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Detection of Lamivudine-Resistant Mutations of HBV DNA Polymerase Gene Using PCR-Direct Sequencing

  • Lee, Kyung-Ok;Lee, Hye-Jung;Byun, Ji-Young;Lee, Sung-Yeun;Kim, Jeong-Sook;Jung, Na-Young;Chung, Soo-Jin;Seong, Hye-Soon;Kim, Kyung-Tae
    • Korean Journal of Clinical Laboratory Science
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    • v.38 no.3
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    • pp.196-202
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    • 2006
  • Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

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Solubility of Hydrogen Sulfide and Methane in Ionic Liquids: 1-Ethy-3-methylimidazolium Trifluoromethanesulfonate and 1-Butyl-1-methylpyrrolidinium Trifluoromethanesulfonate (1-Ethyl-3-methylimidazolium trifluoromethanesulfonate와 1-Butyl-1-methylpyrrolidinium trifluoromethanesulfonate 이온성 액체에 대한 황화수소와 메탄의 용해도)

  • Lee, Byung-Chul
    • Korean Chemical Engineering Research
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    • v.54 no.2
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    • pp.213-222
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    • 2016
  • Solubility data of hydrogen sulfide ($H_2S$) and methane ($CH_4$) in two kinds of ionic liquids with the same anion: 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([emim][TfO]) and 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([bmpyr][TfO]) are presented at pressures up to about 30 MPa and at temperatures between 303 K and 343 K. The gas solubilities in ionic liquids were determined by measuring the bubble point pressures of the gas + ionic liquid mixtures with various compositions at different temperatures using a high-pressure equilibrium apparatus equipped with a variable-volume view cell. The $H_2S$ solubilities in ionic liquid increased with the increase of pressure and decreased with the increase of temperature. On the other hand, the $CH_4$ solubilities in ionic liquid increased significantly with the increase of pressure, but there was little effect of temperature on the $CH_4$ solubility. For the ionic liquds [emim][TfO] and [bmpyr][TfO] with the same anion, the solubility of $H_2S$ as a molality basis was substantially similar, regardless of the temperature and pressure conditions as a molar concentration basis. Comparing the solubilities of $H_2S$ and $CH_4$ in the ionic liquid [emim][TfO], the solubilities of $H_2S$ were much greater than those of $CH_4$. For the same type of ionic liquid, the solubility data of $H_2S$ and $CH_4$ obtained in this study were compared to the solubility data of $CO_2$ from the literature. When compared at the same pressure and temperature conditions, the $CO_2$ solubility was in between the solubility of $H_2S$ and $CH_4$.

Structural Features of Various Trichomes in Vitex negundo during Development (방향성 좀목형(Vitex negundo)모용의 구조적 분화발달)

  • Lee, Seung-Hee;Kim, In-Sun
    • Applied Microscopy
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    • v.36 no.1
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    • pp.35-45
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    • 2006
  • Plants of Vitex negundo are known to develop numerous trichomes throughout their body, where certain trichome types have been believed to be one of the plausible structures for the unique scents. In the current study. structural aspects of the trichomes have been examined in leaves and stems of Vitex negundo using TEM and SEM. Trichome types as well as structural changes that occurred in certain trichomes during secretion have been mainly focused. Three type of glandular trichomes and two types of non-glandular trichomes were developed in the epidermis of young and mature Vitex negundo plants. The glandular trichomes included the peltate type (Type 1), the capitate type (Type 2), and degraded capitate type (Type 3), whereas the non-glandular warty trichomes contained the multicellular (Types 4) and unicellular type (Type 5). Type 1 and 2 consisted of head and stalk cells, but their number and size were different. One secretory cavity was formed from the four head cells in the former, but only two head cells were involved in the latter. The cytoplasmic density in the head cell was quite high and in particular, sER and Golgi bodies were well developed. At initiation of their development, the cuticle layer of the head cells separated from the outer tangential wall to form a secretory cavity. Subsequently the cavity expanded acropetally and a large number of secretory vesicles continuously produced from the head cells until they filled the entire cavity. The cavity contained materials that would be soon discharged into intercellular spaces and/or into the air. The cavity began to decrease the volume by contracting at initial secretion but degrade rapidly within short time. It has been suggested that the mode of secretion in V. negundo is probably the eccrine secretion, since no break or rupture of the cavity has been observed during examination. Contrastingly Type 3 exhibited deterioration of the head cell at early stage. Type 4 was about $110{\sim}190{\mu}m$ long, consisting of $2{\sim}3$ cells, and distributed more in the adaxial epidermis compared to the abaxial surface. However, $20{\sim}30{\mu}m$ long Type 5 was extremely dense in both epidermis. Among several trichome types, Type 1 and 2 probably play an important role in discharging unique aromatic scents in plants of V. negundo.

Effect of Containers on the stability of Malathion emulsion concentrates (E.C.) (Malathion 유제(乳劑)의 포장용기(包裝容器)에 따른 경시변화(經時變化))

  • Lee, D.S.;Lee, J.Y.;Lee, S.H.
    • Applied Biological Chemistry
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    • v.7
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    • pp.15-19
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    • 1966
  • In order to investigate the stability of the major component of malathion E.C. product, dimethyl S-(1, 2-dicarboxyethoxyethyl) dithiophosphate, toward the quality of glasswares as container, the amount of extractable inorganic components, change of pH and decomposition of the major component of the product were examined during the storage in brown-colored bottles of 100 ml. volume from 3 different companies in comparison with that in a Pyrex flask. 1. Malathion E.C. product was put in three containers A,B and C, and any changes occurring in storage were analyzed at three intervals of 60, 120 and 240 days. 2. It was shown that the amounts of Si, Mg, K, Ca, and Na extracted during these periods of storage differed markedly depending on the qualify of container. Container A revealed ten times higher extraction of Na and Ca than container B and C in a 8-month period. 3. Three commercial containers revealed the shift of pH from 6.5 to alkaline reaction in the storage whereas the Pyrex flask did not show any detectable change. In particular, the pH in container A changed to 9.2 in 60 days and 9.9 in 240 days. 4. The decomposition of malathion was the greatest in container A which showed the decomposition of 7.37% in 240 days. On the other hand, 0.5% was decomposed in the Pyrex flask. 5. The decemposition of malathion had a high correlation with the change of pH of water· in the same container, $r^2$ being 0.899. From the above results, it is concluded that about 10% of malathion E.C. product is decomposed in a year due to the alkaline metallic salts extracted from the container when it is stored in glassware bottles of lower quality.

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Properties of Hot Weather Nuclear Power Plant Concrete with Water Cooling Method and Retarding used (배합수 냉각방법 및 지연제 사용에 따른 서중 원전콘크리트의 특성)

  • Lee, Seung-Han;Jung, Yong-Wook;Jang, Seok-Soo;Yeo, In-Dong;Choi, Jong-Oh
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.14 no.9
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    • pp.4602-4609
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    • 2013
  • In summer and winter, the difference between the temperature during the day and that during the night is high, which leads to various problems during concrete placement, such as cracks and defects in the concrete as well as low durability and strength. Although nuclear power plant concrete is widely used for placement in all seasons, particular attention must be paid to its quality during the summer. Therefore, we evaluated the effects of a cooling method for mixing water, which is a commonly used hot weather precooling method, and the use of a retarder, on the characteristics of Nuclear Power Plant concrete. In the cooling method for mixing water, cold water at 5 was used, with 50% of the water content consisting of ice flakes. The effects of using a retarder were evaluated by reviewing the characteristics of the cement at the unset stage and after hardening. To evaluate the characteristics of the unset cement, we measured the slump, air volumes, setting times, and pressure strengths after hardening. Furthermore, we measured the heat of hydration at different temperatures; the loss of heat was minimized using insulation. Both the slump time and the complete ageing time of the air volume were found to be 120 min at $20^{\circ}C$ and 40 min at $40^{\circ}C$. In the case when the cooling method for mixing water was used and in the case when a retarder was used, the initial and final sets by penetration resistance were delayed, and the delay decreased with increasing air temperature. For the heat of hydration, the cooling method for mixing water not only lowered the maximum temperature but also delayed its attainment. However, the use of a retarder had no effect on the maximum temperature. Moreover, in the early ages (e.g., 3 and 7 days), the pressure strength of the concrete was lower than that of plain cement. However, the strength of 28-day concrete met the standard construction specifications.

Environment-friendly and Low-Carbon Agriculture for Demand-Supply Control and Food Security of Korean Rice (쌀 수급안정과 식량안보를 위한 친환경·저탄소 농업 전환방안)

  • Yang, Seung-Koo;Park, Pyung-Sik;Son, Jang-Hwan;An, Kyu-Nam
    • Korean Journal of Organic Agriculture
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    • v.26 no.1
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    • pp.99-128
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    • 2018
  • The cultivation area of rice as staple grains is decreasing in the domestic situation in Korea. Import volume of a duty in foreign rice is 409,000 tons for a year regardless increasing of production per unit area and decreasing of rice consumption. The total stock of rice is increasing cumulatively despite the effort for production mediation of rice. Therefore, maintenance of cultivation area and reduction of production are necessary for national foodstuffs security problems. Development of environment-friendly and low-carbon technology as alternative of global warming and aging of farm labor power is very important responsibility for descendants with creation of sustainable agriculture environment. As alternative for demand and supply stabilization of rice from all angles, first stage: extension of environment-friendly cultivation area as 17% Jeollanam-do level with maintenance of cultivation area under the present circumstances, second stage: extension of environment-friendly cultivation area as 25%, third stage: extension of environment-friendly cultivation area as 35%. From above mentioned scenario, reduction of rice production (60,000 tons), increases of production cost (59,200,000,000 Won), and reduction of income (201,500,000,000 Won) are estimated in first stage. Reduction of rice production (90,000 tons), increases of production cost (122,100,000,000 Won), and reduction of income (313,700,000,000 Won) are estimated in second stage. Reduction of rice production (380,000 tons), increases of production cost (222,000,000,000 Won), and reduction of income (464,500,000,000 Won) are estimated in third stage. From analysis results for partial tillage in transplanting cultivation complex (10ha), rice production is decreased 1.3~1.5 ton by complex. Production cost of rice is decreased and increases of income cultivation type. Gradual extension of environment-friendly agriculture and low-carbon partial tillage could be expected for environment maintenance of the territorial integrity, confidence of consumer, and high-efficiency of low-cost.

The Anti-diabetic Effects of Kocat-D1 on Streptozotocin-Induced Diabetic Rats (Kocat-D1의 streptozotocin으로 유도한 당뇨모델에 대한 항당뇨 활성)

  • Won, Hye-Jin;Lee, Hyun-Sun;Kim, Jong-Tak;Hong, Chung-Oui;Koo, Yun-Chang;Lee, Kwang-Won
    • Korean Journal of Food Science and Technology
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    • v.42 no.2
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    • pp.204-209
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    • 2010
  • This study was conducted to investigate the anti-diabetic activity of Kocat-D1, which is widely used in traditional medicine to treat diabetes in Shandong, China. Sprague Dawley rats (8 weeks of age) were separated into 4 groups: a normal control, streptozotocin (STZ)-induced diabetic rat group (DM control), Kocat-D1-1 (diabetic rat treated with 0.25 g/kg/day hot water extract), and Kocat-D1-2 (diabetic rat treated with 1 g/kg/day hot water extract). After eight weeks of treatment, the fasting blood glucose levels of the Kocat-D1-1 ($334.3{\pm}32.9\;mg/dL$) and Kocat-D1-2 group ($259.5{\pm}35.0\;mg/dL$) were significantly lower when compared to the DM control group ($451{\pm}42.6\;mg/dL$). Furthermore, the levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin and high-density lipoprotein (HDL) cholesterol in the serum of the Kocat-D1-2 group were significantly normalized when compared to the DM control group. However, significant differences were not observed between the Kocat-D1-1 group and the DM control group. Histochemical staining of the liver of the Kocat-D1-2 group revealed no fat accumulation. The insulin level was significantly upregulated in the Kocat-D1-2 group ($0.13{\pm}0.02\;ng/mL$) when compared to the DM control group ($0.05{\pm}0.04\;ng/mL$). The relative volume of $\beta$-cells in the pancreas of the Kocat-D1-2 group ($49.4{\pm}4.2%$) also increased significantly when compared to the DM control group ($12.9{\pm}7.9%$). These results suggest that Kocat-D1 exerts an anti-hyperglycemic effect through the enhancement of insulin secretion.

A Comparative Study on Quantifying Uncertainty of Vitamin A Determination in Infant Formula by HPLC (HPLC에 의한 조제분유 중 비타민 A 함량 분석의 측정불확도 비교산정)

  • Lee, Hong-Min;Kwak, Byung-Man;Ahn, Jang-Hyuk;Jeon, Tae-Hong
    • Korean Journal of Food Science and Technology
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    • v.40 no.2
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    • pp.152-159
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    • 2008
  • The purpose of this study was to determine the accurate quantification of vitamin A in infant formula by comparing two different standard stock solutions as well as various sample weights using high performance liquid chromatography. The sources of uncertainty in measurement, such as sample weight, final smaple vloume, and the instrumental results, were identified and used as parameters to determine the combined standard uncertainty based on GUM(guide to the expression of uncertainty in measurement) and the Draft EURACHEM/CITAC Guide. The uncertainty components in measuring were identified as standard weight, purity, molecular weight, dilution of the standard solution, calibration curve, recovery, reproducibility, sample weight, and final sample volume. Each uncertainty component was evaluated for type A and type B and included to calculate the combined uncertainty. The analytical results and combined standard uncertainties of vitamin A according to the two different methods of stock solution preparation were 627 ${\pm}$ 33 ${\mu}$g R.E./100 g for 1,000 mg/L of stock solution, and 627 ${\pm}$ 49 ${\mu}$g R.E./100 g for 100 mg/L of stock solution. The analytical results and combined standard uncertainties of vitamin A according to the various sample weighs were 622 ${\pm}$ 48 ${\mu}$g R.E./100 g, 627 ${\pm}$ 33 ${\mu}$g R.E./100 g, and 491 ${\pm}$ 23 ${\mu}$g R.E./100 g for 1 g, 2 g, and 5 g of sampling, respectively. These data indicate that the preparation method of standard stock solution and the smaple amount were main sources of uncertainty in the analysis results for vitamin A. Preparing 1,000 mg/L of stock solution for standard material sampling rather than 100 mg, and sampling not more than 2 g of infant formula, would be effective for reducing differences in the results as well as uncertainty.

Validation of Load Calculation Method for Greenhouse Heating Design and Analysis of the Influence of Infiltration Loss and Ground Heat Exchange (온실 난방부하 산정방법의 검증 및 틈새환기와 지중전열의 영향 분석)

  • Shin, Hyun-Ho;Nam, Sang-Woon
    • Horticultural Science & Technology
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    • v.33 no.5
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    • pp.647-657
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    • 2015
  • To investigate a method for calculation of the heating load for environmental designs of horticultural facilities, measurements of total heating load, infiltration rate, and floor heat flux in a large-scale plastic greenhouse were analyzed comparatively with the calculation results. Effects of ground heat exchange and infiltration loss on the greenhouse heating load were examined. The ranges of the indoor and outdoor temperatures were $13.3{\pm}1.2^{\circ}C$ and $-9.4{\sim}+7.2^{\circ}C$ respectively during the experimental period. It was confirmed that the outdoor temperatures were valid in the range of the design temperatures for the greenhouse heating design in Korea. Average infiltration rate of the experimental greenhouse measured by a gas tracer method was $0.245h^{-1}$. Applying a constant ventilation heat transfer coefficient to the covering area of the greenhouse was found to have a methodological problem in the case of various sizes of greenhouses. Thus, it was considered that the method of using the volume and the infiltration rate of greenhouses was reasonable for the infiltration loss. Floor heat flux measured in the center of the greenhouse tended to increase toward negative slightly according to the differences between indoor and outdoor temperature. By contrast, floor heat flux measured at the side of the greenhouse tended to increase greatly into plus according to the temperature differences. Based on the measured results, a new calculation method for ground heat exchange was developed by adopting the concept of heat loss through the perimeter of greenhouses. The developed method coincided closely with the experimental result. Average transmission heat loss was shown to be directly proportional to the differences between indoor and outdoor temperature, but the average overall heat transfer coefficient tended to decrease. Thus, in calculating the transmission heat loss, the overall heat transfer coefficient must be selected based on design conditions. The overall heat transfer coefficient of the experimental greenhouse averaged $2.73W{\cdot}m^{-2}{\cdot}C^{-1}$, which represents a 60% heat savings rate compared with plastic greenhouses with a single covering. The total heating load included, transmission heat loss of 84.7~95.4%, infiltration loss of 4.4~9.5%, and ground heat exchange of -0.2~+6.3%. The transmission heat loss accounted for larger proportions in groups with low differences between indoor and outdoor temperature, whereas infiltration heat loss played the larger role in groups with high temperature differences. Ground heat exchange could either heighten or lessen the heating load, depending on the difference between indoor and outdoor temperature. Therefore, the selection of a reference temperature difference is important. Since infiltration loss takes on greater importance than ground heat exchange, measures for lessening the infiltration loss are required to conserve energy.

Determination and Survey of Fluoroquinolones Residue in Chicken Muscle by HPLC with Fluorescence Detector (액체크로마토그래피-형광검출기를 이용한 닭고기 중 플루오로퀴놀론계 항균물질 정량분석 및 잔류조사)

  • 박은정;임지흔;이성모
    • Journal of Food Hygiene and Safety
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    • v.19 no.1
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    • pp.12-18
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    • 2004
  • Ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin in chicken muscle were seperated by liquid extraction and determined with high performance liquid chromatography (HPLC) with fluorescence detector. Analysis was carried out using following conditions; Cl8 column (250${\times}$4.6 mm i.d. 5 ${\mu}{\textrm}{m}$ particle size), mobile phase composed of D.W. (containing 0.4% triethylamine and phospholic acid): methanol : acetonitrile (800:100:100, v/v/v), isocratic pump at a flow rate of 1.0 $m\ell$/min and 50 ${mu}ell$ of injection volume, fluorescence detector with EX278 nm/EM.456 nm. The calibration curves of four fluoroquinolones showed linearity (${\gamma}$$^2$$\geq$0.999) at concenration range of 0.025-0.6 $\mu\textrm{g}$/ml. The recoveries in fortified chicken muscle represented more than 80% with low coefficient of variation (〈10%) for concentration range of four fluoroquinolones. The detection limits for ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin were 23.5, 3.4, 3.0 and 2.5 ng/g in chicken muscle, respectively. We also monitored fluoroquinolones residue in muscle of chickens (broiler 1:227, Korean native chicken 219, laying chicken 77) using EEC-4-plate screening and HPLC conformation methods. Ten(broiler 5, Korean native chicken 5) out of the fifteen samples which were positively detected by EEC-plate screening method from 1,523 chicken meat were confirmed with ciprofloxacin and enrofloxacin by HPLC. The ranges of residual concentration were 0-0.12 ppm for ciprofloxacin and 0.01-6.79 ppm for enrofloxacin. In conclusion, our method could be applied effectively to determine four fluoroquinolones residues in chicken meat, and further survey for fluoroquinolones residue in chicken meat are needed for more effective control of fluoroquinolones used in livestock.