Annual Report (2020)
No.32
The NOVARTIS Foundation (Japan)
for the Promotion of Science
令和２年度

財団年報 第32号

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Ⅰ．Introduction はじめに ……………………………………………………………………… 6
Kuniaki Takata, Ph.D.
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of Novartis Research Grants

研究報告 ………………………………………………………………………………………… 8
1. Fundamental study for GATA3 transcriptional complex and new insight intofunctional role of type2 innate lymphoid cells to control life-style related disease…… 9
Tomoaki Tanaka
Chiba University, Graduate School of Medicine

2. Chloroplast outer membrane-localized phototropin 2 induces the chloroplast
avoidance response.…………………………………………………………………………… 11
Eiji Gotoh
Kyushu University

3. Machine learning and next generation sequencing-oriented correlation analysis
between gut microbiota and the risk of graft versus-host disease………………………… 13
Yasuyuki Arai
Kyoto University

4. Importance of tissue-resident memory T cells in mucosal tissue………………………… 16
Norifumi Iijima
National Institutes of Biomedical Innovation, Health and Nutrition

5. Analysis of cell lineage and transcriptional regulation during ventriculardevelopment of heart………………………………………………………………………… 19
Yusuke Watanabe
National Cerebral and Cardiovascular Research Center, Research Institute

6. Construction of human complex brain organoids for analysis of brain
development and neurological diseases……………………………………………………… 22
Keiko Muguruma
Kansai Medical University

7. A novel regulatory mechanism of the metabolic signaling complex mTORC1………… 25
Shusaku Shibutani
Yamaguchi University

8. Generation of in vitro heart organ using the mothods of atrial-ventricularcardiomyocyte differentiation ………………………………………………………………… 28
Jun Takeuchi
Medical research Institute, Tokyo Medical and Dental University

9. Development of Catalytic Asymmetric Cycloaddition Reaction of α-Keto Esters
for the Innovative Synthesis of Tetrahydrofuran Compounds…………………………… 31
Akira Sakakura
Graduate School of Natural Science and Technology, Okayama University

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10. Pathophysiologic role of a novel microenvironment that prolongs tissue
damage in acute kidney injury……………………………………………………………… 34
Takayoshi Suganami
Nagoya University, Research Institute of Environmental Medicine

11. Role of a novel activity-dependent gene encoding a lipid modification
enzyme on neuronal and cognitive function……………………………………………… 37
Hiroyuki Okuno
Kagoshima University

12. Synthesis of Phytochrome-type Chromophores Based on Oxidative
Functionalization toward Investigation of their Functions……………………………… 40
Yutaka Ukaji
Kanazawa University

13. Development of a novel base editor………………………………………………………… 43
Nozomu Yachie
Research Center for Advanced Science and Technology, The University of Tokyo

14. Study on the circadian rhythm system regulated by intracellular carbon monoxide…
Hiroaki Kitagishi
Department of Molecular Chemistry and Biochemistry, Doshisha University

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15. Age-related endocrine disruption of skin leads to senescent alopecia………………… 49
Shogo Haraguchi
Showa University School of Medicine

16. The RNA-based mechanism underlying pathophysiology in the neurological diseases…

………………………………………………………………………………………………… 52
Takatoshi Iijima
Department of Molecular Life Science, Division of Basic Medical Science andMolecular Medicine, School of Medicine, Tokai University

17. Elucidation of the molecular basis and physiological significance ofautophagic degradation of the nuclear pore complex…………………………………… 55
Hitoshi Nakatogawa
Tokyo Institute of Technology

18. Comprehensive understanding of intracellular innate immune response
in chronic hepatitis B reactivation………………………………………………………… 58
Takayuki Abe
Kobe University Graduate School of Medicine

19. Identification of glycosyltransferases involved in biosynthesis of pectinin plant cell wall……………………………………………………………………………… 61
Takeshi Ishimizu
College of Life Sciences, Ritsumeikan University

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20. RNA base modification and RNA processing regulate the maintenanceand development of germline stem cells…………………………………………………… 64
Toshie Kai
Graduate School of Frontier Biosciences, Osaka University

21. Elucidation of a novel regulatory mechanism of angiogenesis by intraluminalpressure………………………………………………………………………………………… 66
Shigetomo Fukuhara
Institute of Advanced Medical Sciences, Nippon Medical School

22. Development of new treatment for obesity-related liver cancer, focusing ona Wnt ligand, ACLP, secreted by hepatic stellate cells…………………………………… 69
Kengo Tomita
Division of Gastroenterology and Hepatology, Department of Internal Medicine,National Defense Medical College

23. Integrated analysis of tumor stromal cells using ultra-sensitive
bioluminescence imaging Tg mice………………………………………………………… 71
Shinae Kizaka-Kondoh
School of Life Science and Technology, Tokyo Institute of Technology

24. Novel long-range regulatory mechanisms controlling NKT-cell lineage program…… 74
Nayuta Yakushiji-Kaminatsui
RIKEN Center for Integrative Medical Sciences

25. Molecular mechanisms of extracellular vesicle formations of endogenous retrovirus… 77
Keita Miyoshi
National Institute of Genetics

26. Molecular mechanisms of axis formation in plants: Organization, function
and spatiotemporal dynamics of PIN clusters that regulate polar localizationof auxin efflux carrier PIN proteins………………………………………………………… 79
Satoshi Naramoto
Graduate School of Life Sciences, Tohoku University

27. Physiological roles and molecular mechanisms underlying the synaptic plasticity
in the dentate gyrus…………………………………………………………………………… 82
Yuki Hashimotodani
Doshisha University, Organization for Research Initiatives and Development

28. Genomic analysis of hematopoietic stem cell niche in myelodysplastic syndrome.…
Hidekazu Nishikii
Department of Hematology, Faculty of Medicine, University of Tsukuba

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29. Expression change of desmoplakin by antidepressant treatments and its function
in dentate gyrus of hippocampus…………………………………………………………… 87
Eri Segi-Nishida
Department of Biotechnology, Tokyo University of Science

30. Functional analysis of the GRHL3 factor in epithelial fusion.…………………………… 89
Chiharu Kimura-Yoshida
Department of Molecular Embryology Research Institute, Osaka Women’s and Children’s
Hospital, Osaka Prefectural Hospital Organization

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31. Identification and molecular mechanism of novel calcium transport disease – possible
therapeutic target for bone mineralization defects………………………………………… 93
Yoshiro Suzuki
Department of Physiology, Iwate Medical University

32. Nuclear noncoding RNA degradation regulates innate immune response……………… 96
Nobuyoshi Akimitsu
Isotope Science Center, the University of Tokyo

33. Projection-specific circuit formation in the developing cerebral cortex………………… 99
Yoshiaki Tagawa
Kagoshima University Graduate School of Medical and Dental Sciences

34. Uncovering the Biomarker for Treatment with ATR Inhibitor in Solid Tumor……… 102
Kazumasa Komura
Osaka Medical College

35. Peptide foldamers in drug discovery…………………………………………………… 105
Yosuke Demizu
National Institute of Health Sciences

36. Evaluation of Intra-tumor epigenetic heterogeneity of breast cancer………………… 108
Reo Maruyama
Japanese Foundation for Cancer Research

37. Research on metabolic reprogramming induced by cancer development
and inflammation ………………………………………………………………………… 110
Koji Taniguchi
Keio University School of Medicine

Publication………………………………………………………………………… 113

Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告………………………………………………………………………………… 114
1. The 13th Annual Meeting of the Japanese Society for Epigenetics…………………… 115
Haruhiko Koseki
Deputy Director, RIKEN Center for Integrative Medical Sciences

2. The 60th International Conference on the Bioscience of Lipids………………………… 116
Professor Makoto Arita
RIKEN, Keio University

3. Commemorative International Symposium of the Japan Society of Nucleic
Acids Chemistry (CISNAC 2019)……………………………………………………… 118
Naoki Sugimoto
Director and Professor of Frontier Institute for Biomolecular Engineering Research,
Konan University

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4. The 1st International Symposium on Hybrid Catalysis for Enabling MolecularSynthesis on Demand……………………………………………………………………… 121
Motomu Kanai

5. The 6th meeting of International Society for Zinc Biology (ISZB-2019)“The New Sunrise of Zinc Biology”…………………………………………………… 123
Toshiyuki Fukada

Ⅳ．The 33rd (fiscal year 2019) Promotion Report

第33期（2019年度）助成事業報告 ……………………………………………………… 125

Ⅴ．The 33rd (fiscal year 2019) Financial Report

第33期（2019年度）財務報告 …………………………………………………………… 133

Ⅵ．List of the Trustees, Auditors, Councilors and Grant Selection Committee Members

理事・監事・評議員・選考委員名簿 …………………………………………………… 135

Ⅶ．Information from the Secretariat

事務局便り …………………………………………………………………………………… 139

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The Novartis Foundation(Japan)
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Introduction

Kuniaki Takata, Ph.D.
Chairman of the Board of Trustees
We have compiled here the reports of FY2019 grants (37 research grants and 5 grants forinternational meetings) of the Novartis Foundation (Japan) for the Promotion of Science.
The new coronavirus infection that emerged at the end of last year has spread around theworld. At the beginning of October 2020, as of this writing, it has infected approximately 35
million people around the world and killed more than one million, a situation that is truly aglobal catastrophe. In Japan, more than 80,000 people have been infected and more than 1,600
people have died. There has been a collapse of medical care, urban blockades, and social chaos
in many countries including China, the European countries, and the United States. In Japan
as well, the government has declared a state of emergency and asked people to refrain fromgoing out of their homes and other social activities. We have been forced to live in a world far
from the everyday life we had been used to take for granted before the outbreak. Universities
and research institutes have been greatly affected by the outbreak, including campus closuresor restrictions on the use of their facilities. Because of the closure of the border, interactions
between researchers have been limited to the PC-monitor-basis only. It has become extremely
difficult to hold regular conferences. Many of them have been cancelled or postponed.
Otherwise they were held as web meetings. Under such harsh circumstances researchers
continue to work hard, which I am deeply impressed by their dedication to sciences.
The Foundation was established in 1987 with a donation of 1 billion yen from Ciba-Geigy
AG (now Novartis AG) in Switzerland. The purpose of the Foundation is clearly stated in its
articles of incorporation: “To promote science, and thereby contribute to the health and welfareof the people by encouraging creative research in the natural sciences.” In the document titled“Objectives of the Foundation” written in the year of the Foundation’s establishment, it isstated that the Foundation “will promote and support creative research in the natural sciences,which will form the axis of science in the coming 21st century, and thereby contribute to thewelfare of mankind,” and that it will “provide financial support for research and a forum forcross-border exchange. Under this policy, the Foundation has provided a total of 1,806 grants,
amounting to approximately 2.09 billion yen, over the past 33 years. Today, in the midst of thechaos caused by the corona plague, the Foundation would like to retain this starting point andcontinue to support excellent research in the future.
This annual report summarizes the results of the research supported by the Foundation.
This is a magnificent achievement within a limited time frame of one year. In the list of past
recipients of the Foundation’s support, you will find the names of many leading researchers intheir fields, including Dr. Tasuku Honjo, winner of the Nobel Prize in Physiology or Medicine.
We hope that the recipients of this grant will take the opportunity to make great strides in theirresearch. We would like to express our deepest gratitude to the selection committee members
who selected these excellent researches and to all those who supported the activities of theFoundation, including Novartis Pharma KK, the founder, for their support.

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はじめに

代表理事

高田

邦昭

本年報には、第32回ノバルティス科学振興財団の研究助成金を受けられた方々の研究報告（2019年４月～ 2020年３月の研究：研究奨励金37件、研究集会５件）
を収録しました。
昨年末から始まった新型コロナウイルス感染は世界中に広がり、この原稿を執筆している10月初旬では世界中で約3,500万人が感染し、100万人以上が亡くなるという、まさに地球規模での大惨禍といえる状況を呈しています。日本の感染者数も８万人を超え、死亡者も1,600人を超えています。海外では医療崩壊が起こったり、都市封鎖が行われたりと、社会的に大きな混乱が起こっています。日本でも政府による緊急事態宣言の発出や外出をはじめとする様々な社会活動の自粛要請もあり、感染が起こる前までは当たり前だった日常とはかけ離れた日々が続いています。大学や研究機関においても、キャンパスの閉鎖や使用の制限など大きな影響を受けています。通常の学会開催は困難となり、web 開催や中止・延期となりました。海外への人的な移動ができない中で、研究者間の交流は PC 画面上でのものとなりました。このような様々な困難な状況の中で、研究者の方々が必死に研究を継続している様を見るにつけ、研究者魂を見る思いを改めて深くしています。
本財団は1987年に、スイス、チバガイギー社（現ノバルティスファーマ社）からの10億円のご寄附をもとに設立されたものです。財団の目的は、定款に ｢自然科学における創造的な研究の奨励等を行うことにより、学術の振興を図り、もって国民の健康と福祉の向上に寄与する｣ と明記されています。また、財団設立の年に記された ｢財団設立の趣意｣ と題する文書には、財団が ｢来るべき21世紀の科学の軸となる自然科学の創造的研究の振興助成をはかり、以って人類の福祉に寄与できれば｣ とあり、｢研究のための資金的な助成、並びに国境を越えた交流の場の提供｣ がうたわれています。このような方針のもと、33年間で総計1,806件、金額にして約20.9億円超の助成を行ってきました。コロナ禍で混迷する現在こそ、財団は改めてこの原点に立ち返り、今後とも優れた研究を支援して行きたいと考えています。
この年報には本財団が支援した優れた研究の成果をまとめています。一年間という限られた時間の中で達成した立派な業績です。過去に当財団の助成を受けた方々のリストには、ノーベル医学・生理学賞を受賞した本庶佑博士をはじめ各分野をリードする研究者の名前が多数見られます。今回助成を受けた方々が、この研究成果を契機としてさらに大きく飛躍されることを祈念いたします。これらの優れた研究を選考していただいた選考委員の皆様や、出捐者であるノバルティスファー
マ社をはじめとして財団の活動を支えて下さっている関係者の皆様に深く感謝いたします。

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The Novartis Foundation(Japan)
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Reports from the Recipients ofNovartis Research Grants

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Fundamental study for GATA3 transcriptional complex and
new insight into functional role of type2 innate lymphoid cells tocontrol life-style related disease
Tomoaki Tanaka
tomoaki@restaff.chiba-u.jp
Chiba University, Graduate School of Medicine

Summary Abstract
Recently, type 2 innate lymphocytes (ILC2) have been found in adipose tissue, and their glucose
metabolism regulating action has been attracting attention. However, its existence and functionalrole in the liver are unknown. In this study, KO mice and RNA-seq/ChIP-seq/interactome analysis
were performed to elucidate the physiological role of liver ILC2. For the first time, we showed ablood glucose-improving effect through suppression of hepatic gluconeogenesis, and clarified theimportance of the IL33-IL13 axis through the transcription factor GATA3 complex as a mechanism.
Key Words：type 2 innate lymphocytes, life-style related disease, GATA3 transcriptional factor

Introduction
GATA3, one of GATA family transcription factor, has been shown to control not only T cells but
also the differentiation and function of type 2 ILC cells, and in this physiological context, it has also
been found that active changes are made in the complex mode according to differentiation stages
and functional differences. Although the function of signal-dependent GATA3 transcription complex
plays an important role in T cells and ILC cells in which GATA3 is highly expressed, its control
mechanism is hardly clarified1.
Here, we have conducted research study to clarify its role for disease pathophysiology, particularly
with involving chronic organ inflammation, in life-style related disease including obesity andmetabolic disorders (Fig. 1).

Results
Type 2 innate lymphoid cells (ILC2s) were identified as essential components of innate immunity2.
Besides, beneficial roles of ILC2s in metabolic diseases are
attracting attentions3. Although the liver contains various immune
cells including T cells and ILC2s, little is known about ILC2s
in hepatic gluconeogenesis. We therefore investigated the roleof ILC2s and the effect of IL-33, a potent ILC2 activator, on
hepatic gluconeogenesis in mice. We found that recombinant
IL-33 injection decreased fasting blood glucose levels in wildtype mice. This effect was not observed in NOD/SCID Il2rg null
(NSG) mice lacking certain immune cells including ILC2s, whereas the similar response was seen inanother immune deficient mice Foxnnu/nu (nude mice), which have normal development in ILC2s. IL33 treatment also suppressed hepatic gluconeogenesis assessed by pyruvate tolerance test in wt mice,

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while there was no response in NSG. Moreover, ILC2 transplantation was able to rescue the absenceof glucose response by IL-33 in NSG. We observed a marked increase of IL-13+ ILC2 populationin liver after IL-33 treatment, and Il13-/- mice did not show the decrease of blood glucose or the
suppression of gluconeogenesis by IL-33 treatment. Additionally, the transfer of Il13-/- ILC2 failed
to gain IL-33 induced glucose response in NSG, suggesting that the hypoglycemic effect of IL-33/
ILC2s depends on IL-13. In consistent with these results, the differentially expressed gene analysis
revealed that IL-13 treatment mostly altered the gene expressions of glucose metabolic process inprimary hepatocytes. Notably, we identified GATA3 as a direct regulator of the effecter molecules
such as Il5, Il13 and I1rl1 in hepatic ILC2s. These phenomena were also reproducible in human
ILC2s and primary hepatocyte. In conclusion, these findings indicate that hepatic ILC2s suppress
hepatic gluconeogenesis via the IL-33/GATA3/IL-13 axis, thus reducing blood glucose levels.

Discussion & Conclusion
This is the first study to report the potential role of the IL-33/13 axis in suppressing gluconeogenesis in hepatic ILC2s and lowering blood glucose levels. Several studies have previously suggested
the beneficial roles of ILC2s in metabolic disorders. IL-33 powerfully induces IL-13 production in
hepatic ILC2s, and IL-13 regulates the expression of genes in glucose metabolism by directly acting
on hepatocytes. NAFLD is prevalent among patients with obesity and hyperglycemia, and enhanced
gluconeogenesis exacerbates the pathological conditions of this disease. By further clarifying themechanism of ILC2s to suppress gluconeogenesis in the liver, we could improve our understandingof NAFLD pathogenesis and propose a novel therapeutic target to improve health outcomes for thesepatients.

References
1. Hosokawa H, Romero-Wolf M, Yang Q, Motomura Y, Levanon D, Groner Y, Moro K, Tanaka,
Tomoaki and Rothenberg, E. V. Cell-type specific actions of Bcl11b in early T-lineage and group 2
innate lymphoid cells. J Exp Med. 217(1):e20190972. (2019)
2. Moro, K. et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca1(+) lymphoid cells. Nature 463, 540-544 (2010).
3. Brestoff, J. R. et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue andlimit obesity. Nature 519, 242-246 (2015).

一般の皆様へ
転写因子 GATA3という分子は、免疫細胞である T 細胞の生存や炎症性サイトカイン産生を司る役割が詳細に研究されてきた分子である。一方で、２型自然リンパ球という免疫細胞が糖代謝を改善させる作用にも、GATA3の多面的な役割が新たに見出されている。本研究では、肝臓にいる２型自然リンパ球が、肥満や糖尿病などの代謝疾患病態に良い影響を及ぼすことを明らかにした。今後、これらの詳細な分子メカニズムを明らかにすることで、肥満や糖尿病のインスリン抵抗性に対する新たな治療標的となることが期待される。

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Chloroplast outer membrane-localized phototropin 2 induces thechloroplast avoidance response.
Eiji Gotoheiji.gotoh@agr.kyushu-u.ac.jp
Kyushu University

Summary Abstract
Phototropins are localized mainly on the plasma membrane. However, a substantial amount ofphot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might havedifferent functions. To determine the functions of plasma membrane- and chloroplast envelopelocalized phot2, we tethered it to these structures with their respective targeting signals. Plasma
membrane-localized phot2 regulated chloroplast movements. Chloroplast envelope-localized phot2
failed to mediate chloroplast accumulation response but partially regulated chloroplast avoidance
response. Thus, phot2 localized at the interface between the plasma membrane and the chloroplasts
is required for chloroplast avoidance response.
Key Words：Chloroplast movements, phototropin,

Introduction
Phototropin enhances photosynthesis by controlling phototropism, leaf flattening, stomatal
opening, and chloroplast movements. Arabidopsis thaliana and other seed plants possess the two
redundantly functioning phototropins phot1 and phot2. However, chloroplast avoidance response
induced by strong blue light is regulated mainly by phot2. Most phototropins are localized primarily
to the plasma membrane but phot2 occur on the chloroplast outer envelope. Therefore, differentiallylocalized phot2 might have different functions.

Results
We examined whether chloroplast outer membrane-localized phot2 regulates BL-induced
chloroplast movements. Three-week-old plants were transferred to darkness, weak light, or strong
light and chloroplast distributions were observed under a confocal microscope. In P2G and PMP2G under darkness, chloroplasts accumulated on the cell bottoms. Under weak BL, the chloroplasts
were localized on the upper and lower periclinal walls. Under strong BL, the chloroplasts localizedon the side walls. Thus, P2G and PM-P2G mediated chloroplast dark positioning, accumulation, andavoidance responses. In CP-P2G, chloroplasts localized on the side walls under all light conditions.
Therefore, the CP-P2G line was defective in chloroplast dark positioning and accumulation
response. However, it remains to be determined whether CP-P2G regulates the chloroplast avoidance
response. We assessed chloroplast movements by measuring light-induced transmittance changes(Wada and Kong. 2011). In WT, weak light (3 µmol m-2 s-1) reduced leaf transmittance because
the chloroplast accumulation response was induced. Under strong BL (20 and 50 µmol m-2 s-1), thechloroplast avoidance response was induced and leaf transmittance increased. P2G presented withboth chloroplast accumulation and avoidance responses. The latter was much weaker than that for
the WT but similar to that for phot1 single mutant plants. Thus, P2G rescued the phot2 but not the

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CP-P2G, no chloroplast accumulation response was observed but a weak and significant chloroplast
avoidance response was detected. The latter was dependent on CP-P2G because no light-inducedchanges were detected in the phot1phot2 double mutant plants.

Discussion & Conclusion
Our findings indicated that phot2 proteins at the interface between the chloroplasts and the plasma
membrane are required for the chloroplast avoidance response. However, they do explain why phot1
cannot efficiently mediate this response even though phot1 localizes at the interface between thechloroplasts and the plasma membrane. Thus, substrate specificity between phot1 and phot2 may also
be necessary to determine the precise functions of these phototropins. Domain swapping between
phot1 and phot2 revealed that neither the N-terminal light-sensing- nor the kinase domain determines
the functional specificity of phot2 in chloroplast avoidance response regulation (Aihara et al., 2008).
Phot2 N/phot1- and phot1 N/phot2 kinase chimeras regulated the chloroplast avoidance response(Aihara et al., 2008). To elucidate the functional difference between phot1 and phot2 in chloroplast
avoidance response regulation, a more detailed structure-function analysis is needed.

References
Aihara, Y., Tabata, R., Suzuki, T., Shimazaki, K. and Nagatani, A. (2008) Molecular basis of thefunctional specificities of phototropin 1 and 2. Plant J 56: 364-375.
Wada, M. and Kong, S. -G. (2011) Analysis of chloroplast movement and relocation in Arabidopsis.
Method Mol Biol 774: 87-102

一般の皆様へ
光合成の場である葉緑体は、光の強度に応じて細胞内の空間配置を変化させます。この光に依存した葉緑体の局在変化は、植物特有の青色光受容体フォトトロピンによって制御されます。
フォトトロピンは、細胞膜に加えて葉緑体の周りにも存在することが知られていますが、葉緑体の周りにあるフォトトロピンの機能については分かっていません。そこで私たちは、遺伝子工学技術を利用して、葉緑体のまわりにのみフォトトロピンが存在する植物体を作出し、葉緑体のまわりに存在するフォトトロピンが葉緑体の局在変化を誘導することを明らかにしました。

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Machine learning and next generation sequencing-oriented correlation
analysis between gut microbiota and the risk ofgraft versus-host disease
Yasuyuki Araiysykrai@kuhp.kyoto-u.ac.jp
Kyoto University

Summary Abstract
Acute gut graft-versus-host disease (GVHD) is a devastating adverse event after allogeneic
hematopoietic stem cell transplantation. In this study, new approach from the viewpoint of
microbiota is now undergoing. So far, we have established the system for appropriately categorizing
the microbiota using next generation sequencing.
Key Words：acute gut graft-versus-host disease, allogeneic hematopoietic stem cell transplantation,
microbiota

Introduction
Allogeneic hematopoietic stem cell transplantation is a curative treatment for refractoryhematopoietic tumors such as leukemia and lymphoma. Acute graft-versus-host disease (GVHD) is
a disease in which donor immune cells attack the patient's intestinal mucosal cells and is known asa poor prognostic factor. As with common inflammatory bowel disease, its etiology may be related
to "Changes in gut flora (dysbiosis)". Allogeneic transplants are expected to undergo dramatic shortterm changes in the appearance of gut flora and dysbiosis due to pretreatment chemotherapy andantibiotics, but this has not been thoroughly studied.

Results
The applicant identified intestinal microbiota using next-generation sequencing and analyzed
metabolites using mass spectrograms in the feces of post-transplant patients. These results clarify the
relationship between pretreatment, antibiotic use, and intestinal GVHD. The aim of this study wasto establish characteristic dysbiosis and metabolite patterns as early predictive markers of intestinal
GVHD and to propose an allogeneic transplant regimen optimized for improved prognosis.
This research is divided into (1) collection and identification of intestinal microbiota after bonemarrow transplantation for hematopoietic tumors, (2) classification of intestinal microbiota bymachine learning using AI, and fusion and analysis with clinical database after transplantation,(3) similar study in mouse experimental model and prevention experiment using live bacteria
preparation, and preparation of human clinical trial based on it. Sampling began in August 2018, andfull-scale analysis has been conducted since 2019, with a 3 year plan.
Applicants have started collecting specimens since the latter half of FY 2018. Approximately 21
patients (Total of 100 specimens) have been analyzed. Stool samples were collected weekly (7 times
in total) before and up to 35 days after transplantation to analyze gut flora and other parameters.
Microbiota were identified by sequencing the 16 S ribosomal RNA coding region using a nextgeneration sequencer. Sequence data, metabolite analysis, and residual antibiotic concentration data

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were fused to a clinical database for analysis. The next generation sequence was used to identify thebacterial flora, and it was confirmed that the genus name could be identified. It was also found that
lipid metabolome could be identified in the same way.
A total of 21 patients were enrolled and more than 100 samples were studied. The Shannon
index (Reflecting the diversity of bacterial flora) peaked before transplantation and then gradually
improved after peaking 21 days after transplantation. The Chao1 index followed a similar course.
The Shannon and Chao1 indices were found to be significantly decreased in patients who received
a more intensive conditioning, myeloablative conditioning. Loss of diversity of gut flora (dysbiosis)
was also found to be associated with residual antibiotic concentrations in the gut. In particular, aconcentration-dependent relationship was found for meropenem and piperacillin. It was found that
there was little fermentation by intestinal bacteria in these intestines.
From the results, it was actually confirmed that the disorder of the intestinal flora Dysbiosis andlipid metabolome was generated by the transplantation pretreatment. Based on this, we will proceedwith the research as planned, and hope to lead to new drug discovery in about two years.
Based on the huge amount of metagenome analysis results obtained this time, we will stratify into
30 ~ 50 types by finding common points using AI. The results will be fused to a clinical databaseof allogeneic transplants. Correlations between pretransplant parameters such as pretreatment
and antibiotic use, gut flora dysbiosis, and even GVHD onset events are found and established as
biomarkers. This identifies gut microbiota composition with a lower risk of intestinal GVHD.
After this step, using a mouse bone marrow transplantation model (C57BL6 → BALB/c),
the correlation between the occurrence of GVHD and the mouse intestinal bacterial flora will beanalyzed by combining the next-generation sequence and AI as mentioned above. Intestinal epithelial
cells are collected during the course of the disease, and changes in intracellular signaling molecules
are analyzed in detail to support the mechanism.

Discussion & Conclusion
This study can provide significant information to establish biomarkers and prophylaxis forintestinal GVHD and to elucidate the biological significance of intestinal flora and Dysbiosis. In
addition, the method of combining next-generation sequences and AI, which have become major
breakthroughs in information engineering in recent years, is widely applicable to similar research in
the future. From this new perspective, we hope to improve the prognosis of refractory hematologicalmalignancies.
Pre-transplantation treatment and antibiotic use were found to be associated with dysbiosis andmetabolic abnormalities. This may be a marker of intestinal GVHD and may reduce complications
and improve transplant outcomes. More cases are being analyzed to prove the hypothesis.

References
1. Arai Y, Kondo T, Fuse K, et al. Using a machine learning algorithm to predict acute graft-versushost disease following allogeneic transplantation. Blood Adv. 3(22):3626-3634. 2019.
2. Yasuyuki Arai, Takamitsu Tsukahara, Miki Nagao, Tadakazu Kondo, Akifumi Takaori-Kondo.
Dysbiosis in gut microbiota after allogeneic hematopoietic stem cell transplantation can be
determined by the combination of conditioning regimen and antibiotic usage. 24th European
Hematology Association (Amsterdam, Netherland). 2019.5.14

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一般の皆様へ
近年、「腸内細菌叢」の乱れが様々な疾患や健康状態に関係していることが分かってきました。

白血病やリンパ腫といった、血液疾患に対しても同様のことが言えるのではないか、またそのことが治療にヒントを与えるのではないかと考え、この研究を行っています。将来的には本研究から発展した薬作りに役立てたいと考えています。

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Importance of tissue-resident memory T cells in mucosal tissue
Norifumi Iijima
niijima@nibiohn.go.jp
National Institutes of Biomedical Innovation, Health and Nutrition

Introduction
Sexually transmitted diseases are caused by the invasion of microorganisms into the genitourinary
tracts. One such pathogen, herpes simplex virus type 2 (HSV-2) is known to spread to the neuronaltissues, including the dorsal root ganglia, where it leads to a latent infection and frequent subsequent
cycles of reactivation and latency. To prevent HSV-2 infection of the genital tissues and to treat HSV2-mediated diseases, a large number of prophylactic vaccine trials have been performed; however,none of these trials have succeeded in generating a genital herpes vaccine. To date, it remains unclear
how herpesvirus replication is controlled by a network of peripheral immune surveillance in bothmucosal tissues and neuronal tissues.
Key Words：Tissue-resident memory T cells, gene expression, effector function

Results
In a murine model of genital herpes infection, intravaginal immunization with live attenuatedHSV-2 was highly effective at inducing protective immune responses and protecting against HSV2 infection. Furthermore, at least 4 weeks after thymidine-kinase negative (TK-) HSV-2 intravaginal
immunization, both T cells and IFN-gR signaling were required to inhibit virus replication in murine
vaginal tissues following wild-type (WT) HSV-2 intravaginal challenge. By contrast, intravaginal
immunization with TK- HSV-2 in B cell-deficient mice protected against HSV-2 secondary challenge,
whereas intranasal immunization with attenuated virus in B cell-deficient mice allowed WT HSV2 to invade neuronal tissues and replicate following intravaginal challenge1). Furthermore, HSV2-specific antibody required circulating CD4+ memory T cells (TM) cells to achieve protection inneuronal tissues against WT HSV-2 intravaginal challenge1). These findings suggested that tissueresident immunity in vaginal tissues is critical for rapid viral clearance to block viral spread into
neuronal tissues. Certainly, HSV-specific CD4+ T cells are found to accumulate in the lamina
propria of vaginal tissues 5 weeks after intravaginal immunization with TK- HSV-22). Furthermore,
CD4+ T cells form clusters beneath the vaginal epithelium. To inhibit HSV-2 replication in vaginal
tissues, MHC class II+ APC, including CD11c+ DC and CD20+ B cells, are required for the IFN-γmediated but not cytotoxic function of CD4+ TM following HSV-2 secondary challenge2). To dissect
the mechanism involved in the maintenance of CD4+ TM in vaginal tissues following TK- HSV2 immunization, we performed parabiosis studies. The results revealed that HSV-2-specific CD4+
TM were predominantly a host-derived, not a blood-derived, population3), indicating that these cells
identify as tissue-resident memory T cells (TRM). Furthermore, HSV-2-specific CD4+ TM bearing
TCRVb1 accumulate within memory lymphocyte clusters (MLC)3). To maintain the formationof MLC, CD11b+ macrophages , which secrete CCL5, are required within MLC. Although viralgenomic DNA or RNA transcripts have not been detected in the vaginal tissues of TK- HSV-2
immunized mice, constitutive low level secretion of IFN-γ from CD4+ TRM is observed in vaginal
tissues3), indicating that CD11b+ macrophages that stimulate CD4+ TRM to secrete IFN-γ produce

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CCL5 as a feedback loop mechanism.
To directly elucidate the requirement of tissue-resident immunity, TK- HSV-2 immunized mice
were conjoined with naïve mice to share blood circulation. In naïve pairs, HSV-2-specific cell
populations, including memory T and B cells and antibodies, were observed in blood, whereas TRM
were not established in vaginal tissues. Following WT HSV-2 challenge of the naïve pair, viralclearance was significantly delayed compared with challenge of the immune pair, indicating that TRM,
especially CD4+ TRM, are required for rapid viral clearance3).
Following re-encounter with the same pathogen, CD4+ TRM secrete high levels of IFN-γ within
12 hours 3)4), suggesting that local APC present viral antigens to CD4+ TRM. Furthermore, disruptionof MLC formation by CD11b+ cell depletion reduced the level of CXCL9 expression in vaginal
tissues following WT HSV-2 challenge, causing the failure of memory B cells to migrate into
vaginal tissues to secrete HSV-2-specific IgGs4). This indicates that MLC formation is a platform
for the maintenance of TRM to rapidly exert their effector functions upon reencountering of invading
pathogens.
To this end, we investigated how TRM are retained in MLC by focusing on the non-hematopoietic
cells such as FRC-like cells. Five weeks after TK- HSV-2 immunization, we prepared single cellsuspension from vaginal tissues to analyze CD45+/-, gp38+ and CD31+ cells. Unexpectedly, thenumber of CD45- CD31+ cells was decreased, whereas the number of CD45- gp38+ and CD45- gp38+
CD31+ cells following TK- HSV-2 immunization was comparable to that of naïve mice. These datasuggested that the quantity of FRC network in vaginal tissues would not dictate the retention of TRM
following TK- HSV-2 immunization.

Discussion & Conclusion
To retain mucosal tissues, TRM would receive some retention signal from other types of cells
including macrophages (CCL5+) and non-hematopoietic cells (mainly FRC network). Therefore, we
examined the characteristic of CD45- CD31+ cells, CD45- gp38+ and CD45- gp38+ CD31+ cells. Thistime, we found the quantity of FRC network was not changed following TK- HSV-2 immunization.
Therefore, we are currently examining comprehensive gene expression level of FRC network
following TK- HSV-2 immunization by utilizing single cell RNA-sequencing analysis. From thecomprehensive gene analysis, we would like to elucidate the retention signal from FRC network invaginal tissues in the near future.

References
1) Iijima N, Iwasaki A. Access of protective antiviral antibody to neuronal tissues requires CD4 T
cell help. Nature 533 (7604): 552-556, 2016.
2) Iijima N, Linehan MM, Zamora M, Butkus D, Dunn R, Kehry MR, Laufer TM, Iwasaki A.
Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus. J
Exp Med. 205 (13): 3041-3052, 20083) Iijima N, Iwasaki A. T cell memory. A local macrophage chemokine network sustains protective
tissue-resident memory CD4 T cells.
Science 346 (6205): 93-98, 20144) Iijima N*, Oh JE*, Song E, Lu P, Klein J, Jiang R, Kleinstein SH, Iwasaki A (* equally contributed). Migrant memory B cells secrete luminal antibody in the vagina Nature 571 (7763):122-126,
2019

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一般の皆様へ
様々なウイルスを含む病原体は、多くの粘膜組織より侵入することにより我々の身体機能に重篤な症状を引き起こすことが知られている。これまでワクチンなどの予防を目的とした治療方法が全く効果がないウイルス等に対しては、粘膜などの感染部位で長期間、その機能が維持される組織局在型メモリー T 細胞 (tissue resident memory T cells; TRM) を標的とした治療方法が重要な役割を果たすと考えられる。TRM は、侵入した病原体を感染した粘膜部位で速やかに除去することができるため、重篤な症状を引き起こす前に生体を防御することが可能である。
今後、TRM を標的とした新規治療方法の確立がこれまでにワクチンが確立していない病原体に対しては重要な役割を果たすと確信している。

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Analysis of cell lineage and transcriptional regulation during
ventricular development of heart
Yusuke WATANABE
ywatanabe@ncvc.go.jp
National Cerebral and Cardiovascular Research Center, Research Institute

Summary Abstract
We identified a distal Hey2 enhancer conserved in the mouse and human to possess specifictranscriptional activity in ventricular free wall myocytes at the looping stage of cardiac development.
The ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative
function with cardiac Gata proteins. Our findings delineate a regulatory mechanism of ventricular
Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation andcardiac morphogenesis during embryonic development (Ref 1).
Key Words：Heart development, transcriptional regulation, Hey2

Introduction
Development of multi-chambered heart is associated with spatio-temporal regulation of gene
expression. Combinatorial actions of DNA-binding proteins, co-factors and chromatin remodelingenzymes precisely control gene expression patterns in the embryonic heart, and their gene mutations
often lead to human congenital heart defects. A basic helix-loop-helix transcription factor Hey2
is specifically expressed in the embryonic mouse ventricles and is indispensable for ventricular
myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2transcription is precisely regulated in the heart remains unclear.

Results
To identify an enhancer for Hey2 transcription in the developing ventricles, we analyzed the
ChIP-seq datasets that were previously published and publicly available. Transcriptional activityof the selected genomic regions was evaluated by performing F0 Tg mouse analysis of lacZ reporter
expression. Among tested, the 211k upstream region of the Hey2 gene, which was highly conservedin various species, showed specific transcriptional activity in the ventricular myocardium throughcardiac development (Fig. 1).
To test the importance of the -211k region for endogenous Hey2 expression, we generated a mouseline with its deletion by using the CRISPR/Cas9 genome editing. Expression analysis of embryonichearts revealed that the Hey2 mRNA expression was significantly decreased in the -211k deletionhomozygotes (Fig. 2).
Since the nucleotide sequence of mouse minimal enhancer was highly similar to that of theequivalent region in the human genome, we examined if the enhancer activity was conserved in themouse and human. As a result, a comparable upstream region of the human HEY2 gene showedtranscriptional activity specific to the embryonic ventricles.
In the -211k region enhancer of the mouse Hey2 gene, there were multiple consensus binding
elements for Gata and T-box family members. Mutations of the Gata or a T-box binding sites in

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for the Promotion of Sciencethe -211k region resulted in the loss of transcriptional
activity in lacZ reporter transgenic analyses. Among T-box
transcription factors implicated in cardiac development,a previous study showed that Tbx20 is predominantly
expressed in the ventricular compact layer at the midgestation stage (Ref 2), which is similar to the Hey2
expression pattern. Other study reported that Tbx20
null mice showed severe abnormalities of ventricularmorphogenesis and reduced Hey2 expression (Ref 3).
Based on these lines of evidence, we generated Tbx20 nullmice using the CRISPR/Cas9 genome editing and examined
whether Tbx20 is essential for the Hey2 ventricularenhancer activity. Furthermore, the enhancer activity as
well as endogenous Hey2 expression was completely lostin the heart of Tbx20 knockout embryos. As previously
reported, Tbx20 null embryos had abnormal heart structurewith a single ventricle and hypoplastic outflow tract. Hey2
expression was apparently down regulated in the ventricularmyocardium, although the endocardial expression was
unaltered. Strikingly, Hey2 ventricular enhancer activity
was completely lost with the Tbx20 null background (Fig.
3). These results indicated that Tbx20 was indispensablefor the Hey2 ventricular enhancer activity and that othercardiac transcription factors including Gata proteins were
not able to compensate the loss of Tbx20.
Finally, we performed luciferase reporter assays toestimate how Tbx20 regulated the Hey2 ventricularenhancer activity. T-box factors function either as anactivator, a repressor or both in a context-dependent
manner and often display weak transcriptional activity inluciferase assays. That was the case in our experiments, andthe effect of Tbx20 expression was limited on the luciferase
reporter activity driven by the 649bp Hey2 ventricular enhancer. We therefore used in the following
assays Tbx20 fused with a VP16 transcriptional activation domain (VP16-Tbx20) that activated theluciferase reporter in a dose-dependent manner.  Gata4 significantly induced the enhancer activity
but at a relatively low level. The effect of VP16-Tbx20 on the Hey2 ventricular enhancer activity
was markedly enhanced in the presence of Gata4, whereas Gata4 led to marginal, statisticallyinsignificant change when used alone. Interestingly, the synergy between VP16-Tbx20 and Gata4
was also observed using the Hey2 ventricular enhancer with the T-box site deletion; while VP16Tbx20 failed to stimulate the luciferase activity, it showed significant induction when co-expressedwith Gata4. In contrast, the mutations of Gata binding sites resulted in the loss of such cooperative
activity (Fig. 4). These results suggest that Tbx20 may control the Hey2 ventricular enhancer activity
not only by its binding to the T-box consensus site but also by association with Gata proteins thatbind to the Gata consensus sites.

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Discussion & Conclusion
We demonstrated that a distal Hey2 enhancer conserved in the mouse and human possessedtranscriptional activity specific to the ventricular free wall myocardium at the looping stage of
cardiac morphogenesis. The enhancer was indispensable for the Hey2 expression in the ventricles
of mouse embryos, and its activity was, at least in part, attributable to Tbx20-mediated regulation incollaboration with cardiac Gata proteins.
Since the Hey2 ventricular enhancer is active specifically in the cardiac crescent mesoderm before
heart tube formation, cell lineage analysis of the enhancer active cells will provide new information
on early fate determination of ventricular myocytes. In addition, Hey2 itself strongly supports
ventricular myocyte differentiation of human pluripotent cells (Ref 4). Understanding mechanisms ofHey2 transcriptional control in the course of cell differentiation helps application of stem cell based
therapeutics to cardiovascular diseases.

References(1)Ihara D, Watanabe Y*, Seya D, Arai Y, Isomoto Y, Nakano A, Kubo A, Ogura T, Kawamura T,
Nakagawa O*.
*co-corresponding author
Expression of Hey2 transcription factor in the early embryonic ventricles is controlled through a
distal enhancer by Tbx20 and Gata transcription factors.
Dev Biol, in press. (2020)(2)Zhang, W., et al., J Biol Chem, 2011. 286(42): p. 36820-9.
(3)Singh, M.K., et al., Development, 2005. 132(12): p. 2697-707.
(4)Churko, J.M., et al., Nat Commun, 2018. 9(1): p. 4906.

一般の皆様へ
ヒト新生児の100人に１人が先天性心疾患を発症しており、心臓が正常に形態形成される過程を解明することは、それら疾患発症機構の理解や治療に繋がります。本研究では、心室筋で発現し、その形成に必須である Hey2因子がどのような遺伝子発現制御を受けているかを解析しました。その結果、Tbx20と Gata といった転写因子が Hey2遺伝子の発現に必須であることを明らかにしました。本研究は、心室筋形成における分子機構の１つを解明するものであり、ヒトの心臓形成を理解する上で重要な知見となりました。

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Construction of human complex brain organoids for analysis of brain
development and neurological diseases
Keiko Muguruma
muguruke@hirakata.kmu.ac.jp
Kansai Medical University

Summary Abstract
Stem cell biology utilizing organoid culture technology has gained significant academic andclinical attention as a transformative culture system that resemble the in vivo complexity withapplications in human brain development and drug discovery. The aims in this study is to develop
standardized methods for analyzing the formation of human brain tissues utilizing induced
pluripotent stem (iPS) cells.
Key Words：complex brain organoids, neural development, iPS cells, neurological disease, diseasemodeling

Introduction
The ultimate goal of neuroscience is to understand the structure and functions of the human brain
and to utilize the knowledge to overcome neurological disorders. The research of human brain
had long been limited to non-invasive studies, genomic analyses and studies with autopsied brain
tissues or human cell lines. The emergence of iPS cells and organoid culture technology has enabled
to construct human brain tissues in vitro. We aim to develop versatile methods for construction,measurement and analysis of human brain organoids. With these methods, we will elucidate thestructure and functions of the human brain.

Results
We performed experiments on the development of methods for maturation of brain organoids andnetwork formation of brain organoids.
1.Maturation of brain organoids
We previously reported the methods for formation of brain organoids, cerebellar organoids andcerebral organoids, from human embryonic stem cells and iPS cells by utilizing self-organizing
differentiation techniques (Muguruma et al., Nat Neurosci. 2010; Muguruma et al., Cell Rep 2015;
Ishida et al., Cell Rep 2016; Eguchi et al., Biochem Biophys Res Commun 2018). To elevate thedifferentiation efficiency of a specific kind of neurons, we improved the differentiation culture
methods. By optimizing the treatment with low-molecular chemical compounds and culture protocols
at the early stage of differentiation, we succeeded in significant elevation of neural progenitors.
For facilitating the formation of mature brain tissues, we started to form complex brain organoids
by assembling cerebral cortical neurons and non-neuronal cells such as glial cells, neural crest cells
and vascular endothelial cells. We confirmed that the protocols for individual non-neuronal cell types
are effective and that these protocols can be combined with those are brain organoid suitable forneuronal differentiation. However, we have not yet established optimized combination for the control
of distribution and orientation of individual cell types in the brain organoids.

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2. Network formation by coculturing brain organoids
We tried to form the cerebrocerebellar communication loops and the visual systems in vitro. At
first, cerebral organoids, cerebellar organoids and retinal organoids were separately differentiated by
the specific culture protocols. Then, the cerebral and cerebellar organoids were co-cultured for theformation of the cerebrocerebellar loops. The retinal and cerebral organoids were co-cultured forthe formation of the visual system. In both co-cultures, we observed neuronal connections between
two areas, indicating that long-range neuronal network formation can be recapitulated in vitro. In theactual nervous systems, there exist additional relaying brain regions such as thalamus in the visualsystem and precerebellar nuclei in the cerebrocerebellar systems. Thus, we started to develop the
protocols for differentiation of such relaying brain regions.  

Discussion & Conclusion
This study aimed to develop standardized methods for analyzing the formation, degeneration
and restoration of human brain tissues. Combining 3D brain organoid cultures of human iPSCs, 4D
imaging, and image analysis, we tried to establish efficient methods to faithfully recapitulate and toquantitatively analyze the ontogenetic formation of human brain tissues.
By achievements of this study, we will demonstrate methods integrated for construction ofexperimentally accessible human models of neurological disorders and their applications topathological investigation and effective drug screen. These methods will provide a standard andcomprehensive platform for understanding human brain structure and functions and for overcoming
neurological disorders. The achievement will give great impacts on the biomedical researchers andpharmaceutical developers to accelerate their research and development.

References
1. Muguruma K, Nishiyama A, Ono Y, Miyawaki H, Mizuhara E, Hori S, Kakizuka A, Obata K,
Yanagawa Y, Hirano T, Sasai Y. Ontogeny-recapitulating generation and tissue integration of ES
cell-derived Purkinje cells. Nat Neurosci 2010; 13(10): 1171-80.
2. Muguruma K, Nishiyama A, Kawakami H, Hashimoto K, Sasai Y. Self-organization of polarized
cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep 2015; 10(4): 537-50.
3. Ishida Y, Kawakami H, Kitajima H, Nishiyama A, Sasai Y, Inoue H, Muguruma K. Vulnerabilityof Purkinje cells generated from spinocerebellar ataxia type 6 patient-derived iPSCs. Cell Rep
2016; 17(6): 1482-90.
4. Eguchi N, Sora I, Muguruma K. Self-organizing cortex generated from human iPSCs withcombination of FGF2 and ambient oxygen. Biochem Biophys Res Commun 2018; 498(4): 72935.
5. Matsumoto R, Suga H, Aoi T, Bando H, Iguchi G, Narumi S, Hasegawa T, Muguruma K, Ogawa
W, Takahashi Y. Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2
generation of pituitary progenitor cells. J Clin Invest 2020; 130(2): 641-54.
6. Kanie K, Bando H, Iguchi G, Muguruma K, Matsumoto R, Hideka-Takeno R, Okimura Y,
Tamamoto M, Fujita Y, Fukuoka H, Yoshida K, Suda K, Nishizawa H, Ogawa W, Takahashi Y.
Pathogenesis of anti-PIT-1 antibody syndrome. J Endocr Soc 2019; 3(11): 1969-78.

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一般の皆様へヒト iPS 細胞と自己組織的な分化培養技術を融合させることにより、脳に類似した構造体（脳オルガノイド）を繰り返し何度でも作製できるようになりました。この技術は、ヒト脳の発生や組織構造の形成、神経細胞の機能に関する研究を培養皿の中で行うことを可能にしました。
本研究分野の発展は目覚ましく、病態のモデル化による発症機序の解明が進められ、新規治療法の開発や創薬開発に結びつくことが期待されています。

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A novel regulatory mechanism of the metabolic signaling complex
mTORC1
Shusaku Shibutani
shushibu@yamaguchi-u.ac.jp
Yamaguchi University

Summary Abstract
Recent studies have shown that mTORC1, a major regulator of cellular biosynthesis, is activatedon lysosomes in the presence of amino acids inside lysosomes. We have previously found that
endocytosis of amino acids plays an important role in the activation of mTORC1. Since both
endocytosis and mTORC1 have been linked to cancer activities, we attempted to elucidate themechanism of anti-cancer activity by endocytosis inhibition.
Key Words：mTORC1, amino acid, endocytosis, dynamin

Introduction
The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of
metabolism that is activated by the presence of extracellular amino acids. Previous studies haveshown that mTORC1 is activated when extracellular amino acids are transported into the lumen of
the lysosome. We have previously shown that the endocytosis inhibitor (dynamin inhibitor) Dynasore
suppresses mTORC1 activity, suggesting that the endocytotic uptake pathway of extracellular aminoacids is important for maintaining mTORC1 activity [1]. Previous studies have shown that Dynasore
inhibits cell growth and induces apoptosis in cancer cell lines [2–4]. However, the mechanisms ofthese anti-cancer effects are not well understood.

Results
We first tested the effect of the endocytosis inhibitor Dynasore on cell viability of cancer cell lines.
To this end, a total of 8 cell lines were used: 4 human hematopoietic cancer-derived cell lines (Jurkat,
Raji, K562, and U937) and 4 human epithelial cancer-derived cell lines (HeLa, MCF7, A549, andCaco-2). We employed the MTT assay, which is a method to measure the metabolic activity of cells
and frequently used as a readout of cell viability. In addition to Dynasore, we also tested the effectsof Torin 1, a potent mTORC1/2 inhibitor, because we have previously shown that Dynasore has an
mTORC1 inhibitory effect [1]. Cells were treated with various concentrations of Dynasore and Torin
1 for 24 h, and cell metabolic activity was measured. As a result, both Dynasore and Torin 1 showed
dose-dependent inhibitory effects on all 8 cell lines. Of the four human hematopoietic cancer celllines and four human epithelial cancer cell lines tested in this study, there was a tendency that thehematopoietic cell lines tended to be more sensitive than the epithelial cell lines.
In addition, there was a decrease in phosphorylated S6K (Thr389) by Dynasore and Torin 1 in all
8 cell lines. At the concentrations used in this experiment (1 μM Torin 1 and 40/80 μM Dynasore),
Torin 1 showed stronger or equivalent mTORC1 inhibition than Dynasore in most (7 of 8) cell lines,
whereas Torin 1 showed weaker or equivalent activity to decrease cell viability than Dynasore in all
8 cell lines. These results suggest that the strong viability inhibitory effect of Dynasore cannot be
explained by its mTORC1 suppression ability alone, and therefore other mechanisms exist.

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To determine whether the Dynasore-induced decrease in cell viability is due to a decrease incell proliferation or to an increase in cell death, we used the TUNEL staining method to determine
whether apoptosis is induced. Dynasore markedly increased the number of TUNEL-positive cells.
Furthermore, after 4 and 24 hours of Dynasore treatment, PARP was degraded, which is indicativeof the activation of an apoptosis pathway. Next, to determine how Dynasore and Torin 1 affect thecell cycle, Jurkat cell DNA was stained with propidium iodide and analyzed by flow cytometry.
Cells treated with Dynasore, a sub-G1 population was detected, indicating that Dynasore induced
DNA fragmentation. This result is consistent with the results of the TUNEL method describedabove, reconfirming that Dynasore induces apoptosis. Importantly, induction of sub-G1 cells was
not observed in the non-cancer-derived 293T cells, suggesting that the induction of apoptosis by
Dynasore may be cancer-specific. Furthermore, Dynasore treatment resulted in a relative increasein cells in the early S phase. This suggests that Dynasore treatment may cause a delay or arrest of
S-phase progression (i.e. DNA synthesis during DNA replication). Since stalled DNA synthesis
leads to single-stranded DNA exposure and activates the DNA damage response pathway, we tested
whether the DNA damage response is induced by Dynasore, and found that phospho-Histone H2A.X(Ser139) and phospho-Chk1 (Ser345), which are phosphorylated during the DNA damage response,
were induced in Dynasore-treated cells, but not in Torin 1-treated cells. This suggests that DNA
synthesis delay is caused by Dynasore and that DNA synthesis delay may contribute to the inductionof apoptosis.

Discussion & Conclusion
Dynasore, an inhibitor of dynamin-dependent endocytosis, has been reported to reduce theinvading activity of the human lung cancer cell line H1080 [2] and to induce mitochondria
dysfunction and apoptosis in the human lung cancer cell line A549 [3]. Similarly, it also induced
apoptosis in the human cervical cancer cell line HeLa [4]. However, its effects in non-epithelial
cell lines have not been investigated, and the mechanisms of Dynasore-induced apoptosis andgrowth inhibition were not clear. In this study, we found that the sensitivity to Dynasore tended
to be higher in hematopoietic cell lines than in epithelial cell lines. Possible mechanisms for thedifferences in susceptibility include differences in endocytosis activity, DNA synthesis activity, andthe susceptibility of apoptosis to DNA synthesis inhibition, although these possibilities need to beexperimentally validated. In addition, there was an increase in early S-stage cells in Dynasore-treatedcells, suggesting DNA replication is suppressed by Dynasore. Consistent with this, the activationof the DNA damage response pathways was observed in Dynasore-treated cells. Although further
investigation is required, it is speculated that DNA replication may be inhibited by the depletionof nutrient molecules required for nucleotide synthesis or by the depletion of necessary proteinsrequired for DNA synthesis, because endocytosis inhibition is thought to result in a decrease in the
uptake of membrane-impermeable nutrients. This study has shown that the endocytosis inhibitor
Dynasore induces apoptosis and strong growth inhibition especially in hematopoietic cancer cells.
This study suggests that endocytosis inhibition may be a promising target for cancer treatment.

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References
1. Shibutani, S., Okazaki, H., and Iwata, H. (2017). Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1). J. Biol. Chem. 292, 18052–18061.
2. Yamada, H., Abe, T., Li, S.-A., Masuoka, Y., Isoda, M., Watanabe, M., Nasu, Y., Kumon, H.,
Asai, A., and Takei, K. (2009). Dynasore, a dynamin inhibitor, suppresses lamellipodia formationand cancer cell invasion by destabilizing actin filaments. Biochem. Biophys. Res. Commun. 390,
1142–1148.
3. Shen, F., Gai, J., Xing, J., Guan, J., Fu, L., and Li, Q. (2018). Dynasore suppresses proliferationand induces apoptosis of the non-small-cell lung cancer cell line A549. Biochemical and Biophysical Research Communications 495, 1158–1166.
4. Lee, Y.-Y., Jeon, H.-K., Lee, J., Hong, J.E., Do, I.-G., Choi, C.H., Kim, T.-J., Kim, B.-G., Bae, D.S., Kim, Y.-C., et al. (2016). Dynamin 2 Inhibitors as Novel Therapeutic Agents Against Cervical
Cancer Cells. Anticancer Res. 36, 6381–6388.

一般の皆様へ細胞は周りにアミノ酸が豊富なときにはタンパク質を合成し、逆にアミノ酸が足りないときには自身のタンパク質を壊してアミノ酸を作って飢餓に備えようとします。このように栄養の豊富さに対応するための仕組みは、生物が活動するために必要不可欠なものですが、その詳細はいまだ不明な点が多く残されています。栄養素への細胞応答を解明することは、生物学的に重要なだけでなく、肥満や生活習慣病などの過栄養を原因とする問題に対する取り組みとしても重要であると考えています。

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Generation of in vitro heart organ using the mothods ofatrial-ventricular cardiomyocyte differentiation
Jun Takeuchi
junjunjunta@gmail.com
Medical research Institute, Tokyo Medical and Dental University

Summary Abstract
Functional cardiomyocyte subtypes generated from reprogrammed stem cells or fibroblasts
serve as promising models that aid both researchers and clinicians in understanding the molecularmechanisms of atrial-ventricular cardiomyocyte induction and development of novel therapeutics.
Here, we identified the defined factors required for the induction of atrial or ventricularcardiomyocytes by using bioinformatics analysis. These factors function synergistically to build eachchamber in the heart.
Key Words ： induced pluripotent stem cells, cell fate, reprogramming, atrial-ventricularcardiomyocytes

Introduction
Reprogramming technologies to generate cardiomyocytes (CMs) from fibroblasts revealed thatcell fate is determined by defined factors. These regenerative approaches can repair damaged hearttissues or lost CMs through programmed CMs (induced CMs: iCMs) without transplant rejections.
However, the protocol that controls the induction of atrial or ventricular CMs is not established.
Therefore, several atrium-like iCMs and few ventricle-like iCMs are observed.
In this project, we will screen defined factors determining the atrial or ventricular cell fate.
Using bioinformatics analysis, we will produce atrium-like iCMs (AiCMs) and ventricle-like iCMs
(ViCMs) of the in vivo developing heart in mice and in vitro differentiating heart in human induced
pluripotent stem cells (iPSCs), thus building the functional heart with atrium-ventricle-like chambers.

Results
1: Identification of defined factors for the induction of atrium-like cardiomyocytes (CMs)
To identify the defined factors required for the atrial-CM fate specification, we performed
bioinformatics analysis based on RNA sequences from embryonic mice at the 9th day and fromdifferentiating CMs at 10 days post retinoic acid (RA) treatment in human iPSCs. This analysis
revealed 11 candidate genes, out of which 4 genes were identified to be essential for the atrial CM
specification through exo-utero overexpression analysis.
To address whether these 4 genes are necessary for atrial CM specification, we performed 2
types of analyses: 1) overexpression in the differentiating human iPSCs and 2) knocking out inutero mouse using the CRISPR/Cas9 system. Firstly, we succeeded in the generation of functional
atrium-like CMs from hiPSCs through virus-mediated overexpression of 3 defined factors after
several selections. Secondly, we successfully generated in utero multi-gene knockout mice without
atrial structures. Interestingly, the ventricle-specific genes were clearly observed in these knockout
mice.

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Next, we addressed whether these 3 defined factors are sufficient to reprogram fibroblasts
directly into atrium-like CMs, using an in vitro culture system of mouse skin fibroblasts isolated
from the back. The fibroblasts were cultured for 2 weeks after virus-mediated overexpression of
the factors. Mlc2a-positive CMs (Mlc2a is the most abundant component in atrium CMs) withbeating were observed in the culture dishes, suggesting that these defined factors are sufficient for
specification of the cell fate of atrial CMs in the reprogramming experiment from the fibroblasts
as well as in programming during embryonic development.
2: Identification of defined factors for the induction of ventricle-like CMs
To identify the defined factors required for specification of the ventricular-CM cell fate, we
performed bioinformatics analysis based on RNA sequences from embryonic mice on the 9th day
and differentiating CMs at 10 days post RA inhibitor treatment in human iPSCs. We isolated 11
candidate genes from this analysis, out of which 4 essential genes were identified as essential forthe ventricular CM specification using exo-utero overexpression analysis.
To address whether these 4 genes are necessary for ventricular-CM specification, we performed
2 types of analyses: 1) overexpression of the factors in differentiating human iPSCs and 2)
generation of in utero knockout mouse using the CRISPR/Cas9 system. We succeeded in thegeneration of functional ventricular-like CMs from hiPSCs with virus-mediated overexpressionof 2 defined factors after several selections. We also generated in utero double knockout mice
without ventricle structures. Interestingly, the atrium-specific genes were clearly observed in theseknockout mice.
Next, to address whether these 2 defined factors are sufficient to reprogram fibroblasts directly
into ventricle-like CMs, we generated an in vitro culture system with fibroblasts isolated fromthe back skin in mice. After overexpression of the factors through viral infection, the fibroblasts
were cultured for 2 weeks and Mlc2v-positive CMs (Mlc2v is the most abundant component
in ventricular CMs) with beating were observed in the culture dishes. This suggested thatthese defined factors are sufficient for specification of the cell fate of ventricular CMs in the
reprogramming experiment from fibroblasts as well as in programming during embryonicdevelopment.
3: Development of the functional heart with 2 chambers
To generate a mammalian heart in vitro from human iPSCs, we established a novel protocol
involving treatment with various concentrations of cytokine A and B during differentiation. The
3-dimensional spheroids with 2 chambers from human iPSCs were differentiated at day 10 after their aggregation. Each chamber of these showed high expression levels of either MLC2a or
MLC2v. Unfortunately, we could not establish multi-chamber spheroids from hiPSCs with only
A (MLC2V expression) or B treatment (MLC2A expression) during the aggregation stage. These
results suggest that combinatorial treatment of cytokine A and B is necessary for generating multichamber spheroids with atrium- and ventricle-like structures, and further studies are required toestablish a mammalian-like heart with 4 chambers.

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Discussion & Conclusion
In previous studies from our group, we identified the defined factors required for heart cell fate ina mouse model with both the atrium (Mlc2a) and ventricle (Mlc2v) CMs (Morita et al., JMCC 2016;
Takeuchi & Bruneau, Nature 2009), but we could not control CM cell types in that system.In thisproject, firstly, we identified the defined factors determining their cell fate. Uniquely, combinatorial
functions of 3 factors are necessary to induce the atrium-like CMs and combinatorial functions of 2
factors are necessary for the ventricle-like CM induction. Secondly, we organized the vertebrate-type
heart with two chambers of the atrium- and ventricle-like structures. However, it remains unclear
which signals and factors can select the left-right axis in formation of the atrium and ventricle in the
mammalian heart.
In conclusion, we provided two new insights in heart development and our findings are ofimportance in clinical settings and in drug discovery.

References＊Kakeno et al., The 19th Congress of the Japanese Society for Regenerative Medicine. 18-29th MAY
2020.
＊Kakeno et al., The 42nd Annual Meeting of the Molecular Biology Society of Japan. 3-6th DEC
2019.
＊ Takeuchi, Surugadai mposium 2019. 18th NOV 2019.
＊ Takeuchi, Karolinska Institute Symposium 2019. 4-5th JUN 2019.

一般の皆様へ
本研究は、ヒト・マウス間で保存され、かつ、1心室筋―心房筋の発生運命決定に関わる、

2線維芽細胞から直接心室筋―心房筋への運命転換に関わる共通因子を見出すことを目的としている。本研究期間内に３段階のバイオインフォマティクス解析を経て、各々の細胞運命を決定する共通因子を選出することができた。また、複数遺伝子同時破壊マウスの作成にも成功したことで、マウス発生工学技術開発の発展も見込まれる。本研究結果は、今後、心房筋および心室筋別々の条件下で心筋性質の理解・心筋移植および創薬スクリーニングなどの研究発展に貢献すると期待されることから、今年度中に論文投稿・採択を目指す。

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Development of Catalytic Asymmetric Cycloaddition Reaction of
α-Keto Esters for the Innovative Synthesis of
Tetrahydrofuran Compounds
SAKAKURA, Akira
sakakura@okayama-u.ac.jp
Graduate School of Natural Science and Technology, Okayama University

Introduction
Tetrahydrofuran is a fundamental structure in many biologically active natural products. For thedevelopment of new pharmaceuticals based on these useful compounds, stereoselective methods forthe synthesis of tetrahydrofurans are demanded. Although there have been reported many methods
for the synthesis of tetrahydrofurans, these conventional methods require multiple steps, and thestereoselectivity is not high in some cases. Thus, there is still much room for the improvementin the stereoselective synthesis of tetrahydrofurans. We planned to develop a new method for thestereoselective construction of tetrahydrofurans.
Key Words：Asymmetric Catalysis, Cycloaddition, α-Keto Ester, Tetrahydrofuran

Results
We have reported new chiral Cu(II) complex as a highly efficient Lewis acid catalyst forasymmetric cycloaddition reaction and 1,2-addition of allylic silanes (references 1–3).  Based on
the synthetic method reported by Akiyama and colleagues (reference 4), we examined the reactionof silylmethylcyclopropane compounds with ethyl glyoxylate. As a result, monosubstituted
cyclopropane 1a (R’ = Ph) was found to be less reactive and the desired product was not obtained.
On the other hand, we found that disubstituted cyclopropane 1b did react with ethyl glyoxylate togive 1,2-addition product 2 (15% yield, Scheme 1).

Scheme 1
We next examined the cycloaddition reaction of silylmethylcyclopropane compounds with β,γunsaturated α-keto esters.  The reaction was supposed to give seven-membered ethers that is also
an important structure found in various biologically active compounds. When the reaction of 1a(R’ = i-Pr) with β,γ-unsaturated α-keto ester 3 was conducted in the presence of various Lewis acidcatalyst such as TiCl4, Cu(NTf2)2 and Sc(OTf)3, dihydropyran compound 4 was obtained as a single
diastereomer in 29~41% yield (Scheme 2). This unexpected result was considered to be obtained via
the conversion of 1a into (E)-allylic silane 5 with the aid of Lewis acid catalyst. This result is highly
important since the synthesis of (E)-allylic silane 5 as a pure form is very difficult (see reference 2).  

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The present method is able to use easily available cyclopropane compound 1a instead of 5 for thesynthesis of dihydropyran compound 4.

Scheme 2
The above results showed that the reactivities of silylmethylcyclopropane compounds were nothigh enough to react efficiently with ethyl glyoxylate and β,γ-unsaturated α-keto esters.  Thus, wenext examined the use of stannylmethylcyclopropane compound 6, since the reactivity of 6 was
supposed to be higher than 1a because of high electron-donation nature of the trialkylstannyl group.
When the reaction of 6 with β,γ-unsaturated α-keto ester 3 was conducted at –78 °C in the presenceof Cu(NTf2)2 as a Lewis acid catalyst, 1,2-addition product 7 was obtained in 43% yield (Scheme
3). On the other hand, very interestingly, when the same reaction was conducted in the presence ofIn(OTf)3 instead of Cu(NTf2)2, dihydropyran 8 was obtained as a single diastereomer in 15% yield.
These results suggested that Cu(NTf2)2 catalyst selectively activated β,γ-unsaturated α-keto ester 3 topromote the direct attack of cyclopropane compound 6 toward the α-carbonyl group of 3. In sharp
contrast, In(OTf)3 should selectively activated cyclopropane compound 6. Detailed mechanism of
the generation of dihydropyran 8 is unclear, but it is conceivable that homoallylic stannyl compound
9 was generated with the aid of In(OTf)3, and that regio- and diastereoselective hetero-Diels–Alderreaction of 9 with β,γ-unsaturated α-keto ester 3 would be promoted by In(OTf)3 and gave thecorresponding dihydropyran 8 was produced as a single isomer. These results showed that as we
expected, stannylmethylcyclopropane compound 6 is more reactive than silylmethylcyclopropane
compounds enough to react directly with β,γ-unsaturated α-keto ester 3. In addition, the Lewis acidpromoted cyclopropane ring opening of 6 gave the olefinic compound 9, which was different fromthe compound generated via the Lewis acid-promoted cyclopropane ring opening of 1a.

Scheme 3

Discussion & Conclusion
For the development of stereoselective synthesis of tetrahydrofurans, we have investigatedthe Lewis acid-promoted reaction of silylmethylcyclopropane compounds with ethyl glyoxylate.
Although, as Akiyama reported, SnCl4 and TiCl4 successfully promoted the reaction, the activity of
the chiral copper(II) complex was low, and only 1,2-addition product was obtained. These results
would be attributed to the low reactivity of both silylmethylcyclopropane compounds and ethyl

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The Novartis Foundation(Japan)
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Since we have previously developed the asymmetric hetero-Diels–Alder reaction of allylic
silanes with β,γ-unsaturated α-keto esters (reference 2), we next investigated the reaction of thecyclopropane compounds with β,γ-unsaturated α-keto esters.  As results, we found that the reactivityof β,γ-unsaturated α-keto ester was higher than that of ethyl glyoxylate.  Very interestingly, thereaction pathways highly depended on the structure of the cyclopropane compounds and the Lewis
acid catalysts, and the dihydropyran compounds and 1,2-addition products were selectively generated
as single isomers in each case.

References
1. Sakakura, A.; Kondo, R.; Matsumura, Y.; Akakura, M.; Ishihara, K. J. Am. Chem. Soc. 2009, 131,
17762–17764.  
2. Matsumura, Y.; Suzuki, T.; Sakakura, A.; Ishihara, K. Angew. Chem. Int. Ed. 2014, 53, 6131–6134.  
3. Niwa, Y.; Miyake, M.; Hayakawa, I.; Sakakura, A. Chem. Commun. 2019, 55, 3923–3926.  
4. Fuchibe, K.; Aoki, Y.; Akiyama, T. Chem. Lett. 2005, 34, 538–539.  

一般の皆様へテトラフラン環，すなわち酸素原子を一つ含む五員環構造，は，ポリエーテルイオノフォアやマクロライドなど，様々な生物活性物質に含まれる重要な構造である。テトラフラン環構造をもつ新規医薬品を開発するためには，テトラヒドロフラン環を立体選択的に化学合成する必要がある。本研究では，テトラヒドロフラン環を合成するための新しい方法を開発することを目的として，シリルメチルシクロプロパン化合物とα - ケトエステルとの環化反応の検討を行った。その結果，目的とするテトラヒドロフラン環を合成するには至っていないが，その過程で，1,2- 付加生成物やジヒドロピラン化合物を立体選択的に合成できるという新しい知見を得ることができた。

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Pathophysiologic role of a novel microenvironment that prolongs
tissue damage in acute kidney injury
Takayoshi SUGANAMI
suganami@riem.nagoya-u.ac.jp
Nagoya University, Research Institute of Environmental Medicine

Summary Abstract
Here we show that macrophage-inducible C-type lectin (Mincle) senses renal tubular cell death
to induce sustained inflammation after acute kidney injury in mice. Mincle-deficient mice were
protected against tissue damage and subsequent atrophy of the kidney after ischemia–reperfusion
injury. We also identified β-glucosylceramide as an endogenous Mincle ligand. Notably, freecholesterol markedly enhanced the agonistic effect of β-glucosylceramide on Mincle. Moreover,
β-glucosylceramide and free cholesterol accumulated in dead renal tubules in proximity to Mincleexpressing macrophages, where Mincle was supposed to inhibit clearance of dead cells andincrease proinflammatory cytokine production. This study demonstrates that β-glucosylceramide,in combination with free cholesterol, acts on Mincle as an endogenous ligand to induce cell death–triggered sustained inflammation after acute kidney injury.
Key Words：Mincle, dead cells, acute kidney injury, macrophages, inflammation

Introduction
Mincle is a pattern recognition receptor expressed in innate immune cells such as macrophages(1). Recent studies revealed the role of Mincle in infectious diseases; it recognizes trehalose-6,6’dimycolate (TDM), a mycobacterial cell wall glycolipid, to induce production of proinflammatory
cytokines and chemokines, and thus protects against Mycobacterium tuberculosis infection (2).
Mincle also senses cell death (3), suggesting a role in sterile inflammation. In this regard, we
previously reported that Mincle expression is localized to macrophages surrounding dead or dying
adipocytes during the development of obesity, where Mincle accelerates adipocyte death–triggered
chronic inflammation in visceral adipose tissue to induce systemic insulin resistance (4). However,
little is known about how Mincle recognizes dead cells under pathological conditions in vivo andhow Mincle regulates necroinflammation.

Results
In this study, we induced ischemia–reperfusion injury of the kidney and collected samples atseveral time points corresponding to the acute, reparative, and late phases. We first examined
the mortality of Mincle-deficient (KO) and wild-type mice for up to 6 days after renal ischemia–
reperfusion injury. Although about half of the wild-type mice died during this period, all KO mice
lived. On day 1, although Mincle mRNA expression was markedly increased by AKI, we observed
no apparent difference in renal function, tissue damage, and mRNA expression of proinflammatory
cytokines and chemokines between KO and wild-type mice. On the other hand, on day 3 when the
repair process began, all these parameters were significantly ameliorated in KO mice relative to wildtype mice.

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There are at least two types of macrophages: proinflammatory M1 and anti-inflammatory
M2. Using flow cytometry, we sought to determine which macrophage subset expresses highlevels of Mincle in injured kidneys on day 3 after ischemia–reperfusion. As a result, Mincle waspredominantly expressed in F4/80lo macrophages, which expressed higher levels of Il6 and lower
levels of Mrc1 than F4/80hi macrophages. Moreover, in situ hybridization analysis revealed that
Mincle-expressing cells surrounded the severely damaged tubules with intraluminal debris, andMincle mRNA was positive in only a subset of macrophages infiltrating the injured kidney.
Because Mincle expression persisted in the late phase (on day 7 after ischemia–reperfusion
injury), we further sought to determine whether Mincle is involved in the AKI-to-CKD transition.
The ratio of injured to contralateral kidney weight was significantly higher in KO mice on day 14,
suggesting prevention of renal atrophy in the late phase. Consistent with this, the area positive for
intact proximal tubules in the injured kidney was well preserved, and the number of dead cells was
low in KO mice at this time point. These findings suggest that Mincle contributes to sustained tubularcell death and subsequent renal atrophy in the late phase after renal ischemia–reperfusion injury.
We next examined the role of Mincle using cultured macrophages. It has been reported that Mincle
stimulation increases mRNA levels of inflammation-related genes such as Il6. In addition, we foundthat mRNA expression of the genes related to phagocytosis was markedly suppressed by treatment
with Mincle stimulation. Consistently, Mincle stimulation effectively suppressed phagocytotic
activity. These findings suggest that Mincle signaling in macrophages impairs clearance of deadcells, potently contributing to sustained inflammation after renal ischemia–reperfusion injury.
Finally, we screened for endogenous Mincle ligands released from dead or dying tubular epithelial
cells in the injured kidney after ischemia–reperfusion. Liquid chromatography–mass spectrometry(LC-MS)–based lipidomics analysis revealed thatβ-glucosylceramide was capable of activating
Mincle. Moreover, we found that free cholesterol markedly increased theβ-glucosylceramide-induced
Mincle signaling. Imaging mass spectrometry detected accumulation of β-glucosylceramide mainlyin the corticomedullary junction area. Filipin staining also revealed that intratubular aggregation offree cholesterol was scattered in the injured area and surrounded by macrophages. Collectively, thesefindings indicate that β-glucosylceramide in combination with free cholesterol activates Mincle in
the injured kidney following ischemia–reperfusion.   

Discussion & Conclusion
Non-resolving inflammation is a critical determinant factor in the progression from AKI to CKD.
Following initial insults, inflammatory responses induced by tubular injury provoke maladaptive
tissue repair and additional tubular damage. However, the precise mechanism underlying cell death–triggered sustained inflammation remains to be elucidated. Here we provide evidence that Mincle,
a novel sensor of cell death, is involved in this process. In particular, Mincle senses dead tubules toincrease proinflammatory cytokine production and decrease dead cell clearance, thereby aggravating
a vicious cycle of necroinflammation. Consistent with this, Mincle deficiency markedly preserved
healthy mature proximal tubules in the late phase after renal ischemia–reperfusion injury. In otherwords, the risk of AKI-to-CKD transition was effectively reduced in Mincle KO mice. Although AKI
results from various etiologies, including ischemic and toxic insults, the close interaction between
severely damaged or dead tubules and immune cells is a common pathogenesis in the reparative
phase after an acute destructive event (5). Because it is clinically difficult to intervene in the acutephase of AKI (or initial tubular damage), Mincle is a rational therapeutic target for ameliorating thesubsequent progression from AKI to CKD.

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References
1. Matsumoto, M., T. Tanaka, T. Kaisho, H. Sanjo, N.G. Copeland, D.J. Gilbert, N.A. Jenkins,
and S. Akira. 1999. A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 inmacrophages. J Immunol 163:5039-5048.
2. Ishikawa, E., T. Ishikawa, Y.S. Morita, K. Toyonaga, H. Yamada, O. Takeuchi, T. Kinoshita, S.
Akira, Y. Yoshikai, and S. Yamasaki. 2009. Direct recognition of the mycobacterial glycolipid,
trehalose dimycolate, by C-type lectin Mincle. J Exp Med 206:2879-2888. doi:10.1084/
jem.20091750
3. Yamasaki, S., E. Ishikawa, M. Sakuma, H. Hara, K. Ogata, and T. Saito. 2008. Mincle is an ITAMcoupled activating receptor that senses damaged cells. Nat Immunol 9:1179-1188. doi:10.1038/
ni.1651
4. Tanaka, M., K. Ikeda, T. Suganami, C. Komiya, K. Ochi, I. Shirakawa, M. Hamaguchi, S.
Nishimura, I. Manabe, T. Matsuda, K. Kimura, H. Inoue, Y. Inagaki, S. Aoe, S. Yamasaki, andY. Ogawa. 2014. Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue
fibrosis. Nat Commun 5:4982. doi:10.1038/ncomms5982
5. Huen, S.C., and L.G. Cantley. 2015. Macrophage-mediated injury and repair after ischemic kidney
injury. Pediatr Nephrol 30:199-209. doi:10.1007/s00467-013-2726-y

一般の皆様へ近年、様々な慢性疾患に共通の病態基盤として「慢性炎症」が注目されている。そのメカニズムの１つとして、死細胞が発するメッセージを炎症細胞が受容・応答することが概念的に理解されているが、実際の病態における分子実態は不明の点が多い。我々は、急性腎障害モデルを用いて、新規死細胞センサーの Mincle が、壊死尿細管を感知して炎症慢性化に働くことを見出した。Mincle は、壊死尿細管の周囲に集積するマクロファージに限局して発現し、β - グル

コシルセラミドとコレステロールを認識してマクロファージの炎症性サイトカイン産生を促進するとともに、死細胞貪食を抑制すると考えられた。

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Role of a novel activity-dependent gene encoding a lipid modification
enzyme on neuronal and cognitive function
Hiroyuki Okuno
okuno@m.kufm.kagoshima-u.ac.jp
Kagoshima University

Summary Abstract
Activity-regulated gene expression in neurons play a critical role for our cognitive functions. The
zdhhc18 gene, which encodes a palmitoyl acyltransferase, is identified as one of such activity-regulated genes. Here we aim to investigate its cellular and physiological functions of this gene in mice.
In order to visualize cellular localization of zdhhc18, we adopted a CRISPR/cas9-based protein tagging method, called SLENDR. We also generated a zdhhc18 knockout mouse line using CRISPR/
cas9-based method, called GONAD.
Key Words：Brain, neurons, activity-dependent gene, palmitoyl acyltransferase, cognition

Introduction
In the brain, rapid gene induction following synaptic activities in neurons play a critical role totrigger changes in properties of neural networks, which underlie many types of cognitive function [1].
However, what genes and how they work remain unclear. To address this issue, we have performeda single neuron RNA-seq analysis and identified a list of genes whose expression significantly
changes after synaptic stimulation. In this research, we have focused one of such genes, zdhhc18.
The zdhhc18 gene encodes a palmitoyl acyltransferase (PAT), which catalyze a lipid modification to
yet unknown protein substrates. In humans, there are 24 PAT encoding genes, among which several
are linked with neurodevelopmental and psychiatric disorders in humans [2]. Interestingly, a recent
genome-wide association study has revealed that mutations in zdhhc18 are associated with schizophrenia [3]. However, almost nothing is known so far about biological functions of zdhhc18 in thecentral nervous system.

Results
Aim 1: Visualization of subcellular localization of Zdhhc18 in neurons
Our RNA-Seq analysis revealed that the zdhhc18 geneis up-regulated following strong synaptic stimulationin neurons. However, the site of action and substratesof the Zdhhc18 protein are totally unknown. To obtain
initial clues, we sought for subcellular localization of
the endogenous Zdhhc18 protein. We tried to avoid tosee exogenously overexpressed Zdhhc18, because suchoverexpression often results in unnatural localization due
Fig. 1. SLENDR control experimentto overload for intracellular trafficking systems. Because
no antibodies are commercially available for immunohistochemistry, we decided to introduce a new
method called SLENDR, which enables to insert a tag to endogenous genes using CRISPR/cas9-

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for the Promotion of Sciencebased genome editing [4]. To confirm whether this assay worked in our hands, we first generated
plasmid constructs that target to the β isoform of actin, as a control. When we introduced the constructs into mouse brains, we successfully visualize endogenous β-actin localization via HA-tag immunostaining (Fig. 1). Next, we made constructs to insert an HA-tag to the N-terminus of Zdhhc18.
However, we failed to detect convincing HA signals in SLENDR-treated brains.
Because no detectable HA signals may indicate that the expression level of Zdhhc18 is too low
to detect by the SLENDR method, we then assessed the expression levels of zdhhc18 in several
brain areas of mice by quantitative RT-PCR. A reasonable zdhhc18 expression was found in the
hippocampus, neocortex, olfactory bulbs, and spinal cord of adult mice, suggesting that zdhhc18 may
play a role rather ubiquitously in the central nervous system. Encouraged by these expression data,
we are now trying signal amplification to enhance HA signals. Ongoing experiments are also aiming
to insert an HA tag into the C-terminus of Zdhhc18.
Aim2: Generation of zdhhc18 knockout mice
To generate a zdhhc18 knockout mouse line, we asked technical advice to a local expert, Professor
Masahiro Sato, who has developed an innovative method, called GONAD [4]. In the GONAD
method, fertilized eggs are electroporated with the cas9
protein and sgRNA for genome editing in vivo, without
taking the eggs out from the oviduct. The subject animals
give natural delivery and normally raise pups. This
procedure greatly reduces time, efforts as well as required
skills to obtain genetically modified living animals.
Using this method, we targeted deletion of most part of
Exon 3 and Exon 4 of the mouse zdhhc18 gene (Fig. 2).
After several pilot experiments to determine electropora- Fig. 2. Successful generation of zdhhc18
knockout mice by GONAD.
tion parameters, we successfully obtained nine pups fromfour dams (Fig. 2B). Out of nine, three pups turned out to be non-edited (i.e., wild type), but othersix animals appeared to have the expected deletion at least in one allele. This is highly efficient andtime-saving, given that no in vitro embryo manipulation is required. One mouse seems to be a homozygous knockout. No apparent behavioral abnormalities were found in mutated mice at this moment. Now we are crossing these mutant mice with wild-type mice to confirm germline transmission.
Future experiments we plan include histological and electrophysiological assessments as well as abattery of behavioral tasks to see any abnormal phenotypes in either heterozygous or homozygous
zdhhc18 knockout mice.

Discussion & Conclusion
With the generous support of the Novartis Research Grant, we have conducted SLENDR experiments in order to visualize subcellular localization of Zdhhc18 in mouse neurons. However, so far,
we are unable to see convincing signals. We will keep working on if we could visualize endogenous
zdhhc18. We also carried out GONAD-mediated zdhhc18 knockout in mice. This seems working
well. We will focus on assessing phenotypes of zdhhc18 knockout mice.
In our preliminary experiments, we observed that overexpression of zdhhc18 resulted in augmented complexity of neuronal morphology (Fig. 3). Mutations in zdhhc18 are associated with humanmental disorders [3]. Taken together, we believe that our research on zdhhc18 will contribute to un-

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derstanding yet unknown mechanisms underlying regulation of neuronal properties, and ultimately,
human mental activities.

Fig. 3. Overexpression of
zdhhc18 causes excessive
neurite growth.

References
1. Okuno H, Minatohara K, Bito H: Inverse synaptic tagging: an inactive synapse-specificmechanism to capture activity-induced Arc/arg3.1 and to locally regulate spatial distribution ofsynaptic weights. Semin Cell Dev Biol 77:43-50, 2018.
2. Fukata Y, Fukata M: Protein palmitoylation in neuronal development and synaptic plasticity. Nat
Rev Neurosci 11:161-175, 2010.
3. Zhao Y, He A, Zhu F et al: Integrating genome-wide association study and expression quantitative
trait locus study identifies multiple genes and gene sets associated with schizophrenia. Progress in
Neuropsychopharmco. Biol. Psychi. 81:51-54, 2018.
4. Mikuni T, Nishiyama J, Sun Ye, Kamasawa N, Yasuda R: High-throughput, high-resolution
mapping of protein localization in mammalian brain by in vivo genome editing. Cell 165: 18031817, 2016.
5. Takabayashi S, Aoshima T, Kabashima K, Aoto K, Ohtsuka M, Sato M: i-GONAD (improved genome editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genomeedited rats. Scientific Rep 8:12059, 2018.

一般の皆様へ
我々の認知機能は大脳神経細胞の活動によって担われています。神経細胞が活動すると多数の遺伝子の発現量が変化しますが、その役割は多くが不明です。本研究ではその一つ、
zdhhc18という遺伝子に注目しました。未知である zdhhc18の機能を知るため、ゲノム編集技術を応用した発生工学手法を用いて zdhhc18を欠損したマウスを作出することに成功しました。
今後はこのマウスの大脳や神経細胞の機能・形態異常の有無を調べます。本研究は認知症や精神疾患の治療薬や治療法の開発につながることが期待されます。

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Synthesis of Phytochrome-type Chromophores Based on Oxidative
Functionalization toward Investigation of their Functions
Yutaka Ukaji
ukaji@staff.kanazawa-u.ac.jp
Kanazawa University

Summary Abstract
In order to analyze the structure and function of chromophores in phytochrome, we focused on
the stereochemistry around the C15 position and studied the synthesis of non-natural tetrapyrrole
chromophores. The two-types, 15E-anti-locked and 15E-anti-fixed, sterically locked chromophores
were synthesized based on oxidative functionalization. In the former case, an oxidative
functionalization of C ring aldehyde by DDQ was realized and the following Horner-WadsworthEmmons (HWE) reaction could bind C and D ring. In the latter case, an oxidative functionalizationof CD ring component by using NBS was developed as a key reaction.
Key Words ： phytochrome, tetrapyrrole chromophore, photoconversion, sterically locked
chromophore, oxidative functionalization

Introduction
Phytochromes, photoreceptive chromoproteins, carry a covalently attached linear tetrapyrrole
and mediate developmental processes of plants through the photoconversion between the red lightabsorbing (Pr) and the far-red-light-absorbing (Pfr) forms. It is generally accepted that the first stepof photoconversion is isomerization from Z to E isomer at the C15 double bond between C andD rings. In order to elucidate the mechanism of photoconversion, the sterically locked 15E-anti
chromophore, which corresponds to physiologically active Pfr-form, was planed to be synthesized.
Furthermore, the synthesis of sterically fixed chromophores at the meso-position would be also
useful to explore the dynamic mechanisms of photoconversion.

Results
Initially, the sterically locked 15E-anti-chromophore, which corresponds to physiologically active
Pfr-form, was planned to be synthesized. The new convergent approach via coupling of CD ringusing HWE reaction was designed. In the precedent studies, 15E-anti chromophore incorporated
phycocyanobilin derivative was found to induce increased chlorophyll accumulation, modulationof gravitropism, and induction of side branches in darkness. First, a regioselective oxidative
functionalization of C-ring pyrrole with DDQ was achieved with controlling the three oxidation
states depending on the nucleophiles used. One of the oxidation products, an aldehyde obtained by
the use of MeOH as a nucleophile, could be converted to the one-carbon homologated aldehyde via
Wittig reaction followed by mild hydrolysis of the resulting vinyl ether using oxalyl chloride. To thecontrary, the D ring phosphite could be prepared in short steps procedure starting from commerciallyavailable butyronitrile and dichloroacetone although the previous scheme required many steps ofreactions which often caused the problems on reproducibility. The obtained C-ring aldehydes wereapplied to synthesis of a sterically locked 7- and 8-membered E-anti-CD-ring component. In the case

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The Novartis Foundation(Japan)
for the Promotion of Scienceof 7-membered E-anti-CD-ring component, HWE reaction with the C-ring aldehyde did not proceed
due to low electrophilicity. Then the C-ring aldehyde was protected by Boc group and subjected to
HWE reaction resulting a linkage between C- and D- ring. Subsequently, hydrogenation reaction of
the resulted double bond using palladium carbon, deesterification, formylation, and cyclization using
DBU were successfully carried out to give the 7-membered E-anti CD ring.
In the case of the 8-membered E-anti-CD-ring component, HWE reaction with the homologated
C-ring aldehyde to bind C and D ring proceeded smoothly without Boc protection. Following similartransformations gave 8-membered E-anti-CD-ring component. Finally, a coupling reaction wasachieved with AB ring moiety synthesized separately to afford a 7-membered 15E-anti biliverdin
derivative.

In contrast, the synthesis of sterically fixed chromophores at the meso-position could be crucial,
especially those with configurations and conformations separately locked in order to explore thedetailed dynamic mechanisms of photoconversion of the phytochrome chromophores. For thesynthesis of meso-fixed chromophores, regioselective introduction of an alkyl group at meso-position
was required. We realized an oxidative functionalization of CD ring component by using NBS toafford meso-brominated CD ring regioselectively. Next introduction of an sp3 alkyl chain at the mesoposition as a key step by the use of sp3 hybridized Grignard reagents was carried out. However, thechemical yields were not satisfactory. After several examinations, the desired substitution reaction by
Grignard reagents was improved by the addition of LiCl as an additive to produce the corresponding
E-meso-substituted CD-rings. The present substitution reaction was applied to synthesis a 15E-antifixed tetrapyrrole chromophore. Next, we investigated the cyclization reaction of the alkyl substituent
at the meso-position by the nitrogen atom of the C-ring using a base. When meso-alkylated productwas treated with KOt-Bu, an intramolecular SN2 reaction of the C-ring nitrogen atom to the alkyl
side chain provided the E-anti-fixed CD-ring component. The coupling reaction between the CDand AB-ring components was carried out under acidic conditions to afford the sterically 15E-antifixed tetrapyrrole derivative.

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Discussion & Conclusion
As mentioned above, two types of sterically locked teterapyrrole chromophore derivatives weresuccessfully synthesized based on oxidative functionalization. The synthesis of 8-membered 15E-anti
chromophore will be achieved soon via the coupling with AB-component. In order to pursue thefurther investigation of the stereochemistries and functions of phytochrome chromophores both invitro and in vivo via incorporation into apoprotein, the syntheses in larger scale and deprotection ofester moieties into free acids will be performed. Although we tentatively tried the deprotection underacidic conditions, the desired free acids were not obtained. Ester-exchange strategy from the resulted
propyl ester to allyl ester will be effective to deprotect smoothly under mild conditions.     References
Y. UkajiSynthesis of Sterically Locked Phytochrome Chromophores Based on Oxidative Functionalization,
INTERNATIONAL CONGRESS ON PURE & APPLIED CHEMISTRY (ICPAC Yangon 2019),
August 6, 2019 (Yangon, Myanmar).
R. Sekizawa, H. V. Mai, M. Sisakova, J. Shirakawa, T. Soeta, Y. Ukaji
Synthesis of 15E-anti Phytochrome Chromophore Derivatives, 27th International Society ofHeterocyclic Chemistry Congress (27th ISHC), September 5, 2019 (Kyoto).

一般の皆様へ
“ 植物の眼 ” ともいえる光受容色素タンパク質フィトクロムは，赤色領域の光吸収による立体構造変換により，植物の発芽や生長等の機能制御を行っている。しかし，発色団の立体構造と機能の関係解明はほとんど進んでいなかった。本研究では，「フィトクロム型発色団の化学合成」という独自のアプローチで解明に挑んだ。特にピロール化合物の酸化的官能基化という手法を基盤とした合成を通して，光無しでも発芽機能を発揮すると期待できる立体固定型発色団の基本骨格の合成に成功した。今後，合成した化合物をフィトクロム関連色素タンパク質へ取込みにより，光情報伝達機能解明と光無しでも発芽可能なシステム開発への発展が期待される。

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Development of a novel base editor
Nozomu Yachie
yachie@synbiol.rcast.u-tokyo.ac.jp
Research Center for Advanced Science and Technology, The University of Tokyo

Summary Abstract
We developed base editors that combine both cytosine and adenine base editing functions. A
codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a
Cas9 nickase (Target-ACEmax) showed a high median simultaneous C→T and A→G editing activity
at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax
were similar to those of existing single-function base editors.
Key Words：Genome editing; base editing; high-throughput sequencing; machine learning

Introduction
CRISPR-Cas9 has sparked a revolution in the field of genome editing. In the CRISPR-Cas9
system, a guide RNA (gRNA) recruits Cas9 to a target genomic region. Cas9 then produces a doublestranded DNA break (DSB) at the target region [1,2]. This promotes either gene deletion or transgene
insertion through the induction of different DNA repair pathways. However, there is still room for
improvement in the efficacy and precision of genome editing by Cas9. Furthermore, Cas9 has beenshown to cause cell toxicity due to the generation of DSBs.

Results
By tethering cytidine or adenosine deoxynucleosidedeaminase to a nuclease-deficient or nickase Cas9 (dCas9
or nCas9, respectively), new base editing tools have beendeveloped to induce efficient and direct base substitutionsin the genomic sequence. However, currently available
base editors enable only two transition mutations, C→T
and A→G, and have limited diversity of editing patterns
that they can generate at a target site. To this end, we developed a dual-function base editor Target-ACEmax whichenables simultaneous C→T and A→G on the gRNA targeting sequence (Figure 1). In brief, Target-ACEmax was
derived by fusing a cytidine deaminase PmCDA1 used in a
Fig. 1. Target-ACEmax
previously developed C→T base editor Target-AID [3] andan adenosine deaminase TadA used in a previously developed A→G base editor ABE [4] to nCas9.
To characterize the C→T and A→G base-editing activities of Target-ACEmax and other base editors, we analyzed the base-editing spectra at 47 genomic target sites in human embryonic kidney(HEK293Ta) cells by amplicon sequencing in triplicate (1,833 assays). By taking the average C→Tor A→G editing frequencies at each cytosine or adenine position relative to the PAM, we found thatthe dual-function base editor inherited similar base-editing characteristics from its corresponding

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single-function base editors. The C→T and A→G editing frequencies of 47 endogenous target sites
containing different numbers of cytosines and adenines varied widely. In summary, we found that
Target-ACEmax showed efficient coediting spectra around cytosine at –18 bp and adenine at –15 bp
relative to the PAM with peak heights of 19.2%. Using whole exome and transcriptome sequencing
technologies, we also analyzed off-target effects of Target-ACEmax that induces unwanted mutations
in non-targeting genomic regions and cellular RNA transcripts and found that the DNA/RNA offtarget effects of Target-ACEmax were on par with the previously developed single function base editors.
Similar to the recent machine learning approaches to predict wildtype Cas9-mediated genomeediting outcomes [5-7], we developed a base-editing prediction method that trains ampliconsequencing data and predicts base-editing patterns and their frequencies for a given target sequence.
In brief, we found that our method successfully predicted base-editing outcomes of untrained targets
with Pearson’s correlation coefficient of 0.70 for Target-ACEmax. We also demonstrated that theamplicon sequencing data obtained in this study were sufficient for the training procedure. Because
the machine learning method enabled prediction of multi-nucleotide coediting, we used it to predict
the frequencies of all possible codon conversion patterns in the human genome obtained by thedifferent base-editing methods. When bystander mutations were not allowed to occur, this analysis
showed that Target-ACEmaxhad the highest potentials for diversifying genomic codons. We thenrepeated the same analysis by allowing bystander mutations to occur and estimated bystander risks ofgenerating unwanted mutations for all of the base-editing methods. The bystander mutation risks ofTarget-ACEmax and ACBEmax were within the risk range of commonly used single-function baseeditors of which BE4max was the highest. Finally, our model predicted that Target-ACEmax had thehighest potentials to correct pairs of heterologous disease mutations reported in the ClinVar databaseas a single base editor enzyme.

Discussion & Conclusion
This new genome-editing tool would greatly expand the potential of alternating the genomic DNA
sequence for therapeutics and fundamental biomedical researches. For example, Target-ACEmax
could be applied as a complementary tool for in vivo diversification of targeted sequences formutational scanning analysis of protein functions and directed protein evolution as examples. TargetACEmax could also be a powerful tool for recent cell lineage tracing using CRISPR genome editing[8]. Most of the current implementations employ wildtype Cas9 that induces DSBs. These DSBs
result in cytotoxicity and rapidly saturate the mutation patterns in DNA barcodes following target
site deletions, which theoretically limit the resolution of cell lineage reconstruction [9]. While baseeditors could minimize these detrimental effects caused by DSBs, unidirectional mutations inducedby single-function base editors would also cause saturation in the diversity of mutated DNA barcode
patterns. Target-ACEmax could alleviate this saturation issue because of its reversible C•G↔T•A
activity and contribute to high-resolution cell lineage tracing.

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References[1] Mali, P. et al. Science 339, 823-826 (2013)[2] Cong, L. et al. Science 339, 819-823 (2013)[3] Nishida, K. et al. Science 353, aaf8729 (2016)[4] Gaudelli, N.M. et al. Nature 551, 464-471 (2017)[5] Shen, M.W. et al. Nature 563, 646-651 (2018)[6] Allen, F. et al. Nat Biotechnol, 64-72 (2018)[7] Chen, W. et al. Nucleic Acids Res, gkz487 (2019)[8] Masuyama, N., Mori, H. & Yachie, N. Curr Opin Chem Biol 52, 63-71 (2019)[9] Salvador-Martinez, I., Grillo, M., Averof, M. & Telford, M.J. Elife 8 (2019)

一般の皆様へ近年、細胞内ゲノム DNA 中の特定の狙った配列を自在に編集することができるゲノム編集技術が急速に発展しており、農業、医療分野を含めた生物学分野全体に大きな変革をもたらしつつあります。これまでに、CRISPR–Cas9というゲノム編集ツールや、DNA 配列を編集する塩基編集ツールが開発されてきました。これらは精密なゲノム編集技術として注目を集める一方、ゲノムに書き込まれた生命プログラムを編集するという点においては、その自由度が限られていました。本研究では狙った DNA 配列の C → T および A → G の異種塩基置換を同時に達成できる新たな塩基編集ツール「Target-ACEmax」の開発に成功しました。本新規ゲノム編集ツー
ルは、様々な細胞においてより多様な塩基編集を可能にし、品種改良、遺伝子治療、動物の発生における細胞系譜の追跡など、様々な分野において幅広い応用が期待されます。

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Study on the circadian rhythm system regulated byintracellular carbon monoxide
Hiroaki Kitagishi
hkitagis@mail.doshisha.ac.jp
Department of Molecular Chemistry and Biochemistry, Doshisha University

Summary Abstract
Circadian rhythms are regulated by transcription-translation feedback loops (TTFL) of clock
genes. Previous studies have demonstrated that core transcriptional factors, NPAS2 and CLOCK,in the TTFL can reversibly bind carbon monoxide (CO) in vitro. However, little is known about
whether endogenous CO, which is continuously produced during a heme metabolic process, isinvolved in the circadian system. Here we show that selective removal of endogenous CO in mice
considerably disrupts rhythmic expression of the clock genes.
A highly selective CO scavenger, hemoCD1, which is a supramolecular complex of an iron(II)porphyrin with a per-O-methyl-β-cyclodextrin dimer, was used to remove endogenous CO in mice.
Intraperitoneal administration of hemoCD1 to mice immediately reduced the amount of internal CO.
The removal of CO promoted the bindings of NPAS2 and CLOCK to DNA (E-box) in the murine
liver, resulting in up-regulation of the E-box-controlled clock genes. Within 3 h after the administration, most hemoCD1 in mice was excreted in the urine, and heme oxygenase-1 (HO-1) was graduallyinduced in the liver. Increased endogenous CO production due to the overexpression of HO-1 caused
dissociation of NPAS2 and CLOCK from E-box, which in turn induced down-regulation of the clock
genes. The down-regulation continued over 12 h even after the internal CO level recovered to normal. The late down-regulation was ascribed to an inflammatory response caused by the endogenous
CO reduction. The CO pseudo-knockdown experiments provided the clear evidence that endogenous
CO contributes to regulation in the mammalian circadian clock.
Key Words：carbon monoxide, circadian clock, heme

Introduction
The circadian rhythm is a naturally occurring day-and-night oscillation system that controls
physiological and behavioral cycles, and is regulated in almost all cells [1]. This system involves
a transcription-translation feedback loop for the rhythmical expression of clock components withapproximately 24 h cycles[1]. CLOCK and NPAS2 are the transcriptional factors that play a central
role in the circadian rhythm regulation[1,2]. Both proteins form heterodimers with BMAL1, andbind to a specific DNA sequence called E-box. Transcription of clock genes, such as periods (Per)and cryptochromes (Cry), is enhanced by these heterodimers. The translated PER and CRY proteins
form a heterodimer, which acts on BMAL1:CLOCK(NPAS2) to repress the transcription of Per andCry. PER and CRY are gradually degraded by the ubiquitin proteasome pathway during the night(or the day in nocturnal animals), and then transcription of Per and Cry restarts, thus completing the
feedback loop.
In 2002, Dioum et al. demonstrated that the BMAL1:NPAS2 heterodimer loses its DNA-binding
function in the presence of carbon monoxide (CO) [3]. NPAS2 binds heme as a prosthetic group thatfunctions as the CO-sensing site. The CLOCK protein also possesses a heme-based sensing function

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toward diatomic gases such as CO. Thus, it has been assumed that the circadian clock system might
be affected by internal CO that is generated as an endogenous product of heme oxygenase (HO)catalyzed heme degradation.
In this study, we show that selective removal of endogenous CO in mice significantly affects theexpression levels of the E-box-controlled clock genes in the murine liver. We utilized hemoCD1,
a highly selective CO scavenging agent working in aqueous media. HemoCD1 is a very stable
1:1 supramolecular inclusion complex comprised of 5,10,15,20-tetrakis(4-sulfonatophenyl)
porphinatoiron(II) (Fe IITPPS) encapsulated by a per-O-methylated β-cyclodextrin dimer witha pyridine ligand (Py3CD). Our group has extensively studied hemoCD1 as a water-soluble
hemoprotein model compound. Similar to native hemoglobin and myoglobin, hemoCD1 reversibly
binds oxygen (O2) and CO in aqueous solutions at ambient temperature. It is noteworthy that the CObinding affinity of hemoCD1 is extremely high (Kd = 0.02 nM at 25°C), approximately 100 times
higher than that of hemoglobin in the R-state in aqueous solutions, whereas the O2 binding affinityof hemoCD1 is moderate and close to that of hemoglobin in the T-state. Using hemoCD1, it become
possible to study of the effects of endogenous CO on the circadian clock system. Here, the pseudoknockdown study for CO in vivo provides the clear experimental evidence that endogenous CO
contributes to regulation of the mammalian circadian clock through acting on NPAS2 and CLOCK
and modulating the clock genes related to inflammatory responses.

Results
The solution containing hemoCD1 (1.0 mM) in phosphate buffer saline (PBS, 0.15 mL) was
intraperitoneally (i.p.) administered to mice at circadian time (CT) 7.5. The mice were then housed
under the controlled conditions. The amount of hemoCD1 administered was sufficient to remove
endogenous CO in mice, and CO was excreted in the urine as the CO-bound hemoCD1 within 3 h.
After the administration, the expression levels of the clock genes, Per1, Per2, Cry1, and Cry2, were
measured using real-time PCR at different time points. The buffer (PBS) and the free-base complexof hemoCD1 (Fb-hemoCD1) were independently administered to mice for the negative controls. The
time profile for Per1 mRNA was drastically altered in the liver of hemoCD1-treated mice.
To identify the mechanism causing the circadian rhythm disruption observed in the COdepleted mice, we divided the mRNA expression profiles into four phases. In the first phase (CT7.5–9.5), the administration of hemoCD1 caused higher clock gene mRNA levels and reduced
levels of endogenous CO. These results can be explained in terms of the CO-responsive function of
NPAS2, i.e., the binding of the BMAL1:NPAS2 heterodimer to E-box is enhanced under low COconcentrations. In the second phase (CT 9.5–12.5), the amount of endogenous CO in the liver ofhemoCD1-treated mice was inversely increased. We have previously shown that endogenous CO inmice is quickly produced by inducing HO-1 when endogenous CO is depleted by hemoCD1. HO-1
protein expression was strongly induced in the hemoCD1-treated mice, resulting in the accelerationof endogenous CO production. The increase of endogenous CO might cause down-regulation of theclock genes in the second phase. We used chromatin immunoprecipitation (ChIP) analysis with antiNPAS2 and anti-CLOCK antibodies to quantify the amount of DNA bound by BMAL1:NPAS2 andBMAL1:CLOCK in the murine liver. Indeed, the changes in endogenous CO levels correlated wellwith the quantities of DNA bound by these proteins. The expressions of Npas2 and Clock mRNA
were unaffected by the administration of hemoCD1. Therefore, the significant increase and decreaseof the DNA/protein complexes detected in the ChIP assay indicate that the DNA-binding abilities of
the BMAL1:CLOCK(NPAS2) heterodimers are affected by internal CO levels in vivo.

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Discussion & Conclusion
In conclusion, based on the pseudo-knockdown strategy for CO, we identified the contributionof endogenous CO to regulation of the circadian clock in vivo. Temporal reduction of endogenous
CO in mice by hemoCD1 significantly affected the circadian rhythms of the E-box-controlled clock
genes. The mechanistic study suggested that the CO-dependent transcriptional activity changesof NPAS2 and CLOCK and the subsequent inflammatory response to produce TNF-α were both
responsible for the CO-removal-induced circadian rhythm disruption. In principle, selective depletionof small biomolecules, such as gaseous signaling molecules, by genetic/pharmacological methods
should not be possible without any side effects. We believe that, as demonstrated in this work, thepseudo-knockdown approach based on the highly selective molecular recognition by synthetic
compounds will help to clarify the roles and functions of such the small molecules in biologicalsystems.

References[1] Reviews on the molecular mechanism of circadian clock system: a) S. M. Reppert, D. R. Weaver,Nature, 2002, 418, 935–941; b) U. Albrecht, Neuron, 2012, 74, 246–260; c) E. D. Buhr, J. S.
Takahashi, Handb. Exp. Pharmacol. 2013, 217, 3–27; d) A. Videnovic, A. S. Lazar, R. A. Barker,
S. Overeem, Nat. Rev. Neurol. 2014, 10, 683–693.
[2] The overlapping role of CLOCK and NPAS2 in the circadian clock regulation: a) J. P. DeBruyne,
D. R. Weaver, S. M. Reppert, Nat. Neurosci. 2007, 10, 543–545; b) J. P. Debruyne, J. Genet.
2008, 87, 437–446; c) C. Bertolucci, N. Cavallari, I. Colognesi, J. Aguzzi, Z. Chen, P. Caruso, A.
Foá, G. Tosini, F. Bernardi, M. Pinotti, Mol. Cell. Biol. 2008, 28, 3070–3075.
[3] E. M. Dioum, J. Rutter, J. R. Tuckerman, G. Gonzalez, M.–A. Gilles-Gonzalez, S. L. McKnight,
Science, 2002, 298, 2385–2387.
[4] H. Kitagishi, In Vivo Reactions Triggered by Selective Removal of Endogenous Carbon
Monoxide in Blood Using an Artificial Hemoprotein Model Complex”, Artificial Blood, 27, 44–
52 (2019) (written in Japanese).

一般の皆様へ
本研究では，すべての生物が持つ体内時計システムにおいて，有毒ガスとして知られる一酸

化炭素が生体内において役立つ機能を持つことを実験的に証明した。一酸化炭素は生体内において常に微量ずつ合成されており，何らかの生理機能を持つことが提唱されてきたが，その機能の全容は未解明であった。今回，我々は生体内において合成された一酸化炭素を選択的に除去するオリジナルの試薬 hemoCD1をつかって，動物体内の一酸化炭素を除去したときに起きる体内時計システムの変化について検討を行ったところ，体内時計の制御システムが大きく乱されることを明らかとした。生体内一酸化炭素の新たな生理機能の側面を明らかにした世界で初めての成果が得られた。

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Age-related endocrine disruption of skin leads to senescent alopecia
Shogo Haraguchi
shogo.haraguchi@gmail.com
Showa University School of Medicine

Summary Abstract
With advancing age, circulating androgen levels gradually decline in men due to the deregulationof testicular function. However, the age-associated changes in the cutaneous steroidogenic system
due to the declining sex steroid levels are unknown. Our findings revealed that cutaneous testosterone
levels increased with advancing age, and disrupted zinc homeostasis, thereby disrupting hair follicle
stem cell functions and causing age-associated diffuse hair loss.   
Key Words：Sex steroid, Zinc, Hair loss, Aging, Hair follicle stem cell

Introduction
As the receptors for sex steroids are expressed in the skin, the skin is considered a target organ
for gonadal sex steroids (1,2). However, the skin expresses various steroidogenic enzymes, and iscapable of de novo production of sex steroids (3,4). Thus, the skin is not only a target site of gonadal
sex steroids, but also functions as a local steroidogenic organ.
Circulating androgen levels gradually decline with advancing age in men due to the deregulationof testicular function. However, the age-associated changes in cutaneous steroidogenesis remain
unclear.

Results
To determine the age-associated changes in skin steroidogenesis, we first analyzed the changes in
the levels of sex steroids in young (3 months) and aged (20-22 months) male mice. We demonstrated
that cutaneous testosterone levels were higher in aged mice than in young mice. Nevertheless, serum
and testicular testosterone levels were reduced further in aged mice than in young mice. Testosterone
was mainly localized in the dermis of aged mice, wherein the highest intensity of testosterone wasdistributed in the area adjacent to the hair follicles. Then, we demonstrated that the localization of
Hsd17b3 in the sebaceous glands is responsible for the upregulation of cutaneous testosterone levels
in aged mice. Topical application of sex steroids on the back skin of aged mice was performed for 1
month. Estradiol decreased Hsd17b3 mRNA expression in aged skin after 1 month of treatment (Fig.
1). Furthermore, inhibition of cutaneous testosterone synthesis upon treatment with TC-HSD-21, an
inhibitor of HSD17B3, increased the hair length in aged mice. We subsequently demonstrated thatthe suppression of hair length by elevated testosterone levels was mediated by zinc transporter Zrt/
Irt-like protein 9 (ZIP9), but not by androgen receptor (AR), localized in the Golgi of hair follicle
stem cells of aged mice. ZIP9 was also expressed in hair follicle stem cells derived from head skinin aged mice. In addition, we demonstrated that high testosterone levels induced apoptosis via ZIP9mediated mechanisms in mouse hair follicle stem cells. Elevated testosterone also disrupted theendoplasmic reticulum resident protein 44 (ERp44)-dependent protein quality control system by
disrupting zinc homeostasis. It then induced misfolding of key proteins associated with hair follicle
stem cells and their niche functions.

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Discussion & Conclusion
This study shows that cutaneous testosterone levels increased in the skin with advancing age.
Elevated testosterone levels induced hair follicle stem cell apoptosis via ZIP9. This in turn disrupted
the protein quality control mechanism in hair follicle stem cells by disrupting ERp44 function
through ZIP9-mediated mechanisms. These results can be used to develop novel treatment strategies
based on the regulation of sex steroid levels, HSD17B3 inhibition, zinc supplementation, and theirdownstream pathways. This could lead to improvements in the clinical treatment of age-associateddiffuse hair loss, known as senescent alopecia.     References
1. Choudhry R, Hodgins MB, Van der Kwast TH, Brinkmann AO, Boersma WJ. Localization ofandrogen receptors in human skin by immunohistochemistry: implications for the hormonal
regulation of hair growth, sebaceous glands and sweat glands. J. Endocrinol. 133, 467-475 (1992).
2. Inoue T, Miki Y, Kakuo S, Hachiya A, Kitahara T, Aiba S, Zouboulis CC, Sasano H. Expression ofsteroidogenic enzymes in human sebaceous glands. J. Endocrinol. 222, 301-312 (2014).
3. Chen W, Thiboutot D, Zouboulis CC. Cutaneous androgen metabolism: basic research and clinicalperspectives. J. Invest. Dermatol. 119, 992-1007 (2002).
4. Thiboutot D, Jabara S, McAllister JM, Sivarajah A, Gilliland K, Cong Z, Clawson G. Human skinis a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal
sebocytes, and an immortalized sebocyte cell line (SEB-1). J. Invest. Dermatol. 120, 905-914(2003).

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一般の皆様へ
年齢が進むにつれて精巣や卵巣などの性腺機能が低下し、性ホルモンの血中濃度は減少します。男性ホルモン・女性ホルモンと言われることで、男性には男性ホルモンしかなく、女性には女性ホルモンのみと誤解されている場合も多いですが、Fig. 1に示しているように、そもそも

男性ホルモン・テストステロンは女性ホルモン・エストラジオールを合成する材料にもなり、
男女ともに男性ホルモン・女性ホルモンを体内で合成しています。

本研究では、60歳以降で見られるびまん性（頭部全体に広がる）の脱毛の発症の一因に、性腺機能低下が引き金となって皮膚局所に存在する性ホルモン合成系が乱されてしまう点にあることを解明しました。

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The RNA-based mechanism underlying pathophysiology
in the neurological diseases
Takatoshi IIIJIMA., Ph.D.
takatoshi.iijima@tokai-u.jp
Department of Molecular Life Science, Division of Basic Medical Science and Molecular
Medicine, School of Medicine, Tokai University

Summary Abstract
It has been revealed that dysregulation of alternative pre-mRNA splicing is implicated in several
neurological disorders. Our project aims to understand the neuronal role of alternative splicing to understand the neuronal diseases.
Key Words：Alternative splicing, brain, neurodevelopmental disorder, SAM68, 3’UTR

Introduction
Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity
from a single gene. In the central nervous system (CNS), key neural developmental steps are thought
to be controlled by alternative splicing decisions, including the molecular diversity underlying
synaptic wiring, plasticity, and remodeling. Notably, there is increasing evidence that implicates thedysregulation of neuronal splicing events in several neurological disorders. Therefore, we aim tounderstand the detailed mechanisms of neuronal alternative splicing in the mammalian CNS which
could provide plausible treatment strategies for these diseases.

Results
Characterization of SAM68/SLM1-dependent alternative splicing programs
We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal
alternative splicing in the nervous system. Knockout mice of SAM68, SLM1, and SLM2 exhibit
several morphological and functional defects in adult brains (Ehrmann et al., 2016; Iijima et al.,2014; Iijima et al., 2011; Lukong and Richard, 2008; Traunmuller et al., 2016)., which could be
implicated in several neurodevelopmental disorders. However, the whole aspect of alternative
splicing programs by STARs remains unclear.
Here, we performed a transcriptomic analysis using SAM68 knockout and SAM68/SLM1 double
knockout midbrains. Exon array and RNA-seq analyses revealed different alternative splicing
activity between SAM68 and SLM1; SAM68 preferentially targets alternative 3’UTR exons. SAM68
knockout causes a long-to-short isoform switch of a number of neuronal targets through the alteredselection in alternative 3’UTR exon or alternative polyadenylation. The altered 3’UTR selection of
a novel target, interleukin 1-receptor accessory protein (Il1rap), results in remarkable conversion
from a membrane-bound to a secreted type in Sam68 KO brains, not in Slm1 KO ones, suggesting that
SAM68-specific splicing program is highly involved in alternative choice of 3’UTR exons.

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Proper 3’UTR selection by SAM68 has a critical role in neuronal function
We then examined neuronal effect of aberrant ALE of Il1rap occurring in Sam68 KO brains. It
has been known that IL1RAP is one of the synaptic organizers. We showed that soluble IL1RAP
influences synaptogenic signaling through trans-synaptic IL1RAP-PTPδ interaction. We also
demonstrated that soluble IL1RAP disturbs IL-1-induced Ca2+ influx mediated through NMDA-R
activation. These data suggest that proper SAM68-specific splicing program has a critical effect onsynaptic organization and plasticity.
Tissue-specificity of SAM68 expression determines 3’UTR selection in spatial fashion
We observed that expression of SAM68 exhibited a tissue-specific pattern; SAM68 was highly
expressed in the brain and lung, but not in the liver and spleen. Importantly, 3’UTR selection wascorrelated to the SAM68 expression level. While the brain expressed membrane-bounded IL1RAP,
liver expressed soluble one. Thus, these results demonstrate that SAM68 expression properly
determines 3’UTR isoforms of the targeted transcripts.

Discussion & Conclusion
We showed that neuronal alternative splicing by STAR family proteins is an important mechanism
for functional diversification. We conducted transcriptomic analyses using Slm1 KO and Sam68/
Slm1 DKO brains, and showed a different splicing activity between SAM68 and SLM1. We mainly
demonstrated the neuronal isoform selection in 3’UTR by SAM68 through alternative 3’UTR
selection of Il1rap, a novel target for SAM68. This study further suggests that proper usage of 3’UTR
exons by SAM68-specific splicing is critical for both aspects of synaptic organization and plasticity
in the CNS.
Mutation of Il1rap gene is implicated in intellectual disability and autism. Interestingly,
not only Il1rap, we noticed that targeted transcripts for SAM68 included the risk genes forneurodevelopmental disorders. Thus, the further uncovering of SAM68-mediated splicing
mechanism may provide a profound insight into the pathophysiology of these diseases.

References
1. De Frusco et al., Characterization of Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1
is a Src substrate during mitosis. PNAS (1999)
2. Ehrmann et al., The tissue-specific RNA binding protein T-STAR controls regional splicing
patterns of neurexin pre-mRNAs in the brain. PLoS Genet. (2016)
3. Iijima et al., SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1. Cell
(2011)
4. Iijima et al., Neuronal cell type-specific alternative splicing is regulated by the KH domain protein
SLM1. J. Cell Biol. (2014)

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一般の皆様へ
自閉症をはじめとした脳発達障害や精神疾患などでは RNA スプライシングの異常が見つかっていますが、これらの病気の発症や病態との因果関係はまだはっきりとしていません。本

研究では神経系における時空間的な RNA スプライシング機構の解明をメインに研究を行い、SAM68と呼ばれる神経系スプライシング因子をノックアウトした動物では、IL1RAP と呼ばれる神経分子のスプライシング異常を起因とした神経障害が現れることが示されました。今後このようなスプライシング異常の解明が脳の病気の理解の鍵に繋がることが大いに期待できます。

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Elucidation of the molecular basis and physiological significance ofautophagic degradation of the nuclear pore complex
Hitoshi Nakatogawa
hnakatogawa@bio.titech.ac.jp
Tokyo Institute of Technology

Summary Abstract
In this study, we found that the nuclear pore complex and the components nucleoporins are
selectively degraded by autophagy in yeast when Tor kinase complex 1 is inactivated by nutrient
starvation or the specific inhibitor rapamycin.
Key Words：autophagy, nuclear pore complex, nucleoporin, TORC1, nutrient starvation

Introduction
Autophagy is a major degradation system in eukaryotic cells, in which degradation targets aresequestered within the double-membrane vesicles autophagosomes and transported into lysosomes
in mammals or vacuoles in yeast and plants. Previous studies have revealed that a wide range ofcellular materials, including abnormal proteins, damaged mitochondria, and excess peroxisomes,
are selectively degraded by autophagy, and that the removal of these harmful materials by selective
autophagy is important for preventing a number of human diseases. The nuclear pore complex (NPC)
is a huge protein complex embedded in the nuclear envelope, and mediates transport between the
nucleus and cytoplasm. Previous studies have elucidated the detailed mechanism of the biogenesis of
the NPC, but that of its degradation remained poorly understood.

Results
In this study, we began with examining the possibility that autophagy is involved in degradation of
the NPC by performing immunoblotting analysis and fluorescence microscopy. Indeed, we found that
the NPC is degraded via autophagy in the budding yeast Saccharomyces cerevisiae, when this yeast
is subjected to nitrogen starvation or treated with an inhibitor of Tor kinase complex 1 (TORC1),
rapamycin, which causes cell cycle arrest at G1 phase, and induces different types of autophagy,
including non-selective autophagy and selective autophagy of mitochondria, peroxisomes, theendoplasmic reticulum, and nuclear components. Immunoelectron microscopy revealed that the
NPC is loaded into double-membrane vesicles budded from the nuclear envelope, which areenclosed within autophagosomes. Our results suggested that the NPC is degraded by a selective
type of autophagy. In selective autophagy, degradation targets are recognized by proteins called
autophagy receptors, which interact with Atg11/FIP200 that initiates autophagosome formation on
the targets and also binds to Atg8 family proteins on forming autophagosomal membranes, leading
to the efficient sequestration of the targets into autophagosomes. We showed that ATG11 knockout
or mutations in the Atg8 region responsible for binding to autophagy receptors severely impaired
NPC degradation. We previously reported that the autophagy receptor Atg39 localizes to the outer
nuclear membrane and induces autophagic degradation of parts of the nucleus as nuclear envelopederived double-membrane vesicles (nucleophagy). Given our immunoelectron microscopy resultsthat the NPC is degraded as a component of double-membrane vesicles similar to those degraded via

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Atg39-mediated nucleophagy, we expected that autophagy of the NPC depends on Atg39. However,
ATG39 knockout only partially affected NPC degradation. In addition, knockout of genes encoding
previously described autophagy receptors did not affect NPC degradation. These results suggested
that the NPC is mainly degraded by selective autophagy mediated by an unknown autophagy
receptor. Immunoelectron microscopy detected autophagosomes encapsulating double-membrane
vesicles containing the NPC in ATG39 knockout cells, suggesting that an unknown receptormediated mechanism also induces the autophagic sequestration of the NPC loaded into doublemembrane vesicles generated from the nuclear envelope.
Several targets for selective autophagy are known to directly interact with Atg8 family proteins.
In immunoprecipitation analysis, the NPC was coprecipitated with Atg8. We noticed that three
nucleoporins contain putative Atg8 family interacting motifs (AIMs), and showed that introducing
mutations at a motif in Nup159 abolished the interaction of the NPC with Atg8. These mutations
significantly reduced degradation of Nup159 itself but not that of other nucleoporins, suggestingthat Nup159 not being assembled into the NPC is selectively degraded by autophagy via its directinteraction with Atg8.
In this study, we also investigated the physiological role for NPC degradation by autophagy. A
previous study reported that disruption of NUP116 destabilizes the NPC. We found that degradationof the NPC was promoted in NUP116 knockout cells, suggesting that autophagic degradation of the
NPC is involved in its quality control under TORC1-inactivating conditions.

Discussion & Conclusion
In this study, we provided the first evidence for degradation of the whole NPC and unassembled
Nups by autophagy, which we termed “NPC-phagy” and “nucleoporinophagy”, respectively. In
S. cerevisiae cells, these pathways are triggered when TORC1 activity is attenuated. Our results
suggested that an unknown autophagy receptor mediates NPC degradation. Although we could
not identify such a receptor in this study, we also found that the nucleoporin Nup159 not being
assembled into the NPC directly interacts with Atg8 and degraded by autophagy.
NPC-phagy/nucleoporinophagy can contribute to the quality control of the NPC/nucleoporins
under TORC1-inactivated conditions. The NPC is involved in the replicative life span of yeast. In
addition, a deficiency in the NPC is linked to a number of human diseases and aging. This study
provides a foundation for understanding NPC-phagy and nucleoporinophagy, which will trigger themolecular mechanisms and physiological/pathological significance of these new autophagy pathways
in various organisms.

References
Tomioka Y, Kotani T, Kirisako H, Oikawa Y, Kimura Y, Hirano H, Ohsumi Y, Nakatogawa H.
(2020) TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes. J. Cell
Biol. 219 (7): e201910063.

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一般の皆様へオートファジーは、2016年のノーベル賞の受賞対象となった生命現象ですが、その分子機構及び生理機能にはまだ多くの謎が残されています。また、近年、神経疾患などの病気との直接的関連から、細胞内の特定の成分のオートファジーによる選択的分解が特に注目を集めています。本研究では、モデル生物である出芽酵母を用いて、核膜孔複合体という巨大なタンパク質複合体がオートファジーで選択的に分解されることを明らかにしました。本研究によって得られた成果は、ヒトを含めた他の生物における研究の端緒となり、核膜孔複合体が関与する疾患や老化への対処療法を開発するための基盤情報となることが期待されます。

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Comprehensive understanding of intracellular innate immune response
in chronic hepatitis B reactivation
Takayuki Abe
atakayu@med.kobe-u.ac.jp
Kobe University Graduate School of Medicine

Summary Abstract
The patients with co-infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) are at a highrisk of developing liver cirrhosis and hepatocellular carcinoma. However, it has not been still clear
how the reactivation of HBV occurs in the persistent HCV infection in the liver, and following to the
induction of acute liver failure. To clarify this issue, we attempted to the establishment of novel cell
culture model for HCV/HBV co-infection. Our results indicated that HBV reactivation was occurredin the HCV persistent replicating cells under the treatment of HCV specific inhibitor. As such, theperformance of our proposal experiments would be promising the rational for understanding themechanism of HBV reactivation and acute hepatic failure in chronic HCV/HBV patients.
Key Words：Hepatitis B virus (HBV), Hepatitis C virus (HCV), reactivation, Sodium taurocholate
cotransporting polypeptide (NTCP), Direct acting antiviral (DAA)

Introduction
Direct acting antiviral (DAA) therapy against chronic hepatitis C virus (HCV) are highly effective,
although it has been raised the possibility of an emergency of the drug-resistant viruses in addition
to the implication of virus free-mediated liver pathogenesis following to the viral elimination fromthe liver. On the other hand, hepatitis B virus (HBV) infection usually treated with inhibitors of
nucleotides analogues. However, an eradication of HBV from the infected liver is extremely difficult,because covalently closed circular DNA (cccDNA) remains in the nucleus and is difficult to eliminate
from the infected cells. Recent another concerning is that HBV reactivation in patients with chronic
HCV infection treated with DAAs has been emerged (1). The patients with co-infection of HBVand HCV are at a high risk of developing liver cirrhosis and hepatocellular carcinoma. However, it
has not been still clear how the reactivation of HBV occurs in the persistent HCV infection in theliver, and following to the induction of acute liver failure. This concerning is an urgent issue to besolved in the current situation where interferon (IFN)-free treatment guidelines are mainstream to thechronic HCV infection. The study of molecular mechanism of HBV reactivation is to be stagnant due
to a lacking of the general versatile HBV and HCV co-infection system in vitro.

Results
The HCV sub-genomic replicon (SGR) cells is one of cell culture model for the persistent HCV
replication (2). To investigate the effect of HCV/HBV co-infection in cell culture, we tried toestablish the HCV SGR cells stably expressing Sodium taurocholate cotransporting polypeptide(NTCP) gene, which was identified as a primary HBV entry receptor (3). The expression plasmid
encoding the C-terminal Myc-tagged NTCP was transduced into the HCV SGR cells and was
following to the treatment of puromycin for 3 weeks. The immunoblot analysis using Myc- or NTCP-The Novartis Foundation(Japan)
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specific antibodies indicated that the comparable expressionof NTCP were able to detect in the two independent SGRNTCP cell clones (referred to as SGR-NTCP-myc #1 and #2),
while the expression of HCV proteins such as NS3 and NS5A
were only observed in the SGR-NTCP-myc #1 (Fig.1). The
SGR-NTCP-myc #2 cells showed the undetectable of HCV
proteins due to an unknown reason. Thus, we decided to use#1 cells after following experiments.
Next, to verify the infectivity of HBV on the SGR-NTCPmyc #1 cells, we infected the cells with the recombinant HBVexpressing Nano-Luc (NL) reporter gene (referred to as HBVNL), followed by the measurement of NL activity. As shownin Fig.2A, the high level of NL activity was observed in the
SGR-NTCP-myc #1 cells at the indicated time points (Day-3,
4, and 5 post-infection) compared to that of parental cells (HCV-SGR cells, which is undetectable an
endogenous NTCP expression). This result indicates that HBV-NL can effectively infect in the SGRNTCP-myc #1 cells in a NTCP-dependent manner.
Recent concerning is that HBV reactivation in patients with chronic HCV infection treated with
Direct acting antiviral (DAA) has been emerged, although it remains to be clarified the molecularmechanism. One possibility issuggested that the host innate
immune response might beinvolved in the modulation of
HBV persistent replication. To
investigate the effect of DAAs
on the HBV reactivation with
HCV/HBV co-infection, we
treated the cells with daclatasvir(DCV), which is known as
one of DAA against NS5A
p r o t e i n . I n t e r e s t i n g l y, t h e
HBV-NL activity was shown
to be enhanced in the SGRNTCP cells treated with DCV compared with DMSO treatment (Fig.2B). We also observed theenhancement of NL activity when cells were treated with telaprevir (TPV), which is known as one
of DAA against NS3 protease. These results indicate that the treatment of DAAs may be caused theinduction of HBV reactivation through the HCV clearance in the HCV/HBV replicating cells.
Collectively, these results indicate that the treatment of DAA may cause the induction of HBVreactivation in the HCV/HBV co-infected cells.

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Discussion & Conclusion
In the present study, we established a novel cell culture model for HCV/HBV co-infection.
Our present findings demonstrated that the treatment of DAA may cause the induction of HBVreactivation in the HCV/HBV co-infected cells. Further studies will be needed to clarify themolecular mechanism of HBV reactivation and the development of novel anti-HCV/HBV agentsusing in this cell culture model.

References
1) Pisaturo M et. al., Hepatitis B virus (HBV) reactivation following pharmacological eradication ofhepatitis C virus (HCV). Viruses, 13, 2019 : pii:E850.
2) Lohmann V et. al., Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
Science, 285, 1999 : 110-113.
3) Yan H et. al., Sodium taurocholate cotransporting polypeptide is a functional receptor for humanhepatitis B and D virus. Elife, 13 : eLife.00049.

一般の皆様へ
C 型肝炎ウイルス (HCV) に対する直接作用型の抗ウイルス剤 (Direct Acting Antivirals: DAAs)の開発により、HCV の治療成績の飛躍的向上が期待されている。しかしながら、近年、DAA 製剤の投与による HCV の治療後に、B 型肝炎ウイルス (HBV) の再活性化及び再燃に伴う劇症肝炎の死亡例が報告されているが、その詳細な分子機序は明らかとされていない。この事例は、インターフェロンフリーの治療指針が主流となっている現況においては、解決すべき喫緊の課題である。これまでの HCV 培養系の確立や、近年の HBV 感染受容体の同定に伴い、HCV 及びHBV の個々の感染生活環の概要が明らかにされつつあるが、HCV/HBV 重複感染モデルにおける知見は乏しい研究背景にある。本研究の成果は、肝炎ウイルスの感染生活環を支持する主要な宿主因子及び様々な肝細胞培養系を組み合わせることで、汎用性の高い新たな HCV/HBV 共
培養系を確立できたことにある。今後、この実験系を用いることで、HBV の再活性化及び再燃に伴う劇症肝炎の分子機序の解明に役立つことが期待される。

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Identification of glycosyltransferases involved in biosynthesis of
pectin in plant cell wall
Takeshi Ishimizu
ishimizu@fc.ritsumei.ac.jp
College of Life Sciences, Ritsumeikan University

Summary Abstract
To elucidate the function of pectin, one of the plant cell wall components, we embarked on a study
to identify the genes of pectin biosynthetic enzymes. Of the approximately 30 enzymes involvedin pectin biosynthesis, we focused on two enzymes involved in pectin side chain synthesis. In
this study, a method for detecting the activity of these enzymes was developed and reported in aninternational journal. These results will lead to the identification of pectin biosynthetic enzyme genes
and the elucidation of pectin functions.
Key Words：Enzyme, Glycosyltransferase, Pectin, Plant cell wall, Polysaccharides

Introduction
Plant cells have a cell wall that gives the plant strength. This cell wall is composed ofpolysaccharides such as cellulose, pectin, xyloglucan, and xylan. Among these, the role andfunction of pectin, which accounts for about 30% of the cell wall polysaccharides, remain largely
unexplored. This is because the identification of glycosyltransferase genes involved in thebiosynthesis of pectin has not yet progressed. In this study, we attempted to construct an assay
method for glycosyltransferases that are involved in pectin biosynthesis. This work contributes to theadvancement of the pectin-related biology.

Results
Pectin, a component of plant cell walls, is composed of three regions: homogalacturonan (HG),
rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) (Fig. 1). Approximately 30
glycosyltransferases are thought to be involved in their biosynthesis, but few have been identified.
Our group has previously identified a gene for rhamnosyltransferase involved in pectin RG-I
biosynthesis (Takenaka et al., 2018). Based on this experience, in order to elucidate the functionof pectin, we started the study to identify the genes of two enzymes, apiosyltransferase, which is

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involved in pectin RG-II biosynthesis, and galactosyltransferase, which is involved in pectin RG-I
biosynthesis. Because the genetic approach for identification of genes encoding pectin-biosynthetic
glycosyltransferases has not been successful to date, we attempted to identify these genes bybiochemical approaches.
RG-II biosynthetic apiosyltransferase (Fig. 2) is a critical enzyme that synthesizes RG-II side
chain. The apiose residue of RG-II forms a boron diester bond and is involved in the dimerizationof RG-II. This apiosyltransferase activity has never been detected so far, because no preparation
method for its donor substrate, UDP-apiose, has been established. UDP-apiose is unstable because
the hydroxyl group at the second position of the sugar of UDP-apiose is located close to theadjacent phosphate ester and can easily be nucleophilic attacked to form cyclic phosphate. In thisstudy, the stabilization of UDP-apiose was achieved by orienting triethylamine as a counter ion. By
this method, UDP-apiose could be prepared more than 1 mg. This amount is sufficient to measure
the activity of RG-II apiosyltransferase. The results were published in the journal Carbohydrate
Research (Fujimori et al., 2019). Currently, the gene encoding this enzyme is searched from
Arabidopsis genome using this biochemical method.

RG-I biosynthetic galactosyltransferase (Fig. 3) is a key enzyme in the formation of RG-I side
chain galactans, which interact with cellulose in the cell wall and are thought to play a role in thefirmness and softness of plants. If the gene for this enzyme is identified, it could be used to elucidate
the molecular mechanisms of plant firmness. In addition, RG-I with galactan has been shown tobe an effective medicinal component with immune-enhancing activity in Chinese herbal medicine.
In this study, an acceptor substrate for this enzyme was prepared and a method for measuring theactivity of this enzyme was constructed using the substrate. The RG-I main chain of oligosaccharidesof a certain length (>10 sugars) was found to be the best substrate for this enzyme. We also foundthat there was a superactivation in which the enzyme activity increased up to 40-fold in the presenceof certain cationic surfactants and polyelectrolytes. This would suggest that this enzyme has ahydrophobic region and interacts with other proteins. The results were published in the journal Plant
Physiology and Biochemistry (Matsumoto et al., 2019). As this is the first time that this enzyme
activity has been detected, we have applied to Enzyme Nomenclature to issue an enzyme number(EC number). Using this established method for measuring the activity of this enzyme, we aresearching for the gene encoding this enzyme.

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Discussion & Conclusion
With this research grant, we have established a method for detecting the activity of two enzymes
that are important for pectin biosynthesis. We are currently searching for genes encoding these
two enzymes from the Arabidopsis gene database. Proteins encoded by candidate genes for these
enzymes are expressed in heterologous cells and the enzyme activity is attempted to detect in these
proteins to identify enzyme genes. The identification of these genes will lead to the analysis ofknockout or repressed mutants of these genes, which will lead to the study of the role and function ofpectin in plant physiology.

References
Fujimori, T., Matsuda, R., Suzuki, M., Takenaka, Y., Kajiura, H., Takeda, Y., Ishimizu, T. (2019)
Practical preparation of UDP-apiose and its application for studying apiosyltransferase.
Carbohydr. Res. 477, 20-25.
Matsumoto, N., Takenaka, Y., Wachananawat, B., Kajiura, H., Imai, T., and Ishimizu, T. (2019)
Rhamnogalacturonan I galactosyltransferase: Detection of enzyme activity and its hyperactivation.
Plant Physiol. Biochem. 142, 173-178.
Takenaka, Y., Kato, K., Ogawa-Ohnishi M., Tsuruhama, K., Kajiura, H., Yagyu, K., Takeda,A.,
Takeda, Y., Kunieda, T., Hara-Nishimura, I., Kuroha, T., Nishitani, K., Matsubayashi, Y.,
and Ishimizu, T. (2018) Pectin RG-I rhamnosyltransferases represent a novel plant-specificglycosyltransferase family. Nature Plants 4, 669-676.

一般の皆様へ
植物細胞がもつ細胞壁は、植物の成長、外敵からの防御、植物のしなやかさを生み出すことなどに関わっていると考えられていますが、研究が進んでいません。私は、この助成金により、植物細胞壁を作る酵素を検出する方法を確立しました。この方法を用いて、植物細胞壁を作る
酵素遺伝子を発見することができるようになります。これらの遺伝子が発見されると、植物細胞壁の役割や機能を解明したり、食糧エネルギー資源になる植物の生産に応用したりすることができるようになります。

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RNA base modification and RNA processing regulate the maintenanceand development of germline stem cells
Toshie Kai
toshie_kai@fbs.osaka-u.ac.jp
Graduate School of Frontier Biosciences, Osaka University

Summary Abstract
In this study, we established the analysis procedure for Drosophila mRNA to detect the position
and modification ratio of base modification. The purified RNA library was analyzed by a nanopore
device without converting it to cDNA (Direct-RNA-seq). RNA having an average length of about
1.5 kb could be analyzed for about 500,000 to 1,000,000 molecules. From the obtained raw data, thebase sequence was identified for each RNA molecule. We could identify up to 30 modification candidate sites per gene at the single base level.
Key Words：RNA, base modification, processing, germline cells, nanopore

Introduction
Some mRNA bases undergo various modifications such as methylation, which affects subsequent
processing. As a result, there are known examples in which the isoform of mRNA and translation efficiency are changed. However, there are few cases where the function of base modification has been
elucidated, and the molecular functions of many base modifications remain unclear. The purpose ofthis study is to elucidate the control mechanism of maintenance and differentiation of reproductive
stem cells by the base modification of mRNA using the reproductive stem cells of Drosophila as amodel system.

Results
We prepared the total RNA pools from somatic culture cells (Drosophila S2 cells) and mature
ovaries of Drosophila melanogaster. RNA having a poly A tail was further purified from each celltype. After adding the 3’ adaptor, mRNA library was constructed. The purified RNA library was
analyzed by using a nanopore device without converting it to cDNA (Direct-RNA-seq).
The base-calling analysis was carried out for about 48 hours per sample. RNA having an average
length of about 1.5 kb could be analyzed for about 500,000 to 1,000,000 reads. From the raw dataobtained, the base sequence was identified for each molecule by using a software provided by the
manufacturer, Oxford Nanopore Technologies. The successful ratio of base identification for eachread was around 80%. Reads of the remaining 20% were not analyzed because of the low qualityof the sequencing signal. The base-assigned reads were further mapped on Drosophila genome.
The mapping ratio was about 70% among the base-assigned reads. The unmapped reads may betranscripts from the genome region with high complexity, like a repeated region or transposon
region.
Furthermore, we analyzed the length of the poly A chain, which is important for mRNA stability
and translation efficiency. For the analysis, we introduced a program called “Nanopolish” and used it

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for the Promotion of Sciencein conjunction with our own script. When the poly A chain lengths of several highly expressed genes
were investigated in detail, a short poly A chain gene (about 50 bases on average) and a long gene(about 150 bases on average) were detected. In addition, a program for base modification analysis,
Tombo, was introduced and set up. Modified bases show different measurements than unmodified
bases. Since the Drosophila genomic sequence is well known, it is possible to infer the expectedvalue for the unmodified base as the control. By comparing the measured signal for the purified RNA
and the expected control signals, we could detect more than 10,000 candidate modification sites
having the modification ratio of more than 90%. This analysis found that some RNA isoforms werehighly modified. Up to 30 modification candidate sites per gene could be identified at the single baseresolution. Sap-r, precursor of sphingolipid activator proteins, is one of the top 10 genes which arehighly modified in both libraries from S2 cells and mature ovaries. Some genes were specificallymodified in one of two cell-type libraries, suggesting that base modification is differently regulated
in each cell type.

Discussion & Conclusion
We could set up the analysis protocols of direct RNA-seq. By applying this method to Nanopore
device, we analyzed the poly-A tail length and base modification on each mRNA molecule. As
the results, the distribution of poly-A length was determined for each gene. The potential basemodification sites were also identified and found that up to 30 modification sites per gene.
Currently, large amount of RNA is needed for this analysis. We will optimize the entire protocol
to increase the efficiency of sequencing and reduce the RNA amount required. If we could reduce the
RNA amount required for this analysis, we can analyze cells which are relatively rare, like germline
stem cells.

References
Lewis CJ, Pan T, Kalsotra A., Nat Rev Mol Cell Biol. 2017

一般の皆様へ
ゲノム DNA の配列に変化を起こさず、DNA や RNA の修飾によって、遺伝子発現を変化させる「エピジェネティック制御」は、発生や分化、あるいは環境応答などで重要な役割を担っている。
その中でも、アデノシンのメチル化などの RNA 塩基修飾が注目を集めている。本研究では、最近実用化されている RNA 解析技術を用いて、生殖幹細胞由来 mRNA の塩基修飾部位を解析し、生殖幹細胞の特異的なエピジェネティック制御機構を明らかにする。

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Elucidation of a novel regulatory mechanism of angiogenesis byintraluminal pressure
Shigetomo Fukuharas-fukuhara@nms.ac.jp
Institute of Advanced Medical Sciences, Nippon Medical School

Summary Abstract
In this study, we demonstrate a novel mechanism of angiogenesis regulation during wound healing
mediated by blood flow-driven intraluminal pressure. During wound angiogenesis, elongation of the
injured vessels is preferentially induced downstream from the blood flow, whereas blood flow-driven
intraluminal pressure loading suppresses elongation of the upstream injured vessels by inducing thestretching of endothelial cells (ECs). As the underlying mechanism, we showed that intraluminalpressure load-induced EC stretching prevented localization of Arp2/3 complexes, a key regulator of
actin filament nucleation, at the leading edge, thereby inhibiting formation of actin-based protrusions
and front-rear polarization, leading to impairment of directed EC migration and vessel elongation.
Key Words：Angiogenesis, Wound healing, intraluminal pressure, Live-imaging

Introduction
Angiogenesis refers to the physiological and pathological processes through which new bloodvessels form from pre-existing vessels. Mechanical regulation of angiogenesis is essential tosuccessful wound healing, a complex and dynamic process by which tissue repairs itself after injury(1, 2). However, the dynamics of EC behavior in wound angiogenesis and especially its regulation bymechanical forces remain poorly understood, because methods of analyzing such a highly dynamic
process in vivo have been lacking. In this study, we endeavored to address these questions by
exploiting a recently developed live-imaging system for adult zebrafish (3).

Results
To analyze the behavior of ECs during cutaneous wound healing, we introduced wounds onto
the flanks of adult zebrafish expressing EGFP in ECs. Cutaneous wounding immediately induced
angiogenesis, during which elongation of the severed blood vessels was actively induced at the earlystage, while sprouting from the pre-existing vessels occurred at a relatively late stage. Interestingly,
we noticed that elongation of the severed blood vessels was actively induced at sites downstream
from blood flow, whereas the vessels located upstream did not elongate efficiently. We also observed
preferential elongation of the injured downstream vessels when intersegmental vessels (ISVs) ofzebrafish larvae were severed by laser ablation.
Then, we addressed the cause of the differences in elongation of the injured blood vessels. In thisregard, we assumed that intraluminal pressure acting on the injured blood vessels might accountfor the differences in elongation, because the heart constantly pumps blood only into the upstream
injured vessels. Consistently, we found that the upstream injured vessels in wounded skin began toelongate when IP was diminished by cutting the more upstream site. To confirm our hypothesis, we
originally developed an in vitro angiogenesis system in a microfluidic device, in which hydrostatic

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pressure can be loaded on the lumens of elongating vessels as a collaboration with Dr. Koichi
Nishiyama (Kumamoto University), and showed that an intraluminal pressure load of approximately
1.2 mmHg to the lumens of angiogenic branches significantly suppressed their elongation. These
results indicate intraluminal pressure loading suppresses elongation of upstream injured vessels
during wound angiogenesis.
To investigate how intraluminal pressure loading suppresses vessel elongation, we analyzed
intraluminal pressure load-induced morphological changes of elongating vessels by utilizing an invitro angiogenesis model and by analyzing the morphology of the injured skin vessels and that of
the severed ISVs in adult and larval zebrafish, respectively. As a result, we showed that intraluminalpressure loading induced expansion of the upstream injured vessels, which resulted in stretching of
ECs to restrict the vessel elongation.
Plasma membrane tension reportedly acts as an inhibitor of actin assembly and therefore
maintains the front-rear polarity of migrating cells by confining actin polymerization signals tothe leading edge (4, 5). Thus, we hypothesized that intraluminal pressure load-induced ectopic
stretching of ECs in the angiogenic branches might inhibit actin-based protrusion at the leadingedge, disrupting the front-rear polarity for directional cell migration and thereby leading to inhibitionof vessel elongation. To address this hypothesis, we performed an in vitro angiogenesis assay
and showed that intraluminal pressure load-induced stretching of ECs in the angiogenic branches
prevents leading edge localization of Arp2/3 complexes, a key regulator of actin filament nucleation,
thereby inhibiting actin polymerization and front-rear polarization leading to impaired elongationof blood vessels. Finally, we investigated whether similar mechanism account for the impaired
elongation of upstream injured vessels in zebrafish. As a result, we found that ECs in the downstream
injured vessels established front-rear polarity and extended leading edge protrusions throughArp2/3 complex-mediated actin polymerization to elongate vessel sprouts, whereas in the upstream
injured vessels, intraluminal pressure load-induced EC stretching prevented localization of Arp2/3
complexes at the leading edge, thereby inhibiting formation of actin-based protrusions and front-rear
polarization, leading to impairment of directed EC migration and vessel elongation.

Discussion & Conclusion
Angiogenesis plays a crucial role in wound healing by forming new blood vessels in the injured
tissues, which occurs not only through sprouting from the uninjured vessels but also via elongationof the severed vessels. Herein, surprisingly, we discovered that elongation of the injured vessels ispreferentially induced downstream from the blood flow, whereas blood flow-driven intraluminalpressure loading suppresses elongation of the upstream injured vessels by inducing the stretchingof ECs. As the underlying mechanism, we show that intraluminal pressure load-induced EC
stretching in the upstream injured vessels prevents localization of Arp2/3 complexes at the leadingedge, thereby inhibiting formation of actin-based protrusions and front-rear polarization, leading to
impairment of directed EC migration and vessel elongation. In conclusion, we successfully uncovera novel regulatory mechanism of wound angiogenesis by intraluminal pressure load on the bloodvessels.

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References
1. M. G. Tonnesen, X. Feng, R. A. F. Clark, Angiogenesis in wound healing. J. Invest. Dermatol.
Symposium Proceedings 5, 40-46 (2000).
2. L. Lancerotto, D. P. Orgill, Mechanoregulation of angiogenesis in wound healing. Adv. Wound
Care (New Rochelle) 3, 626-634 (2014).
3. C. Noishiki et al., Live imaging of angiogenesis during cutaneous wound healing in adultzebrafish. Angiogenesis 22, 341-354 (2019).
4. A. R. Houk et al., Membrane tension maintains cell polarity by confining signals to the leadingedge during neutrophil migration. Cell 148, 175-188 (2012).
5. S. Saha, T. L. Nagy, O. D. Weiner, Joining forces: crosstalk between biochemical signalling andphysical forces orchestrates cellular polarity and dynamics. Philos. Trans. R. Soc. Lond. B Biol.
Sci. 373, pii: 20170145 (2018).

一般の皆様へ
生体組織が傷害を受けると、それを修復するため血管新生により新たな血管網が作られます。

今回、私たちはゼブラフィッシュを用いた蛍光イメージングにより、創傷治癒における血管新生を生きた個体で観察し、損傷血管が修復する際、血流に対して下流の損傷血管は伸長するのに対し、上流損傷血管は血流に起因する内腔圧により伸長しないことを発見しました。そのメカニズムとして、内腔圧は上流損傷血管を拡張し、内皮細胞に伸展刺激を負荷することで、内皮細胞の運動を抑え、血管伸長を阻害していることを明らかにしました。本研究により、組織修復における血管新生の新たな制御機構が明らかになり、創傷治癒を促進させるための治療法の開発につながる可能性があります。

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Development of new treatment for obesity-related liver cancer,
focusing on a Wnt ligand, ACLP, secreted by hepatic stellate cells
Kengo Tomita
kengo@ndmc.ac.jp
Division of Gastroenterology and Hepatology, Department of Internal Medicine,National Defense Medical College

Summary Abstract
In the present study, we aimed to elucidate the role of hepatic stellate cell (HSC)-derived aortic
carboxypeptidase-like protein (ACLP) in the onset and progression of obesity-related liver cancer,
using HSC-specific ACLP-deficient mice and an obesity-related liver cancer mouse model. The onsetof liver cancer was significantly suppressed in HSC-specific ACLP-deficient mice compared with
control mice, suggesting the pathological mechanism by which ACLP promotes the development ofobesity-related liver cancer.
Key Words： aortic carboxypeptidase-like protein; hepatic stellate cell; liver cancer; obesity;
nonalcoholic steatohepatitis

Introduction
We have recently found that ACLP is a secretory glycoprotein specifically produced in HSCs
in the liver, and significantly increased in the liver of patients with nonalcoholic steatohepatitis(NASH). Furthermore, we have found that ACLP is a novel Wnt ligand that activates the Wnt/
β-catenin pathway. Those results suggested that ACLP could promote the development of obesityrelated liver cancer.

Results
We conducted this study to analyze the role of ACLP in the pathological mechanism of livercancer associated with obesity, using HSC-specific ACLP-deficient mice. We created Cre-Gfap/Aclpflox mice as HSC-specific ACLP-deficient mice. Aclp-flox mice were used as control mice.
The obesity-related liver cancer model by administration of a high fat diet was performed
according to the method reported in Park-EJ, et al. Cell 2010; 140: 197-208.
Male Cre-Gfap/Aclp-flox mice and Aclp-flox mice were subjected to the obesity-related livercancer model. The onset of liver cancer was significantly suppressed in the Cre-Gfap/Aclp-flox
mice as compared with the Aclp-flox mice. Furthermore, cell proliferation assay by administrationof recombinant ACLP was performed using mouse liver cancer Hepa 1-6 cells. Recombinant ACLP
administration significantly promoted the proliferation of Hepa 1-6 cells.

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Discussion & Conclusion
This study revealed that HSC-derived ACLP not only promotes the development of obesityrelated liver cancer, but also exacerbates the background NASH pathology. Furthermore, it suggested
that HSC-derived ACLP acts as a Wnt ligand on nearby hepatoma cells and promotes obesity-related
hepatocarcinogenesis through enhancement of Wnt/β-catenin signalling.
Hepatocellular carcinoma (HCC) has the characteristic of developing in the form of multicentriccarcinogenesis, which reflects the state of the background liver. Currently, there are various treatment
methods for HCC including surgery, but even if the HCC shown in the image can be treated, it
often repeats recurrence after all, and effective treatment/prevention methods have not yet been
established.
The therapeutic method targeting ACLP signal could suppress liver carcinogenesis and improvethe underlying NASH pathology, and thus may be a promising therapeutic modality for themulticentric carcinogenesis of obesity-related liver cancer.

References
Teratani T, Tomita K (Teratani T and Tomita K share first authorship), Suzuki T, Furuhashi H, Irie R,
Nishikawa M, Yamamoto J, Hibi T, Miura S, Minamino T, Oike Y, Hokari R, Kanai T.
Aortic carboxypeptidase-like protein, a WNT ligand, exacerbates nonalcoholic steatohepatitis.
J Clin Invest.; 128(4): 1581-1596, 2018.

一般の皆様へ
肥満にともなう肝がん患者は急増しており、病態機序の解明と治療法の開発は緊急課題である。私たちは、ACLP (Aortic carboxypeptidase-like protein) が肝星細胞特異的に産生される分泌性糖タンパク質であり、肥満により産生が増強される新規の Wnt リガンドであることを見いだした。本研究では肝星細胞由来の ACLP という見地から、肥満関連肝がん発症・進展の病態機序を解明し、新たな治療法を確立することを目的とした。肥満関連マウス肝がんモデルを作成したところ、肝星細胞特異的 ACLP 欠損マウスでは、有意に肝がん発症が抑制されており、
ACLP が肥満関連肝がんの有望な治療標的であることが明らかとなった。

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Integrated analysis of tumor stromal cells using ultra-sensitive
bioluminescence imaging Tg mice
Shinae Kizaka-Kondoh
skondoh@bio.titech.ac.jp
School of Life Science and Technology, Tokyo Institute of Technology

Summary Abstract
A new cancer treatment strategy is to target the stromal cells that surround the cancer cells. Recent
studies have revealed that stromal cells play a major role in the process of malignant progression
during tumor growth. In this study, we attempted to collect information useful for elucidating themechanism of malignant progression using an ultrasensitive bioluminescence imaging system thatenables non-invasive observation of hypoxia-inducible factor-active stromal cells during tumor
growth. Based on in vivo imaging data, further temporal and spatial analyses of tumors are expected
to lead to the development of new diagnostic markers and therapeutics.
Key Words：Hypoxia inducible factor, stromal cells, in vivo optical imaging, transgenic mice

Introduction
Hypoxia-inducible factor (HIF) is an essential transcription factor that controls the hypoxic response of cells, but it is closely associated with tumor malignancy and poor prognosis [1]. Many
studies have revealed that HIF activity in cancer cells plays an important role in the process of cancer
growth and malignant progression, but its role in stromal cells is unclear. We constructed transgenic(Tg) mice that can be noninvasively analyzed for HIF activity by bioluminescence imaging (BLI),
and in carcinogen-induced cancers, HIF activity is highly correlated with tissues with pathologicalimages of cancer lesions [2]. These analyzes strongly suggest the contribution of HIF activity not
only to cancer cells, but also to stromal cells surrounding cancer cells during the process of cancer
growth and malignant progression.

Results
1. Construction and evaluation of reporter geneIn the analysis for the preexisting HOL-Tg mice [2], analysis of stromal cells was not possible
due to insufficient luminescence intensity and lack of a fluorescent reporter gene suitable for exvivo analysis.
We have recently developed an ultrasensitive in vivo BLI system using AkaLuc [3] andAkaLumine [4] that generates 50 – 1400 times higher bioluminescent signals in vivo than BLI
using firefly luciferase and D-luciferin. Therefore, as an expression plasmid for constructing Tgmice, pHRE/Venus-AkaLuc (HVA) was constructed by connecting cDNA encoding a fusion
protein of AkaLuc and a fluorescent protein Venus [5] downstream of a HIF-dependent promoter(pHRE). This plasmid was transiently introduced into HeLa cells to verify the expression of HIFdependent reporter gene. As a result, Venus fluorescence (green) and high bioluminescence wereobserved in cells with HIF activity that was induced in hypoxia (1% O2) culture, and the plasmid
was used to construct HVA-Tg.

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2. Construction and evaluation of HVA-Tg
Three HVA-Tg lines were created using C57BL/6 at Kyoto University and analyzed in Tokyo
Institute of Technology. All animal experiments were performed with the approval of the Animal
Ethics Committees of Kyoto University and Tokyo Institute of Technology.
Propyl gallate (PG), an antioxidant that stabilizes HIF-1α protein [7], was subcutaneously
administered to confirm HIF responsiveness of the Tg mice, and HIF-dependent bioluminescenceof AkaLuc was observed using bioluminescence imaging device IVIS ™. As a result, PGdependent luminescence was observed only in Tg mice, and HIF activity-dependent
responsiveness of AkaLuc was confirmed. Of the three lines, we decided to perform the transplantmodel experiment using the line with the strongest luminescence.
3. Non-invasive bioluminescence imaging of stromal cells in tumors
We succeeded in noninvasively observing theinvasion of stromal cells with HIF activity into
the tumor in a transplanted tumor model using
5 Tg lines, and obtained the expected results(Figure 1). However, because the fluorescent
Venus reporter did not function sufficiently andanalysis at the cell level was not possible, we arereselecting the Tg mouse lines.
In the meantime, in order to investigate theeffect of HIF-active stromal cells on malignant
progerssion, continuous temporal and spatial Figure 1 in vivo and ex vivo BLI of HIF-active
analysis of HIF-active stromal cells in tumors ofstromal cells in tumors
preexisting Tg mice was continued by ex vivo
bioluminescence imaging. Stromal cells were analyzed by fluorescent immunohistochemistry
staining.
4. Immunohistochemical analysis of stromal cells in tumors
Tumor sections wereprepared from HVA-Tg
tumors. Stromal cells wereanalyzed with antibodies
to known stromal cells andto luciferase, which detects
cells expressing the reporter gene. Double positive
stromal cells were detectedin specific areas of the tumor (Figure 2) and spread Figure 2 Immunohistochemical analysis of stromal cells of HVA-Tg
throughout the tumor as thetumor grew.

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Discussion & Conclusion
By using the ultrasensitive BLI, we were able to noninvasively observe the bioluminescent signal
from HIF-active stromal cells from tumors of HVA-Tg as early as 5 days after transplantation.
However, Venus fluorescence, which is required for FACS sorting of intratumoral stromal cells, was
not detected, so we decided to reconstruct Tg mice.
In the meantime, we analyzed HIF-active intratumoral stromal cells by Immunohistochemical
analysis and found that certain types of stromal cells had high HIF activity and were localized to
specific areas of the tumor, suggesting that the identified stromal cells may contribute to malignant
progression of tumors. Now, we are analyzing the interaction of cancer cells and the stromal cells.
Once Tg mice are constructed, the contribution of HIF-active stromal cells to malignant
progression can be elucidated by single cell-based analysis, leading to more detailed andcomprehensive information of intratumoral stromal cells. The comprehensive information of stromalcells may allow us to identify the stromal cells that are deeply involved in the malignant progression
and development of immunosuppressive environments The findings will lead to the development of
new diagnostic markers and therapeutic strategies.

References[1] Semenza GL. Targeting HIF-1 for Cancer Therapy. Nat Rev. Cancer, 2003 3(10):721-32.
[2] Kadonosono T, Kuchimaru T, Yamada S, Takahashi Y, Murakami A, Tani T, Watanabe H, Tanaka
T, Hirota K, Inoue M, Tsukamoto T, Toyoda T, Urano K, Machida K, Eto T, Ogura T, Tsutsumi
H, Ito M, Hiraoka M, Kondoh G, Kizaka-Kondoh S. Detection of the onset of ischemia andcarcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.
PLoS One. 2011;6(11):e26640.
[3] Iwano S, Sugiyama M, Hama H, Watakabe A, Hasegawa N, Kuchimaru T, Tanaka KZ, Takahashi
M, Ishida Y, Hata J, Shimozono S, Namiki K, Fukano T, Kiyama M, Okano H, Kizaka-Kondoh S,
McHugh TJ, Yamamori T, Hioki H, Maki S, Miyawaki A. Single-cell bioluminescence imaging
of deep tissue in freely moving animals. Science. 2018;359(6378):935-939.
[4] Kuchimaru T, Iwano S, Kiyama M, Mitsumata S, Kadonosono T, Niwa H, Maki S, KizakaKondoh S. A luciferin analogue generating near-infrared bioluminescence achieves highly
sensitive deep-tissue imaging. Nat Commun. 2016, 7:11856.
[5] Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. A variant of yellow fluorescentprotein with fast and efficient maturation for cell-biological applications. Nat Biotechnol. 2002
20(1):87-90.

一般の皆様へ
がんの新たな治療戦略として、がん細胞の周囲に存在する間質細胞を標的にする方法が注目されています。間質細胞は、がん細胞の増殖を助け、転移を促進する悪性化の過程に大きな役割を果たしていることが、最近の研究から明らかになってきました。本研究では、我々が開発した超高感度生体発光イメージング系を用いて、悪性化に関与する間質細胞を腫瘍の成長過程
を通して体の外から観察することで、悪性化機構の解明に有用な情報収集を試みました。生体レベルでの観察を基に、今後、腫瘍内の時間空間的な解析を進めることで、新たな診断マーカー
や治療法の開発に繋がると期待されます。

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Novel long-range regulatory mechanisms controlling
NKT-cell lineage program
Nayuta Yakushiji-Kaminatsui
nayuta.yakushiji@riken.jp
RIKEN Center for Integrative Medical Sciences

Summary Abstract
In this study, we aimed to identify the pivotal enhancers that control NKT cell fate determination
during differentiation from human iPSCs. To do this, we first transferred and optimized the chromatin
profiling technology (CUT&Tag), enabling us to approach at single-cell or low input level. By using
this technology, we identified c.a. 6,800 potential enhancers and found that these potential enhancers
got activated during NKT cell differentiation.
Key Words：human iPSCs, NKT cell, enhancer, CUT&Tag, cancer immunotherapy

Introduction
Natural killer T (NKT) cells have been expected to be utilized for cancer immunotherapy due toits strong anti-tumor effect, and we previously reported that NKT cells derived from human iPSCs
possess significant anti-tumor activity in tumor-bearing mice, providing a first proof of concept
for the clinical application of iPSCs-derived NKT cells for cancer immunotherapy1). Our induction
protocol is a robust, however, it has issues to be improved. To develop next-generation induction
protocol, in this research, we especially focused on to reveal the identification of NKT cell-specificenhancers and how these are regulated during NKT cell fate decision.

Results
The long-term goal of the project is to develop the next-generation NKT cell induction protocol
with shorter culture duration and higher induction efficiency for the future cancer immunotherapy. To
accomplish this, as the short-term goal of the project, we aimed to identify the pivotal enhancers that
control NKT cell fate determination and to reveal how these enhancers are regulated during NKT
cell differentiation from human iPSCs in this research.
We first asked which gene expressions could be controlled by the potential enhancers during NKT
cell differentiation in our current induction protocol. To answer this, we performed transcriptome
analysis (RNA-seq) by using iPSCs (day 0 before induction) and NKT cells (day 40 after induction)and found dynamic changes in gene expression as expected. The genes were divided into two
categories; cluster I contained the genes that are strongly expressed only in iPSCs and grouped into
stem cell maintenance and pluripotency in Gene Ontology term; cluster II included the genes that are
significantly expressed in NKT cells and categorized into the proliferation of immune cells and T cellfate determination.
We next went onto investigate the histone modification status at these genes. The histone proteins
composed of chromatin are well known to control gene expression via various modifications at their
histone tails. In general, trimethylation of H3K27 (H3K27me3) acts on transcriptional repression,
whereas acetylation of H3K27 (H3K27ac) and monomethylation of H3K4 (H3K4me1) work as

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for the Promotion of Sciencetranscriptional activation. Depending on the expression status of gene, H3K27me3 and H3K27ac
are enriched at the transcriptional start site (TSS), while H3K27ac and H3K4me1 are accumulated
at the enhancer. To do this, we first established and optimized the chromatin profiling (Cleavage
Under Targets and Tagmentation, CUT&Tag)2), that can be applied to single cell or low input cells.
By performing CUT&Tag analysis, we found strong enrichments of H3K27ac, H3K4me1 andunexpected H3K27me3 at TSS of genes in cluster I in iPSCs, whereas only high accumulation of
H3K27me3 was seen in NKT cells. On the other hand, the H3K27me3 enrichment at the TSS ofgenes in cluster II was detected in iPSCs, while significant strong accumulations of H3K27ac andH3K4me1 were observed in NKT cells. These results suggest that alterations in gene expression
during NKT cell differentiation were accompanied with dynamic changes of histone modification.
To further identify the potential enhancers that control NKT cell identity, we focused on P300, ahistone acetyltransferase, since histone acetylation of enhancer regions by P300 is required for theenhancer activity3). We checked the binding profiles of P300 by CUT&Tag experiment and identified
approx. 6,800 peaks of P300 in the iPSCs. We further examined whether histone modification status
at the P300-bound regions are changed during differentiation and found that at the P300-boundregions, both levels of H3K4me1 and H3K27ac got dramatically increased, whereas enrichment of
H3K27me3 was decreased as iPSCs differentiated into NKT cells (Fig. 1). These results suggestthat potential enhancers of NKT cell are activated and contribute to control the target genes properly
during NKT cell differentiation.

Discussion & Conclusion
It is still unclear what kinds of cells are proliferating and differentiating into NKT cells in our
culture system. However, the findings obtained from this research clearly revealed what kinds ofgenes are dynamically changed during NKT cell differentiation and showed the well correlation
between gene expression status and histone modification level. In addition, we also found thepotential enhancers that are involved in NKT cell fate decision by P300 chromatin profiling. We
are planning to reveal the nature of cells that eventually differentiate into NKT cells by performing
transcriptome and chromatin accessibility at single-cell resolution. Furthermore, we will dochromatin profiling with time-course analysis during NKT cell determination and screen the pivotal
enhancers of NKT cell identity. All these results will contribute to develop the next-generation NKT
cell induction protocol with shorter culture duration and higher induction efficiency for future cancer
immunotherapy.

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References
1) Yamada D, Iyoda T, Vizcardo R, Shimizu K, Sato Y, Endo TA, Kitahara G, Okoshi M, Kobayashi
M, Sakurai M, Ohara O, Taniguchi M, Koseki H, Fujii SI. Efficient Regeneration of HumanVα24+ Invariant Natural Killer T Cells and Their Anti-Tumor Activity In Vivo. Stem Cells. 2016
Dec;34(12):2852-2860. doi: 10.1002/stem.2465. Epub 2016 Aug 1. PMID: 27422351
2) Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff
S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun.
2019 Apr 29;10(1):1930. doi: 10.1038/s41467-019-09982-5. PMID: 31036827
3) Raisner R, Kharbanda S, Jin L, Jeng E, Chan E, Merchant M, Haverty PM, Bainer R, Cheung T,
Arnott D, Flynn EM, Romero FA, Magnuson S, Gascoigne KE. Enhancer Activity Requires CBP/
P300 Bromodomain-Dependent Histone H3K27 Acetylation. Cell Rep. 2018 Aug 14;24(7):17221729. doi: 10.1016/j.celrep.2018.07.041. PMID: 30110629

一般の皆様へ
がん免疫治療法への利用が期待されるナチュラルキラー T（NKT）細胞は、生体内にはわずかしか存在せず、試験管内で治療に必要な細胞数まで増殖させることは非常に難しい。我々はヒト NKT 細胞由来 iPS 細胞を再度 NKT 細胞へと分化・増殖させることで、この問題に取り組んでいる。今回、我々はヒト iPS 細胞から NKT 細胞への分化運命を制御するような DNA 領域の探索とその制御機構を明らかにすること目指した。その結果、NKT 細胞への分化過程で活性化する約6,800箇所の DNA 領域を見出した。今後この領域に対する解析をさらに進めていくことで、最終的には分化誘導期間の短縮と高い誘導効率を兼ね備えた次世代の分化誘導法の確立を目指していく。

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Molecular mechanisms of extracellular vesicle formations ofendogenous retrovirus
Keita Miyoshi
kmiyoshi@nig.ac.jp
National Institute of Genetics

Summary Abstract
We investigated the molecular mechanisms of the formation and transfer of the extracellular
vesicles derived from endogenous retroviruses to understand the physiological significance ofretroviruses. We identified specific endogenous retrovirus mRNAs in the extracellular vesicles. Our
results suggested that selected endogenous retroviruses might be transferred to different cells via theextracellular vesicles.
Key Words：Endogenous retrovirus, Extracellular vesicles, Drosophila

Introduction
Extracellular vesicles are membrane vesicles that are approximately 30-1,000 nm in diameter thatare secreted by various types of cells and are classified into exosomes, microvesicles, apoptotic bodies, etc. by their size and origin. It has been reported that these extracellular vesicles encapsulate andpropagate nucleic acids, proteins, and lipid-cargos derived from secretory cells and play essential
roles in cell-to-cell communications. In this study, we examined extracellular fractions from our Drosophila cultured cells to identify extracellular vesicles derived from endogenous retroviruses.

Results
The purpose of this study is to understand the particle formation and transfer mechanismof extracellular vesicles derived from endogenous retroviruses in Drosophila melanogaster. It has been reported that extracellular vesicles migrate from ovarian somatic cells to oocytes (1, 2). These extracellular vesicles are derived from endogenous retroviruses (for example, Gypsy and ZAM). So far, it was not easy to purify and analyze those vesicles fromindividual flies. Therefore, it has been unclear the formation and transfer mechanisms ofendogenous retroviruses. We have established the cultured cell line derived from Drosophila follicle cells and could obtain the large fraction containing extracellular vesicles.
Drosophila Ovarian Somatic Cell line (OSC) (3) derived from ovarian follicle cell
was used to collect the extracellular vesicle fraction. First, the extracellular vesicle fraction was collected from the supernatant of the OSC culture medium via filtration. The sizeof vesicles in the fraction was determined by nanoparticle tracking analysis using NanoSight. This result showed the size distribution of extracellular vesicles with three peaks(78, 145, and 226 nm). The size distribution of virus particles is from a few tens to afew hundreds of nm. The most significant peak (78 nm) is similar to the size of viruses.
Second, RNA, which is included in the extracellular vesicle fraction, was examined by RTqPCR and RNA-seq analyses to see whether transposon mRNAs were included. These results
showed that specific transposon mRNAs and their distinct isoforms were contained in the fraction.

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An endogenous retrovirus XXX exists many copies in the genome, including full-length, fragments, and mutants. The full-length XXX mRNA was accumulated in the vesicle fraction, but
the fragments were not. This result suggests that endogenous retroviruses particles are selectively
formed from endogenous retrovirus mRNA in a similar size to general exogenous virus particles.
Unexpectedly, some mRNAs coding genes were identified from the extracellular vesicle fraction.
Third, Proteins in the fraction were identified by the shotgun mass spectrometry analyses.
Many proteins were identified as related proteins in the secretory pathway and translated fromretrovirus mRNAs. The fraction should contain various kinds of vesicles released from OSC. We
need to distinguish which proteins are involved in the formation/transfer of retroviruses particles.
The expression of retroviruses is repressed via the piRNA-Piwi pathway at the transcriptional
level in Drosophila follicle cells. The expression is upregulated to 100 times in the Piwi-knockdown
OSC. Although the extracellular vesicle fraction was obtained from the supernatant of wild-type
OSC in this study, the supernatant of Piwi-knockdown OSC might be more useful for this study.

Discussion & Conclusion
In this project, we obtained the fraction, including the extracellular vesicles from the supernatantof the OSC culture medium. The size of the most abundant particles is 78 nm, which is similar tovirus particles. The extracellular vesicles fraction contains specific mRNAs derived from endogenous
retroviruses. These results suggest that the fraction contains the retrovirus particles. To purify theparticular particle, we need to establish the protocol referring to the protein analyses in this study. If
the necessary foundation for particle formation and transfer of endogenous retroviruses is determinedby this research, we expect to develop an efficient drug delivery technology using it. This study willlead us to understand the physiological significance of retroviruses.

References(1) Pélisson, A., Song, S. U., Prud’homme, N., Smith, P. A., Bucheton, A., & Corces, V. G. (1994).
Gypsy transposition correlates with the production of a retroviral envelope-like protein under thetissue-specific control of the Drosophila flamenco gene. The EMBO journal, 13(18), 4401–4411.
(2) Leblanc, P., Desset, S., Giorgi, F., Taddei, A. R., Fausto, A. M., Mazzini, M., Dastugue,
B., & Vaury, C. (2000). Life cycle of an endogenous retrovirus, ZAM, in Drosophilamelanogaster. Journal of virology, 74(22), 10658–10669.
(3) Saito, K., Inagaki, S., Mituyama, T., Kawamura, Y., Ono, Y., Sakota, E., Kotani, H., Asai, K.,
Siomi, H., & Siomi, M. C. (2009). A regulatory circuit for piwi by the large Maf gene traffic jam
in Drosophila. Nature, 461(7268), 1296–1299.

一般の皆様へ生物は非常に多くの細胞によって構成され、それぞれの細胞は特異的な特徴や性質を持ちます。また、その性質は様々な要因により決定されます。例えば、周りの細胞から放出された微粒子が情報を持ち、受け取った細胞の運命決定を行うことが知られるようになってきました。
本研究では、微粒子を介した細胞間の情報のやり取りを分子レベルで明らかにしたいと考えています。

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Molecular mechanisms of axis formation in plants: Organization,function and spatiotemporal dynamics of PIN clusters that regulate
polar localization of auxin efflux carrier PIN proteins
Satoshi Naramotosatoshi.naramoto.d6@tohoku.ac.jp(Present mail address: satoshi.naramoto@sci.hokudai.ac.jp)
Graduate School of Life Sciences, Tohoku University

Summary Abstract
PIN proteins form cluster-like structures at PMs to maintain their polar localization. However,
how these structures are established is largely unknown. I found that MAB proteins play crucial roles
for establishing PIN clusters. I also found that MAB4 proteins directly interact with PIN proteins at
PMs. These findings suggest that MAB4 protein is one of the components of PIN clusters.
Key Words：auxin, polarity, PIN

Introduction
Polar auxin transport (PAT) determines the vectorial transport of phytohormone auxin within tissues, which act as a basis of body axis formation as well as tropic responses upon light and gravity
stimulus. Direction of PAT is regulated by the polar localization of auxin efflux carrier PIN proteins.
However, the mechanisms that determine the localization of PIN proteins are not sufficiently understood. I have identified that PIN proteins form cluster-like structures at PMs， which is critical for
maintaining the polar localization of PIN proteins. I also found that clustering of PIN proteins is mediated by the unknown function protein MAB4. Here I report the progress of my research regarding
the spatiotemporal dynamics of PIN clusters and MAB4 during cell division and gravitropism. I also
report the current status of another project: identification of molecular components of PIN clusters.

Results
1) Identification of molecular components of PIN clusters
To identify the proteins that compose PIN clusters, immunoprecipitation of PIN1 and PIN2
proteins were conducted. I extracted proteins from the plants expressing either proPIN1::PIN1Venus or proPIN2::PIN2-Venus constructs and added PIN1 or PIN2 antibody to immuno-precipitate
PIN1 and PIN2 protein complexes. However, I failed to precipitate PIN1 and PIN2 proteins,which is presumably due to the lower affinity of antibody. To improve this situation, I also tried toprecipitate PIN1 and PIN2-Venus proteins by using GFP antibody. I could succeed to precipitate
PIN1 and PIN2-Venus proteins but so far, the amount of proteins that are precipitated have notbeen sufficient for the subsequent MS analysis. I also tried to do immno-precipitation experiments
for MAB4-CFP expressing plants by using GFP antibody. However, sufficient amounts of MAB4CFP were not recovered. I am currently optimizing the experimental condition to accomplish theseexperiments.

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for the Promotion of Science2) Observation of spatio-temporal dynamics of PIN clustersa) Spatio-temporal dynamics of PIN clusters during cell division
I established the plants co-expressing PIN2-Venus and MAB4-CFP constructs and observed the
PIN2-Venus during cell division. I found that whereas no clusters are established at the forming
cell plate, PIN clusters gradually appeared at the PMs upon the completion of cell division.
Interestingly, MAB4-GFP are not localized at forming cell plate but began to localize at PMs upon
the completion of cell division, where MAB4 forms clusters in the same way as PIN2-Venus.
b) Spatio-temporal dynamics of PIN clusters during root gravitropism
During the process of root gravitropism, endocytosis activity of PIN2 proteins were known to bechanged. I performed the subcellular localization analysis of PIN2-Venus and MAB4-CFP in root
epidermal cells upon gravity stimulus. I detected enhanced endosomal localization of PIN2-Venus
upon gravity stimulus although PIN2 clusters were still formed at PMs. As for the localization of
MAB4, MAB4-CFP at PMs decreases upon gravity stimulus although MAB4-CFP keep forming
clusters. Importantly, MAB4-CFP proteins were colocalized with PIN2-Venus.
c) In vivo investigation of protein-protein interactions between PIN and MAB4
I found that MAB4 proteins form cluster-like structures, which resembles PIN clusters. In fact, I
found that PIN2-Venus and MAB4-CFP proteins were highly colocalized and they form clusterlike structures at PMs, which implied protein-protein interaction between PIN proteins and
MAB4 proteins. Since immno-precipitation experiments did not work, I utilized the technique,called Forster resonance energy transfer (FRET) to detect their interactions. I performed FRET
experiments, more specifically acceptor photobleaching experiments, at root epidermal cells
and succeeded to detect the direct interaction between PIN2-Venus and MAB4-CFP proteins. I
measured the FRET efficiency during the course of root gravitropism. However, FRET efficiency
varies between samples and thus it was not clear whether FRET efficiency changes upon gravity
stimulus. I also measured the FRET efficiency of PIN2-Venus and MAB4-CFP at the PMs during
cell division and compare the FRET efficiency at the nascent PMs with that at the mature PMs.
Similar to the case with gravitropism, the efficiency varies between cells and thus it was difficult
to draw clear conclusion regarding whether there were changes of FRET efficiency or not during
the maturation process of PMs.

Discussion & Conclusion
1) Identification of molecular components of PIN clusters
To identify the proteins that compose PIN clusters, I performed immunoprecipitation of PINVenus and MAB4-CFP. So far, amount of proteins that are precipitated have not been sufficientfor the subsequent analysis. This suggests low affinity of antibody or low expression levels of
PIN and MAB4 proteins. To conquer these problems, I am planning to increase amount of plants
for extracting proteins. I am also planning to stabilizing protein complexes of PIN and MAB4 byperforming chemical fixation using FAA.
2) Observation of spatio-temporal dynamics of PIN clusters
Spatio-temporal dynamics of PIN clusters
I found that MAB4-GFP proteins begin to localize at PMs upon the completion of cell division,

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for the Promotion of Scienceduring which PIN clusters gradually formed at nascent PMs. Emergence of PIN clusters andthat of MAB4 at PMs appears to take place at the same time. I also detected enhanced PIN2
endocytosis as well as the decreasing amounts of MAB4-CFP at PMs upon gravity stimulus.
These findings suggest the crucial roles of MAB4 in PIN cluster formation as well as inhibiting
endocytosis of PIN proteins.
In vivo investigation of protein-protein interactions between PIN and MAB4
I found that PIN2 and MAB4 proteins were highly colocalized, where they displayed physicalinteractions.

References
Satoshi Naramoto*Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar
auxin transport Curr Opin Plant Biol. 2017 Dec;40; 8-14
Satoshi Naramoto* and Junko Kyozuka.
ARF GTPase machinery at the plasma membrane regulates auxin transport-mediated plant
growth.
Plant Biotechnolol. 2018 Jun 15;35(2):155-159

一般の皆様へオーキシンは植物の発生・成長に重要な役割を果たすことから、農学的に重要とされ、除草剤や単為結果誘導剤として利用されてきた。本研究により得られた知見を発展させ、オーキシンの作用機構の理解を深めることは、将来の持続的な社会を実現する上で有用な、新たな農業方策の礎となると期待される。

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Physiological roles and molecular mechanisms underlying the synaptic
plasticity in the dentate gyrus
Yuki Hashimotodani
yhashimo@mail.doshisha.ac.jp
Doshisha University, Organization for Research Initiatives and Development

Summary Abstract
Mossy cells (MCs) in the dentate gyrus receive inputs from dentate granule cells (GCs)and project back to GCs, making positive feedback loop. We previously found that long term
potentiation (LTP) is induced at MC-GC synapses. This LTP requires postsynaptic BDNF/
TrkB and presynaptic cAMP/PKA signaling. As MC-GC LTP is expressed presynaptically, it
is postulated the presence of retrograde messenger. In this study, we investigated the molecular
identity of the retrograde messenger in MC-GC LTP.
Key Words：Hippocampus, granule cell, dentate gyrus, LTP

Introduction
Excitatory MCs in the dentate gyrus receive inputs from dentate GCs and project back to GCs
locally, contralaterally, and along the longitudinal axis of the hippocampus, thereby establishing
an associative positive-feedback loop and connecting functionally diverse hippocampal areas(ref. 1). We recently reported that MC-GC synapses exhibit presynaptically expressed long-term
potentiation (LTP) (ref. 2). As mechanisms for this LTP, we found that LTP requires postsynaptic
BDNF/TrkB and presynaptic cAMP/PKA signaling. The requirement for postsynaptic signaling
during induction and the presynaptic expression mechanism strongly suggests the involvementof retrograde signaling in MC-GC LTP. However, the molecular identity of the retrograde
messenger remains unknown.

Results

In this study, we tried to identify what molecule(s) mediate retrograde signal in MC-GC LTP.
To this end, we performed whole-cell patch clamp recordings from dentate gyrus GCs in acutehippocampal slices. Excitatory postsynaptic currents (EPSCs) were monitored in the presence of
the GABAA receptor antagonist picrotoxin (100 μM) while presynaptic MC fibers (MCFs) wereactivated by local extracellular stimulation (<150 μm from the recorded GC) in the inner third of thedentate molecular layer. MC-GC LTP was induced by high frequency stimulation of MCFs (5 pulses,
100 Hz, repeated 50 times every 0.5 s).
We first tested the effects of cannabinoid receptor antagonist, as endocannabinoids are the best
characterized retrograde messengers in the central nervous system (ref. 3). We found that MCGC LTP was normally induced in the presence of CB1 receptor antagonist AM251, suggesting that
endocannabinoids are not involved in the induction of MC-GC LTP. Next, we tested the involvementof nitric oxide, well known inter cellular messenger. We found that application of nitric oxide
synthase inhibitor L-NAME did not block induction of LTP, excluding the contribution of nitric oxidein LTP induction. We further tested the effects of several candidate receptor antagonists on MCGC LTP. We found that MC-GC LTP was normally induced in the presence of group I metabotropic

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glutamate receptor antagonist, neurokinin 1 receptor antagonist or muscarinic acetylcholine receptor
antagonist, suggesting that these receptors are not involved in the induction of MC-GC LTP.
As presynaptic cAMP/PKA signaling is required for the induction of MC-GC LTP (ref. 2), we
considered that Gs protein coupled receptors may involve in the induction of MC-GC LTP. In thisregard, we tested the effects of substance X, which is a ligand for substance X receptor and producescAMP through Gs protein. We found that substance X receptor antagonist blocked MC-GC LTP,
suggesting the contribution of substance X receptor to LTP induction. To test whether substance X
receptor activation is sufficient for the induction of MC-GC LTP, we applied substance X and foundthat bath application of substance X potentiated MC-GC EPSCs, indicating that substance X receptor
activation directly triggers LTP induction. In the future study, we will have to further investigate
whether substance X actually works as a retrograde messenger in MC-GC LTP.

Discussion & Conclusion
In this study, we provide the evidence for the molecular identity of retrograde messenger in MCGC LTP. We first excluded the possible involvement of endocannabinoid, nitric oxide, group I
metabotropic glutamate receptor, neurokinin 1 receptor and muscarinic acetylcholine receptor in
MC-GC LTP. We finally found that substance X mimicked MC-GC LTP and blockade of its receptor
abolished MC-GC LTP, suggesting the involvement of substance X in MC-GC LTP. Future studies
will have to investigate how high frequency stimulation triggers postsynaptic release of substance X.

References
1.Scharfman and Myers, Front Neural Circuits 6: 106, 2013.
2.Hashimotodani et al. Neuron 95: 928, 2017.
3.Kano et al. Physiol Rev 89: 309, 2009.

一般の皆様へ
海馬は記憶や学習に重要な役割を担う脳領域であると考えられてきました。海馬の神経回路

は主に歯状回、CA1,CA3という領域で形成され、その間のシナプス結合がこれまで盛んに研究されてきました。本研究では歯状回門に位置する苔状細胞と呼ばれる興奮性ニューロンに焦点を当て、そのシナプス可塑性の分子メカニズムを調べました。苔状細胞は近年注目されるようになり多くの研究成果が発表されつつあります。今後は海馬の神経回路および記憶への寄与を考えるうえで苔状細胞が重要な位置を占める可能性を秘めており、本研究もその一助になると期待されます。

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Genomic analysis of hematopoietic stem cell niche in
myelodysplastic syndrome.
Hidekazu Nishikii
Nishikih66@md.tsukuba.ac.jp
Department of Hematology, Faculty of Medicine, University of Tsukuba

Summary Abstract
Normal hematopoietic system is maintained by the interaction between hematopoietic stem cells(HSCs) and the components of bone marrow microenvironment called HSC niche, such as mesenchymal stem cells, adipocytes, endothelial cells, perivascular stromal cells, and endothelial cells.
Myelodysplastic syndromes (MDS) are both clonal disorders of HSCs in the bone marrow. In current
study, we performed multicolor visualization of the HSC niche in normal samples and its changes
in MDS patients using the tyramide signal amplification (TSA) system. Further, we performed transcriptome analyses of several HSC niche components using leukemic mouse model.
Key Words：Myelodysplastic syndrome, acute leukemia, stem cell niche, stromal cells

Introduction
MDS are clonal disorders of HSCs and frequently progress to acute myeloid leukemia (AML).
These two diseases are occasionally complicated with bone marrow (BM) fibrosis, which is
associated with poor prognosis. Previous literatures showed that C-X-C motif chemokine ligand
12 (CXCL12) -positive mesenchymal cells(1) and glial fibrillary acidic protein (GFAP) -positive
Schwann cells (2) are major components of HSC niche, which plays a crucial role for maintaining
HSCs. In current study, we performed multicolor visualization of the HSC niche in normal samples
and its changes in MDS-with fibrosis (MDS-F) patients. Gene expression analyses of HSC niche inmouse AML model were also performed.

Results
1. Various types of non-hematopoietic cells in normal BM were visualized by TSA staining.
Non-hematopoietic cells, such as endothelial cells, mesenchymal cells, and neural cells
are thought to exist in the BM with low frequencies. To visualize the BM microenvironment,
multi-color TSA staining was performed using several antibodies. Using TSA staining methods
combined with a fluorescence spectrum analyzer, we got simultaneous multi-color images. Thissystem allows multi-color staining with the use of unlabeled primary antibodies derived evenfrom the same species because
residual antibody is removed ineach staining step. We analyzed thefrequencies of PLIN+adipocytes,
CD34+endothelial cells, GFAP
Schwann cells, and heterogenous
mesenchymal cells (Figure 1).
When compared between normal

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The Novartis Foundation(Japan)
for the Promotion of Scienceand MDS-F patient samples, the frequencies of CXCL12+ cells, GFAP+ cells were significantly
increased.
Figure1.Multicolor images of human BM specimen. Representative images of CD146, s100,
CD34, CXCL12, PLIN stained by TSA staining were shown.
2. Transcriptome analyses of HSC niche components in leukemic mice.
To investigate the changes the components of HSC niche in myeloid malignancies, we next
performed the transcriptome analysis of stromal cells in mouse leukemic model. We generated
MLL-AF9 induced AML mouse model using retroviral transduction and isolated endothelial cells(ECs), perivascular stromal cells (PVS), and mesenchymal stromal cells (MSC) by flow cytometry.
Then, we performed RNA sequencing using isolated cells from normal or leukemic mouse (Figure2). In principal component (PCA) or heat map analysis, the gene expression pattern of the samples
from EC, PVS, and MSC from AML or normal mice were distinct. Of note, the expression levelof cxcl12 in PVS was significantly reduced in AML mouse. These data suggested that proliferationof leukemic cells in bone marrow induced not only direct the inhibition of normal hematopoietic
cells but also the defect of niche function.

Figure 2. Transcriptome analysis of HSC niche components from leukemic mouse model.
(i) Representative data of flowcytometry analysis using collagenase treated bone marrow cells.
Hematopoietic cells (CD45 and TER119 positive cells) were eliminated. (ii) (iii) PCA plot data andheat map using the samples from niche components derived from normal or leukemic mice wereshown.

Discussion & Conclusion
In current study, we demonstrated the changes in bone marrow environment in myeloid malignancies using multicolor immunostaining and gene expression profiles. Previous studies suggested that
defects of function in HSC niche might induce hematopoietic failure or hematologic malignancy(3). Our data also suggested that the proliferation of leukemic cells induces the remodeling of HSC
niche, resulting in the impairment of normal HSC maintenance. We are now investigating how leukemic cells affect the function of HSC niche in MDS or AML patients. Further, we are seeking thetherapeutic target for maintenance of HSC niche.

References
1. Ding L, Morrison SJ. Nature 2013 495(7440):231.
2. Yamazaki S. Ema H, Karlsson G. et al. Cell. 2011 147(5):1146.
3. Raaijmakers MH, Mukherjee S, Guo S et al. Nature.2010 464(7290):852.

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一般の皆様へ
骨髄に存在する造血幹細胞は造血幹細胞ニッチと呼ばれる微小環境により維持されると考えられているが、骨髄異形成症候群 (MDS) や急性骨髄性白血病（AML）における骨髄環境の変化の詳細やその病的意義には不明な点が多い。今回の研究で我々は患者サンプル・マウスモデル

を用いて新規多重染色システムを用いた造血幹細胞ニッチの可視化・各ニッチ細胞の遺伝子発現変化の詳細を明らかにし、ニッチ細胞の機能的変化が造血器腫瘍における正常造血障害の一因となっている事を明らかにした。現在この結果をもとに、造血器腫瘍の造血障害に対する治療標的の探索を行っている。

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Expression change of desmoplakin by antidepressant treatments andits function in dentate gyrus of hippocampus
Eri Segi-Nishidaeri.segi.nishida@rs.tus.ac.jp
Department of Biotechnology, Tokyo University of Science

Summary Abstract
Desmoplakin, a component of desmosome, is highly expressed in the dentate gyrus (DG) in thebrain, although the existence of desmosome in neurons is not fully recognized. We found that the expression of desmoplakin protein in DG is decreased by chronic antidepressant treatment. Knockdownof desmoplakin in the DG suggests its function of hippocampal neurogenesis.
Key Words：desmoplakin, antidepressants, hippocampus

Introduction
Desmosome are adhesive intercellular junctions of epithelia and cardiac muscles. Desmoplakin, acomponent of desmosome, is highly expressed in the dentate gyrus (DG) in the brain. However, thefunction of desmoplakin in the DG has been largely unknown. We previously found that the expression of desmoplakin in mouse hippocampus is decreased by antidepressant treatments (Imoto et al.
2017).

Results
We first examined the effect of repeated electroconvulsive seizures(ECS), a model of ECS therapy, and chronic treatment of fluoxetine,
serotonin selective reuptake inhibitor, on the gene expression ofdesmoplakin in the DG. Both treatments significantly decreased theexpression of desmoplakin (Figure). Since it has been suggested thatthe expression of desmoplakin in the brain is restricted in the DG,
we next examined the protein localization of desmoplakin in the DG
by immunohistochemistry. Desmoplakin immunoreactivities weredetected in the cell bodies of granule neurons in the DG, suggesting
a potential role of this molecule in cell body adhesion. Consistent
with the gene expression analysis, the immunostaining signals ofdesmoplakin were dramatically decreased by chronic treatment of ECS. To examine the role ofdesmoplakin in the DG, we tried knockdown of desmoplakin expression using adeno associatedvirus containing artificial miRNA for desmoplakin sequence. Although we could not detect a
decrease of desmoplakin mRNA, the protein expression of desmoplakin was clearly suppressed
4 weeks after AAV injection in the DG. We first examined the expression changes in the DG bydesmoplakin knockdown focusing on the neuronal maturation. We found that the expression of
DG specific neuronal maturational marker, including calbindin (Calb1) and Tdo2 (tryptophan 2,3dioxygenase), was significantly increased in the knockdown samples. This is unexpected, since
we previously reported that the expression of Calb1 and Tdo2 was significantly decreased by ECS
and SSRI treatments. We next examined the influence of desmoplakin knocking down on the adult

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neurogenic process in the DG (Ueno et al. 2019). It has been known that antidepressant treatment
increase multi steps of neurogenic process including neuronal stem/progenitor proliferation, neuronaldifferentiation, survival, and maturation. The numbers of immature neuronal cells were significantly
decreased in the DG of knockdown animals. This result suggests that desmoplakin play a role infacilitating neuronal differentiation in the adult neurogenesis of the DG. In summary, these results
implicate the involvement of desmoplakin in functional modulation of the hippocampus.

Discussion & Conclusion
Desmoplakin interacts with desmosomal cadherins via plakoglobin connecting desmosomes withintermediate filaments of the cytoskeleton. Although there has been no evidence of the presenceof desmosomes in the neurons in the DG, interaction of desmoplakin with plakoglobin might haveinfluence in the cellular signaling, because plakoglobin can translocate cell membrane to nuclear andinteract with a transcription factor TCF. Elucidation of intracellular signaling by desmoplakin could
suggest a novel regulation of neural differentiation via desmosomal adhesion.

References
1) Imoto Y., Segi-Nishida E, Suzuki H., Kobayashi K: Rapid and stable changes in maturationrelated phenotypes of the adult hippocampal neurons by electroconvulsive treatment. Mol Brain,
10:8, 2017 doi: 10.1186/s13041-017-0288-92) Ueno M, Sugimoto M, Ohtsubo K, Sakai N, Endoh A, Shikano K, Imoto Y, Segi-Nishida E. The
effect of electroconvulsive seizure on survival, neuronal differentiation, and expression of thematuration marker in the adult mouse hippocampus. J Neurochem. 149(4):488-498. 2019. doi:
10.1111/jnc.14691

一般の皆様へ海馬は学習・記憶に関わる重要な部位です。海馬の歯状回は、神経細胞が層状に並んでおり、お互いに接着する特徴的な構造を持っています。また、この部位では成体でも神経の新生が起きることが知られています。本研究では、歯状回神経に特異的に発現するデスモプラキンという接着構造を制御する因子に着目し、その機能の解析を行いました。

私たちは、デスモプラキンの機能意義を調べるために、成体マウスでの海馬歯状回でのデスモプラキン発現を減少させました。その結果、神経の成熟マーカーの発現増大や神経新生の減少が観察され、デスモプラキンが海馬の神経機能調節に重要な役割を持つことが明らかになりました。

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Functional analysis of the GRHL3 factor in epithelial fusion.
Chiharu KIMURA-YOSHIDA
chiharu@wch.opho.jp
Department of Molecular Embryology Research Institute, Osaka Women’s and Children’s
Hospital, Osaka Prefectural Hospital Organization

Summary Abstract
Abnormalities affected in a cleft lip or palate, are among most common of all birth defects.
Human GRHL3 is known to cause to Van der Woude syndrome (VWS), which shows thedisorders are born with a cleft lip and cleft palate (Peyrard-Janvid et al., 2014). We tried toshow the same abnormalities by mouse Grhl3-mutation with gene targeting. We identified
that mouse Grhl3-deficiet mice did not display either cleft palate or cleft lip, and led to mild
phenotypes in rugae patterning morphologically and molecularly.
Key Words：Grainyhead gene, cleft palate, rugae, knockout mouse

Introduction
Grainyhead-like (Grhl) genes paly a crucial roles in epithelial development, among animal
kingdoms. In vertebrate, Grhl orthologues are required for the morphogenic development of theepidermal barrier, of the neural tube closure and maintenance of neural cell survival (KimuraYoshida et al., 2018; Mathiyalagan et al., 2019). It remains unknown whether mutations ofmouse Grhl3 lead to clefting of the secondary plate, or not, although it is well established thatoral cleft in in Van der Woude syndrome (VWS) is caused by human GRHL3 (Peyrard-Janvid etal., 2014).

Results
To establish the function of Grhl3 during
cleft palate development, we use the two mousemutant lines of Grhl3-deficient; Grhl3cre-KI and
Grhl3 ∆Exon2 mutant mouse (Fig.1). Grhl3 creKI
mice were generated by crossing between
Grhl-cre-IRES-nlslacZ (IRES; international
ribosome entry site, nls; nuclear localization
signal) knock-in mice, which were obtained
from the Mutant Mouse Regional Resource
Center (MMRRC), and β-actin cre (ubiquitous
cre-expressing) transgenic mice (Lewandoski etal., 1997). And, Grhl3∆Exon2 heterozygous mousewere obtained by crossing Grhl3tm1a(EUCOMM)Wtsi stock (International Mouse Phenotyping Consortium)with Cre-expressing mice. Grhl3 cre-KI and Grhl3 ∆Exon2 mice were maintained on the ICR and
C57BL/6N background respectively. Noon of day in which the vaginal plug was detected was taken
as embryonic day 0.5 (0.5) of development following overnight mating. Mice were housed and bred

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according to standard condition with food andwater ad libitum and were maintained on a 12
h day/night cycle. All animal experiments were
followed fundamental guidelines for the proper
conduct of animal experiments and related
activities in academic research institute under
the jurisdiction of the MEXT in japan, approvedby institutional committees at the Research
institute Osaka Women’s and Children’s
Hospital for animal and recombinant DNA
experiments. We analyzed the palate of Grhl3creKI
homozygous and Grhl3 ∆Exon2 homozygous
embryos at E18.5 (Fig.2 and data not shown).
Phenotypes were determined for at least threeembryos in all cases.
Outlook and gross morphological analysis
revealed correctly formed palate in Grhl3creKI
homozygous and Grhl3 ∆Exon2 homozygous
mutant mice at E18.5 (Fig.2). The palate
shelves from the left and right sides were fusedat the midline along the antero-posterior axis
in wild type embryos. Unexpectedly, both ofmutant embryos, Grhl3 cre-KI homozygous andGrhl3 ∆Exon2 homozygous mutant mouse wereappeared normal development in palates (Fig.2).
These results indicated that in Grhl3-deficientmouse embryos, development of palate was notaffected. mutation.
Thus, mouse models carrying mutations in
the Grhl3 gene may not recapitulate the defectsof the cleft palate, of those observed in humandisease caused by GRHL3 mutation. Palatalrugae are periodic ridges on the hard palateof mammals which are involved in sensing
and holding food. Rugal patterning may be
a sensitive indicator of environmentally or
genetically caused congenital abnormality.
The number of rugae varies between species;
pigs have 21 (Pospieszny et al., 2003), humanhas 4 and mice 8 (Pantalacci et al., 2009).
Studies in the mouse have shown that rugae,
marked initially by Shh (encoding Sonic
hedgehog) expression, appear sequentiallyduring embryonic development (Fig.3). We
examined Shh expression by whole-mount in

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The Novartis Foundation(Japan)
for the Promotion of Sciencesitu hybridization of established methods (Wilkinson, 1998) in each Grhl3-deficient mouse embryos
at E18.5 (Fig.3). In both mutant embryos, the levels of Shh-expression appeared to be lower thanthose of wild type embryos. In addition, Shh is expressed in the hair follicle germ and, subsequently,
in the hair matrix of wild type embryos (Petiot et al., 2003), these expressions showed completely
lost in Grhl3-deficient embryos (Fig.4). Moreover, in Grhl3 cre/cre homozygous mutant embryos
were together with partialy disorganized rugae, ectopic rugae formation. However, in Grhl3∆Exon2
homozygous mutant embryos, rugae appeared almost to be normally patterned and formed properly.
These findings demonstrated that although Grhl3 might be dispensable for the rugal formation, Grhl3
palyed the role of correct Shh expression in the mouse rugal region.

Discussion & Conclusion
This study demonstrated VWS (Van der Wounde Syndrome), which is known to be caused
by mutation human GRHL3, was not appeared in Grhl3-deficient mice (Fig. 1) This suggeststhat some other co-factor, like IRF6, or Grhl-family genes may partially compensate for the
craniofacial disorder (Ingraham et al., 2006). Additionally, Grhl3-deficient embryos did notshow the disorder of rugae patterning (Fig.3). Since recent report demonstrats that SPECC1L,
downstream of IRF6, regulates palate and rugae patterning, Grhl3 might be come activate only
cooperatively with IRF6 for the facial development.
Furthermore, given that Grhl3 expression in the skin might regulate Shh activity, providing a
Shh-Patched signaling network in skin development including hair follicle (Zheng et al., 2019).

References
Ingraham, C. R., Kinoshita, A., Kondo, S., Yang, B., Sajan, S., Trout, K.J., Malik,M.I., Dunnwald,

M., Goudy, S.L., Lovett, M., Murray, J.C. Schutte, B.C. Abnormal skin, limb and craniofacial
morphogenesis in mice deficient for interferon regulatory factor6(Irf6). Nature Genetics. 38(11)1335-1340 (2006).
Kimura-Yoshida, C., Mochida, K., Ellwanger K., Niehrs, C. and Matsuo, I. Fate specificationof neural plate border by canonical Wnt signaling and Grhl3 is crucial for neural tube closure.
EBiomedicine (2) 513-527 (2015).
Kimura-Yoshida, C., Mochida, K., Nakaya M., Mizutani, T. and Matsuo, I. Cytoplasmic localizationof GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis.
Nature Communications. 9 4059 (2018).
Lewandsoki, M., Meyers, E.N and Martin, G. R. Analysis of Fgf8 gene function in vertebrate
development. Cold Spring Harb. Symp. Quant. Biol. 62, 159-168 (1997).
Pantalacci, S., Prochazka, J., Martin, A., Rothova, M., Lambert, L., Bernard, L., Charles, C., Vlriot
L., Peterkova, R and Laudet, V. Patterning of palatal development. BMC Dev. Biol. 8 116 (2008).
Petiot, A., Conti, F.J., Grose, R., Revest, J. M., Hodivala-Dilke, K.M. and Dickson, C. A crucial rolefor Fgfr2-IIIb signaling in epidermal development and hair follicle pattering. Development 130 (22)5493-5501 (2003).

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Peyrard-Janvid M., Leslie E. J., Kousa Y.A., Smith T.L., Dunnwald, M., Magrnusson, M., Lentz,
B.A., Unneberg, P., Fransson, I., Koillinen, H. K. Dominant mutations in GRHL3 cause Van derWoude Syndrome and disrupt oral periderm development. Am. J. Hum. Genet. 94, 23-32 (2014).
Pospieszny, N., Janeczek, M. and Kleckowska, J. Morphology of the incisive papilla (papillaincisive) of pigs during different stages of their prenatal period. EJPAU 6 (1), Veterinary #04 (2003).
Zheng, L., Rui, C., Zhang, H., Chen J., Jia, X and Xia, Y. Sonic hedgehog signaling in epithelial
tissue development. Regen. Med. Res. 7 (3) (2019).

一般の皆様へ
日本人の間で発症頻度が高いことで知られている「口唇口蓋裂」先天異常の実験モデルマウ

スを作製することを目的に、Grhl3 遺伝子欠損マウスの表現型解析を行った。結果、我々が作製した Grhl3 遺伝子欠損マウスでは口唇口蓋裂の異常は見られなかったが、口腔内の横口蓋ヒダに一部形成異常やマーカー遺伝子の発現異常が見られた。

今後、Grhl3 遺伝子と共に働く遺伝子（例えば IRF6遺伝子など）の遺伝子欠損マウスと交配させダブル変異マウスを作製し、口唇口蓋裂の発症の有無を検討する予定である。

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Identification and molecular mechanism of novel calcium transportdisease – possible therapeutic target for bone mineralization defects
Yoshiro Suzuki
yoshiros@iwate-med.ac.jp
Department of Physiology, Iwate Medical University

Summary Abstract
Calcium (Ca2+) is essential for many physiological functions. The blood Ca2+ is tuned by acoordination with small intestine, kidney and bones. Bones act on our structural element as well
as a large reservoir of Ca2+. However, the mechanism by which bone Ca2+ is maintained have notbeen uncovered yet. In this project I investigated the role of TRPV6 in the epithelial Ca2+ absorption
crucial for the bone development, focusing on the variants identified from the individuals withtransient neonatal hyperparathyroidism (TNHP). Results suggest that combination of the variant is a
key determinant for the onset of the bone phenotype.
Key Words：TRP channel, Ca2+ imaging, patch-clamp

Introduction
Blood Ca 2+ is finely maintained by a coordination between intestine, kidney and bone.
During bone development, Ca2+ should be transported through the intestine or the placenta. The
transcellular Ca2+ transport pathway is achieved by components including Ca2+ uptake througha channel. Recently, TRPV6 variants were identified as causative variants of transient neonatal
hyperparathyroidism (TNHP) with an impaired bone mineralization. TRPV6 may act as an apicalCa2+ channel in the placenta for the maternal-fetal Ca2+ transport crucial for fetal bone mineralization.
This project focuses on the association between novel TRPV6 variants and bone phenotypes whichmight be caused by an impaired Ca2+ transport due to a dysregulation of TRPV6 channels.

Results
Previous analysis revealed that TNHP patients possessed a set of different type of TRPV6 variants
which located completely different place of the channel, suggesting a distinct disease mechanism
from previous report (1).
To reveal the functional significance of these variants, I introduced them into TRPV6 cDNAand transfected into HEK293T cells. Patch-clamp recordings indicated that it was not significantly
different between WT and these variants in the aspect of current amplitudes, ion selectivity, as well
as fast inactivation kinetics. However, when I looked at cells more carefully, I found the plasma
membrane Ca2+ permeability(2, 3) in a steady state was significantly higher in variants compared
to WT. Indeed, Western blotting data supported this phenomenon; the band of HEK cells with thevariant was smaller since cells detached from the culture dish due to the Ca2+ overload.
These results suggest that the variant might affect the endogenous inactivation mechanism for
TRPV6, for example, a direct regulation of ATP by binding, or indirect regulation of ATP via

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The Novartis Foundation(Japan)
for the Promotion of Sciencephosphorylation, both of which might affect the TRPV6 channel activity in the plasma membraneof the HEK293T cells. Overexpression of TRPV6 might cause Ca2+ overload and a decrease in
ATP concentration to pump out Ca2+ by the plasma membrane Ca2+-ATPase (PMCA), most likely
resulting in a down-regulation of TRPV6 channel activity(4). We hypothesized that the variant was aphosphorylation-inactivated variant which might not arrow this negative feedback regulation in case
ATP concentration was decreased.
Interestingly, this site seemed to be partially overlapped with a putative ATP binding site (5).
Therefore, future studies should reveal further molecular mechanism of the regulation of TRPV6 bycoupling with intracellular ATP concentration crucial to archive the energy-dependent epithelial Ca2+transport.
Regarding another variant (variant B), since TRPV6 forms
tetramer, I hypothesized that it could not translocate into theplasma membrane. Indeed, Western blotting with biotinylation
showed an impaired trafficking with the variant. When it was
expressed in an epithelial cell, it could result in a disruption of
the unidirectional Ca2+ transport. Future studies could reveal thefunctional significance of the variant B in Ca2+ transport whose
disruption causes bone phenotypes.

Discussion & Conclusion
These results suggest that there are TNHP patients who have different type of TRPV6 mutations.
The disease onset could be completely different from that of previously reported TNHP. Thesemutations could be associated with bone phenotypes most likely because the variant affects theregulation of TRPV6 channel by phosphorylation and another one affects membrane trafficking. I
conclude this kind of combination leads to bone phenotypes at least in part.
In the literatures, bone diseases such as osteogenesis imperfecta are caused by the abnormalitiesof the structural proteins including collagens. My results provide another possibility of the bonedisease: insufficient calcium supply could lead to bone disease as well. TRPV6 might be a goodtherapeutic target for bone mineralization diseases.

References
1. Suzuki Y, Chitayat D, Sawada H, Deardorff MA, McLaughlin HM, Begtrup A, et al. TRPV6
Variants Interfere with Maternal-Fetal Calcium Transport through the Placenta and Cause
Transient Neonatal Hyperparathyroidism. Am J Hum Genet. 2018;102(6):1104-14.
2. Loh NY, Bentley L, Dimke H, Verkaart S, Tammaro P, Gorvin CM, et al. Autosomal dominant
hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5.
PLoS One. 2013;8(1):e55412.
3. Suzuki Y, Watanabe M, Saito CT, Tominaga M. Expression of the TRPM6 in mouse placental
trophoblasts; potential role in maternal-fetal calcium transport. J Physiol Sci. 2017;67(1):151-62.
4. Al-Ansary D, Bogeski I, Disteldorf BM, Becherer U, Niemeyer BA. ATP modulates Ca2+ uptake
by TRPV6 and is counteracted by isoform-specific phosphorylation. FASEB J. 2010;24(2):42535.

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5. Saotome K, Singh AK, Yelshanskaya MV, Sobolevsky AI. Crystal structure of the epithelial
calcium channel TRPV6. Nature. 2016;534(7608):506-11.

一般の皆様へ
私は骨疾患の原因の１つとして Ca2+ 輸送上皮における Ca2+ 輸送機能低下に着目して研究を行った。本研究の結果、体内に Ca2+ を取り込む TRPV6というイオンチャネルの遺伝子変異によって家族性の骨疾患を発症することが明らかになりつつある。骨疾患は骨の土台となるコラーゲ
ンなどのタンパク質成分の異常だけでなく、石灰化のための材料となる Ca2+ の体内動態を含めて解析する必要があることがわかった。今後、その分子メカニズムの詳細を明らかにすることによって、TRPV6が骨疾患の治療の際の良いターゲットになることを示していきたい。

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Nuclear noncoding RNA degradation regulates
innate immune response
Nobuyoshi Akimitsu
akimitsu@ric.u-tokyo.ac.jp
Isotope Science Center, the University of Tokyo

Summary Abstract
In this study, I aimed to reveal the molecular mechanism of diminishment of nuclear RNA
degradation factors, MTR4 and RRP6, in response to infection of Salmonella, an invasive bacteria.
We found that SPI1/2 factors are involved in the diminishment of nuclear RNA degradation factors.
Key Words：Salmonella, nuclear RNA decay, infection

Introduction
Salmonella enterica serovar Typhimurium (Salmonella), a pathogenic bacterium, is a major causeof foodborne diseases worldwide. Salmonella injects multiple virulence factors, called effectors,
into cells and causes multiple rearrangements of cellular biological reactions that are important for
Salmonella proliferation and virulence.
We have shown that loss of MTR4 and RRP6, which are nuclear RNA degradation factors, areoccurred in response to Salmonella infection. The depletion of nuclear RNA degradation factors
causes the stabilization and accumulation of unstable nuclear RNAs. In this study, we examined aseries of Salmonella mutant strains to understand molecular mechanism of depletion of nuclear RNA
degradation factors.

Results
Firstly, Salmonella enterica serovar Typhimurium (Salmonella) mutant strains were constructedbased on wild type Salmonella. Cultured Salmonella was collected by centrifuge and resuspended
with a corresponding volume of 1 × PBS before infection. Hela cells were infected with Salmonella
strains or heat-killed Salmonella at 100 multiplicity of infection (moi). Then we showed that live
Salmonella, but not dead Salmonella, induces MTR4 and RRP6 degradation (Figure1). In addition,
LPS did not decrease MTR4 and RRP6. These show that live Salmonella, but not dead Salmonella,
induces MTR4 and RRP6 degradation.
We hypothesized that MTR4 and RRP6 are not degraded by infection if important effector(s)involved in the degradation of these proteins are mutated. Based on this idea, we constructed 42
Salmonella mutant strains and examined their effect for loss of MTR4
and RRP6. WB analysis was performed to examine the degradation of
MTR4 and RRP6 upon infection of these mutant strains. All 36 strains
induced loss of MTR4 and RRP6, except ΔHilCΔHilD, ΔHilD, ΔPrgI,
ΔFlhDΔFlhC, ΔClpXP and ΔAroA.
Considering the growth condition of these mutant strains, next, we
examined the proliferation of mutant strains in HeLa cells by monitoring

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the amount of the 16S ribosomal RNA gene (16S rRNA gene). Among these mutants, six mutant
strains, ΔHilD, ΔHilCΔHilD, ΔPrgI, ΔFlhDΔFlhC, ΔClpXP, and ΔAroA, did not grow well in thecells.
HilC and HilD, transcriptional regulators encoded in SPI-1, are co-regulated and directly activate
the expression of HilA , the central player of T3SS-1 regulation. In addition, HilD is necessary
for activating regulons of both SPI-1 and SPI-2. PrgI constitutes the needle of the T3SSs and isof great importance to effector translocation. T3SSs derive from flagella and still share regulatory
mechanisms with them, after mutating the gene of the flagellum, the mutant strain ΔFlhDΔFlhC
also showed a poor growth rate. The ClpXP protease, a member of the ATP-dependent proteasefamily, is reported to regulate flagellum synthesis and SPI-1 expression negatively through FlhD4C2
degradation. As an auxotrophic mutation, deletion of aroA is commonly studied for attenuation
without losing the ability of immunostimulation. Felgner et al. found that deletion of aroA affects
flagellin phase variation and the expression of virulence-associated the arnT and ansB genes. These
genes, which show a poor growth condition, may greatly contribute to Salmonella invasion and/or
proliferation in host cells.
The most of which are strains with genes related to effectors translocated by T3SSs encoded on
Salmonella pathogenic islands, SPI-1 and SPI-2, that have been depleted. Among 42 Salmonellamutants, 6 mutants’ infections canceled loss of MTR4 and RRP6. Proliferation assay of Salmonellain the cell revealed that six mutants showed poor proliferation in the host cell, demonstrating thatpoor proliferation contributed to cancellation of MTR4 and RRP6 loss. This result indicates thatcertain events associated with Salmonella proliferation in host cells cause loss of MTR4 and RRP6.

Figure 1. Alive Salmonella, but not heat-killed
Salmonella or LPS, induced loss of MTR4 and RRP6.
(A) MTR4 and RRP6 degradation upon Salmonella
infection. NI: no infection; WT: wild type Salmonella.
(B) LPS and heat-killed Salmonella did not induce lossof MTR4 and RRP6.

Discussion & Conclusion
In this study, although we mainly explored the effectors contributing to the degradation of MTR4
and RRP6, none of the well grown mutant strains canceled the degradation of MTR4 and RRP6.
Several possibilities may contribute to this result. First, there may be no such effector for inducing
loss of MTR4 and RRP6; instead, the loss might be the result of a complex immune response rather
than a specific gene. In addition, a previous study showed that killed Salmonella or its LPS cannot
induce lncRNA or eRNA, which may indicate that only those Salmonellae that are alive and able
to invade the host cells can induce loss of MTR4 and RRP6. Our study indicates that certain events
associated with Salmonella proliferation in the host cell causes loss of MTR4 and RRP6, resulting
in nuclear RNA stabilization. Because limited mutants were examined here, we cannot exclude
the possibility that there might be such genes, but they are not included in the mutants that weconstructed.

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References
Imamura K, Takaya A, Ishida Y, , et al. Diminished nuclear RNA decay upon Salmonella infection
upregulates antibacterial noncoding RNAs. The EMBO Journal. 2018; vol: 37 (13).

一般の皆様へSalmonella enterica serovar Typhimurium (Salmonella), a pathogenic bacterium, is a major causeof foodborne diseases worldwide. In this study, we revealed the molecular mechanism of Salmonella
genes in the regulation of host gene expression through nuclear RNA degradation.

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Projection-specific circuit formation in the developing cerebral cortex
Yoshiaki Tagawa
tagawa@m.kufm.kagoshima-u.ac.jp
Kagoshima University Graduate School of Medical and Dental Sciences

Summary Abstract
Understanding mechanisms underlying circuit formation in the developing brain is crucial to deal
with brain developmental disorders. We found that the specific pattern of spontaneous network activity in neonatal mouse cortex is essential for activity-dependent callosal axon projections, the major
neuronal pathway in the mammalian cerebral cortex. Finding a target molecule that regulates suchactivity is a key to establish an animal model for brain developmental disorders.
Key Words：Cerebral cortex, development, corpus callosum, neuronal activity, sub-network

Introduction
The formation of neural circuits in the mammalian brain depends on activity-dependentand –independent mechanisms. Understanding such mechanisms is crucial to deal with brain
developmental disorders. Callosal projections are among the major neuronal pathways, connecting
the two cortical hemispheres. Defects in callosal projections are frequently reported in brain
developmental disorders. The aims of this study are to reveal how neuronal activity influences
callosal axon projections and how such activity is regulated.

Results
1) How neuronal activity influences callosal axon projections
To study this, we used the mouse as a model. We visualized callosal axons with fluorescentproteins by in utero electroporation. Callosal axons develop through several successive stages(Tagawa and Hirano, 2012). By using Kir2.1 (a potassium channel used as a molecular tool toattenuate neuronal activity), we previously showed that the formation of callosal projections
was activity-dependent (Mizuno et al., 2007). By using temporally controlled Kir2.1 expression
system, we found that neuronal activity in the second postnatal week was critical for the activitydependent callosal projections. To reveal the activity pattern required for callosal axon projections,
we monitored cortical activity by in vivo two-photon Ca2+ imaging in the temporally controlled
Kir2.1 expression experiments (Hagihara et al., 2015). Cortical activity (spontaneous network
activity) in the second postnatal week can be classified into highly synchronous (H) and lesssynchronous (L) events (Siegel et al., 2012). We found that in a condition where H events were
missing but L events were preserved, callosal axon projections could be formed, suggesting that L
events, but not H events, were sufficient for callosal axon projections.
L events are the network activity in which 20-60% of layer 2/3 cortical neurons are active. What
population of the cells are they? Because callosal projection neurons (CPNs) are a subpopulationof neurons in layer 2/3, we hypothesized that a part of L events are the correlated activity of CPNs.
To test this, we performed dual patch clamp recordings from CPN-CPN pairs and CPN-nonCPN
pairs (a collaboration work with Yoshimura Yumiko Lab in the National Institute of Physiological

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Sciences, Okazaki, Japan). We found that CPN-CPN pairs were more likely connected than CPNnonCPN pairs, suggesting that CPNs form sub-networks in the cortical circuits. Indeed, in adultmouse visual cortex, retrograde-labeling experiments with in vivo two-photon Ca2+ imaging
showed that the activity of CPN-CPN pairs (in response to visual stimulation) was more correlated
than that of CPN-nonCPN pairs (bioRxiv: Hagihara et al., https://doi.org/10.1101/795922). These
results suggest projection target specific sub-networks in the cortical circuits. It is still difficult toperform retrograde-labeling experiments with in vivo two-photon Ca2+ imaging in neonatal brain,
but this is essential to test whether the L events reflect the sub-network activity of CPNs during
the callosal axon projections, and we are trying this experiment.
2) How spontaneous neuronal activity is regulated in neonatal cortex
As mentioned above, spontaneous network activity in the developing cortex is criticallyinvolved in network formation such as callosal axon projections. How is such activity regulated?
To study this, we focused on gap junctions and ion channels. Gap junctions mediate electricalcoupling of neurons, and thus are expected to influence the correlated activity. In cortical layer 5,
gap junctions are involved in projection type specific sub-network activity (Maruoka, Nakagawa
et al., Science, 2017). Patch clamp recordings from cortical layer 2/3 CPNs showed that they were
electrically coupled in the early second postnatal week, suggesting that gap junctions exist as inlayer 5. Gene expression and genetic manipulation experiments are underway in the lab, to clarify
which gap junction subtype is involved in CPNs sub-network formation. Ion channels regulate
and modulate the firing pattern of individual neurons and network activity. By using in utero
electroporation, we expressed several disease-causing mutant ion channels in layer 2/3 neurons,
and found that expressing certain mutant channels impairs callosal axon projections. To link theactivity pattern with callosal projections, we will monitor spontaneous activity in the cortex where
those mutant channels are expressed. Our preliminary data suggest that gap junctions and several
ion channels are expressed in neonatal cortical neurons, and that they play an important role inactivity-dependent callosal axon projections.

Discussion & Conclusion
In this study, we sought to reveal how neuronal activity influences callosal axon projections andhow such activity is regulated in neonatal mouse cortex. We found that neuronal activity in thesecond postnatal week was critical for activity-dependent callosal projections. We also found that
L events, but not H events, played a role in callosal axon projections. To our knowledge, this is thefirst to relate the specific activity pattern (i.e. less correlated network activity) to the network wiringin the developing cortex. We have a hypothesis that L events reflect sub-network activity of CPNs
during the callosal axon projections. Experiments to test this hypothesis are underway in the lab. Ourdata also imply that gap junctions and several ion channels play important roles in activity-dependent
callosal axon projections. Finding a specific target molecule (gap junction and ion channel subtype)will lead to establish an animal model for brain developmental disorders.

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References
Mizuno H, Hirano T, Tagawa Y, Journal of Neuroscience, 27, 6760-6770, 2007
Tagawa Y and Hirano T, Neural Plasticity, 797295, 2012
Siegel F et al., Current Biology, 22(3), 253-258, 2012
Hagihara et al., Nature Neuroscience, 18(12), 1780-1788, 2015
Maruoka et al., Science, 358, 610-615, 2017
Hagihara et al., preprint bioRxiv, https://doi.org/10.1101/795922, 2019

一般の皆様へ
この研究は、哺乳類の脳の代表的な神経経路の１つである脳梁神経回路の形成において、発生・発達期に特有の自発神経活動の役割を調べたものです。脳の中には異なった経路に投射する神経細胞が混在していますが、投射先ごとに機能的にまとまり、同期した神経活動をすることで、正しい回路が形成されるメカニズムがあることが明らかになりつつあります。神経回路の形成メカニズムを明らかにし、それを調節する分子を明らかにすることは、神経回路形成・発達に障害が生じる脳疾患のモデル動物作成につながると期待されます。

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Uncovering the Biomarker for Treatment with ATR Inhibitorin Solid Tumor
Kazumasa Komura
uro051@osaka-med.ac.jp
Osaka Medical College

Summary Abstract
Loss of the male specific histone demethylase KDM5D epigenetically modifies histone
methylation marks and causes gene expression resulting in aggressive prostate cancer. Chlp-seq
analysis revealed that KDM5D preferably binds to the promotor region and the loss of KDM5D
expression with dysregulated H3K4me3 transcriptional marks was associated with the accelerationof cell cycle and mitotic entry leading to increased DNA replication stress. We also found stressinduced DNA damage and reliance on ATR signaling with loss of KDM5D. Blocking ATR activity
by its inhibitor enhanced DNA damage, which leads to subsequent apoptosis, especially in cells with
KDM5D loss.
Key Words：Synthetic lethality, ATR inhibition, DNA replication stress

Introduction
We previously reported an association between decreased expression of the histone modifying
enzyme KDM5D (Lysine-Specific Demethylase 5D) and resistance to docetaxel in PC (Komura etal. PNAS, 2016). We demonstrate that loss of KDM5D leads to an aggressive form of prostate cancer
by epigenetic modifications which promotes cell cycling and DNA replication stress. Furthermore,
we show the synthetic lethal approach with ATR inhibition to the cells with the loss of KDM5D
harboring DNA replication stress, which could be a novel treatment strategy in aggressive prostatecancer.

Results
DNA replication and mitotic activity are tightly coordinated, and dysregulation of theseprocesses has been reported as DNA replication stress in various types of cancer. There has also
been accumulating evidence that DNA replication stress causes continuous DNA damage and isassociated with a worse clinical outcome. To test the hypothesis that loss of KDM5D causes DNA
replication stress as well as continuous DNA damage, we assessed several replication stress and
DNA damage markers. We found increased rH2AX (DNA damage marker) and phosphorylated
replication protein A2 (RPA2) (chromatin-bound ssDNA-binding protein) levels in KDM5D knocked
down LNCaP cells. Activated ATR signaling has been shown to have a central role to prevent thecollapse of stalled replication forks against the DNA replication stress, which in turns prevent
massive double strand break leading to cell death. In addition, ATR-CHK1-CDC25C-cyclin B1/
CDK1 pathway is well-studied cell cycle check point. Phosphorylated CHK1 by ATR activation
inactivates CDC25C by its phosphorylation. This leads to an inhibition of CDK1 activity, which
results in preventing mitotic entry, namely G2/M arrest by the activated ATR. To address how thecells with loss of KDM5D simultaneously evade cell cycle arrest in response to the replication stress,

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we have further analyzed the data obtained from our cell line analysis. In our data, 143 genes wereidentified as negatively correlated genes with KDM5D expression. We compared 138 genes listed
in G2/M check point (MsigDB systematic name: M14052) with those 143 genes, and 7 genes wereidentified as upregulated genes by KDM5D for G2/M check point including CDK1 and CDC25C.
Immunoblotting showed increased phosphorylated ATR as well as elevated CDK1 and CDC25C
protein level in knockdown of KDM5D. The DNA damage markers rH2A.X and phospho-histone
H3, a marker of proliferation phosphorylated during mitosis, were co-localized with knocked down
KDM5D, suggesting their cooperation in replication stress-induced DNA damage and ongoingmitotic entry. We further reviewed our ChIP-seq data and validated increased H3K4me3 levels in
the promotor regions of CDK1 and CDC25C by the loss of KDM5D and confirmed that KDM5D
binds to those promotor regions mediating the chromatin status including H3K4me3. Activated ATR
signaling precludes the collapse of stalled replication forks under replication stress. Given the factthat knockdown of KDM5D led to ATR activation, we next tested VE822, which is the first selective
ATR inhibitor to enter clinical development (ClinicalTrials.gov: NCT02157792 in phase 1 and
NCT02567409 in phase 2) and is now known as M6620, to assess its efficacy in KDM5D knocked
down prostate cancer cells. Compared to LNCaP control cells, treatment with M6620 in KDM5D
knocked down decreased cellular proliferation and enhanced DNA damage shown by increased
rH2AX expression as well as increased cleaved PARP expression indicating increased apoptosis.
We next examined the efficacy of M6620 treatment in a panel of PC cell lines with the KDM5D
status. The CNA status of KDM5D in those PC cell lines were shown in our previous study. Cells
with the deletion of KDM5D showed greater sensitivity to M6620 treatment than those expressing
KDM5D. To further validate these results, we next examine the efficacy of M6620 in vivo xenograftmodel using KDM5D expressing cell lines (LNCaP and 22RV1 cells) and KDM5D deficient PC
cells (LNCaP-104R2 and E006AA cells). After the subcutaneous inoculation of these cells, tumor
was allowed to develop the volume of >150mm3 followed by the randomization of gavage treatmentusing M6620 or vehicle. Strikingly, M6620 treatment significantly inhibited the tumor growth inboth KDM5D deficient cell lines, whereas this effect was not observed in KDM5D expressing celllines.

Discussion & Conclusion
Toledo et al firstly showed that ATR inhibitors elicit the breakage of stalled replication forks andthe combination use of hydroxyurea, which promotes fork stalling due to dNTP depletion leading to
DNA replication stress, synthetically creates double strand break. This synthetic lethality exploiting
the stress-sensitization by ATR inhibitor has been recognized as a new therapeutic potential withthe combination use of other DNA damaging agents in various type of cancers. In the current study,
we revealed that cells with DNA replication stress induced by loss of KDM5D were particularly
sensitive to an ATR inhibitor (stress-sensitization) showing increased DNA damage. This indicatesthat detecting the loss of KDM5D could serve as a biomarker predicting the efficacy of ATR
inhibition, which may help select patients for this new targeted therapy.

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References
ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase
KDM5D.
Komura K, Yoshikawa Y, Shimamura T, Chakraborty G, Gerke TA, Hinohara K, Chadalavada K,
Jeong SH, Armenia J, Du SY, Mazzu YZ, Taniguchi K, Ibuki N, Meyer CA, Nanjangud GJ, Inamoto
T, Lee GM, Mucci LA, Azuma H, Sweeney CJ, Kantoff PW.
J Clin Invest. 2018 Jul 2;128(7):2979-2995.
PMID: 29863497

一般の皆様へ
現在、ゲノム医療の普及促進の一つの大きな課題として、解析後のアウトプットとなる臨床治験を含む治療選択肢の拡充が挙げられると申請者らは考えている。癌治療、研究において特定の Key Molecule による多彩な形質変化を明らかにすることは、患者様の発現差異による層別化を可能にし、さらにその変化に伴う治療ターゲットの発見がテーラーメード医療を可能にする。本研究においても ATR inhibitor による治療効果予測因子である DNA 複製ストレスのバイ
オマーカーとして KDM5D の欠失を明らかにしたことにより、個別化医療 Precision Medicineに新たな展開をもたらせるものと期待している。

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Peptide foldamers in drug discovery
Yosuke Demizu
demizu@nihs.go.jp
National Institute of Health Sciences

Summary Abstract
A representative antimicrobial peptide, Magainin 2 (Mag 2) exerts its antimicrobial activity via
microbial membrane disruption. In this study, I revealed that the sequence of 17 amino acid residues
in Mag 2 (peptide 1) is required to exert sufficient activity.  I also designed a set of Mag 2-based
antimicrobial peptide foldamers by incorporation of α,α-disubstituted amino acids, and evaluatedtheir preferred secondary structures and their antimicrobial activities against both Gram-positive andGram-negative bacteria. As a result, peptide 6 formed a stable helical structure, and possessed potent
antimicrobial activities without significant cytotoxicity.
Key Words：Peptide, Non-proteinogenic amino acid, Helical structure, DDS carrier, Cancer

Introduction
Antimicrobial peptides (AMPs) have been paid much attention as the next-generation drugs
against multidrug-resistant bacteria. One of the most studied of the amphipathic helical AMPs is
Magainin 2 (Mag 2), which is composed of 23 amino acid residues, and its helical structure andamphipathic property play a pivotal role to exert the potent antimicrobial activity. However, thelarge molecular weight and the chemical stability of the peptide backbone against digestive enzymes
could be a barrier for clinical use of the AMPs.
In this study, I aimed to develop the novel AMPs by the enhancement of the helicity and theamphipathicity of Mag 2 sequence utilizing non-proteinogenic amino acids, i.e. α,α-disubstituted
amino acids (dAAs).

Results
First of all, I designed and the truncated peptides derived from Mag 2, and evaluated theirantimicrobial activities. These peptides were designed to remain the cationic and hydrophobic
residues to conserve the amphipathic stripe-type helix. Minimum inhibitory concentration (MIC)
was measured against both Gram-positive and Gram-negative bacteria. As a result, the peptide 1
bearing the free N-terminus showed relatively high antimicrobial activity (Figure 1).
Next, I designed and synthesized a series of Mag 2 derivatives based on two different strategies.
That is to say, one is enhancing the amphipathicity of peptides by replacing the amino acids asshown in Figure 1. The helical wheel of Mag 2 insisted that the 3rd-Gly, 7th-His, and 17th-Val residues
positioned on the cationic face and 13th-Gly residue arranged to the hydrophobic face of helical Mag
2. Hence, the peptide 8, 3rd-Gly, 7th-His, and 17th-Val were replaced to Lys, and 13th-Gly replaced
to Val. These substitutions were designed to enhance the amphipathicity. The other strategy isenhancing the helicity by introduction of hydrophobic dAAs such as 2-aminoisobutyric acid (Aib :U) and 1-aminocyclohexylcarboxylic acid (Ac6c : Z) into the sequence instead of two Ala residues(Figure 1 and Table 1).  Based on these strategies, the effects of the amphipathicity and helicity on

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their antimicrobial activities and hemolytic activities were investigated.
After having the synthesized peptides 2–7, the antimicrobial activities of the peptides were
evaluated against several bacteria. Since the antimicrobial peptides target the microbial membranes,
hence it is possible for the antimicrobial peptides to exhibit the cell membrane disruption activity
against even human blood cells. Therefore, I also investigated hemolytic activity of the peptides
2–7 using human blood cells. The results are shown in Table 1. The antimicrobial activity of
peptide 2 was significantly increased compared to that of Mag 2. On the other hand, the peptides 3
and 4 possessing dAA residues also showed the same extent of antimicrobial activity of peptide 2.
Moreover, the peptide 7 exhibited the strong antimicrobial activity against both Gram-positive andGram-negative bacteria. However, peptides 4 and 7 having Ac6c residues, showed potent hemolytic
activity against human red blood cells (3 μM each).  These results indicated that the introduction of
Ac6c residues increased not only antimicrobial activity, but also hemolytic activity. I also performed
WST-8 assay to evaluate the cytotoxicity against HeLa cells. I confirmed that all synthesized peptides
showed no significant cytotoxicity up to 100 μM.   
The preferred secondary structures of the peptides were analyzed using CD spectra, and thetruncated peptide 1 formed a random structure, whereas the peptides 6 and 7 formed stable helicalstructures depending upon dAA residues as a helical promoter.
Finally, I performed the chemical stability assay of the peptides against digestive enzyme. The
peptide backbone could be degraded by digestive enzymes such as protease, resulting in decrease
in its biological activities. The peptides 3, 4, 6, 7 having two dAA residues showed resistanceagainst proteases. I evaluated the half-life (t1/2) of the peptides against proteinase K, a representative
digestive enzyme which recognized hydrophobic and aromatic amino acids such as L-phenylalanine
residues. The peptides wereanalyzed were incubatedwith proteinase K at 37 °C,
and amounts of peptides
at different time intervals
were evaluated using HPLC
analysis. I observed that
Mag 2 composed of only
natural amino acid residues
was totally degraded in 2
h, whereas the peptides 6
and 7 having dAA residues
remained intact at 8-h
incubation.

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Discussion & Conclusion
In this study, I aimed to develop the novel AMPs focusing on their helical structures. As a result,the peptide 1, composed of the essential 17 amino acid residues in the Mag 2 was identified.  Since
the truncated C-terminus of Mag 2 did not contain the cationic amino acid residue, the peptide 1
retained the amphipathic properties and the antimicrobial activity. On the other hand, CD spectra
analysis revealed that the truncation destabilized its helical structure, resulting in decrease of
antimicrobial activity compared to that of Mag 2. To overcome this, I designed and synthesized aset of novel AMPs focusing on amphipathicity and helicity for developing potent AMPs. As a result,the peptide 6 possessing Aib residues exhibited the potent antimicrobial activity against Grampositive and Gram-negative and selectivity over human blood cells, whereas the peptides 4 and 7
possessing Ac6c residues showed strong hemolytic activity. These data suggested that the excessivehydrophobicity of Ac6c induced cytotoxicity against human blood cells. The introduction of Aib
residues into Mag 2 sequence stabilized helical structures and chemically stabilized peptides against
proteinase K. Moreover, the replacement of Gly residues, which is known as helix breaker, with Lys
increased the amphipathicity and helicity simultaneously, resulting in potent antimicrobial activity. I
expect that the peptide 6 can be a promising reagent for infection disease therapy.

References
1. Development of amphipathic antimicrobial peptide foldamers based on Magainin 2 sequence
C. Goto, M. Hirano, K. Hayashi, Y. Kikuchi, Y. Hara-Kudo, T. Misawa,* Y. Demizu*
ChemMedChem, 14, 1911-1916 (2019).
2. Rational design of novel amphipathic antimicrobial peptides focused on distribution of cationicamino acid residues
T. Misawa, C. Goto, N. Shibata, M. Hirano, Y. Kikuchi,* M. Naito,* Y. Demizu*
MedChemComm, 10, 896-900 (2019).
<Selected as Cover Picture>

一般の皆様へペプチド医薬品をはじめとする中分子医薬品は、低分子医薬品と抗体医薬の利点を併せ持つ

新たな医薬品として期待されています。私は本研究で、病気に関連する様々な菌に対して有効な抗菌ペプチドの開発を行いました。本研究を通じて、未だに有効な治療方法が見つかっていない病気に対する、新しい治療薬や治療法の開発に貢献します。

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Evaluation of Intra-tumor epigenetic heterogeneity of breast cancer
Reo Maruyama
reo.maruyama@jfcr.or.jp
Japanese Foundation for Cancer Research

Summary Abstract
In this study, we aim to dissect intra-tumor heterogeneity in breast cancer by utilizing single-cell
ATAC-seq (scATAC-seq) method. We performed scATAC-seq analysis of 10 breast cancer specimens
and analyzed a total of 8,181 cells. Bioinformatics analysis identified 18 distinct cell types in 10
tumors and successfully characterized some of the cell types. The cellular composition of tumors andthe degree of their heterogeneity differed among cases, suggesting that they may reflect differencesin the biological characteristics of tumors. scATAC-seq analysis seems to be useful to analyze theheterogeneity and diversity of clinical cancer samples.
Key Words：heterogeneity, breast cancer, single-cell ATAC-seq

Introduction
Breast cancer is a heterogeneous group of diseases whose biological characteristics vary greatly
from tumor to tumor. Because tumors are composed of diverse populations of cells, it is necessary toaccurately evaluate the cellular composition of tumors and functional properties of each cell to better
understand the biological characteristics of tumors.

Results
In this study, we aimed to dissect intratumor epigenetic heterogeneity in breast cancer byutilizing a single-cell ATAC-seq (scATAC-seq) method. To this end, we first applied the OmniATACseq protocol (Corces MR, 2017, Nat Methods) to cells freshly isolated from needle biopsy
specimens of breast cancer and obtained bulk ATAC-seq profiles of 41 breast cancer cases. ERpositive breast cancer and triple negative breast cancer (TNBC) showed distinct patterns and ERpositive tumors were further divided into two groups and displayed some degree of heterogeneity.
DNA motif enrichment analysis of regulatory elements identified several key transcription factorsthat potentially contribute to the epigenetic heterogeneity in breast cancer. For example, FOXA1
and some other motifs clearly distinguished TNBC and other ER-positive and/or HER2-positivecancers. Furthermore, several other transcription factor motifs were differentially enriched evenin TNBC samples suggesting that these differential patternsof motif enrichment may reflect differences in the biologicalcharacteristics of TNBC.
We then performed scATAC-seq analysis of 10 breast cancer
specimens (three from TNBC and seven from ER-positive cases)
using the droplet-based ddSEQ Single-Cell Isolator (BioRad).
Total of 8,181 cells were passed by quality check and further
analyzed by the snapATAC algorithm (Fang R, bioRxiv, 2019).
Eighteen distinct cell types were identified in 10 tumors and

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these cell types were further classified into four groups. Two groups (8 cell types) were mainly
derived from 3 samples of TNBC and the other two groups (10 cell types) were mainly derived
from ER-positive breast cancer. However, a couple of cell types were derived from both TNBC andER-positive tumors. Some tumors were composed of very homogeneous cell types, while somewere composed of diverse cell types. The cellular composition of tumors and the degree of theirheterogeneity differed among cases, suggesting that they may reflect differences in the biologicalcharacteristics of tumors. Motif enrichment analysis and further bioinformatics analysis successfully
characterized some of the cell types.

Discussion & Conclusion
In this study, we could demonstrate that scATAC-seq is feasible in the clinical setting. Due to thesimplicity of its principle, this method is easy, robust, and cost-effective compared to scRNA-seq
method. In addition, chromatin accessibility profiles obtained by this method potentially include a
wealth of information. At the moment, scATAC-seq seems to be the most suitable method to accessan epigenomic landscape in each cell and to characterize intra-tumor heterogeneity and diversity ofclinical cancer samples.
scATAC-seq analysis of breast cancer specimens revealed that the cellular composition of tumors
and the degree of their heterogeneity differed among cases. The finding suggested that they may
reflect differences in the biological characteristics of tumors, however, further studies will be needed.

References
Corces MR, et al. An Improved ATAC-seq Protocol Reduces Background and Enables
Interrogation of Frozen Tissues. Nat Methods. 2017;14(10):959-962.
Fang R, et al. Fast and Accurate Clustering of Single Cell Epigenomes Reveals Cis-Regulatory
Elements in Rare Cell Types. bioRxiv. 2019.

一般の皆様へがんは１種類の悪性細胞が大量に増殖した単なる「細胞のかたまり」では決してなく、様々な種類の細胞が協調してひとつの「細胞社会」のようなものを形成していることが分かってきました。この「細胞社会」は患者さん一人一人で異なり、さらには時々刻々と変化しています。
がんを克服するためには「がんとは何か？」を知る必要があり、それは腫瘍を構成する細胞一つ一つの「個性」を理解し、それらが作り出す「社会」の成り立ちを理解することであると信じています。本研究はその第一歩であり、まずは患者さんから頂いた乳がんの細胞の特徴を見ることに成功しました。「がん細胞社会」の理解、そしてがん克服を目指し、引き続き努力していきます。

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Research on metabolic reprogramming induced by cancer
development and inflammation
Koji Taniguchi
kotaniguchi@keio.jp
Keio University School of Medicine

Summary Abstract
To investigate metabolic reprogramming induced by cancer development and inflammation,
we investigated how mutations of key driver genes in colorectal cancer and the activation of
inflammatory signaling pathways regulate production of metabolites that affect tumor immunity
and induce metabolic reprogramming of cancer cells using the new technologies, such as organoidculture, CRISPR/Cas9 system, and comprehensive analysis.
Key Words：Cancer, Metabolism, Inflammation

Introduction
During cancer progression, metabolic changes (metabolic reprogramming) in the cancer cells,
including the Warburg effect, occur, and the metabolic changes in the cancer cells contribute to theinvasion and metastasis of some cancers1)-3). However, the detailed mechanisms of how driver genemutations associated with cancer and inflammation cause changes in cancer metabolism and how
metabolic reprogramming contributes to cancer invasion and metastasis are unknown.

Results
In this study, we first established intestinal organoids according to the organoid culture method
established by Dr. Toshiro Sato (Sato et al., Nature 2009). In brief, the intestines of C57BL/6 mice
were washed with PBS, and intestinal crypts were isolated by 2mM EDTA treatment. The isolated
crypts were counted, embedded in Matrigel, and cultured in a medium containing Rspo1 conditioned
medium, noggin (Gold Biotechnology), epidermal growth factor (EGF) (PeproTech), B27
supplement (Thermo Fisher Scientific), and N2 supplement (Thermo Fisher Scientific). For
genetic modification, intestinal organoids were used to perform genome editing with lentivirus andclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, or organoids were
established from the intestines of knockout or flox mice4), 5) (Taniguchi et al. Nature 2015, Taniguchi
et al. Nat Commun 2016, Taniguchi et al. PNAS 2017, etc.). We have genetically modified an
oncogene, Kirsten rat sarcoma viral oncogene homolog (Kras, K) and tumor suppressor genes, suchas ddenomatous polyposis coli (APC, A), tumor protein p53 (p53, P), SMAD family member 4,
Mothers against decapentaplegic homolog 4, or Deleted in Pancreatic Cancer-4 (Smad4, S), all ofwhich are closely associated with colorectal tumorigenesis. We have established wild-type, A (APC
mutated), AK (APC mutated, Kras activated), AKP (APC mutated, Kras activated, p53 mutated),and AKPS (APC mutated, Kras activated, p53 mutated, Smad4 mutated) intestinal organoids. We
have found that the changes of morphology and growth speed occur when such oncogene and/or
tumor suppressor genes are genetically modified, and some of the genetically-modified intestinalorganoids result in engraftment (subcutaneous engraftment or orthotopic engraftment) and liver

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metastasis in mice (Taniguchi et al., Nat Commun 2016, Sakai E and Mimori K et al., Cancer Res.
2018). Recently, we have found that the IL-6 family of cytokines activates the Src family kinase(SFK)-YAP pathway in addition to the JAK-STAT3 pathway, and the SFK-YAP pathway plays anessential role in intestinal regeneration and colorectal tumorigenesis, and links inflammation withtissue regeneration and tumorigenesis (Taniguchi et al. Nature 2015, Taniguchi et al. PNAS 2017.).
Therefore, we also tried to activate these inflammatory signaling pathways, such as the JAK-STAT3
pathway and the SFK-YAP pathway, in the genetically-modified intestinal organoids by using active
mutants of these genes to investigate the role of these inflammatory signaling pathways in metabolic
reprogramming of the genetically-modified intestinal organoids. Next, we tried to establish themethod to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of
the genetically-modified intestinal organoids with Seahorse XF Analyzers (Agilent). We establishedthe protocol and succeeded in measuring OCR and ECAR of the genetically-modified intestinalorganoids. We observed the classical Warburg effect in this system, and this result was confirmed bymeasurement of lactate level in the supernatant of the genetically-modified intestinal organoids. Now
we are investigating how the mutations in the driver genes related to colorectal tumorigenesis andthe activation of inflammatory signaling pathways affect OCR and ECAR of intestinal organoids.
We also performed the metabolome analysis of the genetically-modified intestinal organoids, which
was supported by Human Metabolome Technologies (HMT). We are analyzing the original data
to investigate the effects of each driver gene mutation or the inflammatory signaling pathways on
metabolites.

Discussion & Conclusion
The detailed mechanism of how each mutation of key driver genes and the activation of
inflammatory signaling pathways in colorectal cancer affect the growth and progression of cancer
cells and the tumor microenvironment through metabolic reprogramming has not been clarified.
Therefore, in this study, we are trying to clarify the mechanism by using the new technologies, suchas organoid culture, CRISPR/Cas9 system, and comprehensive analysis. If we can clarify the whole
picture of metabolic reprogramming in genetically-modified intestinal organoids, we would like todevelop a method to normalize cancer metabolism in the future. It will lead to the development of
new treatment methods for cancer metastasis and chemotherapy resistance and can greatly contributeto the improvement of the prognosis of cancer patients.

References
1) Grivennikov SI, Greten FR, Karin M. : Immunity, inflammation, and cancer. Cell. 19;140(6):88399. 2010
2) Taniguchi K, Karin M. : IL-6 and related cytokines as the critical lynchpins between inflammationand cancer. Semin Immunol. ;2 6(1):54-74. 2014
3) Taniguchi K, Karin M. : NF-κB, inflammation, immunity and cancer: coming of age. Nat Rev
Immunol. ;18(5):309-324. 2018
4) Taniguchi K, Wu LW, Grivennikov SI, et al. : A gp130-Src-YAP module links inflammation toepithelial regeneration. Nature. 5;519(7541):57-62. 2015
5) Taniguchi K, Moroishi T, de Jong PR, et al. : YAP-IL-6ST autoregulatory loop activated on APC
loss controls colonic tumorigenesis. Proc Natl Acad Sci U S A. 14;114(7):1643-1648. 2017

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一般の皆様へ
現在の日本人の死亡原因の一位はがんであり、その約 9 割はがんの転移によると考えられています。がん転移の予防や治療に有効な方法がなく、新しい予防・治療方法の開発が強く望まれています。がんが全身に広がった状態では、化学療法や放射線療法に加えて、がん免

疫療法の効果が期待されていますが、まだ効果は十分ではありません。今回の研究ではがん細胞に起こる代謝変化に着目して、新しい観点からのがん転移の予防法と治療法の開発を目指しています。

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Publication

論文掲載

受領者
Recipient

掲載誌 ・ 書誌事項
Journal

論文タイトル
Title

１

渡邉 裕介
（国立循環器病研究センター）

Debelopmental Biology, 461,2,124131 (2020)
https://doi.org/10.1016/
j.ydbio.2020.02.001

Expression of Hey2 Transcription factor
in the early embryonic ventricles iscontrolled through a distal enhancer by
Tbx20 and Ｇａｔａ transcription factors

２

石水 毅（立命館大学）

Plant Physiology andBiochemistry,142,173-178 （2019）https://doi.org/10.1016/
j.plaphy.2019.07.008

Rhamnogalacturonan I
galactosyltransferase: Detection of
enzyme activity and its hyperactivation

３

出水 庸介
（国立医薬品食品
衛生研究所）

ChemMedChem,14,22 （2019）https://doi.org/10.1002/
cmdc.201900460

Development of Amphipathic
Antimicrobial Peptide Foldamers Basedon Magainin 2 Sequence

４

同上MedChemComm,10,6,831-1044（2019）
https://doi.org/10.1039/c9md00166b

Rational design of novel amphipathic
antimicrobial peptides focused on the
distribution of cationic amino acidresidues

5

北岸 宏亮（同志社大学）

Chem.Asia J. 2019.14.3320-3328
https:/doi.org/10.1002/
asia.201900983

Supramolecular Complexationin Biological Media: NMR
Study on Inclusion of an Anionic
Tetraarylporphyrin into a Per‐O ‐Methylated β‐Cyclodextrin Cavity inSerum, Blood, and Urine

6

田川 義晃
（鹿児島大学）

bioRxiv preprint doi:
https://doi.org/10.1101/795922.
posted October 7, 2019

Expression of Hey2 Transcription factor
in the early embryonic ventricles iscontrolled through a distal enhancer by
Tbx20 and Ｇａｔａ transcription factors

７

瀬木（西田）恵里（東京理科大学）

Pharmacology Biochemistry and
Behavior、 Volume 186, November
2019, 172767
https://doi.org/10.1016/
j.pbb.2019.172767

Search for factors contributing toresistance to the electroconvulsive
seizure treatment model using
adrenocorticotrophic hormone-treated
mice

8

甲斐 歳恵
（大阪大学）PLOS ONE、 Published: April 3,
2020
https://doi.org/10.1371/journal.
pone.0231114

Drosophila MARF1 ensures proper
oocyte maturation by regulating nanos
expression

9

田中 知明
（千葉大学）

eNeurologicalSci、 Volume 19, June
2020, 100239
https://doi.org/10.1016/
j.ensci.2020.100239

Monoamine oxidase B rs1799836 G
allele polymorphism is a risk factor forearly development of levodopa-induced
dyskinesia in Parkinson's diseaseNo.

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No.

受領者
Recipient

掲載誌 ・ 書誌事項
Journal

論文タイトル
Title

10

田中 知明
（千葉大学）

Journal of Experimental Medicine
Published: October 25 2019https://doi.org/10.1084/
jem.20190972

Cell type-specific actions of Bcl11b
in early T-lineage and group 2 innate
lymphoid cells

11

谷口 浩二
（慶應義塾大学）

Cancer Science
First published: 15 March 2019https://doi.org/10.1111/cas.13999

The Sprouty/Spred family as tumor
suppressors: Coming of age

12

木村 圭志
（筑波大学）

Nucleic Acids Research, Vol 48,
No.12, 6583-6596, 2020,
doi: 10.1093/nar/gkaa449

Identification of a novel nucleolar
protein complex required for mitotic
chromosome segregation throughcentromeric accumulation of Aurora B

13

山崎小百合
（名古屋市立大学）

The Journal of Immunology, 200:119-129 (2018)
DOI: 10.4049/jimmunol.1701056

Ultraviolet B-Induced Maturation of
CD11b-Type Langerin- Dendritic Cells
Controls the Expansion of Foxp3+
Regulatory T Cells in the Skin
Regulatory T cells expressing abundant
CTLA-4 on the cell surface with aproliferative gene profile are key featuresof human head and neck cancer

Int J Cancer: 144, 2812-2822 (2019)
doi: 10.1002/ijc.32024

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III.
Reports from the Recipients of Grants
for International Meetings

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Report on Research Meeting
1. Name of Research Meeting / Conference
The 13th Annual Meeting of the Japanese Society for Epigenetics
2. Representative
Haruhiko Koseki (Deputy Director, RIKEN Center for Integrative Medical Sciences)
3. Opening period and Place
May 28-29, 2019
Kanagawa Kenmin Hall & Yokohama Sanbo Hall (Yokohama City, Kanagawa)4. Number of participants / Number of participating countries and areas
440 participants from 5 countries (Japan, China, Austria, UK, USA)
5. Total cost
9,507,807 JPY
6. Main use of subsidy
Printing cost for program book
7. Result and Impression
The Japanese Society for Epigenetics (JSE) is a common platform for Japanese scientists who
are doing research on various aspects of epigenetics including gene expression, embryonic andtissue development, cancer, and drug development. JSE organizes an annual meeting every year
in different venues, and this year, the 13th annual meeting was held in Yokohama city on 28-29
May. Dr. Haruhiko Koseki, from RIKEN Yokohama, presided over the meeting as the chairman.
The theme of this year’s meeting was “Epigenetics: From Embryonic Development to Drug
Development”. In recent years, great progress has been made in understanding the mechanisms
by which epigenetic complexes regulate gene expression in different biological contexts. Findings
from such basic research are allowing scientists to explore new avenues of translational researchby which it would be possible to prevent, or to some extent, cure diseases like cancer. To this end,in vitro disease models based on 2D or 3D cell culture systems should be valuable, as these willallow screening for novel epigenetic drugs and chemical/natural compounds targeted to a specificdisease.
With these broad goals in view, in this year’s annual JSE meeting, we invited speakers
from Japan and abroad. The keynote lecture was given by Dr. Hiroyuki Sasaki, from Kyushu
University, who was also the chairman of the 2nd JSE annual meeting in 2008. Dr. Sasaki talkfocused on the epigenetic regulation in mammalian germ cells. The speakers from abroad were
Dr. Rob Klose from Oxford University (UK), Dr. Wei Xie from Tsinghua University (China),
Dr. Fei Lan from Fudan University (China), and Dr. Michael Erb from Scripps Research (USA).
Drs. Klose and Xie talked about embryonic development and stem cell regulation, while Drs. Lan
and Erb discussed chemical modifications involved in epigenetic regulation and their potential astargets for epigenetic therapy.
In addition to the keynote lecture, and presentations from speakers abroad; we also had seven

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oral presentation from distinguished Japanese scientists, and six short talks selected from theposter presenters. This year, nearly 140 posters were presented, and given the high quality of
research in each, it was very challenging to select six posters for the short talks. We also had
four luncheon seminars- two on each day- three among those were sponsored by biomedical
companies, while the remaining one was sponsored by the Japan National Tourism Organization(JNTO). Last but not the least, three young researchers were selected as the recipients of JSE
annual prize, who presented their research as short talks.
The venue, Yokohama, was easily accessible from various parts of Japan, and from abroadespecially via the Haneda airport. The participants were able to enjoy the beautiful early summer
views of the Yamashita Park near the venue, and also of the scenic beauty of the Yokohama bay
area. The Yokohama Chinatown, located only a short distance away from the venue, was a major
attraction for the participants.
It is worthy to note that English was used as the official language of an annual meeting, for the
first time in the history of JSE, which facilitated participation of speakers and posters presentersfrom abroad. In particular, there was abundant discussion and interactions between scientists frombasic (developmental biology) and applied (drug discovery) research areas. Active participationof biomedical companies also provided a great opportunity for scientific discussions between
researchers and personnel from the companies.
In summary, this year’s JSE meeting was a success, thanks to the generous support from manysponsors, most notably the NOVARTIS Foundation (Japan) for the Promotion of Science. The
next JSE meeting will take place in Nagoya in 2020, which we hope will also be very interestingand successful.
8. Additional description

Opening Remarksby Dr. Koseki

Mail Hall

Poster Hall

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Report on Research Meeting
1. Name of Research Meeting / Conference
60th International Conference on the Bioscience of Lipids
2. Representative
Professor Makoto Arita
RIKEN, Keio University
3. Opening period and Place
17-21, June, 2019
Hitotsubashi-Hall, Tokyo, Japan
4. Number of participants / Number of participating countries and areas
450 / 21 countries
5. Total cost
JPY 20,861,926
6. Main use of subsidy
Vanue fee
7. Result and Impression
ICBL is a conference format designed for scientists with common interests in lipid research.
It was founded more than 65 years ago and is a prominent forum for the exchange ofideas, communication of novel developments and discussion of a broad variety of aspects related
to lipid bioscience. ICBL attracts colleagues from all continents to the frontiers of lipid
research presented and discussed during the conferences. Another major aim of ICBL is
supporting research of young scientist, giving them the opportunity to meet renowned
scientists, and thus providing the scientific inspiration for future careers in lipid research.
The 60th ICBL meeting was successfully held between June 17 and 21, 2019 at HitotsubashiHall, Tokyo. The main focus of this meeting was set as “Biology of lipoquality”. Chair was
Makoto Arita, and the co-Chairs were Takehiko Yokomizo, Makoto Murakami, Makoto Ito,
Shinji Yokoyama, and Yukihiko Sugimoto. This conference attracted more than 450 attendees
from 21 countries, and this was the biggest ICBL meeting in the past. More than 200 posters
were exhibited, and totally 77 oral presentations including satellite symposia were provided. The
van Deenen award lecture was presented by Prof. Keizo Inoue, an Honorary Professor of the
University of Tokyo. Special lectures were given by Profs. Takao Shimizu, Yasuyuki Igarashi,
Shigekazu Nagata, and Shu Narumiya. Welcome reception and gala dinner were on 17th and 20th,
June, respectively. In the afternoon of June 20th, foreign attendee enjoyed excursions including
Japanese tea ceremony, tea tasting, Japanese Incense, Sake brewery Terada Honke, and teamlab
Planets Tokyo. Prior to the conference, a satellite symposium consisting with 15 speakers
from Kakenhi “Lipoquality” team was held on June 17. In the closing remarks, Prof. Christian
Wolfrum at ETH Zurich mentioned “This is the biggest and the most successful ICBL meeting,and made the word “lipoquality” famous throuout the world”. Je also announced the ICBL

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meeting in Utrecht in 2020, Montreal in 2021, and Palma, Spain in 2022. After the conference,a satellite workshop, LipoQuality International Workshop on Lipidomics, was held at Riken,
Yokohama on June 22.
8. Additional description
None

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Report on Research Meeting
30th Aug, 2019
1. Name of Research Meeting / Conference
Commemorative International Symposium of the Japan Society of Nucleic Acids Chemistry(CISNAC 2019)
2. Representative
Naoki Sugimoto (Director and Professor of Frontier Institute for Biomolecular Engineering
Research, Konan University)
3. Opening period and Place
22nd July, 2019 – 24th July, 2019 at Port island Campus of Konan University
4

Number of participants / Number of participating countries and areas
130 participants / 31 from abroad (USA, Canada, UK, France, Germany, Slovenia, Czech
Republic, Korea, China, Poland, India)

5 Total cost
9,169,530 yen
6. Main use of subsidy
Hotel charges
7. Result and Impression
We invited 20 famous speakers from abroad and domestic in the field of nucleic acids
chemistry based on the topics (1. Chemistry of Nucleosides, Nucleotides, and Their Analogues,
2. Medicinal Chemistry of Nucleosides and Oligonucleotides, 3. DNA/RNA Chemistry andBiochemistry, 4. DNA/RNA Structure and Recognition, 5. Ribozymes, siRNAs, and miRNAs, 6.
DNA/RNA Materials and Diagnostics). All lectures are listed below;
Shankar Balasubramanian (University of Cambridge, UK), G-Quadruplex secondary structures
and DNA dynamics
Tigran Chalikian (University of Toronto, Canada), Some Physico-Chemical Properties of
Tetraplex DNA Important for Biological Function
Masad J Damha (McGill University, Canada), Conformation, stability and kinetic control ofnon- canonical DNA structures by 2′F-ANA modification
Piet Herdewyn (KU Leuven University, Belgium), Selection of XNA Backbone Motifs inSynthetic Biology
Chris Meier (University of Hamburg, Germany), Design of Nucleotide Prodrugs for Antiviral
Chemotherapy – the TriPPPro-Approach
Jean-Louis Mergny (University of Bordeaux, France), Quadruplexes are everywhere... butwhere exactly?
Janez Plavec (Slovenian NMR Centre at the National Institute of Chemistry, Slovenia),

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Conversion from two- to three-quartet
G-quadruplex
Steven E. Rokita (Johns Hopkins University,USA), Challenging reversible cross-linking of
DNA with biological motors
Lukáš Trantírek (Masaryk University, Czech
Republic), Formation propensity and potential
biological roles of DNA G-hairpins
Sarah Woodson (Johns Hopkins University,USA), Vectorial self-assembly of RNA duringtranscription
Zhen Xi (Nankai University, China), Gene
Nanovector through Branch-PCR

Prof. Shanker BalasubramanianHiroshi Abe (Nagoya University, Japan),Synthetic Oligonucleotide for Drug Discovery
Kazuhiko Nakatani (Osaka University, Japan),
Small Molecules targeting Repeat Sequencescausing Neurological Disorders
Hirohide Saito (Kyoto University, Japan),Prof. Sarah Woodson
Expanding the RNP world in cells
Shinsuke Sando (The University of Tokyo,
Japan), Oligonucleotide-based mimetic ofgrowth factors: A new chemical tool to reveal thefunction of cell membrane receptors
Shigeki Sasaki (Kyushu University, Japan),Site-selective modification of mRNA and its
effect on the translation
Mitsuo Sekine (Tokyo Institute of Technology,
Japan), History of the chemical synthesis of DNA
Prof. Mitsuo Sekine
and RNA in Japan
Hiroshi Sugiyama (Kyoto University, Japan), Chemical Biology of Nucleic Acids: DNA
Origami and Artificial Genetic Switch
Takeshi Wada (Tokyo University of Science, Japan), Development of new moleculartechnologies for oligonucleotide therapeutics
Naoki Sugimoto (Konan University, Japan), To B or not to B : The Watson-Crick world is notenoughTheir talks were really high level, which brought exciting discussions. We also settled theposter presentation for general participants. They enjoyed their presentation and discussion
with all participants including invited speakers. We also encouraged the young scientists tocommunicate with the famous researchers in the symposium. For excellent postgraduate students
and postdoc researchers, CISNAC 2019 Outstanding Poster Award was awarded by reviewing
foreign invited speakers as follows;
Daichi Fushihara (Nagoya Univ.), “Synthesis of Anti-viral Selenium Modified Nucleoside

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Analogues”
Saptarshi Ghosh (Konan Univ.), “Prediction of DNA duplex stability in cell-mimicking
crowded environment using nearest-neighbor model”
Dipanwita Banerjee (Konan Univ.), “Development of prediction method for the stability of
RNA/DNA hybrid duplex under a physiological buffer condition”
Hong-Liang Bao (Miyazaki Univ.), “In-cell 19F NMR: Telomere DNA G-quadruplex structuresin living human cells”
Ji Hoon Han (Kyoto Univ.), “Highly sensitive and selective mercury sensor based on
mismatched base pairing with dioxT”

Winners of Poster Award
All invited speakers from abroad highly evaluated the contents of the symposium. Especially,
one of the speakers, who is a member of IS3NA (International Society for Nucleosides,
Nucleotides, and Nucleic Acids) appreciated the activity and quality of Japan Society of Nucleic
Acids Chemistry (JSNAC). This symposium was really impressing for the important scientists,
which highlights the presence of JASNAC in the field of nucleic acids chemistry all over theworld. For domestic researchers, this symposium was really valuable for their research works,
because they had an opportunity to talk and discuss with 20 famous invited speakers during the
symposium.
8. Additional description
None.

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Report on Research Meeting
Dec. 21, 2018
1. Name of Research Meeting / Conference
The 1st International Symposium on Hybrid Catalysis for Enabling Molecular Synthesis on
Demand
2. Representative
Motomu Kanai
3. Opening period and Place
Date: 30(Thu)-31(Fri), May 2019
Venue: Ito Hall, Ito International Research Center, The University of Tokyo
4. Number of participants / Number of participating countries and areas Japan: 160, USA: 6, UK: 1,
China: 11, Singapore: 2, India:3, Canada:1, Poland:1, Romania:1
5. Total cost
JPY 6,292,000
6. Main use of subsidy
Traveling fee for Prof. Wanbin Zhang (Shanghai Jiao Tong University, China) and Prof. Choon
Hong Tan (NTU, Singapore)
7. Result and Impression
The 1st International Symposium on Hybrid Catalysis for Enabling Molecular Synthesis on
Demand was held at the University of Tokyo Ito International Hall on May 30 and 31, 2019. 8
foreign researchers, F. Dean Toste (UC, Berkeley, USA), Wanbin Zhang (Shanghai Jiao Tong
University, China), Brian M. Stoltz (Caltech, USA), Varinder K. Aggarwal (University of Bristol,UK), Jeffrey N. Johnston (Vanderbilt University, USA), Choon Hong Tan (NTU, Singapore),
Aiwen Lei (Wuhan University, China), and Tamio Hayashi (NTU, Singapore) were invited, and50 posters and 19 oral lectures were presented. Active discussions and interactions were heldamong the presenters and more than 160 participants.
Healthy and civilized human social life is supported by organic molecules, such as medicines,
pesticides, polymers, and functional molecules. The only means of creating and supplying
these organic molecules is organic synthetic chemistry. This international symposium invites
researchers from the world's frontiers in the field, and shares the latest advances in organic
synthetic chemistry and catalytic chemistry, as well as disseminating Japanese research results tothe world.
Organic synthesis has been consistently developed and refined up to the present, but several
important issues remain unresolved. One such issue is the practical synthesis of high-valueadded complex molecules through streamlined multicatalytic reactions starting from readilyavailable, abundant molecules. Nature utilizes multicatalytic (i.e., multienzymatic) systems forthe biosynthesis of natural products. The most effective artificial multicatalyst system in a flask

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for the Promotion of Scienceso far, however, promotes only two or three reactions at most. In order to overcome this problem,the creation of a hybrid catalyst system that makes use of the functions of multiple catalysts withindependent functions in concert has become a global breakthrough. Integrating the functions ofmultiple catalysts, hybrid catalysis will enable molecular synthesis of high efficiency, flexibility,
and adaptability on demand, starting from abundant organic molecules. This international
symposium played a significant role in discussing the issues of this state-of-the-art organic
synthetic chemistry and advancing the field one step toward the future. It was a fusion of a wide
range of fields related to organic synthesis chemistry and catalytic chemistry, and a high-level andmeaningful conference of university-industry bidirectionality.
To conclude, all the agenda of the symposium was closed in a great success.
Photograph (May 31, 2019, invited speakers and advisers)

8. Additional description
The great success of such a highly significant international conference is all thanks to thegenerous support by Novartis foundation. As an organizing chair and one of the participants inthe conference, I sincerely thank the foundation for this support.

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Report on Research Meeting
1. Name of Research Meeting / Conference
The 6th meeting of International Society for Zinc Biology (ISZB-2019)“The New Sunrise of Zinc Biology”
2. Representative
Toshiyuki Fukada
3. Opening period and PlaceSep 9th 2019 ~ Sep 13th 2019
Kyoto Garden Palace
4. Number of participants / Number of participating countries and areas
Total number of participants: 215
Number of participants from foreign countries: 137
Number of Japanese participants: 78
5. Total cost
20,305,000 JPY
6. Main use of subsidy
Discounts of the registration fees for 20 student researchers
Discount for each: 20,000 JPN
Total
400,000 JPN
7. Result and Impression
During the preparation step, we organizers of ISZB-2019 had set the following four goals,
namely 1: Demonstration of Japanese leadership in this field, 2: Promotion of international
collaboration, 3: Training of young investigators, and 4: Establishment of zinc-related life
science research field. To put the goal into practice, we planned 4 plenary lectures by outstanding
scientists, 10 symposiums including a session for young investigators, and a poster session. We
especially took importance on giving many scientific interactions and opportunities to the young
investigators.
We invited the following 4 plenary lecturers, and all of them brought their fascinating researchoutcomes that have impressed all the attendees very much.
Tom O’Hallaran Northwestern University: “Zinc and fertilization”
Nickolas K Tonks Cold Spring Harbor Laboratory: “Metal and signal transduction”
Eric Skaar Vanderbilt University: “Zinc and microbiota”
Ananda Prasad Wayne State University: “Zinc deficiency”
Session topics and chairs of 10 symposiums were described below, which could cover almost
all of the life science fields.
SP1 (Zinc sensor): M. Merkx, C. Fahrni

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The Novartis Foundation(Japan)
for the Promotion of Science

SP2 (Neurology 1): A. Bush, E. Aizenman
SP3 (Physiology and genetics): M. Hershfinkel, S. Kelleher
SP4 (Young investigators): T. Fukada, T. Kambe
SP5 (Immunology and inflammation): L. Rink, D. Knoell
SP6 (Biochemistry): W. Maret, D. Fu
SP7 (Cell biology and cancer): K. Taylor, I. Sekler
SP8 (Diabetes): H. Yasui, L. Huang
SP9 (Neurology 2): A. Takeda, JY. Koh
SP10 (Zinc therapy): H. Kodama, M. Moriyama
We also prepared two awards to honor excellent researches done by young researchers,
including “Metallomics young investigator’s prize”, and “Metallomics poster prize”. We
celebrated 5 awardees for each prize.
After conclusion of ISZB-2019, we have surveyed to know the impression and thoughts thatthe attendees had during and after the conference. The majority of responses were very positive
indeed, especially for usage of the same venue for the conference and accommodation, whichbrought attendees many chances to interact each other. Based on the results of the survey, weorganizers are now proud to conclude that ISZB-2019, supported by the generous fundraisingfrom The NOVARTIS Foundation (Japan) for the Promotion of Science, was a very successfulinternational meeting (see photo, below).

Group photo of ISZB-2019 (Center of the front line: representative of ISZB-2019)
8. Additional description
Our many thanks from the organizers must go to The NOVARTIS Foundation (Japan) for the
Promotion of Science, for providing the generous founding to support ISZB-2019, that brought
the great success into the conference. Without this precious support, we shall say that we could
not have the same success in this conference.

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The Novartis Foundation(Japan)
for the Promotion of Science

33rd Grant Report（FY2019）
The foundation has been conducting public interest activities such as research grant, meeting grant
and international exchange programs since its establishment on Sep. 4, 1987 in Japan underauthorization of the Ministry of Education, Science, Sports and Culture, followed by a transition to a
public interest incorporated foundation on Apr. 1, 2012. The grants conducted in FY 2019 are as
follows.
33rd Novartis Research Grant: 37 Researchers (JPY 1 mil.), Subtotal JPY 37.0 mil.
Research Meeting Grant: 5 Meetings (JPY 0.4 mil.), Subtotal JPY 2.0 mil.
Total
JPY 39.0 mil.

33rd Novartis Research Grant(FY2019)

The Grant is to aim supporting creative research in Japan in the field of Bio, life science andrelevant chemistry.
#

Name

Institution

Title

Research Project1 Nobuaki Takahashi

The Hakubi Center, Kyoto
University

Associate Systematic understanding of oxidative
Professor stress responses in cancer

2 Tomonao Matsushita

Faculty of Agriculture,
Kyushu University

Elucidation of the molecular mechanism
Associateof phytochrome-regulated alternative
Professor
promoter selection

3 Mari Amino

Department of Cardiovascular
Medicine, Tokai University

Effects of carbon beam irradiation
Associate on connexin and sympathetic nerve
Professor expressions and antiarrhythmic effect inrabbits with atrial fibrillation

4 Yosuke Senju

Research Institute forInterdisciplinary Science(RIIS), Okayama University

Assistant Evolution of membrane morphogenesis in
Professor Asgard archaea

5 Eiichi Mizohata

Quantum beam analysis on structure
Department of Applied
Associate
formation of glucuronyltransferase
Chemistry, Graduate School ofProfessor
catalyzing drug metabolism
Engineering, Osaka University

6 Masashi Maekawa

Division of Cell Growth andTumor Regulation, ProteoScience Center (PROS),
Ehime University

Tenuretrack
Novel molecular basis of intracellular
Assistant cholesterol trafficking in endothelial cells
Professor

7 Shunsuke Kon

Research Institute for
Biomedical Sciences, Tokyo
University of Science

Study of the mechanistic dissection
Juniorof cancer incidence caused by agingAssociateassociated dysfunction of cell competition
Lecturer
in intestinal cells

8 Takahiro Shimizu

Endoscopy Unit, Kyoto
University Hospital

Identification of premalignant gastric
Assistant glands in chronic gastritis mucosa
Professor and prophylactics for gastric cancer
development.

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

9 Hitoshi Takizawa

International ResearchCenter for Medical Sciences(IRCMS), Kumamoto
University

Mechanism of inflammatory memory in
Professor hematopoietic stem cell using informatics
unifying multidimensional single cell data

10 Giichi Takaesu

Molecular Microbiology
Group, Tropical Biosphere
Research Center, Universityof the Ryukyus

Molecular mechanisms of mycobacterial
Associate
effector protein for the development ofProfessor
novel anti-TB drugs

11 Teruhiro Okuyama

Institute for Quantitative
Biosciences (IQB), The
University of Tokyo

Associate Functional mechanisms and dynamics ofProfessor oxytocin in autism spectrum disorder

12 Kenichiro Kinouchi

School of Medicine, Baxter
International Endowed
Course for Integrated Renal
Replacement Therapy &Translational Medicine, Keio
University

Assistant Fasting Specific Metabolic Regulation byProfessor the Circadian Clock

13 Kenji Ikeda

Department of Molecular
Endocrinology andMetabolism, Tokyo Medical
and Dental University

JuniorCell senescence effects on transcriptional
Associate regulation and differentiation in beige
Professor preadipocytes

14 Atsushi Hoshino

Department of Cardiovascular
medicine, Kyoto Prefectural
University of Medicine

Identification of molecular network
Assistantbetween mitochondria and nucleus
Professor
underlying mitochondrial biogenesis.

15 Kazuhiro Yoshida

Department of Chemistry,
Graduate School of Science,
Chiba University

Development of Planar Chiral Cyclic
Associate(Amino)(ferrocenyl)carbene Ligands forProfessor
Medicinal Chemistry

16 Tomoko Takahashi

Department of Biological
Sciences, Graduate School of
Science, The University ofTokyo

Regulation of gene expression network
Assistantand cell death by microRNAs during viral
Professor
infection

17 Kazuhiko Yamamuro

Department of Psychiatry,
Nara Medical University

Development of mPFC-pPVT neuronal
Assistantcircuits involved with juvenile social
Professor
experience

18 Takeshi Inoue

Immunology Frontier
Research Center,
Laboratory of Lymphocyte
Differentiation, Osaka
University

Specially
Identification of precursor memory B
Appointed
cells and mechanistic characterization of
Associate
memory B cell generation
Professor

19 Yoshihiro Oonishi

Faculty of Pharmaceutical
Sciences, Hokkaido
University

Rhodium(I)-catalyzed enantioselective
Associatehydroacylation through dynamic kinetic
Professor
resolution

20 Takashi Ebihara

Department of Medical
Biology, Akita University
Graduate School of Medicine

Molecular mechanism underlying
Professor development of memory-like Group 2
innate lymphoid cells

21 Takahiro Yasui

Department of NephroDevelopment of urolithiasis therapy byurology, Nagoya City
Professor
immune response and repair mechanism
University Graduate School of
Medicine

22 Saori Matsuhana

Department of Biology,
Graduate School of Science,
Kobe University

The model chicken of cardiac septum
Assistantdefect；A new tool for analysis ofProfessor
structural heart disease

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

23 Yukinori Hirano

Graduate School of Medicine,
Kyoto University

Associate
Visualizing aging individuality
Professor

24 Yoshishige Miyabe

Institute for Advanced
Medical Sciences, Nippon
Medical School

SeniorIn vivo Imaging of Brain in CNS
Assistant Lupus Defines Unique Roles forProfessor Chemoattractants

25 Daisuke Morito

School of Medicine, Showa
University

Associate Molecular Exploration of Moyamoya
Professor Disease

26 Satoru Okuda

Nano Life Science Institute,
Kanazawa University

Associate Role of actin cytoskeletal fluctuation in
Professor retinal organoid formation

27 Keisuke Nimura

Division of Gene Therapy
Science, Osaka University

Elucidating the system to generate
Associate
dormancy that confer chemotherapyProfessor
resistance to cancer cells

28 Yoku Hayakawa

Department of
Gastroenterology, The
University of Tokyo Hospital

Mechanism and targeting therapy for TGF
Assistantpathway-dependent diffuse-type gastricProfessor
cancer

29 Wataru Ariyoshi

Division of Infections andMolecular Biology, Kyushu
Dental University

Mechanisms involved in regulation ofProfessor osteoclast formation by glucan recognition
via immune receptors

30 Michiko Sekiya

Department of Alzheimer’s
Disease Research, National
Center for Geriatrics andGerontology

Section
Chief

31 Sachihiro Matsunaga

Matsunaga Laboratory,
Department of Applied
Biological Science, Facultyof Science and Technology,
Tokyo University of Science

Elucidation of the promotion mechanism
Professor in plant shoot regeneration byenvironmental stimuli

32 Hiroki Yoshida

Dept Biomol Sci, Faculty of
Medicine, Saga University

Professor

Analyses of pain regulation mechanisms
by IL-27

33 Teiichi Furuichi

Department of Biological
Science, Faculty of Scienceand Technology, Tokyo
University of Science

Professor

Molecular and neural mechanism
associated with social behavior

34 Yusuke Ishigaki

Laboratory of Organic
Development of H2S-Activatable NearChemistry Ⅰ, Department ofAssistant
Infrared-dye Probes: Bioimaging andChemistry, Faculty of Science, Professor
Therapy
Hokkaido University

35 Takatsugu Ishimoto

Division of Translational
Research and Advanced
Treatment Against
Gastrointestinal Cancer,
Kumamoto University
Hospital

Identification of molecular mechanism
Associateunderlying drug resistance by tumorProfessor
stroma derived exosome

36 Koki Makabe

School of science andengineering, Makabe lab.,
Yamagata university

The ultrastabilization mechanism of
Associate
amyloid revealed by a protein engineering
Professor
technique37 Mitsutomo Abe

Graduate School of Arts andSciences, The University ofTokyo

Identification of the lipid species which
Associate
regulate epidermis differentiation in
Professor
Arabidopsis

Weighted gene co-expression network
analysis in Drosophila

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

FY2019 Research Meeting Grant(JPY 400 thousand x5 = 2.0 million)#

Meeting

Date(Place)Institution / Title

Name

1

The 13th Annual Meeting of theJapanese Society for Epigenetics

2019.5.28-29

RIKEN Center for Integrative
Medical Sciences,
Deputy Director

Haruhiko Koseki

260th International Conference on the
Bioscience of Lipids (ICBL)

2019.6.17-21

Dep. Biochemistry, Juntendo
University Medical school
Professor

Takehiko Yokomizo

3

Commemorative International
Symposium of the Japan Society of
Nucleic Acids Chemistry(CISNAC 2019)2019.7.22-24

FIBER, Konan University,
Director, Professor

Naoki Sugimoto4

The 1st International Symposium ofHybrid Catalysis

2019.5.30-31

Graduate School of Pharm.
Sciences, The University ofTokyo, Professor

Motomu Kanai

2019. 9.9-13

Faculty of Pharmaceutical
Sciences, Tokushima Bunri
University,
Professor

Toshiyuki Fukada

The 6th International Society for Zinc
5
Biology Meeting (ISZB-2019)

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

第33期（2019年度）助成事業報告
当財団は、文部大臣の認可を得て1987年9月4日に設立されて以来、研究助成を中心とした公益事業を行って来ました。2012年4月1日には、制度改革に伴い、公益財団法人へ移行
しております。2019年度は、下記の総額3,700万円の助成事業を実施しました。
第33回ノバルティス研究奨励金

37件（1件100万円）

3,700万円

研究集会助成5件（1件 40万円）

200万円

総額

3,900万円

第33回ノバルティス研究奨励金（2019年度）
この事業は、生物・生命科学および関連する化学の領域において、我が国で行われる創造的な研究の助成を目的としています。

（受付順、敬称略、所属職位は申請時、贈呈額：1件 100万円）

No.

氏

名

所

1

髙橋

重成

京都大学白眉センター

特 定
准教授

がん酸化ストレス応答のシステマティッ
クな理解

2

松下

智直

九州大学大学院農学研究院准教授

植物の光受容体フィトクロムによる転写開始点制御の分子機構解明

3

網野

真理

東海大学医学部内科学系
循環器内科

准教授

ウサギ心房細動モデルに対する炭素線照
射がコネキシンおよび交感神経発現と抗不整脈効果に及ぼす影響の検討

4

千住

洋介

岡山大学異分野基礎科学
研究所

助

教

アスガルド古細菌に探る細胞形態の制御機構の分子進化5

溝端栄一

国立大学法人大阪大学大学院
工学研究科 応用化学専攻

講

師

薬物代謝を触媒するグルクロン酸転移酵
素の量子ビームを用いた構造形成の解析

6

前川

大志

愛媛大学 プロテオサイエンス テニュア
血管内皮細胞における細胞内コレステロ
センター
トラック
ール輸送の新規分子基盤
細胞増殖腫瘍制御部門
助教

7

昆

俊亮

東京理科大学生命医科学
研究所

講

師

加齢に伴う腸管細胞の細胞競合機能低下に起因する発がん機序

8

清水

孝洋

京都大学医学部附属病院
内視鏡部

助

教

慢性胃炎粘膜における発癌リスク腺管の同定と発癌予防9滝澤仁

熊本大学国際先端医学研究機構

特別招聘 統合的インフォマティクス解析を用いた
教授
造血幹細胞の炎症記憶のメカニズム

10高江洲 義一琉球大学熱帯生物圏研究センター
分子感染防御学分野

准教授

新規抗結核薬の開発を目指した結核菌病
原因子の作用機序の解明

11

奥山

東京大学
定量生命科学研究所

准教授

自閉症スペクトラムにおける、オキシト
シン作用機序の解明

12

木内謙一郎

慶應義塾大学医学部
バクスター包括的腎代替療法展開医学寄附講座

特任助教 概日時計による空腹代謝の調節

輝大

属

職

位

研究課題

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for the Promotion of Science

131

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

属

職

位

研究課題

13

池田

賢司

東京医科歯科大学大学院
医歯学総合研究科
分子内分泌代謝学分野

講師

肥満に伴う細胞老化がベージュ脂肪前駆細胞の転写調節及び分化に及ぼす影響

14

星野

温

京都府立医科大学
循環器内科

助教

ミトコンドリア生合成の基盤となるミト
コンドリア―核間ネットワークの解明

15

吉田

和弘

千葉大学大学院
理学研究院化学研究部門

准教授

医薬品開発を志向する面性不斉アミノメ
タロセニルカルベン配位子の開発

16

高橋

朋子

東京大学大学院理学系研究科
生物科学専攻助教

マイクロ RNA が制御するウイルス感染細胞の遺伝子発現ネットワークと細胞死誘導機構の解明

17

山室

和彦

奈良県立医科大学
精神医学講座助教

前頭前野－視床室傍核回路の幼少期社会経験による発達

18

井上

毅

19

大西

英博

20

海老原

21

大阪大学免疫学
フロンティア研究センター
分化制御研究室

特任准教 記憶 B 前駆細胞の同定による記憶 B 細胞
授（常勤） 産生機構の解明

北海道大学大学院
薬学研究院

准教授

ロジウム触媒による動的速度論的光学分
割を伴う不斉ヒドロアシル化反応

敬

秋田大学 大学院医学系
研究科微生物学講座

教授

メモリー様 2 型自然リンパ球の分化制御
機構の解析

安井

孝周

名古屋市立大学大学院
医学研究科
腎・泌尿器科学分野

教授

生体内免疫応答と修復機構の統合的解析による尿路結石溶解療法の開発

22

松花

沙織

神戸大学大学院理学研究科
生物学専攻

助教

心臓隔壁欠損モデルを用いた先天性心疾
患機構の解明23平野恭敬

京都大学大学院医学研究科

特定准教授

老化個性の可視化ツールの開発

24

宮部

斉重

日本医科大学先端医学研究所 細胞生物学部門

講師

脳内インビボイメージングシステムによる Central Nervous System Lupus 病態解
明への挑戦

25

森戸

大介

昭和大学医学部講師

もやもや病の分子病態解明

26

奥田

覚

金沢大学
ナノ生命科学研究所

網膜オルガノイド形成におけるアクチン
准教授
細胞骨格の動的ゆらぎ特性とその役割の（Jr.PI）
解明

27

二村

圭祐

大阪大学大学院医学系
研究科・遺伝子治療学

准教授

化学療法抵抗性を示す静止期癌細胞形成システムの解明28

早河翼

東京大学医学部附属病院
消化器内科

助教

TGF 経路依存性浸潤型スキルス胃癌の機序解析と治療応用

29

有吉

渉

九州歯科大学
感染分子生物学分野

教授

免疫受容体による糖鎖認識を介した破骨細胞分化修飾能の分子基盤

30

関谷

倫子

国立長寿医療研究センター
アルツハイマー病研究部
発症機序解析研究室室長

ショウジョウバエの加重共発現遺伝子
ネットワークの構築

31

松永

幸大

東京理科大学理工学部応用生物科学科松永研究室

教授

環境刺激による植物のシュート再生促進
メカニズムの解明

32

吉田

裕樹

佐賀大学医学部分子生命
科学講座

教授

IL-27 による疼痛制御機構の解析

33

古市

貞一

東京理科大学
理工学部応用生物科学科教授

社会性行動に関連する分子・神経機構の
解明

34

石垣

侑祐

北海道大学大学院
理学研究院化学部門
有機化学第一研究室

助教

硫化水素で活性化される近赤外色素プ
ローブの開発 : バイオイメージングと医療への応用The Novartis Foundation(Japan)
for the Promotion of Science132

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

属

職

位

研究課題

35

石本

崇胤

熊本大学病院
消化器癌先端治療開発学

特任准教授

腫瘍間質由来エクソソームによる胃がん
治療抵抗性機構の解明

36

真壁

幸樹

山形大学大学院
理工学研究科真壁研究室

准教授

蛋白質工学によって解明するアミロイド
凝集の異常安定化

37

阿部

光知

東京大学大学院
総合文化研究科准教授

シロイヌナズナの表皮細胞分化を制御す
る脂質種の同定

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for the Promotion of Science

133

The Novartis Foundation(Japan)
for the Promotion of Science

2019年度研究集会助成
この事業は、生物・生命科学および関連する化学の領域において、我が国で開催される国際色豊かな研究集会の助成を目的としています。2019年度は５件の助成を行いました。

（受付順、敬称略、所属・職位は申請時、贈呈額：1件40万円）

No.

氏

名

所

属

職位

開催日 /
開催地

副センター長2019.5.28-29 ／神奈川

第 13 回
エピジェネティクス
研究会年会

研

究

集

会

名

1

古関

明彦

国立研究開発法人
理化学研究所
生命医科学研究
センター

2

横溝

岳彦

順天堂大学医学部
生化学第一講座教授

2019.6.17-21 ／東京

第 60 回
国際脂質生物学会議

3

杉本

直己

甲南大学先端生命工学研究所

所長・教授

2019.7.22-24 ／神戸

日本核酸化学会設立記念
国際シンポジウム4

金井求

東京大学大学院
薬学系研究科教授

2019.5.30-31 ／東京

第１回
ハイブリッド触媒
国際シンポジウム俊幸

徳島文理大学
薬学部薬学科
病態分子薬理学研究室

教授

2019. 9.9-13 ／京都

第６回
国際亜鉛生物学会学
術集会

5

深田

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for the Promotion of Science

134

The Novartis Foundation(Japan)
for the Promotion of Science

33rd Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2020

From April 1st, 2019 to March 31, 2020
（Unit：JP Yen）

Account（Unit：JP Yen）
Account

AmountⅠ Assets

Ⅰ General Net Assets Changes

1. Current Assets

1. Ordinary income & Expenditure

Current Assets Total

15,337,028

2. Fixed Assets(1) Basic Fund
Basic Fund Total

1,100,000,000(2) Specific Assets
Specific Assets Total

Fixed Assets Total
Assets Total

450,594
80,014,094

11,075,113

Donation

40,183,600
623,915

Other Income

51,882,628(2)Ordinaty Expenditure
Project Expense

48,611,540

Grant Expense

39,000,000

Novartis Research Grant

37,000,000

1,195,801,716

Research Meeting Grant

2,000,000

Administrative Expense

4,111,807

Ordinary Expenditure Total

52,723,347

37,089,515

Ordinary Balance without Appraisal Profit or Loss

△ 840,719

37,089,515

2. Nonrecurring Profit & Loss

1. Current Liabilities
Liabilities Total

Interest from basic fund

1,180,464,688

Ⅱ Liabilities
Current Liabilities Total(1) Ordinary income

Ordinary Income Total(3) Other Long - term Assets
Other Long - term Assets Total

AmountⅢ Equity（Net Assets）-

Nonrecurring Balance of Current Period△ 12,381

1. Designated Net Assets-

General Net Assets Ending Balance

158,261,607

1,000,450,594

Ⅱ Designated Net Assets Changes-（Amount Appropriating to basic Fund）(1,000,000,000)

Designated Net Assets Change△ 183,600(Amount Appropriating to specific assets)(450,494)

Designated Net Assets Ending Balance

1,000,450,594

2. General Net Assets

158,261,607

Ⅲ Net Assets Balance Ending Balance

1,158,712,201（Amount Appropriating to）

100,000,000

Designated Net Assets Total

Equity Total（Net Assets）

1,158,712,201

Liabilities & Equity Total

1,195,801,716

The Novartis Foundation(Japan)
for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

第33期（2019年度）財務報告

貸借対照表

正味財産増減計算書

2020 年 3 月 31 日現在

2019 年 4 月 1 日から 2020 年 3 月 31 日まで（単位：円）

科

目

金 額

Ⅰ資産の部

科

15,337,028

⑴

経常収益

基本財産

基本財産合計⑵⑶

固定資産合計
資産合計

450,594
80,014,094

計

51,882,628

経常費用
48,611,540

支払助成金

39,000,000
37,000,000

1,195,801,716

研究集会助成金

2,000,000

管理費
経常費用

4,111,807
計

37,089,515

当期経常増減額

37,089,515

２．経常外増減の部
当期経常外増減額

１ ． 指 定 正 味財 産

一般正味財産期末残高

指定正味財産合計

1,000,450,594（うち基本財産への充当額）(1,000,000,000)（うち特定資産への充当額）(450,494)

２ ． 一 般 正 味財 産

158,261,607（うち基本財産への充当額）

100,000,000

負債及び正味財産合計⑵

623,915

ノバルティス研究奨励金

Ⅲ正味財産の部

正味財産合計40,183,600

1,180,464,688

１．流動負債
負債合計

受取寄付金

事業費

Ⅱ負債の部
流動負債合計11,075,113

経常収益

その他固定資産

その他固定資産合計

基本財産運用益
雑収益

1,100,000,000

特定資産

特定資産合計金 額

１．経常増減の部

２．固定資産⑴

目

Ⅰ一般正味財産増減の部

１．流動資産流動資産合計（単位：円）

52,723,347△ 840,719△ 12,381
158,261,607

Ⅱ指定正味財産増減の部
当期指定正味財産増減額△ 183,600

指定正味財産期末残高

1,000,450,594

Ⅲ正味財産期末残高

1,158,712,201

1,158,712,201
1,195,801,716

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

List of Trustees, Auditors, Councilors and Grant Selection Committee Members[Board of Trustees] 5 trustees, 2 auditors

As of July 17, 2020

Post

Name

Chairman

Kuniaki Takata, Ph.D.

President, Gunma Prefectural Public University Corporation
President, Gunma Prefectural College of Health Sciences

Sadayoshi Ito, M.D., Ph.D.

Special Administrator, Katta General Hospital
Professor Emeritus, Tohoku University

TrusteeAkimichi Kaneko, M.D., Ph.D. Professor Emeritus, Keio University
Fujio Murakami, Ph.D.
Kazunari Tsunaba

Auditor

Title

Professor Emeritus, Osaka University
President, Novartis Pharma K.K.

Tokuzo Nakajima, CPA Representative, Tokuzo Nakajima CPA Firm
Masanori Fuse

Financial Advisor, Novartis Pharma K.K.

[Board of Councilors] 10 councilors

As of July 17, 2020

Post

Name

Title

Chairman

Tsuneyoshi Kuroiwa, Ph.D.

Member of the Japan Academy;
Professor Emeritus, University of Tokyo

Masamitsu Iino, M.D., Ph.D.

Specially Appointed Professor, Nihon University
Professor Emeritus, University of Tokyo

Hiroyuki Kawashima, Ph.D Former Professor, Niigata University
Masakatsu Shibasaki, Ph.D. President, Microbial Chemistry Research Foundation

Councilor

Takao Shimizu, M.D., Ph.D.

Project Head, National Center for Global Health and Medicine
Professor Emeritus, University of Tokyo

Akihiko Nakano, Ph.D.

Deputy Director, Photonics Engineering Research Center & Team Leader,
Live Cell Super-Resolution Imaging Research, RIKEN

Yoichi Nabeshima, M.D., Ph.D.

Trustee, Foundation for Biomedical Research and Innovation at Kobe
Professor Emeritus, Kyoto University

Toyoshi Fujimoto, M.D., Ph.D.

Specially Appointed Professor, Juntendo University
Professor Emeritus, Nagoya University

Miwako Mori, Ph.D.

Professor Emeritus, Hokkaido University

Tohru Hirose, Ph.D.

Head, Global Drug Development, Novartis Pharma K.K.

The Novartis Foundation(Japan)
for the Promotion of Science

137

The Novartis Foundation(Japan)
for the Promotion of Science[Grantee Selection Committee] 20 members
Post
Chairman

Name

As of Oct. 1, 2020
Title

Hiroyuki Takeda, Ph.D. Professor, University of Tokyo Graduate School of Science
Eiji Hara, Ph.D.

Professor, Research Institute for Microbial Diseases, Osaka University

Makoto Hayashi, Ph.D.

Team Leader, Plant Symbiosis Research Team, Center for Sustainable
Resource Science, RIKEN

Hirokazu Hirai, M.D.,Ph.D. Professor, Gunma University Graduate School of Medicine
Kouichi Fukase, Ph.D.

Professor, Osaka University Graduate School of Science

Michisuke Yuzaki, M.D., Ph.D. Professor, Keio University School of Medicine
Akihiko Yoshimura, Ph.D. Professor, Keio University School of Medicine
Hiroshi Ito, M.D., Ph.D. Professor, Keio University School of Medicine,

MemberTakashi Ohshima, Ph.D.

Professor, Kyushu University Graduate School of Pharmaceutical
Sciences

Yoko Hamazaki, Ph.D.

Professor, Center for iPS Cell Research and Application (CiRA), Kyoto
University

Toshiaki Ohteki, D.D.S., Ph.D.

Professor, Medical Research Institute, Tokyo Medical and Dental
University

Yasuyoshi Sakai, Ph.D.

Professor, Kyoto University Graduate School of Agriculture

Taisuke Tomita, Ph.D.

Professor, University of Tokyo Graduate School of Pharmaceutical
Sciences

Fumiko Toyoshima, Ph.D.

Professor, Institute for Frontier Life and Medical Sciences, Kyoto
University

Tohru Minamino, M.D., Ph.D Professor, Juntendo University Graduate School of Medicine
Yasuhiro Yamada, M.D., Ph.D. Professor, The Institute of Medical Science, University of Tokyo
Masaki Ieda, M.D., Ph.D. Professor, University of Tsukuba Faculty of Medicine
Yasuteru Urano, Ph.D.

Professor, University of Tokyo Graduate School of Pharmaceutical
Sciences

Erina Kuranaga, Ph.D.

Professor, Tohoku University Graduate School of Life Sciences

Mitinori Saito, M.D., Ph.D. Professor, Kyoto University Institute for Advanced Study

The Novartis Foundation(Japan)
for the Promotion of Science138

The Novartis Foundation(Japan)
for the Promotion of Science

公益財団法人ノバルティス科学振興財団
役 員 名 簿2020 年 7 月 17 日現在（敬称略）
職

名

氏

名

代表理事

高田

邦昭

群馬県公立大学法人理事長
群馬県立県民健康科学大学長

伊藤貞嘉

公立刈田綜合病院特別管理者
東北大学名誉教授

金子章道

慶應義塾大学名誉教授

理事

監事

現

職

村上富士夫

大阪大学大学院生命機能研究科招聘教授
大阪大学名誉教授

綱場

一成

ノバルティス ファーマ株式会社 代表取締役社長

中嶋德三

公認会計士中嶋德三事務所

布施

正則

ノバルティス ファーマ株式会社
企画管理本部ファイナンシャルアドバイザー

評 議 員 名 簿2020 年 7 月 17 日現在（敬称略）
職

名

氏

名

評議員長

黒岩

常祥

日本学士院会員
東京大学名誉教授

飯野正光

日本大学医学部特任教授
東京大学名誉教授

川島博行

元新潟大学大学院医歯学総合研究科教授

柴崎正勝

微生物化学研究会理事長 微生物化学研究所長
東京大学名誉教授

清水孝雄

国立国際医療研究センター プロジェクト長
東京大学名誉教授

中野明彦

理化学研究所 光量子工学研究センター 副センター長
東京大学名誉教授

鍋島陽一

神戸医療産業都市推進機構 先端医療研究センター長
京都大学名誉教授

藤本豊士

順天堂大学大学院医学研究科特任教授
名古屋大学名誉教授

評議員

森

現

廣瀬

美和子
徹

職

北海道大学名誉教授
ノバルティス ファーマ株式会社

取締役グローバル医薬品開発本部長

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

選 考 委 員 名 簿2020 年 10 月 1 日現在（敬称略）
職

名

選考委員長

選考委員

氏

名

現

武田

洋幸

林

誠

原

英二

大阪大学微生物病研究所教授

平井宏和

群馬大学大学院医学系研究科教授

深瀬浩一

大阪大学大学院理学研究科教授

柚﨑通介

慶應義塾大学医学部教授

吉村昭彦

慶應義塾大学医学部教授

濵﨑洋子

京都大学 iPS 細胞研究所教授

伊藤

裕

大嶋

孝志

九州大学大学院薬学研究院教授

樗木俊聡

東京医科歯科大学難治疾患研究所教授

阪井康能

京都大学大学院農学研究科教授

富田泰輔

東京大学大学院薬学系研究科教授

豊島文子

京都大学ウィルス・再生医科学研究所教授

南野

徹

山田

泰広

東京大学医科学研究所教授

家田真樹

筑波大学医学医療系教授

浦野泰照

東京大学大学院薬学系研究科教授

東京大学大学院理学系研究科教授
理化学研究所環境資源科学研究センター チームリーダー

慶應義塾大学医学部教授

順天堂大学大学院医学研究科教授

倉永英里奈

東北大学大学院生命科学研究科教授

斎藤

京都大学高等研究院教授

通紀

職

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for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

事 務 局 便 り
ご寄付のお願い
当財団は、自然科学における創造的な研究の奨励等を行うことにより、学術の振興を図り、国民の健康と福祉の向上に寄与することを目的に公益事業を行っております。
当財団の事業は、基本財産の運用益並びに寄付金によって賄われており、財団では趣旨にご賛同いただける皆様からのご寄付を募っております。
当財団へのご寄付には、下記の税法上の優遇措置が適用されます。
優遇措置の概略
個人：年間寄付金の合計額もしくは年間所得の40％相当額のいずれか低い方から２千円を引いた金額が、所得税の寄付金控除額となります。
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（資本金等の額×当期の月数/12×0.375%＋所得の金額×6.25%）÷2
ご寄付は、随時受付けております。詳しくは、財団事務局までお問合せください。
（電話：03-6899-2100、E-メール：foundation.japan@novartis.com）

事務局より
本年度もお陰様で、財団年報を発行できることとなりました。今年の初め
より、新型コロナウイルス感染症の影響で、いろんなことが” 非日常” となり、その環境に対応するのに苦労しておりますが、多くの方々のご協力でなんとか事業が継続できております。
当財団は1987年９月の財団設立以来、助成件数は総数で1,806件、総額20億円を超えるものとなりました。当財団は、自然科学の創造的研究への助成によって、日本の学術発展に寄与することを目指しており、助成を受けられた研究成果がすぐに応用につながらなくとも、将来、新分野につながることを夢見て、この事業を継続して参ります。
今まで助成事業が継続できたことも、偏に、助成事業にご理解・ご支援をいただいた方々、助成を受けられた皆様および財団関係者のお力添えの賜物であると、心より感謝申し上げます。引き続きご指導、ご支援の程よろしく
お願い申し上げます。
2020年10月吉日
事務局長 原 健記〒105-6333 東京都港区虎ノ門1-23-1
虎ノ門ヒルズ森タワー
TEL：03-6899-2100 FAX：03-6899-2101
E-メール：foundation.japan@novartis.com
ホームページ：http://japanfoundation.novartis.org/

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for the Promotion of Science141