• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.261-276
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• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

• Korean Journal of Food Science and Technology
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• v.30 no.6
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• pp.1480-1483
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• 1998

To know the probable storage conditions for commercially prepared ‘baked egg’ and ‘rolled egg’ products, microbes and quality changes for samples immediately after the production and stored under the several conditions were tested. Sealed samples were stored at 5, 10, 15, $20^{\circ}C$, respectively and tested at 1 or 2-days intervals, opened samples were stored at 15, 20, 30, $37^{\circ}C$, respectively and tested at 6-hr intervals. Coliform bacteria, E. coli, Salmonella, Staphylococci were not detected, and viable cell counts level was under $10^2\;CFU/g$ for samples immediately after the production. The higher the storage temperature and baked eggs rether than rolled eggs showed more rapid microbial growth for both samples. For sealed condition, 14-days at $5^{\circ}C$ for both samples, 7-days for rolled eggs and 5-days for baked eggs at $10^{\circ}C$ were kept their initial microbial level and favorable flavor. For opened condition, it showed remarkable microbial increase after $18{\sim}24\;hr$ for $15{\sim}20^{\circ}C$ and 12 hr for $30{\sim}37^{\circ}C$. From these results, used egg products samples are able to be stored more than 1-week at below $5^{\circ}C$ and opened samples need to be stored at cold storage. At room temperature, it egg products.

• Journal of Marine Bioscience and Biotechnology
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• v.15 no.2
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• pp.41-48
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• 2023

The objective of the present study was to improve the biomass productivity of newly isolated freshwater green microalga Parachlorella sp. This was accomplished by culture conditions optimization, including CO₂ concentration, superficial gas velocity, and light intensity, in 0.5 L bubble column photobioreactors. The supplied CO₂ concentration and gas velocity varied from 0.032% (air) to 10% and 0.02 m/s - 0.11 m/s, respectively, to evaluate their effects on growth kinetics. Next, to maximize the production rate of Parachlorella sp., a lumostatic operation based on a specific light uptake rate (q_e) was applied. From these results, the optimal CO₂ concentration in the supplied gas and the gas velocity were determined to be 5% and 0.064 m/s, respectively. For the lumostatic operation at 10.2 µmol/g/s, biomass productivity and photon yield showed significant increases of 83% and 66%, respectively, relative to cultures under constant light intensity. These results indicate that the biomass productivity of Parachlorella sp. can be improved by optimizing gas properties and light control as cell concentrations vary over time.

• Korean Journal of Food Science and Technology
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• v.43 no.1
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• pp.98-104
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• 2011

The storage quality of fresh buckwheat sprouts, as influenced by pretreatment and packaging within processing steps, was investigated to establish appropriate postharvest handling treatment for the commodity. After harvest, the sprouts were dipped in chlorine water (100 ppm), rinsed twice with clean water, pre-cooled with iced water, de-watered, and packed in plastic trays. Sprout samples taken from each processing step were stored at $5^{\circ}C$ for 6 days to measure quality attributes. Viable cell counts of mesophilic aerobes and coliform bacteria were lower by about 1 log scale in the postharvest treated samples compared to an untreated control, although the initial microbial reduction due to the postharvest treatments was offset by cell growth during storage. All sprout samples showed a decrease of fresh weight by approximately 4% after 6 days of storage. However, moisture and soluble solid contents were maintained at the initial levels of the sprouts. No significant difference in surface color was observed among sample treatments. For sensory properties including discoloration, wilting, decay, and visual quality, there were no significant differences among sample treatments. The present results suggest that proper postharvest processing treatments can exert positive effects on extending the shelf-life of fresh buckwheat sprout.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1459-1469
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• 2008

Using a rotating biological contactor modified with a sequencing bath reactor system (SBRBC) designed and operated to remove phosphate and nitrogen [58], the microbial community structure of the biofilm from the SBRBC system was characterized based on the extracellular polymeric substance (EPS) constituents, electron microscopy, and molecular techniques. Protein and carbohydrate were identified as the major EPS constituents at three different biofilm thicknesses, where the amount of EPS and bacterial cell number were highest in the initial thickness of 0-100${\mu}m$. However, the percent of carbohydrate in the total amount of EPS decreased by about 11.23%, whereas the percent of protein increased by about 11.15% as the biofilm grew. Thus, an abundant quantity of EPS and cell mass, as well as a specific quality of EPS were apparently needed to attach to the substratum in the first step of the biofilm growth. A FISH analysis revealed that the dominant phylogenetic group was $\beta$- and $\gamma$-Proteobacteria, where a significant subclass of Proteobacteria for removing phosphate and/or nitrate was found within a biofilm thickness of 0-250${\mu}m$. In addition, 16S rDNA clone libraries revealed that Klebsiella sp. and Citrobacter sp. were most dominant within the initial biofilm thickness of 0-250${\mu}m$, whereas sulfur-oxidizing bacteria, such as Beggiatoa sp. and Thiothrix sp., were detected in a biofilm thickness over 250${\mu}m$. The results of the bacterial community structure analysis using molecular techniques agreed with the results of the morphological structure based on scanning electron microscopy. Therefore, the overall results indicated that coliform bacteria participated in the nitrate and phosphorus removal when using the SBRBC system. Moreover, the structure of the biofilm was also found to be related to the EPS constituents, as well as the nitrogen and phosphate removal efficiency. Consequently, since this is the first identification of the bacterial community and structure of the biofilm from an RBC simultaneously removing nitrogen and phosphate from domestic wastewater, and it is hoped that the present results may provide a foundation for understanding nitrate and phosphate removal by an RBC system.

• Korean Journal of Food Science and Technology
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• v.31 no.3
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• pp.788-793
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• 1999

In this study, it was investigated that the isolation and identification of spoilage bacteria from contaminated aseptic packaged cooked rice, the potential for application of acidic electrolyzed saline solution (AESS) as water-for-cooked rice and the microbiological safety of AESS-based cooked rice. Five strains of Bacillus subtilis and a B. cereus strain among the total six isolates were partially identified by biochemical method and by Microbial Identification System (MIS). The bactericidal effect of AESS was similar as 0.1% NaOCl and 70% ethanol solution, or better than that. All of the test microorganisms except Bacillus spp. that were exposed to AESS for five seconds were destructed. The effect of AESS against Bacillus spp. was much better than that of the two solutions and all of them were destructed or inhibited on exposure for five minutes. The pH value of cooked rice prepared using AESS was in the range of 3.6 to 4.3 and was not almost changed through the storage period. Various concentrations of cell suspension of Bacillus isolates were inoculated to cooked rice, which were prepared with tap water and AESS, and stored at $37^{\circ}C$ for two weeks. The result was shown that the bacteria in tap water-based cooked rice appeared normal cell growth, while they were completely repressed in AESS-based cooked rice.

• Journal of Applied Biological Chemistry
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• v.55 no.2
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• pp.117-121
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• 2012

Biological activities of the hot water and ethanol extracts from Opuntia humifusa cladodes were investigated. 1,1-diphenyl-2-picryl hadrazyl (DPPH) electron donating ability of hot water and ethanol extracts was 79.07 and 82.54%, respectively. Hot water extract generally showed better cytotoxic activity than ethanol extract against each cell line. HeLa and AGS cell lines treated with hot water extract had more than 50% cytotoxic activities. Based on the antimicrobial activities against four microbial strains, both extracts inhibited growth of Staphylococcus aureus KCCM 11593, whereas affected cell growth of three other microorganisms, Escherichia coli (KCCM 11234), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhimurium (ATCC 11862), in proportion to the concentration of extracts. The inflammatory activities against hot water extract (34.31%) showed higher than that of ethanol extract (25.59%). The effect of extracts on 3T3-L1 preadipocytes differentiation showed that differentiation of treated group with 80 and 100 ${\mu}g/mL$ of hot and ethanol extracts were increased more than treated group with isobutyl methyl xanthine (IBMX) + dexamethasone. These results indicate that the O. humifusa cladodes extracts can be used as a functional material due to their effective biological activities.

• Economic and Environmental Geology
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• v.42 no.5
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• pp.427-434
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• 2009

Indigenous bacterium which shows a tolerance to high metal toxicity was isolated from soil concomitantly contaminated with oil and heavy metals. The characteristics of the bacterium for Pb and Cd biosorption was investigated under the various experimental conditions such as bacterial growth phase, the initial metal concentration, the input biomass amount, temperature and pH. The Langmuir adsorption isotherm modeling was described to know the capacity and intensity of biosorption. The low initial concentration of heavy metals and high biomass has a maximum heavy metal removal efficiency, but biosorption capacity of Pb and Cd has different values. Biosorption efficiency was highest in the end of the microbial growth stage and under pH 5~9 condition, but was less affected by temperature variation of 25~$35^{\circ}C$. The maximum biosorption capacity for Pb and Cd was 62.11 and 192.31 mg/g, respectively and each $R^2$ was calculated as 0.71 and 0.98 by applying Langmuir isothermal adsorption equation. Biosorption for Cd was considered as monomolecular adsorption to single layer on the surface of cells, whereas biosorption for Pb was considered as accumulation process into the cell by the microbial metabolism and precipitation reaction with anion of bacteria.

• Journal of Life Science
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• v.31 no.5
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• pp.520-526
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• 2021

Microbial protein is one of the sources of protein in the rumen and can also be the source of glutamate production. Glutamic acid is used as fuel in the metabolic reaction in the body and the synthesis of all proteins for muscle and other cell components, and it is essential for proper immune function. Moreover, it is used as a surfactant, buffer, chelating agent, flavor enhancer, and culture medium, as well as in agriculture for such things as growth supplements. Glutamic acid is a substrate in the bioproduction of gamma-aminobutyric acid (GABA). This review provides insights into the role of glutamic acid and glutamic acid-producing microorganisms that contain the glutamate decarboxylase gene. These glutamic acid-producing microorganisms could be used in producing GABA, which has been known to regulate body temperature, increase DM intake and milk production, and improve milk composition. Most of these glutamic acid and GABA-producing microorganisms are lactic acid-producing bacteria (LAB), such as the Lactococcus, Lactobacillus, Enterococcus, and Streptococcus species. Through GABA synthesis, succinate can be produced. With the help of succinate dehydrogenase, propionate, and other metabolites can be produced from succinate. Furthermore, clostridia, such as Clostridium tetanomorphum and anaerobic micrococci, ferment glutamate and form acetate and butyrate during fermentation. Propionate and other metabolites can provide energy through conversion to blood glucose in the liver that is needed for the mammary system to produce lactose and live weight gain. Hence, health status and growth rates in ruminants can be improved through the use of these glutamic acid and/or GABA-producing microorganisms.