Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells

Kö,nig, Alexander,Yang, Jaewon,Jo, Eunji,Park, Kyu Ho Paul,Kim, Hyun,Than, Thoa Thi,Song, Xiyong,Qi, Xiaoxuan,Dai, Xinghong,Park, Soonju,Shum, David,Ryu, Wang-Shick,Kim, Jung-Hee,Yoon, Seung Kew,P Elsevier 2019 Journal of hepatology Vol.71 No.2

<P><B>Background & Aims</B></P> <P>As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible <I>in vitro</I> infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens.</P> <P><B>Methods</B></P> <P>An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed.</P> <P><B>Results</B></P> <P>Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec−. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation.</P> <P><B>Conclusions</B></P> <P>The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients.</P> <P><B>Lay summary</B></P> <P>Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient’s HBV strain to specific antiviral drugs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Cell culture system that mimicks complete HBV life cycle from entry to egress. </LI> <LI> Efficient <I>in vitro</I> infection with crude HBV patient sera. </LI> <LI> Up to 50- and 1,300-fold net amplification of patient- and cell culture-derived input HBV in the supernatant. </LI> <LI> Polyethylene glycol-independent HBV spread to adjacent cells, forming infected cell clusters. </LI> <LI> Evaluation of patient- and cell culture-derived HBV amplification w/wo antivirals over 8 weeks. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Thermopower Enhancement of Bi2Te3 Films by Doping I Ions

Kim, K. C.,Baek, S. H.,Kim, H. J.,Hyun, D. B.,Kim, S. K.,Kim, J. S. Springer Science + Business Media 2014 Journal of electronic materials Vol.43 No.6

The thermoelectric properties of I-doped Bi2Te3 films grown by metal-organic chemical vapor deposition have been studied. I-doped epitaxial (00l) Bi2Te3 films were successfully grown on 4A degrees tilted GaAs (001) substrates at 360 A degrees C. I concentration in the Bi2Te3 films was easily controlled by the variation in a flow rate of H-2 carrier gas for the delivery of an isopropyliodide precursor. As I ions in the as-grown Bi2Te3 films were not fully activated, they did not influence the carrier concentration and thermoelectric properties. However, a post-annealing process at 400 A degrees C activated I ions as a donor, accompanied with an increase in the carrier concentration. Interestingly, the I-doped Bi2Te3 films after the post-annealing process also exhibited enhancement of the Seebeck coefficient at the same electron concentration compared to un-doped Bi2Te3 films. Through doping I ions into Bi2Te3, the thermopower was also enhanced in Bi2Te3, and a high power factor of 5 x 10(-3) W K-2 m(-1) was achieved.

유방암 환자의 말초혈액에서 역전사효소연쇄중합반응을 이용한 Human Mammaglobin 측정의 임상적 유용성

김재홍,강석윤,송정엽,최태영,임홍석,김선경,김영진,박준성,김현수,최진혁,임호영,김효철 대한조혈모세포이식학회 2001 대한조혈모세포이식학회지 Vol.6 No.2

Background: The mammaglobin gene encodes a novel protein that is secreted from the mammalian epithelium of normal breast tissue as well as malignant breast cancer tissues. In order to ascertain the prognostic value of mammaglobin gene in breast cancer patients, we measured the expression of human mammaglobin (hMAM) by RT-PCR method in various stages of breast cancer patients. Methods: Peripheral blood samples from forty healthy volunteers and 114 breast cancer patients were obtained. Peripheral blood stem cells (PBSC) collected for the purpose of autologous stem cell transplantation in five patients with metastatic breast cancer and ten patient with high risk for relapse and no evidence of disease were used for hMAM assay. Results: All samples from peripheral blood of forty healthy individuals (twenty males and twenty females) were negative for hMAM, whereas 43 of 114 samples (38%) from breast cancer patients were positive for hMAM mRNA. All the normal breast tissues were positive for hMAM mRNA. hMAM mRNA expression was detected in 11 of 42 (26%) in breast cancer patients who underwent for curative resection and had no evidence of disease, in 8 of 25 (34%) with chemo-sensitive relapsed disease, and in 16 of 32 (53%) with chemo-refractory progressive disease. Eight (53%) samples from peripheral blood of 15 breast cancer patients with metastatic disease at diagnosis were positive for hMAM. Three (20%) samples from peripheral blood stem cells of 15 breast cancer patients for high dose chemotherapy were positive for hMAM. Conclusion : In contrast to healthy volunteers, hMAM transcripts were detected in the peripheral blood of breast cancer patients. The frequency of hMAM expression in peripheral blood was correlated with the clinical stages of disease, but, was not significant. The contamination of hMAM expressing cells in the stem cell pool warrants additional effective purging method before the transplantation. The clinical relevance of hMAM RT-PCR-based tumor cell detection in the peripheral blood of breast cancer patients should be further evaluated in prospective studies.

포상기태와 융모상피암 환자의 갑상선기능

박기현,김현만,허갑범,이현철,김경래,김한수,김주항,김세광 대한내분비학회 1988 Endocrinology and metabolism Vol.3 No.1

It has been recognized that hyperthyroidism occur in patients with trophoblastic disease, either hydatidiform moles or choriocarcinomas. In the past decade, several lines of evidence have shown that human chorionic gonadotropin, secreted by the trophoblastic disease, is a thyroid stimulator and causes hyperthyroidism. In order to evaluate the relationship between level of thyroid hormone and human chorionic gonadotropin in patients with trophoblastic disease, level of thyroid hormone, serum -HCG and amount of urinary excretion of HCG were measured and analyzed in 24 patients with hydatidiform mole and 11 patients with choriocarcinoma who were admitted to Yonsei University Severance Hospital during the period from January 1981 to December 1986. The results are summarzed as follows: 1) Hyperthyroidism was observed in 33.3% of the patients with hydatidiform mole and in 18.2% of the patients with choriocarcinoma. 2) The amount of 24 hour urinary excretion of HCG in patients with hyperthyroidism was more than that in euthroid patients. 3) There was no difference in the level of thyroid hormone, serum -HCG, and amount of 24 hour urinary excretion of HCG in patients with hydatidifrom mole and choriocarcinoma. 4) The amount of 24 hour urinary excretion of HCG had significant correlation with FT4, whereas no similar correlation was observed between the levels of thyroid hormone and serum -HCG. In conclusion, the occurrence of hyperthyroidism is closely related with the amount of urinary excretion of HCG in patients with trophblastic disease.

전도성 Al-ZnO (AZO) 나노입자의 광경화 투명 코전도성 Al-ZnO(AZO) 나노입자의 광경화 투명 코팅

김현종 ( Hyun Jong Kim ),신치호 ( Chi Ho Shin ),문정호 ( Jung Ho Moon ),한명근 ( M. K. Han ),임병태 ( B. T. Lim ),신태욱 ( T. W. Shin ) 한국공업화학회 2010 응용화학 Vol.14 No.2

Al-doped ZnO (AZO) nanoparticle was synthesized with the size of 100 nm and the sheet resistance of 445 kΩ/□ by polyol precess. The synthesized AZO nanoparticle was directly dispersed in iso-propyl alcohol to form a homogeneous solution. Then, it was mixed with photocurable acrylate monomer which was act as the both of binder and stabilizer, when the AZO was coated on the slide glass, it showed better transparency and conductivity than commercial one.

생쥐의 장내 미생몰로부터 새로운 슬포트란스훼라제 생산균의 분리

김병택(Byung-Taek Kim),김은하(Eun-Ha K im),김동현(Dong-Hyun Kim) 대한약학회 1992 약학회지 Vol.36 No.5

Haemophilus K-12 producing novel sulfotransferase was isolated from mouce intestinal flora. The enzyme catalyzed the transfer of sulfate group from p-nitrophenylsulfate to phenolic compounds. The optimum medium condition for the sulfotransferase production was 0.2% sucrose, 1% yeast extract, Na2HP0 4 and 0.5% NaCl. The enzyme production was induced by donor substrate, but was not by acceptors. When p-nitrophenylsulfate was used as a donor substrate, 1-naphthol was best substrate, followed by phenol, p-acetaminophenol and tyramine.

황산용액 중에서 Na2S2O8에 의한 코발트의 산화 침전

김현호 ( Hyun Ho Kim ),박경호 ( Kyung Ho Park ),남철우 ( Chul Woo Nam ),( P K Parhi ) 대한금속재료학회(구 대한금속학회) 2013 대한금속·재료학회지 Vol.51 No.12

Oxidation and precipitation of Co(II) in sulphate solution using Na2S2O8 as an oxidant was studied. The main factors affecting the oxidation of Co(II), the reaction time, temperature, pH of solution, agitation speed and amount of added Na2S2O8, were investigated. The results indicated that the oxidation rate of Co(II) was not affected by the agitation speed and the amount of the oxidant, but it increased obviously with an increasing pH of the solution and reaction temperature. The kinetics equation was established as .ln(1.X ) = k1.[H+].0.4755.exp ( 3800) .t with a first order reaction. The product precipitated by the oxidation T of Co(II) was CoOOH, and Co2O3 was obtained by calcination of CoOOH at 300 ℃ for 2 hours. The particle size of the precipitated product was influenced by the pH of the solution.

Aerosol delivery of urocanic acid-modified chitosan/programmed cell death 4 complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-ras null mice.

Jin, Hua,Kim, Tae Hee,Hwang, Soon-Kyung,Chang, Seung-Hee,Kim, Hyun Woo,Anderson, Hanjo K,Lee, Han-Woong,Lee, Kee-Ho,Colburn, Nancy H,Yang, Hsin-Sheng,Cho, Myung-Haing,Cho, Chong Su American Association for Cancer Research, Inc 2006 Molecular cancer therapeutics Vol.5 No.4

<P>The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for development of novel treatment options. Although several genes have been investigated for their antitumor activities through gene delivery, problems surrounding the methods used, such as efficiency, specificity, and toxicity, hinder application of such therapies in clinical settings. Aerosol gene delivery as nonviral and noninvasive method for gene therapy may provide an alternative for a safer and more effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in previous study was used as a gene carrier. The efficiency of UAC carrier in lungs was confirmed, and the potential effects of the programmed cell death protein 4 (PDCD4) tumor suppressor gene on three major pathways (apoptosis, cell cycle, and angiogenesis) were evaluated. Aerosol containing UAC/PDCD4 complexes was delivered into K-ras null lung cancer model mice through the nose-only inhalation system developed by our group. Delivered UAC/PDCD4 complex facilitated apoptosis, inhibited pathways important for cell proliferation, and efficiently suppressed pathways important for tumor angiogenesis. In summary, results obtained by Western blot analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay suggest that our aerosol gene delivery technique is compatible with in vivo gene delivery and can be applied as a noninvasive gene therapy.</P>

ECAP 강가공에 의한 마그네슘 AZ31합금의 결정립 미세화 및 미세조직 불안정성

김호경 ( H. K. Kim ),정강 ( K. Chung ),현창용 ( C. Y. Hyun ) 한국열처리공학회 2004 熱處理工學會誌 Vol.17 No.3

N/A Equal channel angular pressing (ECAP) technique had been adapted to the Mg alloy (AZ31) for achieving effective grain refinement through severe deformation. The average grain size of 2.5 ㎛ could be obtained after 4 passes. The stability of the ECAPed structure at elevated temperatures was examined by annealing the ECAPed materials over a wide range of temperature between 473 and 748 K. The average activation energy, Q, for static grain growth of 1, 2 and 3 passes was 33.7 kJ/㏖ (=0.25QL, activation for lattice diffusion). The abnormally low Q value in the lower temperature range may indicate that grain growth occurs in the unrecrystallized microstructure where non-equilibrium grain boundaries containing a large number of extrinsic dislocations exist. The yield stresses of the ECAPed alloys decreased whereas the elongations increased after the ECAP process. These results should be related to the modification of texture for easier slip on basal plane.

Identification and Characterization of a Novel Bacterial ATP-Sensitive K+ Channel

최승범,Jong-Uk Kim,Hyun Joo,Churl K. Min 한국미생물학회 2010 The journal of microbiology Vol.48 No.3

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.