• Title/Summary/Keyword: Q-PCR

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Integrative analysis of cellular responses of Pseudomonas sp. HK-6 to explosive RDX using its xenA knockout mutant (Pseudomonas sp. HK-6의 xenA 돌연변이체를 이용하여 RDX 폭약에 노출된 세포반응들의 통합적 분석)

  • Lee, Bheong-Uk;Choi, Moon-Seop;Seok, Ji-Won;Oh, Kye-Heon
    • Korean Journal of Microbiology
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    • v.54 no.4
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    • pp.343-353
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    • 2018
  • Our previous research demonstrated the essential role of the xenB gene in stress response to RDX by using Pseudomonas sp. HK-6 xenB knockout. We have extended this work to examine the cellular responses and altered proteomic profiles of the HK-6 xenA knockout mutant under RDX stress. The xenA mutant degraded RDX about 2-fold more slowly and its growth and survival rates were several-fold lower than the wild-type HK-6 strain. SEM revealed more severe morphological damages on the surface of the xenA mutant cells under RDX stress. The wild-type cells expressed proportionally-increased two stress shock proteins, DnaK and GroEL from the initial incubation time point or the relatively low RDX concentrations, but slightly less expressed at prolonged incubation period or higher RDX. However the xenA mutant did not produced DnaK and GroEL as RDX concentrations were gradually increased. The wild-type cells well maintained transcription levels of dnaA and groEL under increased RDX stress while those in the xenA mutant were decreased and eventually disappeared. The altered proteome profiles of xenA mutant cells under RDX stress also observed so that the 27 down-regulated plus the 3 up-regulated expression proteins were detected in 2-DE PAGE. These all results indicated that the intact xenA gene is necessary for maintaining cell integrity under the xenobiotic stress as well as performing an efficient RDX degradation process.

Inhibition of miR-128 Abates Aβ-Mediated Cytotoxicity by Targeting PPAR-γ via NF-κB Inactivation in Primary Mouse Cortical Neurons and Neuro2a Cells

  • Geng, Lijiao;Zhang, Tao;Liu, Wei;Chen, Yong
    • Yonsei Medical Journal
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    • v.59 no.9
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    • pp.1096-1106
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    • 2018
  • Purpose: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. Materials and Methods: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. $PPAR-{\gamma}$ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and $NF-{\kappa}B$ activity was determined by a Caspase 3 Activity Assay Kit or $NF-{\kappa}B$ p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and $PPAR-{\gamma}$ 3'UTR. Results: MiR-128 expression was upregulated and $PPAR-{\gamma}$ expression was downregulated in plasma from AD patients and $amyloid-{\beta}$ $(A{\beta})-treated$ primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased $A{\beta}-mediated$ cytotoxicity through inactivation of $NF-{\kappa}B$ in MCN and N2a cells. Moreover, $PPAR-{\gamma}$ was a target of miR-128. $PPAR-{\gamma}$ upregulation attenuated $A{\beta}-mediated$ cytotoxicity by inactivating $NF-{\kappa}B$ in MCN and N2a cells. Furthermore, $PPAR-{\gamma}$ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and $NF-{\kappa}B$ activity in MCN and N2a cells. Conclusion: MiR-128 inhibitor decreased $A{\beta}-mediated$ cytotoxicity by upregulating $PPAR-{\gamma}$ via inactivation of $NF-{\kappa}B$ in MCN and N2a cells, providing a new potential target in AD treatment.

Comparison of the spatial-temporal distributions of the heterotrophic dinoflagellates Gyrodinium dominans, G. jinhaense, and G. moestrupii in Korean coastal waters

  • Lee, Sung Yeon;Jeong, Hae Jin;Kang, Hee Chang;Ok, Jin Hee;You, Ji Hyun;Park, Sang Ah;Eom, Se Hee
    • ALGAE
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    • v.36 no.1
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    • pp.37-50
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    • 2021
  • Heterotrophic dinoflagellates Gyrodinium spp. are one of the major grazers of phytoplankton in many coastal waters. Gyrodinium dominans, G. jinhaense, and G. moestrupii have similar morphologies but different edible prey species. To explore the variations in the ecological niches of these three species, we investigated their spatial-temporal distributions in Korean waters. Because of the high similarity in morphology among these three Gyrodinium species, we used real-time polymerase chain reactions to quantify their abundance in water samples that were seasonally collected from 28 stations along the Korean Peninsula from April 2015 to October 2018. Cells of G. dominans were found at all sampling stations, G. jinhaense at 26 stations, and G. moestrupii at 22 stations, indicating that all three species were widely distributed in Korea. Furthermore, all three species displayed strong seasonal distributions. The largest numbers of the stations where G. dominans and G. jinhaense cells were present were found during the summer (26 and 23 stations, respectively), but that for G. moestrupii was found in the autumn (15 stations). The abundance of G. dominans was positively correlated with that of G. jinhaense, but not with that of G. moestrupii. The highest abundances of G. dominans (202.5 cells mL-1) and G. jinhaense (20.2 cells mL-1) were much greater than that of G. moestrupii (1.2 cells mL-1). The highest abundances of G. dominans and G. jinhaense were found in July, whereas that of G. moestrupii was found in March. The abundances of G. dominans and G. jinhaense, but not G. moestrupii, were positively correlated with water temperature. Therefore, the spatial-temporal distributions of G. dominans and G. jinhaense were closer than those of G. moestrupii and G. dominans or G. jinhaense. This differs from results based on the relative differences in ribosomal DNA sequences and the types of edible prey reported in the literature. Thus, the variations in spatial-temporal distributions and prey species of these three Gyrodinium species suggest that they may have different ecological niches in Korean coastal waters.

Heat Shock Protein 90 Gene Expression in Juvenile Sea Cucumber Apostichopus japonicus (Echinodermata; Holothuroidea) according to Releasing Methods (어린 돌기해삼 Apostichopus japonicus (Echinodermata; Holothuroidea) 방류 방법에 따른 열충격단백질90 유전자의 발현 분석)

  • Lee, Dong-Han;Lee, Seungheon;Jeong, Dong-Bin;Sohn, Young Chang
    • Journal of Marine Life Science
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    • v.7 no.1
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    • pp.29-36
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    • 2022
  • Sea cucumber, Aposticopus japonicus, is a major invertebrate species in the coastal regions of Korea. To evaluate the short-term stress levels according to the releasing methods, this study investigated the gene expression profiles of heat shock protein 90 (HSP90) by real-time quantitative polymerase chain reaction. When the juvenile sea cucumbers were packed in the vinyl bag with oxygen followed by transportation for 30 min or air-exposed for 1 h, the HSP90 gene expression levels in the experimental groups were significantly increased compared to those of the control groups (transported group, p=0.001; air-exposed group, p=0.032). The experimental group at 6 h post-release by seed-spreading method and at 2~6 h post-release by underwater hose-releasing method on board a fishing boat showed that the levels of HSP90 gene expression were not statistically significant but decreased slightly compared to the control group (seed-spreading group, p=0.069; hose-releasing group, p=0.093). On the other hand, the HSP90 gene expression showed an increasing pattern as the time passed (~6 h) after underwater release of juvenile sea cucumbers by divers (p=0.061). These results suggest that HSP90 gene expression can be used to investigate short-term stress response and effective releasing methods of juvenile sea cucumbers.

Effect of Halophyte (Spartina anglica and Calystegia soldanella) Extracts on Skin Moisturizing and Barrier Function in HaCaT Cells (염생식물인 갯끈풀과 갯메꽃 추출물의 HaCaT 세포에서 피부 보습 및 피부 장벽 기능에 미치는 영향)

  • Ha, Yuna;Jeong, JaeWoo;Lee, Won Hwi;Oh, Jun Hyuk;Kim, Youn-Jung
    • Journal of Marine Life Science
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    • v.6 no.2
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    • pp.58-65
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    • 2021
  • As aging progresses, reactive oxygen species (ROS) reduces skin moisturization and collapses skin barrier function. In this study, we evaluated the efficacy of skin moisturizing and skin barrier function enhancement by extracts from halophytes using HaCaT cells. Spartina anglica (S. anglica; SAE) and Calystegia soldanella (C. soldanella; CSE), a kind of halophytes, were collected from Dongmak beach in Incheon, and extracted with 70% ethanol. At the first, we evaluated the cytotoxicity of extracts in HaCaT cell using WST-8 Kit. As a result, the other experiment was conducted by setting the concentration at which the cell viability was 90% or more. SAE and CSE showed high radical scavenging activity through ABTS assay. Expression levels of genes related to skin moisturizing and skin barrier functions, were analyzed by real-time qPCR. As a result, it showed that the expression of aquaporin 3, hyaluronan synthase 2, and transglutaminase 1 was increased by SAE treatment but not changed by CSE. Activation of extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen activated protein kinase was induced by SAE. These results suggest that SAE can be used as functional materials for cosmetics for skin moisturizing and barrier function enhancement.

Gaseous signal molecule SO2 regulates autophagy through PI3K/AKT pathway inhibits cardiomyocyte apoptosis and improves myocardial fibrosis in rats with type II diabetes

  • Zhao, Junxiong;Wu, Qian;Yang, Ting;Nie, Liangui;Liu, Shengquan;Zhou, Jia;Chen, Jian;Jiang, Zhentao;Xiao, Ting;Yang, Jun;Chu, Chun
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.6
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    • pp.541-556
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    • 2022
  • Myocardial fibrosis is a key link in the occurrence and development of diabetic cardiomyopathy. Its etiology is complex, and the effect of drugs is not good. Cardiomyocyte apoptosis is an important cause of myocardial fibrosis. The purpose of this study was to investigate the effect of gaseous signal molecule sulfur dioxide (SO2) on diabetic myocardial fibrosis and its internal regulatory mechanism. Masson and TUNEL staining, Western-blot, transmission electron microscopy, RT-qPCR, immunofluorescence staining, and flow cytometry were used in the study, and the interstitial collagen deposition, autophagy, apoptosis, and changes in phosphatidylinositol 3-kinase (PI3K)/AKT pathways were evaluated from in vivo and in vitro experiments. The results showed that diabetic myocardial fibrosis was accompanied by cardiomyocyte apoptosis and down-regulation of endogenous SO2-producing enzyme aspartate aminotransferase (AAT)1/2. However, exogenous SO2 donors could up-regulate AAT1/2, reduce apoptosis of cardiomyocytes induced by diabetic rats or high glucose, inhibit phosphorylation of PI3K/AKT protein, up-regulate autophagy, and reduce interstitial collagen deposition. In conclusion, the results of this study suggest that the gaseous signal molecule SO2 can inhibit the PI3K/AKT pathway to promote cytoprotective autophagy and inhibit cardiomyocyte apoptosis to improve myocardial fibrosis in diabetic rats. The results of this study are expected to provide new targets and intervention strategies for the prevention and treatment of diabetic cardiomyopathy.

Ginsenoside Rg1 alleviates Aβ deposition by inhibiting NADPH oxidase 2 activation in APP/PS1 mice

  • Zhang, Han;Su, Yong;Sun, Zhenghao;Chen, Ming;Han, Yuli;Li, Yan;Dong, Xianan;Ding, Shixin;Fang, Zhirui;Li, Weiping;Li, Weizu
    • Journal of Ginseng Research
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    • v.45 no.6
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    • pp.665-675
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    • 2021
  • Background: Ginsenoside Rg1 (Rg1), an active ingredient in ginseng, may be a potential agent for the treatment of Alzheimer's disease (AD). However, the protective effect of Rg1 on neurodegeneration in AD and its mechanism of action are still incompletely understood. Methods: Wild type (WT) and APP/PS1 AD mice, from 6 to 9 months old, were used in the experiment. The open field test (OFT) and Morris water maze (MWM) were used to detect behavioral changes. Neuronal damage was assessed by hematoxylin and eosin (H&E) and Nissl staining. Immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction (q-PCR) were used to examine postsynaptic density 95 (PSD95) expression, amyloid beta (Aβ) deposition, Tau and phosphorylated Tau (p-Tau) expression, reactive oxygen species (ROS) production, and NAPDH oxidase 2 (NOX2) expression. Results: Rg1 treatment for 12 weeks significantly ameliorated cognitive impairments and neuronal damage and decreased the p-Tau level, amyloid precursor protein (APP) expression, and Aβ generation in APP/PS1 mice. Meanwhile, Rg1 treatment significantly decreased the ROS level and NOX2 expression in the hippocampus and cortex of APP/PS1 mice. Conclusions: Rg1 alleviates cognitive impairments, neuronal damage, and reduce Aβ deposition by inhibiting NOX2 activation in APP/PS1 mice.

Comparison of Laboratory Tests Applied for Diagnosing the SARS-CoV-2 Infection (SARS-CoV-2 감염의 진단에 이용되는 검사실 테스트의 비교)

  • Lee, Chang-Gun;Lee, Dongsup
    • Korean Journal of Clinical Laboratory Science
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    • v.54 no.2
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    • pp.79-94
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    • 2022
  • Due to the highly contagious nature and severity of the respiratory diseases caused by COVID-19, economical and accurate tests are required to better monitor and prevent the spread of this contagion. As the structural and molecular properties of SARS-CoV-2 were being revealed during the early stage of the COVID-19 pandemic, many manufacturers of COVID-19 diagnostic kits actively invested in the design, development, validation, verification, and implementation of diagnostic tests. Currently, diagnostic tests for SARS-CoV-2 are the most widely used and validated techniques for rapid antigen, and immuno-serological assays for specific IgG and IgM antibody tests and molecular diagnostic tests. Molecular diagnostic assays are the gold standard for direct detection of viral RNA in individuals suspected to be infected with SARS-CoV-2. Antibody-based serological tests are indirect tests applied to determine COVID-19 prevalence in the community and identify individuals who have obtained immunity. In the future, it is necessary to explore technical problems encountered in the early stages of global or regional outbreaks of pandemics and provide future directions for better diagnostic tests. This article evaluates the commercially available and FDA-approved molecular and immunological diagnostic assays and analyzes their performance characteristics.

A Study on Inflammation and Itching of Cyperus rotundus Ethyl Acetate Fractions (향부자 에틸아세테이트 분획물의 염증 및 가려움증에 관한 연구)

  • Do Gyu, Kim;Ji Yeon, Lee;Sohyun, Mun;Yukyung, Kim;Nari, Kim;Ah Reum, Jung;Jun-Hwan, Jang;Jae-Soeb, Lee;Sang Bae, Han;Jun-Tae, Bae
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.48 no.4
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    • pp.303-312
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    • 2022
  • In this study, Cyperus rotundus (C. rotundus) was fractionated into ethyl acetate to identify α-cyperone, a representative indicator, and its ethyl acetate fraction were evaluated to confirm the possibilityas a functional cosmetic ingredient. As a result of HPLC analysis, it was confirmed that the content of α-cyperone was 5.243%. In order to verify the inflammatory relief effect of the ethyl acetate fraction from C. rotundus (EAFC) and α-cyperone, it was confirmed that nitric oxide (NO) production inhibitory ability in RAW 264.7 macrophages induced an inflammatory reaction with lipopolysaccharide (LPS). Real-time qPCR analysis confirmed inhibition of mRNA expression level of inflammatory factors, IL-1β, IL-6, TNF-α, COX-2 and iNOS. As a results of conducting a clinical study using a simple cosmetic formulation containing EAFC, it was confirmed that the skin irritation stimulated by sodium lauryl sulfate (SLS) was calming and relieving itching. Through these results, it is believed that the C. rotundus can be used as a natural cosmetic ingredient that has the effect of inhibiting inflammation and relieving itching.

Comparative metabolomic analysis in horses and functional analysis of branched chain (alpha) keto acid dehydrogenase complex in equine myoblasts under exercise stress

  • Jeong-Woong, Park;Kyoung Hwan, Kim;Sujung, Kim;Jae-rung, So;Byung-Wook, Cho;Ki-Duk, Song
    • Journal of Animal Science and Technology
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    • v.64 no.4
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    • pp.800-811
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    • 2022
  • The integration of metabolomics and transcriptomics may elucidate the correlation between the genotypic and phenotypic patterns in organisms. In equine physiology, various metabolite levels vary during exercise, which may be correlated with a modified gene expression pattern of related genes. Integrated metabolomic and transcriptomic studies in horses have not been conducted to date. The objective of this study was to detect the effect of moderate exercise on the metabolomic and transcriptomic levels in horses. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we analyzed the concentrations of metabolites in muscle and plasma; we also determined the gene expression patterns of branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK), which encodes the key regulatory enzymes in branched-chain amino acid (BCAA) catabolism, in two breeds of horses, Thoroughbred and Jeju, at different time intervals. The concentrations of metabolites in muscle and plasma were measured by 1H NMR (nuclear magnetic resonance) spectroscopy, and the relative metabolite levels before and after exercise in the two samples were compared. Subsequently, multivariate data analysis based on the metabolic profiles was performed using orthogonal partial least square discriminant analysis (OPLS-DA), and variable important plots and t-test were used for basic statistical analysis. The stress-induced expression patterns of BCKDK genes in horse muscle-derived cells were examined using quantitative reverse transcription polymerase chain reaction (qPCR) to gain insight into the role of transcript in response to exercise stress. In this study, we found higher concentrations of aspartate, leucine, isoleucine, and lysine in the skeletal muscle of Jeju horses than in Thoroughbred horses. In plasma, compared with Jeju horses, Thoroughbred horses had higher levels of alanine and methionine before exercise; whereas post-exercise, lysine levels were increased. Gene expression analysis revealed a decreased expression level of BCKDK in the post-exercise period in Thoroughbred horses.