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A Simple Carbamidomethylation-Based Isotope Labeling Method for Quantitative Shotgun Proteomics

  • Oh, Donggeun;Lee, Sun Young;Kwon, Meehyang;Kim, Sook-Kyung;Moon, Myeong Hee;Kang, Dukjin
    • Mass Spectrometry Letters
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    • v.5 no.3
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    • pp.63-69
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    • 2014
  • In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

DC23, a Triazolothione Resorcinol Analogue, Is Extensively Metabolized to Glucuronide Conjugates in Human Liver Microsomes

  • Shon, Jong Cheol;Joo, Jeongmin;Lee, Taeho;Kim, Nam Doo;Liu, Kwang-Hyeon
    • Mass Spectrometry Letters
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    • v.9 no.1
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    • pp.24-29
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    • 2018
  • DC23, a triazolothione resorcinol analogue, is known to inhibit heat shock protein 90 and pyruvate dehydrogenase kinase which are up-regulated in cancer and diabetes, respectively. This study was performed to elucidate the metabolism of DC23 in human liver microsomes (HLMs). HLMs incubated with DC23 in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA) and/or ${\beta}$-nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the formation of four metabolites, M1-M4. M1 was identified as DC23-N-Oxide, on the basis of LC-MS/MS analysis. DC23 was further metabolized to its glucuronide conjugates (M2, M3, and M4). In vitro metabolic stability studies conducted with DC23 in HLMs revealed significant glucuronide conjugation with a $t_{1/2}$ value of 1.3 min. The inhibitory potency of DC23 on five human cytochrome P450s was also investigated in HLMs. In these experiments, DC23 inhibited CYP2C9-mediated tolbutamide hydroxylase activity with an $IC_{50}$ value of $8.7{\mu}M$, which could have implications for drug interactions.

Active Compounds and Antimicrobial Effects from Castanea crenata Leaf (밤나무 잎의 항미생물 효과 및 활성물질)

  • Choi Ok-Beom
    • The Korean Journal of Food And Nutrition
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    • v.18 no.4
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    • pp.367-372
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    • 2005
  • Antimicrobial effects of the extracts from Castanea crenata leaf were investigated. The antimicrobial effects of methanol extract (8 mg, 20 mg) of 0.2 g and 0.5 g. eq. of Castanea crenata leaf was stronger than that of 0.65 mg of benzoic acid against Gram(+) bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Leuconostoc mesenteroides and Bacillus subtilig and Gram(-) bacteria such as Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. Growth inhibition of various microorganisms was observed in Castanea crenata leaf, therefor the Castanea crenata leaf were solvent fractionated. The ethyl acetate-soluble acidic and phenolic fraction were showed remarkable antimicrobial activity against microorganisms tested. The acidic fraction was purified with silica gel adsorption column chromatography, Sephadex LH-20 column chromatography and HPLC, subsequantly. The antimicrobial active substance isolated from the acid fraction of Castanea crenata leaf was characterized as stigmast-5-en-3-ol($\beta$-sitosterol) by MS and NMR analysis.

Design and Implementation of an Android Application for Real-time Motion Control (실시간 정밀 모션 제어를 위한 안드로이드 응용 설계 및 구현)

  • Kim, Dohyeon;Kang, Hyeongseok;Kang, Jeongnam;Lee, Eungyu;Kim, Kanghee
    • KIISE Transactions on Computing Practices
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    • v.21 no.4
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    • pp.315-319
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    • 2015
  • This paper addresses the design and implementation of an Android application for real-time precise motion control. To provide stable real-time performance, we implemented the application in two parts: Android service in the form of a daemon process, which periodically transfers a set of position commands for all motors through a real-time fieldbus, and Android UI application, which generates and delivers the set of position commands to the Android service. To support such a real-time motion control application, we use multi-core partitioning, which partitions the processor cores into a real-time partition to be used by the real-time motion control service and a non-real-time partition to be used by the Android application, and set up a shared buffer between them for communication. Our experiments show that we can obtain a motion control period of 2 ms with 99% task activation jitters less than ${\pm}55{\mu}s$ for a configuration where each of the four threads controls two motors in a group.

Qualitative Analysis for Metabolites of Pharmaceuticals Formed in Daphnia magna and Gammarus pulex Using Liquid Chromatogram-High Resolution Mass Spectrometry (LC-HRMS) (LC-HRMS를 이용한 Daphnia magna 및 Gammarus pulex 생체내 의약품 대사체 정성분석)

  • Jeon, Junho
    • Journal of Environmental Analysis, Health and Toxicology
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    • v.21 no.4
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    • pp.243-251
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    • 2018
  • Pharmaceuticals in wastewater effluents have been recognized as emerging pollutants threatening freshwater organisms. To extend understanding for bioaccumulation and toxicity in those organisms, information on biotransformation products (or metabolites) and their metabolic pathway are crucial. The aim of the present study is to identify and elucidate metabolites of pharmaceuticals formed in exposed organisms using suspect and nontarget screening approach using LC-HRMS. As the target pharmaceuticals, carbamazepine, ketoprofen, metoprolol, propranolol, and verapamil were selected whereas Daphnia magna and Gammarus pulex were used as test organisms. After 24h exposure, metabolites formed in the organisms were identified using LC-HRMS. The structures of metabolites were elucidated via analysis of MS/MS fragment pattern and the comparison with fragment database. As the results, a total of 10 metabolites were identified for 5 parent compounds (C253/C356 for carbamazepine, K211 for ketoprofen, M256 for metoprolol, P218/P276/P306 for propranolol, V196/V291/V441 for verapamil). Among them, the presence of C253 and V291 was confirmed using standard materials. Most of the identified metabolites were formed through oxidative reactions such as hydroxylation, N-demethylation, and dealkylation. Cysteine conjugation (phase II reaction) metabolite (C356) for carbamazepine was found in daphnia. The metabolic pathway of verapamil showed similar metabolic pathways and metabolic pathways for both species. Although the toxicological information on the identified metabolites could not be confirmed, the molecular structure information of the proposed metabolites can be used for future evaluation and prediction of toxicity.

Ionic liquid coated magnetic core/shell CoFe2O4@SiO2 nanoparticles for the separation/analysis of trace gold in water sample

  • Zeng, Yanxia;Zhu, Xiashi;Xie, Jiliang;Chen, Li
    • Advances in nano research
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    • v.10 no.3
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    • pp.295-312
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    • 2021
  • A new ionic liquid functionalized magnetic silica nanoparticle was synthesized and characterized and tested as an adsorbent. The adsorbent was used for magnetic solid phase extraction on ICP-MS method. Simultaneous determination of precious metal Au has been addressed. The method is simple and fast and has been applied to standard water and surface water analysis. A new method for separation/analysis of trace precious metal Au by Magnetron Solid Phase Extraction (MSPE) combined with ICP-MS. The element to be tested is rapidly adsorbed on CoFe2O4@SiO2@[BMIM]PF6 composite nano-adsorbent and eluted with thiourea. The method has a preconcentration factor of 9.5-fold. This method has been successfully applied to the determination of gold in actual water samples. Hydrophobic Ionic Liquids (ILs) 1-butyl-3-methylimidazole hexafluorophosphate ([BMIM]PF6) coated CoFe2O4@SiO2 nanoparticles with core-shell structure to prepare magnetic solid phase extraction agent (CoFe2O4@SiO2@ILs) and establish a new method of MSPE coupled with inductively coupled plasma mass spectrometry for separation/analysis of trace gold. The results showed that trace gold was adsorbed rapidly by CoFe2O4@SiO2@[BMIM]PF6 and eluanted by thiourea. Under the optimal conditions, preconcentration factor of the proposed method was 9.5-fold. The linear range, detection limit, correlation coefficient (R) and relative standard deviation (RSD) were found to be 0.01~1000.00 ng·mL-1, 0.001 ng·mL-1, 0.9990 and 3.4% (n = 11, c = 4.5 ng·mL-1). The CoFe2O4@SiO2 nanoparticles could be used repeatedly for 8 times. This proposed method has been successfully applied to the determination of trace gold in water samples.

Effect of pasture and intensive feeding systems on the carcass and meat quality of buffalo

  • Conto, Michela;Cifuni, Giulia Francesca;Iacurto, Miriam;Failla, Sebastiana
    • Animal Bioscience
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    • v.35 no.1
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    • pp.105-114
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    • 2022
  • Objective: This work was carried out to evaluate the effect of pasture (PA) feeding on buffalo meat quality compared with buffaloes reared intensively with the use of corn silage as a forage base or alternatively with polyphite meadow hay (PH). Methods: Thirty Mediterranean bull buffaloes were distributed into three experimental diet groups: maize silage (MS), PH, and PA. The animals were slaughtered at a live weight of 250 kg, and carcass and meat quality were evaluated. After 7 days of ageing, physical and chemical parameters of longissimus thoracis muscle were determined. To evaluate lipid oxidation the thiobarbituric acid reactive substances was tested at 7 and 14 days, and also the fatty acid profile was recorded by gas chromatography. Results: The PA group, even if it showed carcass parameters lower than those of the silage maize group, reported a good meat percentage (60.59% vs 58.46%, respectively) and lower fat percentage (p<0.001). PA-fed animals showed meat redness, and even if only on raw meat, shear force was higher than the others. Low values of conjugate linoleic acid, polyunsaturated fatty acids, and n-3 were reported in the silage maize group. Principal component analysis (PCA) clearly showed the influence of different diets on meat quality, and PCA1 and PCA2 explained 82% of the variability. Conclusion: Buffaloes reared on PA had meat with high nutritional value even if they showed poor carcass performance compared to the animals fed on MS. Buffaloes fed on polyphite hay were in an intermediate position, similar to grazing animals, according to the same nutritional determinations.

Elicitation of Innate Immunity by a Bacterial Volatile 2-Nonanone at Levels below Detection Limit in Tomato Rhizosphere

  • Riu, Myoungjoo;Kim, Man Su;Choi, Soo-Keun;Oh, Sang-Keun;Ryu, Choong-Min
    • Molecules and Cells
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    • v.45 no.7
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    • pp.502-511
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    • 2022
  • Bacterial volatile compounds (BVCs) exert beneficial effects on plant protection both directly and indirectly. Although BVCs have been detected in vitro, their detection in situ remains challenging. The purpose of this study was to investigate the possibility of BVCs detection under in situ condition and estimate the potentials of in situ BVC to plants at below detection limit. We developed a method for detecting BVCs released by the soil bacteria Bacillus velezensis strain GB03 and Streptomyces griseus strain S4-7 in situ using solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS). Additionally, we evaluated the BVC detection limit in the rhizosphere and induction of systemic immune response in tomato plants grown in the greenhouse. Two signature BVCs, 2-nonanone and caryolan-1-ol, of GB03 and S4-7 respectively were successfully detected using the soil-vial system. However, these BVCs could not be detected in the rhizosphere pretreated with strains GB03 and S4-7. The detection limit of 2-nonanone in the tomato rhizosphere was 1 µM. Unexpectedly, drench application of 2-nonanone at 10 nM concentration, which is below its detection limit, protected tomato seedlings against Pseudomonas syringae pv. tomato. Our finding highlights that BVCs, including 2-nonanone, released by a soil bacterium are functional even when present at a concentration below the detection limit of SPME-GC-MS.

Comparative Analysis of the Phyto-compounds Present in the Control and Experimental Peels of Musa paradisiaca used for the Remediation of Chromium Contaminated Water

  • Kaniyappan, Vidhya;Rathinasamy, Regina Mary;Manivanan, Job Gopinath
    • Mass Spectrometry Letters
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    • v.13 no.4
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    • pp.166-176
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    • 2022
  • Banana peels are also widely used as bio-adsorbent in the removal of chemicals contaminants and heavy metals from water and soil. GC-MS plays an essential role in the phytochemical analysis and chemo taxonomic studies of medicinal plants containing biologically active components. Intrinsically, with the use of the flame ionization detector and the electron capture detector which have very high sensitivities, Gas chromatography can quantitatively determine materials present at very low concentrations and most important application is in pollution studies. In the present study banana peels were used as bio-adsorbent to remediate the heavy metal contaminated water taken from three different stations located around the industrial belts of Ranipet, Tamilnadu, India. The AAS analysis of the samples shows a decrement of chromium concentration of 98.93%, 96.16% and 96.5% in Station 1, 2 and 3 respectively which proves the efficiency of the powdered peels of Musa paradisiaca. The GC-MS analysis of the control and treated peels of Musa paradisiaca reveals the presence of phytochemicals like Acetic Acid, 1-Methylethyl Ester, DL-Glyceraldehyde Dimer, N-Hexadecanoic Acid, 3-Decyn-2-Ol, 26-Hydroxy, Cholesterol, Ergost-25-Ene-3,5,6,12-Tetrol, (3.Beta.,5.Alpha.,6.Beta.,12.Beta.)-, 1-Methylene-2b-Hydroxymethyl-3, and 3-Dimethyl-4b-(3-Methylbut-2-Enyl)-Cyclohexane in the control banana peels. The banana peels which were used for the treatment reveals the changes and alteration of the phytochemicals. It is concluded that the alteration in phytochemicals of the experimental banana peels were due to adsorption of chromium heavy metal from the sample.

In vitro Micropropagation and Root Induction of Pear Genetic Resources

  • Jae-young Song;Jinjoo Bae;Woohyung Lee;Jung-ro Lee;Munsup Yoon
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2022.09a
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    • pp.63-63
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    • 2022
  • Pear (Pyrus spp.) is a typical fruit and grown in the temperate climate regions throughout the world. Development of appropriate methods for in vitro propagation and root induction are important to increase the production rate and plant quality rapidly. This study was conducted to find the most appropriate media conditions for in vitro propagation and rooting of three pear cultivars, 'Barttlett', 'BaeYun No.3' and 'Oharabeni'. In vitro propagation was induced on Murashige and Skoog medium (MS) with 2.0 mg/L N6-benzyladenine (BA) and 0.2 mg/L indole-3-butyric acid (IBA) medium. For root induction of these cultivars, the shoot explants of the propagated plants were cultured on two different media containing 1/2 MS medium containing 0.2 mg/L IBA with 15 g/L Sucrose (Rooting Medium 1, RM1) and 1/4 Linsmaier and Skoog medium (LS) medium containing 1 mg/L IBA and 1 mg/L NAA hormone with 7.5 g/L glucose (Rooting Medium, RM2) and after 2 weeks, the plants on the RM2 medium are transferred on RM1 medium (RM2 condition). After nearly seven weeks, percentage of rooting formation were 22.2% in RM1 and 30% in RM2 conditions for Barttlett and 70% in RM1 and 60% in RM2 conditions for Oharabeni cultivars. No differences in these cultivars were observed between RM1 and RM2 conditions. However, BaeYun No.3 cultivar was observed 0% in RM1 and 72.7% in RM2 conditions. This study will help to propagation and root induction of in vitro plants for various pear cultivars.

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