduction by shuttle operation

NASA Technical Reports Server (NTRS)

Potter, A. E.

1978-01-01

Previous calculations by five different modeling groups of the effect of space shuttle operations on the ozone layer yielded an estimate of 0.2 percent ozone reduction for the Northern Hemisphere at 60 launches per year. Since these calculations were made, the accepted rate constant for the reaction between hydroperoxyl and nitric oxide to yield hydroxyl and nitrogen dioxide, HO2 + NO yields OH + NO2, was revised upward by more than an order of magnitude, with a resultant increase in the predicted ozone reduction for chlorofluoromethanes by a factor of approximately 2. New calculations of the shuttle effect were made with use of the new rate constant data, again by five different modeling groups. The new value of the shuttle effect on the ozone layer was found to be 0.25 percent. The increase resulting from the revised rate constant is considerably less for space shuttle operations than for chlorofluoromethane production, because the new rate constant also increases the calculated rate of downward transport of shuttle exhaust products out of the stratosphere.

Hydrodynamic effects on microcapillary motility and chemotaxis assays of Methylosinus trichosporium OB3b.

PubMed Central

Shonnard, D R; Taylor, R T; Tompson, A; Knapp, R B

1992-01-01

A study of the random motility and chemotaxis of Methylosinus trichosporium OB3b was conducted by using Palleroni-chamber microcapillary assay procedures. Under the growth conditions employed, this methanotroph was observed qualitatively with a microscope to be either slightly motile or essentially nonmotile. However, the cells did not not respond in the microcapillary assays in the manner expected for nonmotile Brownian particles. As a consequence, several hydrodynamic effects on these Palleroni microcapillary assays were uncovered. In the random-motility microcapillary assay, nondiffusive cell accumulations occurred that were strongly dependent upon cell concentration. An apparent minimal random-motility coefficient (mu) for this bacterial cell of 1.0 x 10(-7) cm2/s was estimated from microcapillary assays. A simple alternative spectrophotometric assay, based upon gravitational settling, was developed and shown to be an improvement over the Palleroni microcapillary motility assay for M. trichosporium OB3b in that it yielded a more-accurate threefold-lower random-motility coefficient. In addition, it provided a calculation of the gravitational-settling velocity. In the chemotaxis microcapillary assay, the apparent chemotactic responses were strongest for the highest test-chemical concentrations in the microcapillaries, were correlated with microcapillary fluid density, and were strongly dependent upon the microcapillary volume. A simple method to establish the maximal concentration of a chemical that can be tested and to quantify any contributions of abiotic convection is described. Investigators should be aware of the potential problems due to density-driven convection when using these commonly employed microcapillary assays for studying cells which have low motilities. PMID:1444383

A novel reporter gene assay for recombinant human erythropoietin (rHuEPO) pharmaceutical products.

PubMed

Yang, Yushuai; Zhou, Yong; Yu, Lei; Li, Xiang; Shi, Xinchang; Qin, Xi; Rao, Chunming; Wang, Junzhi

2014-11-01

Accurate determination of in vitro biological activity of therapeutic erythropoietin is essential in quality control of recombinant human erythropoietin (rHuEPO) pharmaceutical products. However, most of currently-used methods leave much to be desired so that a simpler, quicker and more accurate method is urgently needed. The bioassay described here utilizes a sub clone of UT-7/epo cell line stably transfected with luciferase gene under the control of sis inducible element and interferon γ-activated sequence element promoter. Active erythropoietin could induce the expression of luciferase by signaling through the erythropoietin receptor and the dose-response curve showed good linearity, yielding a coefficient of determination of 0.99 or higher. The optimized assay was simpler with the operation completed within 24h and more sensitive with EC50 being 0.077IU/mL. The accuracy estimates ranged from 81.7% to 102.4%, and both intra-assay and inter-assay precision was below 15.0%. The robustness of the assay was demonstrated by no effect of passage levels of the cells on the performance of the assay (p values: 0.772 for sample 1 and 0.943 for sample 2). Besides, Bland-Altman analysis showed a high consistency of the new assay with in vivo reticulocyte assay in results. These results suggested that the new reporter gene assay can be a viable supplement to the traditional reticulocyte assay and employed in potency determination of rHuEPO pharmaceutical products. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Drag reduction of a hairy disk

NASA Astrophysics Data System (ADS)

Niu, Jun; Hu, David L.

2011-10-01

We investigate experimentally the hydrodynamics of a hairy disk immersed in a two-dimensional flowing soap film. Drag force is measured as a function of hair length, density, and coating area. An optimum combination of these parameters yields a drag reduction of 17%, which confirms previous numerical predictions (15%). Flow visualization indicates the primary mechanism for drag reduction is the bending, adhesion, and reinforcement of hairs trailing the disk, which reduces wake width and traps "dead water." Thus, the use of hairy coatings can substantially reduce an object's drag while negligibly increasing its weight.

An automated real-time free phenytoin assay to replace the obsolete Abbott TDx method.

PubMed

Williams, Christopher; Jones, Richard; Akl, Pascale; Blick, Kenneth

2014-01-01

Phenytoin is a commonly used anticonvulsant that is highly protein bound with a narrow therapeutic range. The unbound fraction, free phenytoin (FP), is responsible for pharmacologic effects; therefore, it is essential to measure both FP and total serum phenytoin levels. Historically, the Abbott TDx method has been widely used for the measurement of FP and was the method used in our laboratory. However, the FP TDx assay was recently discontinued by the manufacturer, so we had to develop an alternative methodology. We evaluated the Beckman-Coulter DxC800 based FP method for linearity, analytical sensitivity, and precision. The analytical measurement range of the method was 0.41 to 5.30 microg/mL. Within-run and between-run precision studies yielded CVs of 3.8% and 5.5%, respectively. The method compared favorably with the TDx method, yielding the following regression equation: DxC800 = 0.9**TDx + 0.10; r2 = 0.97 (n = 97). The new FP assay appears to be an acceptable alternative to the TDx method.

Texas Native Plants Yield Compounds with Cytotoxic Activities against Prostate Cancer Cells.

PubMed

Shaffer, Corena V; Cai, Shengxin; Peng, Jiangnan; Robles, Andrew J; Hartley, Rachel M; Powell, Douglas R; Du, Lin; Cichewicz, Robert H; Mooberry, Susan L

2016-03-25

There remains a critical need for more effective therapies for the treatment of late-stage and metastatic prostate cancers. Three Texas native plants yielded three new and three known compounds with antiproliferative and cytotoxic activities against prostate cancer cells with IC50 values in the range of 1.7-35.0 μM. A new sesquiterpene named espadalide (1), isolated from Gochnatia hypoleuca, had low micromolar potency and was highly effective in clonogenic assays. Two known bioactive germacranolides (2 and 3) were additionally isolated from G. hypoleuca. Dalea frutescens yielded two new isoprenylated chalcones, named sanjuanolide (4) and sanjoseolide (5), and the known sesquiterpenediol verbesindiol (6) was isolated from Verbesina virginica. Mechanistic studies showed that 1-4 caused G2/M accumulation and the formation of abnormal mitotic spindles. Tubulin polymerization assays revealed that 4 increased the initial rate of tubulin polymerization, but did not change total tubulin polymer levels, and 1-3 had no effects on tubulin polymerization. Despite its cytotoxic activity, compound 6 did not initiate changes in cell cycle distribution and has a mechanism of action different from the other compounds. This study demonstrates that new compounds with significant biological activities germane to unmet oncological needs can be isolated from Texas native plants.

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

2015-01-01

Background Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. Methodology/Principal Findings Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. Conclusions/Significance During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. PMID:26075598

Plausible rice yield losses under future climate warming.

PubMed

Zhao, Chuang; Piao, Shilong; Wang, Xuhui; Huang, Yao; Ciais, Philippe; Elliott, Joshua; Huang, Mengtian; Janssens, Ivan A; Li, Tao; Lian, Xu; Liu, Yongwen; Müller, Christoph; Peng, Shushi; Wang, Tao; Zeng, Zhenzhong; Peñuelas, Josep

2016-12-19

Rice is the staple food for more than 50% of the world's population 1-3 . Reliable prediction of changes in rice yield is thus central for maintaining global food security. This is an extraordinary challenge. Here, we compare the sensitivity of rice yield to temperature increase derived from field warming experiments and three modelling approaches: statistical models, local crop models and global gridded crop models. Field warming experiments produce a substantial rice yield loss under warming, with an average temperature sensitivity of -5.2 ± 1.4% K -1 . Local crop models give a similar sensitivity (-6.3 ± 0.4% K -1 ), but statistical and global gridded crop models both suggest less negative impacts of warming on yields (-0.8 ± 0.3% and -2.4 ± 3.7% K -1 , respectively). Using data from field warming experiments, we further propose a conditional probability approach to constrain the large range of global gridded crop model results for the future yield changes in response to warming by the end of the century (from -1.3% to -9.3% K -1 ). The constraint implies a more negative response to warming (-8.3 ± 1.4% K -1 ) and reduces the spread of the model ensemble by 33%. This yield reduction exceeds that estimated by the International Food Policy Research Institute assessment (-4.2 to -6.4% K -1 ) (ref. 4). Our study suggests that without CO 2 fertilization, effective adaptation and genetic improvement, severe rice yield losses are plausible under intensive climate warming scenarios.

[Comparison of the performance of the ECLusys anti-HCV reagent with the Lumipulse f and HISCL 2000-i HCVAb assays].

PubMed

Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

2012-09-01

We compared the ECLusys Anti-HCV (ECL) reagent to the Lumipulse f (LPf) and HISCL (HIS) HCV assays. In a correlation test using 210 routine clinical specimens measured using the Lumipulse method (96 positive and 114 negative), most of the results were consistent for all specimens. In a dilution sensitivity test using three different routine positive specimens, the ECL assay enabled detection at higher levels of sensitivity than either the LPf or the HIS assay. Moreover, when the distribution of the cut-off index (C.O.I.) values of the routine LPf negative specimens were compared to those on the ECL and HIS assays, it was found that on the ECL assay, most of the specimens had cut-off index values < 0.1, indicating a more clear-cut distribution. In a specificity test using high RF positive specimens(n = 33), pregnancy specimens (n = 35), cytomegalovirus (CMV) antibody positive specimens (n = 36), and high M protein positive specimens (n = 21), the ECL assay yielded positive results for a CMV antibody positive specimen and three high M protein positive specimens. Further testing using samples from the same patients collected on different days than these four samples resulted in a second positive result for the CMV positive specimen, and single antigen measurement yielded a Core/NS3 positive result, as well, suggesting past infection. However, since negative results were obtained for the three M protein positive specimens, the possibility of this being a ECLusys non-specific reaction could not be ruled out. The above results confirmed that the ECL assay provides superior fundamental performance, and possesses test performance nearly identical to that of the existing measurement methods that are widely used at a large number of facilities, and would therefore be a suitable assay for use in routine HCV antibody screening.

Skin friction drag reduction in turbulent flow using spanwise traveling surface waves

NASA Astrophysics Data System (ADS)

Musgrave, Patrick F.; Tarazaga, Pablo A.

2017-04-01

A major technological driver in current aircraft and other vehicles is the improvement of fuel efficiency. One way to increase the efficiency is to reduce the skin friction drag on these vehicles. This experimental study presents an active drag reduction technique which decreases the skin friction using spanwise traveling waves. A novel method is introduced for generating traveling waves which is low-profile, non-intrusive, and operates under various flow conditions. This wave generation method is discussed and the resulting traveling waves are presented. These waves are then tested in a low-speed wind tunnel to determine their drag reduction potential. To calculate the drag reduction, the momentum integral method is applied to turbulent boundary layer data collected using a pitot tube and traversing system. The skin friction coefficients are then calculated and the drag reduction determined. Preliminary results yielded a drag reduction of ≍ 5% for 244Hz traveling waves. Thus, this novel wave generation method possesses the potential to yield an easily implementable, non-invasive drag reduction technology.

A new assay system for guinea pig interferon biological activity.

PubMed

Yamamoto, Toshiko; Jeevan, Amminikutty; Ohishi, Kazue; Nojima, Yasuhiro; Umemori, Kiyoko; Yamamoto, Saburo; McMurray, David N

2002-07-01

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation 1

PubMed Central

Hardy, R. W. F.; Holsten, R. D.; Jackson, E. K.; Burns, R. C.

1968-01-01

The methodology, characteristics and application of the sensitive C2H2-C2H4 assay for N2 fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N2ase-catalyzed reduction of C2H2 to C2H4, gas chromatographic isolation of C2H2 and C2H4, and quantitative measurement with a H2-flame analyzer. As little as 1 μμmole C2H4 can be detected, providing a sensitivity 103-fold greater than is possible with 15N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C2H2-reducing activity in the field. Applications to field samples included an evaluation of N2 fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg N2 fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N2 fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N2 fixing activity of soybeans. The validity of measuring N2 fixation in terms of C2H2 reduction was established through extensive comparisons of these activities using defined systems, including purified N2ase preparations and pure cultures of N2-fixing bacteria. With this assay it now becomes possible and practicable to conduct comprehensive surveys of N2 fixation, to make detailed comparisons among different N2-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N2 fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N2

Cross-reactions in specific Brachyspira spp. PCR assays caused by "Brachyspira hampsonii" isolates: implications for detection.

PubMed

Aller-Morán, Luis M; Martínez-Lobo, F Javier; Rubio, Pedro; Carvajal, Ana

2016-11-01

An emerging novel spirochete in swine, provisionally designated "Brachyspira hampsonii," has been detected worldwide. It has been associated with swine dysentery and cannot be differentiated from B. hyodysenteriae, the classical etiologic agent of this disease, using standard phenotypic methods. We evaluated cross-reactions of "B. hampsonii" isolates recovered from avian species in some of the currently available species-specific polymerase chain reaction (PCR) assays for the identification of swine Brachyspira species. Ten avian "B. hampsonii" isolates recovered from wild waterfowl were used. No false-positive results were recorded with a B. pilosicoli-specific PCR based on the amplification of a fragment of the 16S rRNA gene. However, the percentage of false-positive results varied, with a range of 10-80%, in the evaluated B. hyodysenteriae-specific assays based on the amplification of the 23S rRNA, nox, and tlyA genes. Similarly, results of the B. intermedia-specific PCR assays yielded poor specificity, with up to 80% of the "B. hampsonii" isolates tested giving false-positive results. Finally, 2 "B. hampsonii" avian isolates yielded a positive result in a B. innocens- and B. murdochii-specific PCR. This result should be interpreted very cautiously as these 2 isolates could represent a recombinant genotype. © 2016 The Author(s).

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

PubMed

Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

PubMed

Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

2016-02-01

Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P<0.0001), while minor quantitative biases existed. The Lumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases. Copyright © 2015 Elsevier B.V. All rights reserved.

Impacts of climate change on peanut yield in China simulated by CMIP5 multi-model ensemble projections

NASA Astrophysics Data System (ADS)

Xu, Hanqing; Tian, Zhan; Zhong, Honglin; Fan, Dongli; Shi, Runhe; Niu, Yilong; He, Xiaogang; Chen, Maosi

2017-09-01

Peanut is one of the major edible vegetable oil crops in China, whose growth and yield are very sensitive to climate change. In addition, agriculture climate resources are expected to be redistributed under climate change, which will further influence the growth, development, cropping patterns, distribution and production of peanut. In this study, we used the DSSAT-Peanut model to examine the climate change impacts on peanut production, oil industry and oil food security in China. This model is first calibrated using site observations including 31 years' (1981-2011) climate, soil and agronomy data. This calibrated model is then employed to simulate the future peanut yield based on 20 climate scenarios from 5 Global Circulation Models (GCMs) developed by the InterSectoral Impact Model Intercomparison Project (ISIMIP) driven by 4 Representative Concentration Pathways (RCPs). Results indicate that the irrigated peanut yield will decrease 2.6% under the RCP 2.6 scenario, 9.9% under the RCP 4.5 scenario and 29% under the RCP 8.5 scenario, respectively. Similarly, the rain-fed peanut yield will also decrease, with a 2.5% reduction under the RCP 2.6 scenario, 11.5% reduction under the RCP 4.5 scenario and 30% reduction under the RCP 8.5 scenario, respectively.

Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

PubMed

Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

2017-09-05

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

PubMed Central

Leach, L.; Zhu, Y.

2017-01-01

ABSTRACT Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris. The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities. PMID:29187562

A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

PubMed Central

Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

2014-01-01

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

Effects of aqueous uranyl speciation on the kinetics of microbial uranium reduction

DOE PAGES

Belli, Keaton M.; DiChristina, Thomas J.; Van Cappellen, Philippe; ...

2015-02-16

The ability to predict the success of the microbial reduction of soluble U(VI) to highly insoluble U(IV) as an in situ bioremediation strategy is complicated by the wide range of geochemical conditions at contaminated sites and the strong influence of aqueous uranyl speciation on the bioavailability and toxicity of U(VI) to metal-reducing bacteria. In order to determine the effects of aqueous uranyl speciation on uranium bioreduction kinetics, incubations and viability assays with Shewanella putrefaciens strain 200 were conducted over a range of pH and dissolved inorganic carbon (DIC), Ca 2+, and Mg 2+ concentrations. A speciation-dependent kinetic model was developedmore » to reproduce the observed time series of total dissolved uranium concentration over the range of geochemical conditions tested. The kinetic model yielded the highest rate constant for the reduction of uranyl non-carbonate species (i.e., the ‘free’ hydrated uranyl ion, uranyl hydroxides, and other minor uranyl complexes), indicating that they represent the most readily reducible fraction of U(VI) despite being the least abundant uranyl species in solution. In the presence of DIC, Ca 2+, and Mg 2+ is suppressed during the formation of more bioavailable uranyl non-carbonate species and resulted in slower bioreduction rates. At high concentrations of bioavailable U(VI), however, uranium toxicity to S. putrefaciens inhibited bioreduction, and viability assays confirmed that the concentration of non-carbonate uranyl species best predicts the degree of toxicity. The effect of uranium toxicity was accounted for by incorporating the free ion activity model of metal toxicity into the bioreduction rate law. These results demonstrate that, in the absence of competing terminal electron acceptors, uranium bioreduction kinetics can be predicted over a wide range of geochemical conditions based on the bioavailability and toxicity imparted on U(VI) by solution composition. Finally, these findings also

Reduction of aldehydes and hydrogen cyanide yields in mainstream cigarette smoke using an amine functionalised ion exchange resin

PubMed Central

2011-01-01

Background Cigarette smoking is a well recognized cause of diseases such as lung cancer, chronic obstructive pulmonary disease and cardiovascular disease. Of the more than 5000 identified species in cigarette smoke, at least 150 have toxicological activity. For example, formaldehyde and acetaldehyde have been assigned as Group 1 and Group 2B carcinogens by IARC, and hydrogen cyanide has been identified as a respiratory and cardiovascular toxicant. Active carbon has been shown to be an effective material for the physical adsorption of many of the smoke volatile species. However, physical adsorption of acetaldehyde, formaldehyde and also hydrogen cyanide from smoke is less effective using carbon. Alternative methods for the removal of these species from cigarette smoke are therefore of interest. A macroporous, polystyrene based ion-exchange resin (Diaion®CR20) with surface amine group functionality has been investigated for its ability to react with aldehydes and HCN in an aerosol stream, and thus selectively reduce the yields of these compounds (in particular formaldehyde) in mainstream cigarette smoke. Results Resin surface chemistry was characterized using vapour sorption, XPS, TOF-SIMS and 15N NMR. Diaion®CR20 was found to have structural characteristics indicating weak physisorption properties, but sufficient surface functionalities to selectively remove aldehydes and HCN from cigarette smoke. Using 60 mg of Diaion®CR20 in a cigarette cavity filter gave reductions in smoke formaldehyde greater than 50% (estimated to be equivalent to >80% of the formaldehyde present in the smoke vapour phase) independent of a range of flow rates. Substantial removal of HCN (>80%) and acetaldehyde (>60%) was also observed. The performance of Diaion®CR20 was found to be consistent over a test period of 6 months. The overall adsorption for the majority of smoke compounds measured appeared to follow a pseudo-first order approximation to second order kinetics. Conclusions This

A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)

PubMed Central

Deshpande, Kanchanmala; Mishra, Rupesh K.; Bhand, Sunil

2010-01-01

A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg·mL−1 with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%. PMID:22163555

Quantitative non-destructive assay of PuBe neutron sources

NASA Astrophysics Data System (ADS)

Lakosi, László; Bagi, János; Nguyen, Cong Tam

2006-02-01

PuBe neutron sources were assayed, using a combination of high resolution γ-spectrometry (HRGS) and neutron correlation technique. In a previous publication [J. Bagi, C. Tam Nguyen, L. Lakosi, Nucl. Instr. and Meth. B 222 (2004) 242] a passive neutron well-counter was reported with 3He tubes embedded in a polyamide (TERRAMID) moderator (lined inside with Cd) surrounding the sources to be measured. Gross and coincidence neutron counting was performed, and the Pu content of the sources was found out from isotope analysis and by adopting specific (α, n) reaction yields of the Pu isotopes and 241Am in Be, based on supplier's information and literature data. The method was further developed and refined. Evaluation algorithm was more precisely worked out. The contribution of secondary (correlated) neutrons to the total neutron output was derived from the coincidence (doubles) count rate and taken into account in assessing the Pu content. A new evaluation of former results was performed. Assay was extended to other PuBe sources, and new results were added. In order to attain higher detection efficiency, a more efficient moderator was also applied, with and without Cd shielding around the assay chamber. Calibration seems possible using neutron measurements only (without γ-spectrometry), based on a correlation between the Pu amount and the coincidence-to-total ratio. It is expected that the method could be used for Pu accountancy and safeguards verification as well as identification and assay of seized, found, or not documented PuBe neutron sources.

Automation of the anthrone assay for carbohydrate concentration determinations.

PubMed

Turula, Vincent E; Gore, Thomas; Singh, Suddham; Arumugham, Rasappa G

2010-03-01

Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

The LabTube - a novel microfluidic platform for assay automation in laboratory centrifuges.

PubMed

Kloke, A; Fiebach, A R; Zhang, S; Drechsel, L; Niekrawietz, S; Hoehl, M M; Kneusel, R; Panthel, K; Steigert, J; von Stetten, F; Zengerle, R; Paust, N

2014-05-07

Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-on-time to one minute per extraction.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

PubMed Central

Dreier, Jens; Störmer, Melanie; Mäde, Dietrich; Burkhardt, Sabine; Kleesiek, Knut

2006-01-01

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities. PMID:16891482

A Novel, Rapid Assay for Detection and Differentiation of Serotype-Specific Antibodies to Venezuelan Equine Encephalitis Complex Alphaviruses

DTIC Science & Technology

2005-01-01

Research Center Detachment, Lima, Peru Abstract. An epitope-blocking enzyme-linked immunosorbent assay was developed for the rapid differentiation of...subtype and variety of antibodies to VEEV in equines, humans, or rodent reservoir hosts can be critical for determining the potential of a naturally...of human sera from Mexico and Peru using a blocking enzyme-linked immunosorbent assay and plaque reduction neutralization tests* Serum number Country

Identification of target genes to control acetate yield during aerobic fermentation with Saccharomyces cerevisiae.

PubMed

Curiel, José Antonio; Salvadó, Zoel; Tronchoni, Jordi; Morales, Pilar; Rodrigues, Alda Joao; Quirós, Manuel; Gonzalez, Ramón

2016-09-15

Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines.

Use of viscoelastic haemostatic assay in emergency and elective surgery.

PubMed

Yeung, Maximus C F; Tong, Steven Y T; Tong, Paul Y W; Cheung, Billy H H; Ng, Joanne Y W; Leung, Gilberto K K

2015-02-01

To review the current evidence for the use of viscoelastic haemostatic assays in different surgical settings including trauma, cardiac surgery, liver transplantation, as well as the monitoring of antiplatelet agents and anticoagulants prior to surgery. PubMed database. Key words for the literature search were "thromboelastography" or "ROTEM" in combination with "trauma", "antiplatelet", "cardiac surgery", "liver transplantation" or "anticoagulants". Original and major review articles related to the use of viscoelastic haemostatic assays. Haemostatic function is a critical factor determining patient outcomes in emergency or elective surgery. The increasing use of antiplatelet agents and anticoagulants has potentially increased the risks of haemorrhages and the need for transfusion. Conventional coagulation tests have limitations in detecting haemostatic dysfunctions in subgroups of patients and are largely ineffective in diagnosing hyperfibrinolysis. The viscoelastic haemostatic assays are potentially useful point-of-care tools that provide information on clot formation, clot strength, and fibrinolysis, as well as to guide goal-directed transfusion and antifibrinolytic therapy. They may also be used to monitor antiplatelet and anticoagulant therapy. However, standardisation of techniques and reference ranges is required before these tests can be widely used in different clinical settings. Viscoelastic haemostatic assays, as compared with conventional coagulation tests, are better for detecting coagulopathy and are the only tests that can provide rapid diagnosis of hyperfibrinolysis. Goal-directed administration of blood products based on the results of viscoelastic haemostatic assays was associated with reduction in allogeneic blood product transfusions in trauma, cardiac surgery, and liver transplantation cases. However, there is currently no evidence to support the routine use of viscoelastic haemostatic assays for monitoring platelet function prior to surgery.

Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction.

PubMed

Shokri-Afra, H; Ostovar-Ravari, A; Rasouli, M

2016-10-01

Acid digestion of animal tissues yields two fractions of glycogen, acid soluble (ASG) and insoluble (AIG). The current study was performed to improve the assay method for glycogen fractions in rat liver in different physiological states. All steps of the assay were manipulated and optimized to measure the content of ASG and AIG in fed and starved rat liver. In postmortem liver tissue, total glycogen was decreased slowly at 4°C and rapidly at 25°C but was well stabilized at -20°C and -70°C. At room temperature, ASG underwent autolysis at the rate of 1.3% and decreased by half at 35 min, while AIG increased slightly. The yield of the recovery of ASG during four successive extractions depends on the tissue concentration, and at the ratio of 50 mg tissue per 2 mL perchloric acid (PCA) was about 93.2%, 6.3%, 0.3% and 0.05% respectively. The increase in the time and extent of homogenization of the tissue with cold PCA and using ultrasonication had not any significant effect on the extraction yield of ASG. The time of centrifugation of the tissue extract could be reduced from 15 to 7.5 minutes with no significant decrease in the recovery of ASG. On extraction with ethanol, the yield of recovery of ASG reached the maximal level of 97.5% at a final ethanol concentration of 60%. The recovery of ASG was not improved in the presence of KCl. During 24 starvation, total glycogen depleted completely and the change occurred entirely in ASG, while AIG did not change significantly. The CV% was less than 5% for the optimized assays of glycogen fractions. ASG is the main and metabolically active portion of glycogen in rat liver.

Correlation between electron spin resonance spectra and oil yield in eastern oil shales

USGS Publications Warehouse

Choudhury, M.; Rheams, K.F.; Harrell, J.W.

1986-01-01

Organic free radical spin concentrations were measured in 60 raw oil shale samples from north Alabama and south Tennessee and compared with Fischer assays and uranium concentrations. No correlation was found between spin concentration and oil yield for the complete set of samples. However, for a 13 sample set taken from a single core hole, a linear correlation was obtained. No correlation between spin concentration and uranium concentration was found. ?? 1986.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Modified in vivo comet assay detects the genotoxic potential of 14-hydroxycodeinone, an α,β-unsaturated ketone in oxycodone.

PubMed

Pant, Kamala; Roden, Nicholas; Zhang, Charles; Bruce, Shannon; Wood, Craig; Pendino, Kimberly

2015-12-01

14-Hydroxycodeinone (14-HC) is an α,β-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay. © 2015 Wiley Periodicals, Inc.

Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

2013-02-05

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

Implications of Measurement Assay Type in Design of HIV Experiments.

PubMed

Cannon, LaMont; Jagarapu, Aditya; Vargas-Garcia, Cesar A; Piovoso, Michael J; Zurakowski, Ryan

2017-12-01

Time series measurements of circular viral episome (2-LTR) concentrations enable indirect quantification of persistent low-level Human Immunodeficiency Virus (HIV) replication in patients on Integrase-Inhibitor intensified Combined Antiretroviral Therapy (cART). In order to determine the magnitude of these low level infection events, blood has to be drawn from a patients at a frequency and volume that is strictly regulated by the Institutional Review Board (IRB). Once the blood is drawn, the 2-LTR concentration is determined by quantifying the amount of HIV DNA present in the sample via a PCR (Polymerase Chain Reaction) assay. Real time quantitative Polymerase Chain Reaction (qPCR) is a widely used method of performing PCR; however, a newer droplet digital Polymerase Chain Reaction (ddPCR) method has been shown to provide more accurate quantification of DNA. Using a validated model of HIV viral replication, this paper demonstrates the importance of considering DNA quantification assay type when optimizing experiment design conditions. Experiments are optimized using a Genetic Algorithm (GA) to locate a family of suboptimal sample schedules which yield the highest fitness. Fitness is defined as the expected information gained in the experiment, measured by the Kullback-Leibler Divergence (KLD) between the prior and posterior distributions of the model parameters. We compare the information content of the optimized schedules to uniform schedules as well as two clinical schedules implemented by researchers at UCSF and the University of Melbourne. This work shows that there is a significantly greater gain information in experiments using a ddPCR assay vs. a qPCR assay and that certain experiment design considerations should be taken when using either assay.

Assays for estimating HIV incidence: updated global market assessment and estimated economic value.

PubMed

Morrison, Charles S; Homan, Rick; Mack, Natasha; Seepolmuang, Pairin; Averill, Megan; Taylor, Jamilah; Osborn, Jennifer; Dailey, Peter; Parkin, Neil; Ongarello, Stefano; Mastro, Timothy D

2017-11-01

Accurate incidence estimates are needed to characterize the HIV epidemic and guide prevention efforts. HIV Incidence assays are cost-effective laboratory assays that provide incidence estimates from cross-sectional surveys. We conducted a global market assessment of HIV incidence assays under three market scenarios and estimated the economic value of improved incidence assays. We interviewed 27 stakeholders, and reviewed journal articles, working group proceedings, and manufacturers' sales figures. We determined HIV incidence assay use in 2014, and estimated use in 2015 to 2017 and in 5 to 10-years under three market scenarios, as well as the cost of conducting national and key population surveys using an HIV incidence assay with improved performance. Global 2014 HIV incidence assay use was 308,900 tests, highest in Asia and mostly for case- and population-based surveillance. Estimated 2015 to 2017 use was 94,475 annually, with declines due to China and the United States discontinuing incidence assay use for domestic surveillance. Annual projected 5 to 10 year use under scenario 1 - no change in technology - was 94,475. For scenario 2 - a moderately improved incidence assay - projected annual use was 286,031. Projected annual use for scenario 3 - game-changing technologies with an HIV incidence assay part of (a) standard confirmatory testing, and (b) standard rapid testing, were 500,000 and 180 million, respectively. As HIV incidence assay precision increases, decreased sample sizes required for incidence estimation resulted in $5 to 23 million annual reductions in survey costs and easily offset the approximately $3 million required to develop a new assay. Improved HIV incidence assays could substantially reduce HIV incidence estimation costs. Continued development of HIV incidence assays with improved performance is required to realize these cost benefits. © 2017 The Authors. Journal of the International AIDS Society published by John Wiley & sons Ltd on

Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

PubMed

Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

2012-04-01

Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Harmonization of the Bayer ADVIA Centaur and Abbott AxSYM automated B-type natriuretic peptide assay in patients on hemodialysis.

PubMed

Barak, Mira; Weinberger, Ronit; Marcusohn, Jerom; Froom, Paul

2005-01-01

There are two fully automated high-throughput clinical instruments for brain natriuretic peptide (BNP) assays, the Bayer ADVIA Centaur assay, and the Abbott AxSYM assay. Although both recommend a cut-off value of 100 pg/mL, we are unaware of previous studies that have compared the unadjusted results of the two methods, required for proper evaluation of patients undergoing this test on different platforms. From 43 hemodialysis patients, 80 paired samples were collected by venipuncture into plastic evacuated tubes containing EDTA. The Bayer assay yielded lower values than the Abbott assay, with linear regression of 0.53 x Abbott assay (95% confidence interval, 0.50-0.56) being forced through 0, demonstrating an r(2)-value of 0.954. Regression for the Abbott assay was 1.79 x Bayer assay (95% CI, 1.69-1.89). The cut-off values for abnormal BNP results analyzed on the Abbott system are not identical to those on the Bayer system, and this needs to be taken into account when comparing studies on the clinical utility of these systems.

A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

PubMed

Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

2009-08-01

A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

Synergistic interactions of ecosystem services: florivorous pest control boosts crop yield increase through insect pollination

PubMed Central

Albrecht, Matthias

2016-01-01

Insect pollination and pest control are pivotal functions sustaining global food production. However, they have mostly been studied in isolation and how they interactively shape crop yield remains largely unexplored. Using controlled field experiments, we found strong synergistic effects of insect pollination and simulated pest control on yield quantity and quality. Their joint effect increased yield by 23%, with synergistic effects contributing 10%, while their single contributions were 7% and 6%, respectively. The potential economic benefit for a farmer from the synergistic effects (12%) was 1.8 times greater than their individual contributions (7% each). We show that the principal underlying mechanism was a pronounced pest-induced reduction in flower lifetime, resulting in a strong reduction in the number of pollinator visits a flower receives during its lifetime. Our findings highlight the importance of non-additive interactions among ecosystem services (ES) when valuating, mapping or predicting them and reveal fundamental implications for ecosystem management and policy aimed at maximizing ES for sustainable agriculture. PMID:26865304

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

PubMed Central

Khan, Mehran; Li, Benjin; Jiang, Yue; Weng, Qiyong; Chen, Qinghe

2017-01-01

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics. PMID:29051751

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene.

PubMed

Khan, Mehran; Li, Benjin; Jiang, Yue; Weng, Qiyong; Chen, Qinghe

2017-01-01

Late blight, caused by the oomycete Phytophthora infestans , is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10 -4 ng μL -1 ), being 10 times more sensitive than nested PCR (1.28 × 10 -3 ng μL -1 ), 100 times more sensitive than real-time PCR (1.28 × 10 -2 ng μL -1 ) and 10 3 times more sensitive than the conventional PCR assay (1.28 × 10 -1 ng μL -1 ). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

PubMed

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

2015-01-01

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

Dynamics and yielding of binary self-suspended nanoparticle fluids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agrawal, Akanksha; Yu, Hsiu-Yu; Srivastava, Samanvaya

Yielding and flow transitions in bi-disperse suspensions of particles are studied using a model system comprised of self-suspended spherical nanoparticles. An important feature of the materials is that the nanoparticles are uniformly dispersed in the absence of a solvent. Addition of larger particles to a suspension of smaller ones is found to soften the suspensions, and in the limit of large size disparities, completely fluidizes the material. We show that these behaviors coincide with a speeding-up of de-correlation dynamics of all particles in the suspensions and are accompanied by a reduction in the energy dissipated at the yielding transition. Wemore » discuss our findings in terms of ligand-mediated jamming and un-jamming of hairy particle suspensions.« less

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Results Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. Conclusions This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further

Yield gaps and yield relationships in US soybean production systems

USDA-ARS?s Scientific Manuscript database

The magnitude of yield gaps (YG) (potential yield – farmer yield) provides some indication of the prospects for increasing crop yield to meet the food demands of future populations. Quantile regression analysis was applied to county soybean [Glycine max (L.) Merrill] yields (1971 – 2011) from Kentuc...

Conformationally selective biophysical assay for influenza vaccine potency determination.

PubMed

Wen, Yingxia; Han, Liqun; Palladino, Giuseppe; Ferrari, Annette; Xie, Yuhong; Carfi, Andrea; Dormitzer, Philip R; Settembre, Ethan C

2015-10-05

Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of KRAS genotype: therascreen assay vs. LNA-mediated qPCR clamping assay.

PubMed

Chang, Shao-Chun; Denne, Jonathan; Zhao, Luping; Horak, Christine; Green, George; Khambata-Ford, Shirin; Bray, Christopher; Celik, Ilhan; Van Cutsem, Eric; Harbison, Christopher

2013-09-01

Kirsten rat sarcoma virus (KRAS) wild-type status determined using a locked nucleic acid (LNA)-mediated quantitative polymerase chain reaction (qPCR) clamping assay (LNA assay) predicted response to therapy in the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study. A companion KRAS diagnostic tool has been developed for routine clinical use (QIAGEN therascreen kit) (QIAGEN Manchester Ltd, Manchester, UK). We wanted to assess the concordance between the validated US Food and Drug Administration (FDA)-approved therascreen assay and the LNA assay in determining the KRAS status of a subset of patients enrolled in the CRYSTAL study. DNA extracted from paraffin-embedded tumor sections was tested for KRAS status using the therascreen assay. Efficacy data from the CRYSTAL study were assessed to determine if the overall survival (OS) hazard ratio for cetuximab in patients identified as having KRAS wild-type status using the therascreen assay was equivalent to that in patients identified as KRAS wild-type using the LNA assay. This was determined by assessing if the concordance between the therascreen assay and the LNA assay met the minimum threshold (prespecified as 0.8) to achieve a significant difference in the OS hazard ratio in favor of the cetuximab + FOLFIRI (5-fluorouracil, leucovorin [folinic acid], irinotecan) arm in the KRAS wild-type population as identified using the therascreen assay. Of the 148 samples determined to be KRAS wild-type (therascreen assay), 141 (95.3%) samples were also KRAS wild-type (LNA assay) and 7 samples (4.7%) were KRAS mutant (LNA assay). The prespecified primary concordance measure p was 141/148 = 0.953 (95% confidence interval [CI], 0.905-0.981). The concordance was statistically significantly higher than the prespecified threshold of 0.8 for concordance between the therascreen assay and the LNA assay. Consistent with the concordance exceeding the prespecified threshold, the OS

Decrease in milk yield associated with exposure to bluetongue virus serotype 8 in cattle herds.

PubMed

Nusinovici, S; Souty, C; Seegers, H; Beaudeau, F; Fourichon, C

2013-02-01

Decreased milk yield and reduced fertility are the primary consequences of infection by bluetongue virus serotype 8 (BTV-8). These effects must be quantified to fully assess the economic benefit of vaccination. This can be estimated by measuring the effect of BTV-8 exposure on milk yield and fertility for all cows belonging to an infected herd. The objectives of this study were (1) to quantify the mean effect of exposure to BTV-8 on milk yield following natural challenge for cows in herds previously naïve, (2) to determine the duration of reduced milk yield before and after the date disease was first detected in the herd to estimate the cumulative loss of milk yield during this period, and (3) to evaluate the influence of the proportion of infected neighboring herds on the reduction in milk yield following exposure to BTV-8. The effects of exposure to BTV-8 during the French outbreak of 2007 were assessed using mixed linear models, which allow adjustment for factors known to influence milk yield. Exposure to BTV-8 was associated with a sharp decrease in milk yield over a period of 6 mo (2 mo before to 4 mo after the reported date of disease detection in the herd). The cumulative loss of milk yield was more than 3% of annual production. The relatively earlier reduction in milk yield in infected herds detected later in the outbreak period suggests that detection of clinical signs was delayed in these herds. Finally, the greatest decrease in milk yield was observed in herds detected early during the outbreak period and located in areas with the highest disease incidence. This may be due to a greater within-herd incidence or to a greater amount of virus injected by midges to individual cows in these herds. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Detection of Zaire ebolavirus in swine: Assay development and optimization.

PubMed

Pickering, B S; Collignon, B; Smith, G; Marszal, P; Kobinger, G; Weingartl, H M

2018-02-01

Ebolaviruses (family Filoviridae, order Mononegavirales) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low-level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7 dpi, 3 days earlier than virus neutralization tests (VNT). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results. © 2017 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of Health and Agriculture, Canadian Food Inspection Agency.

Quantification of ozone exposure- and stomatal uptake-yield response relationships for soybean in Northeast China.

PubMed

Zhang, Weiwei; Feng, Zhaozhong; Wang, Xiaoke; Liu, Xiaobing; Hu, Enzhu

2017-12-01

High ground-level O 3 is a new threat to agricultural production in Northeast China with the increasing ambient O 3 concentration. Little is known about its impacts on soybean production in this key agricultural region. Accumulated O 3 exposure-response and stomatal O 3 flux-response relationships were developed during two continuous growing seasons to evaluate O 3 -induced yield reduction of four typical soybean cultivars in Northeast China. Results showed that critical levels of AOT40 (accumulated hourly O 3 concentrations over a threshold of 40nmol·mol -1 ), SUM06 (sum of all hourly average O 3 concentrations over 0.06μmol·mol -1 ) and W126 (sum of O 3 concentrations weighted by a sigmoidal function) in relation to 5% reduction in relative seed yield were 4.2, 7.6 and 6.8μmol·mol -1 ·h, respectively. The effect of O 3 on plants was influenced by leaf position in canopy. An improved Jarvis stomatal conductance model including leaf (node) position fitted well with field measurements. The best linear relationship between stomatal O 3 flux and relative soybean yield was obtained when phytotoxic ozone dose was integrated over a threshold of 9.6nmol·m -2 ·s -1 (POD 9.6 ) to represent the detoxification capacity of soybean. POD 9.6 and the commonly used POD 6 in relation to 5% reduction in relative seed yield of soybean were 0.9mmol·m -2 and 1.8mmol·m -2 , respectively. O 3 concentrations above ~38nmol·mol -1 contributed to POD 9.6 and caused seed yield loss in soybean. Current annual yield loss of soybean at ambient O 3 was estimated to range between 23.4% and 30.2%. The O 3 dose-response relationships and corresponding thresholds obtained here will benefit regional O 3 risk assessment on soybean production in Northeast China. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential sensitivity of von Willebrand factor (VWF) 'activity' assays to large and small VWF molecular weight forms: a cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays.

PubMed

Favaloro, E J; Bonar, R; Chapman, K; Meiring, M; Funk Adcock, D

2012-06-01

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura. © 2012 International Society on Thrombosis and Haemostasis.

Carbon dioxide reduction by the Bosch process

NASA Technical Reports Server (NTRS)

Manning, M. P.; Reid, R. C.

1975-01-01

Prototype units for carrying out the reduction of carbon dioxide to elementary carbon have been built and operated successfully. In some cases, however, startup difficulties have been reported. Moreover, the recycle reactor product has been reported to contain only small amounts of water and undesirably high yields of methane. This paper presents the results of the first phase of an experimental study that was carried out to define the mechanisms occurring in the reduction process. Conclusions are drawn and possible modifications to the present recycle process are suggested.

Spatial variability of climate change impacts on yield of rice and wheat in the Indian Ganga Basin.

PubMed

Mishra, Ashok; Singh, R; Raghuwanshi, N S; Chatterjee, C; Froebrich, Jochen

2013-12-01

Indian Ganga Basin (IGB), one of the most densely populated areas in the world, is facing a significant threat to food grain production, besides increased yield gap between actual and potential production, due to climate change. We have analyzed the spatial variability of climate change impacts on rice and wheat yields at three different locations representing the upper, middle and lower IGB. The DSSAT model is used to simulate the effects of climate variability and climate change on rice and wheat yields by analyzing: (i) spatial crop yield response to current climate, and (ii) impact of a changing climate as projected by two regional climate models, REMO and HadRM3, based on SRES A1B emission scenarios for the period 2011-2040. Results for current climate demonstrate a significant gap between actual and potential yield for upper, middle and lower IGB stations. The analysis based on RCM projections shows that during 2011-2040, the largest reduction in rice and wheat yields will occur in the upper IGB (reduction of potential rice and wheat yield respectively by 43.2% and 20.9% by REMO, and 24.8% and 17.2% by HadRM3). In the lower IGB, however, contrasting results are obtained, with HadRM3 based projections showing an increase in the potential rice and wheat yields, whereas, REMO based projections show decreased potential yields. We discuss the influence of agro-climatic factors; variation in temperature, length of maturity period and leaf area index which are responsible for modeled spatial variability in crop yield response within the IGB. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples.

PubMed

Leach, L; Zhu, Y; Chaturvedi, S

2018-02-01

Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 ( ITS 2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities. Copyright © 2018 Leach et al.

Optimizing rice yields while minimizing yield-scaled global warming potential.

PubMed

Pittelkow, Cameron M; Adviento-Borbe, Maria A; van Kessel, Chris; Hill, James E; Linquist, Bruce A

2014-05-01

To meet growing global food demand with limited land and reduced environmental impact, agricultural greenhouse gas (GHG) emissions are increasingly evaluated with respect to crop productivity, i.e., on a yield-scaled as opposed to area basis. Here, we compiled available field data on CH4 and N2 O emissions from rice production systems to test the hypothesis that in response to fertilizer nitrogen (N) addition, yield-scaled global warming potential (GWP) will be minimized at N rates that maximize yields. Within each study, yield N surplus was calculated to estimate deficit or excess N application rates with respect to the optimal N rate (defined as the N rate at which maximum yield was achieved). Relationships between yield N surplus and GHG emissions were assessed using linear and nonlinear mixed-effects models. Results indicate that yields increased in response to increasing N surplus when moving from deficit to optimal N rates. At N rates contributing to a yield N surplus, N2 O and yield-scaled N2 O emissions increased exponentially. In contrast, CH4 emissions were not impacted by N inputs. Accordingly, yield-scaled CH4 emissions decreased with N addition. Overall, yield-scaled GWP was minimized at optimal N rates, decreasing by 21% compared to treatments without N addition. These results are unique compared to aerobic cropping systems in which N2 O emissions are the primary contributor to GWP, meaning yield-scaled GWP may not necessarily decrease for aerobic crops when yields are optimized by N fertilizer addition. Balancing gains in agricultural productivity with climate change concerns, this work supports the concept that high rice yields can be achieved with minimal yield-scaled GWP through optimal N application rates. Moreover, additional improvements in N use efficiency may further reduce yield-scaled GWP, thereby strengthening the economic and environmental sustainability of rice systems. © 2013 John Wiley & Sons Ltd.

Resist process optimization for further defect reduction

NASA Astrophysics Data System (ADS)

Tanaka, Keiichi; Iseki, Tomohiro; Marumoto, Hiroshi; Takayanagi, Koji; Yoshida, Yuichi; Uemura, Ryouichi; Yoshihara, Kosuke

2012-03-01

Defect reduction has become one of the most important technical challenges in device mass-production. Knowing that resist processing on a clean track strongly impacts defect formation in many cases, we have been trying to improve the track process to enhance customer yield. For example, residual type defect and pattern collapse are strongly related to process parameters in developer, and we have reported new develop and rinse methods in the previous papers. Also, we have reported the optimization method of filtration condition to reduce bridge type defects, which are mainly caused by foreign substances such as gels in resist. Even though we have contributed resist caused defect reduction in past studies, defect reduction requirements continue to be very important. In this paper, we will introduce further process improvements in terms of resist defect reduction, including the latest experimental data.

Analytical evaluation of the automated galectin-3 assay on the Abbott ARCHITECT immunoassay instruments.

PubMed

Gaze, David C; Prante, Christian; Dreier, Jens; Knabbe, Cornelius; Collet, Corinne; Launay, Jean-Marie; Franekova, Janka; Jabor, Antonin; Lennartz, Lieselotte; Shih, Jessie; del Rey, Jose Manuel; Zaninotto, Martina; Plebani, Mario; Collinson, Paul O

2014-06-01

Galectin-3 is secreted from macrophages and binds and activates fibroblasts forming collagen. Tissue fibrosis is central to the progression of chronic heart failure (CHF). We performed a European multicentered evaluation of the analytical performance of the two-step routine and Short Turn-Around-Time (STAT) galectin-3 immunoassay on the ARCHITECT i1000SR, i2000SR, and i4000SR (Abbott Laboratories). We evaluated the assay precision and dilution linearity for both routine and STAT assays and compared serum and plasma, and fresh vs. frozen samples. The reference interval and biological variability were also assessed. Measurable samples were compared between ARCHITECT instruments and between the routine and STAT assays and also to a galectin-3 ELISA (BG Medicine). The total assay coefficient of variation (CV%) was 2.3%-6.2% and 1.7%-7.4% for the routine and STAT assays, respectively. Both assays demonstrated linearity up to 120 ng/mL. Galectin-3 concentrations were higher in plasma samples than in serum samples and correlated well between fresh and frozen samples (R=0.997), between the routine and STAT assays, between the ARCHITECT i1000 and i2000 instruments and with the galectin-3 ELISA. The reference interval on 627 apparently healthy individuals (53% male) yielded upper 95th and 97.5th percentiles of 25.2 and 28.4 ng/mL, respectively. Values were significantly lower in subjects younger than 50 years. The galectin-3 routine and STAT assays on the Abbott ARCHITECT instruments demonstrated good analytical performance. Further clinical studies are required to demonstrate the diagnostic and prognostic potential of this novel marker in patients with CHF.

Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay.

PubMed

Brawner, Andrew; Hinrichs, Steven H; Larson, Marilynn A; Lockridge, Oksana

2016-04-05

The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min(-1) and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Selenium isotope fractionation during reduction by Fe(II)-Fe(III) hydroxide-sulfate (green rust)

USGS Publications Warehouse

Johnson, T.M.; Bullen, T.D.

2003-01-01

We have determined the extent of Se isotope fractionation induced by reduction of selenate by sulfate interlayered green rust (GRSO4), a Fe(II)-Fe(III) hydroxide-sulfate. This compound is known to reduce selenate to Se(0), and it is the only naturally relevant abiotic selenate reduction pathway documented to date. Se reduction reactions, when they occur in nature, greatly reduce Se mobility and bioavailability. Se stable isotope analysis shows promise as an indicator of Se reduction, and Se isotope fractionation by various Se reactions must be known in order to refine this tool. We measured the increase in the 80Se/76Se ratio of dissolved selenate as lighter isotopes were preferentially consumed during reduction by GRSO4. Six different experiments that used GRSO4 made by two methods, with varying solution compositions and pH, yielded identical isotopic fractionations. Regression of all the data yielded an instantaneous isotope fractionation of 7.36 ?? 0.24???. Selenate reduction by GRSO4 induces much greater isotopic fractionation than does bacterial selenate reduction. If selenate reduction by GRSO4 occurs in nature, it may be identifiable on the basis of its relatively large isotopic fractionation. ?? 2003 Elsevier Science Ltd.

Spiral Salmonella assay: validation against the standard pour-plate assay.

PubMed

Diehl, M; Fort, F

1996-01-01

The spiral Ames assay, an automated approach to bacterial mutagenicity testing which simplifies the test procedure and reduces the amount of drug required to generate mutagenic dose-response information, has been evaluated and validated for routine screening. The spiral plater delivers the Salmonella bacteria, exogenous metabolic activation system and drug to the surface of a rotating agar plate one on top of another in such a way that a uniform density of bacteria is exposed to a logarithmically decreasing volume of drug. Following an incubation of 48 hr at 37 degrees C, the plates are scanned by a laser counter, and the data are subjected to a computerized analysis. Petri plates of 15 cm diameter were used to provide a concentration range of about 250-fold per plate. The Salmonella were concentrated 20-fold to increase sensitivity. Thirty-eight compounds from a variety of chemical classes, including both pharmaceuticals and known mutagens of moderate to strong potency, were tested in both the spiral and the standard pour-plate assays. There was overall test agreement on positive or negative results for 82% of the compounds tested. When only the results from strains TA98 plus TA100 were considered, the agreement was 87%. When positive results were obtained, the fold increase over vehicle control was on average twice as great for the spiral assay compared to the pour-plate assay. It was concluded that the two assay procedures generally provided comparable results, with the spiral assay being somewhat more sensitive in terms of dose-response than the pour-plate assay.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield.

PubMed

Guo, Mei; Rupe, Mary A; Wei, Jun; Winkler, Chris; Goncalves-Butruille, Marymar; Weers, Ben P; Cerwick, Sharon F; Dieter, Jo Ann; Duncan, Keith E; Howard, Richard J; Hou, Zhenglin; Löffler, Carlos M; Cooper, Mark; Simmons, Carl R

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield

PubMed Central

Guo, Mei

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays ARGOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer’s modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer’s female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance. PMID:24218327

Honey is cytotoxic towards prostate cancer cells but interacts with the MTT reagent: Considerations for the choice of cell viability assay.

PubMed

Abel, Sean D A; Baird, Sarah K

2018-02-15

Honey is a complex biological substance, consisting mainly of sugars, phenolic compounds and enzymes. Using five quick and accessible assays for measuring honey's cytotoxicity in vitro, we found honey is cytotoxic towards prostate cancer cells PC3 and DU145. However, the level of cell death varied with assay. The MTT assay was confounded by the reduction of the MTT reagent by honey's reducing sugars and phenolic compounds, and the lactate dehydrogenase assay was invalidated by honey oxidising the enzyme cofactor NADH. The sulforhodamine B assay gave valid results, but measures only protein content, providing no information about cell death in the remaining cells. The trypan blue assay and a microscope-based propidium iodide/Hoechst staining assay assess only late stage membrane permeability. However, the propidium iodide/Hoechst assay gives morphological information about cell death mechanism. A combination of the sulforhodamine B and propidium iodide/Hoechst assays would provide the most accurate quantification of honey cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Determination of paralytic shellfish toxins in shellfish by receptor binding assay: collaborative study.

PubMed

Van Dolah, Frances M; Fire, Spencer E; Leighfield, Tod A; Mikulski, Christina M; Doucette, Gregory J

2012-01-01

hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1-93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r(2)) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r(2) of 0.92. When samples were sorted according to increasing toxin concentration (microg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 microg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

Development of a high-throughput assay for rapid screening of butanologenic strains.

PubMed

Agu, Chidozie Victor; Lai, Stella M; Ujor, Victor; Biswas, Pradip K; Jones, Andy; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2018-02-21

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP + to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

PubMed

Shalaby, Mohamed A; El-Deeb, Ayman; El-Tholoth, Mohamed; Hoffmann, Donata; Czerny, Claus-Peter; Hufert, Frank T; Weidmann, Manfred; Abd El Wahed, Ahmed

2016-11-02

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Utility and limitations of a peptide reactivity assay to predict fragrance allergens in vitro.

PubMed

Natsch, A; Gfeller, H; Rothaupt, M; Ellis, G

2007-10-01

A key step in the skin sensitization process is the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. A published peptide depletion assay was used to relate the in vitro reactivity of fragrance molecules to LLNA data. Using the classical assay, 22 of 28 tested moderate to strong sensitizers were positive. The prediction of weak sensitizers proved to be more difficult with only 50% of weak sensitizers giving a positive response, but for some compounds this could also be due to false-positive results from the LLNA. LC-MS analysis yielded the expected mass of the peptide adducts in several cases, whereas in other cases putative oxidation reactions led to adducts of unexpected molecular weight. Several moderately sensitizing aldehydes were correctly predicted by the depletion assay, but no adducts were found and the depletion appears to be due to an oxidation of the parent peptide catalyzed by the test compound. Finally, alternative test peptides derived from a physiological reactive protein with enhanced sensitivity for weak Michael acceptors were found, further increasing the sensitivity of the assay.

Capsaicin-capped silver nanoparticles: its kinetics, characterization and biocompatibility assay

NASA Astrophysics Data System (ADS)

Amruthraj, Nagoth Joseph; Preetam Raj, John Poonga; Lebel, Antoine

2015-04-01

Capsaicin was used as a bio-reductant for the reduction of silver nitrate to form silver nanoparticles. The formation of the silver nanoparticles was initially confirmed by color change and Tyndall effect of light scattering. It was characterized with UV-visible spectroscopy, FTIR and TEM. Hemagglutination (H) test and H-inhibition assay were performed in the presence of AgNPs-capsaicin conjugates. The silver colloid solution after complete reduction turned into pale gray color. The characteristic surface plasmon resonance of silver nanoparticles (SNPs) was observed at 450 nm. Time taken for complete bio-reduction of silver nitrate and capping was found to be 16 hours. The amount of capsaicin required to reduce 20 ml of 1 mM silver nitrate solution was found to be 40 μg approximately. The FTIR results confirmed the capping of capsaicin on the silver metal. The particle size was within the range of 20-30 nm. The hemagglutination and H-inhibition test was negative for all the blood groups. The capsaicin-capped silver nanoparticles were compatible with blood cells in hemagglutination test implying biocompatibility as future therapeutic drug.

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

PubMed Central

Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M. Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

2016-01-01

Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

Evaluation of the BD Max StaphSR Assay for Rapid Identification of Staphylococcus aureus and Methicillin-Resistant S. aureus in Positive Blood Culture Broths

PubMed Central

Hofko, Marjeta; Hamilton, Fiona; Mackenzie, Laura; Zimmermann, Stefan; Templeton, Kate

2015-01-01

We evaluated the performance of the BD Max StaphSR assay for the direct detection of Staphylococcus aureus from blood culture medium. In a two-center trial, 155 blood cultures from the BD Bactec FX system and 212 from the bioMérieux BacT/Alert system were tested; 170 bottles yielded S. aureus, and all were identified correctly by the BD Max StaphSR assay. The assay required approximately 2.5 h, thus allowing rapid identification of blood cultures flagged positive. PMID:26292311

Drought effects on composition and yield for corn stover, mixed grasses, and Miscanthus as bioenergy feedstocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emerson, Rachel; Hoover, Amber; Ray, Allison

Drought conditions in 2012 were some of the most severe in recent history. The purpose of this study is to examine the impact of drought on quality, quantity, and theoretical ethanol yield (TEY) of three bioenergy feedstocks, corn stover, mixed grasses from Conservation Reserve Program lands, and Miscanthus × giganteus. To assess drought effects on these feedstocks, samples from 2010 (minimal to no drought) and 2012 (severe drought) were compared from multiple locations in the US. In all feedstocks, drought significantly increased extractives and reduced structural sugars and lignin; subsequently, TEYs were reduced 10–15%. Biomass yields were significantly reduced formore » M. × giganteus and mixed grasses. When reduction in quality and quantity were combined, TEYs decreased 26–59%. Drought negatively affected biomass quality and quantity that resulted in significant TEY reductions. As a result, such fluctuations in biomass quality and yield may have significant consequences for developing lignocellulosic biorefineries.« less

Drought effects on composition and yield for corn stover, mixed grasses, and Miscanthus as bioenergy feedstocks

DOE PAGES

Emerson, Rachel; Hoover, Amber; Ray, Allison; ...

2014-07-04

Drought conditions in 2012 were some of the most severe in recent history. The purpose of this study is to examine the impact of drought on quality, quantity, and theoretical ethanol yield (TEY) of three bioenergy feedstocks, corn stover, mixed grasses from Conservation Reserve Program lands, and Miscanthus × giganteus. To assess drought effects on these feedstocks, samples from 2010 (minimal to no drought) and 2012 (severe drought) were compared from multiple locations in the US. In all feedstocks, drought significantly increased extractives and reduced structural sugars and lignin; subsequently, TEYs were reduced 10–15%. Biomass yields were significantly reduced formore » M. × giganteus and mixed grasses. When reduction in quality and quantity were combined, TEYs decreased 26–59%. Drought negatively affected biomass quality and quantity that resulted in significant TEY reductions. As a result, such fluctuations in biomass quality and yield may have significant consequences for developing lignocellulosic biorefineries.« less

A review on photocatalytic CO2 reduction using perovskite oxide nanomaterials

NASA Astrophysics Data System (ADS)

Zeng, Sheng; Kar, Piyush; Thakur, Ujwal Kumar; Shankar, Karthik

2018-02-01

As the search for efficient catalysts for CO2 photoreduction continues, nanostructured perovskite oxides have emerged as a class of high-performance photocatalytic materials. The perovskite oxide candidates for CO2 photoreduction are primarily nanostructured forms of titanates, niobates, tantalates and cobaltates. These materials form the focus of this review article because they are much sought-after due to their nontoxic nature, adequate chemical stability, and tunable crystal structures, bandgaps and surface energies. As compared to conventional semiconductors and nanomaterial catalysts, nanostructured perovskite oxides also exhibit an extended optical-absorption edge, longer charge carrier lifetimes, and favorable band-alignment with respect to reduction potential of activated CO2 and reduction products of the same. While CO2 reduction product yields of several hundred μmol-1 h-1 are observed with many types of perovskite oxide nanomaterials in stand-alone forms, yield of such quantities are not common with semiconductor nanomaterials of other types. In this review, we present current state-of-the-art synthesis methods to form perovskite oxide nanomaterials, and procedures to engineer their bandgaps. This review also presents a comprehensive summary and discussion on crystal structures, defect distribution, morphologies and electronic properties of the perovskite oxides, and correlation of these properties to CO2 photoreduction performance. This review offers researchers key insights for developing advanced perovskite oxides in order to further improve the yields of CO2 reduction products.

Differential Effect of Irradiance and Nutrient Nitrate on the Relationship of in Vivo and in Vitro Nitrate Reductase Assay in Chlorophyllous Tissues 1

PubMed Central

Jones, Richard Wyn; Sheard, Robert W.

1977-01-01

Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity. PMID:16659888

Ammonia Formation by the Reduction of Nitrite/Nitrate by FeS: Ammonia Formation Under Acidic Conditions

NASA Technical Reports Server (NTRS)

Summers, David P.; DeVincenzi, Donald (Technical Monitor)

2000-01-01

FeS reduces nitrite to, ammonia at pHs lower than the corresponding reduction by aqueous Fe+2. The reduction follows a reasonable first order decay, in nitrite concentration, with a half life of about 150 min (room temperature, CO2, pH 6.25). The highest ammonia product yield measured was 53%. Under CO2, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate at higher pH interferes with the reaction. Bicarbonate interference is shown by comparing runs under N2 and CO2. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe+2, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears as if nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe+2 (which also can be sensitive to binding by certain species).

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

PubMed

Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

2015-08-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

PubMed

Catalano, Valentina; Moreno-Sanz, Paula; Lorenzi, Silvia; Grando, Maria Stella

2016-09-21

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.

Reducing uncertainties for short lived cumulative fission product yields

DOE PAGES

Stave, Sean; Prinke, Amanda; Greenwood, Larry; ...

2015-09-05

Uncertainties associated with short lived (halflives less than 1 day) fission product yields listed in databases such as the National Nuclear Data Center’s ENDF/B-VII are large enough for certain isotopes to provide an opportunity for new precision measurements to offer significant uncertainty reductions. A series of experiments has begun where small samples of 235U are irradiated with a pulsed, fission neutron spectrum at the Nevada National Security Site and placed between two broad-energy germanium detectors. The amount of various isotopes present immediately following the irradiation can be determined given the total counts and the calibrated properties of the detector system.more » The uncertainty on the fission yields for multiple isotopes has been reduced by nearly an order of magnitude.« less

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

PubMed Central

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

PubMed

Cozma, Claudia; Eichler, Sabrina; Wittmann, Gyula; Flores Bonet, Alba; Kramp, Guido Johannes; Giese, Anne-Katrin; Rolfs, Arndt

2015-01-01

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance. We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone. The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays. The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

PubMed

Visser, M; Stephan, D; Jaynes, J M; Burger, J T

2012-06-01

Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. © 2012 The Authors. Letters in Applied Microbiology © 2012

Marker assisted pyramiding of drought yield QTLs into a popular Malaysian rice cultivar, MR219.

PubMed

Shamsudin, Noraziyah Abd Aziz; Swamy, B P Mallikarjuna; Ratnam, Wickneswari; Sta Cruz, Ma Teressa; Raman, Anitha; Kumar, Arvind

2016-01-27

Three drought yield QTLs, qDTY 2.2, qDTY 3.1, and qDTY 12.1 with consistent effect on grain yield under reproductive stage drought stress were pyramided through marker assisted breeding with the objective of improving the grain yield of the elite Malaysian rice cultivar MR219 under reproductive stage drought stress. Foreground selection using QTL specific markers, recombinant selection using flanking markers, and background selection were performed. BC1F3-derived lines with different combinations of qDTY 2.2 , qDTY 3.1, and qDTY 12.1 were evaluated under both reproductive stage drought stress and non-stress during the dry seasons of 2013 and 2014 at IRRI. The grain yield reductions in the stress trials compared to non-stress trials ranged from 79 to 93%. In the stress trials, delay in days to flowering and reduction in plant height were observed. In both seasons, MR219 did not produce any yield under stress, however it produced a yield of 5917 kg ha(-1) during the 2013 dry season and 8319 kg ha(-1) during the 2014 dry season under non-stress. Selected introgressed lines gave a yield advantage of 903 to 2500 kg ha(-1) over MR219 under reproductive stage drought stress and a yield of more than 6900 kg ha(-1) under non-stress during the 2014 dry season. Among lines with single qDTY, lines carrying qDTY 2.2 provided a higher yield advantage under reproductive stage drought stress in the MR219 background. Two-qDTY combinations (qDTY 3.1+ qDTY 2.2 and qDTY 3.1+ qDTY 12.1) performed better than lines with three qDTY combinations, indicating the absence of positive interactions between the three qDTYs. We successfully developed drought-tolerant MR219 pyramided lines with a yield advantage of more than 1500 kg ha(-1). Differential yield advantages of different combinations of the qDTYs indicate a differential synergistic relationship among qDTYs. This is the first report on the successful effect of qDTYs in increasing the yield under drought in genetic backgrounds other

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

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 tomore » 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.« less

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.

A combined approach to investigate the toxicity of an industrial landfill's leachate: Chemical analyses, risk assessment and in vitro assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baderna, D., E-mail: diego.baderna@marionegri.it; Maggioni, S.; Boriani, E.

2011-05-15

Solid wastes constitute an important and emerging problem. Landfills are still one of the most common ways to manage waste disposal. The risk assessment of pollutants from landfills is becoming a major environmental issue in Europe, due to the large number of sites and to the importance of groundwater protection. Furthermore, there is lack of knowledge for the environmental, ecotoxicological and toxicological characteristics of most contaminants contained into landfill leacheates. Understanding leachate composition and creating an integrated strategy for risk assessment are currently needed to correctly face the landfill issues and to make projections on the long-term impacts of amore » landfill, with particular attention to the estimation of possible adverse effects on human health and ecosystem. In the present study, we propose an integrated strategy to evaluate the toxicity of the leachate using chemical analyses, risk assessment guidelines and in vitro assays using the hepatoma HepG2 cells as a model. The approach was applied on a real case study: an industrial waste landfill in northern Italy for which data on the presence of leachate contaminants are available from the last 11 years. Results from our ecological risk models suggest important toxic effects on freshwater fish and small rodents, mainly due to ammonia and inorganic constituents. Our results from in vitro data show an inhibition of cell proliferation by leachate at low doses and cytotoxic effect at high doses after 48 h of exposure. - Research highlights: {yields} We study the toxicity of leachate from a non-hazardous industrial waste landfill. {yields} We perform chemical analyses, risk assessments and in vitro assays on HepG2 cells. {yields} Risk models suggest toxic effects due to ammonia and inorganic constituents. {yields} In vitro assays show that leachate inhibits cell proliferation at low doses. {yields} Leachate can induce cytotoxic effects on HepG2 cells at high doses.« less

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

PubMed

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Comparison of different methods for extraction of Cinnamomi ramulus: yield, chemical composition and in vitro antiviral activities.

PubMed

Zhou, Jing; Yuan, Xiurong; Li, Ling; Zhang, Tong; Wang, Bing

2017-12-01

Hydrodistillation (HD), supercritical fluid extraction (SFE) and reflux extraction (RE) were applied to obtain Cinnamomi ramulus extracts. The yields, chemical compositions and antiviral activities of the extracts were investigated. Extracts were analysed using gas chromatography-mass spectrometry and the antiviral activities were evaluated using cytopathic effect inhibition assay. HD, SFE and RE afforded 0.376, 1.227 and 5.914% yields, respectively. Cinnamaldehyde (CA), SFE and ethanol extracts exhibited antiviral activities against herpes simplex virus type 1. Moreover, CA and other three extracts had inhibition efficacy against respiratory syncytial virus. The most efficient antiviral activities were obtained with SFE.

Assay of potency of the proposed Fifth International Standard for Gas-Gangrene Antitoxin (Perfringens)

PubMed Central

Prigge, R.; Micke, H.; Krüger, J.

1963-01-01

As part of a collaborative assay of the proposed Fifth International Standard for Gas-Gangrene Antitoxin (Perfringens), five ampoules of the proposed replacement material were assayed in the authors' laboratory against the then current Fourth International Standard. Both in vitro and in vivo methods were used. This paper presents the results and their statistical analysis. The two methods yielded different results which were not likely to have been due to chance, but exact statistical comparison is not possible. It is thought, however, that the differences may be due, at least in part, to differences in the relative proportions of zeta-antitoxin and alpha-antitoxin in the Fourth and Fifth International Standards and the consequent different reactions with the test toxin that was used for titration. PMID:14107746

Comparison of human immunodeficiency virus assays in window phase and elite controller samples: viral load distribution and implications for transmission risk

PubMed Central

Vermeulen, Marion; Coleman, Charl; Mitchel, Josephine; Reddy, Ravi; van Drimmelen, Harry; Fickett, Tracy; Busch, Michael; Lelie, Nico

2016-01-01

BACKGROUND After 3 years of individual-donation nucleic acid test (ID-NAT) screening by the South African National Blood Service (SANBS), a repository of 73 human immunodeficiency virus antibody (anti-HIV)-negative window period (WP)-yield samples and 28 anti-HIV–positive, HIV-RNA–negative elite controllers (ECs) became available for comparison of a p24 antigen (p24 Ag) assay (Innogenetics), two viral load assays (Siemens branch DNA [bDNA] 3.0 and Abbott real-time polymerase chain reaction [RT-PCR]), and three triplex NAT assays (Novartis Diagnostics Ultrio and Ultrio-Plus and Roche TaqScreen) by replicate testing of dilutions. STUDY DESIGN AND METHODS Viral loads were assessed by bDNA and RT-PCR assays and if below 100 copies (cps)/mL, by Ultrio limiting dilution probit analysis. The probability of virus transmission by WP and EC donations was estimated for different levels of the 50% minimum infectious dose (ID50) using Poisson distribution statistics. RESULTS The equal distribution of WP donations plotted by log HIV-RNA levels indicated a random appearance of donors in the ramp-up phase. The HIV p24 Ag assay detected 45% of WP samples and the cutoff crossing point was estimated at 8140 (bDNA)/ 22,710 (RT-PCR) cps/mL. On replicate retesting of 40 HIV p24 Ag–negative ID-NAT WP-yield samples Ultrio minipool (MP)8, Ultrio-Plus MP8, and TaqScreen MP6 detected 79, 81, and 78%, respectively. Modeling with an estimated ID50 of 31.6 virions/RBC indicated that 15% of p24 Ag–negative ID-NAT WP-yield donations would have transmitted HIV if MP6–8 NAT had been used. Only 2% of RBC transfusions from ECs are estimated to be infectious with a worst-case ID50 estimate of 316 virions. CONCLUSION Our analysis of viremia and infectivity of WP and EC donations enables comparison of the efficacy of NAT options in preventing HIV transmission risk. PMID:23445273

Comparison of human immunodeficiency virus assays in window phase and elite controller samples: viral load distribution and implications for transmission risk.

PubMed

Vermeulen, Marion; Coleman, Charl; Mitchel, Josephine; Reddy, Ravi; van Drimmelen, Harry; Fickett, Tracy; Busch, Michael; Lelie, Nico

2013-10-01

After 3 years of individual-donation nucleic acid test (ID-NAT) screening by the South African National Blood Service (SANBS), a repository of 73 human immunodeficiency virus antibody (anti-HIV)-negative window period (WP)-yield samples and 28 anti-HIV-positive, HIV-RNA-negative elite controllers (ECs) became available for comparison of a p24 antigen (p24 Ag) assay (Innogenetics), two viral load assays (Siemens branch DNA [bDNA] 3.0 and Abbott real-time polymerase chain reaction [RT-PCR]), and three triplex NAT assays (Novartis Diagnostics Ultrio and Ultrio-Plus and Roche TaqScreen) by replicate testing of dilutions. Viral loads were assessed by bDNA and RT-PCR assays and if below 100 copies (cps)/mL, by Ultrio limiting dilution probit analysis. The probability of virus transmission by WP and EC donations was estimated for different levels of the 50% minimum infectious dose (ID50 ) using Poisson distribution statistics. The equal distribution of WP donations plotted by log HIV-RNA levels indicated a random appearance of donors in the ramp-up phase. The HIV p24 Ag assay detected 45% of WP samples and the cutoff crossing point was estimated at 8140 (bDNA)/22,710 (RT-PCR) cps/mL. On replicate retesting of 40 HIV p24 Ag-negative ID-NAT WP-yield samples Ultrio minipool (MP)8, Ultrio-Plus MP8, and TaqScreen MP6 detected 79, 81, and 78%, respectively. Modeling with an estimated ID50 of 31.6 virions/RBC indicated that 15% of p24 Ag-negative ID-NAT WP-yield donations would have transmitted HIV if MP6-8 NAT had been used. Only 2% of RBC transfusions from ECs are estimated to be infectious with a worst-case ID50 estimate of 316 virions. Our analysis of viremia and infectivity of WP and EC donations enables comparison of the efficacy of NAT options in preventing HIV transmission risk. © 2013 American Association of Blood Banks.

Synergistic interactions of ecosystem services: florivorous pest control boosts crop yield increase through insect pollination.

PubMed

Sutter, Louis; Albrecht, Matthias

2016-02-10

Insect pollination and pest control are pivotal functions sustaining global food production. However, they have mostly been studied in isolation and how they interactively shape crop yield remains largely unexplored. Using controlled field experiments, we found strong synergistic effects of insect pollination and simulated pest control on yield quantity and quality. Their joint effect increased yield by 23%, with synergistic effects contributing 10%, while their single contributions were 7% and 6%, respectively. The potential economic benefit for a farmer from the synergistic effects (12%) was 1.8 times greater than their individual contributions (7% each). We show that the principal underlying mechanism was a pronounced pest-induced reduction in flower lifetime, resulting in a strong reduction in the number of pollinator visits a flower receives during its lifetime. Our findings highlight the importance of non-additive interactions among ecosystem services (ES) when valuating, mapping or predicting them and reveal fundamental implications for ecosystem management and policy aimed at maximizing ES for sustainable agriculture. © 2016 The Author(s).

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

USGS Publications Warehouse

Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

2013-01-01

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system

PubMed Central

Nie, Jianhui; Wu, Xiaohong; Ma, Jian; Cao, Shouchun; Huang, Weijin; Liu, Qiang; Li, Xuguang; Li, Yuhua; Wang, Youchun

2017-01-01

Pseudoviruses are useful virological tools because of their safety and versatility; however the low titer of these viruses substantially limits their wider applications. We developed a highly efficient pseudovirus production system capable of yielding 100 times more rabies pseudovirus than the traditional method. Employing the high-titer pseudoviruses, we have developed robust in vitro and in vivo neutralization assays for the evaluation of rabies vaccine, which traditionally relies on live-virus based assays. Compared with current rapid fluorescent focus inhibition test (RFFIT), our in vitro pseudovirus-based neutralization assay (PBNA) is much less labor-intensive while demonstrating better reproducibility. Moreover, the in vivo PBNA assay was also found to be superior to the live virus based assay. Following intravenous administration, the pseudovirus effectively infected the mice, with dynamic viral distributions being sequentially observed in spleen, liver and brain. Furthermore, data from in vivo PBNA showed great agreement with those generated from the live virus model but with the experimental time significantly reduced from 2 weeks to 3 days. Taken together, the effective pseudovirus production system facilitated the development of novel PBNA assays which could replace live virus-based traditional assays due to its safety, rapidity, reproducibility and high throughput capacity. PMID:28218278

Polyphosphoester-Camptothecin Prodrug with Reduction-Response Prepared via Michael Addition Polymerization and Click Reaction.

PubMed

Du, Xueqiong; Sun, Yue; Zhang, Mingzu; He, Jinlin; Ni, Peihong

2017-04-26

Polyphosphoesters (PPEs), as potential candidates for biocompatible and biodegradable polymers, play an important role in material science. Various synthetic methods have been employed in the preparation of PPEs such as polycondensation, polyaddition, ring-opening polymerization, and olefin metathesis polymerization. In this study, a series of linear PPEs has been prepared via one-step Michael addition polymerization. Subsequently, camptothecin (CPT) derivatives containing disulfide bonds and azido groups were linked onto the side chain of the PPE through Cu(I)-catalyzed azidealkyne cyclo-addition "click" chemistry to yield a reduction-responsive polymeric prodrug P(EAEP-PPA)-g-ss-CPT. The chemical structures were characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography, Fourier transform infrared, ultraviolet-visible spectrophotometer, and high performance liquid chromatograph analyses, respectively. The amphiphilic prodrug could self-assemble into micelles in aqueous solution. The average particle size and morphology of the prodrug micelles were measured by dynamic light scattering and transmission electron microscopy, respectively. The results of size change under different conditions indicate that the micelles possess a favorable stability in physiological conditions and can be degraded in reductive medium. Moreover, the studies of in vitro drug release behavior confirm the reduction-responsive degradation of the prodrug micelles. A methyl thiazolyl tetrazolium assay verifies the good biocompatibility of P(EAEP-PPA) not only for normal cells, but also for tumor cells. The results of cytotoxicity and the intracellular uptake about prodrug micelles further demonstrate that the prodrug micelles can efficiently release CPT into 4T1 or HepG2 cells to inhibit the cell proliferation. All these results show that the polyphosphoester-based prodrug can be used for triggered drug delivery system in cancer treatment.

Specific yield: compilation of specific yields for various materials

USGS Publications Warehouse

Johnson, A.I.

1967-01-01

Specific yield is defined as the ratio of (1) the volume of water that a saturated rock or soil will yield by gravity to (2) the total volume of the rock or soft. Specific yield is usually expressed as a percentage. The value is not definitive, because the quantity of water that will drain by gravity depends on variables such as duration of drainage, temperature, mineral composition of the water, and various physical characteristics of the rock or soil under consideration. Values of specific yields nevertheless offer a convenient means by which hydrologists can estimate the water-yielding capacities of earth materials and, as such, are very useful in hydrologic studies. The present report consists mostly of direct or modified quotations from many selected reports that present and evaluate methods for determining specific yield, limitations of those methods, and results of the determinations made on a wide variety of rock and soil materials. Although no particular values are recommended in this report, a table summarizes values of specific yield, and their averages, determined for 10 rock textures. The following is an abstract of the table. [Table

Yield and yield gaps in central U.S. corn production systems

USDA-ARS?s Scientific Manuscript database

The magnitude of yield gaps (YG) (potential yield – farmer yield) provides some indication of the prospects for increasing crop yield. Quantile regression analysis was applied to county maize (Zea mays L.) yields (1972 – 2011) from Kentucky, Iowa and Nebraska (irrigated) (total of 115 counties) to e...

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

PubMed

Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

2015-06-01

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster

PubMed Central

Bauer, Johannes H.; Goupil, Stephan; Garber, Graham B.; Helfand, Stephen L.

2004-01-01

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. PMID:15328413

Use of Enzymes in Organic Synthesis: Reduction of Ketones by Baker's Yeast Revisited

ERIC Educational Resources Information Center

Patterson, James; Sigurdsson, Snorri Th.

2005-01-01

The reduction of ethyl acetoacetate using common baker's yeast is a traditional experiment that shows the stereoselective power of a biochemical system. Addition of organic solvents to aqueous reaction system increased the yields and reproducibility of the experiment thus overcoming the two problems associated with the experiment, low yield, and…

Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

PubMed

O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

2017-01-27

Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

A reductive aminase from Aspergillus oryzae

NASA Astrophysics Data System (ADS)

Aleku, Godwin A.; France, Scott P.; Man, Henry; Mangas-Sanchez, Juan; Montgomery, Sarah L.; Sharma, Mahima; Leipold, Friedemann; Hussain, Shahed; Grogan, Gideon; Turner, Nicholas J.

2017-10-01

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l-1 d-1.

Photodissociable dimer reduction products of 2-thiopyrimidine derivatives.

PubMed Central

Wrona, M; Giziewicz, J; Shugar, D

1975-01-01

Both 4,6-dimethyl-2-thipyrimidine and its 1-methyl derivative undergo polarographic reduction in aqueous medium, via a 1e/1H+ reduction to a free radical which rapidly dimerizes to products isolates and identified as 4,4'-bis-(4,6-dimethyl-3,4-dihydropyrimidin-2-thione) and the corresponding 1-methyl dimer. The dimers may be oxidized electrolytically to regenerate the parent monomers. Both dimers also undergo photodissociation to quantitatively regenerate the parent monomers, in high quantum yield, 0.23 and 0.35 M/Einstein. The correlation between electrochemical and photochemical reductions of 2-thiopyrimidines are discussed, as well as the significance of the dimer photodissociation reactions in relation to nucleic acid photochemistry. PMID:28516

Ammonia Formation By The Reduction Of Nitrite/Nitrate By Fes: Ammonia Formation Under Acidic Conditions

NASA Astrophysics Data System (ADS)

Summers, David P.

2005-08-01

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe+ 2. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO2, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO2, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N2 CO2 shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe+ 2, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe+ 2 (which also can be sensitive to binding by certain species)

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

PubMed Central

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-01-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

PubMed

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

A cluster pattern algorithm for the analysis of multiparametric cell assays.

PubMed

Kaufman, Menachem; Bloch, David; Zurgil, Naomi; Shafran, Yana; Deutsch, Mordechai

2005-09-01

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.

Split-luciferase complementary assay: applications, recent developments, and future perspectives.

PubMed

Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman

2014-09-01

Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.

Determination of Microbial Growth by Protein Assay in an Air-Cathode Single Chamber Microbial Fuel Cell.

PubMed

Li, Na; Kakarla, Ramesh; Moon, Jung Mi; Min, Booki

2015-07-01

Microbial fuel cells (MFCs) have gathered attention as a novel bioenergy technology to simultaneously treat wastewater with less sludge production than the conventional activated sludge system. In two different operations of the MFC and aerobic process, microbial growth was determined by the protein assay method and their biomass yields using real wastewater were compared. The biomass yield on the anode electrode of the MFC was 0.02 g-COD-cell/g- COD-substrate and the anolyte planktonic biomass was 0.14 g-COD-cell/g-COD-substrate. An MFC without anode electrode resulted in the biomass yield of 0.07 ± 0.03 g-COD-cell/g-COD-substrate, suggesting that oxygen diffusion from the cathode possibly supported the microbial growth. In a comparative test, the biomass yield under aerobic environment was 0.46 ± 0.07 g-COD-cell/g-COD-substrate, which was about 3 times higher than the total biomass value in the MFC operation.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

“Life-like” assessment of antimicrobial surfaces by a new touch transfer assay displays strong superiority of a copper alloy compared to silver containing surfaces

PubMed Central

Tofern, Sabrina; Kunz, Wladimir; Schütze, Sara; Riecke, Michael; Solbach, Werner; Wuske, Thomas

2017-01-01

Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. In this study we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces, with confirmed antibacterial activity by a modified ISO 22196 (JIS Z 2801) assay to test such surfaces under more realistic conditions. Bacteria were taken up from a dry standardized primary contaminated surface (PCS) with disinfected fingers or fingers covered with sterile and moistened cotton gloves. Subsequently, bacteria were transferred by pressing on secondary contaminated surfaces (SCS) with or without potential antibacterial activity and the relative reduction rate was determined after 24 h. A stable transmission rate between PCS and SCS was observed using moistened sterile gloves. A copper containing alloy displayed at least a tenfold reduction of the bacterial load consistently reaching less than 2.5 cfu/cm2. In contrast, no significant reduction of bacterial contamination by silver containing surfaces and matured pure silver was observed in the touch transfer assay. With the touch transfer assay we successfully established a new reproducible method modeling cross contamination. Using the new method we were able to demonstrate that several surfaces with confirmed antimicrobial activity in a modified ISO 22196 (JIS Z 2801) assay lacked effectiveness under defined ambient conditions. This data indicate that liquid based assays like the ISO 22196 should be critically reviewed before claiming antibacterial activity for surfaces in the setting of contamination of dry surfaces by contact to the human skin. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

PubMed

Grosvenor, A L; Feltus, A; Conover, R C; Daunert, S; Anderson, K W

2000-06-01

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

PubMed Central

2013-01-01

Background Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance. Results Although lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield. Conclusions Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell

Iron Reduction and Carbonate Precipitation by Shewanella oneidensis

NASA Astrophysics Data System (ADS)

Zeng, Z.; Tice, M. M.

2011-12-01

This study is to contribute to better understanding of how Archean microbes induced carbonate diagenesis in mats and stromatolites. Previous studies showed sulfate reduction, a common promoter of carbonate precipitation in modern mats[1], is likely to have been less effective in Archean mats in marine fluids lower in sulfate[2]. Alternatively, iron reduction produces far more alkalinity per unit carbon respired than sulfate reduction. Therefore, we hypothesize iron reduction can promote much more carbonate precipitation than sulfate reduction. Our study might also have some relevance to banded iron formation on which microbial iron reduction played a potential role[3]. To test our hypothesis, Shewanella oneidensis MR-1, a dissimilatory iron reducing bacterium will be cultured anaerobically (79%N2, 20%CO2 and 1%H2) in basal medium to trigger iron reduction. Lactate will be used as electron donor, and the electron acceptor will be fresh ferrihydrite. Culture medium will be added with various metal ions, such as Ca2+ and Mg2+, to obtain potential carbonate precipitate. Escherichia coli (with fumarate added as an electron acceptor) will be used to provide a comparison to live but non-iron- reduction cells. After 20 days incubation, precipitate will be collected, washed and identified by X-ray diffraction (XRD). Besides, iron reduction rate (ferrozine assay)[4], PH and amount of precipitate (carbonate and oxidize fractions)[5] will be measured over time to well understand how S. oneidensis drives carbonate precipitation.

Development of a colourimetric assay for glycosynthases.

PubMed

Hayes, Marc R; Bochinsky, Kevin A; Seibt, Lisa S; Elling, Lothar; Pietruszka, Jörg

2017-09-10

The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of geoengineering on crop yields

NASA Astrophysics Data System (ADS)

Pongratz, J.; Lobell, D. B.; Cao, L.; Caldeira, K.

2011-12-01

The potential of "solar radiation management" (SRM) to reduce future climate change and associated risks has been receiving significant attention in scientific and policy circles. SRM schemes aim to reduce global warming despite increasing atmospheric CO2 concentrations by diminishing the amount of solar insolation absorbed by the Earth, for example, by injecting scattering aerosols into the atmosphere. Climate models predict that SRM could fully compensate warming at the global mean in a high-CO2 world. While reduction of global warming may offset a part of the predicted negative effects of future climate change on crop yields, SRM schemes are expected to alter regional climate and to have substantial effects on climate variables other than temperature, such as precipitation. It has therefore been warned that, overall, SRM may pose a risk to food security. Assessments of benefits and risks of geoengineering are imperative, yet such assessments are only beginning to emerge; in particular, effects on global food security have not previously been assessed. Here, for the first time, we combine climate model simulations with models of crop yield responses to climate to assess large-scale changes in yields and food production under SRM. In most crop-growing regions, we find that yield losses caused by climate changes are substantially reduced under SRM as compared with a non-geoengineered doubling of atmospheric CO2. Substantial yield losses with SRM are only found for rice in high latitudes, where the limits of low temperatures are no longer alleviated. At the same time, the beneficial effect of CO2-fertilization on plant productivity remains active. Overall therefore, SRM in our models causes global crop yields to increase. We estimate the direct effects of climate and CO2 changes on crop production, and do not quantify effects of market dynamics and management changes. We note, however, that an SRM deployment would be unlikely to maintain the economic status quo, as

Simultaneous saccharification and fermentation of Eastern redcedar heartwood and sapwood using a novel size reduction technique.

PubMed

Ramachandriya, Karthikeyan D; Wilkins, Mark; Pardo-Planas, Oscar; Atiyeh, Hasan K; Dunford, Nurhan T; Hiziroglu, Salim

2014-06-01

This study investigated the effect of two wood zones (sapwood versus heartwood) and size reduction techniques [Crumbles® (Crumbles® is a registered trademark of Forest Concepts, LLC, Auburn, WA, USA) particles versus ground particles] on wood glucan-to-ethanol yield after acid bisulfite pretreatment and simultaneous saccharification and fermentation (SSF) of Eastern redcedar. SSFs were conducted at 8% solids loading (w/w dry basis) using Accellerase® 1500 at a loading of 46FPU/g glucan and Saccharomyces cerevisiae D5A for ethanol fermentation. The size reduction technique had no effect on ethanol yield. However, sapwood glucan-to-ethanol yields were significantly greater than heartwood yields. The highest wood glucan-to-ethanol yield of 187L/dryMg (95% of theoretical) was achieved with sapwood crumbled particles in 240h. Ground sapwood, crumbled heartwood and ground heartwood achieved ethanol yields of 89%, 81% and 80% of theoretical in 240h, respectively. Preliminary mass balances showed 100% glucan recovery with crumbled sapwood and extensive (72%) delignification. Copyright © 2014 Elsevier Ltd. All rights reserved.

Charge Yield at Low Electric Fields: Considerations for Bipolar Integrated Circuits

NASA Technical Reports Server (NTRS)

Johnston, A. H.; Swimm, R. T.; Thorbourn, D. O.

2013-01-01

A significant reduction in total dose damage is observed when bipolar integrated circuits are irradiated at low temperature. This can be partially explained by the Onsager theory of recombination, which predicts a strong temperature dependence for charge yield under low-field conditions. Reduced damage occurs for biased as well as unbiased devices because the weak fringing field in thick bipolar oxides only affects charge yield near the Si/SiO2 interface, a relatively small fraction of the total oxide thickness. Lowering the temperature of bipolar ICs - either continuously, or for time periods when they are exposed to high radiation levels - provides an additional degree of freedom to improve total dose performance of bipolar circuits, particularly in space applications.

Magnesium fluoride reduction-vessel liners. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latham-Brown, C.E.

1986-03-26

The work described in this report details a program that demonstrated a method by which magnesium fluoride, the by-product of the reduction reaction of uranium tetrafluoride to uranium metal could be used to replace the present graphite used to line the reduction vessel. Utilization of magnesium fluoride (MgF2) as a reduction-vessel liner has the potential to decrease carbon contamination and thereby reduce DU derby rejects due to chemistry. Additionally, there would be the elimination of the cost of the graphite crucible liner and the associated disposal costs by replacement with the by-product of the reduction reaction, which is magnesium fluoride.more » The process would ultimately result in reduced manufacturing costs for derby metal and higher yield of finished penetrators. This was to be accomplished in such a manner as to produce uranium metal derbies which would be accommodated into the present Nuclear Metals-Carolina Metals penetrator production process with minimal changes in equipment and procedures.« less

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.

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Kinetics and yields of pesticide biodegradation at low substrate concentrations and under conditions restricting assimilable organic carbon.

PubMed

Helbling, Damian E; Hammes, Frederik; Egli, Thomas; Kohler, Hans-Peter E

2014-02-01

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter(-1). We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.

Monocytes and macrophages in malignant melanoma. III. Reduction of nitroblue tetrazolium by peripheral blood monocytes.

PubMed Central

Hedley, D. W.; Currie, G. A.

1978-01-01

Peripheral-blood monocytes from normal individuals and from patients with malignant melanoma reduce nitroblue tetrazolium (NBT). A quantitative assay for dye reduction was applied to 25 healthy donors and 31 patients with malignant melanoma. NBT reduction expressed as dye reduction per monocyte was significantly impaired in patients with disseminated disease, and they responded poorly to a phagocytic stimulus. Monocytes from patients with micrometastatic disease, however, showed normal resting NBT reduction but, following exposure to a suspension of latex-polystyrene, showed significantly greater NBT reduction than those from normal individuals. Since NBT reduction is an indirect measure of intracellular hexose-monophosphate-shunt activity we conclude that the monocytes from patients with minimal disease are in some way activated. PMID:656304

Discordance between MTB/RIF and Real-Time Tuberculosis-Specific Polymerase Chain Reaction Assay in Bronchial Washing Specimen and Its Clinical Implications.

PubMed

Jo, Yong Suk; Park, Ju-Hee; Lee, Jung Kyu; Heo, Eun Young; Chung, Hee Soon; Kim, Deog Kyeom

2016-01-01

The prevalence and clinical implications of discordance between Xpert MTB/RIF assays and the AdvanSure TB/NTM real-time polymerase chain reaction (PCR) for bronchial washing specimens have not been studied in pulmonary TB (PTB) patients. The discordant proportion and its clinical impact were evaluated in 320 patients from the bronchoscopy registry whose bronchial washing specimens were tested simultaneously with Xpert MTB/RIF and the TB/NTM PCR assay for three years, and the accuracy of the assays, including the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), were studied. The clinical risk factors for discordance and false positivity of assays were also studied. Among 130 patients who were clinically diagnosed with PTB, 64 patients showed positive acid-fast bacilli culture results, 56 patients showed positive results in molecular methods and clinician diagnosed PTB without results of microbiology in 10 patients. The sensitivity, specificity, PPV, and NPV were 80.0%, 98.95%, 98.1%, and 87.9%, respectively, for Xpert MTB/RIF and 81.5%, 92.6%, 88.3%, and 88.0%, respectively, for TB/NTM PCR. The discordant proportion was 16.9% and was higher in culture-negative PTB compared to culture-confirmed PTB (24.3% vs. 9.4%, p = 0.024). However, there were no significant differences in the clinical characteristics, regardless of the discordance. The diagnostic yield increased with an additional assay (7.7% for Xpert MTB/RIF and 9.2% for TB/NTM PCR). False positivity was less common in patients tested with Xpert MTB/RIF (1.05% vs. 7.37%, p = 0.0035). No host-related risk factor for false positivity was identified. The Xpert MTB/RIF and TB/NTM PCR assay in bronchial washing specimens can improve the diagnostic yields for PTB, although there were considerable discordant results without any patient-related risk factors.

Measurement of fission yields and isomeric yield ratios at IGISOL

NASA Astrophysics Data System (ADS)

Pomp, Stephan; Mattera, Andrea; Rakopoulos, Vasileios; Al-Adili, Ali; Lantz, Mattias; Solders, Andreas; Jansson, Kaj; Prokofiev, Alexander V.; Eronen, Tommi; Gorelov, Dimitri; Jokinen, Ari; Kankainen, Anu; Moore, Iain D.; Penttilä, Heikki; Rinta-Antila, Sami

2018-03-01

Data on fission yields and isomeric yield ratios (IYR) are tools to study the fission process, in particular the generation of angular momentum. We use the IGISOL facility with the Penning trap JYFLTRAP in Jyväskylä, Finland, for such measurements on 232Th and natU targets. Previously published fission yield data from IGISOL concern the 232Th(p,f) and 238U(p,f) reactions at 25 and 50 MeV. Recently, a neutron source, using the Be(p,n) reaction, has been developed, installed and tested. We summarize the results for (p,f) focusing on the first measurement of IYR by direct ion counting. We also present first results for IYR and relative yields for Sn and Sb isotopes in the 128-133 mass range from natU(n,f) based on γ-spectrometry. We find a staggering behaviour in the cumulative yields for Sn and a shift in the independent fission yields for Sb as compared to current evaluations. Plans for the future experimental program on fission yields and IYR measurements are discussed.

Validation of a next-generation sequencing assay for clinical molecular oncology.

PubMed

Cottrell, Catherine E; Al-Kateb, Hussam; Bredemeyer, Andrew J; Duncavage, Eric J; Spencer, David H; Abel, Haley J; Lockwood, Christina M; Hagemann, Ian S; O'Guin, Stephanie M; Burcea, Lauren C; Sawyer, Christopher S; Oschwald, Dayna M; Stratman, Jennifer L; Sher, Dorie A; Johnson, Mark R; Brown, Justin T; Cliften, Paul F; George, Bijoy; McIntosh, Leslie D; Shrivastava, Savita; Nguyen, Tudung T; Payton, Jacqueline E; Watson, Mark A; Crosby, Seth D; Head, Richard D; Mitra, Robi D; Nagarajan, Rakesh; Kulkarni, Shashikant; Seibert, Karen; Virgin, Herbert W; Milbrandt, Jeffrey; Pfeifer, John D

2014-01-01

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Solutions to decrease a systematic error related to AAPH addition in the fluorescence-based ORAC assay.

PubMed

Mellado-Ortega, Elena; Zabalgogeazcoa, Iñigo; Vázquez de Aldana, Beatriz R; Arellano, Juan B

2017-02-15

Oxygen radical absorbance capacity (ORAC) assay in 96-well multi-detection plate readers is a rapid method to determine total antioxidant capacity (TAC) in biological samples. A disadvantage of this method is that the antioxidant inhibition reaction does not start in all of the 96 wells at the same time due to technical limitations when dispensing the free radical-generating azo initiator 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH). The time delay between wells yields a systematic error that causes statistically significant differences in TAC determination of antioxidant solutions depending on their plate position. We propose two alternative solutions to avoid this AAPH-dependent error in ORAC assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Reductions in Perceived Injustice are Associated With Reductions in Disability and Depressive Symptoms After Total Knee Arthroplasty.

PubMed

Yakobov, Esther; Scott, Whitney; Stanish, William D; Tanzer, Michael; Dunbar, Michael; Richardson, Glen; Sullivan, Michael J L

2018-05-01

Perceptions of injustice have been associated with problematic recovery outcomes in individuals with a wide range of debilitating pain conditions. It has been suggested that, in patients with chronic pain, perceptions of injustice might arise in response to experiences characterized by illness-related pain severity, depressive symptoms, and disability. If symptoms severity and disability are important contributors to perceived injustice (PI), it follows that interventions that yield reductions in symptom severity and disability should also contribute to reductions in perceptions of injustice. The present study examined the relative contributions of postsurgical reductions in pain severity, depressive symptoms, and disability to the prediction of reductions in perceptions of injustice. The study sample consisted of 110 individuals (69 women and 41 men) with osteoarthritis of the knee scheduled for total knee arthroplasty (TKA). Patients completed measures of perceived injustice, depressive symptoms, pain, and disability at their presurgical evaluation, and at 1-year follow-up. The results revealed that reductions in depressive symptoms and disability, but not pain severity, were correlated with reductions in perceived injustice. Regression analyses revealed that reductions in disability and reductions in depressive symptoms contributed modest but significant unique variance to the prediction of postsurgical reductions in perceived injustice. The present findings are consistent with current conceptualizations of injustice appraisals that propose a central role for symptom severity and disability as determinants of perceptions of injustice in patients with persistent pain. The results suggest that the inclusion of psychosocial interventions that target depressive symptoms and perceived injustice might augment the impact of rehabilitation programs made available for individuals recovering from TKA.

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase.

PubMed

Zhou, Min; Kitagawa, Yoshinori; Yamaguchi, Mayu; Uchiyama, Chika; Itoh, Masae; Gotoh, Bin

2013-01-01

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types

PubMed Central

Yek, Christina; Massanella, Marta; Peling, Tashi; Lednovich, Kristen; Nair, Sangeetha V.; Worlock, Andrew; Vargas, Milenka; Gianella, Sara; Ellis, Ronald J.; Strain, Matthew C.; Busch, Michael P.; Nugent, C. Thomas

2017-01-01

ABSTRACT The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma (n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings. PMID:28592548

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types.

PubMed

Yek, Christina; Massanella, Marta; Peling, Tashi; Lednovich, Kristen; Nair, Sangeetha V; Worlock, Andrew; Vargas, Milenka; Gianella, Sara; Ellis, Ronald J; Strain, Matthew C; Busch, Michael P; Nugent, C Thomas; Richman, Douglas D

2017-08-01

The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma ( n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.

A microfluorometric assay for cholinesterases, suitable for multiple kinetic determinations of picomoles of released thiocholine.

PubMed

Parvari, R; Pecht, I; Soreq, H

1983-09-01

A highly sensitive microfluorometric assay for cholinesterases has been developed. Enzymatic activity is measured by monitoring the thiocholine produced by specific hydrolysis of acetylthiocholine. This is carried out by reacting the thiocholine formed with the fluorogenic compound N-(4(7 diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide to yield an intensely fluorescent product. The assay is linear over a range extending from a few picomoles to nanomoles of thiocholine. The specificity and accuracy of this microfluorometric assay were examined using microgram quantities of rat brain tissue as a source for cholinesterases. The specific activities and the Km values determined by this new method for both cholinesterase activities present in the brain (acetylcholine hydrolase, EC 3.1.1.7, and "nonspecific" cholinesterase-acylcholine acylhydrolase, EC 3.1.1.8) were identical to those reported earlier using the less sensitive spectrophotometric and radiometric methods. The background emission caused by nonenzymatic hydrolysis of the substrate is relatively low, and does not exceed background values encountered in other methods. The assay may be used for monitoring the kinetics of enzymatic activities in microscale reaction mixtures, providing a linear determination of the thiocholine produced over a period of at least 30 h at room temperature. The method can also be adapted for use in other enzymatic assays where reagents containing thiol groups can be produced or consumed.

Ensemble brightening and enhanced quantum yield in size-purified silicon nanocrystals

DOE PAGES

Miller, Joseph B.; Van Sickle, Austin R.; Anthony, Rebecca J.; ...

2012-07-18

Here, we report on the quantum yield, photoluminescence (PL) lifetime and ensemble photoluminescent stability of highly monodisperse plasma-synthesized silicon nanocrystals (SiNCs) prepared though density-gradient ultracentrifugation in mixed organic solvents. Improved size uniformity leads to a reduction in PL line width and the emergence of entropic order in dry nanocrystal films. We find excellent agreement with the anticipated trends of quantum confinement in nanocrystalline silicon, with a solution quantum yield that is independent of nanocrystal size for the larger fractions but decreases dramatically with size for the smaller fractions. We also find a significant PL enhancement in films assembled from themore » fractions, and we use a combination of measurement, simulation and modeling to link this ‘brightening’ to a temporally enhanced quantum yield arising from SiNC interactions in ordered ensembles of monodisperse nanocrystals. Using an appropriate excitation scheme, we exploit this enhancement to achieve photostable emission.« less

Secondary electron emission yield from high aspect ratio carbon velvet surfaces

NASA Astrophysics Data System (ADS)

Jin, Chenggang; Ottaviano, Angelica; Raitses, Yevgeny

2017-11-01

The plasma electrons bombarding a plasma-facing wall surface can induce secondary electron emission (SEE) from the wall. A strong SEE can enhance the power losses by reducing the wall sheath potential and thereby increasing the electron flux from the plasma to the wall. The use of the materials with surface roughness and the engineered materials with surface architecture is known to reduce the effective SEE by trapping the secondary electrons. In this work, we demonstrate a 65% reduction of SEE yield using a velvet material consisting of high aspect ratio carbon fibers. The measurements of SEE yield for different velvet samples using the electron beam in vacuum demonstrate the dependence of the SEE yield on the fiber length and the packing density, which is strongly affected by the alignment of long velvet fibers with respect to the electron beam impinging on the velvet sample. The results of SEE measurements support the previous observations of the reduced SEE measured in Hall thrusters.

Effects of ambient and elevated level of ozone on Brassica campestris L. with special reference to yield and oil quality parameters.

PubMed

Tripathi, Ruchika; Agrawal, S B

2012-11-01

Tropospheric ozone (O(3)) has become a serious threat to growth and yield of important agricultural crops over Asian regions including India. Effect of elevated O(3) (ambient+10ppb) was studied on Brassica campestris L. (cv. Sanjukta and Vardan) in open top chambers under natural field conditions. Eight hourly mean ambient O(3) concentration varied from 26.3ppb to 69.5ppb during the growth period. Plants under O(3) exposure showed reductions in photosynthetic rate, reproductive parameters, yield as well as seed and oil quality. Cultivar Sanjukta showed more reduction in photosynthetic characteristics, reproductive structures and seed and oil quality. However, total yield was more affected in Vardan. Exposure of O(3) increased the degree of unsaturation and level of PUFA, ω-6fatty acid, linolenic acid and erucic acid in oil indicating the deterioration of its quality. The study further confirmed that there is a correspondence between O(3) induced change in photosynthetic processes, reproductive development and yield and did not find any compensatory response in the final yield. Copyright © 2012 Elsevier Inc. All rights reserved.

Very high CO2 reduces photosynthesis, dark respiration and yield in wheat

NASA Technical Reports Server (NTRS)

Reuveni, J.; Bugbee, B.

1997-01-01

Although terrestrial CO2 concentrations, [CO2] are not expected to reach 1000 micromoles mol-1 for many decades, CO2 levels in closed systems such as growth chambers and glasshouses, can easily exceed this concentration. CO2 levels in life support systems in space can exceed 10000 micromoles mol-1 (1%). Here we studied the effect of six CO2 concentrations, from ambient up to 10000 micromoles mol-1, on seed yield, growth and gas exchange of two wheat cultivars (USU-Apogee and Veery-l0). Elevating [CO2] from 350 to 1000 micromoles mol-1 increased seed yield (by 33%), vegetative biomass (by 25%) and number of heads m-2 (by 34%) of wheat plants. Elevation of [CO2] from 1000 to 10000 micromoles mol-1 decreased seed yield (by 37%), harvest index (by 14%), mass per seed (by 9%) and number of seeds per head (by 29%). This very high [CO2] had a negligible, non-significant effect on vegetative biomass, number of heads m-2 and seed mass per head. A sharp decrease in seed yield, harvest index and seeds per head occurred by elevating [CO2] from 1000 to 2600 micromoles mol-1. Further elevation of [CO2] from 2600 to 10000 micromoles mol-1 caused a further but smaller decrease. The effect of CO2 on both wheat cultivars was similar for all growth parameters. Similarly there were no differences in the response to high [CO2] between wheat grown hydroponically in growth chambers under fluorescent lights and those grown in soilless media in a glasshouse under sunlight and high pressure sodium lamps. There was no correlation between high [CO2] and ethylene production by flag leaves or by wheat heads. Therefore, the reduction in seed set in wheat plants is not mediated by ethylene. The photosynthetic rate of whole wheat plants was 8% lower and dark respiration of the wheat heads 25% lower when exposed to 2600 micromoles mol-1 CO2 compared to ambient [CO2]. It is concluded that the reduction in the seed set can be mainly explained by the reduction in the dark respiration in wheat heads

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster.

PubMed

Bauer, Johannes H; Goupil, Stephan; Garber, Graham B; Helfand, Stephen L

2004-08-31

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. Copyright 2004 The National Academy of Sciencs of the USA

Ammonia formation by the reduction of nitrite/nitrate by FeS: ammonia formation under acidic conditions.

PubMed

Summers, David P

2005-08-01

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe+ 2. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO2, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO2, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N2 CO2 shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe+ 2, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe+ 2 (which also can be sensitive to binding by certain species).

Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae

PubMed Central

McNabb, David S.; Reed, Robin; Marciniak, Robert A.

2005-01-01

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements. PMID:16151247

Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

PubMed

Krug, Anne K; Balmer, Nina V; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel

2013-12-01

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays.

PubMed

Durtschi, Jacob D; Stevenson, Jeffery; Hymas, Weston; Voelkerding, Karl V

2007-02-01

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

Slope Controls Grain Yield and Climatic Yield in Mountainous Yunnan province, China

NASA Astrophysics Data System (ADS)

Duan, X.; Rong, L.; Gu, Z.; Feng, D.

2017-12-01

Mountainous regions are increasingly vulnerable to food insecurity because of limited arable land, growing population pressure, and climate change. Development of sustainable mountain agriculture will require an increased understanding of the effects of environmental factors on grain and climatic yields. The objective of this study was to explore the relationships between actual grain yield, climatic yield, and environmental factors in a mountainous region in China. We collected data on the average grain yield per unit area in 119 counties in Yunnan province from 1985 to 2012, and chose 17 environmental factors for the same period. Our results showed that actual grain yield ranged from 1.43 to 6.92 t·ha-1, and the climatic yield ranged from -0.15 to -0.01 t·ha-1. Lower climatic yield but higher grain yield was generally found in central areas and at lower slopes and elevations in the western and southwestern counties of Yunnan province. Higher climatic yield but lower grain yield were found in northwestern parts of Yunnan province on steep slopes. Annual precipation and temperature had a weak influence on the climatic yield. Slope explained 44.62 and 26.29% of the variation in grain yield and climatic yield. The effects of topography on grain and climatic yields were greater than climatic factors. Slope was the most important environmental variable for the variability in climatic and grain yields in the mountainous Yunnan province due to the highly heterogeneous topographic conditions. Conversion of slopes to terraces in areas with higher climatic yields is an effective way to maintain grain production in response to climate variability. Additionally, soil amendments and soil and water conservation measures should be considered to maintain soil fertility and aid in sustainable development in central areas, and in counties at lower slopes and elevations in western and southwestern Yunnan province.

Extreme Cost Reductions with Multi-Megawatt Centralized Inverter Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwabe, Ulrich; Fishman, Oleg

2015-03-20

The objective of this project was to fully develop, demonstrate, and commercialize a new type of utility scale PV system. Based on patented technology, this includes the development of a truly centralized inverter system with capacities up to 100MW, and a high voltage, distributed harvesting approach. This system promises to greatly impact both the energy yield from large scale PV systems by reducing losses and increasing yield from mismatched arrays, as well as reduce overall system costs through very cost effective conversion and BOS cost reductions enabled by higher voltage operation.

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.

Characteristics of sludge reduction in an integrated pretreatment and aerobic digestion process.

PubMed

Hwang, S; Jang, H; Lee, M; Song, J; Kim, S

2006-01-01

In this study, integrated pretreatments and aerobic digestion processes were investigated in order to provide a feasible alternative that can achieve effective sludge reduction. An ozone treatment in the presence of ionic manganese, a catalyst, increased the sludge reduction ratio three times higher than that of a single ozonation, presumably due to an increase in OH radical production. The ozone treatment yielded the effective sludge reduction ratio with an increasing ozone dosage, and an effective dosage of the catalyst was found to be 4 mg-Mn/g-TS. When a mechanical pretreatment and an ozone/catalyst were applied in a series, the integrated process, even at a half mechanical intensity and a half level of ozone dosage, showed higher and faster sludge reduction than each single process did. In addition, the integrated pretreatment process showed the highest dewaterability of the treated sludges. A ratio of sludge cake generation, which was newly introduced to quantify overall performance of sludge treatment processes, showed that the integrated pretreatment followed by the aerobic digestion yielded approximately a half of the sludge cake volume compared to the single aerobic digestion. Therefore, the integrated pretreatment can be a feasible method for the effective reduction of total suspended solid and the final volume.

Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction.

PubMed Central

Dilworth, M J; Thorneley, R N

1981-01-01

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3. PMID:7030315

The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

PubMed

Kimber, Ian; Dearman, Rebecca J; Betts, Catherine J; Gerberick, G Frank; Ryan, Cindy A; Kern, Petra S; Patlewicz, Grace Y; Basketter, David A

2006-04-01

The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.

High content evaluation of shear dependent platelet function in a microfluidic flow assay

PubMed Central

Hansen, Ryan R.; Wufsus, Adam R.; Barton, Steven T.; Onasoga, Abimbola A.; Johnson-Paben, Rebecca M.; Neeves, Keith B.

2012-01-01

The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50–920 s−1). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring and dosing antiplatelet agents. PMID:23001359

Biofuel manufacturing from woody biomass: effects of sieve size used in biomass size reduction.

PubMed

Zhang, Meng; Song, Xiaoxu; Deines, T W; Pei, Z J; Wang, Donghai

2012-01-01

Size reduction is the first step for manufacturing biofuels from woody biomass. It is usually performed using milling machines and the particle size is controlled by the size of the sieve installed on a milling machine. There are reported studies about the effects of sieve size on energy consumption in milling of woody biomass. These studies show that energy consumption increased dramatically as sieve size became smaller. However, in these studies, the sugar yield (proportional to biofuel yield) in hydrolysis of the milled woody biomass was not measured. The lack of comprehensive studies about the effects of sieve size on energy consumption in biomass milling and sugar yield in hydrolysis process makes it difficult to decide which sieve size should be selected in order to minimize the energy consumption in size reduction and maximize the sugar yield in hydrolysis. The purpose of this paper is to fill this gap in the literature. In this paper, knife milling of poplar wood was conducted using sieves of three sizes (1, 2, and 4 mm). Results show that, as sieve size increased, energy consumption in knife milling decreased and sugar yield in hydrolysis increased in the tested range of particle sizes.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

NASA Technical Reports Server (NTRS)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

Some short-term effects of changing to lower yield cigarettes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minty, B.D.; Royston, D.; Jones, J.G.

The rate of clearance from the lung of the hydrophilic tracer molecule /sup 99m/Tc DTPA was used to investigate the short-term effects on lung epithelial function when smokers switched to cigarettes with lower yields of tobacco smoke constituents. Two separate studies were performed. In the first study, subjects smoked conventional mid- and low-tar cigarettes. The second study used two specially manufactured cigarettes with similar tar and nicotine yields, but differing carbon monoxide yields. Neither study demonstrated any significant improvement in /sup 99m/Tc DTPA clearance. The yields of carbon monoxide determined under standard machine smoking conditions implied that there would bemore » a 44 percent reduction in exposure to carbon monoxide when subjects switched from smoking conventional mid-tar to low-tar cigarettes. However, measurements of carboxyhemoglobin showed that the smokers compensated for the lower yields and their exposure was reduced by only 11 percent. Similarly, in the second study, the subjects reduced their exposure by 7 percent instead of the expected 44 percent. Urine nicotine/cotinine excretion measurements in this study indicated that there was no complimentary increase in nicotine absorption suggesting the possibility that subjects may be able to regulate their intake of individual components of the cigarette smoke. Thus, the unexpected result from this study was the finding that cigarette smokers could, in some way, regulate their intake of smoke from cigarettes of different composition so as to maintain a constant exposure of smoke constituents.« less

Reductive amination with zinc powder in aqueous media

PubMed Central

Imperio, Daniela; Penoni, Andrea; Palmisano, Giovanni

2011-01-01

Summary Zinc powder in aqueous alkaline media was employed to perform reductive amination of aldehydes with primary amines. The corresponding secondary amines were obtained in good yields along with minor amounts of hydrodimerization byproducts. The protocol is a green alternative to the use of complex hydrides in chlorinated or highly flammable solvents. PMID:21915212

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

PubMed Central

Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

2013-01-01

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

NASA Astrophysics Data System (ADS)

Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

2011-05-01

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

Tradeoffs between water requirements and yield stability in annual vs. perennial crops

NASA Astrophysics Data System (ADS)

Vico, Giulia; Brunsell, Nathaniel A.

2018-02-01

Population growth and changes in climate and diets will likely further increase the pressure on agriculture and water resources globally. Currently, staple crops are obtained from annuals plants. A shift towards perennial crops may enhance many ecosystem services, but at the cost of higher water requirements and lower yields. It is still unclear when the advantages of perennial crops overcome their disadvantages and perennial crops are thus a sustainable solution. Here we combine a probabilistic description of the soil water balance and crop development with an extensive dataset of traits of congeneric annuals and perennials to identify the conditions for which perennial crops are more viable than annual ones with reference to yield, yield stability, and effective use of water. We show that the larger and more developed roots of perennial crops allow a better exploitation of soil water resources and a reduction of yield variability with respect to annual species, but their yields remain lower when considering grain crops. Furthermore, perennial crops have higher and more variable irrigation requirements and lower water productivity. These results are important to understand the potential consequences for yield, its stability, and water resource use of a shift from annual to perennial crops and, more generally, if perennial crops may be more resilient than annual crops in the face of climatic fluctuations.

Linkages among climate change, crop yields and Mexico-US cross-border migration.

PubMed

Feng, Shuaizhang; Krueger, Alan B; Oppenheimer, Michael

2010-08-10

Climate change is expected to cause mass human migration, including immigration across international borders. This study quantitatively examines the linkages among variations in climate, agricultural yields, and people's migration responses by using an instrumental variables approach. Our method allows us to identify the relationship between crop yields and migration without explicitly controlling for all other confounding factors. Using state-level data from Mexico, we find a significant effect of climate-driven changes in crop yields on the rate of emigration to the United States. The estimated semielasticity of emigration with respect to crop yields is approximately -0.2, i.e., a 10% reduction in crop yields would lead an additional 2% of the population to emigrate. We then use the estimated semielasticity to explore the potential magnitude of future emigration. Depending on the warming scenarios used and adaptation levels assumed, with other factors held constant, by approximately the year 2080, climate change is estimated to induce 1.4 to 6.7 million adult Mexicans (or 2% to 10% of the current population aged 15-65 y) to emigrate as a result of declines in agricultural productivity alone. Although the results cannot be mechanically extrapolated to other areas and time periods, our findings are significant from a global perspective given that many regions, especially developing countries, are expected to experience significant declines in agricultural yields as a result of projected warming.

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

PubMed

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

PubMed Central

2013-01-01

Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

Immunochromatographic strip assay for the rapid and sensitive detection of Salmonella Typhimurium in artificially contaminated tomato samples.

PubMed

Shukla, Shruti; Leem, Hyerim; Lee, Jong-Suk; Kim, Myunghee

2014-06-01

This study was designed to confirm the applicability of a liposome-based immunochromatographic assay for the rapid detection of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) in artificially contaminated tomato samples. To determine the detection limit and pre-enrichment incubation time (10, 12, and 18 h pre-enrichment in 1% buffered peptone water), the tests were performed with different cell numbers of Salmonella Typhimurium (3 × 10(0), 3 × 10(1), 3 × 10(2), and 3 × 10(3) CFU·mL(-1)) inoculated into 25 g of crushed tomato samples. The assay was able to detect as few as 30 Salmonella Typhimurium cells per 25 g of tomato samples (1.2 cells·g(-1)) after 12 h pre-enrichment incubation. Moreover, when the developed assay was compared with traditional morphological and biochemical culture-based methods as well as colloidal gold nanoparticle-based commercial test strips, the developed assay yielded positive results for the detection of Salmonella Typhimurium within a shorter period time. These findings confirm that the developed assay may have practical application for the sensitive detection of Salmonella Typhimurium in various food samples, including raw vegetables, with a relatively low detection limit and shorter analysis time.

Inhibition of Methanogenesis in Marine Sediments by Acetylene and Ethylene: Validity of the Acetylene Reduction Assay for Anaerobic Microcosms

PubMed Central

Oremland, Ronald S.; Taylor, Barrie F.

1975-01-01

Methanogenesis was irreversibly inhibited in sediments by concentrations of acetylene employed in nitrogen fixation assays (1 to 20%, vol/vol). Ethylene, but not ethane, also stopped methane production, and the inhibition was reversed by gassing with hydrogen. PMID:1190767

Detecting crop yield reduction due to irrigation-induced soil salinization in South-West Russia

NASA Astrophysics Data System (ADS)

Argaman, E.; Beets, W.; Croes, J.; Keesstra, S.; Verzandvoort, S.; Zeiliguer, A.

2012-04-01

The South-European part of the Russian Federation has experienced serious land degradation in the form of soil salinization since the 1960s. This land degradation was caused by intensive, large-scale irrigation on reclaimed land in combination with the salt-rich nature of the substrate. Alkaline soil salinity is believed to be an important factor decreasing crop yield in this area. A large research effort has been directed to the effects of soil salinity on crops, there is a need for simple, easily determinable indicators of crop health and soil salinity in irrigated systems, that can help to detect crop water stress in an early stage. The objectives of this research were to study the effects of soil salinity and vegetation water stress on the performance of alfalfa crop yield and physiological crop properties, and to study the possibility to measure soil salinity and alkalinity and the crop water stress index at plot level using a thermal gun and a regular digital camera. The study area was located in Saratov District, in the South-West part of Russia. Variables on the surface energy balance, crop properties, soil properties and visible reflectance were measured on plots with alfalfa cultures in two fields with and without signs of alkaline soil salinity, and with and without irrigation in July 2009. The research showed no clear adverse effects of soil salinity and soil alkalinity on crop yield and physiological crop properties. Soil salinity, as reflected by the electric conductivity, positively affected the root biomass of alfalfa in the range of 0.15 to 1.52 dS/m . This was a result of EC levels being below the documented threshold to negatively affect Alfalfa, as would be the case in truly saline soils. The soil pH also showed a positive correlation with root biomass within the range of pH 6.2 and 8.5 . From the literature these pH values are generally believed to be too high to exhibit a positive relationship with root biomass. No relationship was found

Fast and inexpensive synthesis of pentacene with high yield using 6,13-pentacenequinone as precursor

NASA Astrophysics Data System (ADS)

Mota, María L.; Rodriguez, Bibiana; Carrillo, Amanda; Ambrosio, Roberto C.; Luque, Priscy A.; Mireles, Marcela; Vivaldo, Israel; Quevedo, Manuel A.

2018-02-01

Pentacene is an important semiconductor in the field of organic electronics. In this work is presented an alternative synthesis procedure to obtain pentacene from 6,13-pentacenequinone as a precursor. Synthesis of pentacene was performed in two reactions, Diels-Adler cycloaddition of 6,13-pentacenequinone followed by 6,13-pentacenequinone reduction to pentacene, employing LiAlH4 as reducing agent. The products were characterized by Fourier Transform Infrared Spectroscopy (FTIR), 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR), X-Ray Diffraction (XRD), Thermogravimetric Analysis (TGA) and Ultraviolet-Visible Spectroscopy (UV-VIS). In this work, 6,13-pentacenequinone was synthetized with a high yield (55%) using an alternative method. The optimization process resulted in an overall reduction of reaction time while exhibiting high yield. The method presented here provides an affordable pentacene synthesis route with high purity, which can be further applied for research and development of organic electronic applications.

Low Carbon Rice Farming Practices in the Mekong Delta Yield Significantly Higher Profits and Lower Greenhouse Gas Emissions

NASA Astrophysics Data System (ADS)

Rudek, J.; Van Sanh, N.; Tinh, T. K.; Tin, H. Q.; Thu Ha, T.; Pha, D. N.; Cui, T. Q.; Tin, N. H.; Son, N. N.; Thanh, H. H.; Kien, H. T.; Kritee, K.; Ahuja, R.

2014-12-01

The Vietnam Low-Carbon Rice Project (VLCRP) seeks to significantly reduce GHG emissions from rice cultivation, an activity responsible for more than 30% of Vietnam's overall GHG emissions, while improving livelihoods for the rice farmer community by decreasing costs and enhancing yield as well as providing supplemental farmer income through the sale of carbon credits. The Mekong Delta makes up 12% of Vietnam's land area, but produces more than 50% of the country's rice, including more than 90% of the rice for export. Rice cultivation is the main source of income for 80% of farmers in the Mekong Delta. VLCRP was launched in late 2012 in the Mekong Delta in two major rice production provinces, Kien Giang and An Giang. To date, VLCRP has completed 11 crop seasons (in Kien Giang and An Giang combined), training over 400 farmer households in applying VLCRP's package of practices (known as 1 Must - 6 Reductions) and building technical capacity to its key stakeholders and rice farmer community leaders. By adopting the 1 Must- 6 Reductions practices (including reduced seeding density, reduced fertilizer and pesticide application, and alternative wetting and drying water management), rice farmers reduce their input costs while maintaining or improving yields, and decreasing greenhouse gas emissions. The VLCRP package of practices also deliver other environmental and social co-benefits, such as reduced water pollution, improved habitat for fishery resources and reduced health risks for farmers through the reduction of agri-chemicals. VLCRP farmers use significantly less inputs (50% reduction in seed, 30% reduction in fertilizer, 40-50% reduction in water) while improving yields 5-10%, leading to an increase in profit from 10% to as high as 60% per hectare. Preliminary results indicate that the 1 Must- 6 Reductions practices have led to approximately 40-65% reductions in greenhouse gas emissions, equivalent to 4 tons of CO2e/ha/yr in An Giang and 35 tons of CO2e/ha/yr in Kien

Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

PubMed Central

2015-01-01

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs. PMID:25226846

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

PubMed

Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

2012-04-20

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

Sensitivity and Specificity of Human Immunodeficiency Virus Rapid Serologic Assays and Testing Algorithms in an Antenatal Clinic in Abidjan, Ivory Coast

PubMed Central

Koblavi-Dème, Stéphania; Maurice, Chantal; Yavo, Daniel; Sibailly, Toussaint S.; N′guessan, Kabran; Kamelan-Tano, Yvonne; Wiktor, Stefan Z.; Roels, Thierry H.; Chorba, Terence; Nkengasong, John N.

2001-01-01

To evaluate serologic testing algorithms for human immunodeficiency virus (HIV) based on a combination of rapid assays among persons with HIV-1 (non-B subtypes) infection, HIV-2 infection, and HIV-1–HIV-2 dual infections in Abidjan, Ivory Coast, a total of 1,216 sera with known HIV serologic status were used to evaluate the sensitivity and specificity of four rapid assays: Determine HIV-1/2, Capillus HIV-1/HIV-2, HIV-SPOT, and Genie II HIV-1/HIV-2. Two serum panels obtained from patients recently infected with HIV-1 subtypes B and non-B were also included. Based on sensitivity and specificity, three of the four rapid assays were evaluated prospectively in parallel (serum samples tested by two simultaneous rapid assays) and serial (serum samples tested by two consecutive rapid assays) testing algorithms. All assays were 100% sensitive, and specificities ranged from 99.4 to 100%. In the prospective evaluation, both the parallel and serial algorithms were 100% sensitive and specific. Our results suggest that rapid assays have high sensitivity and specificity and, when used in parallel or serial testing algorithms, yield results similar to those of enzyme-linked immunosorbent assay-based testing strategies. HIV serodiagnosis based on rapid assays may be a valuable alternative in implementing HIV prevention and surveillance programs in areas where sophisticated laboratories are difficult to establish. PMID:11325995

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Ni-Catalyzed Carbon-Carbon Bond-Forming Reductive Amination.

PubMed

Heinz, Christoph; Lutz, J Patrick; Simmons, Eric M; Miller, Michael M; Ewing, William R; Doyle, Abigail G

2018-02-14

This report describes a three-component, Ni-catalyzed reductive coupling that enables the convergent synthesis of tertiary benzhydryl amines, which are challenging to access by traditional reductive amination methodologies. The reaction makes use of iminium ions generated in situ from the condensation of secondary N-trimethylsilyl amines with benzaldehydes, and these species undergo reaction with several distinct classes of organic electrophiles. The synthetic value of this process is demonstrated by a single-step synthesis of antimigraine drug flunarizine (Sibelium) and high yielding derivatization of paroxetine (Paxil) and metoprolol (Lopressor). Mechanistic investigations support a sequential oxidative addition mechanism rather than a pathway proceeding via α-amino radical formation. Accordingly, application of catalytic conditions to an intramolecular reductive coupling is demonstrated for the synthesis of endo- and exocyclic benzhydryl amines.

Tar yields from low-temperature carbonization of coal facies from the Powder River Basin, Wyoming, USA

USGS Publications Warehouse

Stanton, Ronald W.; Warwick, Peter D.; Swanson, Sharon M.

2005-01-01

Tar yields from low-temperature carbonization correlate with the amount of crypto-eugelinite in samples selected to represent petrographically distinct coal facies of the Wyodak-Anderson coal zone. Tar yields from Fischer Assay range from <1 to 11 wt.% on a dry basis and correspond (r = 0.72) to crypto-eugelinite contents of the coal that range from 15 to 60 vol.%. Core and highwall samples were obtained from active surface mines in the Gillette field, Powder River Basin, Wyoming. Because the rank of the samples is essentially the same, differences in low-temperature carbonization yields are interpreted from compositional differences, particularly the crypto-eugelinite content. In the Wyodak-Anderson coal zone, crypto-eugelinite probably was derived from degraded humic matter which absorbed decomposition products from algae, fungi, bacteria, and liptinitic plant parts (materials possibly high in hydrogen). Previous modeling of the distribution of crypto-eugelinite in the discontinuous Wyodak-Anderson coal zone indicated that tar yields should be greater from coal composing the upper part and interior areas than from coal composing the lower parts and margins of the individual coal bodies. It is possible that hydrocarbon yields from natural coalification processes would be similar to yields obtained from laboratory pyrolysis. If so, the amount of crypto-eugelinite may also be an important characteristic when evaluating coal as source rock for migrated hydrocarbons.

Confined ion energy >200 keV and increased fusion yield in a DPF with monolithic tungsten electrodes and pre-ionization

NASA Astrophysics Data System (ADS)

Lerner, Eric J.; Hassan, Syed M.; Karamitsos, Ivana; Von Roessel, Fred

2017-10-01

To reduce impurities in the dense plasma focus FF-1 device, we used monolithic tungsten electrodes with pre-ionization. With this new set-up, we demonstrated a three-fold reduction of impurities by mass and a ten-fold reduction by ion number. FF-1 produced a 50% increase in fusion yield over our previous copper electrodes, both for a single shot and for a mean of ten consecutive shots with the same conditions. These results represent a doubling of fusion yield as compared with any other plasma focus device with the same 60 kJ energy input. In addition, FF-1 produced a new single-shot record of 240 ± 20 keV for mean ion energy, a record for any confined fusion plasma, using any device, and a 50% improvement in ten-shot mean ion energy. With a deuterium-nitrogen mix and corona-discharge pre-ionization, we were also able to reduce the standard deviation in the fusion yield to about 15%, a four-fold reduction over the copper-electrode results. We intend to further reduce impurities with new experiments using microwave treatment of tungsten electrodes, followed by the use of beryllium electrodes.

Increasing selectivity for TNT-based explosive detection by synchronous luminescence and derivative spectroscopy with quantum yields of selected aromatic amines.

PubMed

Sheaff, Chrystal N; Eastwood, Delyle; Wai, Chien M

2007-01-01

The detection of explosive material is at the forefront of current analytical problems. A detection method is desired that is not restricted to detecting only explosive materials, but is also capable of identifying the origin and type of explosive. It is essential that a detection method have the selectivity to distinguish among compounds in a mixture of explosives. The nitro compounds found in explosives have low fluorescent yields or are considered to be non-fluorescent; however, after reduction, the amino compounds exhibit relatively high fluorescence. We discuss how to increase selectivity of explosive detection using fluorescence; this includes synchronous luminescence and derivative spectroscopy with appropriate smoothing. By implementing synchronous luminescence and derivative spectroscopy, we were able to resolve the reduction products of one major TNT-based explosive compound, 2,4-diaminotoluene, and the reduction products of other minor TNT-based explosives in a mixture. We also report for the first time the quantum yields of these important compounds. Relative quantum yields are useful in establishing relative fluorescence intensities and are an important spectroscopic measurement of molecules. Our approach allows for rapid, sensitive, and selective detection with the discrimination necessary to distinguish among various explosives.

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality.

PubMed

Lee, Scott J; Warnick, Thomas A; Pattathil, Sivakumar; Alvelo-Maurosa, Jesús G; Serapiglia, Michelle J; McCormick, Heather; Brown, Virginia; Young, Naomi F; Schnell, Danny J; Smart, Lawrence B; Hahn, Michael G; Pedersen, Jeffrey F; Leschine, Susan B; Hazen, Samuel P

2012-02-08

There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality

PubMed Central

2012-01-01

Background There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. Results We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). Conclusion Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality. PMID:22316115

Effect of treated tannery effluent with domestic wastewater and amendments on growth and yield of cotton.

PubMed

Jagathjothi, N; Amanullah, M Mohamed; Muthukrishnan, P

2013-11-15

Pot culture and field experiments were carried out at the Common Effluent Treatment Plant (CETP), Dindigul during kharif 2011-12 to investigate the influence of irrigation of treated tannery effluent along with domestic wastewater on growth, yield attributes and yield of cotton. The pot culture was in a factorial completely randomized design and field experiment laid out in factorial randomized block design with four replications. The results revealed that the mixing proportion of 25% Treated Tannery Effluent (TTE)+75% domestic wastewater (DWW) application recorded taller plants, higher dry matter production, number of sympodial branches plant(-1), number of fruiting points plant(-1), number of bolls plant(-1) and seed cotton yield with yield reduction of 15.28 and 16.11% compared to normal water irrigation under pot culture and field experiment, respectively. Regarding amendments, gypsum application registered higher seed cotton yield followed by VAM.

Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

PubMed

Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

2015-01-01

Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies.

Evidence for a weakening strength of temperature-corn yield relation in the United States during 1980-2010.

PubMed

Leng, Guoyong

2017-12-15

Temperature is known to be correlated with crop yields, causing reduction of crop yield with climate warming without adaptations or CO 2 fertilization effects. The historical temperature-crop yield relation has often been used for informing future changes. This relationship, however, may change over time following alternations in other environmental factors. Results show that the strength of the relationship between the interannual variability of growing season temperature and corn yield (R GST_CY ) has declined in the United States between 1980 and 2010 with a loss in the statistical significance. The regression slope which represents the anomalies in corn yield that occur in association with 1 degree temperature anomaly has decreased significantly from -6.9%/K of the first half period to -2.4%/K--3.5%/K of the second half period. This implies that projected corn yield reduction will be overestimated by a fact of 2 in a given warming scenario, if the corn-temperature relation is derived from the earlier historical period. Changes in R GST_CY are mainly observed in Midwest Corn Belt and central High Plains, but are partly reproduced by 11 process-based crop models. In Midwest rain-fed systems, the decrease of negative temperature effects coincides with an increase in water availability by precipitation. In irrigated areas where water stress is minimized, the decline of beneficial temperature effects is significantly related to the increase in extreme hot days. The results indicate that an extrapolation of historical yield response to temperature may bias the assessment of agriculture vulnerability to climate change. Efforts to reduce climate impacts on agriculture should pay attention not only to climate change, but also to changes in climate-crop yield relations. There are some caveats that should be acknowledged as the analysis is restricted to the changes in the linear relation between growing season mean temperature and corn yield for the specific study period

Optimal design of compact spur gear reductions

NASA Technical Reports Server (NTRS)

Savage, M.; Lattime, S. B.; Kimmel, J. A.; Coe, H. H.

1992-01-01

The optimal design of compact spur gear reductions includes the selection of bearing and shaft proportions in addition to gear mesh parameters. Designs for single mesh spur gear reductions are based on optimization of system life, system volume, and system weight including gears, support shafts, and the four bearings. The overall optimization allows component properties to interact, yielding the best composite design. A modified feasible directions search algorithm directs the optimization through a continuous design space. Interpolated polynomials expand the discrete bearing properties and proportions into continuous variables for optimization. After finding the continuous optimum, the designer can analyze near optimal designs for comparison and selection. Design examples show the influence of the bearings on the optimal configurations.

Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions

PubMed Central

Marques, Sara S.; Magalhães, Luís M.; Tóth, Ildikó V.; Segundo, Marcela A.

2014-01-01

Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples. PMID:24968275

Evaluation of a novel delayed-type hypersensitivity assay to Candida albicans in adult and neonatal rats.

PubMed

Thorn, Mitchell; Hudson, Adam W; Kreeger, John; Kawabe, Thomas T; Bowman, Christopher J; Collinge, Mark

2015-01-01

Delayed-type hypersensitivity (DTH) is a T-cell-mediated immune response that may be used for immunotoxicity testing in non-clinical species. However, in some cases DTH assays using T-dependent antigens may be confounded by the production of antibodies to the antigen. The authors have previously modified a DTH assay, initially validated in the mouse, for use in juvenile rats to assess the effect of immunosuppressive drugs on the developing rat immune system. The assay measures footpad swelling induced by subcutaneous footpad injection of Candida albicans (C. albicans) derived-chitosan in rats previously sensitized with C. albicans. Antibodies to chitosan are not produced in this model. However, considerable inter-animal variability inherent in the footpad swelling assay can make it difficult to precisely quantify the magnitude of the immune response and inhibition by immunosuppressants, particularly if complete suppression is not observed. This report describes the development of an ex vivo assay to assess DTH in rats using interferon (IFN)-γ production by splenocytes, obtained from rats sensitized with C. albicans, as the quantifiable measure of the DTH response. Adult and neonatal rats administered dexamethasone (DEX), a known immunosuppressant, exhibited immunosuppression as evidenced by a reduction in ex vivo IFNγ production from splenocytes challenged with C. albicans-derived chitosan. Current data indicate that the ex vivo based DTH assay is more sensitive than the conventional footpad swelling assay due to a lower background response and the ability to detect a response as early as post-natal day (PND) 12. The ex vivo based rat DTH assay offers a highly sensitive and quantitative alternative to the footpad swelling assay for the assessment of the immunotoxic potential of drugs. The increased sensitivity of the ex vivo DTH assay may be useful for identifying smaller changes in response to immunotoxic drugs, as well as detecting responses earlier in animal

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis

PubMed Central

Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-01-01

ABSTRACT Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. PMID:28202794

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

PubMed

Robert-Gangneux, Florence; Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-05-01

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations ( P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. Copyright © 2017 American Society for Microbiology.

Enhancing photoluminescence yields in lead halide perovskites by photon recycling and light out-coupling.

PubMed

Richter, Johannes M; Abdi-Jalebi, Mojtaba; Sadhanala, Aditya; Tabachnyk, Maxim; Rivett, Jasmine P H; Pazos-Outón, Luis M; Gödel, Karl C; Price, Michael; Deschler, Felix; Friend, Richard H

2016-12-23

In lead halide perovskite solar cells, there is at least one recycling event of electron-hole pair to photon to electron-hole pair at open circuit under solar illumination. This can lead to a significant reduction in the external photoluminescence yield from the internal yield. Here we show that, for an internal yield of 70%, we measure external yields as low as 15% in planar films, where light out-coupling is inefficient, but observe values as high as 57% in films on textured substrates that enhance out-coupling. We analyse in detail how externally measured rate constants and photoluminescence efficiencies relate to internal recombination processes under photon recycling. For this, we study the photo-excited carrier dynamics and use a rate equation to relate radiative and non-radiative recombination events to measured photoluminescence efficiencies. We conclude that the use of textured active layers has the ability to improve power conversion efficiencies for both LEDs and solar cells.

Enhancing photoluminescence yields in lead halide perovskites by photon recycling and light out-coupling

PubMed Central

Richter, Johannes M.; Abdi-Jalebi, Mojtaba; Sadhanala, Aditya; Tabachnyk, Maxim; Rivett, Jasmine P.H.; Pazos-Outón, Luis M.; Gödel, Karl C.; Price, Michael; Deschler, Felix; Friend, Richard H.

2016-01-01

In lead halide perovskite solar cells, there is at least one recycling event of electron–hole pair to photon to electron–hole pair at open circuit under solar illumination. This can lead to a significant reduction in the external photoluminescence yield from the internal yield. Here we show that, for an internal yield of 70%, we measure external yields as low as 15% in planar films, where light out-coupling is inefficient, but observe values as high as 57% in films on textured substrates that enhance out-coupling. We analyse in detail how externally measured rate constants and photoluminescence efficiencies relate to internal recombination processes under photon recycling. For this, we study the photo-excited carrier dynamics and use a rate equation to relate radiative and non-radiative recombination events to measured photoluminescence efficiencies. We conclude that the use of textured active layers has the ability to improve power conversion efficiencies for both LEDs and solar cells. PMID:28008917

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

PubMed

Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

2014-11-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

Agreement between commercial assays for haptoglobin and serum amyloid A in goats.

PubMed

Czopowicz, Michał; Szaluś-Jordanow, Olga; Mickiewicz, Marcin; Moroz, Agata; Witkowski, Lucjan; Markowska-Daniel, Iwona; Reczyńska, Daria; Bagnicka, Emilia; Kaba, Jarosław

2017-10-02

Haptoglobin (Hp) and serum amyloid A (SAA) are considered as the major acute phase proteins (APPs) in goats. These APPs have been investigated in several studies during the last decade. In most studies, a colorimetric assay for Hp and a solid phase sandwich ELISA for SAA have been used for quantification. In 2015, reference intervals for APPs were determined using a new type of assay, the competitive ELISA (cELISA). Results obtained by the cELISA differed significantly from results obtained by previously used assays. The present study aimed to assess the agreement between so far used assays and cELISAs. Sera of 152 female dairy goats of two Polish national breeds were analysed. The concentration of Hp was determined using a colorimetric assay (Hp-CA) and the cELISA (Hp-cELISA), while a solid phase sandwich ELISA (SAA-sELISA) and the cELISA (SAA-cELISA) were used to measure SAA. Agreement between test results was assessed by preparing Bland-Altman plots, and analyzing 95% limits of agreement (LoA). Finally, the assays for Hp and SAA were compared using 147 and 138 serum samples, respectively, as 5 and 14 paired measurements, respectively, were excluded from agreement analyses to avoid extrapolation of Hp and SAA concentration. Measurements obtained by the Hp-CA and Hp-cELISA showed weak positive correlation (r = 0.24, P = 0.003). Limits of agreement (LoA) ranged from + 1.6 (95% CI 1.4 to 1.8) g/L to - 1.5 (95% CI - 1.7 to - 1.3) g/L. Measurements yielded by the SAA-sELISA and SAA-cELISA did not correlate (r = - 0.01, P = 0.855). LoA ranged from + 14.5 mg/L (95% CI 12.9 to 16.1) to - 8.5 mg/L (95% CI - 10.1 to - 6.9). Agreement between the two types of commercial assays for determination of Hp and SAA concentrations in goats is poor and cELISAs tend to underrate both Hp and SAA concentrations.

A comparative study of tissue factor and kaolin on blood coagulation assays using rotational thromboelastometry and thromboelastography.

PubMed

Peng, Henry T; Grodecki, Richard; Rizoli, Sandro; Shek, Pang N

2016-01-01

Rotational thromboelastometry (ROTEM) and thromboelastography (TEG) have been increasingly used to diagnose acute coagulopathy and guide blood transfusion. The tests are routinely performed using different triggering activators such as tissue factor and kaolin, which activate different pathways yielding different results. To optimize the global blood coagulation assays using ROTEM and TEG, we conducted a comparative study on the activation methods employing tissue factor and kaolin at different concentrations as well as standard reagents as recommended by the manufacturer of each device. Key parameter values were obtained at various assay conditions to evaluate and compare coagulation and fibrinolysis profiles of citrated whole blood collected from healthy volunteers. It was found that tissue factor reduced ROTEM clotting time and TEG R, and increased ROTEM clot formation time and TEG K in a concentration-dependent manner. In addition, tissue factor affected ROTEM alpha angle, and maximum clot firmness, especially in the absence of kaolin activation, whereas both ROTEM and TEG clot lysis (LI30, CL30, and LY30) remained unaffected. Moreover, kaolin reduced ROTEM clotting time and TEG R and K, but to a lesser extent than tissue factor, in-tem and ex-tem. Correlations in all corresponding parameters between ROTEM and TEG were observed, when the same activators were used in the assays compared with lesser correlations between standard kaolin TEG and ROTEM (INTEM/EXTEM). The two types of viscoelastic point-of-care devices provide different results, depending on the triggering reagent used to perform the assay. Optimal assay condition was obtained to reduce assay time and improve assay accuracy.

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption.

PubMed

Yu, Mi-Ji; Yoon, Sun-Hee; Kim, Young-Wan

2016-11-01

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other's activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

Chiral symmetry and N*(1440){yields}N{pi}{pi} decay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamano, H.; Morishita, M.; Arima, M.

2005-04-01

The N*(1440){yields}N{pi}{pi} decay is studied by making use of the chiral reduction formula. This formula suggests a scalar-isoscalar pion-baryon contact interaction that is absent in the recent study of Hernandez et al. [Phys. Rev. C 66, 065201 (2002)]. The contact interaction is introduced into their model and is found to be necessary for the simultaneous description of g{sub RN{pi}}{sub {pi}} and the {pi}{pi} and {pi}N invariant mass distributions.

Optimising yield and resource utilisation of summer maize under the conditions of increasing density and reducing nitrogen fertilization.

PubMed

Wei, Shanshan; Wang, Xiangyu; Zhu, Qicen; Jiang, Dong; Dong, Shuting

2017-10-05

The inefficient use of resources always poses risks of maize (Zea mays L.) yield reduction in China. We performed this research to monitor the effects of increasing plant density and reducing nitrogen (N) rate on radiation-use efficiency (RUE), N efficiency traits, grain yield (GY) and their inter-relationships. Besides, whether GY and resource-use efficiency can both be maximized was examined. Hence, a 2-year field experiment was conducted using a widely grown variety "Denghai 618" in Shandong, China. Treatments contained two different plant densities [67,500 (D 1 ) and 97,500 (D 2 ) plant ha -1 ] and three N levels [0 (N -2 ), 180 (N -1 ), 360 (N ck ) kg ha -1 ], set D 1 N ck as control. Significant increases in grain yield, biomass, RUE, above-ground N uptake (AGN) and N efficiency were observed when density increased from D 1 to D 2 . Declining N application was accompanied by reductions in yield, RUE and AGN especially under high density, yet an obvious improvement in N recovery efficiency (NRE), agronomic N efficiency and N partial factor productivity. The increased GY was positive related with population biomass (r = 0.895**), RUE (r = 0.769**) and AGN (r = 0.923**), whereas it has no significant correlation with N efficiency. In this study, D 2 N ck obtained 18.8, 17.9, 24.8 and 29.7% higher grain yield, RUE, AGN and NRE respectively, compared to control, optimizing both yield and the efficiencies of radiation and N use. Furthermore, higher yield and RUE with more desirable N efficiency may be possible via optimizing density and N rate combination.

Optimising yield and resource utilisation of summer maize under the conditions of increasing density and reducing nitrogen fertilization

NASA Astrophysics Data System (ADS)

Wei, Shanshan; Wang, Xiangyu; Zhu, Qicen; Jiang, Dong; Dong, Shuting

2017-12-01

The inefficient use of resources always poses risks of maize ( Zea mays L.) yield reduction in China. We performed this research to monitor the effects of increasing plant density and reducing nitrogen (N) rate on radiation-use efficiency (RUE), N efficiency traits, grain yield (GY) and their inter-relationships. Besides, whether GY and resource-use efficiency can both be maximized was examined. Hence, a 2-year field experiment was conducted using a widely grown variety "Denghai 618" in Shandong, China. Treatments contained two different plant densities [67,500 (D1) and 97,500 (D2) plant ha-1] and three N levels [0 (N-2), 180 (N-1), 360 (Nck) kg ha-1], set D1Nck as control. Significant increases in grain yield, biomass, RUE, above-ground N uptake (AGN) and N efficiency were observed when density increased from D1 to D2. Declining N application was accompanied by reductions in yield, RUE and AGN especially under high density, yet an obvious improvement in N recovery efficiency (NRE), agronomic N efficiency and N partial factor productivity. The increased GY was positive related with population biomass ( r = 0.895**), RUE ( r = 0.769**) and AGN ( r = 0.923**), whereas it has no significant correlation with N efficiency. In this study, D2Nck obtained 18.8, 17.9, 24.8 and 29.7% higher grain yield, RUE, AGN and NRE respectively, compared to control, optimizing both yield and the efficiencies of radiation and N use. Furthermore, higher yield and RUE with more desirable N efficiency may be possible via optimizing density and N rate combination.

Initial experience with GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.

PubMed

Patil, Naveen; Saba, Hamida; Marco, Asween; Samant, Rohan; Mukasa, Leonard

2014-01-01

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay. To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance. Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas.Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas.Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation. Between June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Löwenstein-Jensen (LJ) slants. Surveillance data for January 2009-June 2013 was analysed to characterise sputum smear-negative but culture-positive cases. Seventy-one TB cases were reported from June 2012- June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports. GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one

Cell oxidation-reduction imbalance after modulated radiofrequency radiation.

PubMed

Marjanovic, Ana Marija; Pavicic, Ivan; Trosic, Ivancica

2015-01-01

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800 MHz, strength of 30 V/m on oxidation-reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60 min, specific absorption rate was calculated to be 1.6 W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2',7'-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p < 0.05) increased after 10 min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation-reduction equilibrium within the growing cells.

Evaluation of the Luminex xTAG Respiratory Viral Panel FAST v2 assay for detection of multiple respiratory viral pathogens in nasal and throat swabs in Vietnam

PubMed Central

Thi Ty Hang, Vu; Thi Han Ny, Nguyen; My Phuc, Tran; Thi Thanh Tam, Pham; Thao Huong, Dang; Dang Trung Nghia, Ho; Tran Anh Vu, Nguyen; Thi Hong Phuong, Pham; Van Xang, Nguyen; Dong, Nguyen; Nhu Hiep, Pham; Van Hung, Nguyen; Tinh Hien, Tran; Rabaa, Maia; Thwaites, Guy E.; Baker, Stephen; Van Tan, Le; van Doorn, H.Rogier

2018-01-01

Background: Acute respiratory infections (ARI) are among the leading causes of hospitalization in children ≤5 years old. Rapid diagnostics of viral pathogens is essential to avoid unnecessary antibiotic treatment, thereby slowing down antibiotic-resistance. We evaluated the diagnostic performance of the Luminex xTAG Respiratory Viral Panel FAST v2 against viral specific PCR as reference assays for ARI in Vietnam. Methods: Four hundred and forty two nose and throat swabs were collected in viral transport medium, and were tested with Luminex xTAG Respiratory Viral Panel FAST v2. Multiplex RT-PCR and single RT-PCR were used as references.    Results: Overall, sensitivity of the Luminex against reference assays was 91.8%, 95% CI 88.1-94.7 (270/294), whilst 112/6336 (1.8%, 95% CI, 1.4-2.1) of pathogens were detected by the Luminex, but not by reference assays. Frequency of pathogens detected by Luminex and reference assays was 379 and 292, respectively. The diagnostic yield was 66.7% (295/442, 95%CI 62.1-71.1%) for the Luminex assay and 54.1% (239/442, 95% CI, 49.3-58.8%) for reference assays. The Luminex kit had higher yields for all viruses except influenza B virus, respiratory syncytial virus, and human bocavirus. High agreements between both methods [mean (range): 0.91 (0.83-1.00)] were found for 10/15 viral agents. Conclusions: The Luminex assay is a high throughput multiplex platform for rapid detection of common viral pathogens causing ARI. Although the current high cost may prevent Luminex assays from being widely used, especially in limited resource settings where ARI are felt most, its introduction in clinical diagnostics may help reduce unnecessary use of antibiotic prescription. PMID:29503874

Mercury removal from contaminated water by ultrasound-promoted reduction/vaporization in a microscale reactor.

PubMed

Gil, Sandra; Lavilla, Isela; Bendicho, Carlos

2008-03-01

A new method is described for the removal of Hg(II) at trace level from waters using an ultrasound-promoted reduction/volatilization process. The method is accomplished in a sonoreactor (100 W power; 20 kHz frequency) by adding formic acid to induce the reduction of Hg(II) to Hg(0). In contrast to other treatments, it does not introduce further foreign substances for water decontamination. A reduction mechanism is proposed, which relies on the sonolytic decomposition of formic acid to yield reducing gases such as H(2) and CO, which in turn, causes the reduction of Hg(II). After the formation of Hg(0), its removal is facilitated by the degassing effect caused by ultrasound irradiation. Hg at 100 ng/mL concentration can be removed within 30 min with a yield of 90% from a 10 mL water volume. The presence of stabilizing anions or oxidants in waters may preclude the Hg removal. Effects of experimental variables such as treatment time, amplitude of the ultrasonic probe vibration, formic acid concentration and sample volume were investigated.

Water yield issues in the jarrah forest of south-western Australia

NASA Astrophysics Data System (ADS)

Ruprecht, J. K.; Stoneman, G. L.

1993-10-01

The jarrah forest of south-western Australia produces little streamflow from moderate rainfall. Water yield from water supply catchments for Perth, Western Australia, are low, averaging 71 mm (7% of annual rainfall). The low water yields are attributed to the large soil water storage available for continuous use by the forest vegetation. A number of water yield studies in south-western Australia have examined the impact on water yield of land use practices including clearing for agricultural development, forest harvesting and regeneration, forest thinning and bauxite mining. A permanent reduction in forest cover by clearing for agriculture led to permanent increases of water yield of approximately 28% of annual rainfall in a high rainfall catchment. Thinning of a high rainfall catchment led to an increase in water yield of 20% of annual rainfall. However, it is not clear for how long the increased water yield will persist. Forest harvesting and regeneration have led to water yield increases of 16% of annual rainfall. The subsequent recovery of vegetation cover has led to water yields returning to pre-disturbance levels after an estimated 12-15 years. Bauxite mining of a high rainfall catchment led to a water yield increase of 8% of annual rainfall, followed by a return to pre-disturbance water yield after 12 years. The magnitude of specific streamflow generation mechanisms in small catchments subject to forest disturbance vary considerably, typically in a number of distinct stages. The presence of a permanent groundwater discharge area was shown to be instrumental in determining the magnitude of the streamflow response after forest disturbance. The long-term prognosis for water yield from areas subject to forest thinning, harvesting and regeneration, and bauxite mining are uncertain, owing to the complex interrelationship between vegetation cover, tree height and age, and catchment evapotranspiration. Management of the forest for water yield needs to acknowledge