• Journal of Veterinary Science
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• v.22 no.6
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Microbiology and Biotechnology Letters
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• v.43 no.2
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• pp.134-141
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• 2015

An agarolytic marine bacterium (H9) was isolated from the coastal seawater of the West Sea, South Korea. The isolate, H9, was gram-negative and rod-shaped with a smooth surface and polar flagellum. Cells grew at 20-30℃, between pH 5.0 and 9.0, and in ASW-YP (Artificial Sea Water-Yeast extract, Peptone) media containing 1-5% (w/v) NaCl. The G+C content was 41.56 mol%. The predominant isoprenoid quinone in strain H9 was ubiquinone-8. The major fatty acids (>10%) were C_16:1ω7c (34.3%), C_16:0 (23.72%), and C_18:1ω7c (13.64%). Based on 16S rRNA gene sequencing, and biochemical and chemotaxonomic characterization, the strain was designated as Pseudoalteromonas sp. H9 (=KCTC23887). In liquid culture supplemented with 0.2% agar, the cell density and agarase activity reached a maximum level of OD = 4.32 (48 h) and OD = 3.87 (24 h), respectively. The optimum pH and temperature for the extracellular crude agarases of H9 were 7.0 and 40℃, respectively. Thin-layer chromatography analysis of the agarase hydrolysis products revealed that the crude agarases hydrolyze agarose into neoagarotetraose and neoagarohexaose. Therefore, the new agar-degrading strain, H9, can be applicable for the production of valuable neoagarooligosaccharides and for the complete degradation of agar in bio-industries.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9313-9317
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• 2014

Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG island hypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. It may play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study was to observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNA methylation of FHIT, serum folate level and HPV16 status were measured using methylation-specific polymerase chain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 women with a diagnosis of normal cervix (NC, n=109), cervical intraepithelial neoplasia (CIN, n=101) and squamous cell carcinoma of the cervix (SCC, n=101). There were significant differences in HPV16 status (${\chi}^2=36.64$, P<0.001), CpG island methylation of FHIT (${\chi}^2=71.31$, P<0.001) and serum folate level (F=4.57, P=0.011) across the cervical histologic groups. Interaction analysis showed that the ORs only with FHIT methylation (OR=11.47) or only with HPV 16 positive (OR=4.63) or with serum folate level lower than 3.19ng/ml (OR=1.68) in SCC group were all higher than the control status of HPV 16 negative and FHIT unmethylation and serum folate level more than 3.19ng/ml (OR=1). The ORs only with HPV 16 positive (OR=2.58) or with serum folate level lower than 3.19ng/ml (OR=1.28) in CIN group were all higher than the control status, but the OR only with FHIT methylation (OR=0.53) in CIN group was lower than the control status. HPV 16 positivity was associated with a 7.60-fold increased risk of SCC with folate deficiency and with a 1.84-fold increased risk of CIN. The patients with FHIT methylation and folate deficiency or with FHIT methylation and HPV 16 positive were SCC or CIN, and the patients with HPV 16 positive and FHIT methylation and folate deficiency were all SCC. In conclusion, HPV 16 infection, FHIT methylation and folate deficiency might promote cervical cancer progression. This suggests that FHIT may be an effective target for prevention and treatment of cervical cancer.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.558-563
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• 2013

The present study was conducted to estimate energy requirements for maintenance in laying hens by using indirect calorimetry and energy balance. A total of 576 28-wk-old Nongda-3 laying hens with dwarf gene were randomly allocated into four ME intake levels (86.57, 124.45, 166.63 and 197.20 kcal/kg body weight $(BW)^{0.75}$ per d) with four replicates each. After a 4 d adaptation period, 36 hens from one replicate were maintained in one of the two respiration chambers to measure the heat production (HP) for 3 d during the feeding period and subsequent 3 d fast. Metabolizable energy (ME) intake was partitioned between heat increment (HI), HP associated with activity, fasting HP (FHP) and retained energy (RE). The equilibrium FHP may provide an estimate of NE requirements for maintenance (NEm). Results showed that HP, HI and RE in the fed state increased with ME intake level (p<0.05). Based on the regression of HP on ME intake, the estimated ME requirements for maintenance (MEm) was 113.09 kcal/kg $BW^{0.75}$ per d when ME intake equals HP. The FHP was decreased day by day with the lowest value on the third day of starvation. Except for lowest ME intake level, the FHP increased with ME intake level on the first day of starvation (p<0.05). The FHP at the two higher ME intake levels were greater than that at the two lower ME intake levels (p<0.05) but no difference was found between the two lower ME intake levels. Linear regression of HP from the fed state to zero ME intake yielded a value of 71.02 kcal/kg $BW^{0.75}$ per d, which is higher than the extrapolated FHP at zero ME intake (60.78, 65.23 and 62.14 kcal/kg $BW^{0.75}$ per d for the first, second and third day of fasting, respectively). Fasting time, lighting schedules, calculation methods and duration of adaptation of hens to changes in ME intake level should be properly established when using indirect calorimetry technique to estimate dietary NE content, MEm and NEm for laying hens.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.4
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• pp.560-563
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• 2004

The study was undertaken to evaluate the effect of naked neck gene on mortality, cell mediated and humoral immune response in white plumage broiler population. The mortality of homozygous naked neck (Na/Na) broilers (11.71%) was comparatively lower than that of heterozygous naked neck (Na/na) (12.28%) and normally feathered (na/na) (13.59%) broilers. The humoral immune response was measured against (1% v/v) sheep red blood cells (SRBC) for total haemagglutinin (HA) antibody, 2-mercaptoethanol resistance (MER) or (IgG) antibody and 2-mercaptoethanol sensitive (MES) or (IgM) antibody titre on 7 days post-immunization. The titre was expressed as log2 of the highest dilution which shows complete haemagglutination. Total HA titers of Na/Na and Na/na (11.05$\pm$0.53 and 11.09$\pm$0.38) were comparatively higher than that of na/na (10.26$\pm$0.42). The MES antibody titre of Na/Na (8.50$\pm$0.53) and Na/na (7.63$\pm$0.45) broilers were significantly higher as compared to na/na (6.11$\pm$0.32) broilers. The MER titre of na/na genetic group (4.15$\pm$0.42) was significantly higher than Na/Na (2.55$\pm$0.37) and comparatively higher than Na/na (3.45$\pm$0.38) broilers. In vivo cell response to phytohaemagglutinin-P (PHA-P), measured as Foot Index (FI) in mm expressed significantly higher response in Na/na (0.473$\pm$0.05) and Na/Na (0.413$\pm$0.04) broilers as compared to na/na (0.304$\pm$0.03) broilers. The result of present study suggested that white plumage naked neck broilers had better immune response as compared to normally feathered broilers.

• Nutrition Research and Practice
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• v.13 no.1
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• pp.58-63
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• 2019

BACKGROUND/OBJECTIVES: Telomeres are located at the chromosomal ends and progressively shortened during each cell cycle. Telomerase, which is regulated by hTERT and c-MYC, maintains telomeric DNA sequences. Especially, telomerase is active in cancer and stem cells to maintain telomere length for replicative immortality. Recently we reported that walnut phenolic extract (WPE) can reduce cell viability in a colon cancer stem cell (CSC) model. We, therefore, investigated the effect of WPE on telomere maintenance in the same model. MATERIALS AND METHODS: $CD133^+CD44^+$ cells from HCT116, a human colon cancer cell line, were sorted by Fluorescence-activated cell sorting (FACS) and treated with WPE at the concentrations of 0, 10, 20, and $40{\mu}g/mL$ for 6 days. Telomere lengths were assessed by quantitative real-time PCR (qRT-PCR) using telomere specific primers and DNA extracted from the cells, which was further adjusted with single-copy gene and reference DNA ($ddC_t$). Telomerase activity was also measured by qRT-PCR after incubating the PCR mixture with cell protein extracts, which was adjusted with reference DNA ($dC_t$). Transcriptions of hTERT and c-MYC were determined using conventional RT-PCR. RESULTS: Telomere length of WPE-treated cells was significantly decreased in a dose-dependent manner ($5.16{\pm}0.13$ at $0{\mu}g/mL$, $4.79{\pm}0.12$ at $10{\mu}g/mL$, $3.24{\pm}0.08$ at $20{\mu}g/mL$ and $3.99{\pm}0.09$ at $40{\mu}g/mL$; P = 0.0276). Telomerase activities concurrently decreased with telomere length ($1.47{\pm}0.04$, $1.09{\pm}0.01$, $0.76{\pm}0.08$, and $0.88{\pm}0.06$; P = 0.0067). There was a positive correlation between telomere length and telomerase activity (r = 0.9090; P < 0.0001). Transcriptions of both hTERT and c-MYC were also significantly decreased in the same manner. CONCLUSION: In the present cell culture model, WPE reduced telomere maintenance, which may provide a mechanistic link to the effect of walnuts on the viability of colon CSCs.

• Journal of Life Science
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• v.33 no.4
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• pp.357-362
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• 2023

This report looks at an agar-degrading marine bacterium and characterization of its agarase. Agar-degrading marine bacterium JS-1 was isolated with Marine agar 2216 media from seawater from the seashore of Sojuk-do, Changwon in Gyeongnam Province, Korea. The agar-degrading bacterium was named as Agarivorans sp. JS-1 by phylogenetic analysis based on 16S rRNA gene sequencing. The extracellular agarase was prepared from the culture media of Agarivorans sp. JS-1 and used for characterization. Relative activities at 20℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ were 70%, 74%, 78%, 83%, 87%, 100%, 74%, and 66%, respectively. Relative activities at pH 5, 6, 7, and 8 were 91%, 100%, 90%, and 89%, respectively. Its extracellular agarase showed maximum activity (207 units/l) at pH 6.0 and 50℃ in 20 mM Tris-HCl buffer. The residual activity after heat treatment at 20℃, 30℃, and 50℃ for 30 minutes was 90%, 70%, and 50% or more, respectively. After a 2-hour heat treatment at 20℃, 30℃, 35℃, 40℃, and 45℃, the residual activity was 80%, 68%, 65%, 63%, and 57%, respectively. At 50℃ and above, after heat treatment for 30 minutes, the residual activity was below 60%. Thin layer chromatography analysis suggested that Agarivorans sp. JS-1 produces extracellular β-agarases as they hydrolyze agarose to produce neoagarooligosaccharides such as neoagarohexaose (20.6%), neoagarotetraose (58.5%), and neoagarobiose (20.9%). Agarivorans sp. JS-1 and its thermotolerant β-agarase would be useful in the production of neoagarooligosaccharides, showing functional activity such as inhibition of bacterial growth and delay of starch degradation.

• Journal of the Korean Wood Science and Technology
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• v.34 no.3
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• pp.56-63
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• 2006

In order to evaluate the mechanism of cellulose hydrolysis, the complementary DNA encoding Glycoside Hydrolase Family (GHF)74 was cloned from Phanerochaete chrysosporium. Depending on the presence of Cellulose Binding Module (CBM), it can be classified as GHF74A or GHF74B. The GHF74A gene from P. chrysosporium (PcGHF74A) consists of 2163 bp encoding a protein of 721 amino acid residues. The PcGHF74A showed homology of 70~77% compared with the GHF74 from other filamentous fungi. The PcGHF74B, which contains CBM and is a member of family 1, was transcribed to various transcripts depending on the nature of carbon sources and their concentration. To study the possible presence of splice variants in GHF74B transcripts in P. chrysospoium, we carried out RT-PCR analysis using primers that designed based on the annotation data and sequenced data. Our result indicated that PcGHF74B was transcribed to several splicing variants in various culture conditions. Especially in the culture of 2% cellulose, three transcript products were observed. First transcript was presumed to be a full length ORF that contained 11th intron with stop codon at position 2562 bp. The second one consisted of 12 exons and 11 introns with stop codon at position 1187 bp with 7th exon. The shortest transcript consisted of 10 exons and 9 introns with stop codon at 910 bp in the 7th exon. These premature stop codon might prevent the synthesis of fully active GHF74 or contribute for the production of protein with distinct function depending on the ambient carbon sources.

• Applied Biological Chemistry
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• v.37 no.1
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• pp.56-63
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• 1994

Oligonucleotides for a conserved region of the coat protein gene of cucumber mosaic virus (CMV) and a hammerhead structure ribozyme against CMV RNA were synthesized using a DNA synthesizer. Both strands of oligonucleotides were annealed and restricted with BamHI/SacI, then cloned into a plasmid pBS SK (+). The cloned CMV substrate and ribozyme were sequenced to verify correct constructions. In vitro transcriptions were carried out by using T7 RNA polymerase with BssHII or SspI digests of $1\;{\mu}g$ of substrate and ribozyme clones. The size of substrate RNA was 176 nucleotides (nt) containing 50 nt of CMV RNA sequence, 6 nt of XbaI restriction site and 120 nt of vector-derived sequence in the case of BssHII digest. The size of ribozyme RNA was 164 nt containing 40 nt of ribozyme RNA sequence and same sequences of substrate. Substrate RNA was efficiently cleaved into two fragments (96 nt and 80 nt) by ribozyme RNA. This endonucleolytic cleavage occurred more efficiently at $55^{\circ}C$ than $37^{\circ}C$. SspI digest-derived substrate RNA (2234 nt) was also cleaved into two fragments by the same ribozyme. SspI digest-derived ribozyme RNA (2222 nt) cleaved the above substrate to two fragments. In vitro-tested ribozyme construct is being cloned into a plant transformation vector to develop virus-resistant plants.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.22
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• pp.9739-9745
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• 2014

Purpose: We aimed to evaluate the effects of hormone receptor, HER2, and epidermal growth factor receptor (EGFR) expression on epithelial ovarian cancer (EOC) prognosis and investigate whether or not phenotypic subtypes might exist. Materials and Methods: The medical records of 82 patients who were diagnosed with EOC between 2003 and 2012 and treated by platinum-based chemotherapy were retrospectively evaluated. Expression of EGFR, oestrogen (ER), progesterone (PR), and cerbB2 (HER2) receptors were assessed immunohistochemically on paraffin-embedded tissues of these patients. Three phenotypic subtypes were defined according to ER, PR, and HER2 expression and associations of these with EGFR expression, clinicopathologic features, platinum sensitivity, and survival were investigated. Results: When we classified EOC patients into three subtypes, 63.4% had hormone receptor positive (HR(+)) (considering breast cancer subtypes, luminal A), 18.3% had triple negative, and 18.3% had HER2(+) disease. EGFR positivity was observed in 37 patients (45.1%) and was significantly more frequent with advanced disease (p=0.013). However, no significant association with other clinicopathologic features and platinum sensitivity was observed. HER2(+) patients had significantly poorer outcomes than HER2(-) counterparts (triple negative and HR positive patients) (p=0.019). Multivariate analysis demonstrated that the strongest risk factor for death was residual disease after primary surgery. Conclusions: Triple negative EOC may not be an aggressive phenotype as in breast cancer. The HER2 positive EOC has more aggressive behaviour compared to triple negative and HR(+) phenotypes. EGFR expression is more frequent in advanced tumours, but is not related with poorer outcome. Additional ovarian cancer molecular subtyping using gene expression analysis may provide more reliable data.