• Asian-Australasian Journal of Animal Sciences
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• v.32 no.2
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• pp.241-248
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• 2019

Objective: To isolate and identify new methanogens from the rumen of Holstein steers in Korea. Methods: Representative rumen contents were obtained from three ruminally cannulated Holstein steers ($793{\pm}8kg$). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. Results: One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to $0.5{\mu}m$. KOR-2 utilized $H_2+CO_2$ and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were $38^{\circ}C$ and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. Conclusion: Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2.

• Fisheries and Aquatic Sciences
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• v.9 no.2
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• pp.70-74
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• 2006

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products. This sequencing strategy relies on 'nested' or 'tailed' primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. $5{\mu}L$) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.

• Korean Journal of Microbiology
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• v.33 no.2
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• pp.149-156
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• 1997

We have employed comparative RFL,P(Restriction Fragment Ixngth Pol~iniorphism) analysis and molecular phylogenetic techniques to investigate the diversity of uncultured microorganisms associated with the anaerobic PCP degradation in PCP-adapted enrichment cultures inoculated by samples from anaerobic cewage sludgc(Jangrim, Pusan) and leachate of landfill site(Kimhae). 16s rDNA cloncs were obtairted by PCR amplification of mixed population DNAs extracted directly from the nonactive and active stage ol each PCP-adapted culture. After three rounds of comparative RFLP analyses. two RFLP types. designated as Ala and Hld, were found prevalent and common in both active stage samples. Thc analysis of phylogenctic diversity bawd on the 5'-terminal 180 nt of sequences from whole clones of the Ala and Bld RFLP types showed close similarity among themselves. In case of Bld clones, 7XQ of them shared identical sequences. Thcse resuliq suggest that the clones of both RFLP types wcre originated from highly affiliated microorganisms which are e~iriched as a result of metabolic activity to PCP. The full-length 16s rRNA sequence of each representative clone from both RFLP types was determined. and an Ala clone w i n found to he related to Clo.strrdiurn ulfutzac~(Genk~ank No. Z69203) and a Bld clone to Thermobacteroides proteolyticus(Genbank No. X09335), with sequence similarities of 89%' and 97%. respectively.

• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.95-104
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• 2012

Purpose: The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat ($600^{\circ}C$) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. Methods: Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (${\alpha}1$), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. Results: Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. Conclusions: The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

• Parasites, Hosts and Diseases
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• v.56 no.6
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• pp.559-565
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• 2018

The identification and characterization of pathogenic and zoonotic tick-borne diseases like granulocytic anaplasmosis are essential for developing effective control programs. The differential diagnosis of pathogenic Anaplasma phagocytophilum and non-pathogenic A. phagocytophilum-like Anaplasma spp. is important for implementing effective treatment from control programs. The objective of the present study was to investigate the prevalence of Anaplasma spp. in horses in Korea by nucleotide sequencing and restriction enzyme fragment length polymorphism assay. Of the 627 horses included in the study, only 1 (0.2%) was infected with A. phagocytophilum. Co-infection with A. phagocytophilumlike Anaplasma spp. was not detected in the study. The 16S rRNA sequence of A. phagocytophilum was similar (99.5-100%) to A. phagocytophilum 16S rRNA isolated from horses in other countries. PCR adapted to amplify A. phagocytophilum groEL and msp2 genes failed to generate amplicons, suggesting genetic diversity in these genes. This study is the first molecular detection of A. phagocytophilum in horses in Korea. Human granulocytic anaplasmosis and animal infection of A. phagocytophilum have been reported in Korea recently. Because of vector tick distribution, global warming, and the increase of the horse industry, horses should be considered as a potential reservoir for A. phagocytophilum, and cross infectivity should be evaluated even though a low prevalence of infection was detected in this study. Furthermore, continuous surveillance and effective control measures for A. phagocytophilum should be established to prevent disease distribution and possible transmission to humans.

• Journal of Microbiology and Biotechnology
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• v.32 no.10
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• pp.1292-1298
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• 2022

Soil autotrophic bacterial communities play a significant role in the soil carbon (C) cycle in paddy fields, but little is known about how rhizosphere soil microorganisms respond to different long-term (35 years) fertilization practices under double rice cropping ecosystems in southern China. Here, we investigated the variation characteristics of rhizosphere soil RubisCO gene cbbL in the double rice ecosystems of in southern China where such fertilization practices are used. For this experiment we set up the following fertilizer regime: without any fertilizer input as a control (CK), inorganic fertilizer (MF), straw returning (RF), and organic and inorganic fertilizer (OM). We found that abundances of cbbL, 16S rRNA genes and RubisCO activity in rhizosphere soil with OM, RF and MF treatments were significantly higher than that of CK treatment. The abundances of cbbL and 16S rRNA genes in rhizosphere soil with OM treatment were 5.46 and 3.64 times higher than that of CK treatment, respectively. Rhizosphere soil RubisCO activity with OM and RF treatments increased by 50.56 and 45.22%, compared to CK treatment. Shannon and Chao1 indices for rhizosphere soil cbbL libraries with RF and OM treatments increased by 44.28, 28.56, 29.60, and 23.13% compared to CK treatment. Rhizosphere soil cbbL sequences with MF, RF and OM treatments mainly belonged to Variovorax paradoxus, uncultured proteobacterium, Ralstonia pickettii, Thermononospora curvata, and Azoarcus sp.KH33C. Meanwhile, cbbL-carrying bacterial composition was obviously influenced by soil bulk density, rhizosphere soil dissolved organic C, soil organic C, and microbial biomass C contents. Fertilizer practices were the principal factor influencing rhizosphere soil cbbL-carrying bacterial communities. These results showed that rhizosphere soil autotrophic bacterial communities were significantly changed under conditions of different long-term fertilization practices Therefore, increasing rhizosphere soil autotrophic bacteria community with crop residue and organic manure practices was found to be beneficial for management of double rice ecosystems in southern China.

• Animal Bioscience
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• v.35 no.3
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• pp.367-376
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• 2022

Objective: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry as well as the economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for HPAIV resistance. Therefore, in this study, we investigated gene expression related to cytokine-cytokine receptor interactions by comparing resistant and susceptible Ri chicken lines for avian influenza virus infection. Methods: Ri chickens of resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) lines were selected by genotyping the Mx dynamin like GTPase (Mx) and major histocompatibility complex class I antigen BF2 genes. These chickens were then infected with influenza A virus subtype H5N1, and their lung tissues were collected for RNA sequencing. Results: In total, 972 differentially expressed genes (DEGs) were observed between resistant and susceptible Ri chickens, according to the gene ontology and Kyoto encyclopedia of genes and genomes pathways. In particular, DEGs associated with cytokine-cytokine receptor interactions were most abundant. The expression levels of cytokines (interleukin-1β [IL-1β], IL-6, IL-8, and IL-18), chemokines (C-C Motif chemokine ligand 4 [CCL4] and CCL17), interferons (IFN-γ), and IFN-stimulated genes (Mx1, CCL19, 2'-5'-oligoadenylate synthase-like, and protein kinase R) were higher in H5N1-resistant chickens than in H5N1-susceptible chickens. Conclusion: Resistant chickens show stronger immune responses and antiviral activity (cytokines, chemokines, and IFN-stimulated genes) than those of susceptible chickens against HPAIV infection.

• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1427-1434
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• 2022

Two novel, halotolerant strains of Martelella soudanensis, NC18^T and NC20, were isolated from deep subsurface sediment, deeply sequenced, and comparatively analyzed with related strains. Based on a phylogenetic analysis using 16S rRNA gene sequences, the two strains grouped with members of the genus Martelella. Here, we sequenced the complete genomes of NC18^T and NC20 to understand the mechanisms of their halotolerance. The genome sizes and G+C content of the strains were 6.1 Mb and 61.8 mol%, respectively. Moreover, NC18^T and NC20 were predicted to contain 5,849 and 5,830 genes, and 5,502 and 5,585 protein-coding genes, respectively. Both strains contain the identically predicted 6 rRNAs and 48 tRNAs. The harboring of halotolerant-associated genes revealed that strains NC18^T and NC20 might tolerate high salinity through the accumulation of potassium ions in a "salt-in" strategy induced by K⁺ uptake protein (kup) and the K⁺ transport system (trkAH and kdpFABC). These two strains also use the ectoine transport system (dctPQM), the glycine betaine transport system (proVWX), and glycine betaine uptake protein (opu) to accumulate "compatible solutes," such as ectoine and glycine betaine, to protect cells from salt stress. This study reveals the halotolerance mechanism of strains NC18^T and NC20 in high salt environments and suggests potential applications for these halotolerant and halophilic strains in environmental biotechnology.

• Genomics & Informatics
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• v.19 no.3
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• pp.29.1-29.10
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• 2021

In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H₂O₂. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: -15 vs. -13.8 kcal/mol; and dissociation constant, K_d of wild-type vs. mutant: 7.2E^-12 vs. 7.0E^-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG(0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.