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Development of mcyB-specific Ultra-Rapid Real-time PCR for Quantitative Detection of Microcystis aeruginosa (Microcystis aeruginosa의 정량을 위한 mcyB 특이 초고속 실시간 유전자 증폭법의 개발)

  • Jung, Hyunchul;Yim, Byoungcheol;Lim, Sujin;Kim, Byounghee;Yoon, Byoungsu;Lee, Okmin
    • Journal of Korean Society on Water Environment
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    • v.34 no.1
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    • pp.46-56
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    • 2018
  • A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

Utilization of qPCR Technology in Water Treatment (수질분석에 사용되는 qPCR기술)

  • Kim, Won Jae;Hwang, Yunjung;Lee, Minhye;Chung, Minsub
    • Applied Chemistry for Engineering
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    • v.33 no.3
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    • pp.235-241
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    • 2022
  • According to the World Water Development Report 2015 released by the United Nations, drinking water is expected to decrease by 40% by 2030. This does not mean that the amount of water decreases, but rather that the water source is contaminated due to environmental pollution. Because microbes are deeply related to water quality, the analysis of microbe is very important for water quality management. While the most common method currently used for microbial analysis is microscopic examination of the shape and feature after cell culture, as the gene analysis technology advances, quantitative polymerase chain reaction (qPCR) can be applied to the microscopic microbiological analysis, and the application method has been studied. Among them, a reverse transcription (RT) step enables the analysis of RNA by RT-PCR. Integrated cell culture (ICC)-qPCR shortens the test time by using it with microbial culture analysis, and viability qPCR can reduce the false positive errors of samples collected from natural water source. Multiplex qPCR for improved throughput, and microfluidic qPCR for analysis with limited amount of sample has been developed In this paper, we introduce the case, principle and development direction of the qPCR method applied to the analysis of microorganisms.

Geochemical Reactive Experimental and Modeling Studies on Caprock in the Pohang Basin (포항분지 덮개암에 대한 지화학적 반응 실험 및 모델링 연구)

  • Kim, Seon-ok;Wang, Sookyun;Lee, Minhee
    • Economic and Environmental Geology
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    • v.49 no.5
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    • pp.371-380
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    • 2016
  • This study aims to identify the mineraloical and petrographical characteristics of caprock from drilling cores of Pohang basin as a potential $CO_2$ storage site. Experiments and modeling were conducted in order to investigate the geochemical and mineralogical caprock effects of carbon dioxide. A series of autoclave experiments were conducted to simulate the interaction in the $scCO_2$-caprock-brine using a high pressure and temperature cell at $50^{\circ}C$ and 100 bar. Geochemical and mineralogical alterations after 15 days of $scCO_2$-caprock-brine sample reactions were quantitatively examined by XRD, XRF, ICP-OES investigation. Results of mineralogical studies, together with petrographic data of caprock and data on the physicochemical parameters of brine were used for geochemical modeling. Modelling was carried out using the The Geochemist's Workbench 11.0.4 geochemical simulator. Results from XRD analysis for caprock sample showed that major compositional minerals are quartz, plagioclase, and K-feldspar, and muscovite, pyrite, siderite, calcite, kaolinite and montnorillonite were included on a small scale. Results from ICP-OES analysis for brine showed that concentration of $Ca^{2+}$, $Na^+$, $K^+$ and $Mg^{2+}$ increased due to dissolution of plagioclase, K-feldspar and muscovite. Results of modeling for the period of 100 years showed that the recrystallization of kaolinite, dawsonite and beidellite, at the expense of plagioclase and K-feldspar is characteristic. Volumes of newly precipitation minerals and minerals passing into brine were balanced, so the porosity remained nearly unchanged. Experimental and modeling results indicate the interaction between caprock and $scCO_2$ during geologic carbon sequestration can exert significant impacts in brine pH and solubility/stability of minerals.

Comparative Compressional Behavior of Zeolite-W in Different Pressure-transmitting Media (제올라이트-W의 압력전달매개체에 따른 체적탄성률 비교 연구)

  • Seoung, Donghoon;Kim, Hyeonsu;Kim, Pyosang;Lee, Yongmoon
    • Korean Journal of Mineralogy and Petrology
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    • v.34 no.3
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    • pp.169-176
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    • 2021
  • This study aimed to fundamentally understand structural changes of zeolite under pressure and in the presence of different pressure-transmitting media (PTM) for application studies such as immobilization of heavy metal cation or CO2 storage using pressure. High-pressure X-ray powder diffraction study was conducted on the zeolite-W (K6.4Al6.5Si25.8O64× 15.3H2O, K-MER) to understand linear compressibility and the bulk moduli in different PTM conditions. Zeolite-w is a synthetic material having the same framework as natural zeolite merlinoite ((K, Ca0.5, Ba0.5, Na)10 Al10Si22O64× 22H2O). The space group of the sample was identified as I4/mmm belonging to the tetragonal crystal system. Water, carbon dioxide, and silicone-oil were used as pressure-transmitting media. The mixture of sample and each PTM was mounted in a diamond anvil cell (DAC) and then pressurized up to 3 GPa with an increment of ca. 0.5 GPa. Pressure-induced changes of powder diffraction patterns were measured using a synchrotron X-ray light source. Lattice constants, and bulk moduli were calculated using the Le-Bail method and the Birch-Murnaghan equation. In all PTM conditions, linear compressibility of c-axis (𝛽c) was 0.006(1) GPa-1 or 0.007(1) GPa-1. On the other hand, the linear compressibility of a(b)-axis (𝛽a) was 0.013(1) GPa-1 in silicone-oil run, which is twice more compressible than the a(b)-axis in water and carbon dioxide runs, 𝛽a = 0.006(1) GPa-1. The bulk moduli were measured as 50(3) GPa, 52(3) GPa, and 29(2) GPa in water, carbon dioxide, and silicone-oil run, respectively. The orthorhombicities of ac-plane in the water, and carbon dioxide runs were comparatively constant, near 0.350~0.353, whereas the value decreased abruptly in the silicone-oil run following formula, y = -0.005(1)x + 0.351(1) by non-penetrating pressure fluid condition.

Diversity and Biomass of Benthic Diatoms in Hampyeong Bay Tidal Flats (함평만 갯벌 저서규조류의 다양성과 생물량)

  • Lee, Hak Young
    • Korean Journal of Environmental Biology
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    • v.31 no.4
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    • pp.295-301
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    • 2013
  • The diversity and biomass distribution of benthic diatom flora at tidal flats of Hampyeong Bay were studied from 2006 to 2013 as a part of KLTER Program. A total of 83 species (77 strains in 2006, 65 strains in 2007, 41 strains in 2008, 45 strains in 2009, 54 strains in 2010, 55 strains in 2011, 56 strains in 2012 and 40 strains in 2012) were identified as benthic diatoms of Hampyeong Bay tidal flats. The most dominant species were Paralia sulcata and Cyclotella litoralis. Cyclotella sp., Diploneis sp., Entomoneis alata, Gyrosigma sp., Navicula abunda, Navicula gregaria, Navicula spp. and Nitzschia palea were the other common species which contributed to the high benthic diatom biomass in the Bay. The diversity of benthic diatoms varied according to the sample stations and seasons. The highest diversity was observed in August samples. The range of chlorophyll- a concentration in sediments of tidal flats for 8 years was 21~65 mg $m^{-2}$. The standing crops of benthic diatoms varied according to the studied stations and sampled seasons. The number of taxa and standing crops showed decreasing tendency year after year from all sampled stations. The distributions of standing crops and cell volumes of benthic diatoms showed similar pattern. The taxa and biomass of benthic diatoms showed low correlation coefficients with temperature variables expressed as the following equations $Y=-0.0208X^2+0.5264X+19.529(r^2=0.0269)$ and $Y=-0.9181X^2+27.011X+310.07(r^2=0.0797)$ respectively.

Comparison of Cytotoxin and Immune Activities between Natural and Tissue Cultured Plant in Artemisia capillaris Thunb. (자연산 및 조직배양 사철쑥의 세포독성 및 면역활성 비교)

  • Kim, Jung-Hwa;Kim, Dae-Ho;You, Jin-Hyun;Kim, Cheol-Hee;Kwon, Min-Chul;Hwang, Baik;Lee, Hyeon-Yong
    • Korean Journal of Medicinal Crop Science
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    • v.13 no.4
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    • pp.154-160
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    • 2005
  • This study was performed to compare anticancer and immune activities between natural Artemisia capillaris Thunb. extract and tissue cultured plant extract (hairy root, in vitro culture, callus). The inhibitory effect of cancer cell growth, human B cell growth and productivity of cytokines were examined. Furthermore, HPLC analysis was performed to confirm the components. The anticancer activities increased by more than 55% with the cultured callus of Artemisia capillaris T. for four cancer cell lines(Lung carcunoma, Stomach adenocarcinoma, Hepatocillular carcinoma, Breast adenocarcinoma), showing higher effect than natural Artemisia capillaris T. The extracts from hairy root and in vitro culture of Artemisia capillaris T. significantly increased the immune B cell growth. The immune B cell growth effect of natural Artemisia capillaris T. was higher than that of the tissue culture plants such as hairy root, in vitro culture and callus. Both natural and tissue cultured plants showed similar effects on cytokine secretion. The similar peak size was observed between natural Artemisia capillaris T. and cultured callus in HPLC analysis. As a results, the biological activities were not observed the difference between natural Artemisia capillaris T. and cultured callus. Thus, the cultured callus will be altered natural Artemisia capillaris T. in the environmental side and the resources preservative side

Adhesion Characteristics and the High Pressure Resistance of Biofilm Bacteria in Seawater Reverse Osmosis Desalination Process (역삼투 해수담수화 공정 내 바이오필름 형성 미생물의 부착 및 고압내성 특성)

  • Jung, Ji-Yeon;Lee, Jin-Wook;Kim, Sung-Youn;Kim, In-S.
    • Journal of Korean Society of Environmental Engineers
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    • v.31 no.1
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    • pp.51-57
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    • 2009
  • Biofouling in seawater reverse osmosis (SWRO) desalination process causes many problems such as flux decline, biodegradation of membrane, increased cleaning time, and increased energy consumption and operational cost. Therefore biofouling is considered as the most critical problem in system operation. To control biofouling in early stage, detection of the most problematic bacteria causing biofouling is required. In this study, six model bacteria were chosen; Bacillus sp., Flavobacterium sp., Mycobacterium sp., Pseudomonas aeruginosa, Pseudomonas fluorescens, and Rhodobacter sp. based on report in the literature and phylogenetic analysis of seawater intake and fouled RO membrane. The adhesion to RO membrane, the high pressure resistance, and the hydrophobicity of the six model bacteria were examined to find out their fouling potential. Rhodobacter sp. and Mycobacterium sp. were found to attach very well to RO membrane surface compared to others used in this study. The test of hydrophobicity revealed that the bacteria which have high hydrophobicity or similar contact angle with RO membrane ($63^{\circ}$ of contact angle) easily attached to RO membrane surface. P. aeruginosa which is highly hydrophilic ($23.07^{\circ}$ of contact angle) showed the least adhesion characteristic among six model bacteria. After applying a pressure of 800 psi to the sample, Rhodobacter sp. was found to show the highest reduction rate; with 59-73% of the cells removed from the membrane under pressure. P. fluorescens on the other hand analyzed as the most pressure resistant bacteria among six model bacteria. The difference between reduction rates using direct counting and plate counting indicates that the viability of each model bacteria was affected significantly from the high pressure. Most cells subjected to high pressure were unable to form colonies even thought they maintained their structural integrity.

Interleukin 10 rs1800872 T>G Polymorphism was Associated with an Increased Risk of Esophageal Cancer in a Chinese Population

  • Sun, Jia-Ming;Li, Qiong;Gu, Hai-Yong;Chen, Yi-Jang;Wei, Ji-Shu;Zhu, Quan;Chen, Liang
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.6
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    • pp.3443-3447
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    • 2013
  • Aim: Esophageal cancer is the eighth most common cancer and sixth leading cause of cancer associated death worldwide. The 5 year survival rate for esophageal cancer patients is very poor and accounts for only 12.3%. Besides environmental risk factors, genetic factors might play an important role in the esophageal cancer carcinogenesis. Methods: We conducted a hospital based case-control study to evaluate the genetic effects of functional single nucleotide polymorphisms (SNPs): interleukin 9 (IL9) rs31563 C>T, IL9 rs31564 G>T, IL10 rs1800872 T>G, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T on the development of esophageal cancer. A total of 380 esophageal squamous cell carcinoma (ESCC) cases and 380 controls were recruited for this study. The genotypes were determined using a custom-by-design 48-Plex SNPscan$^{TM}$ Kit. Results: The IL10 rs1800872 T>G polymorphism was associated with an increased risk of ESCC. However, there were no significant links with the other five SNPs. Stratified analyses indicated no significant risk of ESCC associated with the IL10 rs1800872 T>G polymorphism evident among any subgroups. Conclusion: These findings indicated that functional polymorphism IL10 rs1800872 T>G might contribute to ESCC susceptibility. However, our results were obtained with a limited sample size, so that the power of our analysis was low. Future larger studies with more rigorous study designs of other ethnic populations are required to confirm the current findings.

Comparison of the Morphological Characteristics and the 24S rRNA Sequences of Cochlodinium polykrikoides and Gyrodinium impudicum (Cochlodinium polykrikoides와 Gyrodinium impudicum 형태특성과 24S rRNA 유전자 염기서열 비교)

  • Park, Jong-Gyu;Park, Young-Shik
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.4 no.4
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    • pp.363-370
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    • 1999
  • When the first red tide alert by Cochlodinium polykrikoides was alarmed around the Oenarodo coast on Aug. 27, 1997, there co-occurred two chain-forming naked dinoflagellates which were different sized but looked fairly similar. The analyses of 24S rRNA sequences of these species showed that their gene sequences had only 74.9% identity. This low value implies that they are quite different species. After isolation and cultivation of each species, the morphological characteristics were observed. This revealed that the larger species ranging from 20 to 35 ${\mu}m$ was the well known, Cochlodinium polykrikoides and the smaller one ranging from 12 to 25 ${\mu}m$ was Gyrodinium impudicum which had not been reported in Korea. As their 24S rRNA sequences had not been analysed yet, we deposited the sequences in Genbank. At that time of the investigation. the red tide was caused by G. impudicum of which maximum cell counts reached up to 30,000 cells $ml^{-1}$. In this study we describe the morphological characteristics and the behavioral patterns of each species which can be easily observed with light microscope or stereomicroscope. In addition, their morphology transformed by the fixation with Lugol's solution are also characterized. which can help to discriminate each one in the fixed sample.

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Effects of Gamma Irradiation on the Shelf Stability of Whole Baked Egg (구운 계란의 감마선 조사에 따른 미생물학적 유통기한 설정)

  • Kim Dong-Ho;Song Hyun-Pa;Lee You-Seok;Cha Bo-Suk;Kim Byung-Keun;Byun Myung-Woo
    • Food Science and Preservation
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    • v.11 no.3
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    • pp.394-399
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    • 2004
  • The effects of gamma irradiation to improve the hygienic quality and microbiological shelf stability of whole baked egg were investigated by comparison with autoclaving process. The contamination levels of coliform, total aerobic bacteria and fungal group in a fresh egg were 10$^{5}$ CFU/g, 10$^{2}$ CFU/g and 10$^{1}$ CFU/g, respectively. After baking process, total aerobic bacteria and fungi were not exceeded to 10$^{1}$ CFU/g. Also, coliform was not detected under the aseptic process. However, cell counts of the baked egg after packaging reached to 10$^{4}$ CFU/g of total aerobic bacteria, 10$^{1}$ CFU/g of coliform, and 10$^{2}$ CFU/g of fungi. Therefore, it was assumed that microbial contamination of baked and packaged egg was mainly originated from an environmental uptake during packaging process. Microbiological shelf stability of the non sterilized control was about a week. Whereas, the baked eggs irradiated at more than 5 kGy were stable over 12 weeks at ambient condition as like those being autoclaved. Analytical texture profIle was stable within 10 kGy, but it became hardened in the sample treated with autoclaving. About 67$\%$ of panelists identified a sensory difference between non-irradiated and 10 kGy-irradiated sample. The baked egg irradiated at 10 kGy and autoclaved had lower acceptability than the control or samples irradiated lower than 5 kGy. Therefore, it was considered that optimal irradiation dose for radiation sterilization of baked and packaged egg was 5 kGy. At that point, it was recommended that appropriate microbiological shelf-life was 12 weeks at ambient condition.