基本情報
- 所属
- 自治医科大学 医学部 感染・免疫学講座ウイルス学部門
- 学位
- 博士(医学)(2011年9月 京都大学)学士(獣医学)(2008年3月 岩手大学)
- ORCID ID
- https://orcid.org/0000-0002-0888-204X
- J-GLOBAL ID
- 201601002536816808
- Researcher ID
- AAD-1553-2020
- researchmap会員ID
- B000256217
研究キーワード
2経歴
10-
2022年10月 - 現在
-
2021年4月 - 2022年9月
-
2019年4月 - 2021年3月
-
2017年10月 - 2019年3月
-
2017年4月 - 2017年9月
学歴
2-
2008年4月 - 2011年9月
-
2002年4月 - 2008年3月
受賞
8論文
26-
The Journal of Infectious Diseases 2024年7月11日 査読有り筆頭著者責任著者Abstract Background Approximately 296 million people suffer from chronic hepatitis B (CHB) caused by hepatitis B virus (HBV). Current standard treatment, nucleos(t)ide analogs, are not efficient enough to eradicate HBV from the hepatocytes. Thus, developing new drugs for CHB is desired to achieve complete cure. Methods Here we established a novel HBV reporter system, HBV-HiBiT-PS2, to screen new drugs for CHB. HBV-HiBiT-PS2 was constructed by introducing a HiBiT-tag at the 5’-end of PreS2 and introduced into HepG2-NTCP cells. Culture supernatant containing HBV-HiBiT-PS2 virions was fractionated by a sucrose density gradient ultracentrifugation to characterize their components. Replication kinetics and reporter function of HBV-HiBiT-PS2 were determined by analyzing the parameters for HBV replication in the presence or absence of HBV inhibitors. Results HBV-HiBiT-PS2 could be used for monitoring most of the replication cycle of HBV. The effects of well-characterized HBV inhibitors could be evaluated by the HiBiT activity. HBV-HiBiT-PS2 could be specialized for screening secretion inhibitors for hepatitis B surface antigen (HBsAg) because most of the HiBiT activity was derived from subviral particles which are the multimers of HBsAg. Conclusions We demonstrated that HBV-HiBiT-PS2 would be a robust tool for screening novel drugs, especially HBsAg secretion inhibitors, targeted for CHB.
-
Hepatology Communications 8(1) 2024年1月 査読有りBackground: Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. Methods: The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. Results: NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. Conclusions: NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.
-
Scientific Reports 13(1) 13584 2023年8月21日 査読有り筆頭著者責任著者Abstract Chronic hepatitis B virus (HBV) infection is a major medical concern worldwide. Current treatments for HBV infection effectively inhibit virus replication; however, these treatments cannot cure HBV and novel treatment-strategies should be necessary. In this study, we identified tripartite motif-containing protein 26 (TRIM26) could be a supportive factor for HBV replication. Small interfering RNA-mediated TRIM26 knockdown (KD) modestly attenuated HBV replication in human hepatocytes. Endogenous TRIM26 physically interacted with HBV core protein (HBc), but not polymerase and HBx, through the TRIM26 SPRY domain. Unexpectedly, TRIM26 inhibited HBc ubiquitination even though TRIM26 is an E3 ligase. HBc was degraded by TRIM26 KD in Huh-7 cells, whereas the reduction was restored by a proteasome inhibitor. RING domain-deleted TRIM26 mutant (TRIM26ΔR), a dominant negative form of TRIM26, sequestered TRIM26 from HBc, resulting in promoting HBc degradation. Taking together, this study demonstrated that HBV utilizes TRIM26 to avoid the proteasome-dependent HBc degradation. The interaction between TRIM26 and HBc might be a novel therapeutic target against HBV infection.
-
BMC Microbiology 22(1) 300 2022年12月12日 査読有り筆頭著者Abstract Background Although conventional polymerase chain reaction (PCR) methods are widely used in diagnosis, the titer of the pathogenic virus is difficult to determine based on the PCR. In our prior report, a long-range reverse-transcription quantitative PCR (LR-RT-qPCR) assay was developed to assess the titer of UV-irradiated influenza A virus (IAV) rapidly. In this research, we focused on whether the LR-RT-qPCR assay could evaluate the titer of IAV inactivated by other methods. Methods IAV was inactivated by: heating at 100 °C for periods ranging from 1 to 15 min, treating with 0.12% sodium hypochlorite for periods ranging from 3 to 30 min, or treating with 70% ethanol for periods ranging from 10 to 30 min. Fifty percent tissue culture infectious dose (TCID50) assay was performed to confirm the efficacy of the inactivation methods, followed by LR-RT-qPCR to investigate the correlation between infectivity and copy number. Results One minute heating, 3 min sodium hypochlorite treatment, or 10 min ethanol treatment was sufficient to deactivate IAV. Changes before and after the inactivations in the copy numbers on LR-RT-qPCR were significantly different among the inactivation methods. Heat-inactivation drastically decreased the copy number to below the cutoff value around 5 copies/μL after 5 min treatment. The inactivation time of heating estimated using LR-RT-qPCR was marginally higher than that determined using TCID50. However, the treatments with sodium hypochlorite or ethanol moderately and minimally affected the copy numbers obtained using LR-RT-qPCR (~ 1 digit or no copy number decrease), respectively. Conclusions In addition to good applicability in UV-irradiation previously reported, the LR-RT-qPCR method is suitable for evaluating the effect of heat-inactivation on IAV infectivity. However, minor modifications may be made and investigated in the future to reduce the time intervals with TCID50. Although this method is not applicable for the ethanol inactivation, rapid evaluation of the effects of chlorination on IAV can be determined by comparing copy numbers before and after treatment using the LR-RT-qPCR method.
-
BMC INFECTIOUS DISEASES 20(1) 585 2020年8月 査読有り筆頭著者責任著者BackgroundThe polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation.MethodsIAV was irradiated with 253.7nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays.ResultsA long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3 termini of each genomic segment and subsequent qPCR of the 5 ' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R-2=0.931, P=0.000066).Conclusions p id=Par This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.
MISC
34-
肝臓 64(Suppl.2) A530-A530 2023年9月 査読有り筆頭著者
-
電気学会研究会資料. OQD = The papers of technical meeting on optical and quantum devices, IEE Japan / 光・量子デバイス研究会 [編] 2020(41-45) 25-28 2020年10月30日
-
「センサ・マイクロマシンと応用システム」シンポジウム論文集 電気学会センサ・マイクロマシン部門 [編] 35 4p 2018年10月30日
-
生物科学 = Biological science / 日本生物科学者協会 編 67(1) 28-37 2015年12月 査読有り招待有り筆頭著者
-
BIOLOGY OF REPRODUCTION 85 2011年7月
-
日本繁殖生物学会 講演要旨集 104 228-228 2011年【目的】内在性レトロウイルス関連遺伝子は、ヒト、マウスにおいて胎盤、栄養膜細胞の分化に必要であるとの報告がある。我々はこれまでに、ウシ胎盤、栄養膜細胞に発現する内在性レトロウイルス関連遺伝子が存在することを明らかにした。しかしウシにおいて、栄養膜細胞の分化過程に、内在性レトロウイルス関連遺伝子が関連するかどうかは不明である。本研究では、ウシ培養栄養膜細胞を用い、二核細胞分化誘導時に内在性レトロウイルス関連遺伝子がどのような発現変化をみせるのか明らかにすることを目的とした。<BR>【方法】我々が樹立したウシ培養栄養膜細胞(BT-B、BT-C、BT-K)を用い、コラーゲンゲル上培養、DHSを用いた血清飢餓培養、マウスEHS腫瘍由来基底膜様物質であるマトリゲルを用いたゲル上培養を8日間行うことで二核細胞を誘導した。またこれらの細胞を材料に定量的PCR法を行い、各細胞系列における二核細胞特異的遺伝子 (bCSH1、bPRP-1、bPAG-1)、IFNT並びに内在性レトロウイルス関連遺伝子(BERV-K1 env、BERV-K2 env、bERVE-A、P1x)のRNA発現変化を検討した。<BR>【結果】マトリゲル上で培養したBT-B、BT-Cでは、二核細胞特異的遺伝子の発現上昇が生じたが、BT-Kでは発現の変化は確認できなかった。コラーゲンゲル上で培養した場合、いずれの細胞系でもIFNTの発現上昇が認められた。このことから、BT-B、BT-Cのマトリゲル上培養において二核細胞への分化誘導が生じていると考えられた。また内在性レトロウイルス関連遺伝子の発現変化として、BT-B、BT-Cのマトリゲル上培養においてBERV-K1 env、bERVE-Aの発現上昇が、血清飢餓培養でbERVE-Aの発現上昇がそれぞれ認められた。一方、BERV-K2 env、P1xについては培養条件による発現変化は確認されなかった。これらの結果より、二核細胞への分化もしくは、二核細胞の形質に内在性レトロウイルス関連遺伝子が関与することが示唆された。
-
日本繁殖生物学会 講演要旨集 104 5-5 2011年【目的】祖先動物の生殖細胞に感染し、子孫にゲノムの一部として遺伝したレトロウイルスは内在性レトロウイルス(ERV)と定義される。多くのERVは変異や欠失によりそのオープンリーディングフレーム(ORF)を失っているが、一部のERVにはORFが保存され、宿主の生理機能を担うものが存在する。ヒトやマウスでは、胎盤形成時に必須である栄養膜細胞同士の融合をERVのエンベロープ(遺伝子;env,タンパク;Env)が担うと考えられている。ウシ胎盤においては、栄養膜二核細胞(BNC)と子宮内膜細胞(EMC)との融合細胞の存在が報告されているが、その形成に関わる機構は明らかになっていない。本研究では、最近我々が同定したウシ胎盤で発現する、ウシERV(BERV)-K1およびBERV-K2 envの融合細胞形成への関与を明らかにすべく実験を行った。<BR>【方法】FLAGタグ付BERV-K Env発現プラスミドを構築し、Cos-7細胞に導入後、イムノブロット(IB)により細胞溶解液中における両Envの検出を試みた。各Env発現Cos-7細胞を、初代培養ウシEMCと共培養し、細胞融合依存的ルシフェラーゼアッセイにより両Envの細胞融合活性を調べた。さらに、妊娠約30、90、150、230日齢のウシ胎盤組織における、両envmRNAならびにBERV-K1 Envの発現を、in situハイブリダイゼーション(ISH)ならびに免疫組織化学染色(IHC)により調べた。<BR>【結果と考察】IBの結果、BERV-K1およびBERV-K2 Envともにその発現を確認できたことから、両envはEnv産生能を有することが明らかとなった。さらに、BERV-K1 Envの細胞融合活性は、陽性対照と同様に高い値を示したのに対し、BERV-K2 Envの値は低かった。ISHの結果、ウシ胎盤ではBERV-K1 envのみ検出でき、その発現はBNCに特異的であった。そこで、BERV-K1 Envに対する抗体を作製し、IHCを行ったところ、ISHの結果と同様に、BERV-K1 EnvはBNC特異的に発現していることが明らかとなった。以上の結果より、BNCとEMCとの細胞融合過程に、BERV-K1 Envが中心的な役割を担うことが強く示唆された。
-
J-vet : jounral for veterinary practitioner 23(12) 21-33,96 2010年12月記事種別: 翻訳
-
BIOLOGY OF REPRODUCTION 295-296 2008年
-
BIOLOGY OF REPRODUCTION 104-105 2007年
講演・口頭発表等
2-
10th Korea-Japan-Taiwan HBV RESEARCH SYMPOSIUM 2024年4月27日
-
HEPATITIS B VIRUS HIJACK HOST TRIM26 TO AVOID PROTEASOME-DEPENDENT DEGRADATION OF VIRAL CORE PROTEINThe Liver Meeting® 2023 2023年
共同研究・競争的資金等の研究課題
11-
公益財団法人 武田科学振興財団 2024年度 武田科学振興財団 医学系研究助成(感染領域) 2024年8月 - 2029年3月
-
日本学術振興会 科学研究費助成事業 2024年4月 - 2027年3月
-
日本学術振興会 科学研究費助成事業 2022年4月 - 2025年3月
-
国立研究開発法人日本医療研究開発機構 令和4年度 肝炎等克服緊急対策研究事業 2022年4月 - 2025年3月
-
日本学術振興会 科学研究費助成事業 2021年4月 - 2024年3月