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Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness

  • Kim, Eun-Ha;Kwon, Hyeok-Il;Park, Su-Jin;Kim, Young-Il;Si, Young-Jae;Lee, In-Won;Kim, Se mi;Kim, Soo-In;Ahn, Dong-Ho;Choi, Young-Ki
    • Journal of Microbiology and Biotechnology
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    • v.28 no.6
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    • pp.997-1006
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    • 2018
  • As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over $10^8PFU/ml$. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells ($PB1_{D153N}$, $M1_{A137T}$, and $NS1_{N176S}$). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than $10^7PFU/ml$) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Effects of High Temperature Heating on the Some Physicochemical Properties of Korean Red Ginseng (Panax ginseng C.A. Meyer) Water Extract (고온 열처리가 홍삼물추출물의 이화학적 특성에 미치는 영향)

  • Kwak, Yi-Seong;Choi, Keum-Hee;Kyung, Jong-Soo;Won, Jun-Yeon;Rhee, Man-Hee;Lee, Jae-Gon;Hwang, Mi-Sun;Kim, Seok-Chang;Park, Chae-Kyu;Song, Kyung-Bin;Han, Gyeong-Ho
    • Journal of Ginseng Research
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    • v.32 no.2
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    • pp.120-126
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    • 2008
  • This study was carried out to investigate the some physicochemical properties of Korean red ginseng (Panax ginseng C.A. Meyer) water extract (RGWE) after heated with high temperatures above $100^{\circ}C$ for 2 hours. RGWEs were heated at 100, 110 and $120^{\circ}C$ for 2 hours by using autoclave. After RGWEs were heated at high temperature for 2 hours without not adjustment of pH, the changes of saponin, free sugars, mineral and color in the RGWEs were investigated. Total ginsenoside content in control was 1.99%, while those of RGWE were 1.65, 1.49 and 1.29% when treated at 100, 110 and $120^{\circ}C$, respectively. The contents of total ginsenoside showed decreased tendency as heating temperatures were increased. The ginsenoside-$Rh_{2}$ and $-Rg_{3}$, which have been reported as very stable red ginseng ginsenosides, showed relatively strong spots on TLC when RGWEs were heated at 110 and $120^{\circ}C$. In case of free sugars in RGWEs, fructose, glucose and maltose showed high contents when compared with control, while Fe, Ca and Mg ions showed very low contents. Value of L in RGWE treated with high temperature was almost the same with control, while values of a and b were increased. Values of a were increased from -0.86 of control to +0.04, +0.05 and +1.14 when treated with 100, 110 and $120^{\circ}C$, respectively. Values of b also were increased from 27.68 of control to 33.61, 33.61 and 37.42 when treated with 100, 110 and $120^{\circ}C$, respectively. Values of total color in RGWEs treated with high temperatures, E, were finally increased by values of a and b.

A Study on the Implementation of Dose Constraints in Occupational Dose According to ICRP 103 Recommendations in Korea (ICRP신권고에 따른 직무피폭에서의 선량제약치 국내 적용 방안 연구)

  • Kim, Yong-Min;Cho, Kun-Woo
    • Journal of Radiation Protection and Research
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    • v.36 no.3
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    • pp.127-133
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    • 2011
  • In 2007, the International Commission on Radiological Protection (ICRP) published Recommendations of the International Commission on Radiological Protection. Accordingly IAEA safety standards committees have reviewed and revised the BSS. The process of the implementation of the ICRP 103 into Korean radiation protection regulations has been continued. Although the new recommendations retain the fundamental protection principles, the impact of the new ICRP recommendations will necessarily be greater than ever before. ICRP recommends the application of dose constraint in planned situations and reference level in existing & emergency situations for strengthening of the principle of optimization. Dose constraints and reference level play a criterion on the level of individual dose as prospective and source-related values. Therefore it is necessary to apply dose constraints and reference levels to all nuclear and RI&RG facilities in Rep. of Korea. Dose constraints and reference level of occupational exposure will be set-up by the stakeholder itself with the cooperation of regulatory body. In this study, the implementation method was discussed to apply the dose constraints and reference level as the procedure for the optimization, not the tool of the regulation.

Changes in Growth, Active Ingredients, and Rheological Properties of Greenhouse-cultivated Ginseng Sprout during its Growth Period (하우스에서 재배된 새싹인삼의 재배시기별 생육, 유효성분 및 물성의 변화)

  • Seong, Bong Jae;Kim, Sun Ick;Jee, Moo Geun;Lee, Hee Chul;Kwon, A Reum;Kim, Hyun Ho;Won, Jun Yeon;Lee, Ka Soon
    • Korean Journal of Medicinal Crop Science
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    • v.27 no.2
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    • pp.126-135
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    • 2019
  • Background: The ginseng ginsenosides, which have various physiological activities, are known to be more abundant in the leaves than in the roots, and the consumers' interest in ginseng sprout as a functional vegetable has been increasing. Methods and Results: The aim of this study was to investigate the effects of growth period on growth properties, active ingredients and rheology of ginseng sprouts cultivated in a non-heated greenhouse equipped with a shade net for 60 days, starting from the end of May to the middle of July. The chlorophyll content of the leaves decreased, but their length and width increased with increasing cultivation days. In particular, growth increased significantly until 40 days, but only slightly after 50 days. The stem length did not increase greatly from the 20 th to the 30 th day of cultivation, but increased significantly from the 30 th to the 40 th day, and then further increased gradually. The weight of the leaves, stems, and roots increased slightly, but not change significantly. After 40 days of cultivation, the total ginsenoside content increased by 1.07 times in the leaves and decreased by 0.80 times in the roots with increasing cultivation days. The leaf contents of ginsenosides $Rg_1$, Re, $Rb_1$, Rc, $F_3$ and $F_4$ increased with increasing cultivation days. The rheological properties of ginseng sprout showed the greatest influence on stem hardening with increasing cultivation days. Conclusions: Therefore, based on the growth characteristics, active ingredients and physical properties, 40 days after sowing was considered to be an appropriate harvesting time for ginseng sprouts.

Major ginsenosides from Panax ginseng promote aerobic cellular respiration and SIRT1-mediated mitochondrial biosynthesis in cardiomyocytes and neurons

  • Huang, Qingxia;Lou, Tingting;Lu, Jing;Wang, Manying;Chen, Xuenan;Xue, Linyuan;Tang, Xiaolei;Qi, Wenxiu;Zhang, Zepeng;Su, Hang;Jin, Wenqi;Jing, Chenxu;Zhao, Daqing;Sun, Liwei;Li, Xiangyan
    • Journal of Ginseng Research
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    • v.46 no.6
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    • pp.759-770
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    • 2022
  • Background: Aerobic cellular respiration provides chemical energy, adenosine triphosphate (ATP), to maintain multiple cellular functions. Sirtuin 1 (SIRT1) can deacetylate peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) to promote mitochondrial biosynthesis. Targeting energy metabolism is a potential strategy for the prevention and treatment of various diseases, such as cardiac and neurological disorders. Ginsenosides, one of the major bioactive constituents of Panax ginseng, have been extensively used due to their diverse beneficial effects on healthy subjects and patients with different diseases. However, the underlying molecular mechanisms of total ginsenosides (GS) on energy metabolism remain unclear. Methods: In this study, oxygen consumption rate, ATP production, mitochondrial biosynthesis, glucose metabolism, and SIRT1-PGC-1α pathways in untreated and GS-treated different cells, fly, and mouse models were investigated. Results: GS pretreatment enhanced mitochondrial respiration capacity and ATP production in aerobic respiration-dominated cardiomyocytes and neurons, and promoted tricarboxylic acid metabolism in cardiomyocytes. Moreover, GS clearly enhanced NAD+-dependent SIRT1 activation to increase mitochondrial biosynthesis in cardiomyocytes and neurons, which was completely abrogated by nicotinamide. Importantly, ginsenoside monomers, such as Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, and Rb3, were found to activate SIRT1 and promote energy metabolism. Conclusion: This study may provide new insights into the extensive application of ginseng for cardiac and neurological protection in healthy subjects and patients.

Qualities and Anti-inflammatory Activity of Kyungokgos Sold in Local Markets (국내 시판 경옥고 제품의 품질 특성 및 항염증 활성)

  • Lee, Ka-Soon;Kim, Gwan-Hou;Kim, Hyun-Ho;Seong, Bong-Jae;Kim, Sun-Ick;Han, Seung-Ho;Kang, Eun Ju;Yoo, Yung Choon
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.42 no.3
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    • pp.335-341
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    • 2013
  • Kyungokgos purchased in local markets in Korea vary in their combination and mixing ratios during processing. This study was investigated qualities of Kyungokgos manufactured traditionally to evaluating its qualities. The general components of Kyungokgos were moisture (18.62~49.78%), ash (0.198~1.211%), protein (0.89~3.58%), lipid (0.16~1.14%) and carbohydrates (47.95~77.08%). The color values of L, a, and b were 26.49~73.87, 16.51~38.64, and 45.41~88.94, respectively. The viscosity was classified into three non-Newtonian type groups: high, medium, and non-dilatant, according to the increase of loop execution times. Three extracts (KOG-1, -7, and -8, in a 30-fold dilution) showed no cytotoxicity toward RAW 264.7 cells, while the extracts of KOG-2, -4, and -5 showed a low cytotoxic effect. KOG-1 and -2 extracts with low cytotoxicity markedly inhibited the production of the inflammatory mediators-nitric oxide (NO) and tumor necrosis factor-alpha (TNF-${\alpha}$) in LPS-stimulated RAW 264.7 cells. These results indicate that KOG-1 and -2 extracts have anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages.

Fertilizing Effects of Swine Compost Fermented with Sawdust on Mixed Pastures (혼파초지에 대한 톱밥발효돈분의 시용효과)

  • Shin, J. Soon;Cho, Young-Mu;Lee, Hyo-Ho;Yoon, Sea-Hung;Park, Geun-Je;Choi, Ki-Chun
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.24 no.3
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    • pp.245-252
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    • 2004
  • Experiment was carried out to find the fertilizing effects of 8 different application rates of swine compost fermented with sawdust(SCS) including Chemical fertilizer(CF) on forage yield and soil chemical characteristics of mixed pastures sown in Sep. 1993 at National Livestock Research Institute, RDA., in Suwon during low years. It was arranged in a randomized complete block design with three replicates. Dry matter yield were shown at similar among treatments except Control and $50\%$ SCS of standard amount plot. In botanical composition, the legume and weeds percentages of each treatment were increased as advancing year. The final year's legume percentage were high in line with SCS fertilizing plots($39\%{\sim}43\%$), SCS + CF plots($30\%{\sim}41\%$) and CF plot($32\%$). In productions of TDN, NE and crude protein yield, SCS or SCS($75\%$) + CF($25\%$) were nearly same comparing those of CF, respectively. Phosphate, potassium, magnesium contents and K/(Ca + Mg) except calcium contents of those SCS fertilizing plots in plant were generally high with comparing CF. Those contents were proportional according to the fertilizing amount These result indicate the possibility to substitute chemical fertilizer for SCS($75\%$, 25ton/ha) + CF, $25\%$) as manure-N 210 kg/ha, but might be considered accumulation phosphate in the soil.

Characteristics of Ginseng Extract and Its Effects on Sensory Properties of Pork Sausage (인삼 추출물의 사포닌 조성 특성과 추출물 첨가가 포크소시지 관능 특성에 미치는 영향)

  • Park, Kyoung-Mi;Hwang, In-Ho
    • Food Science of Animal Resources
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    • v.26 no.4
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    • pp.418-425
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    • 2006
  • This study was conducted to evaluate extraction properties of crude saponin and ginsenosides, and their effects on sensory properties of emulsified pork sausage. Non-dried ginseng root was boiled in 0 (e.g., 100% distilled water), 20, 40, 60, 80 or 100% ethanol, and powdered by a freezing dry method. Weight of dried powder for the 0% ethanol extraction was 20% of initial non-dried ginseng weight, while $20{\sim}80%$ and 100% ethanol extractions resulted in approximately 15 and 10% of their initial weights, respectively. On the other hand, crude saponin content in the dried powder was linearly increased for a higher ethanol content where 100% ethanol extraction resulted in 123.52 mg/g. LC/MS analysis of crude saponin for quantifying ginsenosides showed that Rb1, Rb2 and Rc were significantly (p<0.05) higher levels for both 80 and 100% ethanol extractions. In the case of Rg1 ginsenoside, 60, 80 and 100% ethanol extractions resulted in significantly (p<0.05) higher levels. Emulsified pork sausages containing 0, 1 or 2% ginseng extracts were smoked or non-smoked and their sensory characteristics and preference were evaluated. Smoking process significantly (p<0.05) decreased juiciness and tenderness, but the treatment significantly (p<0.05) improved flavor and consumer preference. It was particularly noticed that a 2% addition of ginseng extract prevented the adverse effects of smoking process on juiciness and tenderness while the 2% addition significantly (p<0.05) improved consumer preference. The current results implied that addition of ginseng extract in emulsified pork sausage could improve sensory quality.

Definition of Tumor Volume Based on 18F-Fludeoxyglucose Positron Emission Tomography in Radiation Therapy for Liver Metastases: An Relational Analysis Study between Image Parameters and Image Segmentation Methods (간 전이 암 환자의 18F-FDG PET 기반 종양 영역 정의: 영상 인자와 자동 영상 분할 기법 간의 관계분석)

  • Kim, Heejin;Park, Seungwoo;Jung, Haijo;Kim, Mi-Sook;Yoo, Hyung Jun;Ji, Young Hoon;Yi, Chul-Young;Kim, Kum Bae
    • Progress in Medical Physics
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    • v.24 no.2
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    • pp.99-107
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    • 2013
  • The surgical resection was occurred mainly in liver metastasis before the development of radiation therapy techniques. Recently, Radiation therapy is increased gradually due to the development of radiation dose delivery techniques. 18F-FDG PET image showed better sensitivity and specificity in liver metastasis detection. This image modality is important in the radiation treatment with planning CT for tumor delineation. In this study, we applied automatic image segmentation methods on PET image of liver metastasis and examined the impact of image factors on these methods. We selected the patients who were received the radiation therapy and 18F-FDG PET/CT in Korea Cancer Center Hospital from 2009 to 2012. Then, three kinds of image segmentation methods had been applied; The relative threshold method, the Gradient method and the region growing method. Based on these results, we performed statistical analysis in two directions. 1. comparison of GTV and image segmentation results. 2. performance of regression analysis for relation between image factor affecting image segmentation techniques. The mean volume of GTV was $60.9{\pm}65.9$ cc and the $GTV_{40%}$ was $22.43{\pm}35.27$ cc, and the $GTV_{50%}$ was $10.11{\pm}17.92$ cc, the $GTV_{RG}$ was $32.89{\pm}36.8$4 cc, the $GTV_{GD}$ was $30.34{\pm}35.77$ cc, respectively. The most similar segmentation method with the GTV result was the region growing method. For the quantitative analysis of the image factors which influenced on the region growing method, we used the standardized coefficient ${\beta}$, factors affecting the region growing method show GTV, $TumorSUV_{MAX/MIN}$, $SUV_{max}$, TBR in order. The result of the region growing (automatic segmentation) method showed the most similar result with the CT based GTV and the region growing method was affected by image factors. If we define the tumor volume by the auto image segmentation method which reflect the PET image parameters, more accurate and consistent tumor contouring can be done. And we can irradiate the optimized radiation dose to the cancer, ultimately.

Mass Culture and Ginsenoside Production of Ginseng Hairy Root by Two-Step Culture Process (2계단 배양방법을 이용한 인삼 모상근의 대량배양과 Ginsenoside 생산)

  • Ko, Kyeong-Min;Yang, Deok-Chun;Park, Ji-Chang;Choi, Kang-Ju;Choi, Kwang-Tae;Hwang, Baik
    • Journal of Plant Biology
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    • v.39 no.1
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    • pp.63-69
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    • 1996
  • A hairy root clone of Panax ginseng C.A. Meyer, HRB-15 was cultured iu various conditions with 3 L bubble type bioreactor to enhance both growth and ginsenoside production. The hairy roots were more rapidly grown under the dark condition than under the light condition. However, total amount of ginsenoside of hairy roots cultured under the light for 30 days increased 2 folds as compared with the dark condition and was 1.10% based on 6 ginsenosides. Especially, ginsenoside-Re was significantly increased and some ginsenosides except for ginsenoside-Re was slightly reduced. Also, the growth of hairy roots decreased about 30% as compared with the dark condition. In contrast, addition of sodium acetate led to decreased production of ginsenoside and growth of hairy roots under light condition. The influence of potassium dihydrogenphosphate concentration was examined in MS medium and a 1.25 mM concentration was found to be the most appropriate for growth and ginsenoside production under light condition. Two-step process of hairy roots culture with yeast elicitation or without ammonia in culture medium was developed to enhance growth and giusenoside synthesis. $50\;\mu\textrm{g}$ of yeast elicitor per g of fresh weight showed a synergistic effect on the ginsenoside synthesis of hairy roots on 20 days after culture. At that time, the content of total ginsenoside was 1.15%, while the growth of hairy roots decreased 21 % as compared with the dark condition. In addition, when elimination of ammonia on 20 days after culture, the content of total ginsenoside was 1.26% with significant increment of ginsenoside-Rd (0.27%) in addition to ginsenoside-Re and the growth of hairy roots decreased 10% as compared with the dark condition. In this system, we have demonstrated a unique two-step process of hairy root cultures to maximize biomass and secondary metabolites. It has found possibility to enhance ginsenosides production by growing hairy roots in this method.

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