Publications by authors named "In-Won Lee"

28 Publications

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Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Cell Reprogram 2021 Apr;23(2):89-98

Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, Republic of Korea.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased ( < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.
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http://dx.doi.org/10.1089/cell.2020.0088DOI Listing
April 2021

Acentriolar microtubule organization centers and Ran-mediated microtubule formation pathways are both required in porcine oocytes.

Mol Reprod Dev 2019 08 28;86(8):972-983. Epub 2019 May 28.

Department of Animal Sciences, Chungbuk National University, Cheong-Ju, Chungcheongbuk-do, Republic of Korea.

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.
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http://dx.doi.org/10.1002/mrd.23172DOI Listing
August 2019

Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

FASEB J 2019 03 17;33(3):4432-4447. Epub 2018 Dec 17.

Department of Animal Science, Chungbuk National University, Cheongju, North Chungcheong, South Korea.

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis, and depletion of zinc impairs cell cycle control, asymmetric division, and cytokinesis in oocyte. We report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte impaired cortical and cytoplasmic actin formation. Spire is colocalized with zinc-containing vesicles via its zinc finger-containing Fab1, YOTB, Vac 1, EEA1 (FYVE) domain. Improper localization of Spire by zinc depletion or mutations in the FYVE domain impair cytoplasmic actin mesh formations and asymmetric division and cytokinesis of oocyte. All 3 major domains of the Spire are required for its proper localization and activity. After fertilization or parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire by controlling its localization in mammalian oocyte.-Jo, Y.-J., Lee, I.-W., Jung, S.-M., Kwon, J., Kim, N.-H., Namgoong, S. Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.
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http://dx.doi.org/10.1096/fj.201801905RDOI Listing
March 2019

Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness.

J Microbiol Biotechnol 2018 Jun;28(6):997-1006

Microbiology Department, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju 28644, Republic of Korea.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 10⁸ PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1, M1, and NS1). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 10⁷ PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.
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http://dx.doi.org/10.4014/jmb.1712.12007DOI Listing
June 2018

Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016-2017 winter season.

Emerg Microbes Infect 2018 Mar 14;7(1):29. Epub 2018 Mar 14.

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, KS001, Korea.

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus-type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.
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http://dx.doi.org/10.1038/s41426-018-0029-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849756PMC
March 2018

Comparison of the virulence and transmissibility of canine H3N2 influenza viruses and characterization of their canine adaptation factors.

Emerg Microbes Infect 2018 Mar 7;7(1):17. Epub 2018 Mar 7.

College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju, KS001, Republic of Korea.

Recent canine influenza outbreaks have raised concerns about the generation of pathogenic variants that may pose a threat to public health. Here, we examine avian-like H3N2 canine influenza viruses (CIVs) isolated from 2009 to 2013 in South Korea from dogs. Phylogenetic analysis revealed that these viruses are closely related to strains previously isolated from dogs in Korea and China. However, molecular characterization demonstrated non-synonymous mutations between the canine viruses, particularly in the putative H3 antigenic sites, NA stalk regions, and in the internal genes of the 2012-2013 isolates compared with the 2009 isolate. Animal experiments showed that three representative isolates (A/canine/Korea/AS-01/2009(AS-01/09), A/canine/Korea/AS-05/2012(AS-05/12) and A/canine/Korea/AS-11/2013(AS-11/13), were readily droplet transmitted between dogs, whereas AS-05/12 induced more severe clinical disease and was lethal in dogs compared with AS-01/09. Although all viruses were able to infect ferrets, AS-05/12 consistently yielded higher nasal wash titers and was transmissible to ferrets via airborne droplets. Using reverse genetics, we show that the NA, NP, and M genes of CIV are critical for the adaptation of avian H3N2 viruses, and the resulting reassortant genotypes promote viral growth in dogs in a manner similar to that of the wild-type AS-01/09 virus. Taken together, these results demonstrate that CIVs continuously evolve in dogs thereby allowing them to gain a foothold in mammalian hosts. Importantly, we elucidated the genetic contributions of the NA, NP, and M genes to the adaptability of CIVs derived from the avian H3N2 virus.
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http://dx.doi.org/10.1038/s41426-017-0013-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841232PMC
March 2018

The Effect of Precursor Composition on the Structural Properties of Nanocrystalline Diamond Films.

J Nanosci Nanotechnol 2018 Mar;18(3):1901-1904

Department of Nanomechatronics Engineering, Pusan National University, Busan 46241, Korea.

Nanocrystalline diamond (NCD) films were grown by hot filament CVD and the precursor composition dependence of the structural properties was examined. Films grown at 1 and 2 CH4 Vol% were found to be NCD layers with grain sizes of ~23-25 nm while films grown at 3-5 Vol% were identified as the mixtures of microcrystalline diamond and graphitic phase. The sp2/sp3 bonded carbon ratio in the grown films increased as the CH4 content increased up to 3 Vol% and then decreased beyond 4 Vol%. Microstructure and deposition rate were also found to be affected by the precursor composition and the NCD film grown at 1 CH4 Vol% showed a very dense microstructure and the highest deposition rate of ~3 nm/min.
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http://dx.doi.org/10.1166/jnn.2018.14936DOI Listing
March 2018

Identification of New Potential APE1 Inhibitors by Pharmacophore Modeling and Molecular Docking.

Genomics Inform 2017 Dec 29;15(4):147-155. Epub 2017 Dec 29.

Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an enzyme responsible for the initial step in the base excision repair pathway and is known to be a potential drug target for treating cancers, because its expression is associated with resistance to DNA-damaging anticancer agents. Although several inhibitors already have been identified, the identification of novel kinds of potential inhibitors of APE1 could provide a seed for the development of improved anticancer drugs. For this purpose, we first classified known inhibitors of APE1. According to the classification, we constructed two distinct pharmacophore models. We screened more than 3 million lead-like compounds using the pharmacophores. Hits that fulfilled the features of the pharmacophore models were identified. In addition to the pharmacophore screen, we carried out molecular docking to prioritize hits. Based on these processes, we ultimately identified 1,338 potential inhibitors of APE1 with predicted binding affinities to the enzyme.
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http://dx.doi.org/10.5808/GI.2017.15.4.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769857PMC
December 2017

Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.

FASEB J 2018 02 4;32(2):625-638. Epub 2018 Jan 4.

Department of Animal Science, Chungbuk National University, Cheongju, South Korea.

Mammalian oocytes lack a centriole that acts as a microtubule organization center (MTOC) in most somatic cells. During oocyte maturation, MTOCs undergo remodeling processes, including decondensation, fragmentation, and self-organization. However, the underlying mechanisms of MTOC remodeling in mouse oocytes are not well understood. We showed that two pericentriolar proteins, Cep192 and Cep152, play crucial roles during MTOC remodeling in mouse oocytes. Cep192 is present in MTOCs at all stages of oocyte maturation, and its depletion induces ablation of MTOCs, delay in spindle formation, and abnormal chromosomal alignment in spindles. In the case of Cep152, its localization on MTOCs is limited at the germinal vesicle stage and then disappears from the MTOCs after the germinal vesicle breakdown stage. Cep152 exclusion from MTOCs is involved in the fragmentation of MTOCs, and it is regulated by cyclin-dependent kinase 1 activity. Our results demonstrate the different roles of Cep192 and Cep152 in MTOC remodeling and a novel regulatory mechanism during meiotic spindle formation in mouse oocytes.-Lee, I.-W., Jo, Y.-J., Jung, S.-M., Wang, H.-Y., Kim, N.-H., Namgoong, S. Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.
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http://dx.doi.org/10.1096/fj.201700559RRDOI Listing
February 2018

CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation.

Development 2017 10 21;144(20):3829-3839. Epub 2017 Sep 21.

Department of Genetic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 16419, Korea

In somatic cells spindle microtubules are nucleated from centrosomes that act as major microtubule organizing centers (MTOCs), whereas oocytes form meiotic spindles by assembling multiple acentriolar MTOCs without canonical centrosomes. Aurora A and Plk1 are required for these events, but the underlying mechanisms remain largely unknown. Here we show that CIP2A regulates MTOC organization by recruiting aurora A and Plk1 at spindle poles during meiotic maturation. CIP2A colocalized with pericentrin at spindle poles with a few distinct cytoplasmic foci. Although CIP2A has been identified as an endogenous inhibitor of protein phosphatase 2A (PP2A), overexpression of CIP2A had no effect on meiotic maturation. Depletion of CIP2A perturbed normal spindle organization and chromosome alignment by impairing MTOC organization. Importantly, CIP2A was reciprocally associated with CEP192, promoting recruitment of aurora A and Plk1 at MTOCs. CIP2A was phosphorylated by Plk1 at S904, which targets CIP2A to MTOCs and facilitates MTOC organization with CEP192. Our results suggest that CIP2A acts as a scaffold for CEP192-mediated MTOC assembly by recruiting Plk1 and aurora A during meiotic maturation in mouse oocytes.
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http://dx.doi.org/10.1242/dev.158584DOI Listing
October 2017

Altered virulence of Highly Pathogenic Avian Influenza (HPAI) H5N8 reassortant viruses in mammalian models.

Virulence 2018 01 21;9(1):133-148. Epub 2017 Sep 21.

a Department of Microbiology , College of Medicine and Medical Research Institute, Chungbuk National University , Cheongju , Republic of Korea.

Recently identified highly pathogenic avian influenza (HPAI) H5N8 viruses (clade 2.3.4.4) are relatively low to moderately pathogenic in mammalian hosts compared with HPAI H5N1 viruses. In this study, we generated reassortant viruses comprised of A/MD/Korea/W452/2014(H5N8) with substitution of individual genes from A/EM/Korea/W149/2006(H5N1) to understand the contribution of each viral gene to virulence in mammals. Substituting the PB2 gene segment or the NA gene segment of the H5N8 virus by that from the H5N1 virus resulted in significantly enhanced pathogenicity compared with the parental H5N8 virus in mice. Of note, substitution of the PB2 gene segment of the H5N8 virus by that from the H5N1 virus resulted in a 1000-fold increase in virulence for mice compared with the parental virus (MLD decreased from 10 to 10 EID). Further, the W452 virus also induced the highest virus titers in lungs at all time points and the highest levels of inflammatory cytokine responses among all viruses tested. This high virulence phenotype was also confirmed by high viral titers in the respiratory tracts of infected ferrets. Further, a mini-genome assay revealed that W452 has significantly increased polymerase activity (p < 0.001). Taken together, our study demonstrates that a single gene substitution from other avian influenza viruses can alter the pathogenicity of recent H5N8 viruses, and therefore emphasizes the need for intensive monitoring of reassortment events among co-circulating avian and mammalian viruses.
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http://dx.doi.org/10.1080/21505594.2017.1366408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955454PMC
January 2018

Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea.

Infect Genet Evol 2017 09 3;53:56-67. Epub 2017 May 3.

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju 361-763, Republic of Korea. Electronic address:

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.
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http://dx.doi.org/10.1016/j.meegid.2017.05.001DOI Listing
September 2017

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

J Virol 2017 06 12;91(11). Epub 2017 May 12.

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses. Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.
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http://dx.doi.org/10.1128/JVI.02259-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432866PMC
June 2017

Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016.

Euro Surveill 2017 Jan;22(1)

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).
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http://dx.doi.org/10.2807/1560-7917.ES.2017.22.1.30434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388099PMC
January 2017

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

J Virol 2017 03 28;91(6). Epub 2017 Feb 28.

Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, South Korea

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed and growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses. Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.
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http://dx.doi.org/10.1128/JVI.01693-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331804PMC
March 2017

Centriolin, a centriole-appendage protein, regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes.

Cell Cycle 2017 Oct 11;16(19):1774-1780. Epub 2017 Jan 11.

a Department of Animal Sciences , Chungbuk National University , Cheongju , Korea.

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.
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http://dx.doi.org/10.1080/15384101.2016.1264544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628640PMC
October 2017

Genetic diversity and pathogenic potential of low pathogenic H7 avian influenza viruses isolated from wild migratory birds in Korea.

Infect Genet Evol 2016 11 9;45:268-284. Epub 2016 Sep 9.

College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea. Electronic address:

To detect the circulation of H7 avian influenza viruses, we characterized H7 viruses found in migratory birds and live poultry markets of South Korea from 2005 to 2014. Phylogenic analysis revealed that while all viruses clustered into the Eurasian-lineage of H7 avian viruses, at least 12 distinct genotypes were represented. Most H7 viruses contained at least one gene segment from the highly-pathogenic A/Sck/Hong Kong/YU100/02(H5N1)-like avian virus, and they could be separated into at least two antigenic groups. Although we did not detect genetically identical strains, HI assay demonstrated close cross-reactivity of some isolates with the H7N9 viruses from China. Animal studies revealed that most of the genotypes could replicate in the lungs of mice and chickens without prior adaptation and some, particularly H7N4 and H7N7 subtypes, induced mortality in mice. These results reinforce growing pandemic concerns regarding recent H7 viruses and emphasize the importance of continued surveillance of avian influenza viruses in the wild.
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http://dx.doi.org/10.1016/j.meegid.2016.09.005DOI Listing
November 2016

Genetic characteristics of highly pathogenic H5N8 avian influenza viruses isolated from migratory wild birds in South Korea during 2014-2015.

Arch Virol 2016 Oct 16;161(10):2749-64. Epub 2016 Jul 16.

College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, 361-763, Republic of Korea.

The continuous worldwide spread of highly pathogenic avian influenza (HPAI) H5N8 viruses among wild birds and poultry is a potential threat to public health. In the present study, we investigated the genetic characteristics of recent H5N8 viruses continuously isolated from migratory birds over two winters (2013-2014 and 2014-2015) in South Korea. Genetic and phylogenetic analysis demonstrated that the 2014-2015 HPAI H5N8 viruses are closely related to the 2013-2014 viruses, including virulence markers; however, all eight gene segments of 2014-2015 H5N8 viruses clustered in different phylogenetic branches from 2013-2014 H5N8 viruses, except the A/Em/Korea/W492/2015 virus. The H5N8 viruses of Europe and North America belong to sublineages of the 2013-2014 Korean H5N8 viruses but differ from the 2014-2015 Korean H5N8 viruses. Further hemagglutination inhibition (HI) assay results showed that there were 2-to-4 fold differences in HI titer between 2013-2014 and 2014-2015 H5N8 viruses. Taken together, our results suggested that the 2014-2015 Korean H5N8 viruses were genetically and serologically different from those of 2013-2014 winter season H5N8 viruses, including those from Europe and North America.
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http://dx.doi.org/10.1007/s00705-016-2979-4DOI Listing
October 2016

Depletion of the LINC complex disrupts cytoskeleton dynamics and meiotic resumption in mouse oocytes.

Sci Rep 2016 Feb 4;6:20408. Epub 2016 Feb 4.

Department of Animal Science, Chungbuk National University, Cheongju, Korea.

The SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) proteins constitute the linker of nucleoskeleton and cytoskeleton (LINC) complex on the nuclear envelope. To date, the SUN1/KASH5 complex is known to function as meiotic-specific factors. In this study, gene-silencing methods were used to explore the roles of SUN1 and KASH5 in mouse oocytes after prophase. SUN1 was detected throughout the nucleus; however, KASH5 was dispersed through the cell. After germinal vesicle breakdown (GVBD), SUN1 and KASH5 migrated during spindle formation and localized to the spindle poles at the MII stage. Most oocytes were arrested at the germinal vesicle (GV) stage after depletion of either SUN1 or KASH5. The DNA damage response was triggered in SUN1-depleted oocytes and thus gave rise to the G2/M checkpoint protein, p-CHK1. Oocytes that underwent GVBD had relatively small and abnormal spindles and lower levels of cytoplasm F-actin mesh. Immunofluorescence results also indicated the dislocation of pericentrin and P150(Glued) after SUN1 or KASH5 depletion. Furthermore, KASH5 localized exclusively near the oocyte cortex after SUN1 depletion, but SUN1 localization was unaffected in KASH5-depleted oocytes. Taken together, the results suggest that SUN1 and KASH5 are essential factors in the regulation of meiotic resumption and spindle formation.
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http://dx.doi.org/10.1038/srep20408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740751PMC
February 2016

Environmental Contamination and Viral Shedding in MERS Patients During MERS-CoV Outbreak in South Korea.

Clin Infect Dis 2016 Mar 17;62(6):755-60. Epub 2015 Dec 17.

Microbiology, College of Medicine and Medical Research Institute, and Zoonotic Infectious Diseases Research Center, Chungbuk National University, Seowon-Gu, Cheongju, Republic of Korea.

Background: Although Middle East Respiratory Syndrome coronavirus (MERS-CoV) is characterized by a risk of nosocomial transmission, the detailed mode of transmission and period of virus shedding from infected patients are poorly understood. The aims of this study were to investigate the potential role of environmental contamination by MERS-CoV in healthcare settings and to define the period of viable virus shedding from MERS patients.

Methods: We investigated environmental contamination from 4 patients in MERS-CoV units of 2 hospitals. MERS-CoV was detected by reverse transcription polymerase chain reaction (PCR) and viable virus was isolated by cultures.

Results: Many environmental surfaces of MERS patient rooms, including points frequently touched by patients or healthcare workers, were contaminated by MERS-CoV. Viral RNA was detected up to five days from environmental surfaces following the last positive PCR from patients' respiratory specimens. MERS-CoV RNA was detected in samples from anterooms, medical devices, and air-ventilating equipment. In addition, MERS-CoV was isolated from environmental objects such as bed sheets, bedrails, IV fluid hangers, and X-ray devices. During the late clinical phase of MERS, viable virus could be isolated in 3 of the 4 enrolled patients on day 18 to day 25 after symptom onset.

Conclusions: Most of touchable surfaces in MERS units were contaminated by patients and health care workers and the viable virus could shed through respiratory secretion from clinically fully recovered patients. These results emphasize the need for strict environmental surface hygiene practices, and sufficient isolation period based on laboratory results rather than solely on clinical symptoms.
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http://dx.doi.org/10.1093/cid/civ1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108026PMC
March 2016

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos.

PLoS One 2015 10;10(11):e0142561. Epub 2015 Nov 10.

Department of Animal Sciences, Chungbuk National University, Naesudong-ro, Seowon-gu, Cheongju-si, 362-763, Chungcheongbuk-do, Korea.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640854PMC
June 2016

Generating nearly single-cycle pulses with increased intensity and strongly asymmetric pulses of petawatt level.

Phys Rev E Stat Nonlin Soft Matter Phys 2012 Feb 16;85(2 Pt 2):026405. Epub 2012 Feb 16.

Department of Photonics and Applied Physics, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.

Generation of petawatt-class pulses with a nearly single-cycle duration or with a strongly asymmetric longitudinal profile using a thin plasma layer are investigated via particle-in-cell simulations and the analytical flying mirror model. It is shown that the transmitted pulses having a duration as short as about 4 fs (1.2 laser cycles) or one-cycle front (tail) asymmetric pulses with peak intensity of about 10^{21}W/cm^{2} can be produced by optimizing system parameters. Here, a new effect is found for the shaping of linearly polarized laser pulses, owing to which the peak amplitude of the transmitted pulse becomes larger than that of the incoming pulse, and intense harmonics are generated. Characteristics of the transmitting window are then studied for different parameters of laser pulse and plasma layer. For a circular polarization, it is shown that the flying mirror model developed for shaping laser pulses with ultrathin foils can be successfully applied to plasma layers having a thickness of about the laser wavelength, which allows the shape of the transmitted pulse to be analytically predicted.
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http://dx.doi.org/10.1103/PhysRevE.85.026405DOI Listing
February 2012

Phase-controlled switching by interference between incoherent fields in a double-Λ system.

Opt Express 2011 Feb;19(5):4113-9

Quantum optics Laboratory, Advanced Photonics Research Institute, GIST, Gwangju 500-712, South Korea.

We showed experimentally interference could be occurred between incoherent lights in a double-Λ lambda transition implemented with rubidium atomic vapor. Switching of probe transmission was controlled by the phases of two` independent probe lasers with low light intensity. More than 70% of the probe transmission could be switched by ultra-weak incoherent field. We suggested optically cryptic information could be delivered by the phase-controlled switching with incoherent fields in a double-Λ system.
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http://dx.doi.org/10.1364/OE.19.004113DOI Listing
February 2011

Femtosecond laser and arc discharge induced microstructuring on optical fiber tip for the multidirectional firing.

Opt Express 2010 Sep;18(19):19755-60

Advanced Photonics Research Institute(APRI), Gwangju Institute of Science and Technology (GIST), Buk-gu, Gwangju, South Korea.

Most optical fibers are designed for forward firing i.e. the light is emitted at the distal end along the optical axis of the fiber. In some applications such as the laser surgery and laser scanners, side firing of the optical fiber is required. In this paper, we present the microstructuring of an optical fiber tip using the femtosecond laser and an arc discharging process for the multidirectional firing of the beam. The distal end of the optical fiber with diameter of 125 μm was machined into a conical structure using a femtosecond laser. The surface of the machined tip was exposed to the arc discharge using a fiber splicer. The arc discharge leads to the melting and re-solidification of the fiber tip. This results in a smoothing of laser-induced conical microstructure at the tip of the fiber. We were able to demonstrate the multidirectional (circumferential) emission of the light from the developed fiber tip.
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http://dx.doi.org/10.1364/OE.18.019755DOI Listing
September 2010

Testing of steep convex aspheric surface with a Hartmann sensor by using a CGH.

Opt Express 2006 Apr;14(8):3247-54

Most aspheric surfaces have been tested by interferometer with some null correctors. This approach, however, often fails when there are many aspherical terms or test surface is very steep because it is not easy to design the conventional null lens or CGH (Computer Generated Hologram). On the other hand, 3-D profilometer can measure aspheric surfaces without any null correctors; however, it takes some time to measure, which makes it unsuitable for the production line in the factory. In this paper, we apply the Hartmann test to the measurement of steep convex aspheric surfaces of which diameter is about 16 mm. In order to increase the measurement accuracy, we calibrated the test setup using a CGH that simulates the ideal test surface. We demonstrated that the significant amount of error in the test setup could be removed by this calibration process. The test results showed only 2 nm rms WFE (wave front error) difference even though the WFE of test setup was worsened by more than 0.13 mum rms. Since this method makes it possible to measure highly aspheric surface quickly and accurately, it can be used in the production line.
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http://dx.doi.org/10.1364/oe.14.003247DOI Listing
April 2006

Null Hartmann test for the fabrication process of large aspheric surfaces.

Opt Express 2005 Mar;13(6):1839-47

Most aspheric mirrors have been tested by the null lens or computer-generated hologram method. This approach, however, requires that the shape of the surface be similar to the target shape; otherwise testing may not be possible or correct. The Hartmann test has an advantage in that it has a larger dynamic range than a general interferometer, which means that the surface can be tested beginning at an early stage of the polishing process. We suggest use of the null Hartmann test in conjunction with a phase-shifting interferometer for the measurement of a 0.9-m aspheric concave mirror. This setup was able to measure the surface with a large surface form error as well as with a small error without sacrificing any measurement accuracy. Using this setup, we have successfully polished a surface to remove approximately 1 microm of peak-to-valley wave-front error of a total of 39 microm of error during 1 month of polishing.
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http://dx.doi.org/10.1364/opex.13.001839DOI Listing
March 2005

Simple phase-shifting method in a wedge-plate lateral-shearing interferometer.

Appl Opt 2004 Jul;43(20):3989-92

Photometry & Imaging Optics Group, Korea Research Institute of Standards and Science, P.O. Box 102, Yusong, Daejeon, 305-600, Korea.

A simple phase-shifting method in a wedge-plate lateral shearing interferometer is described. Simply moving the wedge plate in an in-plane parallel direction gives the amount of phase shift required for phase-shifting interferometry because the thickness of a wedge plate is not constant and varies along the wedge direction. This method requires only one additional linear translator to move the wedge plate. The required moving distance for a phase shift of the wave front with this method is of the order of a millimeter, whereas the typical moving distance for another method that uses a piezoelectric transducer is of the order of a wavelength. This method yields better precision in controlling the moving distance than do the other methods.
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http://dx.doi.org/10.1364/ao.43.003989DOI Listing
July 2004