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New surveillance concepts in food safety in meat producing animals: the advantage of high throughput 'omics' technologies - A review

  • Pfaffl, Michael W.;Riedmaier-Sprenzel, Irmgard
    • Asian-Australasian Journal of Animal Sciences
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    • v.31 no.7
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    • pp.1062-1071
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    • 2018
  • The misuse of anabolic hormones or illegal drugs is a ubiquitous problem in animal husbandry and in food safety. The ban on growth promotants in food producing animals in the European Union is well controlled. However, application regimens that are difficult to detect persist, including newly designed anabolic drugs and complex hormone cocktails. Therefore identification of molecular endogenous biomarkers which are based on the physiological response after the illicit treatment has become a focus of detection methods. The analysis of the 'transcriptome' has been shown to have promise to discover the misuse of anabolic drugs, by indirect detection of their pharmacological action in organs or selected tissues. Various studies have measured gene expression changes after illegal drug or hormone application. So-called transcriptomic biomarkers were quantified at the mRNA and/or microRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology or by more modern 'omics' and high throughput technologies including RNA-sequencing (RNA-Seq). With the addition of advanced bioinformatical approaches such as hierarchical clustering analysis or dynamic principal components analysis, a valid 'biomarker signature' can be established to discriminate between treated and untreated individuals. It has been shown in numerous animal and cell culture studies, that identification of treated animals is possible via our transcriptional biomarker approach. The high throughput sequencing approach is also capable of discovering new biomarker candidates and, in combination with quantitative RT-qPCR, validation and confirmation of biomarkers has been possible. These results from animal production and food safety studies demonstrate that analysis of the transcriptome has high potential as a new screening method using transcriptional 'biomarker signatures' based on the physiological response triggered by illegal substances.

NMR Signal Assignments of Human Adenylate Kinase 1 (hAK1) and its R138A Mutant (hAK1R138A)

  • Kim, Gilhoon;Chang, Hwanbong;Won, Hoshik
    • Journal of the Korean Magnetic Resonance Society
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    • v.20 no.2
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    • pp.56-60
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    • 2016
  • Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of $^1H-^{15}N$ HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

The effect of nanoemulsified methionine and cysteine on the in vitro expression of casein in bovine mammary epithelial cells

  • Kim, Tae-Il;Kim, Tae-Gyun;Lim, Dong-Hyun;Kim, Sang-Bum;Park, Seong-Min;Lim, Hyun-Joo;Kim, Hyun-Jong;Ki, Kwang-Seok;Kwon, Eung-Gi;Kim, Young-Jun;Mayakrishnan, Vijayakumar
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.2
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    • pp.257-264
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    • 2019
  • Objective: Dairy cattle nutrient requirement systems acknowledge amino acid (AAs) requirements in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. Regulation of mammary protein synthesis may be associated with AA input and milk protein output. The aim of this study was to evaluate the effect of nanoemulsified methionine and cysteine on the in-vitro expression of milk protein (casein) in bovine mammary epithelial cells (MAC-T cells). Methods: Methionine and cysteine were nonionized using Lipoid S 75 by high-speed homogenizer. The nanoemulsified AA particle size and polydispersity index were determined by dynamic light scattering correlation spectroscopy using a high-performance particle sizer instrument. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity effect of AAs with and without nanoionization at various concentrations (100 to $500{\mu}g/mL$) in mammary epithelial cells. MAC-T cells were subjected to 100% of free AA and nanoemulsified AA concentration in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) for the analysis of milk protein (casein) expression by the quantitative reverse transcription polymerase chain reaction method. Results: The AA-treated cells showed that cell viability tended to decrease (80%) in proportion to the concentration before nanogenesis, but cell viability increased as much as 90% after nanogenesis. The analysis of the expression of genetic markers related to milk protein indicated that; ${\alpha}_{s2}$-casein increased 2-fold, ${\kappa}$-casein increased 5-fold, and the amount of unchanged ${\beta}$-casein expression was nearly doubled in the nanoemulsified methionine-treated group when compared with the free-nanoemulsified methionine-supplemented group. On the contrary, the non-emulsified cysteine-administered group showed higher expression of genetic markers related to milk protein ${\alpha}_{s2}$-casein, ${\kappa}$-casein, and ${\beta}$-casein, but all the genetic markers related to milk protein decreased significantly after nanoemulsification. Conclusion: Detailed knowledge of factors, such nanogenesis of methionine, associated with increasing cysteine and decreasing production of genetic markers related to milk protein (casein) will help guide future recommendations to producers for maximizing milk yield with a high level of milk protein casein.

Glass Dissolution Rates From MCC-1 and Flow-Through Tests

  • Jeong, Seung-Young
    • Proceedings of the Korean Radioactive Waste Society Conference
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    • 2004.06a
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    • pp.257-258
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    • 2004
  • The dose from radionuclides released from high-level radioactive waste (HLW) glasses as they corrode must be taken into account when assessing the performance of a disposal system. In the performance assessment (PA) calculations conducted for the proposed Yucca Mountain, Nevada, disposal system, the release of radionuclides is conservatively assumed to occur at the same rate the glass matrix dissolves. A simple model was developed to calculate the glass dissolution rate of HLW glasses in these PA calculations [1]. For the PA calculations that were conducted for Site Recommendation, it was necessary to identify ranges of parameter values that bounded the dissolution rates of the wide range of HLW glass compositions that will be disposed. The values and ranges of the model parameters for the pH and temperature dependencies were extracted from the results of SPFT, static leach tests, and Soxhlet tests available in the literature. Static leach tests were conducted with a range of glass compositions to measure values for the glass composition parameter. The glass dissolution rate depends on temperature, pH, and the compositions of the glass and solution, The dissolution rate is calculated using Eq. 1: $rate{\;}={\;}k_{o}10^{(ph){\eta})}{\cdot}e^{(-Ea/RT)}{\cdot}(1-Q/K){\;}+{\;}k_{long}$ where $k_{0},\;{\eta}$ and Eaare the parameters for glass composition, pH, $\eta$ and temperature dependence, respectively, and R is the gas constant. The term (1-Q/K) is the affinity term, where Q is the ion activity product of the solution and K is the pseudo-equilibrium constant for the glass. Values of the parameters $k_{0},\;{\eta}\;and\;E_{a}$ are the parameters for glass composition, pH, and temperature dependence, respectively, and R is the gas constant. The term (1-Q/C) is the affinity term, where Q is the ion activity product of the solution and K is the pseudo-equilibrium constant for the glass. Values of the parameters $k_0$, and Ea are determined under test conditions where the value of Q is maintained near zero, so that the value of the affinity term remains near 1. The dissolution rate under conditions in which the value of the affinity term is near 1 is referred to as the forward rate. This is the highest dissolution rate that can occur at a particular pH and temperature. The value of the parameter K is determined from experiments in which the value of the ion activity product approaches the value of K. This results in a decrease in the value of the affinity term and the dissolution rate. The highly dilute solutions required to measure the forward rate and extract values for $k_0$, $\eta$, and Ea can be maintained by conducting dynamic tests in which the test solution is removed from the reaction cell and replaced with fresh solution. In the single-pass flow-through (PFT) test method, this is done by continuously pumping the test solution through the reaction cell. Alternatively, static tests can be conducted with sufficient solution volume that the solution concentrations of dissolved glass components do not increase significantly during the test. Both the SPFT and static tests can ve conducted for a wide range of pH values and temperatures. Both static and SPFt tests have short-comings. the SPFT test requires analysis of several solutions (typically 6-10) at each of several flow rates to determine the glass dissolution rate at each pH and temperature. As will be shown, the rate measured in an SPFt test depends on the solution flow rate. The solutions in static tests will eventually become concentrated enough to affect the dissolution rate. In both the SPFt and static test methods. a compromise is required between the need to minimize the effects of dissolved components on the dissolution rate and the need to attain solution concentrations that are high enough to analyze. In the paper, we compare the results of static leach tests and SPFT tests conducted with simple 5-component glass to confirm the equivalence of SPFT tests and static tests conducted with pH buffer solutions. Tests were conducted over the range pH values that are most relevant for waste glass disssolution in a disposal system. The glass and temperature used in the tests were selected to allow direct comparison with SPFT tests conducted previously. The ability to measure parameter values with more than one test method and an understanding of how the rate measured in each test is affected by various test parameters provides added confidence to the measured values. The dissolution rate of a simple 5-component glass was measured at pH values of 6.2, 8.3, and 9.6 and $70^{\circ}C$ using static tests and single-pass flow-through (SPFT) tests. Similar rates were measured with the two methods. However, the measured rates are about 10X higher than the rates measured previously for a glass having the same composition using an SPFT test method. Differences are attributed to effects of the solution flow rate on the glass dissolution reate and how the specific surface area of crushed glass is estimated. This comparison indicates the need to standardize the SPFT test procedure.

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Bioanalytical method validation for determination of arsenic speciation in dog plasma using HPLC-ICP/MS (Dog 혈장 중 HPLC-ICP/MS를 이용한 비소 화학종 분석법 검증)

  • Kim, Jong-Hwan;Kwon, Young Sang;Shin, Min-Chul;Kim, Su Jong;Seo, Jong-Su
    • Analytical Science and Technology
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    • v.29 no.5
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    • pp.234-241
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    • 2016
  • The approach presented in this article refers to the bioanalytical method validation for the detection and quantitative determination of arsenic species including arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) in dog plasma by high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP/MS). The arsenic species were separated using an agilent As speciation column by a mobile phase of 2 mM sodium phosphate monobasic, 0.2 mM ethylenediaminetetraacetic acid disodium salt dehydrate, 10 mM sodium acetate, 3 mM sodium nitrate and 1 % ethyl alcohol at pH 11 (adjusted with 1M NaOH). The method validation experiment was obtained selectivity, linearity, accuracy, precision, matrix effect, recovery, system suitability, dilution integrity and various stabilities. All calibration curves showed good linearity (R2>0.999) within test ranges. The lower limit of quantitation (LLOQ) was 5 ng/mL for As(III), As(V) and DMA, and 20 ng/mL for MMA. The system suitability and dilution values were within 6.5 % and 7.7 %. Subsequently, the developed and validated HPLC-ICP/MS method was also successfully applied to determine the arsenic speciation in dog plasma samples, and the recoveries for the spiked samples were in the range of 91.5–102.2 %. Therefore, this method could be applied to the evaluation of arsenic exposure, health effect assessment and other bio-monitoring studies in biological samples.

New Analytical Method to Identify Chromium Species, Cr(III) and Cr(VI), and Characteristic Distribution of Chromium Species in the Han River (한강수계해서의 크롬(III,VI) 종(species) 분포 및 분석방법 정립)

  • Jeong, Gwan-Jo;Kim, Dok-Chan;Park, Hyeon
    • Journal of Korean Society of Environmental Engineers
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    • v.27 no.6
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    • pp.590-598
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    • 2005
  • An adequate method to identify chromium separation, Cr(III) and Cr(VI), in water samples were studied by using High Performance Liquid Chromatography(HPLC) coupled with Inductively Coupled Plasma Mass Spectometer(ICP-MS) equipped with Dynamic Reaction Cell(DRC). The characteristic distribution of Cr(III) and Cr(VI) in the raw water taken at the six water intake stations in Seoul, was analyzed by the method developed by the authors. The chromium species separated by HPLC was isocratically conducted by using tetrabutylammonium phosphate monobasic(1.0 mM TBAP), ethylenediaminetetraacetic acid(0.6 mM EDTA) and 2% v/v methanol as the mobile phase. 5% v/v methanol was used as flushing solvent. A reactive ammonia($NH_3$) gas was used to eliminate the potential interference of $ArC^+$. Several Parameters such as solvent ratio, pH, flow rate and sample injection volume were optimized for the successful separation and reproducibility. Although it has been reported thai the separation sensitivity of Cr(III) is superior to that of Cr(VI), the authors observed Cr(VI) was more sensitive than Cr(III) when ammonia($NH_3$) gas was used as the reaction gas. It took less than 3 minutes to analyze chromium species with this method and the estimated detection limits were $0.061\;{\mu}g/L$ for Cr(III) and $0.052\;{\mu}g/L$, for Cr(VI). According to the results from the analysis on chromium species in the raw water of the six intake stations, the concentrations of Cr(III) ranged from 0.048 to $0.064\;{\mu}g/L$(ave. $0.054\;{\mu}g/L$) while that of Cr(VI) ranged from 0.014 to $0.023\;{\mu}g/L$(ave. $0.019\;{\mu}g/L$). Recovery ratio was very high($90.1{\sim}94.1%$). There were two or three times more Cr(III) than Cr(VI) in the raw water.