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A Fluorescence-based cDNA-AFLP Method for Identification of Differentially Expressed Genes

  • Park, Sook-Young;Jwa, Nam-Soo;Chi, Myoung-Hwan;Lee, Yong-Hwan
    • The Plant Pathology Journal
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    • v.25 no.2
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    • pp.184-188
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    • 2009
  • Identification of differently expressed genes under specific tissues and/or environments provides insights into the nature and underlying mechanisms of cellular processes. Although cDNA-AFLP (Amplified Fragment Length Polymorphism) is a powerful method for analyzing differentially expressed genes, its use has been limited to the requirement of radioactive isotope use and the difficulty of isolating the bands of interest from a gel. Here, we describe a modified method for cDNA-AFLP that uses a fluorescence dye for detection and isolation of bands directly from a small size polyacrylamide gel. This method involves three steps: (i) preparation of cDNA templates, (ii) PCR amplification and differential display, and (iii) identification of differentially expressed genes. To demonstrate its utility and efficiency, differentially expressed genes during vegetative growth and appressorial development of Magnaporthe oryzae were analyzed. This method could be applied to compare gene expression profiles in a diverse array of organisms.

Characterization of the Vertical Position of the Trapped Charge in Charge-trap Flash Memory

  • Kim, Seunghyun;Kwon, Dae Woong;Lee, Sang-Ho;Park, Sang-Ku;Kim, Youngmin;Kim, Hyungmin;Kim, Young Goan;Cho, Seongjae;Park, Byung-Gook
    • JSTS:Journal of Semiconductor Technology and Science
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    • v.17 no.2
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    • pp.167-173
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    • 2017
  • In this paper, the characterization of the vertical position of trapped charges in the charge-trap flash (CTF) memory is performed in the novel CTF memory cell with gate-all-around structure using technology computer-aided design (TCAD) simulation. In the CTF memories, injected charges are not stored in the conductive poly-crystalline silicon layer in the trapping layer such as silicon nitride. Thus, a reliable technique for exactly locating the trapped charges is required for making up an accurate macro-models for CTF memory cells. When a programming operation is performed initially, the injected charges are trapped near the interface between tunneling oxide and trapping nitride layers. However, as the program voltage gets higher and a larger threshold voltage shift is resulted, additional charges are trapped near the blocking oxide interface. Intrinsic properties of nitride including trap density and effective capture cross-sectional area substantially affect the position of charge centroid. By exactly locating the charge centroid from the charge distribution in programmed cells under various operation conditions, the relation between charge centroid and program operation condition is closely investigated.

Effects of Searing Cooking on Sensory and Physicochemical Properties of Beef Steak

  • Yoo, Ji Hyun;Kim, Ji Won;Yong, Hae In;Baek, Ki Ho;Lee, Hyun Jung;Jo, Cheorun
    • Food Science of Animal Resources
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    • v.40 no.1
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    • pp.44-54
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    • 2020
  • The purpose of the present study was to evaluate the sensory and instrumental quality of thawed beef steak prepared by searing and oven cooking. Beef purchased in the local market was divided into two groups; one group was cooked in a 180℃ oven until the internal temperature reached 60℃, and the other group was oven cooked until 35℃, then cooked in a 250℃ pan until the internal temperature reached 60℃. Despite a noticeable change in appearance due to the high temperature of the searing, there was no significant difference in juiciness, water content, and cooking loss between the searing-cooked and the oven-cooked steaks. However, in searing cooking, both scores of overall flavor and roast meat flavor were significantly higher than those of oven cooking. In the searing-cooked steak, the reducing sugar, which is a reactant of the Maillard reaction, was lower and Maillard-reaction products were higher than oven-cooked steak. From our results, it can be concluded that searing does not improve juiciness of the steak, but improves the flavor of beef steak due to higher levels of Maillard reaction products.

Clinical Analysis of Epidural Fluid Collection as a Complication after Cranioplasty

  • Kim, Seung Pil;Kang, Dong Soo;Cheong, Jin Hwan;Kim, Jung Hee;Song, Kwan Young;Kong, Min Ho
    • Journal of Korean Neurosurgical Society
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    • v.56 no.5
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    • pp.410-418
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    • 2014
  • Objective : The epidural fluid collection (EFC) as a complication of cranioplasty is not well-described in the literature. This study aimed to identify the predictive factors for the development of EFC as a complication of cranioplasty, and its outcomes. Methods : From January 2004 to December 2012, 117 cranioplasty were performed in our institution. One-hundred-and-six of these patients were classified as either having EFC, or not having EFC. The two groups were compared to identify risk factors for EFC. Statistical significance was tested using the t-test and chi-square test, and a logistic regression analysis. Results : Of the 117 patients undergoing cranioplasty, 59 (50.4%) suffered complications, and EFC occurred in 48 of the patients (41.0%). In the t-test and chi-test, risk factors for EFC were size of the skull defect (p=0.003) and postoperative air bubbles in the epidural space (p<0.001). In a logistic regression, the only statistically significant factor associated with development of EFC was the presence of postoperative air bubbles. The EFC disappeared or regressed over time in 30 of the 48 patients (62.5%), as shown by follow-up brain computed tomographic scan, but 17 patients (35.4%) required reoperation. Conclusion : EFC after cranioplasty is predicted by postoperative air bubbles in the epidural space. Most EFC can be treated conservatively. However, reoperation is necessary to resolve about a third of the cases. During cranioplasty, special attention is required when the skull defect is large, since EFC is then more likely.

Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

  • Min, Hophil;Han, Dohyun;Kim, Yikwon;Cho, Jee Yeon;Jin, Jonghwa;Kim, Youngsoo
    • Molecules and Cells
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    • v.37 no.6
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    • pp.457-466
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    • 2014
  • Proteomic analysis is helpful in identifying cancerassociated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine metastatic process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials - NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage IV). We identified 2130 proteins, 1355 of which were common to both cell lines. In the label-free quantitative analysis, we used the NSAF normalization method, resulting in 242 differential expressed proteins. For the N-terminal proteome analysis, 325 N-terminal peptides, including 45 novel fragments, were identified in the 2 cell lines. Based on two proteomic analysis, 11 quantitatively expressed proteins and 8 N-terminal peptides were enriched for the focal adhesion pathway. Most proteins from the quantitative analysis were upregulated in metastatic cancer cells, whereas novel fragment of CRKL was detected only in primary cancer cells. This study increases our understanding of the NSCLC metastasis proteome.

Identification and Functional Analysis of Vibrio vulnificus SmcR, a Novel Global Regulator

  • Lee, Jeojng-Hyun;Rhee, Jee-Eun;Park, U-Ryung;Ju, Hyun-Mok;Lee, Byung-Cheol;Kim, Tae-Sung;Jeong, Hye-Sook;Choi, Sang-Ho
    • Journal of Microbiology and Biotechnology
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    • v.17 no.2
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    • pp.325-334
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    • 2007
  • Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the smcR mutant was approximately $10^2$ times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.

Nurses' Job Analysis in a Regional Emergency Center Using the DACUM (데이컴 기법을 이용한 일개 권역응급의료센터 간호사의 직무 분석)

  • Yun, Hee Je;Jung, Eun Hee;Jung, Woon Seon;Lee, Sang Rim;Kwon, Young Ok
    • Journal of Korean Clinical Nursing Research
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    • v.26 no.1
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    • pp.26-46
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    • 2020
  • Purpose: This study aimed to establish the role of and to analyze the job of nurses in a regional emergency center using the Developing a Curriculum(DACUM) process, a job analysis technique. Methods: The DACUM committee was organized, and three workshops were held from June 1st to July 4th, 2016. One hundred two nurses in a regional emergency center participated in a survey from October 3rd to 10th, 2016. The frequency, importance and difficulty of each tasks and task elements in the DACUM were investigated on a Likert scale. Results: The roles of nurses in a regional emergency center were identified and the DACUM chart was developed with 6 duties, 29 tasks and 153 task elements. According to the survey, some tasks such as 'CPR,' 'acute respiratory distress care,' and 'drug administration' showed high frequency and importance or difficulty in all nursing units in common, and different results reflected the characteristics of each nursing unit were also found. Conclusion: The results showed the feature of the regional emergency center. Based on these results, it is necessary to develop educational programs for practice.

Effect of p38 inhibitor on the proliferation of chicken muscle stem cells and differentiation into muscle and fat

  • Minkyung, Ryu;Minsu, Kim;Hyun Young, Jung;Cho Hyun, Kim;Cheorun, Jo
    • Animal Bioscience
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    • v.36 no.2
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    • pp.295-306
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    • 2023
  • Objective: Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Methods: Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. Results: The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. Conclusion: p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.

Pro-inflammatory Cytokine Expression Through NF-${\kappa}B/I{\kappa}B$ Pathway in Lung Epithelial Cells (폐 상피세포에서 NF-${\kappa}B/I{\kappa}B$ 경로에 의한 염증매개 사이토카인의 발현)

  • Park, Gye-Young;Lee, Seung-Hee;HwangBo, Bin;Yim, Jae-Joon;Lee, Choon-Taek;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo;Yoo, Chul-Gyu
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.3
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    • pp.332-342
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    • 2000
  • Background : The importance of pro-inflammatory cytokines, especially tumor necrosis factor $\alpha$ (INF-$\alpha$) and interleukin-1$\beta$ (IL-1$\beta$), have been extensively documented in the generation of inflammatory lung disease. Lung epithelial cells are also actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. Understanding the mechanism of pro-inflammatory cytokine expression in lung epithelial cells is crucial to the development of new therapeutic modalities for inflammatory lung disease. Transcription of most pro-inflammatory cytokines is dependent on the activation of NF-${\kappa}B$. However, the relationship between pro-inflammatory cytokine expression and NF-${\kappa}B/I{\kappa}B$ pathway in lung epithelial cells is not clear. Methods : BEAS-2B, A549, Na-H157, NCI-H719 cells were stimulated with IL-$1{\beta}$ or TNF-$\alpha$ at various times, and then IL-8 and TNF-$\alpha$mRNA expressions were assayed by Northern blot analysis. IL-$1{\beta}$ or TNF-$\alpha$-induced NF-${\kappa}B$ activation was assessed by the nuclear translocation of p65 NF-${\kappa}B$ subunit. The degradation of $I{\kappa}B{\alpha}$ and $I{\kappa}B{\beta}$ by IL-$1{\beta}$ or TNF-$\alpha$stimulation was assayed by Western blot analysis. The phosphorylation of $I{\kappa}B{\alpha}$ was evaluated by Western blot analysis after pre-treating cells with proteasome inhibitor followed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation. The basal level of IKK $\alpha$ expression was evaluated by Western blot analysis. Results: $I{\kappa}B{\alpha}$ and $I{\kappa}B{\alpha}$ was rapidly degraded after 5 minutes of incubation with IL-$1{\beta}$ or TNF-$\alpha$ in BEAS-2B, A549, and NCI-H157 cells. The activation of NF-${\kappa}B{\alpha}$ and the induction of IL-8 and TNF-$\alpha$ mRNA expression were observed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in these cells. In contrast, neither the changes in NF-${\kappa}B/I{\kappa}B$ pathway nor IL-8 and TNF-$\alpha$mRNA expression was induced by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in NCI-H719 cells. IL-$1{\beta}$ and TNF-$\alpha$-induced $I{\kappa}B$ phosphorylation was observed in BEAS-2B, A549, and NCI-H157 cells, but not in NCI-H719 cells. The basal level of IKK$\alpha$ expression was not different between cell. Conclusion : NF-${\kappa}B/I{\kappa}B$ pathway plays an important role in the expression of pro-inflammatory cytokine in most lung epithelial cells. The absence of the effect on NF-${\kappa}B/I{\kappa}B$ pathway in NCI-H719 cells sæms to be due to the defect in the intracellular signal transduction pathway upstream to IKK.

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Transforming growth factor $(TGF)-{\beta}1$ conjugated chitosan film for enhanced osteoblastic activity (변형성장인자가 고정된 키토산 필름의 골아세포 활성에 미치는 영향)

  • Park, Yoon-Jeong;Lee, Jue-Yeon;Kim, Kyung-Hwa;Kim, Tae-Il;Lee, Myung-Hee;Shin, Seung-Yoon;Seol, Yang-Jo;Lee, Yong-Moo;Rhyu, In-Chul;Ku, Young;Han, Soo-Boo;Min, Byung-Moo;Lee, Seung-Jin;Chung, Chong-Pyoung
    • Journal of Periodontal and Implant Science
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    • v.34 no.4
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    • pp.781-790
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    • 2004
  • 골아세포의 생물학적 기능을 증진시키기 위해 키토산의 표면개질에 대하여 연구하였다. 생체적합성 천연고분자인 키토산은 1차 아미노기를 소유하고 있으므로 적정한 공유결합제를 사용하여 세포성장인자와 같은 생리활성을 지닌 단백질을 키토산의 표면에 고정시킬 수 있다. 본 연구에서는 키토산을 필름형태로 제조하여 세포성장인자 중 형질전환성장인자를 고정하고 골아세포의 부착, 성장 및 분화를 증가시키고자 하였다. 형태전환성장인자의 고정화 효율은 단순한 흡착방법에 비해 높았으며, 표면에 형성된 공유결합은 매우 안정하였다. 골아세포를 배양하여 초기세포부착능에 대한 영향을 연구한 결과, 배양 후 4시간, 1일째, 형질전환성장인자를 고정한 키토산 표면에서 고정하지 않은 키토산의 표면에 비해 더 많은 수의 골아세포가 부착되었고, 더 많이 신장된 부착형태를 보였다. 세포활성정도와 배양 후 4주일째의 칼슘축적량을 측정한 결과, 형질전환성장인자를 고정한 키토산 표면에서 고정하지 않은 키토산의 표면에 비해 더 높았다. 위의 결과는 키토산 표면에 형태전환성장인자의 고정이 성공적으로 이루어졌으며, 또한 실제로 활성이 있는 것이 증명되었다. 위의 연구 결과에서 형질전환성장인자로 고정된 키토산은 골아세포의 초기 부착 및 분화를 촉진시켰음을 알 수 있었던 바 성장인자의 표면고정은 임플란트 및 조직공학용 지지체에도 적용하여 생체적합성과 세포기능을 증진시키는데 이용할 수 있음을 알 수 있었다.