• Animal Bioscience
• /
• v.34 no.5
• /
• pp.801-810
• /
• 2021

Objective: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. Methods: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. Results: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. Conclusion: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.32 no.7
• /
• pp.913-921
• /
• 2019

Objective: The objectives of this study were to compare identified informative regions through two genome-wide association study (GWAS) approaches and determine the accuracy and bias of the direct genomic value (DGV) for milk production traits in Korean Holstein cattle, using two genomic prediction approaches: single-step genomic best linear unbiased prediction (ss-GBLUP) and Bayesian Bayes-B. Methods: Records on production traits such as adjusted 305-day milk (MY305), fat (FY305), and protein (PY305) yields were collected from 265,271 first parity cows. After quality control, 50,765 single-nucleotide polymorphic genotypes were available for analysis. In GWAS for ss-GBLUP (ssGWAS) and Bayes-B (BayesGWAS), the proportion of genetic variance for each 1-Mb genomic window was calculated and used to identify informative genomic regions. Accuracy of the DGV was estimated by a five-fold cross-validation with random clustering. As a measure of accuracy for DGV, we also assessed the correlation between DGV and deregressed-estimated breeding value (DEBV). The bias of DGV for each method was obtained by determining regression coefficients. Results: A total of nine and five significant windows (1 Mb) were identified for MY305 using ssGWAS and BayesGWAS, respectively. Using ssGWAS and BayesGWAS, we also detected multiple significant regions for FY305 (12 and 7) and PY305 (14 and 2), respectively. Both single-step DGV and Bayes DGV also showed somewhat moderate accuracy ranges for MY305 (0.32 to 0.34), FY305 (0.37 to 0.39), and PY305 (0.35 to 0.36) traits, respectively. The mean biases of DGVs determined using the single-step and Bayesian methods were $1.50{\pm}0.21$ and $1.18{\pm}0.26$ for MY305, $1.75{\pm}0.33$ and $1.14{\pm}0.20$ for FY305, and $1.59{\pm}0.20$ and $1.14{\pm}0.15$ for PY305, respectively. Conclusion: From the bias perspective, we believe that genomic selection based on the application of Bayesian approaches would be more suitable than application of ss-GBLUP in Korean Holstein populations.

• Animal Bioscience
• /
• v.34 no.1
• /
• pp.74-84
• /
• 2021

Objective: Feed additives that modify rumen fermentation can be used to prevent metabolic disturbances such as acidosis and optimize beef cattle production. The study evaluated the effects of liquid and powdered forms of polyclonal antibody preparation (PAP) against Streptococcus bovis and Fusobacterium necrophorum on rumen fermentation parameters in ruminally cannulated non-lactating dairy cows that were adapted or unadapted to a high concentrate diet. Methods: A double 3×3 Latin square design was used with three PAP treatments (control, powdered, and liquid PAP) and two adaptation protocols (adapted, unadapted; applied to the square). Adapted animals were transitioned for 2 weeks from an all-forage to an 80% concentrate diet, while unadapted animals were switched abruptly. Results: Interactions between sampling time and adaptation were observed; 12 h after feeding, the adapted group had lower ruminal pH and greater total short chain fatty acid concentrations than the unadapted group, while the opposite was observed after 24 h. Acetate:propionate ratio, molar proportion of butyrate and ammonia nitrogen concentration were generally greater in adapted than unadapted cattle up to 36 h after feeding. Adaptation promoted 3.5 times the number of Entodinium protozoa but copy numbers of Streptococcus bovis and Fibrobacter succinogens genes in rumen fluid were not affected. However, neither liquid nor powdered forms of PAP altered rumen acidosis variables in adapted or unadapted animals. Conclusion: Adaptation of cattle to highly fermentable carbohydrate diets promoted a more stable ruminal environment, but PAP was not effective in this study in which no animal experienced acute or sub-acute rumen acidosis.

• Journal of Plant Biotechnology
• /
• v.48 no.1
• /
• pp.34-43
• /
• 2021

Targeted genome editing using the CRISPR/Cas9 system is a ground-breaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoprotein-mediated transfection approach is useful for the production of transgene-free genome-edited tree plants.

• Journal of Microbiology and Biotechnology
• /
• v.34 no.5
• /
• pp.1146-1153
• /
• 2024

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.30 no.1
• /
• pp.25-34
• /
• 2010

Although whole crop barley (Hordeum vulgare L.) is now widely grown as a silage crop in Korea, forage production and silage quality of that for organic farm have not been published. Therefore, this experiment was conducted to evaluate the effect of harvest stage on forage production and quality of organic barley, and effect of field wilting and crushed rice for shortening of harvest date and improvement of forage quality. The experiment was split-plot design with three replications. Main plots were heading, milking and yellow stages, and sub-plot were field wilting for I day, crushed rice 10% (CR10%) and 15% (CR15%) treatments. The dry matter (DM) contents of heading, milking and yellow stages were 12.8%, 21.9% and 29.8%, respectively. The DM yields of heading, milking and yellow stages were 10,346, 15,819 and 18,336 kg/ha, respectively, and the total digestible nutrients (TDN) of these were 6,288, 9,550 and 10,178 kg/ha, respectively. The pH of milking stage showed low 4.00 pH values. The crude protein, ether extract, crude ash, non-fiber carbohydrate (NFC) and TDN contents were decreased as harvest stage progressed, while neutral detergent fiber (NDF) and acid detergent fiber (ADF) were increased. The crude ash (CA) of milking stage showed the lowest among harvest stages. Field wilting and crushed rice treatments decreased CA, NDF and ADF contents, and increased NFC and TDN contents. In vitro dry matter digestibility (IVDMD) decreased with progressed harvest stage, while field wilting and crushed rice treatments increased that of barley silage. Lactic acid and total organic acid contents of milking stage were the highest, and butyric acid of milking stage was the lowest among harvest stage. The good effect of field wilting and crushed rice was observed in heading stage. The experiment results indicate that optimum harvest stage of barley silage for organic was milking stage. The field wilting and crushed rice additive could be recommended as effective method for shortening harvest date and increasing forage quality of organic barley silage

• The Korean Journal of Nuclear Medicine
• /
• v.34 no.3
• /
• pp.252-259
• /
• 2000

Purpose: The purpose of this study was to compare the radiation adaptive response (RAR) in peripheral lymphocytes (PL) of patients induced by Tc-99m MDP and Tc-99m DTPA scintigraphies. Materials and Methods: Lymphocytes from 45 patients (25 males, 20 females, mean age $44{\pm}18$ years) were collected before and after scintigraphies using 740 MBq Tc-99m MDP (n=22) or Tc-99m DTPA (n=23). Lymphocytes from 20 controls (12 males, 8 females, mean age $43{\pm}7$ years) were also collected. They were exposed to challenge dose of 2 Gy ${\gamma}-rays$ using a Cs-137 cell irradiator Number of ring-form (R) and dicentric (D) chromosomes was counted under the light microscope. From them a representative score, Ydr, was calculated as Ydr=(D+R)/cells. Adaptation index (AI) was defined as difference of Ydr between unconditioned and conditioned lymphocytes. Ydr was also measured after an administration of cycloheximide (CHM), a protein synthesis inhibitor, before challenge dose. Results: RAR was induced in both groups of patients. CHM abolished the adaptive response in both groups. AI of Tc-99m MDP group was significantly higher than that of Tc-99m BTPA group. Conclusion: Tc-99m MDP induced RAR was more prominent than those induced by Tc-99m DTPA.

• Applied Microscopy
• /
• v.34 no.1
• /
• pp.31-42
• /
• 2004

Acute respiratory distress syndrome (ARDS) is a kind of acute lung injury characterized by inflammatory disruption of alveolar-capillary barrier and notorious for its high mortality. Neutrophils cause cell damage through the production of free radicals, inflammatory mediators, and proteases in ARDS. $PLA_2$ might serve a primary regulatory role in the activation of neutrophils. This present study was performed to elucidate the effect of rutin known as $PLA_2$ inhibitor on ARDS induced by endotoxin. Endotoxin had increased lung myeloperoxidase (MPO) activity, BAL (bronchoalveolar lavage) protein content, numbers of neutrophils in BALF (bronchoalveolar lavage fluid) compared with those of control rat (p<0.001). In addition, histological evidence of lung injury was correlated with neutrophil influx into alveolar space and cerrous perhydroxide granules were found in lining of endothelial cell, alveolar type I, II cells. In contrast, pretreated group of rutin had significantly decreased all of the parameters (p<0.001). These data suggest that inhibition of $PLA_2$ is one step approach that block the process of ARDS. Accordingly, we conclude that rutin can be used as the prophylactic agent for ARDS on the bases of these experimental results.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.21 no.4
• /
• pp.353-366
• /
• 1992

This study was devised to purify the compound from tuna that have cytotoxic activities against various cancer cell lines and to observe its immunopotentiating activities. The cytotoxic compound was partially purified 277 fold, from petroleum ehter extract (crude extract) of tuna by silicic acid column chromatography (fraction D) and thin layer chromatography (Spot I). Cytotoxic activity was monitored using human colon cancer cell, HCT-48. The active compound (Spot I) was composed of seven materials which are fatty acids of four kinds ($C_{14:0},\;C_{16:0},\;C_{17:1},\;and\;C_{18:0}$) and unknown three fat materials. The active compound has cytotoxic activities against various cancer cell lines, that is, murine leukemic lymphocytes (L1210, P388) and human rectal (HRT-18) and colon cancer cells (HCT-48, HT-29). The patterns of size distribution of HCT-48 cells in the medium containing tuna extract were shifted to direction of the small size region. Also, the microscopic shape of HCT-48 cells were shrinked and distracted. The number of plaque forming cell and immunoglobin fraction of serum protein obtained from tuna-treated mice were increased, but natural killer cell activity was not affected.

• Food Science and Preservation
• /
• v.31 no.3
• /
• pp.423-432
• /
• 2024

In this study, a vacuum tumbler with 4 impellers (DVT) was designed and applied for thawing frozen pork (vacuum -60 kPa, jacket 35℃, 1 rpm). Quality characteristics of the thawed pork were compared with those of industrially thawed meat by natural air at room temperature (NAT) and imported vacuum tumbler (IVT). The thawing time for frozen pork (303.36 kg) using DVT (165 min) was much shorter than that of NAT (4,200 min). DVT-thawed pork had lower drip loss (0.85%) than NAT (2.08%). DVT-thawed pork showed a pH of 5.92, a total bacterial count of 1.96±0.02 log CFU/g and no coliforms. Deteriorations in fat (TBARS 0.31±0.01 MDA mg/kg) and protein (VBN 5.67±1.98 mg%) in DVT-thawed pork were significantly lower than those of NAT (p<0.05). DVT-thawed pork had a high water-holding capacity (WHC, 97.5%). The hardness (34.59±0.46 N) and chewiness (188.21±0.17) of cooked DVT-thawed pork were about 5-6 times lower than those of NTA. Microstructure (SEM) showed myofibrillar damage in NAT-thawed pork, whereas dense myofibrillar structure was observed in DVT-thawed pork. DVT was better or similar to IVT in all evaluation parameters. The designed DVT is expected to be used as an efficient thawing method in terms of processing time and yield and to produce thawed meat with high WHC, soft texture, and low spoilage by minimizing tissue damage.