• 한국어병학회지
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• 제33권2호
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• pp.103-109
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• 2020

In order to use nervous necrosis virus (NNV) virus-like particles (VLPs) as a delivery tool for heterologous antigens or plasmids, we attempted to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs displaying a partial region of viral hemorrhagic septicemia virus (VHSV) glycoprotein at the surface and VLPs that are harboring DNA vaccine plasmids within the VLP. A peptide encoding 105 amino acids of VHSV glycoprotein was genetically inserted in the loop region of NNV capsid gene, and VLPs expressing the partial part of VHSV glycoprotein were successfully produced. However, in the transmission electron microscope analysis, the shape and size of the partial VHSV glycoprotein-expressing NNV VLPs were irregular and variable, respectively, indicating that the normal assembly of capsid proteins was inhibited by the relatively long foreign peptide (105 aa) on the loop region. To encapsulate by simultaneous transformation with both NNV capsid gene expressing plasmids and DNA vaccine plasmids (having an eGFP expressing cassette under the CMV promoter), NNV VLPs containing plasmids were produced. The encapsulation of plasmids in the NNV VLPs was demonstrated by PCR and cells exposed to the VLPs encapsulating DNA vaccine plasmids showed fluorescence. These results suggest that the encapsulation of plasmids in NNV VLPs can be done with a simple one-step process, excluding the process of disassembly-reassembly of VLPs, and NNV VLPs can be used as a delivery tool for DNA vaccine vectors.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.233-239
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• 2021

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λabs.max = 520 nm, λfluor.max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1722-1731
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• 2021

The genus Streptomyces is intensively studied due to its excellent ability to produce secondary metabolites with diverse bioactivities. In particular, adequate precursors of secondary metabolites as well as sophisticated post modification systems make some high-yield industrial strains of Streptomyces the promising chassis for the heterologous production of natural products. However, lack of efficient genetic tools for the manipulation of industrial strains, especially the episomal vector independent tools suitable for large DNA fragment deletion, makes it difficult to remold the metabolic pathways and streamline the genomes in these strains. In this respect, we developed an efficient deletion system independent of the episomal vector for large DNA fragment deletion. Based on this system, four large segments of DNA, ranging in length from 10 kb to 200 kb, were knocked out successfully from three industrial Streptomyces strains without any marker left. Notably, compared to the classical deletion system used in Streptomyces, this deletion system takes about 25% less time in our cases. This work provides a very effective tool for further genetic engineering of the industrial Streptomyces.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.621-629
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• 2022

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

• Journal of the Korean Wood Science and Technology
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• 제51권4호
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• pp.270-282
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• 2023

Lower termites need symbiotic microbes for cellulose digestion. Elizabethkingia miricola strain BM10 has been proposed as a symbiotic microbe that assists in low-temperature digestion and metabolism of Reticulitermes speratus KMT1, a termite on Bukhan Mountain, Seoul, Korea. In E. miricola strain BM10, β-glucosidase genes expressed at 10℃ were identified, and the psychrophilic enzymatic characteristic was confirmed by heterogeneously expressed proteins. Crude β-glucosidase in the culture broth of E. miricola strain BM10 showed specific enzymatic properties, and its substrate affinity was 4.69 times higher than that of Cellic CTec2. Among the genes proposed as β-glucosidase, two genes, bglB_1 and bglA_2, whose gene expression was more than doubled at 10℃ than at 30℃, were identified. They were heterogeneously expressed in Escherichia coli and identified as psychrophilic enzymes with an optimal reaction temperature of about 20℃-25℃. In this study, E. miricola strain BM10, a symbiotic bacterium of lower termites, produced psychrophilic β-glucosidases that contribute to the spread of the low-temperature habitat of a lower termite, R. speratus KMT1.

• Clinical and Experimental Vaccine Research
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• 제12권2호
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• pp.172-175
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• 2023

Coronavirus disease 2019 (COVID-19) has become a part of our lives now and we have no more effective way of coping than a vaccine. COVID-19 is a disease that causes severe thrombosis outside the respiratory tract. Vaccines also protect us in this respect, but in some rare cases, thrombosis has been found to develop after vaccination (much less frequently than COVID-19). What was interesting in our case was that it showed how a disaster could happen under three factors that predispose to thrombosis. A 65-year-old female patient with disseminated atherosclerosis was admitted to the intensive care unit with complaints of dyspnea and dysphasia. In the evening of the day, the patient had the vaccination 2 weeks ago, she had active COVID-19. On examination, lower extremity pulses could not be detected. The patient's imaging and blood tests were performed. Multiple complications such as embolic stroke, venous and arterial thrombosis, pulmonary embolism, and pericarditis were observed in the patient. This case may give consideration to anticoagulant therapy studies. We give effective anticoagulant therapy in the presence of COVID-19 in patients at risk of thrombosis. Can anticoagulant therapy be considered after vaccination in patients at risk of thrombosis such as disseminated atherosclerosis?

• Journal of Plant Biotechnology
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• 제50권
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• pp.225-231
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• 2023

Cold stress is one of the most vulnerable environmental stresses that affect plant growth and crop yields. With the recent advancements in genetic approaches using Arabidopsis and other model systems, genes involved in cold-stress response have been identified and the key cold signaling factors have been characterized. Exposure to low-temperature stress triggers the activation of a set of genes known as cold regulatory (COR) genes. This activation process plays a crucial role in enhancing the resistance of plants to cold and freezing stress. The inducer of the C-repeatbinding factor (CBF) expression 1-CBF module (ICE1-CBF module) is a key cold signaling pathway regulator that enhances the expression of downstream COR genes; however, this signaling module in Panax ginseng remains elusive. Here, we identified cold-signaling-related genes, PgCBF1, PgCBF3, and PgICE1 and conducted functional genomic analysis with a heterologous system. We confirmed that the overexpression of cold- PgCBF3 in the cbf1/2/3 triple Arabidopsis mutant compensated for the cold stress-induced deficiency of COR15A and salt-stress tolerance. In addition, nuclearlocalized PgICE1 has evolutionarily conserved phosphorylation sites that are modulated by brassinsteroid insensitive 2 (PgBIN2) and sucrose non-fermenting 1 (SNF1)-related protein kinase 3 (PgSnRK3), with which it physically interacted in a yeast two-hybrid assay. Overall, our data reveal that the regulators identified in our study, PgICE1 and PgCBFs, are evolutionarily conserved in the P. ginseng genome and are functionally involved in cold and abiotic stress responses.

• Molecules and Cells
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• 제46권11호
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• pp.710-724
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• 2023

The plant defense responses to microbial infection are tightly regulated and integrated with the developmental program for optimal resources allocation. Notably, the defense-associated hormone salicylic acid (SA) acts as a promoter of flowering while several plant pathogens actively target the flowering signaling pathway to promote their virulence or dissemination. Ralstonia pseudosolanacearum inject tens of effectors in the host cells that collectively promote bacterial proliferation in plant tissues. Here, we characterized the function of the broadly conserved R. pseudosolanacearum effector RipL, through heterologous expression in Arabidopsis thaliana. RipL-expressing transgenic lines presented a delayed flowering, which correlated with a low expression of flowering regulator genes. Delayed flowering was also observed in Nicotiana benthamiana plants transiently expressing RipL. In parallel, RipL promoted plant susceptibility to virulent strains of Pseudomonas syringae in the effector-expressing lines or when delivered by the type III secretion system. Unexpectedly, SA accumulation and SA-dependent immune signaling were not significantly affected by RipL expression. Rather, the RNA-seq analysis of infected RipL-expressing lines revealed that the overall amplitude of the transcriptional response was dampened, suggesting that RipL could promote plant susceptibility in an SA-independent manner. Further elucidation of the molecular mechanisms underpinning RipL effect on flowering and immunity may reveal novel effector functions in host cells.

• International Journal of Oral Biology
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• 제33권1호
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• pp.1-5
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• 2008

Adenosine 5'-triphosphate (ATP) is an important extracellular signaling molecule which is involved in a variety of physiological responses in many different tissues and cell types, by acting at P2 receptors, either ionotropic (P2X) or G protein-coupled metabotropic receptors (P2Y). P2X receptors have seven isoforms designated as $P2X_{1^-}P2X_7$. In this study, we investigated the electrophysiological and pharmacological properties of rat $P2X_{1^-}P2X_4$ currents by using whole-cell patch clamp technique in a heterologous expression system. When ATP-induced currents were analyzed in human embryonic kidney (HEK293) cells following transient transfection of rat $P2X_{1^-}P2X_4$, the currents showed different pharmacological and electrophysiological properties. ATP evoked inward currents with fast activation and fast desensitization in $P2X_{^1-}$ or $P2X_{3^-}$ expressing HEK293 cells, but in $P2X_{2^-}$ or $P2X_{4^-}$ expressing HEK293 cells, ATP evoked inward currents with slow activation and slow desensitization. While PPADS and suramin inhibited $P2X_2$ or $P2X_3$ receptor-mediated currents, they had little effects on $P2X_4$ receptor-mediated currents. Ivermectin potentiated and prolonged $P2X_4$ receptor-mediated currents, but did not affect $P2X_2$ or $P2X_3$ receptor-mediated currents. We suggest that distinct pharmacological and electrophysiological properties among P2X receptor subtypes would be a useful tool to determine expression patterns of P2X receptors in the nervous system including trigeminal sensory neurons and microglia.

• 미생물학회지
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• 제51권3호
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• pp.187-193
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• 2015

Candida (Pseudozyma로도 알려짐) antarctica lipase B(CAL-B)는 학문적으로 그리고 산업적으로 많이 활용되고 있다. CAL-B 자체에 대한 연구는 많이 진행되어온 반면, CAL-B 상동체에 관한 연구는 그리 알려진 바가 없다. 본 연구에서는 단백질 유사성 검색을 통해서 CAL-B의 상동체 탐색을 수행하였고, 6종의 단백질 서열을 찾았다. 해당하는 유전자들을 대장균에 대한 코돈 최적화를 수행하였고, 이를 바탕으로 유전자 합성을 진행하였다. 이들 유전자를 대장균 발현용 벡터에 클로닝한 후, 대장균 내에서 단백질 발현을 시도하여 이들 중 4종의 단백질이 성공적으로 발현되었다. 이들 단백질들이 가수분해 효소로서의 활성이 있는지 확인하기 위해서, 4-nitrophenyl acetate와 4-nitrophenyl butyrate를 반응기질로 하여 가수분해 반응성을 확인하였다. 이들 단백질들의 비활성(specific activity)값은 $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$로 측정되었고, 이는 CAL-B의 비활성 수치보다는 다소 낮은 값에 해당하였다. (${\pm}$)-1-phenylethyl acetate의 가수분해 반응에 대한 입체선택성은 이들 상동체 효소들 중에서 Pseudozyma hubeiensis SY62에서 유래된 효소만이 CAL-B의 입체선택성과 유사함이 확인되었다.