• Research in Plant Disease
• /
• v.21 no.2
• /
• pp.74-81
• /
• 2015

The specific and sensitive nested-PCR method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-PCR primer set (Ac-ORF 21F/Ac-ORF 21R) were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-PCR primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of $1^{st}$ PCR detection limit (10 ng genomic DNA/PCR). The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection) of the artificially inoculated watermelon seeds with $10^1cfu/ml$ and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection) of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test) used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-PCR developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds.

• Korean Journal of Veterinary Research
• /
• v.42 no.1
• /
• pp.1-6
• /
• 2002

The frequencies of gamma-ray-induced micronuclei (MN) in cytokinesis-blocked (CB) lymphocytes at several doses were measured in three donors of three species (human, rabbit, dog). Measurements performed after irradiation showed a dose-related increases in MN frequency in each of the donors studied. When analysed by linear-quadratic model the line of best fit was : human : $y=0.1184D+0.01867D^2+0.01$, rabbit : $y=0.0387D+0.00528D^2+0.01$ (y = number of MN/CB cells and D = irradiation dose in Gy). The relative sensitivity of rabbit lymphocytes compared with human lymphocytes was estimated by best fitting linear-quadratic model based on the radiation-induced MN data over the range from 0 Gy to 4 Gy. In the case of MN frequency with 0.2, the relative sensitivities of rabbit lymphocytes was 0.39. These data indicate that the induction of MN in rabbit CB cells following irradiation was much less sensitive to the MN induction effects of gamma-irradiation than those from human. The MN assay with dog lymphocytes was very difficult and time-consumed because the dog PHA-stimulated lymphocytes yielded cultures with very low level of CB cells formation in the condition of this experiment. Our in vitro radiobiological study confirmed that the cytogenetic response obtained in blood from rabbit can be utilized for application in environmental studies.

• Research in Plant Disease
• /
• v.12 no.2
• /
• pp.144-147
• /
• 2006

Egg yolk immunoglobulin (IgY) is much widely used in medical fields, but its use in serology of plant viruses is much limited. We produced an IgY against pepper mild mottle virus (PMMoV) and applied it to several serological tests. Polyclonal antibodies were obtained from the egg yolk of chicken immunized with a total of 2mg of purified PMMoV over 2 months. The titers of antibodies were measured with the ring-test over six months after the first injection. The highest.titers of IgY was 1/2,560 at 2 months after the first injection. Approximately 60-80 mg of IgY were obtained from one egg yolk. Using the IgY, 1ng/ml of purified PMMoV was detected with the indirect ELISA. Gelrite gel double diffusion test, ELISA and tissue immuno-binding assay employing IgY gave similar sensitivity and specificity to those of IgG developed in rabbit. Therefore, the IgY which can be obtained in large quantities from a chicken, might be useful for the antibody production and the serology of plant viruses.

• Journal of The Korean Society of Inherited Metabolic disease
• /
• v.3 no.1
• /
• pp.86-93
• /
• 2003

Background : The SCL began screening of newborns and high risk group blood spots with tandem mass spectrometry (MS/MS) in April 2001. Our goal was to determine approximate prevalence of metabolic disorders, optimization of decision criteria for estimation of preventive effect with early diagnosis. This report describes the ongoing effort to identify more than 30 metabolic disorders by MS/MS in South Korea. Methods : Blood spot was collected from day 2 to 30 (mostly from day 2 to 10) after birth for newborn. Blood spot of high risk group was from the pediatric patients in NICU, developmental delay, mental retardation, strong family history of metabolic disorders. One punch (3.2 mm ID) of dried blood spots was extracted with $150{\mu}L$ of methanol containing isotopically labelled amino acids (AA) and acylcarnitines (AC) internal standards. Butanolic HCl was added and incubated at $65^{\circ}C$ for 15 min. The butylated extract was introduced into the inlet of MS/MS. Neutral loss of m/z 102 and parent ion mode of m/z 85 were set for the analyses of AA and AC, respectively. Diagnosis was confirmed by repeating acylcarnitine profile, urine organic acid and plasma amino acid analysis, direct enzyme assay, or molecular testing. Results : Approximately 31,000 neonates and children were screened and the estimated prevalence (newborn/high risk group), sensitivity, specificity and recall rate amounted to 1:2384/1:2066, 96.55%, 99.98%, and 0.73%, respectively. Confirmed 28 (0.09%) multiple metabolic disorders (newborn/high risk) were as follows; 13 amino acid disorders [classical PKU (3/4), BH4 deficient-hyperphenylalaninemia (0/1), Citrullinemia (1/0), Homocystinuria (0/2), Hypermethioninemia (0/1), Tyrosinemia (1/0)], 8 organic acidurias [Propionic aciduria (2/1), Methylmalonic aciduria (0/1), Isovaleric aciduria (1/1), 3-methylcrotonylglycineuria (1/0), Glutaric aciduria type1 (1/0)], 7 fatty acid oxidation disorders [LCHAD def. (2/2), Mitochondrial TFP def. (0/1), VLCAD def. (1/0), LC3KT def. (0/1). Conclnsion : The relatively normal development of 10 patients with metabolic disorders among newborns (except for the expired) demonstrates the usefulness of newborn screening by MS/MS for early diagnosis and medical intervention. However, close coordination between the MS/MS screening laboratory and the metabolic clinic/biochmical geneticists is needed to determine proper decision of screening parameters, confirmation diagnosis, follow-up scheme and additional tests.

• Journal of Gastric Cancer
• /
• v.23 no.3
• /
• pp.476-486
• /
• 2023

Purpose: The optimal tumor mutational burden (TMB) value for predicting treatment response to programmed cell death-1 (PD-1) checkpoint inhibitors in advanced gastric cancer (AGC) remains unclear. We aimed to investigate the optimal TMB cutoff value that could predict the efficacy of PD-1 checkpoint inhibitors in AGC. Materials and Methods: Patients with AGC who received pembrolizumab or nivolumab between October 1, 2020, and July 27, 2021, at Samsung Medical Center in Korea were retrospectively analyzed. The TMB levels were measured using a next-generation sequencing assay. Based on receiver operating characteristic curve analysis, the TMB cutoff value was determined. Results: A total 53 patients were analyzed. The TMB cutoff value for predicting the overall response rate (ORR) to PD-1 checkpoint inhibitors was defined as 13.31 mutations per megabase (mt/Mb) with 56% sensitivity and 95% specificity. Based on this definition, 7 (13.2%) patients were TMB-high (TMB-H). The ORR differed between the TMB-low (TMB-L) and TMB-H (8.7% vs. 71.4%, P=0.001). The progression-free survival and overall survival (OS) for 53 patients were 1.93 (95% confidence interval [CI], 1.600-2.268) and 4.26 months (95% CI, 2.992-5.532). The median OS was longer in the TMB-H (20.8 months; 95% CI, 2.292-39.281) than in the TMB-L (3.31 months; 95% CI, 1.604-5.019; P=0.049). Conclusions: The TMB cutoff value for predicting treatment response in AGC patients who received PD-1 checkpoint inhibitor monotherapy as salvage treatment was 13.31 mt/Mb. When applying the programmed death ligand-1 status to TMB-H, patients who would benefit from PD-1 checkpoint inhibitors can be selected.

• Tuberculosis and Respiratory Diseases
• /
• v.42 no.3
• /
• pp.302-313
• /
• 1995

Background: Tumor necrosis factor(TNF) showed antitumor cytolytic effects on sensitive tumor cells in numerous in vivo and in vitro studies. But it could not be administered systemically to human because of severe systemic adverse effects at effective concentrations against tumor cells. Many studies showed that a high concentrations of TNF in the local milieu may evoke in vivo TNF-responsive mechanisms sufficient to suppress tumor growth. Recently developed technique of TNF gene transfer to tumor cells using retrovirus vector could be a good candidate for local TNF administration. TNF is also known to synergistically enhance in vitro cytotoxicity of chemotherapeutic drugs targeted to DNA topoisomerase II against TNF-sensitive tumor cell lines. In this study the in vitro chemosensitivity against DNA topoisomerase II targeted chemotherapeutic drugs was evaluated using some respiratory cancer cell lines to which TNF gene had been transferred. Method: NCI-H2058, a human mesothelioma cell line, A549, a human lung adenocarcinoma cell line and WEHI 164 cell line, a murine fibrosarcoma cell line were treated with etoposide and doxorubicin, which are typical topoisomerase II - targeted chemotherapeutic agents, at different concentration. The resultant cytotoxicity was measured by MIT assay. Then the cytotoxicity of the same chemotherapeutic agents was measured after TNF-$\alpha$ gene-transfer and the two results were compared. Results: The cytotoxicity was not increased significantly in WEHI164 cell line and A549 cell line but statistically significant increase was observed in H2058 cell line when TNF-$\alpha$ gene was transferred(p<0.05). Conclusion: These findings show that TNF-$\alpha$ gene transfer to respiratory cancer cell lines results in variable effects on chemosensitivity against topoisomerase II inhibitor among different cell lines in vitro and can be additively cytotoxic in certain selective tumor cell lines.

• Parasites, Hosts and Diseases
• /
• v.30 no.4
• /
• pp.299-307
• /
• 1992

An antigenic protein in cystic quid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal quid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminthes extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRl confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after prasiquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently, These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in 505-PAGE before and after immunoprecipitation.

• Clinical and Experimental Pediatrics
• /
• v.49 no.1
• /
• pp.87-92
• /
• 2006

Purpose : It is difficult to make a distinction between lower urinary tract infection(UTI) and acute pyelonephritis(APN) during the acute phase of febrile UTI due to nonspecific clinical symptoms and laboratory findings, especially among young children. We measured the serum procalcitonin(PCT) in children with UTI to distinguish between acute pyelonephritis and lower UTI, and to determine the accuracy of PCT measurement compared with other inflammatory markers. Methods : Serum samples were taken from children who admitted with unexplained fever or were suspected of having UTI. 51 children(mean $12.2{\pm}11.4$ months) were enrolled in this study. Leukocyte counts, erythrocyte sedimentation rates(ESR) and C-reactive protein(CRP) were also measured. Renal parenchymal involvement was assessed by $^{99m}Tc$ DMSA scintigraphy in the first 7 days after admission. PCT was measured by immunoluminometric assay. Results : PCT values were significantly correlated with the presence of renal defects in children with UTI(n=16)($5.06{\pm}12.97{\mu}g/L$, P<0.05). However, PCT values were not significantly different between children with UTI without renal damage(n=18) and children without UTI(n=17). Using a cutoff of $0.5{\mu}g/L$ for PCT and 20 mm/hr for ESR, 20 mg/L for CRP, sensitivity and specificity in distinguishing between UTI with and without renal involvement were 81.3 percent and 88.9 percent for PCT 87.5 percent and 72.2 percent for ESR, and 87.5 percent and 55.6 percent for CRP, respectively. Positive and negative predictive values were 86.7 percent and 84.2 percent for PCT and 60.9 percent and 81.8 percent for CRP, respectively. Conclusion : In febrile UTI, PCT values were more specific than CRP, ESR and leukocyte count for the identification of patients who might develop renal defects.

• Asian Journal of Turfgrass Science
• /
• v.20 no.1
• /
• pp.65-76
• /
• 2006

Scz1, an isolate of Sclerotinia homoeocarpa, was recently reported as a novel pathogen responsible for dollar spot disease in Zoysiagrass, a warm season turfgrass. Scz1 possessed different characteristics on mycelial pigment, mycelial affinity and host pathogenecity compared to those of Scb1, a typical isolate, obtained from creeping bentgrass, a cool season turfgrass. In this study, only three isolates, Scz1, Scz2(another analogous isolate of Sclerotinia homoeocarpa from zoysiagrass), and Scb1, were examined at the molecular level using the internal transcribed spacer(ITS) and random amplified polymorphic DNA(RAPD) assays to verify their identification and genetic variation. As a result of ITS assay, partial ITS sequences of three isolates showed 94-97% similarity with a standardized ITS sequence of S. homoeocarpa registered on BLAST. In the analysis of RAPD, range value through similarity matrix was 0.167 between Scz1 and Scb1, 0.139 between Scz2 and Scb1, and 0.713 between Scz1 and Scz2, respectively. Furthermore, tendegram analysis indicated that Scz1 and Scz2, unlike Scb1, were clustered together as accompanying a high genetic similarity. In in vitro fungicide bioassay, $EC_{50}$ value representing the sensitivity degree to propiconazole, a well-known fungicide for dollar spot disease, was 0.012 ${\mu}g/ml$ for Sczl, 0.003 ${\mu}g/ml$ for Scz2, and 0.030 ${\mu}g/ml$ for Scb1. From all data taken, we concluded that both Scz1 and Scz2 belonged to one group of S. homoeocarpa, since they exhibit the same host range and high level of genetic similarity, whereas their chemical competences to a fungicide were different. This study would provide further approach for assessing genetic diversity of S. homoeocarpa isolates as well as characterizing individual isolate against chemical exposure.

• Pediatric Infection and Vaccine
• /
• v.15 no.1
• /
• pp.52-58
• /
• 2008

Purpose : Whole blood interferon-$\gamma$ assay was developed and many studies showed its usefulness in diagnosing tuberculosis (TB) including latent tuberculosis infection (LTBI). However, assessment in children has been limited. This study was undertaken to evaluate the usefulness of QuantiFERON-TB Gold for the diagnosis of LTBI in children exposed to pulmonary TB. Methods : Children who visited Seoul National University Bundang Hospital with a history of TB exposure were enrolled from January 2006 to December 2007. They were evaluated with chest x-rays, tuberculin skin test (TST) and QuantiFERON-TB Gold test. TST was retested 3 months later for those with initial negative reactivity. Definition of LTBI was made on the basis of the TST reactivity. Results : Among the 103 children with a history of TB exposure, 49 children were tested with chest x-ray, TST, and QuantiFERON-TB Gold. Twenty-two were males. Median age was 7.5 years (range; 3 months to 14.7 years). According to TST reactivity, LTBI was in 8 (19%), no infection was in 21 (50%), possible LTBI was in 13 (31%). QuantiFERON-TB Gold test was positive in 5 of the 49 subjects (10%); 3 of the 13 subjects (23.1%) in unknown status, 1 of the 8 subjects (13%) in LTBI, and 1 of the 21 subjects (5%) without infection. The agreement between the QuantiFERON-TB Gold and the TST was poor (${\kappa}=0.101$). Conclusion : QuantiFERON-TB Gold showed poor sensitivity for the diagnosis of LTBI in children with exposure to TB. QuantiFERON-TB Gold alone does not seem to be useful in the diagnosis of LTBI in children.