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Enhanced intracellular survival of Brucella abortus in RAW 264.7 cells by mutagenesis at BruAb2_1031
( Myunghwan Jung ),( Soo Jin Shim ),( Young Bin Im ),( Woo Bin Park ),( Hansang Yoo ) 대한인수공통전염병학회 2016 창립총회 및 학술대회 초록집 Vol.2016 No.1
Introduction: Brucella abortus(B. abortus) , known as intracellular bacteria, can invade and replicate in professional and nonprofessional phagocytic cells. Pathogenesis of B. abortus is more related to strategies for intracellular survival such as modulation of normal host functions than production of classical virulence factors such as exotoxin, cytolysins, capsules, fimbria, plasmids, lysogenic phage, and endotoxic lipopolysaccharide (LPS) molecules. Therefore, identification of the intracellular survival strategies of B. abortus is important in a study on pathogenicity of brucellosis. As the first step to find out the intracellular survival strategies, functions of Brucella genes were analyzed using transposon mutagenesis based on differences in the transcriptional responses between macrophages infected with the B. abortus mutant strains and the B. abortus wild strain. Materials and methods: Mutant strains derived from B. abortus 1119-3 strain, C3, C24, and C30, were generated by Tn5 transposome complexes.Identification of transposon insertion site was conducted using randomly primed PCR and Southern blot. Macrophages were infected with B. abortus wild and mutant strains of MOI 10. Total RNA was extracted from the cells at 0, 12, 24 h after infection using RNeasy mini kit as described by the manufacturers. Following purity and integrity check, RNA samples were subjected to microarray hybridization. The results of microarray were validated using quantitative real-time PCR. The genes showing change of expression level were analyzed based on the pathway, gene networks, and biological process. Gentamicin assay was also carried out using RAW 264.7 cells infected with each strain at MOI 100 to demonstrate levels of internalization, intracellular survival, and replication in RAW 264.7 cells. Results: The single insertion of transposon was confirmed by randomly primed PCR and Southern blot in the mutant strains. Following infection, wild strain infected RAW 264.7 cells showed up-regulated inflammatory responses and down-regulated phagolysosome formation process. The genes involved in apoptosis showed both up- and down-regulation. When compared to the transcriptional responses of wild strain infected cells, down-regulation of genes associated with cytokine responses and apoptosis was observed in RAW 264.7 cells infected with C3 mutant strain, in which mutation was confirmed at the site of the ATP-binding cassette transporter permease (BruAb2_1031). However, RAW 264.7 cells infected with C24 and C30 mutant strains did not show notable differences when compared to the wild strain infected cells. Although B. abortus wild strain showed high level of internalization, a steep decrease in CFU number of intracellular brucellae was observed in wild strain infected RAW 264.7 cells when compared to C3 mutant (p< 0.01). In real-time PCR to verify the microarray results, all genes determined by PCR showed the same direction in expression levels as the microarray results. Conclusion: C3 mutant strain infected RAW 264.7 cells showed down-regulation in genes associated with protective immune responses to Brucella infection. This result suggested that C3 mutant strain has more enhanced strategies for intracellular survival than the wild strain. Therefore, it is plausible that transposon insertion at BruAb2_1031 of Brucella abortus could enhance the intracellular survival in RAW 264.7 cells. Acknowledgements: This work was supported by NRF grant funded by MSIP (No. 2014R1A2A2A01007291), Bk21 PLUS and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.
Salmonella spp. 특이적인 검출을 위한 SYBR Green real-time PCR 기법 적용
신승원,차승빈,이원정,신민경,정명환,유안나,정병열,유한상,Shin, Seung Won,Cha, Seung Bin,Lee, Won-Jung,Shin, Min-Kyoung,Jung, Myunghwan,Yoo, Anna,Jung, Byeng Yeal,Yoo, Han Sang 대한수의학회 2013 大韓獸醫學會誌 Vol.53 No.1
The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle ($C_T$) of Salmonella spp. was $11.83{\pm}0.78$ while non-Salmonella spp. was $30.86{\pm}1.19$. Correlation coefficients of standard curves constructed using $C_T$ versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity ($R^2=0.993$; slope = 3.563). Minimum level of detection with the method was > $10^2$ colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.
Jung, Jaehan,Byun, Myunghwan,Chang, Mincheol Elsevier 2019 APPLIED SURFACE SCIENCE - Vol.475 No.-
<P><B>Abstract</B></P> <P>Intimately contact organic-inorganic nanocomposites were crafted by capitalizing on robust click coupling between judiciously synthesized functionalized nanocrystals (NCs) and end-functionalized organics. The functionalized NCs were synthesized with no need for ligand exchange by directly employing bifunctional ligand (i.e., 4-bromomethyl benzoic acid) during NC synthesis steps. The synthetic condition was first explored by judiciously tailoring the ratio between aliphatic ligands and bifunctional ligands to control its morphology. Subsequent substitution of bromine moiety at the NCs into azide yielded azide-terminated NCs. Finally, ethynyl-terminated molecules were grafted onto azide-functionalized NCs surface via click coupling, forming intimately contact organic-inorganic hybrid nanocomposites. The success of grafting of CPs with NCs was substantiated by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Hybrid inorganic-organic core-shell elongated architecture may enhance the device performance if the proper alignment of NRs can be achieved. To this end, the synthetic procedure to crafting organic-inorganic hybrid possibly can be employed to prepare variety of promising building block which can be served as a lot of applications including lasers, solar cells, and LEDs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Mobile nature of monomers results in anisotropic structure. </LI> <LI> Surface engineering with bifunctional ligands introduces functional moieties. </LI> <LI> Functional moieties such as bromide and azide enable chemical coupling. </LI> <LI> Catalyst-free click chemistry yields organic-inorganic nanocomposites. </LI> </UL> </P>
A comprehensive overview of coccidiosis in chicken
( Myunghwan Yu ),( Jung Min Heo ) 한국축산학회 2021 축산기술과 산업 Vol.8 No.2
Coccidiosis is the main parasitic disease resulting from the intracellular protozoan that targets each different part of the intestinal tract leading to destroy in poultry. For this reason, coccidiosis induces an enormous economic loss in the poultry industry. Eimeria life cycle is complicated and comprised of exogenous and endogenous stages inducing an inflammatory response which results in enteric damage associated with diarrheal hemorrhage, disorder digestion of feed and nutrient absorption, dehydration, blood loss, mortality. Hence, it is very important to understand the information of Eimeria parasites for elimination and treatment. This disease has been controlled by various anticoccidial drugs and vaccines as the most common management practices. However, not only the occurrence of drug resistance due to anticoccidial drugs but lack of a guarantee of safety with vaccine use, has led to the development of alternative strategies to control coccidiosis. For these reasons, phytogenic compounds are emerging for the control and prevention of poultry coccidiosis to alternate previous methods. The main aim of this review is to provide an overview of coccidiosis including etiology, morphology, life cycle, pathogenicity, clinical sign, diagnosis, control and prevention.