• Annals of Clinical Microbiology
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• v.18 no.2
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• pp.37-43
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• 2015

Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. Methods: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. Results: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

• Tuberculosis and Respiratory Diseases
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• v.70 no.1
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• pp.21-27
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• 2011

Background: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of $EGFR$ mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to $EGFR$ mutation status using both methodologies. Methods: Clinical specimens from 112 NSCLC patients were analyzed for $EGFR$ mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to $EGFR$-TKIs were compared. Results: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). Conclusion: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in $EGFR$ gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of $EGFR$ mutations in clinical setting.

• IMMUNE NETWORK
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• v.16 no.1
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• pp.52-60
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• 2016

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that bridge innate and adaptive immune responses, thereby leading to immune activation. DCs have been known to recognize pathogen-associated molecular patterns such as lipopolysaccharides (LPS) and nucleic acids via their pattern recognition receptors, which trigger signaling of their maturation and effector functions. Furthermore, DCs take up and process antigens as a form of peptide loaded on the major histocompatibility complex (MHC) and present them to T cells, which are responsible for the adaptive immune response. Conversely, DCs can also play a role in inducing immune suppression under specific circumstances. From this perspective, the role of DCs is related to tolerance rather than immunity. Immunologists refer to these special DCs as tolerogenic DCs (tolDCs). However, the definition of tolDCs is controversial, and there is limited information on their development and characteristics. In this review, we discuss the current concept of tolDCs, cutting-edge methods for generating tolDCs in vitro, and future applications of tolDCs, including clinical use.

• Tuberculosis and Respiratory Diseases
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• v.69 no.4
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• pp.271-278
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• 2010

Background: Recent studies have demonstrated that the epidermal growth factor receptor (EGFR) genotype is the most important predictive marker to EGFR-tyrosine kinase inhibitors (TKIs) and first-line gefitinib treatment will be approved in the near future for use in non-small cell lung cancer (NSCLC) patients with the EGFR mutation. Direct sequencing is known to be the standard for detecting EGFR mutations; however, it has limited sensitivity. Peptide nucleic acids (PNA)-mediated PCR clamping method is a newly introduced method for analyzing EGFR mutations with increased sensitivity and stability. Methods: A total of 71 NSCLC patients were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. Sixty-nine patients were analyzed for clinicopathologic correlation with EGFR genotype; 2 patients with indeterminate results were excluded. In order to determine EGFR-TKI drug response, 57 patients (42 gefitinib, 15 erlotinib) were included in the analysis. Results: The EGFR mutation rate was 47.8%. Being female, a non-smoker, and having adenocarcinoma were favorable clinicopathologic factors, as expected. However, more than a few smokers (33.3%), male (28.1%), and patients with non-adenocarcinoma (28.6%) had the EGFR mutation. Having a combination of favorable clinicopathologic factors did not increase the EGFR mutation rate significantly. Drug response to EGFR-TKIs showed significant differences depending on the EGFR genotype; ORR was 14.3% for wild type vs 69.0% for mutant type; DCR is 28.6% for wild type vs 96.6% for mutant type. The median EGFR-TKI treatment duration is 7.6 months for mutant type group and 1.4 months for wild type group. Conclusion: EGFR genotype determined using the PNA-mediated PCR clamping method is significantly correlated with the clinical EGFR-TKI responses and PNA-mediated PCR.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1774-1783
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• 2015

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector $pPICZ{\alpha}A$ and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.37 no.5
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• pp.396-402
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• 2011

Introduction: Epidermal growth factor is a single-chain polypeptide consisting of 53 amino acids with potent mitogenic activity that stimulates the proliferation of a range of normal and neoplastic cells through an interaction with its specific receptor (epidermal growth factor receptor, EGFR). This interaction plays a key role in tumor progression including the induction of tumor cell proliferation. An increased EGFR copy number have been associated with a favorable response to EGFR tyrosine kinase inhibitors therapy. In contrast, K-ras mutations tend to predict a poor response to such therapy. The aim of this study was to determine the correlation between the clinicopathological factors and the up-regulation of EGFR expression and Kras mutations in oral squamous cell carcinoma. Materials and Methods: This study examined the immunohistochemical staining of EGFR, K-ras mutation detection with peptide nucleic acid (PNA)-based real-time polymerase chain reaction (PCR) clamping in 20 specimens from 20 patients with oral squamous cell carcinoma. Results: 1. In the immunohistochemical study of poorly differentiated and invasive oral squamous cell carcinoma, a high level of EGFR staining was observed. The correlation between immunohistochemical EGFR expression and histological differentiation, as well as the tumor size of the specimens was significant (Pearson correlation analysis, significance [r] >0.5, P<0.05). 2. In PNA-based real-time PCR clamping analysis, a K-ras mutation was not detected in all specimens. Conclusion: These findings suggest that the up-regulation of the EGFR may play a role in the progression and invasion of oral squamous cell carcinoma that is, independent of a K-ras mutation.

• KSBB Journal
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• v.24 no.1
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• pp.80-88
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• 2009

Human prostatic acid phosphatase (PAP), with comprehensive homology to glandular kallikrein, are representative serum biomarkers of prostate cancer. Dendritic cell (DC), which is the potent antigen-presenting cells(APC) in the immune system, can induce strong T cell responses against viruses, microbial pathogens, and tumors. Therefore, the immunization using DC loaded with tumor-associated antigens is a powerful method for inducing anti-tumor immunity. The CTP (Cytoplasmic Transduction Peptide) technology developed by Creagene which can transport attached bio-polymers like nucleic acids or proteins into the cell with high permeation efficiency. As the active forms of PAP can mediate apoptotic processing, we used multimer forms of PAP as an inactive form for antigen pulsing of DCs. In this study, multimeric forms of CTP-rhPAP was obtained according to the advanced purification process and subsequently confirmed by gel filtration chromatography, western blot and Dynamic Light Scattering. Therefore, CTP-conjugated PA multimers transduced into the cytoplasm were efficiently presented on the cell surface without any harm effect on cells via MHC class I molecules and result in induction of a large number of effector cell.

• Journal of Life Science
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• v.30 no.9
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• pp.772-782
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• 2020

Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.