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Development of Synthetic Unimolecular Chaperon

Research Project

Project/Area Number 19H02831
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 37010:Bio-related chemistry
Research InstitutionTokyo Institute of Technology

Principal Investigator

Kinbara Kazushi  東京工業大学, 生命理工学院, 教授 (30282578)

Co-Investigator(Kenkyū-buntansha) 田中 良和  東北大学, 生命科学研究科, 教授 (20374225)
Project Period (FY) 2019-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2021: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2020: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2019: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Keywordsイオン輸送 / 構造化PEG / 大環状化合物 / 有機金属触媒 / 分子シャペロン / タンパク質凝集抑制 / オリゴエチレングリコール / リフォールディング / 凝集抑制 / タンパク質 / 両親媒性分子 / 分子動力学シミュレーション / ポリエチレングリコール / タンパク質凝集 / フォールディング / 変性 / 熱応答性 / AFM
Outline of Research at the Start

本研究課題は,研究代表者がこれまで開発してきた構造化PEG分子をもとに,生体で実現されているような「単分子」でタンパク質のリフォールディングを促進する合成分子,すなわち合成分子シャペロンの創成を目的としている。これを通じて,バイオ医薬をはじめとする機能化タンパク質の安定的供給を可能とする方法論の確立を目指す。

Outline of Final Research Achievements

Based on the structured PEGs consisting of monodisperse short oligoethylene glycols, which suppress thermal protein aggregation, we planned to introduce ligand units for organometallic catalysts in the molecular skeleton. Indeed, we have succeeded in developing a systematic method for the systematic construction of structured PEG molecules, and also in synthesizing ligand molecules bearing imidazolinium moieties in the molecular skeleton. The ligand molecule was serendipitously found to act as an anion transporter across the lipid bilayer membranes, where interaction between the cationic C-H and anion was suggested to play an essential role for the transport capability . Also, we got results suggesting that the ligand molecules can be used for the synthesis of the desired organometallic catalysts.

Academic Significance and Societal Importance of the Research Achievements

本研究の成果は,触媒開発の意味ついて,物質合成ではない新たな機能に着目するという観点から,学術的,社会的に貢献できるものと期待される。また,偶然発見されたアニオン輸送体としてのはたらきについては,医学的観点からも意義のある結果である。

Report

(3 results)
  • 2021 Final Research Report ( PDF )
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • Research Products

    (59 results)

All 2021 2020 2019 Other

All Int'l Joint Research (3 results) Journal Article (17 results) (of which Int'l Joint Research: 5 results,  Peer Reviewed: 17 results,  Open Access: 10 results) Presentation (38 results) (of which Int'l Joint Research: 11 results,  Invited: 12 results) Book (1 results)

  • [Int'l Joint Research] IECB(フランス)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] ボルドー大学(フランス)

    • Related Report
      2019 Annual Research Report
  • [Int'l Joint Research] アイルランド国立大学(アイルランド)

    • Related Report
      2019 Annual Research Report
  • [Journal Article] Synthetic Ion Channel Formed by Multiblock Amphiphile with Anisotropic Dual-Stimuli-Responsiveness2021

    • Author(s)
      Sasaki Ryo、Sato Kohei、Tabata Kazuhito V.、Noji Hiroyuki、Kinbara Kazushi
    • Journal Title

      Journal of the American Chemical Society

      Volume: 143 Issue: 3 Pages: 1348-1355

    • DOI

      10.1021/jacs.0c09470

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope2021

    • Author(s)
      Makabe Koki、Yokoyama Takeshi、Uehara Shiro、Uchikubo-Kamo Tomomi、Shirouzu Mikako、Kimura Kouki、Tsumoto Kouhei、Asano Ryutaro、Tanaka Yoshikazu、Kumagai Izumi
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 5790-5790

    • DOI

      10.1038/s41598-021-84171-3

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Reversible formation of multiple stimuli-responsive polymeric materials through processing control of trifunctional amphiphilic molecules2020

    • Author(s)
      Muraoka Takahiro、Honda Hidetaka、Nabeya Kota、Kinbara Kazushi
    • Journal Title

      Chemical Communications

      Volume: 56 Issue: 57 Pages: 7881-7884

    • DOI

      10.1039/d0cc02716b

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A synthetic ion channel with anisotropic ligand response2020

    • Author(s)
      Muraoka Takahiro, Noguchi Daiki, Kasai Rinshi, Sato Kohei, Sasaki Ryo, Tabata Kazuhito, Ekimoto Toru, Ikeguchi Mitsunori, Kamagata Kiyoto, Hoshino Norihisa, Noji Hiroyuki, Akutagawa Tomoyuki, Ichimura Kazuaki, Kinbara Kazushi
    • Journal Title

      Nature Communications

      Volume: 11 Issue: 1 Pages: 2924-2924

    • DOI

      10.1038/s41467-020-16770-z

    • NAID

      120006878529

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Thermo-driven self-assembly of a PEG-containing amphiphile in a bilayer membrane2020

    • Author(s)
      Li Rui、Muraoka Takahiro、Kinbara Kazushi
    • Journal Title

      RSC Advances

      Volume: 10 Issue: 43 Pages: 25758-25762

    • DOI

      10.1039/d0ra03920a

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Imidazolinium‐based Multiblock Amphiphile as Transmembrane Anion Transporter2020

    • Author(s)
      Mori Miki、Sato Kohei、Ekimoto Toru、Okumura Shinichi、Ikeguchi Mitsunori、Tabata Kazuhito V.、Noji Hiroyuki、Kinbara Kazushi
    • Journal Title

      Chemistry - An Asian Journal

      Volume: 16 Issue: 2 Pages: 147-157

    • DOI

      10.1002/asia.202001106

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The N-terminal amino-latch region of Hlg2 component of staphylococcal bi-component γ-haemolysin is dispensable for prestem release to form β-barrel pores2020

    • Author(s)
      Takeda Kein、Tanaka Yoshikazu、Kaneko Jun
    • Journal Title

      The Journal of Biochemistry

      Volume: 168 Issue: 4 Pages: 349-354

    • DOI

      10.1093/jb/mvaa052

    • NAID

      40022393358

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Association behavior and control of the quality of cancer therapeutic bispecific diabodies expressed in Escherichia coli2020

    • Author(s)
      Hikaru Nakazawa, Tomoko Onodera-Sugano, Aruto Sugiyama, Yoshikazu Tanaka, Takamitsu Hattori, Teppei Niide, Hiromi Ogata, Ryutaro Asano, Izumi Kumagai, Mitsuo Umetsu
    • Journal Title

      Biochemical Engineering Journal

      Volume: 160 Pages: 1-9

    • DOI

      10.1016/j.bej.2020.107636

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes2020

    • Author(s)
      Wakui Hajime、Tanaka Yoshikazu、Ose Toyoyuki、Matsumoto Isamu、Kato Koji、Min Yao、Tachibana Taro、Sato Masaharu、Naruchi Kentaro、Martin Fayna Garcia、Hinou Hiroshi、Nishimura Shin-Ichiro
    • Journal Title

      Chemical Science

      Volume: 11 Issue: 19 Pages: 4999-5006

    • DOI

      10.1039/d0sc00317d

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] タンパク質を模倣した人工発動分子の創製に向けて -膜タンパク質を模倣した合成化学的アプローチ-2020

    • Author(s)
      金原 数
    • Journal Title

      化学と工業

      Volume: 73 Pages: 457-459

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Aromatic Fluorination of Multiblock Amphiphile Enhances Its Incorporation into Lipid Bilayer Membranes2020

    • Author(s)
      Sasaki Ryo, Sato Kohei, Kinbara Kazushi
    • Journal Title

      ChemistryOpen

      Volume: 9 Issue: 3 Pages: 301-303

    • DOI

      10.1002/open.201900374

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] New Modified Deoxythymine with Dibranched Tetraethylene Glycol Stabilizes G-quadruplex Structures2020

    • Author(s)
      H. Tateishi-Karimata, T. Ohyama, T. Muraoka, S. Tanaka, K. Kinbara, and N. Sugimoto
    • Journal Title

      Molecules

      Volume: 25 Issue: 3 Pages: 705-705

    • DOI

      10.3390/molecules25030705

    • NAID

      120006811187

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus2020

    • Author(s)
      Minghao Chen, Masato Ishizaka, Shun Narai, Masaki Horitani, Naoki Shigi, Min Yao & Yoshikazu Tanaka
    • Journal Title

      Communications Biology

      Volume: 3 Issue: 1 Pages: 168-168

    • DOI

      10.1038/s42003-020-0895-3

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Introduction: Molecular Motors2020

    • Author(s)
      Iino Ryota、Kinbara Kazushi、Bryant Zev
    • Journal Title

      Chemical Reviews

      Volume: 120 Issue: 1 Pages: 1-4

    • DOI

      10.1021/acs.chemrev.9b00819

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Session 2SDA―Nonequilibrium energetics of biological molecular machines2020

    • Author(s)
      Toyabe Shoichi, Li Chun-Biu, Kinbara Kazushi
    • Journal Title

      Biophysical Reviews

      Volume: 12 Issue: 2 Pages: 273-274

    • DOI

      10.1007/s12551-020-00634-3

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand2019

    • Author(s)
      Tsubasa Hashimoto, Yuxin Ye, Mihoko Ui, Tohohisa Ogawa, Takashi Matsui, and Yoshikazu Tanaka
    • Journal Title

      Biochem. Biophys. Res. Commun.

      Volume: 印刷中 Issue: 1 Pages: 31-36

    • DOI

      10.1016/j.bbrc.2019.04.062

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Crystal structure of the catalytic unit of GH87-type α-1,3-glucanase Agl-KA from Bacillus circulans2019

    • Author(s)
      S. Yano, W. Suyotha, N. Oruro, T. Matsui, S. Shiga, T. Itoh, T. Hibi, Y. Tanaka, M. Wakayama, K. Makabe
    • Journal Title

      Scientific Reports

      Volume: 9 Issue: 1 Pages: 15259-15259

    • DOI

      10.1038/s41598-019-51822-5

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] 発光性マルチブロック両親媒性分子の合成と環境応答特性2021

    • Author(s)
      足立 惇弥 , 佐藤 浩平 , 金原 数
    • Organizer
      日本化学会 第101春季年会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Synthetic Ion Channel with Dual Stimuli-responsiveness2021

    • Author(s)
      Ryo Sasaki , Kohei Sato , Kazuhito Tabata , Hiroyuki Noji , Kazushi Kinbara
    • Organizer
      日本化学会 第101春季年会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Transmembrane Anion Transport by Imidazolinium-based Multiblock Amphiphile2021

    • Author(s)
      Miki Mori, Kohei Sato, Toru Ekimoto, Shinichi Okumura, Mitsunori Ikeguchi, Kazuhito Tabata, Hiroyuki Noji, Kazushi Kinbara
    • Organizer
      日本化学会 第101春季年会
    • Related Report
      2020 Annual Research Report
  • [Presentation] 新規レクチン様タンパク質の結晶構造解析と糖との結合解析2021

    • Author(s)
      影山大夢, 小野寺かこ, 小川智久, 横山武司, 田中良和
    • Organizer
      2020年度量子ビームサイエンスフェスタ
    • Related Report
      2020 Annual Research Report
  • [Presentation] 生体分子を模倣した人工発動分子 の設計に向けて2020

    • Author(s)
      金原 数
    • Organizer
      「細胞を創る」研究会13.0
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 単分散PEGを利用した熱応答性機能分子の創成2020

    • Author(s)
      金原 数
    • Organizer
      第69回高分子討論会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] Design of stimuli-responsive molecules mimicking dynamic functions of transmembrane proteins2020

    • Author(s)
      Kazushi Kinbara
    • Organizer
      SPIE Optics + Photonics 2020
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 両親媒性ポリサルコシンの合成と物性2020

    • Author(s)
      林 虹太 , 佐藤 浩平 , 金原 数
    • Organizer
      第30回バイオ・高分子シンポジウム
    • Related Report
      2020 Annual Research Report
  • [Presentation] Development of PEG-based Stimuli-Responsive Molecules2020

    • Author(s)
      Kazushi Kinbara
    • Organizer
      2nd International Conference on Materials Design and Applications
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの結晶中の空隙への生体分子の包摂2020

    • Author(s)
      田中良和
    • Organizer
      化学工学会第51回秋季大会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 蛋白質の立体構造解析 -X線結晶構造解析とクライオ電顕単粒子解析-2020

    • Author(s)
      田中良和
    • Organizer
      酵素工学研究会第84回講演会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの構造解析2020

    • Author(s)
      田中良和
    • Organizer
      農芸化学会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] Saframycin A生合成関連蛋白質のクライオ電子顕微鏡単粒子解析2020

    • Author(s)
      本多葵一、松井崇、小松玲子、谷藤涼、大栗博毅、横山武司、田中良和
    • Organizer
      日本生物物理学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] クライオ電子顕微鏡単粒子解析による百日咳壊死毒の構造解析2020

    • Author(s)
      次田篤史、松井崇、堀口安彦、横山武司、田中良和
    • Organizer
      日本生物物理学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Crystal structure analysis of BIR3 domain of ubiquitin ligase in complex with its specific ligand for designing potent ligand2020

    • Author(s)
      Makoto Okabe, Shinichi Sato, Takeshi Yokoyama, Shusuke Tomoshige, Minoru Ishikawa, and Yoshikazu Tanaka
    • Organizer
      日本分子生物学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Structural analysis of LanK, a protein associated with the production of antibiotics in actinomycetes2020

    • Author(s)
      Shiro Uehara, Iryna Ostash, Bohdan Ostash, Takashi Matsui, Tomohisa Ogawa, Yoshikazu Tanaka
    • Organizer
      日本分子生物学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Fluorinated multiblock amphiphiles that transport ions across lipid bilayers2020

    • Author(s)
      Kohei Sato, Kota Nabeya, Takahiro Muraoka, Norihisa Hoshino, Tomoyuki Akutagawa, Kazushi Kinbara.
    • Organizer
      ACS Spring 2020 National Meeting & Expo
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Transmembrane ion transport by asymmetric multiblock amphiphiles2020

    • Author(s)
      Ryo Sasaki, Kohei Sato, Kazuhito Tabata, Hiroyuki Noji, Kazushi Kinbara
    • Organizer
      ACS Spring 2020 National Meeting & Expo
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Development of amphiphilic linker for protein labeling and polymerization2020

    • Author(s)
      Sanghun Han, Yusuke Aoki, Kohei Sato, Kazushi Kinbara
    • Organizer
      日本化学会第100春季年会
    • Related Report
      2019 Annual Research Report
  • [Presentation] カチオン部位を導入した交互両親媒性分子による膜間イオン輸送2020

    • Author(s)
      森 未妃・佐藤浩平・田端和仁・ 野地博行・金原 数
    • Organizer
      日本化学会第100春季年会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Rectified Ion Transport by Asymmetric Multiblock Amphiphiles2019

    • Author(s)
      Ryo Sasaki, Kohei Sato, Kazuhito V. Tabata, Hiroyuki Noji, Kazushi Kinbara
    • Organizer
      CEMSupra 2019
    • Related Report
      2019 Annual Research Report
  • [Presentation] 交互両親媒性分子のオリゴマー化2019

    • Author(s)
      三栖 花梨, 佐藤 浩平, 金原 数
    • Organizer
      第68回高分子討論会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 大環状両親媒性 PEG によるタンパク質の凝集抑制2019

    • Author(s)
      中川 優大, アダム ヴァヴロ, 佐藤 浩平, 金原 数
    • Organizer
      第68回高分子討論会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Asymmetric Multiblock Amphiphiles for Transmembrane Ion Transport2019

    • Author(s)
      Ryo Sasaki, Kohei Sato, Kazushi Kinbara
    • Organizer
      第68回高分子討論会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 屈曲型交互両親媒性分子の開発2019

    • Author(s)
      森 未妃, 佐藤 浩平, 田端 和仁, 野地 博行, 金原 数
    • Organizer
      第13回バイオ関連化学シンポジウム
    • Related Report
      2019 Annual Research Report
  • [Presentation] Transmembrane Ion Transport by Multiblock Arene-Perfluoroarene Macrocycle2019

    • Author(s)
      Kohei Sato, Kota Nabeya, Takahiro Muraoka, Norihisa Hoshino, Tomoyuki Akutagawa, Kazushi Kinbara
    • Organizer
      第68回高分子討論会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 主鎖置換によるタンパク質のサイボーグ化2019

    • Author(s)
      青木 祐輔, 宇井 美穂子, 松井 崇, 田中 良和, 村岡 貴博, 佐藤 浩平, 金原 数
    • Organizer
      第68回高分子学会年次大会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 構造化された大環状ポリアミド誘導体の開発2019

    • Author(s)
      玉田 浩之, 佐藤 浩平, 金原 数
    • Organizer
      第68回高分子学会年次大会
    • Related Report
      2019 Annual Research Report
  • [Presentation] リン酸基を有する双頭型両親媒性分子の開発2019

    • Author(s)
      清水 友輔, 佐藤 浩平, 金原 数
    • Organizer
      第68回高分子学会年次大会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Design of Engineered PEGs for Biomaterial Developments2019

    • Author(s)
      Kazushi Kinbara
    • Organizer
      10th International Conference on Materials for Advanced Technologies
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Development of Amphiphilic Multiblock Molecule Bearing Cationic Moiety2019

    • Author(s)
      Miki Mori, Kohei Sato, Kazushi Kinbara
    • Organizer
      10th International Conference on Materials for Advanced Technologies
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 膜タンパク質を模倣したマルチブロック分子の機能 開拓2019

    • Author(s)
      金原 数
    • Organizer
      第68回高分子討論会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Synthetic Multiblock Molecules Mimicking Structure and Function of Membrane Proteins2019

    • Author(s)
      Kazushi Kinbara
    • Organizer
      2nd International Conference on Molecular Systems Engineering
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Design of synthetic chiral receptor molecules inspired by structure and function of membrane proteins2019

    • Author(s)
      Kazushi Kinbara, Takahiro Muraoka, Daiki Noguchi, Takahiro Endo
    • Organizer
      Chirality2019
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Design of PEG-Based Functional Molecules for Biorelated Applications2019

    • Author(s)
      Kazushi Kinbara
    • Organizer
      Japan-BIOCEV Symposium
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Development of PEG-based Stimuli-Responsive Molecules2019

    • Author(s)
      Kauzhi Kinbara
    • Organizer
      2nd International Conference on Materials Design and Applications
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Structure analysis of protein using both electron microscopy and X-ray crystallography2019

    • Author(s)
      Yoshikazu Tanaka
    • Organizer
      Forefront and Future of Electron Microscopic Imaging
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの構造解析 ―結晶構造と電顕構造―2019

    • Author(s)
      田中良和
    • Organizer
      2019年度 日本生化学会九州支部例会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Book] MolluscanHemocyanins in Vertebrate and Invertebrate Respiratory Proteins, Lipoproteins and other Body Fluid Proteins, Chapter 72019

    • Author(s)
      Sanae Kato, Takashi Matsui and Yoshikazu Tanaka
    • Total Pages
      24
    • Publisher
      Springer
    • ISBN
      9783030417697
    • Related Report
      2019 Annual Research Report

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Published: 2019-04-18   Modified: 2023-12-25  

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