Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We previously demonstrated that ribosomal RNA (rRNA) of Bombyx mon BM-N cells is rapidly degraded upon infection with heterologous nucleopolyhedroviruses (NPVs), including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV and S. litura MNPV, and that this response is triggered by viral P143 proteins. The transient expression of P143 proteins from heterologous NPVs was also shown to induce apoptosis and caspase-3-like protease activation in BM-N cells. In the present study, we conducted a transient expression assay using BM-N cells expressing mutant AcMNPV P143 (Ac-P143) proteins and demonstrated that five amino acid residues cooperatively participate in Ac-P143 protein-triggered apoptosis of BM-N cells. Notably, these five residues were previously shown to be required for triggering rRNA degradation in BM-N cells. As rRNA degradation in BM-N cells does not result from apoptosis, the present results suggest that Ac-P143-triggered rRNA degradation is the upstream signal for apoptosis induction in BM-N cells. We further showed that P143 protein-triggered apoptosis does not occur in S. frugiperda Sf9 or Lymantria dispar Ld652Y cells, indicating that apoptosis induction by heterologous P143 proteins is a BM-N cell-specific response. In addition, the observed induction of apoptosis in BM-N cells was found to be mediated by activation of the initiator caspase Bm-Dronc. Taken together, these results suggest that BM-N cells evolved a unique antiviral system that recognizes heterologous NPV P143 proteins to induce rRNA degradation and caspase-dependent apoptosis. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2017.03.012

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Recent advances in genome-wide surveys have revealed a number of lepidopteran insect homologs of mammalian and Drosophila genes that are responsible for apoptosis regulation. However, the underlying molecular mechanisms for apoptosis regulation in lepidopteran insect cells remain poorly understood. In the present study, we demonstrated that the transfection of Bombyx mori BM-N cells with dsRNA against the B. mori cellular iap1 gene (cbm-iap1) induces severe apoptosis that is accompanied by an increase of caspase-3-like protease activity. In these apoptotic cells, the cleaved form of the endogenous initiator caspase Dronc (Bm-Dronc) was detected, indicating that cBm-IAP1 protein depletion by RNAi silencing resulted in the activation of Bm-Dronc. In transient expression assays in BM-N cells, cBm-IAP1 suppressed the apoptosis triggered by Bm-Dronc overexpression and depressed the elevation of caspase-3-like protease activity, but also increased the cleaved form of Bm-Dronc protein. cBm-IAP1 also suppressed the caspase-3-like protease activity stimulated by Bm-caspase-1 overexpression. Co-immunoprecipitation experiments demonstrated that cBm-IAP1 strongly interacts with Bm-Dronc, but only has weak affinity for Bm-caspase-1. Transient expression analyses showed that truncated cBm-IAP1 proteins defective in the BIR1, BIR2 or RING domain were unable to suppress Bm-Dronc-induced apoptosis. In addition, BM-N cells expressing truncated cBm-IAP1 proteins underwent apoptosis, suggesting that intact cBm-IAP1, which has anti-apoptotic activity, was replaced or displaced by the over expressed truncated cBm-IAP1 proteins, which are incapable of interfering with the apoptotic caspase cascade. Taken together, the present results demonstrate that cBm-IAP1 is a vital negative regulator of apoptosis in BM-N cells and functions by preventing the activation and/or activity of Bm-Dronc and Bm-caspase-1. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ibmb.2016.10.012

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Language：English   Publisher：The Japanese Society of Sericultural Science  

We previously found that rRNA of BM-N cells derived from the silkworm <i>Bombyx mori</i> undergoes rapid and extensive degradation through site-specific cleavage during abortive infection with nucleopolyhedroviruses (NPVs) of <i>Autographa californica</i> (AcMNPV), <i>Hyphantria cunea</i> (HycuMNPV), <i>Spodoptera exigua</i> and <i>S. litura</i>. Here, we demonstrated that rRNA degradation also occurs in Bme21 and Bm-aff3 cells, which are derived from <i>B. mori</i> embryo and fat body, respectively, during infection with AcMNPV. rRNA degradation in Bme21 cells was also observed following HycuMNPV infection, but was not detected in Bm-aff3 cells. We further showed that rRNA in a cell line derived from <i>B. mandarina</i>, an ancestor of <i>B. mori</i>, underwent degradation in response to cellular infection with AcMNPV and HycuMNPV. In contrast, no rRNA degradation was observed in a cell line derived from <i>Antheraea pernyi</i>. Taken together, these results indicate that NPV-triggered rRNA degradation represents a mechanism of innate antiviral immunity that is unique to <i>B. mori</i> and <i>B. mandarina</i> cells.<br>

DOI： 10.11416/jibs.85.3_073

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We previously demonstrated that rRNA undergoes rapid and extensive degradation in Bombyx mori BM-N cells upon infection with AcMNPV, which is triggered by AcMNPV P143 (Ac-P143) protein. Here, we showed that six amino acid residues of Ac-P143 protein, distributing between positions 514 and 599, are involved in rRNA degradation in BM-N cells. The six residues are highly conserved among P143 proteins from AcMNPV, HycuMNPV, SeMNPV and SpItMNPV, which trigger rRNA degradation in BM-N cells upon infection, but are only partially conserved in Bm-P143 protein, which does not induce rRNA degradation in BM-N cells. We also demonstrated that substitution of only two selected residues (N565S/L578F) of Bm-P143 protein with the corresponding Ac-P143 protein residues generates a mutant Bm-P143 protein that is capable of triggering rRNA degradation in BM-N cells. These results indicate that BmNPV evolved a unique P143 protein to evade the antiviral response and allow replication in B. mori cells. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2015.08.008

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Language：English   Publisher：WILEY  

Baculoviruses, members of the family Baculoviridae, are large, enveloped viruses that contain a double-stranded circular DNA genome of 80-180kbp, encoding 90-180 putative proteins. These viruses are exclusively pathogenic for arthropods, particularly insects, and have been developed, or are being developed, as environmentally sound pesticides and eukaryotic vectors for foreign protein expression, surface display, gene delivery for gene therapy, vaccine production and drug screening. The baculoviruses contain a set of approximately 30 core genes that are conserved among all baculovirus genomes sequenced to date. Individual baculoviruses also contain a number of lineage- or species-specific genes that have greatly impacted the diversification and evolution of baculoviruses. In this review, we first describe the general properties and biology of baculoviruses and then focus on the baculovirus genes and mechanisms involved in the replication, spread and survival of baculoviruses within the context of their diversity, evolution and insect manipulation.

DOI： 10.1111/ens.12105

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Language：English   Publisher：The Japanese Society of Sericultural Science  

We previously showed that rRNA cleavage and degradation occur in <i>Bombyx mori</i> BM-N cells upon infection with heterologous NPVs, including <i>Autographa californica</i> multiple NPV (AcMNPV), <i>Hyphantria cunea</i> MNPV, <i>Spodoptera exigua</i> MNPV, and <i>Spodoptera litura</i> MNPV. Additionally, transient expression assay analyses suggested that viral P143s, which are baculovirus DNA helicases that are essential for viral DNA replication, are responsible for the observed rRNA cleavage and degradation. Here, we examined whether viral DNA replication is related to rRNA cleavage and degradation in AcMNPV-infected BM-N cells using an AcMNPV temperature-sensitive-mutant (ts8) defective in P143 function at non-permissive temperature. Electrophoretic analyses of total RNAs from ts8-infected BM-N cells revealed that rRNA cleavage and degradation still occurred in the absence of viral DNA replication at non-permissive temperature. In addition, transfection analysis with an <i>ac-p143</i> null AcMNPV bacmid demonstrated that the <i>p143</i> gene is responsible for rRNA cleavage and degradation in AcMNPV-infected BM-N cells. Taken together, these results indicate the possibility that the viral DNA replication-related function of P143 is not associated with rRNA cleavage and degradation in NPV-infected BM-N cells.<br>

DOI： 10.11416/jibs.83.1_019

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Other Link： https://jlc.jst.go.jp/DN/JALC/10041327660?from=CiNii

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We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells. © 2013, American Society for Microbiology.

DOI： 10.1128/JVI.02065-13

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Cell lines derived from the silkworm, Bombyx mori, are only permissive for B. mori nucleopolyhedrovirus (NPV), with other NPVs generally resulting in abortive infection. Here, we demonstrate that rRNA of B. mori BM-N cells undergoes rapid degradation through site-specific cleavage upon infection with NPVs from Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua (SeMNPV) and Spodoptera litura (SpltMNPV). No significant decreases in cellular RNA were observed in Ld652Y, Se301, Sf9, Splm and S2 cells infected with AcMNPV or HycuMNPV, indicating the response is unique to BM-N cells. A transient expression assay using a cosmid library of the HycuMNPV genome demonstrated that HycuMNPV P143 is responsible for rRNA degradation, which was also detected in BM-N cells transfected with plasmids expressing the P143 proteins from AcMNPV, SeMNPV and SpltMNPV. These results indicate that B. mori evolved to acquire a unique antiviral immune mechanism that is activated by P143 proteins from heterologous NPVs.

DOI： 10.1099/vir.0.053645-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329 bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706 Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAc Delta p35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAc Delta p35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2013.05.103

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Innate immunity is essential for insects to survive infectious pathogens. In baculovirus-infected lepidopteran cells, apoptosis and global protein synthesis shutdown are major mechanisms of intracellular innate immunity that inhibit viral replication. In contrast, baculoviruses have evolved diverse genes and mechanisms to counter the antiviral immunity activated in infected cells. In this review, we summarize the current knowledge of the cellular antiviral pathways and the baculovirus genes that modulate antiviral immunity. The studies highlighted illustrate a high degree of diversity in both the cellular responses against viral infections and viral responses against intracellular antiviral immunity, providing an important basis of further studies in this field. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2012.10.016

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission.IMPORTANCEThe efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

DOI： 10.1128/spectrum.02859-23

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The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

DOI： 10.1038/s41598-022-05036-x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

Rotaviruses (RVs) are an important cause of acute gastroenteritis in young children. Recently, versatile plasmid-based reverse genetics systems were developed for several human RV genotypes; however, these systems have not been developed for all commonly circulating human RV genotypes. In this study, we established a reverse genetics system for G2P[4] human RV strain HN126. Nucleotide sequence analysis, including that of the terminal ends of the viral double-stranded RNA genome, revealed that HN126 possessed a DS-1-like genotype constellation. Eleven plasmids, each encoding 11 gene segments of the RV genome, and expression plasmids encoding vaccinia virus RNA capping enzyme (D1R and D12L), Nelson Bay orthoreovirus FAST, and NSP2 and NSP5 of HN126, were transfected into BHK-T7 cells, and recombinant strain HN126 was generated. Using HN126 or simian RV strain SA11 as backbone viruses, reassortant RVs carrying the outer and intermediate capsid proteins (VP4, VP7 and VP6) of HN126 and/or SA11 (in various combinations) were generated. Viral replication analysis of the single, double and triple reassortant viruses suggested that homologous combination of the VP4 and VP7 proteins contributed to efficient virus infectivity and interaction between other viral or cellular proteins. Further studies of reassortant viruses between simian and other human RV strains will contribute to developing an appropriate model for human RV research, as well as suitable backbone viruses for generation of recombinant vaccine candidates.

DOI： 10.1099/jgv.0.001816

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The cell line NISES-AnPe-428 (AnPe), derived from the Chinese oak silkworm Antheraea pernyi, was characterized for its permissiveness and productivity for six different nucleopolyhedrovirus (NPV) species. These NPVs included homologous Antheraea pernyi NPV (AnpeNPV) and heterologous Autographa californica multiple NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Spodoptera exigua MNPV (SeMNPV), and Lymantria dispar MNPV (LdMNPV), representing viruses that had been isolated from insect species belonging to five different families (Saturniidae, Noctuidae, Bombycidae, Arctiidae, and Lymantriidae). We found that AnPe cells supported productive replication of AnpeNPV, AcMNPV, BmNPV, HycuMNPV, and SeMNPV to varying degrees. Upon infection with SeMNPV, a subset of AnPe cell population in the culture underwent apoptosis, while remaining cells produced limited amounts of progeny virions and polyhedra. AnPe cells were refractory to LdMNPV infection and failed to support replication of viral DNA, indicating that viral replication was restricted at or prior to the step of viral DNA replication. These results indicated that AnPe cells have the potential to provide excellent systems for studying the molecular mechanisms underlying cellular permissiveness for NPV replication and host-range determination of NPVs.

DOI： 10.1007/s10616-021-00485-0

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High-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.

DOI： 10.1038/s41467-021-24001-2

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Ld652Y cells from the gypsy moth, Lymantria dispar, are highly sensitive to apoptotic stimuli compared to BM-N cells from the silkworm, Bombyx mori, and undergo apoptosis upon infection with various nucleopolyhe-droviruses (NPVs). We previously cloned and characterized Dronc homologues from L. dispar Ld652Y and B. mori BM-N cells and demonstrated that L. dispar Dronc (Ld-Dronc) undergoes proteolytic processing more rapidly and extensively than does B. mori Dronc (Bm-Dronc) upon infection with vAcΔp35, a p35-defective Autographa californica multiple NPV. Here, we comparatively examined expression and proteolytic processing of the Ld- and Bm-Dronc proteins in the transient expression assays using three different insect cell lines, Ld652Y, BM-N and Spodoptera frugiperda Sf9. We demonstrate that even in the transient expression assays, Ld-Dronc undergoes proteolytic processing and activates caspase-3-like protease more rapidly and extensively than Bm-Dronc in a cell-line independent manner. These results indicate that intrinsic properties of Dronc are involved in the differing capacity of Ld652Y and BM-N cells to induce apoptosis in response to apoptotic stimuli, including NPV infection.

DOI： 10.11416/jibs.82.2_049

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Event date： 2014.10

Presentation type：Oral presentation (general)  

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Event date： 2014.10

Presentation type：Oral presentation (general)  

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Event date： 2014.9

Presentation type：Poster presentation  

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Event date： 2014.9

Presentation type：Poster presentation  

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Event date： 2014.9

Presentation type：Poster presentation  

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Event date： 2014.4

Presentation type：Oral presentation (general)  

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Event date： 2014.4

Presentation type：Oral presentation (general)  

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Event date： 2014.4

Presentation type：Oral presentation (general)  

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Event date： 2013.11

Presentation type：Oral presentation (general)  

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Event date： 2013.11

Presentation type：Oral presentation (general)  

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Event date： 2013.9

Presentation type：Oral presentation (general)  

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Event date： 2013.9

Presentation type：Oral presentation (general)  

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Event date： 2013.3

Presentation type：Oral presentation (general)  

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Event date： 2012.11

Presentation type：Oral presentation (general)  

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Event date： 2012.11

Presentation type：Oral presentation (general)  

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Event date： 2012.9

Presentation type：Poster presentation  

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Event date： 2012.8

Language：English   Presentation type：Poster presentation  

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Event date： 2012.3

Presentation type：Oral presentation (general)  

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Event date： 2011.11

Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (invited, special)  

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