Annual Report (2021)
No.33
The NOVARTIS Foundation (Japan)
for the Promotion of Science
2021年度

財団年報 第33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 the 33rd Novartis Research Grants
第33回研究奨励報告 ………………………………………………………………………… 9
1. Systematic understanding of oxidative stress responses in cancer …………… 10
Nobuaki Takahashi
Kyoto University

2. Elucidation of the molecular mechanism of phytochrome-regulated alternative
promoter selection ……………………………………………………………………… 13
Tomonao Matsushita
Kyoto University

3. Carbon beam induced upregulation of cardiac gap junction proteins andprevention of sympathetic nerve sprouting in hypercholesterolemic rabbits,
leading to reduction of arrhythmia vulnerability …………………………………… 16
Mari Amino
Department of Cardiology, Tokai University, Isehara, Japan
National Institute for Quantum and Radiological Science and Technology, Inage,Japan

4. Evolution of membrane morphogenesis in Asgard archaea …………………… 20
Yosuke Senju
Okayama University

5. Quantum beam analysis on structure formation of glucuronosyltransferase
catalyzing drug metabolism …………………………………………………………… 23
Eiichi Mizohata
Osaka University

6. Novel molecular basis of intracellular cholesterol trafficking in endothelial cells

25

Masashi Maekawa
Proteo-Science Center, Ehime University

7. Study of the mechanistic dissection of cancer incidence caused by agingassociated dysfunction of cell competition in intestinal cells …………………… 27
Shunsuke Kon
Tokyo University of Science

8. Identification of premalignant gastric glands in chronic gastritis mucosa andprophylactics for gastric cancer development. …………………………………… 30
Takahiro Shimizu
Kyoto University Hospital

9. Mechanism of inflammatory memory in hematopoietic stem cells using
informatics unifying multidimensional single cell data …………………………… 33
Hitoshi Takizawa
International Research Center for Medical Sciences, Kumamoto University

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10. Molecular mechanisms of mycobacterial effector protein
for the development of novel anti-TB drugs ……………………………………… 36
Giichi Takaesu
Tropical Biosphere Research Center, University of the Ryukyus

11. Functional mechanisms and dynamics of oxytocin in autism spectrum disorder 39
Teruhiro Okuyama
The University of Tokyo

12. Fasting Specific Metabolic Regulation by the Circadian Clock ……………… 41
Kenichiro Kinouchi
Keio University School of Medicine

13. Cell senescence effects on transcriptional regulation and differentiation inbeige preadipocytes …………………………………………………………………… 44
Kenji Ikeda
Department of Molecular Endocrinology and Metabolism,
Tokyo Medical and Dental University

14. Identification of molecular network between mitochondria and nucleus
underlying mitochondrial biogenesis ……………………………………………… 48
Atsushi Hoshino
Kyoto Prefectural University of Medicine

15. Development of Planar Chiral Cyclic (Amino)(ferrocenyl)carbene Ligands for
Medicinal Chemistry …………………………………………………………………… 51
Kazuhiro Yoshida
Chiba University

16. Regulation of gene expression network and cell death by microRNAs
during viral infection …………………………………………………………………… 54
Tomoko Takahashi
Saitama University

17. Functional analysis of the paraventricular nucleus of the thalamus involved insocial behavior …………………………………………………………………………… 57
Kazuhiko Yamamuro
Department of Psychiatry, Nara Medical University

18. Identification of precursor memory B cells and mechanistic characterization ofmemory B cell generation …………………………………………………………… 60
Takeshi Inoue
Immunology Frontier Research Center, Osaka University

19. Rhodium(I)-catalyzed enantioselective hydroacylation through dynamic kinetic
resolution ………………………………………………………………………… 63
Yoshihiro Oonishi
Faculty of Pharmaceutical Sciences Hokkaido University

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20. Molecular mechanism underlying development of memory-like Group 2 innate
lymphoid cells …………………………………………………………………………… 66
Takashi Ebihara
Department of Medical Biology, Akita University Graduate School of Medicine

21. Development of urolithiasis therapy by immune response and repair
mechanism ………………………………………………………………………………… 69
Takahiro Yasui
Nagoya City University Graduate School of Medical Sciences

22. The model chicken of cardiac septum defect ; A new tool for analysis of
structural heart disease ………………………………………………………………… 72
Saori Tani-Matsuhana
Kobe University

23. Visualizing aging individuality ………………………………………………………… 75
Yukinori Hirano
Kyoto University

24. In vivo imaging the brain defines new mechanisms of leukocytes
migration cascade in Central Nervous System Lupus ………………………… 78
Yoshishige Miyabe
Department of Cell Biology, Institute for Advanced Medical Sciences,
Nippon Medical School

25. Molecular Pathology of Moyamoya Disease ……………………………………… 81
Daisuke Morito
Showa University School of Medicine

26. Role of actin cytoskeletal fluctuation in retinal organoid formation………… 84

Satoru Okuda
Kanazawa University

27. Elucidating the system to generate dormancy that confer chemotherapyresistance to cancer cells …………………………………………………………… 86
Keisuke Nimura
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University

28. Mechanism and targeting therapy for TGF pathway-dependent diffuse-type
gastric cancer …………………………………………………………………………… 89
Yoku Hayakawa
The University of Tokyo Hospital

29. Mechanisms involved in regulation of osteoclast formation by glucan
recognition via immune receptors ………………………………………………… 91
Wataru Ariyoshi
Division of Infections and Molecular Biology, Kyushu Dental University

30. Weighted gene co-expression network analysis in Drosophila ……………… 96
Michiko Sekiya
National Center for Geriatrics and Gerontology

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31. Elucidation of the promotion mechanism in plant shoot regeneration byenvironmental stimuli. ………………………………………………………………… 99
Sachihiro Matsunaga
Department of Integrated Biosciences, Graduate School of Frontier Sciences
The University of Tokyo

32. Analyses of pain regulation mechanisms by IL-27 ………………………… 102
Hiroki Yoshida
Saga University Faculty of Medicine

33. Molecular and neural mechanism associated with social behavior ……… 105
Teiichi Furuichi
Tokyo University of Science

34. Development of H2S-Activatable Near-Infrared-dye Probes:
Bioimaging and Therapy …………………………………………………………… 108
Yusuke Ishigaki
Hokkaido University

35. Identification of molecular mechanism underlying drug resistance bytumor-stroma derived exosome ………………………………………………… 112
Takatsugu Ishimoto
The International Research Center for Medical Sciences (IRCMS), Kumamoto
University

36. Stabilization mechanism of amyloid aggregates revealed by protein
engineering techniques …………………………………………………………… 115
Koki Makabe
Yamagata university

37. Identification of the lipid species which regulate epidermis differentiation in
Arabidopsis. …………………………………………………………………………… 118
Mitsutomo Abe
Department of Life Sciences, Graduate School of Arts and Sciences,
The University of Tokyo

Publication - the 33rd (FY2019) Grant Recipients ………………………………… 121

Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告 ……………………………………………………………………………… 127
1. The International Centenarian Consortium: ICC 2021 ………………………… 128
Yasumichi Arai, Professor, Faculty of Nursing and Medical Care, Keio University/
Center for Supercentenarian Medical Research, Keio University School of Medicine

2. 3rd Mini-symposium on the blood-brain barrier: from basic to clinical research

130

Yoichi Morofuji Department of Neurosurgery, Nagasaki University

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Ⅳ．The 34th (fiscal year 2020) Promotion Report

第34期 （2020年度） 助成事業報告 …………………………………………………… 132

Ⅴ．The 34th (fiscal year 2020) Financial Report

第34期 （2020年度） 財務報告 …………………………………………………………… 144

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 ………………………………………………… 146

Ⅶ．Information from the Secretariat
事務局便り …………………………………………………………………………………… 150

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Introduction

Kuniaki Takata, Ph.D.
Chairman of the Board of Trustees
This annual report contains the research reports of the recipients of the 33rd Novartis
Research Grants. (research from April 2020 to March 2021: 37 research grants and 2
research meetings).
The COVID-19, which began in 2019, has been raging around the world, and as of thiswriting in early October, a cumulative total of more than 200 million people worldwide havebeen infected and nearly 5 million people have died, a situation that has shaken the very
foundations of the world. The cumulative number of infected people in Japan has also
exceeded 1.7 million, and more than 17,000 people have died. During this time, the days
are far from what they were before the COVID-19 outbreak. Universities and research
institutes have also been greatly affected, with campus closures and restrictions on theiruse. It has become difficult to hold regular conferences and we have been forced to holdwebinars, cancel or postpone them. In the absence of physical human exchanges withoverseas countries, researchers continue to communicate with each other on PC screens.
In spite of all these difficulties, the spirit of the researchers is again evident in the way they
continue to promote their research.
The NOVARTIS Foundation (Japan) for the Promotion of Science was establishedin 1987 with a donation of one billion yen from Ciba-Geigy AG (now Novartis AG) of
Switzerland. The purpose of the Foundation is clearly stated in its Articles of Incorporation:“To contribute to the improvement of the health and welfare of the people through thepromotion of science by encouraging creative research in the natural sciences. In addition,a document titled “Prospectus for the Establishment of the Foundation,” written in the yearof the foundation’s establishment, states that the foundation “hopes to contribute to thewelfare of mankind by promoting and subsidizing creative research in the natural sciences,which will be the axis of science in the coming 21st century. It also states that it will “provide
financial support for research and opportunities for cross-border exchange. Based on
this policy, our Foundation has provided financial assistance to a total of 1,854 projects
over the past 34 years, amounting to approximately 2.14 billion yen. Now, in the midst of
the turmoil caused by the COVID-19 disaster, our Foundation would like to return to thisstarting point and continue to support outstanding research.
This annual report summarizes the results of the excellent research supported by ourFoundation. It is an impressive accomplishment that was achieved within the limited timeframe of one year. The list of past recipients of the Foundation’s support includes manyof the leading researchers in their fields, including Professor Tasuku Honjo, who wasawarded the Nobel Prize in Physiology or Medicine. I hope that the recipients of thesegrants will use the results of their research as an opportunity to make even greater strides.
I would like to express my deepest gratitude to the members of the selection committee
who selected these outstanding research projects and to Novartis Pharma K.K, the donorof the donation, as well as all those who support the activities of our Foundation.

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はじめに代表理事 高田 邦昭
本年報には、 第33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 （2020年４月～ 2021年３月の研究 ： 研究奨励金37件、 研究集会２件） を収録しました。
2019年に始まった新型コロナウイルス （COVID-19） は世界中で猛威をふるい、 この原稿を執筆している10月初旬では世界中で累計２億人以上の人が感染し、 500万人近い人が亡くなるという、 世界の根幹を揺るがす事態になっています。 日本の累計感
染者数も170万人を超え、 17,000人を超える方が亡くなりました。 この間、 COVID-19
感染が起こる前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きました。 通常の学会開催は困難となり、 web 開催や中止 ・ 延期を余儀なくされました。 海外との物理的な人的な交流ができない中で、 研究者は PC 画面上で交流を続けています。 このような様々な困難な状況にもかかわらず、研究者が変わりなく、あるいは一層研究を推進している様には、あらためて研究者魂を見る思いです。
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、 34年間で総計1,854件、 金額にして約21.4億円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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Ⅱ.
Reports from the Recipients ofNovartis Research Grants

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Systematic understanding of oxidative stress responses
in cancer
Nobuaki Takahashi
takahashi@sbchem.kyoto-u.ac.jp
Kyoto University

Summary Abstract
Accumulating evidence has shown that cancer cells exhibit higher levels of toxic H2O2 compared with normal cells by through metabolic and signaling aberrations. However, there are
no reports visualizing H2O2 levels at a single cell level in in vivo tumor. Here we developeda H2O2 probe that can be specifically accumulated in tumor cells and found that H2O2 levels
are very heterogeneous within tumors. Interestingly, tumor cells with high levels of H2O2 exhibit some unique characteristics that are not observed in tumor cells with low levels of H2O2.
These findings could provide the basis of oxidative stress beyond the role in the just toxic effects on cells in tumor biology and could be exploited for targeted cancer therapies.
Key Words ： Cancer, Oxidative Stress

Introduction
Cancer cells are exposed to numerous cellular insults during tumorigenesis, leading to
the generation of toxic reactive oxygen species, including H2O2, through metabolic andsignaling aberrations1-3. Therefore, oxidative-stress defense programs are critically involvedin tumor progression and therapy resistance and represent an “Achilles heel” or vulnerabilityof tumor cells. Despite the tight association between H2O2 and cancer cell survival, there
are no reports visualizing H2O2 levels at a single cell level in in vivo tumor.

Results
In this study, we developed a H2O2 probe that can be specifically accumulated in tumor
cells through the conjugation of a H2O2 probe with the antibody that binds to proteinsspecifically expressed in tumor cells. One important feature of this probe is that the probe
allows us to know the tumor cells that experienced H2O2 exposure because the H2O2 probe
irreversibly reacts with H2O2; therefore, this probe enables us to detect the tumor cells thatexperienced H2O2 exposure even after the isolation of tumor tissues, which is distinct fromthe currently used technologies for the detection of oxidative stress.
Using this innovative probe, we injected the probe to stomach cancer-carrying micethrough i.v., and 12-hour after administration of the probe, we dissected the tumor,
prepared tumor sectioning, and assessed fluorescent signals of the probe using confocal
microscopy. Notably, we uncovered that H2O2 levels are very heterogeneous within tumors.
Heterogeneity of H2O2 levels was also detected in in vivo tumors directly observed using
a two-photon microscopy without the dissection of tumors in live animals. In addition tostomach cancer, we also assess intratumor H2O2 levels in mice carrying breast cancer byusing the H2O2 probe and confirmed heterogeneity of H2O2 levels within breast tumor cells.

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While substantial evidence has shown that tumor cells overall exhibit high levels of H2O2
compared with normal tissue cells, our study is the first to demonstrate the heterogeneity of
H2O2 levels at a single cell level within tumors.
We next investigated the mechanism by which specific tumor cells exhibit high levels of
H2O2. We examined the correlation between the localization of tumor cells with high H2O2levels and that of stroma cells as well as immune cells and found that tumor cells exhibit
high levels of H2O2 are surrounded by a type of immune cells, suggesting that the enhanced
H2O2 levels are at least in part due to the exposure of H2O2 or cytokines/chemokines thatare produced from the immune cells. Indeed, depletion of the immune cells suppressed
H2O2 levels in tumor cells in mice. While we found that the immune cells are critical for H2O2
generation within tumor, the mechanisms underlying H2O2 generation in the immune cells or
the tumor cells are still unknown.
In order to characterize tumor cells with high levels of H2O2, we separated tumor cellswith high levels of H2O 2 and the cells with low levels of H2O 2 through cell sorter andperformed RNA-seq analyses. We found that tumor cells with high levels of H2O2 exhibit
some unique characteristics, including changes in expression of extracellular matrix
proteins and cytoskeletal proteins as well as signaling proteins. We show that the signaling
protein is activated by H2O2 and regulate the expression levels of extracellular matrix
proteins and cytoskeletal proteins. We are currently looking at the phenotypic changes intumor cells with high levels of H2O2 in terms of tumorigenesis and metastasis.
These findings could provide the basis of oxidative stress beyond the role in the just toxic
effects on cells in tumor biology and could be exploited for targeted cancer therapies.

Discussion & Conclusion
Our study reveals that tumor cells exhibit heterogeneous H2O2 levels within in vivo tumor
and that the tumor cells with high levels of H2O2 show unique characteristics, which isdistinct from tumor cells with low levels of H2O2.
There is now compelling evidence that oxidative-stress defense programs represent avulnerability of tumor cells. Therefore, the development of therapies targeting oxidativestress defense represents an attractive strategy to enhance the sensitivity of tumor cells toexisting therapies. Our finding on the unique characteristics of tumor cells with high levelsof H2O2 could be exploited for targeted cancer therapies that enhance the sensitivity oftumor cells to existing therapies.

References
1. Gorrini, C., Harris, I. S. & Mak, T. W. Modulation of oxidative stress as an anticancer
strategy. Nat. Rev. Drug Discov. 12, 931−947 (2013).
2. Takahashi, N. et al. Cancer Cells Co-opt the Neuronal Redox-Sensing Channel TRPA1 to
Promote Oxidative-Stress Tolerance. Cancer Cell 33, 985−1003 (2018).
3. Takahashi, N. et al. 3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2
as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.
Mol. Cell 80, 828−844 (2020).

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一般の皆様へかつて数十年もの間、 ビタミン C やビタミン E をはじめとした抗酸化サプリメントは癌抑制効果があると信じられてきましたが、 近年行われた大規模疫学調査により、 抗酸化サプリメントの摂取はむしろ癌の発生率を上昇させることが明らかにされました。
癌細胞は、 正常細胞が本来存在している場所を逸脱して生存 ・ 増殖することが知られていますが、 近年の研究により、 このようなニッチの逸脱は高いレベルの酸化ストレスにさらされることになり、 抗酸化を含めた強固な酸化ストレス防御機能の獲得が癌化および癌の成長過程において必須であることが分かってきました。 実際、 放射線治療や一部の癌化学療法は酸化ストレスを亢進させることで癌細胞を攻撃することが知られており、 癌細胞は正常細胞に比べて酸化ストレスに対して脆弱であるといえます。
このように、 癌と酸化ストレスとの間には密接な関連性がありますが、 腫瘍内1細胞レベルで酸化ストレスを可視化した例はこれまで存在しませんでした。
本研究では腫瘍内酸化ストレスの度合いを１細胞レベルで検出することに成功し、 また強い
酸化ストレスにされされているがん細胞は他とは違う特徴的な性質を有することが明らかになりました。
本研究による成果は、 酸化ストレス防御を標的とした新しいがん治療薬の開発につながることが期待できます。

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Elucidation of the molecular mechanism of phytochromeregulated alternative promoter selection
Tomonao Matsushita
mat@gr.bot.kyoto-u.ac.jp
Kyoto University

Summary Abstract
It is widely believed that the plant photoreceptor phytochrome regulates the transcriptionof light-responsive genes by modulating the activity of several transcription factors.
However, we recently found that phytochrome controls not only transcription, but also
alternative promoter selection, to allow plants to adapt to light environment in Arabidopsis.
In order to reveal the molecular mechanism of phytochrome-regulated alternative promoter
selection, we conducted a forward genetic screen and isolated several mutants withaberrant patterns of light-responsive changes in alternative promoter selection. Moreover,
we also performed cis element analysis and found that some non-G-box motif was enrichedin the promoters of the target genes.
Key Words ： phytochrome, alternative promoter, gene expression, light signaling,
Arabidopsis

Introduction
Our group has found that phytochrome controls not only transcription, but also alternative
splicing, at a similar genomic scale to mediate light responses in Arabidopsis (Shikata etal., PNAS 2014). Recently, we found that phytochromes also directly induce the selectionof alternative promoters in more than 2,000 genes in Arabidopsis, resulting in lightdependent production of protein isoforms with different subcellular localizations (Ushijima
et al. Cell 2017). Moreover, we physiologically demonstrated that this mechanism indeed
allows plants to respond metabolically to fluctuating light conditions for more efficientphotosynthesis (Ushijima et al. Cell 2017). The purpose of this research is to reveal themolecular mechanism of how phytochrome regulates alternative promoter selection without
affecting total amount of mRNA in each target gene.

Results
One of the target genes, GLYCERATE KINASE (GLYK), encodes proteins that show
light-dependent changes in subcellular localization through phytochrome-mediatedalternative promoter selection. The T-DNA insertion null mutant of GLYK was transformed
with the GLYK genomic DNA fragment fused with GFP at its 3’ end. Then, after confirmingthat the mutant phenotype was complemented and that the GFP subcellular localization
pattern exhibited light-dependent alterations, this transgenic line was mutated with EMS
and a forward genetic screen was conducted in the M2 population. As a result, we isolated
several mutants with aberrant patterns of light-responsive changes in GFP subcellular
localization. Then, we identified candidate responsible genes for each mutant after high-

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The Novartis Foundation(Japan)
for the Promotion of Sciencethroughput mapping by re-sequencing of genomic DNA using next-generation sequencer.
Further analysis of these candidate responsible genes will lead to the identification of transacting factors required for phytochrome-regulated alternative promoter selection.
Phytochrome is known to regulate transcription by inhibiting basic helix-loop-helix
transcription factors PIFs, which specifically bind to some DNA sequence motif called G-box
both in vitro and in vivo. Consistently, G-box motif is shown to be overrepresented in the
promoters of those genes whose mRNA levels are regulated by phytochrome. Therefore,
cis element analysis was performed to see whether G-box motif is also enriched in the
promoters of the genes under the control of phytochrome-mediated alternative promoter
selection. If the G-box motif is enriched in these target genes, it is most likely that PIFs arealso involved in phytochrome-mediated alternative promoter selection. However, surprisingly,
certain cis element other than G-box motif was identified in the analysis, suggesting thattrans-acting factors other than PIFs are involved in this regulatory mechanism. As someparticular trans-acting factor can be inferred from the nucleotide sequence of the identified
cis element, the following experiments are now being conducted. 1) 5’ RACE experiments
will be performed to see if red light-responsive changes in alternative promoter patternsof some target genes are impaired in the mutants deficient in the trans-acting factor. 2)
Direct interaction between the inferred trans-acting factor and DNA fragments containing
the non-G-box motif will be examined in the AlphaScreen system. 3) ChIP analysis will be
performed to examine whether the trans-acting factor bind to the non-G-box motifs in vivo.
4) the non-G-box element will be mutated in the context of genomic DNA fragments andthese mutated constructs will be introduced into wild-type plants to see if the non-G-box
motif is required for the binding of the trans-acting faoctor and for phytochrome-mediatedalternative promoter control.

Discussion & Conclusion
enerality and biological impact of alternative promoter control has been somewhat
underestimated. However, given that alternative promoter changes of a similar scale
could be potentially induced, not only by phytochrome but also by any kinds of signals,
alternative promoters are likely to represent another universal aspect of gene expression
regulation in eukaryotes. Therefore, the mechanistic elucidation of phytochrome-regulated
alternative promoter selection would not only reveal novel mechanism of light signaling inplants, but also force us to reconsider the general concept of gene expression regulation in
eukaryotes.

References
Shikata H, Hanada K, Ushijima T, Nakashima M, Suzuki Y, and Matsushita T. Phytochrome
controls alternative splicing to mediate light responses in Arabidopsis.
Proc Natl Acad Sci USA 111: 18781-18786 (2014)
Ushijima T, Hanada K, Gotoh E, Yamori W, Kodama Y, Tanaka H, Kusano M, Fukushima
A, Tokizawa M, Yamamoto YY, Tada Y, Suzuki Y, and Matsushita T. Light controls protein
localization through phytochrome-mediated alternative promoter selection.
Cell 171: 1316-1325 (2017)

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一般の皆様へ
同規模の転写開始点変化は、 フィトクロムシグナルに限らず、 ありとあらゆるシグナル ・ 事象に伴って、 真核生物において共通の分子機構で起こるものである可能性が高いと考えられるため、 その分子機構が本研究により解明されれば、 1 つの遺伝子から機能の異なる複数のタンパ
ク質を生み出す普遍的な仕組みを世界に先駆けて明らかにすることとなり、 生物学上の大きな進
歩となることは間違いないと考えられます。

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Carbon beam induced upregulation of cardiac gap junction proteinsand prevention of sympathetic nerve sprouting in hypercholesterolemic
rabbits, leading to reduction of arrhythmia vulnerability
Mari Amino
mariam@is.icc.u-tokai.ac.jp
Department of Cardiology, Tokai University, Isehara, Japan
National Institute for Quantum and Radiological Science and Technology, Inage, Japan

Summary Abstract
Development of noninvasive cardiac radioablation therapy for ventricular tachycardia
attracts scientific attention as a novel antiarrhythmic method. To examine the effects oftargeted heavy ion irradiation (THIR) in hypercholesterolemia (HC) rabbits with gap junctional remodeling and nerve sprouting. A single 15-Gy THIR was applied to eight rabbitswith high fat/cholesterol chow (HC+THIR group). Another eight rabbits who did not undergo
THIR were used as controls (HC group). THIR reduced the vulnerability to atrial/ventriculartachyarrhythmias by improving cardiac conductivity. This mechanism may be attributed tothe upregulation of gap junction proteins and, partly, to sympathetic growth suppression.
Key Words ： radioablation therapy, late potential, growth-associated protein 43, tyrosinehydroxylase, sympathetic denervation

Introduction
A single 25-Gy X-ray application for patients with refractory ventricular tachycardia (VT)using stereotactic body radiation therapy markedly reduced the VT burden.1,2 A recent
review on previous animal experiments reported that the antiarrhythmic effect occurredwith not only depend on the replacement with fibrosis but other radiobiological changes.3
We previously reported that targeted heavy ion irradiation (THIR; 15 Gy) to rabbit or
canine hearts with myocardial infarction (MI) caused connexin 43 (Cx43) upregulation in
the ventricles 2 weeks thereafter.4,5,6 However, changes in atrial vulnerability, Cx40 levels,or relation with the autonomic nervous system remain unknown. We aimed to examine
the effects of THIR on in-vivo electrophysiology and vulnerability to atrial/ventriculartachyarrhythmias in aged hypercholesterolemia (HC) rabbits, which were reported as themodel of electrical- and neural-remodeling involved in proarrhythmic propensities,7,8 with
gap junctional remodeling and nerve sprouting.

Results
Animals and Serum cholesterol levels
Sixteen 3-year-old rabbits were fed with high fat/cholesterol chow for 14 weeks. A single
15-Gy THIR was applied to eight rabbits (HC+THIR group) using a heavy ion medical
accelerator. Eight rabbits who did not undergo THIR were used as controls (HC group). In
the serum cholesterol levels, there was no significant difference in the cholesterol levels
between the two groups.

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Signal averaged ECGs (SAECGs), UCGs, and 12-lead ECGs
The atrial late potentials (LPs) were characterized by RMS20ms increase in HC+THIR
rabbits compared to HC rabbits (n=4 for both groups, p<0.01). There was no significant
difference in the fPd between the two groups. The ventricular LP in HC+THIR rabbits wascharacterized by a decrease in the fQRSd and LAS40μV, and an increase in the RMS40ms,
compared to the HC group (p<0.05). Regarding the hemodynamic status estimated by
UCG, LV systolic and diastolic functions were not different between the two groups. There
were no differences in the ECG parameters, including RR and PQ intervals and QRS
durations. The QT interval was prolonged moderately in the HC+THIR group, but QTc wasunaffected.
In-vivo electrophysiology
Sustained AT (≥60 s) was elicited spontaneously in the absence of ACh infusion in 2/4
HC rabbits. In the remaining 2, sustained AT or AF was induced by programmed stimulationor burst stimulation in the presence of ACh infusion. AT or AF lasting for ≥60 sec wasterminated by direct current shock application. The incidence rate and average duration of
the HC group were 100% and 62.4 s, respectively, while in the HC+THIR group, the mean
duration was very short (5.1 s). AT was induced only in one rabbit by burst stimulation in
the presence of ACh infusion. Atrial arrhythmia inducibility and durations were higher andlonger in the HC+THIR group than in the HC group (p<0.05).
Non-sustained or sustained VT was elicited spontaneously in two HC rabbits in theabsence of NE infusion. Additionally, VF was induced by programmed LV stimulation or
LSS in the remaining two rabbits. Sustained VT or VF lasting for ≥30 s was terminated bydirect current shock application. The incidence rate and average duration in the HC groupwere 100% and >30 s, respectively, while in the HC+THIR group, the mean duration wasvery short (2.2 s). NST was induced in one rabbit by LSS in the presence of NE infusion.
Ventricular arrhythmia inducibility and durations were higher and longer in the HC+THIR
group than in the HC group (p<0.05).
Cardiac gap junction proteins
The cardiac gap junction protein expressions were examined in four rabbits of the HC and
HC+THIR groups, respectively. Cx40 formed clusters of brown punctate immunoreacted
domains at the periphery of atrial myocytes in abutment with neighbor myocytes. The
total cell area proportion occupied by Cx40 signals indicated that THIR resulted in an
increase by 59.7% and 43.6% in the RA and LA, respectively, compared with the HC
group (p<0.01). Cx43 formed clusters of brown punctate immunoreacted domains at the
periphery of ventricular myocytes; some of them were confined to intercalated disks running
perpendicular to the longitudinal axis, and others were on the lateral cell abutment. THIR
resulted in an increase by 48.1% and 40.3% in the RV and LV, respectively, compared with
the HC group (all p<0.01).
Sympathetic nerve sprouting
Immunolabeling of the growth-associated protein 43 and tyrosine hydroxylase (GAP43and TH-) positive nerves in four rabbits of the HC and HC+THIR groups, respectively. In thetissue sections of HC+THIR rabbits, the GAP43 positive nerves were less prominent. The
proportion of total area indicated that the irradiation resulted in a reduction of the GAP43immunopositive nerve density by 56.0%, 62.8%, 68.3%, and 70.6% in the RA, LA, RV and
LV, respectively, compared with the HC group (p<0.01). Contrarily, the TH-immunopositive

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domains were less abundant in the HC+THIR rabbits. The irradiation resulted in a reductionof the TH-immunopositive nerve density by 29.1%, 61.0%, 66.1%, and 64.8% in the RA,
LA, RV and LV, respectively, compared with the HC group (p<0.01).

Discussion & Conclusion
We examined the electrophysiological effects of radiation in terms of gap junction andneural remodeling using HC rabbits. In hypotheses that the arrhythmogenic substrate of HC
rabbits could be ameliorated by 15-Gy THIR. We found that SAECG showed better atrial/
ventricular LPs, electrophysiological study resulted in lower inducibility of atrial/ventriculararrhythmias, and immunohistochemistry indicated higher Cx40 and Cx43 expressions andlower sympathetic nerve densities.
THIR may improve cardiac conductivity in favor of reduction of vulnerability to AT/AF and
VT/VF. Moreover, we showed that this antiarrhythmic effect is attributed to gap junction
protein upregulation and, in part, to sympathetic denervation.
To our knowledge, our work was the first to identify the suppression of atrial/ventriculararrhythmias with the underlying mechanism of suppression efficacy by using heavy ion
beams. Next steps will be required to investigate these proposed mechanisms in humans,
using LP assessment by SAECGs and functional evaluation for sympathetic/vagus nerves
by nuclear medicine study or heart rate variability analysis.

References
1. Cuculich PS, Schill MR, Kashani R, et al. Noninvasive cardiac radiation for ablation ofventricular tachycardia. N Engl J Med 2017;377:2325-2336.
2. Robinson CG, Samson PP, Moore KMS, et al. Phase I/II trial of electrophysiology-guided
noninvasive cardiac radioablation for ventricular tachycardia. Circulation 2019;139:313321.
3. van der Ree MH, Blanck O, Limpens J, et al. Cardiac radioablation-A systematic review.
Heart Rhythm 2020;17:1381-1392.
4. Amino M, Yoshioka K, Tanabe T, et al. Heavy ion radiation up-regulates Cx43 andameliorates arrhythmogenic substrates in hearts after myocardial infarction. Cardiovasc
Res 2006;72:412-421.
5. Amino M, Yoshioka K, Fujibayashi D, et al. Year-long upregulation of connexin43 in rabbit
hearts by heavy ion irradiation. Am J Physiol Heart Circ Physiol 2010;98:1014-1021.
6. Amino M, Yoshioka K, Furusawa Y, et al. Inducibility of ventricular arrhythmia 1 year
following treatment with heavy ion irradiation in dogs with myocardial infarction. Pacing
Clin Electrophysiol 2017;40:379-390.
7. Lee KT, Hsieh CC, Tsai WC, et al. Characteristics of atrial substrates for atrial
tachyarrhythmias induced in aged and hypercholesterolemic rabbits. Pacing Clin
Electrophysiol 2012;35:544-552.
8. Liu YB, Wu CC, Lu LS, et al. Sympathetic nerve sprouting, electrical remodeling, andincreased vulnerability to ventricular fibrillation in hypercholesterolemic rabbits. Circ Res
2003;92:1145-1152.

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一般の皆様へ
体幹部定位放射線治療などの放射線技術を “難治性致死性心室不整脈 （心室頻拍）” に対して応用する発想により、 「X 線を体外から照射して不整脈基質をわずか15分で焼灼する」 という新しい治療法が2017年にワシントン大学にて実施され、 その成果は瞬く間に循環器領域のトップニュースとなりました （NEJM， 2017）。 東海大学でも2019年11月、 日本人で第一例目となる臨床治療を実施し （jRCTs032190041）、 その効果につき論文報告を行っています （Heart
Rhythm Case Reports 2021）。 しかし、 なぜ放射線が不整脈治療に有効であるかの理由は十分に明らかではありません。 我々は世界に先駆けて1997年より 「重粒子線 （放射線のなかで健常組織への影響が少ない） を利用し、 心臓に及ぼす電気生理学的効果について」 基礎研究を実施してきました （放射線医学総合研究所との共同研究）。 放射線による抗不整脈作用の主要なメカニズムは、 「ギャップ結合蛋白コネキシン43 （Cx43） の亢進による興奮伝導の回復」 に起因することを発見しましたが、 今回の研究ではさらに、 「心表面の過剰な交感神経増生の除神経作用を呈する」 可能性を明らかにしました。 現在は重粒子以外に X 線についても基礎実験を施行し、 放射性臓器障害を最小限にとどめつつ、 最大効果を得るためのデータを蓄積中です。

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Evolution of membrane morphogenesis in Asgard archaea
Yosuke Senju
yosuke.senju@okayama-u.ac.jp
Okayama University

Summary Abstract
Metagenomic analysis of the novel superphylum Asgard archaea, considered phylogenetically close to eukaryotes, has revealed genes that encode protein homologs to thosein eukaryotes that regulate morphological changes of cell membrane. We propose solving
this novel protein’s three-dimensional structure and elucidate its function by combining biophysical and cell biology methods. We will show that membrane properties such as membrane curvature and tension are essential, evolutionarily conserved features that can trigger
downstream signals and maintain cell morphogenesis and biological functions. Additionally,
we will elucidate further insights into the origin of life in prokaryotes by identifying any conserved eukaryotic-like cellular functions.
Key Words ： membrane, cytoskeleton, evolution, liposome, structural biology

Introduction
Cells are defined as the basic units of life. A key function is to compartment cellular contentsfrom their surroundings by cell membranes. Besides this, morphological changes in cell membranes are essential for biological functions, including cell motility, cell division, and differentiation, which are pathologically relevant in cancer cell invasion and metastasis (1).
How are signal transduction mechanisms that originate with changes in cell membrane
morphology acquired and selected during evolution? Recently, metagenomic reconstructionof Asgard archaea superphylum, considered closely related to eukaryotes, revealed several
genes that encode proteins with eukaryotic homologs (2-4). These include proteins that
control changes in cell membrane morphology, actin cytoskeleton, and regulators of actin
polymerization, previously thought to be absent in prokaryotes.

Results
This study elucidates how the ability to facilitate and respond to morphological changes
in the cell membrane of a representative biological system has evolved by solving the crystal structures of novel proteins found in Asgard archaea
Based on metagenomic analysis results, novel proteins with eukaryotic homologs
have been identified in Asgard archaea, suggesting that a mechanism for altering membrane morphogenesis is conserved in both prokaryotes and eukaryotes. Asgard archaea may have obtained this mechanism by phagocytosis of a bacterium with these novel
proteins and actin polymerization.
We are pursuing the following six lines of research.
Line 1
We have identified novel proteins in Asgard archaea using bioinformatics and created a

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molecular evolutionary phylogenetic tree (unpublished). Genes
have been synthesized for these and are for further studies.
Line 2
We have successfully expressed and purified the novel proteins. Crystals were obtained by screening for protein crystallization conditions (Fig.1), and a 2.9-Å resolution diffraction
dataset has been collected at Spring-8 (unpublished). We are
now introducing selenomethionine to determine its phase.
Fig. 1. Crystals of a novel
Line 3protein from Asgard archaea
X-ray crystal structure analysis will be attempted using the(unpublished)beamlines of the Spring-8 synchrotron radiation facilities. From
the XRD data, we will elucidate the three-dimensional structures of the proteins and compare them with those of known eukaryotic proteins to predict intracellular functions. Additionally, we will examine the conservation of amino acid residues necessary for phospholipid binding.
Line 4
Purified archaeal proteins will be labeled with fluorescent dyes and applied to giant unilamellar vesicles (GUVs) to clarify whether they can deform cell membranes.
Line 5
We will express Asgard archaeal proteins fused with green fluorescent protein (GFP) incells to determine whether they can deform cell membranes and identify their subcellular
localizations.
Line 6
Correlative light and electron microscopy (CLEM) has allowed us to identify protein functions and elucidate morphology, and high spatial resolution fluorescence and electron microscopy have revealed structure information (unpublished).
The laboratory in which the applicant is currently working has published remarkable accomplishments in protein purification, crystallization, and structural analyses and has accumulated significant knowledge and resources. Additionally, the applicant has sufficientexperience in biophysics using GUVs, and cell biological experiments, and has already prepared samples and equipment (5-6).
From the above series of studies, we will further elucidate whether the regulation of Asgard archaeal proteins in cell morphogenesis are essential for maintaining biological systems preserved throughout evolution.

Discussion & Conclusion
Functional analysis of proteins that regulate cell membrane morphogenesis has been
performed mainly in eukaryotic endocytosis and filopodia formation studies. Recent
metagenomic studies have shown that Asgard archaea contain protein homologs
involved in cell membrane morphogenesis, including cytoskeletal actin and regulatorsof actin polymerization. A professor in the applicant's laboratory has succeeded in X-ray
crystallographic analysis of complex actin and Asgard profilin, an actin-binding protein
regulating actin polymerization (7). Interestingly, Asgard profilins bind to actin and regulate

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The Novartis Foundation(Japan)
for the Promotion of Scienceactin polymerization. Thus, actin polymerization is essential for life and is conserved
throughout the evolutionary process from prokaryotes to eukaryotes. According to thisbackground, we are convinced that we can find in Asgard archaea how the functions ofproteins in cell morphogenesis have been conserved and developed during evolution.

References
1: Senju Y, Kalimeri M, Koskela EV, Somerharju P, Zhao H, Vattulainen I, Lappalainen
P. Mechanistic principles underlying regulation of the actin cytoskeleton byphosphoinositides. Proc Natl Acad Sci U S A. 2017 Oct 24;114(43):E8977-E8986.
2: Spang A, Saw JH, Jørgensen SL, Zaremba-Niedzwiedzka K, Martijn J, Lind AE, van
Eijk R, Schleper C, Guy L, Ettema TJG. Complex archaea that bridge the gap between
prokaryotes and eukaryotes. Nature. 2015 May 14;521(7551):173-179.
3: Zaremba-Niedzwiedzka K, Caceres EF, Saw JH, Bäckström D, Juzokaite L, Vancaester
E, Seitz KW, Anantharaman K, Starnawski P, Kjeldsen KU, Stott MB, Nunoura T, Banfield
JF, Schramm A, Baker BJ, Spang A, Ettema TJ. Asgard archaea illuminate the origin of
eukaryotic cellular complexity. Nature. 2017 Jan 19;541(7637):353-358.
4: Imachi H, Nobu MK, Nakahara N, Morono Y, Ogawara M, Takaki Y, Takano Y, Uematsu
K, Ikuta T, Ito M, Matsui Y, Miyazaki M, Murata K, Saito Y, Sakai S, Song C, Tasumi E,
Yamanaka Y, Yamaguchi T, Kamagata Y, Tamaki H, Takai K. Isolation of an archaeon at
the prokaryote-eukaryote interface. Nature. 2020 Jan;577(7791):519-525.
5: Senju Y, Zhao H. Fluorescence Assays to Study Membrane Penetration of Proteins.
Methods Mol Biol. 2021;2251:215-223.
6: Senju Y, Lappalainen P, Zhao H. Liposome Co-sedimentation and Co-flotation Assays to
Study Lipid-Protein Interactions. Methods Mol Biol. 2021;2251:195-204.
7: Akıl C, Robinson RC. Genomes of Asgard archaea encode profilins that regulate actin.
Nature. 2018 Oct;562(7727):439-443.

一般の皆様へ
真核生物に近い系統と考えられている、 アスガルドと命名された新規古細菌のメタゲノム解析
から、 細胞膜の形態変化を制御するタンパク質をコードする遺伝子が発見された。 本研究課題
では、 アスガルド古細菌の新規タンパク質の立体構造を解き、 生物物理的手法と細胞生物学を組み合わせてその機能を解明する。 さらに、 原核生物にも保存された真核生物と同様な細胞機能を見いだしていくことで、 生命の起源の一端を解明していく。

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Quantum beam analysis on structure formation of
glucuronosyltransferase catalyzing drug metabolism
Eiichi Mizohata
mizohata@chem.eng.osaka-u.ac.jp
Osaka University

Summary Abstract
Glucuronosyltransferase plays a central role in detoxification of toxic insolublecompounds by promoting their removal from the body. This study elucidated the interactionof glucuronosyltransferase and GroEL by observing the binary complex structure using
cryo-EM single particle analysis at 3 Å resolution.
Key Words ： glucuronosyltransferase; cryo-electron microscopy; protein folding

Introduction
Glucuronosyltransferase catalyzes the transfer of UDP-glucuronic acid as a donor
substrate to insoluble compounds such as bilirubin as an acceptor substrate. Determinationof the structure of the enzyme and understanding of its structure-function relationshipare important for future medical applications. However, structural studies of the enzyme
have been difficult because it has never been functionally isolated and purified using
any recombinant protein expression system. I attempted to purify a mutant enzyme
by expressing it in E. coli, and succeeded in isolating a complex of the enzyme andE. coli GroEL. The aim of this study is to elucidate the mechanism of interaction of
glucuronosyltransferase and GroEL by observing the complex structure using cryo-EM.

Results
1.2 million particle images from 2,951 micrographs were processed using the RELION
software package and successfully classified into 38 reference-free 2-dimensional classes
with multiple particle orientations with flexible substrate densities clearly visible in theside view in the ring cavity. Scrutiny of these particle images resulted in an asymmetricreconstruction of two GroEL complexes containing bound glucuronosyltransferase
substrates with a resolution of 3 Å, evaluated at the criterion of FSC = 0.143. The modelingof the individual subunits was done by rigid fitting of the atomic coordinates of the previously
solved structure to the EM density map using COOT and final refinement of the map
model in Phenix. As in previous studies, the density of glucuronosyltransferase is found tobe localized in the hydrophobic substrate-binding helix of the apical domain covering thefolding chamber cavity. It is conspicuously in contact with 2-3 subunits on one side of thering.
The capture of non-native substrates by GroEL is performed by exposed hydrophobic
residues covering the open cavity of the seven-membered ring. Such non-specific binding of
non-native polypeptides is required for the folding protein of GroEL, which requires contact
with the apical domains of the 2-3 contiguous subunits that make up the ring. Previous

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for the Promotion of Sciencestudies of non-native polypeptide binding have revealed several structures detailing theessential residues of GroEL binding, various low-energy substrate binding conformational
states, hydrophobic and amphiphilic binding motifs, and the initial substrate binding state.
In complete agreement with these studies, the substrate density was formed around two or
three subunits in the ring. Based on previous biochemical and structural studies, this study
developed a new approach to 3-dimensional classification using a signal mask to confirm
this observation. A tailor-made masking strategy was created, combining a cylindricalvolume containing a cavity region and specific residues of the apical domains of the threesubunits (two adjacent and one laterally opposite in the ring).
Focused classification (masked 3D classification) on the refined map allowed toseparate the particle images from the 3 Å consensus map based on compositional andconformational heterogeneities contained only in the masked regions, while maintaining theorientation of the original particle images.
The analysis resulted in a three-dimensional volume that accounted for 96% of thetotal particle image. This volume clearly showed the density formed between the two tip
domains, while very little was present in the tip domain across the cavity.
Unexpectedly, this method resulted in a classified volume (about 2% of the particle
image) where the density in the cavity was not visible. When the mask was removed andthe rings with and without substrate were compared, it was found that the ring cavity had
expanded by about 4 Å as the substrate bound to it. Closer examination of the results
revealed that the conformation of the seven-membered ring was different at the domain
level of the subunit.

Discussion & Conclusion
This study demonstrated that there are 2-3 consecutive subunit binding motifs in theseven-membered ring, which explains the unexpected ring cavity extension in the ringoccupied by glucuronosyltransferase.
Furthermore, the study analyzed the single-ring (sR) and double-ring (dR) complexes
and were able to model the continuous movement of subunits upon substrate binding.
These data provide an in vivo and near-atomic resolution snapshot of the early stages of
the GroEL-glucuronosyltransferase folding reaction cycle.
This allows us to reveal the recognition and occupancy of non-native substrates by
GroEL, as well as to annotate intramolecular changes.

一般の皆様へ
生 物 の 細 胞 は タ ン パ ク 質 で で き て い ま す。 タ ン パ ク 質 は 様 々 な 構 造 を も ち、 こ の 構 造 はGroEL と呼ばれる巨大なタンパク質の中で形づくられます。 今回、 体内の有毒物質や薬剤の解毒を担うタンパク質であるグルクロン酸転移酵素が、 GroEL にどのように認識されて構造が形成されるのか、 そのメカニズムの一端を、 クライオ電子顕微鏡を用いて可視化することができました。
将来、 がんや黄疸などの病気の治療につながる成果です。

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Novel molecular basis of intracellular cholesterol trafficking inendothelial cells
Masashi Maekawa
masashim@m.ehime-u.ac.jp
Proteo-Science Center, Ehime University

Summary Abstract
Endothelial cells lining along inside vessels receive a variety of stimulations leading totheir patho-physiological responses (e.g. angiogenesis). Cholesterol is an essential lipid
for cell survival including endothelial functions. In this study, we identified a ubiquitin ligase
complex, cullin-3 (CUL3)/BTBP/substrates, that requires both localization of cholesterol at
the plasma membrane and angiogenesis.
Key Words ： Endothelial cells, Cholesterol, Cullin-3, BTBP

Introduction
Cholesterol is an essential component of cell membranes. Although angiogenesis ofhuman umbilical endothelial cells (HUVECs) requires plasmalemmal cholesterol (ref 1),
the molecular mechanisms for the determination of proper cholesterol localization at theplasma membrane remain unclear. Cullin-3 (CUL3) is a scaffold protein for a RING-type
ubiquitin E3 ligase complex. The CUL3-mediated ubiquitination requires recruitment oftarget substrates through its recognition by substrate recognition receptors, BTBPs. Human
genome encodes 183 BTBPs and the repertoire of BTBP exerts various cellular functionsof the CUL3 system. We have found that CUL3 knockdown inhibited angiogenesis as well
as dissociation of D4H, a cholesterol biosensor (ref 2), from the plasma membrane. These
data suggest the existence of BTBP/substrates axes that are essential for cholesterol
trafficking to the plasma membrane leading to proper angiogenesis. This study aims
to identify the BTBPs and ubiquitinated substrates in the CUL3 system, which regulatecholesterol trafficking in endothelial cells.

Results
Identification of a BTBP that is responsible for plasmalemmal localization ofcholesterol in HUVEC
To identify BTBPs that regulate cholesterol localization at the plasma membrane, wesilenced 183 BTBP genes in HUVECs using BTBP siRNA library (ref 3). The mCherrytagged D4H cholesterol biosensor was expressed in HUVECs. As a readout, we observed
D4H localization at the plasma membrane by an A1R confocal microscopy. As a result, we
identified one BTBP (BTBP-X), by which knockdown caused the disappearance of D4Hfrom the plasma membrane (unpublished data). Importantly, knockdown of BTBP-X in
HUVECs drastically inhibited tube formation that mimics angiogenesis in vitro. These datasuggest that BTBP-X is essential for cholesterol trafficking and angiogenesis.

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A screen of BTBP-interacting proteins by AlphaScreen using human protein arrays
We next sought to identify the ubiquitinated substrate by the CUL3/BTBP-X axis. To thisaim, we made use of AlphaScreen that detects the direct protein-protein interactions invitro. The human protein array (approximately 20,000 of FLAG-GST tagged proteins) was
produced by wheat germ extracts. We also prepared biotinylated BTBP-X. As a result, we
identified approximately 100 proteins that interact with BTBP-X (unpublished data).

Discussion & Conclusion
We still need to assess candidate substrates of the CUL3/BTBP-X axis. First, we
will prepare siRNA library for the candidate substrates (~100 genes). Genes by whichknockdown exhibits mislocalization of D4H at the plasma membrane as well as inhibition ofangiogenesis could be target substrates the CUL3/BTBP-X axis. Second, we will validate
the CUL3/BTBP-X-dependent ubiquitination of the substrates by in vitro ubiquitination
assay. We have recently identified novel molecular machineries that regulates endothelial
functions (ref. 4, 5). The crosstalk of the CUL3/BTBP-X/substrate complex with otherpathways should be examined. In future, inhibitors of the formation of CUL3/BTBP-X/
substrate complex would be novel anti-angiogenic drugs. Since the agents do not target
VEGF pathways, the combination with existing anti-VEGF drugs would promise better antiangiogenic efficacy.

References(1) Fang et al., Nature. 498 (7452). 118-122. 2013
(2) Maekawa and Fairn. J. Cell Sci. 128 (7). 1422-1433. 2015.
(3) Maekawa et al., Biol. Open. 6 (11). 1707-1719. 2017.
(4) Kovacevic et al., J. Cell Biol. 217 (3). 1015-1032. 2018.
(5) Tanigawa, Maekawa et al., 234 (10). 17280-17294. 2019.

一般の皆様へ全身に行き渡る血管は人体最大の臓器の一つです。 血管の内側に存在し、 血流と直に接する
血管内皮細胞は炎症反応や血管新生等の生理機能発揮に重要な役割を持ちます。 近年の研究で、 血管内皮細胞内でコレステロールが細胞膜に適切に存在する事が血管内皮細胞の機能に重要である事が分かってきました。 一方で、 血管内皮細胞内のコレステロールを適材適所に配置させる仕組みは良く分かっていません。 本研究では、 この仕組み （= コレステロールの細胞内輸送） の分子機構の一端を解明する事に成功しました。 今後は当該分子機構を標的とする新
しい医薬品 （血管新生阻害剤等） 開発への応用が期待されます。

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Study of the mechanistic dissection of cancer incidence caused byaging-associated dysfunction of cell competition in intestinal cells
Shunsuke Kon
kon44@rs.tus.ac.jp
Tokyo University of Science

Summary Abstract
In this study, we exploited the cell competition mouse model and examined how
sequential accumulation of gene mutations which mimic the aging-associated genomic
alteration affects the function of cell competition. The directionality of cellular extrusionof RasV12-transformed cells in the Wnt-activated background conferred by APC ablationor β-catenin activation is reversed, and the transformed cells were outcompeted to basalside through non-cell autonomous MMP21 upregulation. Furthermore, we found that theincreased production of MMP21 was mediated at least, in part, via NF-κB signal, andhampering NF-κB signal restored apical elimination of transformed cells. Collectively, theseresults demonstrate that Wnt activation promotes diffuse invasion of transformed cells
through malfunctional cell competition.
Key Words ： Cell competition, multi-sequential carcinogenesis, diffuse invasion,

Introduction
Living organisms implement the self-defense systems to fight against cancer, withcell competition being one of tumor-surveillance systems to remove the newly emerging
transformed cells. In general, the progressive accumulation of genetic aberrations by aging
is strongly associated with onset of tumorigenesis. This prompted us to investigate whether
the sequential genetic mutations affect the behavior of transformed cells in the competitiveenvironment. We herein engineer the mice to sustain infrequent somatic activation of Ras in
the background of APC silencing in a manner reminiscent of human familial adenomatous
polyposis and examine the cell fates of transformed cells.

Results
1.Aberrant Wnt-activation disturbs
apical extrusion of RasV12-transformed
cells and induces non-cell autonomous
diffuse invasion.
To examine the effects of APC-deficiency on efficiency of cell competition,
we exploited APCmin mice harboring heterozygous loss-of-functional mutation in
APC gene and crossed with the cell competition mouse model where RasV12transformed cells are mosaically proFigure 1. Frequency of basal invasion

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duced by administration of the low dose tamoxifen (1). The substantial number of RasV12expressing cells surrounded by normal cells were apically eliminated in single RasV12mutated mice, while tiny fraction of transformed cells appeared basally extruded. We thenfound that number of basally extruded cells in APC/RasV12-mutated mice were significantly
higher than single RasV12-mutated mice. This suggests that Wnt activation leads to malfunction of cell competition, resulting in promotion of basal extrusion of RasV12-transformed
cells (Figure 1). At 28 days after tamoxifen administration, APC/RasV12-transformed cells
were confined in the stroma of the upper villi. Yet, microscopically detectable deformities tothe tissue architecture in the form of benign tumors were not observed in surrounding tissues, indicating that cancer cells were directly generated from normal mucous membrane
through diffuse invasion of transformants.
2. MMP21 regulates the non-cell autonomous basal invasion of β-cat ΔN/RasV12 cells
in vitro.
With the aim of understanding molecular mechanism underlying the non-cell autonomousbasal extrusion of transformed cells, we established MDCK or MDCK pTR-RasV12 cells
stably expressing a mcherry-conjugated β-catenin mutant (β-catΔN) to mimic Wnt-induced
basal extrusion of RasV12 cells in vitro. When β-catΔN/RasV12-transformed cells were
co-cultured with β-cat ΔN cells at a ratio of 50:1, a sizeable fraction of β-cat ΔN/RasV12
cells were basally invaded into the collagen matrix over time, successfully recapitulating
the salient feature of non-cell autonomous basal extrusion of APC/RasV12 cells in vitro. To
reveal the underlying molecular mechanism whereby Wnt activation promotes the diffuse
invasion of RasV12-transformed cells, we conducted a microarray analysis to search formolecules whose expression is changed in β-cat ΔN/RasV12 cells co-cultured with β-cat
ΔN cells. Among the top listed highly expressing genes, we found one member of matrix
metalloproteases (MMPs) superfamily named MMP21 was highly upregulated. Furthermore,
MMP21 knockout prevented the basal extrusion of β-catΔN/RasV12 cells, highlighting anactive role for MMP21 in non-cell autonomous basal invasion of transformed cells.
3. NF-κB signal regulates the upregulation of MMP21.
A gene-set enrichment analysis (GSEA) revealed that NF-κB pathway was activated in
β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells. Accordingly, the reporter assay using
the plasmid in which luciferase expression is under the control of tandem repeats of the NFκB transcriptional response element showed the non-cell autonomous activation of NF-κB
signal. To study functional relevance of NF-κB
signal in MMP21-mediated diffuse invasion oftransformed cells, we treated cells with inhibitorof NF-κB, BAY11-7082. As a result, additionof BAY 11-7082 substantially diminished thenon-cell autonomous upregulation of MMP21,
suggesting that NF-κB signal functions as an
upstream regulator for enhancing the MMP21
expression. Furthermore, inhibition of NFκB signal profoundly suppressed the basal
extrusion of β-cat ΔN/RasV12 cells co-cultured
Figure 2. Summary of this study

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with β-cat ΔN cells, or even enhanced apical elimination of them. These results suggestthat NF-κB signal plays a central role for redirecting transformed cells to basal invasion via
MMP21 upregulation (Figure 2).

Discussion & Conclusion
I n t h i s s t u d y, w e f o u n d t h e a b e r r a n t a c t i v a t i o n o f W n t s i g n a l i n g a l t e r e d
the balance in cell competition-induced cellular extrusion, turning RasV12transformed cells to invade basally. The basal invasion of RasV12 cells was
induced, at least partly, by MMP21 activity intrinsic to the translocating cells,
and involved NF-κB signal. The diffuse-type cancers generated in the digestive
tract are hardly treatable due to inconspicuous early symptoms, highly invasive
characters of cancer cells. Therefore, it is of clinical significance to delineate
the nature of pathogenesis of diffuse-type cancer, and there is a requirement forsensitive novel treatments. The present study is indicative of NF-κB-MMP21 axis
as a novel therapeutic target for the treatment of diffuse-type carcinoma. This
study also points to NF-κB signal as one of key determinants of switching thepredominant direction of extrusion from apical to basal. Yet, it remains elusive
which factor(s) determine the divergent fate or APC/RasV12-transfomed mutants.
It needs to be addressed in the future how the molecular landscape is profoundly
remodeled to rewire the direction of extrusion by Wnt activation, which could be
harnessed for therapeutic applications.

References
1) Kon, S., Ishibashi, K., Katoh, H., et al. 2017. Cell competition with normal epithelial cells
promotes apical extrusion of transformed cells through metabolic changes. Nat Cell Biol
19:530-541.

一般の皆様へ
本研究成果より、 細胞競合の機能変容によって、 がん変異細胞が基底膜へとびまん性に浸潤する分子機構の一端を明らかにすることができました。 具体的には、 Wnt シグナルの活性化によって、 細胞非自律的に NF- κ B シグナルが活性化され、 さらにマトリックスメタロプロテアーゼ
である MMP21の発現が増加することにより、 がん細胞が基底膜浸潤能を獲得することが分かりました。 びまん性の悪性腫瘍は一般的に予後が悪く、 革新的な治療法が希求されているため、 本研究により明らかとなった NF- κ B-MMP21経路が治療標的となることが今後期待されます。

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Identification of premalignant gastric glands in chronic gastritis
mucosa and prophylactics for gastric cancer development.
Takahiro Shimizu
shimy@kuhp.kyoto-u.ac.jp
Kyoto University Hospital

Summary Abstract
To explore the susceptibility of pure gastric intestinal metaplasia (IM) to cancer development, we investigated the landscape of genetic alterations in gastric single glands. Whole
exome sequencing of gastric single glands revealed significantly high accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Targeted
capture deep sequencing and copy number analyses in gastric clustered glands showedthat IM glands polyclonally expanded to aggregates, which commonly had copy number
aberrations (CNAs) similar to those of intramucosal gastric carcinoma (IGC). These results
suggest that in the gastric mucosa inflamed with Helicobacter pylori (H. pylori) infection, IM
glands expand via acquisition of CNAs comparable to those of gastric cancer, contributing
to field cancerization.
Key Words ： gastric cancer, intestinal metaplasia, copy number aberration

Introduction
Gastric IM is considered a risk factor for gastric cancer in gastric mucosa with H. pylori
infection. However, whether IM is a true precursor lesion or just a confounding factor for
gastric cancer remains unknown. To explore the susceptibility of pure gastric IM to cancer
development, we investigated the landscape of genetic alterations in gastric single glands
with IM.

Results
We isolated 42 single glands with or without IM in inflamed gastric mucosa from 11
patients with IGC and in non-inflamed gastric mucosa from 3 people. Then, we performed
whole-exome sequencing (WES) of each single gland after the whole amplification process.
The mean number of mutations in single normal glands was 18.5, whereas those in single
non-IM glands and single IM glands were 32.2 and 81, respectively. Somatic mutations
in single normal glands and single non-IM glands were within the range predicted to beacquired spontaneously in normal cells of various tissues, whereas single IM glands had
more spontaneous mutations than those predicted in normal cells. In addition, mutation
signature analysis of single glands revealed that C to T substitutions at the CpG site,or signature 1, was clearly dominant in single IM glands, suggesting that increased cell
turnover could be related to the induction of mutations in IM glands. Intramucosal gastriccarcinoma with microsatellite stability phenotype in the same cases had averages of 89.65
mutations. Limited to cancer-related genes, single IM glands showed 3.8 non-synonymous
mutations on average, whereas single non-IM glands and normal glands showed 1.2 andThe Novartis Foundation(Japan)
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1.0 non-synonymous mutations on average, respectively. However, no mutations weredetected in the driver genes crucial for gastric cancer development, such as TP53, ARID1A,
APC, CDH1 and RHOA. Phylogenetic analyses of IM aggregates showed that IM glands
expand to form clusters sharing some mutations.
Next, we performed targeted-capture deep sequencing in 96 gastric-clustered glands.
The number of somatic mutations in targeted genes was similar between clustered IM
glands and clustered non-IM glands (1.92 vs 1.67 on average, respectively, P = 0.60).
Regarding cancer-related genes, clustered IM glands had more mutations than didclustered non-IM glands, but the difference was not significant (0.27 vs 0.13 per sample,
respectively, P = 0.39). No mutations were detected in crucial driver genes, including TP53,
ARID1A, APC, CDH1 and RHOA.
Furthermore, we performed autosomal CN analyses in 96 clustered glands of inflamed
gastric mucosa. CNAs were identified in 33 of 72 clustered IM glands, whereas only
2 glands among 24 clustered non-IM glands had CNAs. The CNA score revealed thatclustered IM glands had significantly broader CN gain than did clustered non-IM glands(BCS 1.62 vs 0.17, P = 0.0121), whereas no difference was observed between clustered
IM and non-IM glands in terms of CN loss (BCS 0.11 vs 0, P = 0.61. FCS 0.51 vs 0, P =0.757) and focal CN gain (FCS 0.56 vs 0, P = 0.535). To compare the CNAs of IM or nonIM glands with those of gastric cancer, we calculated the autosomal CNA score for 41 IGC
from our previous study, and of 10 submucosal gastric carcinoma at Stage IA and 34 gastricadenocarcinoma at Stage IB from TCGA database. Broad CNAs showed similar trends
between IM and IGC and were significantly increased in submucosal gastric carcinoma
and stages that are more progressive. On the contrary, focal CNAs were increased in IGC
compared to those in IM glands. CNAs frequently observed in clustered IM glands showedbroad gains of chr 2, 8 and 20. The distribution of CN gain in IM glands was quite similar
to that in IGC and more progressive stages. CN loss was frequently observed in Stage IA
and IB gastric carcinoma, whereas it was rarely observed in gastric IM glands and non-IM
glands. These results suggest that broad CN gains, including gain of chr 8 and 20, wereacquired by IM before malignant transformation, and that focal CNAs and broad CN loss
emerged from the early stage of gastric cancer.

Discussion & Conclusion
We demonstrated that gastric IM is susceptible to somatic mutations and CNAs andthat it expands multi-focally with acquisition of CNAs commonly found in gastric cancer.
This suggests that the chronic inflamed stomach has a number of small fields where IM
glands with CNAs expand, resulting in field cancerization. In addition, the similarity of CNA
patterns and the number of mutations in gastric IM and gastric cancer in the early stage
indicated that most intestinal-type gastric cancers might originate from IM glands. Thesefindings could be a clear evidence that gastric cancer risk exists, especially, in people withgastric IM even after eradication of H pylori. Thus, to prevent gastric cancer development,
H pylori eradication should be performed early before the development of IM. For people
with gastric IM, the IM region should be evaluated carefully during short-interval endoscopy
as surveillance for cancer development. To determine approaches for further prevention ofgastric cancer development, it would also be important to unveil the mechanism by which
IM develops during chronic gastric inflammation and to develop therapeutic and preventive

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approaches targeting IM specifically.

References
Shimizu T, Marusawa H, Matsumoto Y, et al. Accumulation of somatic mutations in TP53
in gastric epithelium with Helicobacter pylori infection. Gastroenterology 2014;147:407–17.
e3.
Huang KK, Ramnarayanan K, Zhu F, et al. Genomic and Epigenomic Profiling of HighRisk Intestinal Metaplasia Reveals Molecular Determinants of Progression to Gastric
Cancer. Cancer Cell 2018;33:137-150.e5.
Kakiuchi N, Yoshida K, Uchino M, et al. Frequent mutations that converge on the NFKBIZ
pathway in ulcerative colitis. Nature 2020;577:260–265.

一般の皆様へ
H.pylori 感染による慢性胃炎粘膜に発生する腸上皮化生は前癌病変であるのか傍癌病変であるのかは議論の分かれるところである。 そこで、 本研究では、 慢性胃炎粘膜から腸上皮化生と非腸上皮化生を取り分け、 網羅的なゲノム解析を行うことで、 その点を明らかにしようとした。
腸上皮化生には、 早期胃癌とほぼ同じ数の遺伝子変異を認め、 また早期胃癌でよく見られるコ
ピー数異常も認めた。 本研究の結果は、 腸上皮化生から胃癌が発生することを直接証明したわけではないが、 腸上皮化生が前癌病変である可能性を示唆する結果であった。 腸上皮化生の発生予防や、 腸上皮化生をターゲットとした治療に向けて現在研究を継続中である。

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Mechanism of inflammatory memory in hematopoietic stem cells
using informatics unifying multidimensional single cell data
Hitoshi Takizawa
htakizawa@kumamoto-u.ac.jp
International Research Center for Medical Sciences, Kumamoto University

Summary Abstract
Inflammation is the cardinal immune response for defense against pathogens and forfacilitating tissue repair. Long-lived hematopoietic stem cells (HSCs) are maintained in
adult bone marrow (BM) with little immune reactions upon immunological challenges, andproduce all blood and immune cells. This project aims at understanding how HSCs canmemorize inflammation, and if it is possible to enhance HSC function by modulating its
inflammatory memory.
Key Words ： Hematopoietic stem cells, Inflammatory memory, single cell data

Introduction
Adult bone marrow (BM) had been long thought to be an immune-privileged organ
and thus, to form an advantageous environment to preserve long-lived hematopoietic
and immune cells. However, recent studies indicate that the BM is in fact an active siteof various types of immunological and inflammatory reactions which largely influencequiescent blood-forming hematopoietic stem cells (HSCs). We have previously shown thatsystemic bacterial infection modulates BM-resident HSC identity through innate immune
signal activation in a HSC-intrinsic and -extrinsic (niche-dependent) manner.

Results
Recent works have proposed that innate immunity is sensitized (“trained”) upon infection
and immune cells are epigenetically inscribed with innate immune memory to resist future
infectious insults (Netea MG, Science 2016). Likewise, HSC and their progenitor can also
memorize infection by changing their metabolism and epigenetics (Sezaki M, Front Immunol2020). Our study has previously shown with bacterial infection models, that gram negative
bacterial infection activates HSCs to proliferation but eventually impairs their hematopoietic
repopulating ability through direct activation of innate immune signaling (Takizawa H, Cell
Stem Cell 2017).
Since microorgansims coexist in our body, we studied how hematopoiesis responds tomicrobial signals upon their infiltration to the body, by using inflammatory bowel disease
mouse model. We have found that intestinal tissue damage activates early hematopoiesis in
BM via microbial signals and direct early hematopoietic progenitors to inflamed mesentericlymph node to locally produce myeloid cells and promote tissue repair, suggesting crossorgan communication-driven tissue repair (unpublished data). However, chronic tissue
invasion impinges on lifelong hematopoietic repopulation without disturbance of daily
hematopoiesis, and decreased their responsiveness to immunological challenges.

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To understand the mechanism by which microbial signals regulate HSC function towardtissue repair, we performed single cell RNA-/ATAC-sequencing with HSCs that had been invivo pre-stimulated with lipopolysaccharide (LPS), a gram-negative bacterial component,
and a microbacterial species (Bac). Although the analysis is still ongoing, a preliminary
result showed innate immune signal activation as a control, metabolic regulators, and many
genes involved in immune activation and epigenetics. We also performed a mass cytometerto analyze a number of epigenetic marks like histon H3 K27 acetylation, and found somehiston marks got activated at 16 hours after the stimulation and stayed active even at 1
month after, indicating that the innate immune signals imprint on HSCs. To confirm thischromatin change and its impact on locus specific gene regulation, we will do cut-tag
sequencing, an alternative of ChIP-seq for smaller cell number, and see what genes are
regulated by the specific epigenetic marks found here. We are still on the way to develop abioinformatics analytic pipeline that unifies all three single cell data set to predict key factorsthat control inflammatory memory in HSCs. Given successful selection of those candidate
genes, we will overexpress or knockdown the genes of our interest in HSCs and transplant
the genetically modified HSCs to lethally irradiated mice, to reconstitute hemato-immune
system and test immune activation in response to infection re-challenge.

Discussion & Conclusion
How long-lived tissue stem cells as HSCs respond to hematopoietic challenges, andhow the response relates to the functional alteration of hemato-immune system andhematopoietic abnormalities (e.g. aplasia/neoplasis) is a focus of ongoing research.
Emerging evidences have demonstrated that in response to tissue invasion such as
infection, inflammation, HSCs and their progenies are activated to migrate, proliferateand differentiate via immune cell-secreted cytokine- or pathogen-associated molecule
pattern signaling. We have shown that tissue-insulting bacteria or LPS directly cause
functional attenuation, while commensal bacteria, i.e, microbiota instructs hematopoiesis topromote tissue repair. Thus, Uncovering the molecular mechanism by which hematopoietic
adaptation is formed and maintained in early hematopoietic cells, which reside at the apexof hemato-immune system, will dissociate beneficial signals from unwanted ones.
The project will converge to the development of a new strategy for vaccination against
existing and emerging infection, or for regenerative medicine, especially for infants orelderly who have reduced tissue function. Adequate management of the hematopoiesisniche cross-talk during tissue invasion is a key to success in treating patients with chronic
inflammatory diseases, autoimmunity, and cancer, which incidence increase with age(Kovtonyuk LV, Front Immunol 2016).

References
1. Netea MG, Joosten LA, Latz E, Mills KH, Natoli G, Stunnenberg HG, O’Neill LA, Xavier
RJ. Trained immunity: A program of innate immune memory in health and disease.
Science. 2016 Apr 22;352(6284):aaf1098. doi: 10.1126/science.aaf1098. Epub 2016 Apr
21.
2. Sezaki M, Hayashi Y, Wang Y, Johansson A, Umemoto T, Takizawa H. Immunomodulation of hematopoietic stem and progenitor cells in inflammation, Front Immunol.,

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for the Promotion of Science2020 Nov 24;11 doi: 10.3389/fimmu.2020.585367
3. Takizawa H, Fritsch K, Kovtonyuk LV, Saito Y, Yakkala C, Jacobs K, Ahuja AK, Lopes
M, Hausmann A, Hardt WD, Gomariz Á, Nombela-Arrieta C and Manz MG. Pathogeninduced TLR4-TRIF innate immune signaling in hematopoietic stem cells promotes
proliferation but reduces competitive fitness. Cell Stem Cell., 2017 Aug 3;21(2):225-240
4. Kovtonyuk LV, Fritsch K, Feng X, Manz MG, Takizawa H. Inflamm-Aging of
Hematopoiesis, Hematopoietic Stem Cells and the Bone Marrow Microenvironment.
Front Immunol., 2016, Nov 14;7;502

一般の皆様へ
炎症は病原体に対する生体防御および組織修復を促進するための主要な免疫応答です。 骨髄はほとんど免疫応答が起こらない免疫特権臓器の一つとして、 長命の血液細胞および免疫細胞を保存するのに有利な環境を形成すると考えられてきました。 しかしながら、 近年、 骨髄では実に活発で多様な免疫応答が行われており、 血液の源泉である血液幹細胞の機能特性に多大な影響を及ぼすことが分かりつつあります。 本研究では、 炎症が血液幹細胞機能を適応させ、感染症、 炎症および血液悪性腫瘍などに対してより応答性のある血液 ・ 免疫系を構築するという “血液幹細胞の炎症記憶” という概念を検証します。 得られる知見は、 様々な感性症に対するワクチン戦略や老化に伴い増加する炎症疾患の制御や治療に対して非常に有用な知見をもたらすものと期待されます。

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Molecular mechanisms of mycobacterial effector protein for thedevelopment of novel anti-TB drugs
Giichi Takaesu
takaesug@comb.u-ryukyu.ac.jp
Tropical Biosphere Research Center, University of the Ryukyus

Summary Abstract
A mycobacterial virulence factor, Zn2+ metalloprotease 1 (Zmp1), is known to suppress IL1β production by inhibiting caspase-1 resulting in phagosome maturation arrest. However,
the molecular mechanism of caspase-1 inhibition by Zmp1 remains obscure. In this study,
we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial
respiratory chain complex I, as a novel Zmp1-binding protein. Using CRISPR/Cas9mediated gene knockout cells, we found a previously unrecognized role of GRIM-19 as an
essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1mediated suppression of IL-1β production during mycobacterial infection.
Key Words ： Tuberculosis, Inflammasome, Mitochondrial respiratory complex I

Introduction
Mycobacterium tuberculosis (Mtb), a causative agent of human tuberculosis, is an
intracellular pathogen that primarily infects macrophages. Macrophages cultured in thepresence of anti-IL-1β neutralizing antibody lose control of intracellular Mtb replication.
Conversely, the addition of recombinant IL-1β in the macrophage culture restricts theintracellular Mtb replication, indicating the critical role of IL-1β for the bactericidal activity ofmacrophages against Mtb1,2. It has been shown that the mycobacterial Rv0198c gene that
encodes Zmp1 is required for phagosome maturation arrest and intracellular survival of
Mtb in macrophages by inhibiting IL-1β production3. However, the molecular mechanisms
underlying the inhibition of IL-1β production by Zmp1 are currently unclear.

Results
To investigate the molecular mechanisms of the Zmp1-mediated inhibition of IL-1β
production, we performed yeast two-hybrid screening using the full-length Zmp1 as abait and isolated a cDNA encoding the C-terminal portion (86-144 amino acids) of murine
GRIM-19. The interaction of Zmp1 with GRIM-19 in mammalian cells was confirmed byimmunoprecipitation assay using cell extracts of HEK293T cells transfected with T7-tagged
GRIM-19 and FLAG-tagged Zmp1. Since GRIM-19 functions as an accessory subunitof mitochondrial respiratory chain complex I4-7, we examined the subcellular localizationof Zmp1 in macrophages. We found that endogenous GRIM-19 is mainly expressed in
mitochondria, and colocalization of Zmp1 with GRIM-19 was detected in mouse bonemarrow-derived macrophages infected with BCG. Collectively, these data indicate that
Zmp1 interacts with GRIM-19 in mitochondria during mycobacterial infection.
It is currently unclear whether GRIM-19 is involved in IL-1β production. To understand

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the role of GRIM19 in macrophages, we generated Grim-19-deficient J774.1 mouse
macrophage cells using the CRISPR/Cas9 system. IL-1β production was barely detected
when J774.1 cells were infected with wild-type BCG, but it was significantly increased
when these cells were infected with ∆zmp1 BCG, confirming that Zmp1 suppresses IL1β production in BCG-infected J774.1 cells. The IL-1β production induced by ∆zmp1 BCG
infection was abolished in Grim-19-deficient cells, suggesting that GRIM-19 plays anessential role in IL-1β production in macrophages upon mycobacterial infection. We also
confirmed that ∆zmp1 BCG-induced IL-1β production was dependent on NLRP3 since
pretreatment of these cells with MCC950 blunted the production of IL-1β upon ∆zmp1 BCG
infection.
Next, we analyzed the mechanism of GRIM-19-mediated regulation of the NLRP3
inflammasome. Wild-type and Grim-19-deficient J774.1 cells were primed with LPS to
induce expression of pro-IL-1β and treated with ATP or nigericin to activate the NLRP3
inflammasome. We found that mature IL-1β protein was detected in culture supernatantsof wild-type, but not Grim-19-deficient, cells treated with ATP or nigericin. Pro-IL-1β protein
was detected from the cell lysates of both wild-type and Grim-19-deficient cells stimulatedwith LPS. We observed a 10-kDa fragment of caspase-1, which is generated uponactivation of caspase-1, in the culture supernatant of wild-type cells treated with ATP or
nigericin. This fragment was almost completely diminished in Grim-19-deficient cells. Since
the components of the NLRP3 inflammasome, NLRP3, pro-Caspase-1, and ASC wereexpressed in Grim-19-deficient cells to a similar extent to wild-type cells, it is suggested that
GRIM-19 is required for activation of the NLRP3 inflammasome.
Because mitochondria-derived reactive oxygen species (mtROS) is known to induce
NLRP3 inflammasome activation 8, we next examined whether GRIM-19 is involved in
the production of mtROS. The level of mtROS was significantly increased when LPSprimed wild-type J774.1 cells were treated with ATP, whereas the level of mtROS was
not increased in Grim-19-deficient cells under the same condition. We also examined theactivity of mitochondrial respiratory complex I in these cells and found that the complex I
activity was markedly reduced in Grim-19-deficient cells compared to wild-type cells. These
results indicate that GRIM-19 is necessary for mtROS production, and the diminished
mtROS production in Grim-19-deficient cells is most likely due to the impaired complex I
activity.
An intact mitochondrial respiratory chain generates mitochondrial membrane potential(∆Ψm). Wild-type and Grim-19-deficient J774.1 cells were stained with a cell-permeable
fluorescent probe, JC-1, and analyzed by fluorescence microscopy. We found that thefluorescence ratio of red/green (JC-1 aggregates/monomer) was significantly lower in the
Grim-19-deficient cells than in wild-type cells, suggesting that GRIM-19 is essential for
maintaining ∆Ψm in macrophages. We also found that a transient expression of Zmp1 in
HEK293T cells decreased the ∆Ψm to a similar extent as the cells treated with rotenone,
a selective inhibitor of the mitochondrial respiratory chain complex I, implying that Zmp1
decreases ∆Ψm presumably by modulating the complex I activity.

Discussion & Conclusion
In this study, we identified GRIM-19, an essential accessory subunit of mitochondrial
respiratory chain complex I, as a novel Zmp1-interacting host protein. CRISPR/Cas9-

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mediated gene knockout of Grim-19 in a murine macrophage cell line revealed that
GRIM-19 is required for IL-1β production during mycobacterial infection. Further analysis
suggested that GRIM-19 is critically involved in NLRP3 inflammasome activation via
the production of mtROS. Either loss of GRIM-19 or expression of Zmp1 resulted in asignificant decrease in mitochondrial membrane potential. Collectively, our results indicatethat Zmp1 inhibits IL-1β production, presumably by targeting GRIM-19 which is necessary
for NLRP3 inflammasome activation. Our work provides new insight into the mechanism
underlying the activation and modulation of the NLRP3 inflammasome during mycobacterial
infection. The development of a drug that blocks the interaction between Zmp1 and GRIM19 could potentially be a novel host-directed therapeutic option for tuberculosis treatment.
In addition, our study also provides clues to enhance the effect of trained immunity, leading
to the development of novel drugs for infectious diseases.

References
1 Jayaraman, P. et al. IL-1β promotes antimicrobial immunity in macrophages by regulating
TNFR signaling and caspase-3 activation. J Immunol 190, 4196-4204 (2013).
2 Jayaraman, P. et al. Tim3 binding to galectin-9 stimulates antimicrobial immunity. J Exp
Med 207, 2343-2354 (2010).
3 Master, S. S. et al. Mycobacterium tuberculosis prevents inflammasome activation. Cell
Host Microbe 3, 224-232 (2008).
4 Fearnley, I. M. et al. GRIM-19, a cell death regulatory gene product, is a subunit of bovine
mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Biol Chem 276, 3834538348 (2001).
5 Huang, G. et al. GRIM-19, a cell death regulatory protein, is essential for assembly andfunction of mitochondrial complex I. Mol Cell Biol 24, 8447-8456 (2004).
6 Stroud, D. A. et al. Accessory subunits are integral for assembly and function of humanmitochondrial complex I. Nature 538, 123-126 (2016).
7 Guerrero-Castillo, S. et al. The Assembly Pathway of Mitochondrial Respiratory Chain
Complex I. Cell Metab 25, 128-139 (2017).
8 Zhou, R., Yazdi, A. S., Menu, P. & Tschopp, J. A role for mitochondria in NLRP3
inflammasome activation. Nature 469, 221-225 (2011).

一般の皆様へ
本研究では、 結核菌 (Mycobacterium tuberculosis) が産生する炎症抑制因子の作用機序の解明を目的として、 まず宿主側標的タンパク質を同定し、 その遺伝子を破壊した細胞を作製して、この宿主タンパク質が炎症誘導に重要な役割を果たす新規制御因子であることを見出しました。
本研究の成果は、 宿主免疫応答を増強するタイプの新たな抗結核薬、 新しい結核ワクチン、 さらには近年注目されている 「訓練免疫」 を増強することによる感染症の治療法や予防法の創出に繋がるものと期待されます。

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Functional mechanisms and dynamics of oxytocin
in autism spectrum disorder
Teruhiro Okuyama
okuyama@iqb.u-tokyo.ac.jp
The University of Tokyo

Summary Abstract
The ability to recognize familiar individuals is essential for social animals including
humans, and an impairment of this ability is a hallmark of autism spectrum disorders(ASDs). We previously showed that neurons in the ventral CA1 (vCA1) of the hippocampus
store social memory for familiar individuals. However, how neural activities of vCA1 neurons
are coordinated for social memory formation remained unclear. Here, we found that social
memory in the vCA1 is replayed during sleep period, which is disrupted in autism-modelmice.
Key Words ： Autism spectrum disorder, social memory, hippocampus, Oxytocin, physiology

Introduction
Autism spectrum disorder (ASD) is a developmental disability that can cause significant
difficulties with social communication and interaction and may also exhibit restricted,
repetitive behaviors and interests. To understand the molecular basis underlying ASD,
large-scale genetic screenings of ASD-related genes have been vigorously conducted [1],and a large number of genes, including shank3 and neuroligin, were identified. Most ofthese genes are involved in forming the dendritic spine and synapse, which function as aphysical scaffold for neurons to process information [2]. However, the essential question ofwhich brain region(s) and neural circuit(s) were impaired in ASD remains unanswered.

Results
Along with social communication impairment, clinical studies have reported that autistic
patients also exhibit social memory impairment, which is the inability to remember others
accurately [3]. This evidence came up with the idea that the information processing
abnormalities in the neurons involved in social memory itself, or the social neurons locatedupstream or downstream of those neurons, may cause autism symptoms.
On the other hand, we previously
showed that ventral CA1 neurons in
the hippocampus store social memory(Okuyama et al., Science 2016). Using
the microendoscopy to detect the neuralactivity of ventral CA1 neurons duringsocial behavior, we found that a particular population of ventral CA1 neurons is enormously
excited only when recalling a specific memorized individual. However, how neural activities

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for the Promotion of Scienceof this social memory ensemble are coordinated remained unclear. Here, we found thatsocial memory in the vCA1 is replayed during sleep period, which is disrupted in autismmodel mice.
Our in vivo electrophysiological recordings allow us to compare neural activities during
awake social behavior and during high-frequency oscillations called sharp-wave ripples(SPW-Rs) observed during sleep periods with high temporal resolution. In this study, we
showed that vCA1 social memory neurons, characterized by enhanced activity in response
to memorized individuals, were preferentially reactivated during sharp-wave ripples. Also,
we revealed that these features are impaired in autism-associated Shank3 mutant mice [4].

Discussion & Conclusion
These results suggest that SPW-R-mediated sequential reactivation of memory
ensembles is a general mechanism for coordinating hippocampus-dependent social
memories and spatial memory. Importantly, we found that its disruption may underlie thepathophysiology of ASD-related social memory defects. Our findings provide a first-everneuroscientific answer to the question of why autistic patients are unable to exhibit social
familiarity, which is expected to provide a breakthrough in the therapeutic development of
ASDs.

References[1] A de novo convergence of autism genetics and molecular neuroscience.
Niklas Krumm, Brian J O’Roak, Jay Shendure, Evan E Eichler. Trends in
Neurosci. 37:95(2014)[2] Progress toward treatments for synaptic defects in autism. Richard Delorme,
Elodie Ey, Roberto Toro, Marion Leboyer, Christopher Gillberg, Thomas
Bourgeron. Nat. Med.19:685 (2013)[3] Face identity recognition in autism spectrum disorders: a review of behaviorals t u d i e s . S a r a h W e i g e l t , K a m i K o l d e w y n , N a n c y K a n w i s h e r. N e u r o s c i .
Biobehav. Rev. 36:1060 (2012)[4] Disrupted social memory ensembles in the ventral hippocampus underlie social
amnesia in autism-associated Shank3 mutant mice. Kentaro Tao, Myung
Chung, Akiyuki Watarai, Ziyan Huang, Mu-Yun Wang, Teruhiro Okuyama.
bioRxiv (2021)

一般の皆様へ
自閉症スペクトラムは、 社会性コミュニケーションなどに異常を示す発達障害で、 その病態神
経メカニズムの解明は喫緊の課題の一つです。 私たちの研究では、 自閉症を引き起こす事が既に報告されている自閉症関連遺伝子の一つである Shank3遺伝子に注目し、 脳内のどのような神経回路がどのように変容することで自閉症病態を引き起こすのかを明らかにしました。

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Fasting Specific Metabolic Regulation by the Circadian Clock
Kenichiro Kinouchi
ken-k@keio.jp
Keio University School of Medicine
Key Words ： Circadian rhythms, Fasting, Metabolism, Epigenetics, Circadian clock

Introduction
Circadian rhythms are organismal innate oscillation to preserve homeostasis in an
anticipation of day-night cycle. The intrinsic rhythms are driven by the circadian clock,which orchestrates daily rhythms of metabolism, physiology, and behavior. Feeding-fasting
rhythms entrain peripheral circadian clocks, emphasizing the notion that temporal patternsof nutrient availability impact clock functions. While a number of molecular mechanisms are
responsible for clock integration of metabolic states, it remains to be defined whether clockmediated transcriptional regulation is altered by fasting.

Results
To explore whether the circadian clock impinges
on genomic response to fasting, 8-12 weeks-old,male clock deficient (Bmal1 -/-) mice and control
mice were subjected to ad libitum feeding (FED) or
24hr fasting (FAST) under a 12hr/12hr light-dark
condition. Liver, skeletal muscle, and serum werecollected at two different time points (at zeitgeber
time (ZT) 8 and 20, wherein light turns on at ZT0
and off at ZT12). qPCR revealed differentially
Figure 1
expressed transcripts in the liver and skeletal
muscle between FED and FAST conditions (Figure1). Ablation of Bmal1 has been first validated. We previously demonstrated that genomic
response to fasting is categorized into three groups: Class I, BMAL1 target and repressedby fasting; Class II, BMAL1 target and induced by fasting; Class III, non-BMAL1 target andinduced by fasting 1. Consistent with the previous study, expression of hepatic Dbp gene
was repressed by fasting and virtually abolished in Bmal1-/- mice (Class I). Hepatic Por
expression was induced by fasting and attenuated in Bmal1-/- mice (Class II). Hepatic Fgf21
expression was induced by fasting and this induction was comparable between WT and
Bmal1-/- mice (Class III). Similarly, expression of skeletal muscle Dbp gene was repressedby fasting and virtually abolished in Bmal1-/- mice (Class I). Skeletal muscle Per1 expression
was induced by fasting and attenuated in Bmal1-/- mice (Class II). Thus, distinct classes ofgenes display differential genomic response to fasting.
Circadian gene expression is controlled by rhythmic activation of clock and non-clock,nutrient-sensitive transcription factors (metabolic clock) such as SREBP, PPAR, andCREB2. Fasting-specific gene regulation is achieved by cyclic coordination of circadian and

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metabolic clocks. To advance our understanding of underlying transcriptional mechanisms,
high-throughput transcriptomic analysis was performed by RNA sequencing (RNAseq). After the process of mapping, alignment, annotation and quantitation, differentiallyexpressed genes were identified using CyberT analysis3. BMAL1 target genes were definedby genes whose expression was repressed by BMAL1 ablation. 816 and 879 genes
were identified as the class I gene in the liver and skeletal muscle, respectively. 269 and221 genes were found as the class II gene in the liver and skeletal muscle, respectively.
2293 and 2981 genes in the class III were discovered in the liver and skeletal muscle,
respectively. 96 genes in the class I, 20 genes in the class II, and 879 genes in the class
III were in common between liver and skeletal muscle, respectively. This observation
likely represents tissue specificity of the clock regulation upon fasting. KEGG (Kyoto
Encyclopedia of Genes and Genomes) pathway analysis revealed that pentose andglucuronate interconversions is enriched in the hepatic class I genes, AMPK signaling,
FoxO signaling pathways in the hepatic class II genes, and Proteasome, PPAR signaling
pathway in the hepatic class III genes. Meanwhile, PI3K-Akt signaling pathway are
overrepresented in the muscle class I genes, circadian rhythm in the muscle class II genes,
Proteasome in the muscle class III genes. Thus fasting seems to induce a transcriptionalswitch of the circadian clock, whereby achieving different classes of fasting-specific gene
regulation in the liver and skeletal muscle.

Discussion & Conclusion
Circadian transcription is subject to temporal patterns of nutrient availability. There
are a number of underlying mechanisms which contribute to such receptivity of the clockmachinery to nutritional states. For instance, feeding drives Per2 gene expression throughinsulin signaling, while fasting induces Per1 gene activation by glucagon and glucocorticoid
via phosphorylated CREB and GR, respectively. Although it becomes more evident thatclock components are subject to regulation or modification by nutrient sensing pathways,
it remains to be clarified whether changes in chromatin landscape by nutritional states
dictate clock-mediated transcription in concert with nutrient sensitive transcription factors.
Preliminary results from our study suggest that fasting imparts a switch to clock-mediatedtranscription both in the liver and skeletal muscle. Future experiments will define how clocktranscription factors are conditioned by fasting at the chromatin level.

References
1 Kinouchi, K. et al. Fasting Imparts a Switch to Alternative Daily Pathways in Liver and
Muscle. Cell reports 25, 3299-3314 e3296 (2018).
2 Vollmers, C. et al. Time of feeding and the intrinsic circadian clock drive rhythms inhepatic gene expression. Proceedings of the National Academy of Sciences of the UnitedStates of America 106, 21453-21458 (2009).
3 Kayala, M. A. & Baldi, P. Cyber-T web server: differential analysis of high-throughput data.
Nucleic acids research 40, W553-559 (2012).

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一般の皆様へ
体内時計は昼夜の明暗の変化に予測して適応する分子であり、 光や食事で時刻合わせをします。 本研究では、 体内時計がどのように空腹に対する応答を調節することで、 臓器間の代謝協調性に寄与するのかを研究目的としています。 これまでの検討で、 肝臓や筋肉の絶食に対する応答は体内時計で調節されていることが明らかになっています。 今後詳細な分子機序を検討することで、 空腹によって分泌されるホルモンの効果が、 ホルモン分泌の日内変動に加えて、 効き
目にも日内変化が存在し、 それが体内時計によって調節されている可能性を明らかにしていきた
いと考えています。

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Cell senescence effects on transcriptional regulation anddifferentiation in beige preadipocytes
Kenji Ikeda
kikeda.mem@tmd.ac.jp
Department of Molecular Endocrinology and Metabolism,
Tokyo Medical and Dental University

Summary Abstract
Beige fat was induced by thermal stress adaptation and increase energy expenditure asheat. Aging, obesity, cell senescence strongly inhibits the induction of beige adipocytes.
Still, how cell senescence regulate differentiation of beige adipocytes remain incompletely
understood. The complicated reasons to study their mechanism, because beige adipocytes
are heterogeneity.
Here, we analyzed single cells with adipose tissue in young and aging mice using single
cell RNA sequencing. We identified new cell populations of subtype of preadipocytes.
These findings provides understanding the beige adipocytes differentiation in aging.
Key Words ： Beige adipocytes, Senescence, cell differentiation

Introduction
Beige fat was induced by thermal stress adaptation and increase energy expenditure asheat. Aging, cell senescence strongly inhibits the induction of beige adipocytes. Still, how cellsenescence regulate differentiation of beige adipocytes remain incompletely understood. The
complicated reasons to study their mechanism, because beige adipocytes are heterogeneity.
Recently our study reveals a new subtype of beige adipocytes and preadipocytes . This new
subtype is distinct from conventional beige fat in developmental origin and regulation and cellfunction.
We analyzed single cell RNA sequencing with adipose tissue in young and aging micewith cold simulation. We identified new cell populations of subtypes of preadipocytes.

Results
1. Aging mice showed cell senescent in inguinal adipose tissue.
To examine aging effects in inguinal adipose tissue, we analyzed young and old micein inguinal adipose tissue. Old mice showed higher mRNA expression of several cellsenescent marker genes, p19 and p16(Figure1).

Figure1. Aging mice showed cell senescent in inguinal adipose tissue.

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2. Aging mice showed impaired beige fat induction.
Next, we investigated the beige fat induction in old mice at cold exposure. Old mice
showed a significantly decrease beige fat induction at cold exposure(Figure2).

Figure2. Aging mice showed impaired beige fat induction.
3. Chronic cold exposure induced beige fat in old mice.
In contrast to this, longer cold stimulation can induce beige fat induction in aging mice.

Figure3. Chronic cold exposure induced beige fat in old mice.
4. Identification and Characterization of inguinal adipose tissue by scRNA-seq.
Given the differences in beige fat induction in inguinal adipose tissue between young
and aged mice.We next explored the inguinal adipose tissue, we run scRNA-seq analysis.
We isolated single cell suspension in inguinal adipose tissue by collagenase digestion
as previously described(1). Next, single cell cDNA libraries were generated using 10X
Genomics platform to run scRNA-seq in inguinal adipose tissue from young mice andold mice housed room temperature (RT) or Cold. To classify the cell types in the inguinal
adipose tissue, we performed uniform manifold approximation and projection(UMAP)
analysis using Seurat.
We identified 40 cell clusters, which are displayed using UMAP dimension reduction
technique after integration datasets. Clusters were annotated based on canonical genes,for example adipocytes progeneitor, preadipocytes(Pdgfra) We identify preadipocytes are
multiple cluster and subpopulations (Figure4).
We compared to clusters among datasets, notably we identified cell clusters specificallyin cold exposure (Figure5).
Cluster 23 and 30 are expressed preadipocytes marker Pdgfra and uniquely enrichedin cold exposure condition, it suggest that these cell cluster are associated with beige fat
induction even in aging condition.

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Figure4. Single cell RNA sequencing identifies preadipocytes cell cluster(Pdgfra expressed) in adipose tissue.

Figure5. Single cell RNA sequencing identifies cell clusters in adipose tissue.

Discussion & Conclusion
Cell senescent impairs beige fat induction2). Obese condition cause cell senescence inadipose tissue. Aging and Obesity storongly inhibit beige induction.
However, our findings suggest that even in aging mice, chronic cold stimulation can
induce beige adipocytes. Aging mice have potential beige fat induction.
Recently our study reveals a new subtype of beige adipocytes and preadipocytes3). These findigs suggest that subtypes of beige adipocytes are different under aging
conditions. By single-cell analysis in inguinal adipose tissue, we identified new cell
populations in cold stimulation. These new cell populations are likely associated with beige
fat induction in cell senescence. Future work will further elucidate the role of the new cell
population in beige fat induction, even in aging and obesity.

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References
1. Ikeda K, Kang Q, Yoneshiro T, Camporez JP, Maki H, Homma M, et al. UCP1independent signaling involving SERCA2b-mediated calcium cycling regulates beige fatthermogenesis and systemic glucose homeostasis. Nat Med. 2017;23(12):1454-65.
2. Berry DC, Jiang Y, Arpke RW, Close EL, Uchida A, Reading D, et al. Cellular Aging
Contributes to Failure of Cold-Induced Beige Adipocyte Formation in Old Mice andHumans. Cell Metab. 2017;25(1):166-81.
3. Chen Y, Ikeda K, Yoneshiro T, Scaramozza A, Tajima K, Wang Q, Kim K, Shinoda K,
Sponton CH, Brown Z, Brack A, Kajimura S.
Thermal stress induces glycolytic beige fat formation via a myogenic state. Nature.
2019;565:180-185.

一般の皆様へ
熱産生脂肪組織であるベージュ脂肪組織は、 エネルギー消費量を増やし抗糖尿病、 抗肥満作用を示すことから肥満症や２型糖尿病への治療開発が期待されています。 ベージュ脂肪組織
は、 長期の寒冷暴露や運動などの刺激によって誘導されることが特徴です。 ベージュ脂肪細胞
は肥満や老化に伴い、 その誘導が低下することが知られておりますが、 その仕組みはこれまで
明らかになっておりません。 ベージュ脂肪細胞は脂肪前駆細胞から分化することが知られておりますが、 その詳細は未だ不明な点が多いです。 最近になってベージュ脂肪細胞、 前駆細胞には様々なサブタイプが存在することがわかってきました。 本研究では、 ベージュ脂肪細胞の誘導機構を一細胞毎に調べることで、 その仕組みを明らかにし、 加齢や肥満状態でもエネルギー消費量を亢進する肥満症 ・ ２型糖尿病に対する新しい治療法開発に繋げることを目指します。

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Identification of molecular network between mitochondria andnucleus underlying mitochondrial biogenesis
Atsushi Hoshino
a-hoshi@koto.kpu-m.ac.jp
Kyoto Prefectural University of Medicine

Summary Abstract
Mitochondrial degradation and biogenesis are coordinately regulated to maintainmitochondrial homeostasis. Genome-wide CRISPR library screen and subsequent perturbsequence identified p53 and RREB1 as novel regulators for mitochondrial biogenesis after
mitophagy.
Key Words ： mitochondrial biogenesis, heart failure

Introduction
Mitochondria play a critical role in energy production, redox and calcium metabolism
and apoptosis, all of which are involved in cardiac hypertrophy and fibrotic change during
heart failure progression. Mitochondria are dynamic organelle, repeating fission andfusion and damaged ones are degraded by autophagy machinery, called “mitophagy”.
Usually, mitophagy are coupled with biogenesis to maintain mitochondrial content. We
and other groups reported that mitophagy was down-regulated by p53 and others andmitochondrial biogenesis was also impaired subsequently in heart failure. The dysregulatedbalance between degradation and biogenesis induced low mitochondrial content, energy
compromise, oxidative stress and impaired homeostasis. In this study, we aim to reveal
mitochondria-nucleus signal network to maintain the balance of mitochondrial degradation
and biogenesis.

Results
We employed the mitochondrial biogenesis in response
to forced mitophagy in cultured C2C12 cells. At first,
we performed CRISPR library screening to identify key
players in mitophagy-mediated mitochondrial biogenesis.
Lentivirus based GeCKO library was transduced to Parkin
expressing cells. Mitophagy was induced by CCCP orOXPHOS inhibitor cocktail treatment. Mitochondrial
content was analyzed by MitoTracker Green. High or
low MitoTracker signal population was sorted and thenanalyzed by deep sequencing. This screening identified TNFα-NFkB pathway including
Traf3, Traf2, Birc2, RBCK1, ubiquitin-related genes and nuclear protein-coding genes
including Rreb1, p53, Bmi1, Atxn7l3 as positive regulators for mitochondrial biogenesis after
mitochondrial degradation. Based on these regulators, single cell CRISPR screening was
performed in the Perturb-seq platform. gRNAs targeting 9 genes as well as non-targeting

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control were transduced to Parkin expressing cells. Samples were harvested 4 time points
before and after mitophagy induction with parkin overexpression and CCCP treatment.
Single cell analysis was performed by 10X Genomics platform. After quality control filtering,
we obtained 16,616 cells from 4 time points around mitophagy induction. In the clustering
analysis, there were seven clusters with two fate directions; successful mitochondria
biogenesis (cluster 6) and failure of biogenesis (cluster 7). Cluster 6 was characterized bygene ontology of mitochondrial respiratory electron transport chain, whereas cluster 7 had
upregulated genes in related with p53-mediated transduction and cell cycle arrest. Amongthe perturbed genes, Rreb1 knockout shifted the population from cluster 6 to 7, and p53
knockout decreased cluster 6 with increased cluster 4. These results indicated that Rreb1
knockout failed mitochondrial biogenesis, whereas p53 knockout just delayed biogenesis.
ATAC sequence was performed to analyze the epigenetic alteration in mitochondrial
biogenesis after mitophagy induction. High accessibility was associated with regulation oftranscription, cell differentiation, and regulation of ion transmembrane transport. In contrast,
low accessibility was related with DNA methylation, DNA replication, positive regulationof gene expression, and nucleosome assembly. In the motif analysis, SP1, SP2, Sp3,
Sp8, KLF5, KLF16, RREB1, and ZNF 740 motifs become more accessible in the phase of
mitochondrial biogenesis. These results indicated that RREB1 binding site becomes open
and contributes to mitochondrial biogenesis.
As the downstream of RREB1, RREB1 motif binding peak was analyzed with RNA
sequence data. There were about 50 genes matched with ATAC-seq peaks annotatedas promoter. Among them, several genes including ESR1 ware involved in mitochondrial
biogenesis and functional maturation.
PGC1a is well-known master regulator of mitochondrial biogenesis. Its expression level
was elevated and partially involved in biogenesis after mitophagy. To find upstream of PCG1a expression, we also performed CRISPR library screening using PGC-1a reporter cells.
P2A-mCherry fragment was knocked-in in the front of stop codon. Several COMM (copper
metabolism gene MURR1) domain containing genes were identified to positive regulator for
PGC-1a expression. Together with mitochondrial biogenesis regulators screening, autophagyrelated gene, AMBRA1 and cell cycle-related gene, PPP1R15A were common hit genes in both
PGC-1a expression and biogenesis, indicating that AMBRA1 and PPP1R15A were promoted
biogenesis through PGC-1a expression.

Discussion & Conclusion
Our current impressive result is that RREB1 was identified in both CRIPR library
screening and ATAC sequence. RREB1 is responsible for mitochondrial biogenesis andits binding site becomes more accessible after mitophagy. Single cell RNA sequencewith perturbed genes also suggested RREB1 knockout failed to promote mitochondrial
biogenesis after mitochondrial degradation. One of the candidates of downstream is
estrogen receptor to drive biogenesis. RREB1 seemed to regulate other mitochondrial
genes. To obtain more convincing data, we are now setting to perform Cut&Tag. Thismethod can provide direct evidence of RREB1 binding sites. And RREB1-flox mouse isunder generation to analyze the physiological and pathological importance of RREB1 in
associated with mitochondrial biogenesis.

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一般の皆様へ
ミトコンドリアは細胞のエネルギー産生の中心であるとともに酸化ストレスや慢性炎症、 細胞死の制御に関連しておりその機能は細胞の恒常性維持に非常に重要です。 不良ミトコンドリアはオートファジーによる分解され、 新たなミトコンドリアが合成されますが、 それらがどのようにして協調的に制御されているかはよく分かっていません。 本研究では多面的オミクス解析で RREB1という転写因子がミトコンドリア分解後の生合成に重要であることが分かりました。 今後さらにミトコンド
リアと RREB1をつなぐ因子の同定に取り組んでいきます。

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Development of Planar Chiral Cyclic (Amino) (ferrocenyl)
carbene Ligands for Medicinal Chemistry
Kazuhiro Yoshidakyoshida@faculty.chiba-u.jp
Chiba University

Summary Abstract
New cyclic (amino)(ferrocenyl)carbene (CAFeC) ligand containing the C5(Ph)5 grouphas been developed as a modification of their prototype Cp version. The new carbene
was generated from a salt by simple deprotonation with a strong base and identified by acarbene trapping experiment with sulfur.
Key Words ： asymmetric synthesis, chiral N-heterocyclic carbene (NHC), planar chirality

Introduction
Chiral N-heterocyclic carbenes (NHCs) have received much recent attention for theirimportance as asymmetric organocatalysts and chiral ligands in transition metal catalysis.
Very recently, we developed a new type of planar chiral cyclic (amino)(ferrocenyl)carbene(Rp)-1. The purpose of this study was to develop Cp(Ph)5 version (Rp)-2 in order to create amore attractive chiral environment than 1 as chiral ligands because of the bulkiness of the
C5(Ph)5 group.

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Results
Scheme 1.

The synthetic route to carbene precursor (Rp)-[2H]OTf is shown in Scheme 1. Here,the same strategy as that established by us for prototype ligand (Rp)-1 was employed.
First, known chiral formylferrocene 3, which was obtained by a diastereoselective orthofunctionalization strategy, was subjected to the Wittig reaction with [Ph3PCH2CN]Br toform 4. Then, resulting 4 was treated with LiAlH4 to give primary amine 5. Then, 5 wastreated with hydrochloric acid for acetal hydrolysis, and this was followed by a spontaneous
intramolecular cyclization reaction to yield aldimine 6. Desired iminium salt (Rp)-[2H]OTf
was obtained by reacting 6 with isopropyl iodide, followed by an anion exchange with
AgOTf.
Because it was confirmed that CAFeC 1 was not isolable and decomposed into a mixture
even at low temperature, the generation of 1 was indirectly confirmed by carbene trapping
experiments with sulfur in our previous study. The same trapping experiment was also
conducted to confirm the generation of 2. As a result, the addition of LiHMDS as a base toa mixture of iminium salt (Rp)-[2H]OTf and sulfur in THF gave desired sulfur adduct (Rp)-7 in
26% yield (Scheme 2).
Scheme 2.

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Discussion & Conclusion
We have developed novel CAFeC ligand 2 with C5(Ph)5 group by the modification of
its prototype version, 1. The existence of the carbene at low temperature was confirmed
by a carbene trapping experiment with sulfur. Encouraged by these results, we are now
preparing its metal complexes to apply this novel ligand in asymmetric reactions.

References
1. Planar Chiral Cyclic (Amino)(ferrocenyl)carbene as Ligand for Transition Metals Adv.
Synth. Catal. 2017, 359, 255-259. Risa Yasue, Masaru Miyauchi, and Kazuhiro Yoshida
2. Coordination Behavior of a Planar Chiral Cyclic (Amino)(Ferrocenyl)Carbene Ligand in
Iridium Complexes Chem. Eur. J. 2017, 23, 16806-16812. Yuta Shikata, Risa Yasue, andKazuhiro Yoshida
3. A Planar Chiral Six-Membered Cyclic (Amino)(Ferrocenyl)Carbene and Its Sulfur
Adduct Tetrahedron: Asymmetry 2017, 28, 824-829. Risa Yasue, Masaru Miyauchi, andKazuhiro Yoshida
4. Planar-Chiral Ferrocene-Based N-Heterocyclic Carbene Ligands Chem. Eur. J. 2018, 24,
18575-18586. Kazuhiro Yoshida and Risa Yasue
5. Synthesis and Application of Planar Chiral Cyclic (Amino)(ferrocenyl)carbene Ligands
Bearing FeCp* Group Organometallics 2019, 38, 2211-2217. Risa Yasue and Kazuhiro
Yoshida6. Development of Planar Chiral Five-membered Cyclic (Amino)(ferrocenylene)carbene
Ligand and Its Iridium Dicarbonyl Complex Bull. Chem. Soc. Jpn. 2020, 92, 200-204.
Waka Takagaki, Risa Yasue, and Kazuhiro Yoshida

一般の皆様へ中心不斉や軸不斉を持つことにより、 その鏡像と重ね合わすことのできない化合物は鏡像異性体と呼ばれています。 鏡像異性体は互いにその生物活性を異にするため、 これらを区別して合成することは医薬化学及び有機合成化学における重要課題となっています。 本研究は、 このような鏡像異性体を作り分ける技術の中で、 最も理想的とされている 「触媒的不斉合成 （微量の不斉源より大量の光学活性化合物の合成を可能とする技術）」 分野における研究です。 将来の産業に役立つ、 力量ある触媒の開発を目指しています。

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Regulation of gene expression network and cell death by
microRNAs during viral infection
Tomoko Takahashi
takahas@mail.saitama-u.ac.jp
Saitama University

Summary Abstract
RNA silencing is a posttranscriptional gene silencing mechanism directed by endogenous
small non-coding RNA, microRNAs (miRNAs). By contrast, the type-I interferon (IFN) system is an innate immune system activated by exogenous RNA such as viral RNAs. RNA silencing and IFN system are considered as independent pathways previously. We found thatthe crosstalk between RNA silencing and IFN system regulates gene expression networkand functions as an antiviral defense system in mammalian cells.
Key Words ： microRNA, IFN system, antiviral immunity

Introduction
RNA silencing is a posttranscriptional gene silencing mechanism directed by endogenous
small non-coding RNAs called microRNAs (miRNAs). The human genome encodes > 2,000
miRNAs, and the deletion or mutation, as well as upregulation or downregulation, of miRNAcauses serious diseases including cancer, neurodegenerative diseases, and diabetes.
By contrast, the type-I interferon (IFN) system is an innate immune system activated byexogenous RNAs, such as viral RNAs.

Results
During viral infection, viral RNAs are detected by virus sensor proteins, including Tolllike receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in theendosome and cytoplasm, respectively. The activated sensor proteins transfer signals tothe downstream molecules to induce IFN production. Secreted IFN induces hundreds of
IFN-stimulated genes (ISGs), which represses viral replications.
Among RLRs, RIG-I recognizes 5’-triphosphate- or 5’-diphosphate RNA and small RNA
duplexes, and MDA5 recognizes long RNA duplexes as exogenous RNAs. They transfer signals to downstream molecules through their caspase recruitment domains (CARDs). LGP2
is a member of the RLRs, but does not have a CARD, rendering it incapable of transferring
signals downstream. We identified that LGP2 modulates RNA silencing by interacting with
the RNA silencing enhancer, TRBP. LGP2 interacts with TRBP via the dsRNA-binding sitesof TRBP through competition with TRBP-bound pre-miRNAs. This competitive binding inhibits the binding of 40 types of pre-miRNAs with TRBP in HeLa cells, as well as the maturationof those miRNAs by Dicer/TRBP, and their subsequent RNA silencing activities.
To identify the spe- cific structural characteristics of TRBP-bound and -non- bound premiRNAs, we calculated the base-pairing prob- ability (BPP), which provides the probability
for each base- pair with respect to an ensemble of RNA secondary struc- tures for predict-

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for the Promotion of Scienceing accurate RNA secondary structure. The mean BPP values at stem regions of all premiRNAs, TRBP-bound and non-bound pre-miRNAs were calculated using the max valuesof fluctuating BPPs at each nucleotide, calculated by CentroidFold. The results clearly distinguished be- tween pre-miRNA that preferably bound to TRBP and pre- miRNA not boundto TRBP in the context of miRNA sec- ondary structures. The stem region of TRBP-boundpre- miRNA had tight base-pairing, while that of TRBP-non- bound pre-miRNA had weakbase-pairing.
Thus, LGP2 has been demonstrated to inhibit RNA silencing directed by TRBP-bound
miRNAs and upregulate expression of their target genes. However, the biological implications of this activity remained unclear. We generated TRBP or LGP2 knock-out cell lines
using CRISPR/Cas9 genome engineering. The Gene expression profiling using these celllines revealed that apoptosis regulatory genes were upregulated during SeV infection: caspases-2, -8, -3 and -7, four cysteine proteases with key roles in apoptosis, were upregulated directly or indirectly through the repression of a typical TRBP-bound miRNA, miR-106b.
LGP2 upregulated apoptosis regulatory genes by inhibition of TRBP-bound pre-miRNAmaturation by competitive binding with TRBP, and enhanced apoptosis during Sendai virus(SeV) infection. SeV is a single-stranded RNA virus recognized by RIG-I, and its infection
induces IFN production, which strongly induces LGP2 expression. Induced LGP2 protein interacted with TRBP and reduced RNA silencing directed by a typical TRBP-bound miRNA,miR-106b, resulting in upregulation of its target genes, including initiator or executioner caspases, directly or indirectly.
We showed that caspase-2 is a direct target of miR-106b. Although we could not clarify
whether caspase-8 is a direct or indirect target gene of miR-106b, caspase-2 is reported
to be activated along with caspase-8 in response to multiple triggers including DNA damage, heat shock, endoplasmic reticulum stress, and oxidative stress. The initiator caspases,
caspases-2 and -8, form a complex with Fas-associated protein with death domain (FADD),
and this interaction induces the downstream activation of executioner caspase-3, and finallyinduces apoptosis. Thus, LGP2 may efficiently enhance apoptosis by regulating initiatorcaspase(s).

Discussion & Conclusion
The function of one of RLRs, LGP2, was controversial. We revealed that LGP2 functions
as a modulator of RNA silencing by interacting with an RNA silencing enhancer, TRBP. The
biological implication of modulation of RNA silencing by LGP2 induction during viral infection was unclear in previous reports, but we found that LGP2 upregulates apoptosis regulatory genes mediated by the repression of TRBP- bound miRNAs and enhances apoptosis
during SeV infection. Our finding showed that the crosstalk between RNA silencing, which
is directed by endogenous miRNAs, and IFN system, which is activated by exogenous viralRNAs, functions to induce apoptosis as an antiviral defense system in mammalian cells.

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References
Takahashi T, Ui-Tei K.
Mutual regulation of RNA silencing and the IFN response as an antiviral defense system inmammalian cells.
Int. J. Mol. Sci. 21, E1348 (2020)
aTakahashi T, Nakano Y, Onomoto K, Yoneyama M, Ui-Tei K.
LGP2 virus sensor enhances apoptosis by upregulating apoptosis regulatory genes through
TRBP-bound miRNAs during viral infection.
Nucleic Acids Res. 48, 1494-1507. (2019)
Takahashi T, Nakano Y, Onomoto K, Murakami F, Komori C, Suzuki Y, Yoneyama M, Ui-Tei
K. LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs. Nucleic Acids Res., 46, 9134-9147. (2018)

一般の皆様へウイルスが細胞に感染すると、 生体を防御するための免疫応答が誘導されます。 我々は、 細胞内ウイルスセンサータンパク質のひとつであるとされながらも機能が不明であった 「LGP2」 がRNA サイレンシングの促進因子である 「TRBP」 と相互作用することで、 TRBP が結合する特定のマイクロ RNA の成熟過程を阻害し、 ウイルス感染細胞の細胞死を促進することを明らかにしました。
LGP2と TRBP によるマイクロ RNA を介した細胞死の制御は、 ウイルス感染細胞における新しい生体防御機構として機能していると考えられ、 抗ウイルス治療や核酸医薬開発への応用が期待されます。

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Functional analysis of the paraventricular nucleus of thethalamus involved in social behavior
Kazuhiko Yamamuro
muro@naramed-u.ac.jp
Department of Psychiatry, Nara Medical University

Summary Abstract
Whether oxytocin neurons in posterior paraventricular thalamus (pPVT) are related tosociability are poorly understand. Chemogenetic suppression of oxytocin neurons in pPVT
activity in adulthood was sufficient to induce sociability deficits without affecting anxietyrelated behaviors. However, optogenetic activation of oxytocin neurons in pPVT activity inadulthood did not increase sociability. Our study identifies oxytocin neurons in pPVT activity
required for sociability.
Key Words ： Posterior paraventricular thalamus, Oxytocin, Sociability

Introduction
My previous study showed 2 weeks of social isolation immediately following weaningleads to a failure to activate medial prefrontal cortex neurons projecting to the posteriorparaventricular thalamus (mPFC→pPVT) during social exposure in adulthood1). However,whether oxytocin neurons in posterior paraventricular thalamus (pPVT) are related tosociability are poorly understand. Here, we sought to identify the specific types of neurons
in pPVT that requires sociability.

Results
To examine the extent to which activity of oxytocin neurons in pPVT activity is necessary
for sociability, we chemogenetically suppressed the activity of oxytocin neurons in pPVT
expressing inhibitory DREADD (iDREADD). Cre-dependent iDREADD or mCherry vector
were injected into the pPVT, respectively, to express iDREADD in pPVT neurons in
oxtr mice. Suppression of oxytocin neurons in pPVT activity through clozapine N-oxide
dihydrochloride (CNO) treatment in adult mice reduced sociability in the three-chamber test((two-way repeated-measures (RM) ANOVA, drug (CNO or SAL) × stimulus (social or object)interaction F1,24 = 12.900, **P = 0.001; effect of drug F1,24 = 1.905, P = 0.180; effect of stimulus
F1,24 = 0.419, P = 0.524), with no impact on motor activity or measurements of anxiety-related
behavior (Left, two-tailed paired t-test, t12 = 0.005, P = 0.996, Middle, two-tailed paired t-test,
t12 = 0.864, P = 0.405, Right, two-tailed paired t-test, t12 = 0.368, P = 0.719). Moreover, wefound reduced social contact in suppression of oxytocin neurons in pPVT activity through
AR-LABO (Animals Behavior Observing-system). CNO injection in mCherry-expressing
control mice produced no significant differences in behavior compared with saline (SAL)
injection in sociablity(two-way RM ANOVA, drug (CNO or SAL) × stimulus (social or object)interaction F1,18 = 0.428, P = 0.415; effect of light F1,18 = 0.204, P = 0.657; effect of stimulus
F1,18 = 6.135, P = 0.023) and motor activity or anxiety-related behaviors (Left: two-tailed

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for the Promotion of Sciencepaired t-test, t9 = 0.796, P = 0.447, Middle: two-tailed paired t-test, t9 = 1.267, P = 0.237, Right:
two-tailed paired t-test, t9 = 0.255, P = 0.804)..
To examine whether oxytocin neurons in pPVT activity is sufficient to increase sociability
in adult mice, we optogenetically activated oxytocin neurons in pPVT virally expressing
channelrhodopsin (ChR2) under the CaMKII promotor by wirelessly illuminating a blue
LED implanted over the pPVT while mice performed the three-chamber test. CaMKII-ChR2
AAV1 was injected into the pPVT and a wireless blue LED was inserted above the pPVT in
oxtr mice. However, we did not find that optogenetic activation of oxytocin neurons in pPVT
within the social-interaction chamber led to an increase in sociability (two-way RM ANOVA,
light (ON or OFF) × stimulus (social or object) interaction F1,18 = 0.428, P = 0.415; effect oflight F1,18 = 0.204, P = 0.657; effect of stimulus F1,18 = 6.135, P = 0.023). On the other hand,
optogenetic activation of oxytocin neurons in pPVT in one of two empty corral interaction
zones was sufficient to increase time spent in that zone (ChR2+ mice with optogenetic
stimulation showed real-time preference to the stimulation zone (S) over the nonstimulation
zone (N) as indicated by increased discrimination score (calculated as (S − N)/(S + N), twotailed paired t-test, t9 = 3.197, *P = 0.011) as well as investigation time (two-way RM ANOVA,
time (OFF, ON) × zone (stimulation zone or nonstimulation zone) interaction F1.18 = 8.500,**P = 0.009; effect of time F1.18 = 3.185, P = 0.091; effect of zone F1.18 = 2.879, P = 0.107).

Discussion & Conclusion
Overall, our study identifies oxytocin neurons in pPVT activity required for normal adultsociability. Given that the paraventricular thalamus (PVT) projects to various rewardassociated areas including the nucleus accumbens, the bed nucleus of the stria terminalis
and the central amygdala through collateralized projections2)3). In contrast to other mPFC
projections that have been reported to reduce sociability upon activation, such as the
projections to the lateral habenula (located in close proximity to the PVT), activation ofmPFC→PVT projections promotes sociability4)5). Future studies are warranted to reveal how
different populations of mPFC projection neurons influence subcortical networks that control
social behavior and are impacted by previous social experience.

References
1) Yamamuro K et al. A prefrontal–paraventricular thalamus circuit requires juvenilesocial experience to regulate adult sociability in mice. Nature Neuroscience volume 23,
pages1240–1252 (2020)
2) Kirouac, G. J. Placing the paraventricular nucleus of the thalamus within the brain circuitsthat control behavior. Neurosci. Biobehav. Rev. 56, 315–329 (2015).
3) Dong, X., Li, S. & Kirouac, G. J. Collateralization of projections from the paraventricular
nucleus of the thalamus to the nucleus accumbens, bed nucleus of the stria terminalis,and central nucleus of the amygdala. Brain Struct. Funct. 222, 3927–3943 (2017).
4) Benekareddy, M. et al. Identification of a corticohabenular circuit regulating sociallydirected behavior. Biol. Psychiatry 83, 607–617 (2018).
5) Murugan, M. et al. Combined social and spatial coding in a descending projection fromthe prefrontal cortex. Cell 171, 1663–1677.e16 (2017).

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一般の皆様へ
オキシトシンが社会行動に関わることが知られているが、 その機序はいまだに不明のままである。 私達は前頭全皮質－視床室傍核の神経回路が者かい行動に関わることを報告した。 その視床室傍核にはオキシトシン受容体が発現している神経細胞が豊富にあることが知られており、今回は視床室傍核のオキシトシン受容体が発現している神経細胞が社会行動に関わることを明らかにした。 今後はさまざまな精神疾患に関わる社会行動への薬物治療への応用が期待される。

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Identification of precursor memory B cells and mechanistic
characterization of memory B cell generation
Takeshi Inoue
inoue@ifrec.osaka-u.ac.jp
Immunology Frontier Research Center, Osaka University

Summary Abstract
Memory B cells and long-lived plasma cells are the critical components of humoral immune memory. In this study, we analyzed the memory B cells differentiation mechanisms
during germinal center reaction using single cell RNA-seq techniques. These results may
contribute to the understanding how immunological memory establishes in our body during
infection or vaccination.
Key Words ： memory B cell, vaccine, single cell RNA-seq

Introduction“Immunological memory” is the ability of the immune system of our body to quickly andspecifically recognize the previously encountered antigens, such as viruses and bacteria.
Memory B cells and long-lived plasma cells are the cellular components which have central
roles in humoral immune memory. In this study, we have tried to identify and characterize
precursor memory B cells during germinal center B cell selection using single cell RNA-seq
techniques.

Results
During germinal center (GC) reaction, light zone B cells are selected to differentiate intomemory B cells, plasma cells, or to re-enter into dark zone. We have previously identified
and characterized a small GC population of precursor memory B cells, and found that GC B
cells with lower mTORC1 activity resulted from weak T cell help and higher survival signal
from surface B cell receptors favor a memory B cell fate (Fig. 1) (Inoue et al., J Exp Med,2021).

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To further clarify the detailed trajectory path of the GC B cell differentiation into memory
B cell, I first performed a high throughput droplet-based single cell RNA-seq (scRNAseq) analysis on sorted splenic murine B cells after T cell dependent antigen (protein)
immunization using the 10x Genomics Chromium platform. After quality control, 9,972
cells with 2,086 median genes per cell were retained. Unsupervised clustering revealed 14
clusters on t-SNE plot, which were assigned to each B cell subsets (GC light zone, darkzone, plasma cells, memory B cells, and naïve B cells) using known landmark genes (Cd38,
Bcl6, Zeb2, Cd83, Sdc1, and Hmmr) (Fig. 2). Validation of this assignment was achieved bycomparing the results with bulk RNA-seq data we obtained before (Inoue et al., J Exp Med,2021).

scRNA-seq analysis of samples prepared as above was also performed using micro-wellbased BD Rhapsody platform, which provided a comparable or a slightly better throughput
and sensitivity than with 10x Chromium. These results showed that scRNA-seq analysis
of murine activated B cells has been successfully set up with two independent platforms.
Interestingly, these scRNA-seq data identified putative dying GC B cells (not dead cells)
which could not be observed in the conventional flow cytometry analysis, suggesting
the possibility of the existence of GC B cell-specific population which failed to receive
appropriate T cell help and therefore are destined to apoptosis and to be outcompeted
during selection. Differential gene expression analysis to identify the specific genes in this
apoptosis-prone GC population is now under investigation.

Discussion & Conclusion
scRNA-seq analysis of primary immune cells, such as B cells and T cells, is generally
difficult due to their extremely low mRNA abundance, resulted in only few studies
published so far (Laidlaw et al., Nat Immunol, 2020). In this study, I have successfully
obtained scRNA-seq data using ex vivo murine GC B cells and assigned each clusters toknown B cell subsets and also identified uncharacterized dying GC B cells. Clarifying theheterogeneity in GC B cells will be a key to elucidate the memory B cell generation from theGC.
Given the importance of memory B cells in protecting us against re-infection,
understanding how memory B cells are produced will provide us the basal information
required for novel vaccine design against Influenza infection or COVID-19. Our studies
may contribute better understanding how immunological memory against these viruses or
vaccines establishes in our body.

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References
Inoue T, Shinnakasu R, Kawai C, Ise W, Kawakami E, Sax N, Oki T, Kitamura T,
Yamashita K, Fukuyama H, Kurosaki T. Exit from germinal center to become quiescentmemory B cells depends on metabolic reprograming and provision of a survival signal. J
Exp Med. 218(1):e20200866, 2021
Laidlaw BJ, Duan L, Xu Y, Vazquez SE, Cyster JG. The transcription factor Hhexcooperates with the corepressor Tle3 to promote memory B cell development. Nat
Immunol. 21(9):1082-93, 2020

一般の皆様へ
免疫記憶応答はワクチン療法の根幹となる免疫反応であり、 記憶 B 細胞の産生、 活性化原理の解明は、 インフルエンザ ・ 新型コロナウイルスなどに対する効果的なワクチン開発において重要な課題である。 我々は様々なマウスモデルを駆使して記憶 B 細胞の分化メカニズムの解明に注力し、 エネルギー代謝や生存シグナルの獲得など記憶 B 細胞への分化運命決定に必要な様々な細胞特性を明らかにしてきた。 このような基礎研究による知見の蓄積が新規ワクチン開発
戦略に貢献できれば幸いである。

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Rhodium(I)-catalyzed enantioselective hydroacylation throughdynamic kinetic resolution
Yoshihiro Oonishioonishi@pharm.hokudai.ac.jp
Faculty of Pharmaceutical Sciences Hokkaido University

Summary Abstract
Rhodium(I)-catalyzed enantioselective intermolecular hydroacylation of an alkyne througha dynamic kinetic resolution was investigated. It was found that the reaction of a racemic
aldehyde having a sulfur atom in the tether with an alkyne gave the α,β-unsaturated ketone
in good yield with moderate ee.
Key Words ： rhodium, hydroacylation, enantioselective, alkyne, aldehyde

Introduction
Transition metal-catalyzed hydroacylation is one of the most promising methods for thesynthesis of ketones from an aldehyde and a C-C multiple bond.1 In particular, rhodiumcatalyzed intermolecular hydroacylation of an alkyne has been received much attention
from chemists because various α,β-unsaturated ketones can be prepared by using it. 1
However, there is only one report on the enantioselective variant through a kinetic resolution
so far.2 In this context, I investigated rhodium(I)-catalyzed enantioselective intermolecularhydroacylation of an alkyne through a dynamic kinetic resolution.

Results
Initially, the reaction of racemic aldehyde 1a having a sulfur atom in the tether with alkyne
2a was investigated (Table 1). The reaction was carried out in the presence of 10 mol% of[Rh(cod)2]ClO4 and (R,R)-Me-duphos at 0 °C in acetone, and the desired α,β-unsaturated
ketone 3aa showed moderate yield and low ee (entry 1). Next, the effects of the reaction
temperature and additives were examined. As a result, it was found that when the reaction
was carried out at -20°C, the yield of 3aa was improved to 78%, albeit with 10% ee (entry2). On the other hand, the use of alcohol and phenol derivative as an additive showed theimprovement of the ee of 3aa (entries 3-6). On the basis of these results, the reaction was
carried out using various ligands. When (R,R)-Et-duphos was used as a ligand, the reaction
proceeded to give 3aa in 43% yield with 60% ee (entry 7). On the other hand, when biarytype ligands were employed in this reaction, both yield and ee of 3aa were decreased(entries 8,9).

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Next, the reaction of 1a with various alkyne using (R,R)-Me-duphos as a ligand was
examined (Scheme 1). As a result, it was found that alkyne 2b having an alkyl moiety (R= n-Bu) at the terminus afforded 3ab in 49% yield with 65% ee. When alkyne 2c having
t-Butyl moiety was used, the yield of 3ac was decreased to 19%. On the other hand, theuse of the internal alkyne 2d gave α,β-unsaturated ketone 3ad in 45% yield with 20% ee.
These results suggest that the steric and electric property of alkyne 2 is influenced on thereactivity in this hydroacylation.

Furthermore, the reaction of aldehyde 1b having an aromatic ring at the α-position withalkyne 2a was employed in this hydroacylation (Scheme 2). The reaction proceeded to giveα,β-unsaturated ketone 3ba in 79% yield with 26% ee.

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Discussion & Conclusion
I investigated rhodium(I)-catalyzed enantioselective intermolecular hydroacylation of analkyne. As a result, it was found that this hydroacylation proceeded smoothly even at -20°C, giving the α,β-unsaturated ketone in good yield with moderate ee when phenol wasused as an additive. At this stage, this reaction cannot be expanded to the enantioselectivehydroacylation through a dynamic kinetic resolution. Further investigation of improvementof the ee is in progress.

References
1. (a) Willis M. C. Chem. Rev. 2010,110, 725. (b) Davison, R. T.; Dong, V. M. Acc. Chem.
Res. 2021, 54, 1236.
2. Gonzalez-Rodriguez, C.; Parsons, S. R.; Thompson, A. L.; Willis, M. C. Chem. Eur. J.
2010, 16, 10950.

一般の皆様へロジウム触媒を用いた分子間ヒドロアシル化反応は、 アルデヒドとアルキンから不飽和ケトンを合成する効率的な手法です。 しかしながら、 この方法を利用して光学活性な不飽和ケトンを合成した報告はわずか一例しかありません。 そこで、 本研究では、 動的速度論的光学分割を利用して、 この反応を不斉合成へと応用すべく検討しました。 その結果、 フェノールを添加剤として用いると、 ラセミ体のアルデヒドとアルキンから良好な収率及び中程度の不斉収率で不飽和ケトンが得られることがわかりました。 不斉収率は未だ満足するものではありませんが、 フェ
ノールのような弱酸性の物質を添加すると、 ヒドロアシル化反応に大きく影響を与えるという現象を初めて見出しました。

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Molecular mechanism underlying development of memory-likeGroup 2 innate lymphoid cells
Takashi Ebihara
tebihara@med.akita-u.ac.jp
Department of Medical Biology, Akita University Graduate School of Medicine

Summary Abstract
Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells that contribute to thepathogenesis of allergy by producing innate type 2 cytokines. Allergy-experienced ILC2s
can live long and exhibit memory-like features upon second allergen challenge. However,
it is not clear what activated ILC2s differentiate into memory-like ILC2s or die due to overactivation. Here, our new fate-mapping mice demonstrated that ILC2s extremely activatedduring chronic allergic airway inflammation express Tigit and are destined to die either inthe middle of chronic allergy or in the convalescent phase of allergy. We here show the firstevidence of activation-induced cell death of ILC2s during chronic allergy.
Key Words ： memory-like ILC2, trained ILC2, Tigit, activation-induced cell death

Introduction
Group 2 innate lymphoid cells (ILC2s) are responsible for innate allergic inflammation1.
ILC2s can proliferate well in vitro for over a year. However, physiological conditions do
not allow ILC2s to grow forever. There should be a machinery of cell death caused byoveractivation. We previously showed that activated ILC2s successively express aninhibitory receptor PD-1, an inhibitory cytokine IL-10, and an inhibitory receptor Tigit, andthat highly activated ILC2s express all these molecules2-4. However, as expression of Tigit
by ILC2s is not so high, it is hard to isolate Tigit+ ILC2s for research. In addition, fate of theactivated Tigit+ ILC2s remains elusive.

Results
To clearly identify Tigit+ ILC2s and follow the fate of Tigit+ ILC2s, we generated TigitCreERT2
mice where IRES-Cre-ERT2 is inserted into 3’ UTR of Tigit gene and crossed the
Cre-ERT2
mice to Rosa26-flox-stop-flox-tdTomato mice. To validate tdTomato expression
Tigit
in Tigit+ cells, we gave tamoxifen to the TigitCre-ERT2: Rosa26-flox-stop-flox-tdTomato mice
(Tigitfm mice) and confirmed that tdTomato expression correlates Tigit expression in Treg
and NK cells both of which are known to express Tigit. The Lung ILC2s did not express
tdTomato as steady state ILC2s do not express Tigit. Thus, our fate mapping system works
well to label Tigit-expressing cells by tdTomato expression.
To induce tdTomato+ ILC2s in the lung, we intranasally administered papain and gave
tamoxifen twice a week until day 21. One-to-two % of lung ILC2s expressed tdTomato since
day 9. As the tdTomato+ ILC2s produced more IL-5 and IL-10 than tdTomato− ILC2s, they
can be recognized as more activated ILC2s than tdTomato− ILC2s. Although number of lung
ILC2s dramatically increased during repeated papain treatments, frequencies of tdTomato+

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for the Promotion of Sciencecells among the lung ILC2s were quite stable, suggesting that Tigit+ ILC2s do not havea tendency to accumulate at the site of chronic inflammation like exhausted-T cells. To
address the question whether Tigit+ ILC2s live long in the convalescent phase of airway
allergy, we examined tdTomato+ lung ILC2s a week or a month after cessation of papain
treatment. Number of tdTomato+ ILC2s reduced by half a week after the last treatment andcompletely disappear another three weeks later. Thus, chronic allergy is required for the
emergence of tdTomato+ ILC2s that never differentiate to memory-like ILC2s.
Given chronic allergy constantly gives rise to highly activated tdTomato+Tigit+ ILC2s
that neither die nor leave the lung during inflammation, such tdTomato+ ILC2s would
accumulate in the lung with chronic allergy. However, frequency of tdTomato+ lung ILC2s
did not goes up as allergic inflammation deteriorated. To examine the possibility that
tdTomato+ ILC2s migrate to lymph node or other organ through blood stream, we sought
to identify tdTomato+ ILC2s in the lymph node and peripheral blood. However, tdTomato+
ILC2s did not exist there, suggesting that tdTomato+ ILC2s may not leave the lung. We
then hypothesized that tdTomato+ ILC2s are destined to die due to overactivation. To
explore whether tdTomato+ ILC2s tend to die, we sorted tdTomato− ILC2s and tdTomato+
ILC2s from the lung with chronic allergy and cultured the cells with IL-2/IL-7/IL-33 to keep
activation status. More tdTomato+ ILC2s were dead than tdTomato− ILC2s. These datasuggest that tdTomato+ ILC2s are in a high activation status just before cell death.
To determine physiological roles of activation-induced cell death of Tigit+ ILC2s during
chronic allergy, we injected blocking antibody against Tigit to the Tigitfm mice and induced
chronic allergy as described above. Blockade of Tigit increased tdTomato+ ILC2s that wereassociated with increased eosinophil recruitment to the lung. Therefore, activation-induced
cell death through Tigit+ ILC2s is an important regulatory mechanism during chronic airway
allergy.

Discussion & Conclusion
Our new mouse system for fate-mapping of Tigit+ cells revealed that Tigit+ ILC2s
represent a highly activated subpopulation of ILC2s destined to die during chronic allergy
and in recovery period of allergy. Blockade of Tigit during chronic pulmonary allergy
increased Tigit+ ILC2s in the lung. This is striking resemblance to exhausted Tigit+ CD8 T
cells whose apoptosis is reduced by Tigit blockade. To clarify decisive factors of activationinduced cell death, we are currently investigating transcriptional and epigenetic changes in
tdTomato+ ILC2s.

References
1. Ebihara T*. Dichotomous Regulation of Acquired Immunity by Innate Lymphoid Cells.
Cells. 11;9(5):1193.2020.
2. Miyamoto C, Ebihara T*. Runx/Cbfβ complexes protect group 2 innate lymphoid cells
from exhausted-like hyporesponsiveness during allergic airway inflammation. Nat
Commun. 10:447, 2019.
3. Ebihara T* and Taniuchi I. Exhausted-like Group 2 Innate Lymphoid Cells in Chronic
Allergic Inflammation. Trends Immunol. 40:1095-1104, 2019.

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4. Ebihara T* et al. Trained innate lymphoid cells in allergic diseases. Allergology
International. 70: 174-180. 2021.

一般の皆様へアレルギー炎症は増悪していく疾患傾向を持ちますが、 その原因については未だ分かっていないことが多いです。 私達は、 免疫細胞の中でアレルギー炎症の記憶を持ち、 アレルギー疾患の増悪に寄与する２型自然リンパ球 （ILC2） の研究を行いました。 ILC2 は炎症を経験した後、多様な細胞集団へと変化しますが、 炎症の後も生き残り炎症の記憶を持つようになる細胞集団と死にゆく運命の細胞集団の区分は明らかになっていませんでした。 私達は、 Tigit という抑制性受容体を発現した ILC2 は活性化により常に死んでいく運命にあることを明らかにしました。

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Development of urolithiasis therapy by immune response
and repair mechanism
Takahiro Yasui
yasui@med.nagoya-cu.ac.jp
Nagoya City University Graduate School of Medical Sciences

Summary Abstract
Urolithiasis, a complex multifactorial disease, results from interactions between environmental and genetic factors. Recent literature has shown that oxidative stress and reactive
oxygen species could be one such mechanistic pathway. We attempted to elucidate themechanism of urethral stone formation by focusing on the in vivo immune response and repair mechanism. (1) Deregulated MTOR is responsible for autophagy defects exacerbatingkidney stone development. (2) M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development. (3) Fatty acid-binding protein 4 downregulation drives calcificationin the development of kidney stone disease
Key Words ： Urolithiasis, macrophage, autophagy, transcriptomics

Introduction
Kidney stone disease has a high prevalence and recurrence rate, so that a number ofpatients would suffer from this illness. The recent studies with Randall’s plaque, urinary
components, as in vivo/vitro experiments, including gene manipulation, tried to unveil its
pathogenesis; however, pieces of evidence were yet complicated to integrate to develop anovel therapy to cure this disease of its origin. One of the promising findings in renal andperipheral macrophages' involvement via the inflammatory process, which potentially leads
to the therapeutic target.

Results(1) Autophagy in renal tubular cells
As cellular damage in renal tubular cells (RTCs) is responsible for the disease, here,
we focused on the role of macroautophagy/autophagy in RTCs. We found that autophagic
activity was significantly decreased in mouse RTCs exposed to calcium oxalate (CaOx)
monohydrate crystals and in the kidneys of GFP-conjugated MAP1LC3B (microtubuleassociated protein 1 light chain 3 beta) transgenic mice with CaOx nephrocalcinosis
induced by glyoxylate. This caused accumulation of damaged intracellular organelles, suchas mitochondria and lysosomes, the normal functioning of which is mediated by functional
autophagy. An impairment of autophagy was also observed in the mucosa with plaques of
CaOx kidney stone formers. We determined that the decrease in autophagy was causedby an upregulation of MTOR (mechanistic target of rapamycin kinase), which consequently
resulted in the suppression of the upstream autophagy regulator TFEB (transcription
factor EB). Furthermore, we showed that an MTOR inhibitor could recover a decrease in
autophagy and alleviate crystal-cell interactions and the formation of crystals associated

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with increased inflammatory responses.
(2) Macrophage phenotypes for renal calcium oxalate crystal development
We investigated the role of M1/M2s in crystal development by using in vitro and in vivo
approaches. The crystal phagocytic rate of bone marrow-derived M2Mφs was higher thanthat of bone marrow-derived Mφs and M1Mφs and increased on co-culture with renal
tubular cells (RTCs). However, the amount of crystal attachment on RTCs reduced on coculture with M2Mφs. In six hyperoxaluric C57BL/6J mice, M1Mφ transfusion and induction
by LPS and IFN-γ facilitated renal crystal formation, whereas M2Mφ transfusion andinduction by IL-4 and IL-13 suppressed renal crystal formation compared with the control.
These M2Mφ treatments reduced the expression of crystal-related genes, such as
osteopontin and CD44, whereas M1Mφ treatment increased the expression of proinflammatory and adhesion-related genes such as IL-6, inducible NOS, TNF-α, C3, andVCAM-1.
(3) Fatty acid-binding protein 4 (FABP4) for calcification in the development of kidney stonedisease
We aimed to utilize a transcriptomics approach to discover the missing link between
these two epidemic diseases. We investigated gene expression profiling of nephrolithiasis
patients by two RNA-sequencing approaches: comparison between renal papilla tissue withand without the presence of calcified Randall's plaques (RP), and comparison between thepapilla, medulla, and cortex regions from within a single recurrent stone forming kidney.
Results were overlaid between differentially expressed genes found in the patient cohortand in the severely lithogenic kidney to identify common genes. Overlay of these two RNAsequencing datasets demonstrated there is impairment of lipid metabolism in renal papillatissue containing RP linked to downregulation of FABP4. Immunohistochemistry of humankidney specimens and microarray analysis of renal tissue from a nephrolithiasis mousemodel confirmed that FABP4 downregulation is associated with renal stone formation. In a
FABP4 knockout mouse model, FABP4 deficiency resulted in the development of both renal
and urinary crystals.

Discussion & Conclusion(1) We conclude that autophagy compromised by MTOR deregulation is a fundamental
feature in the pathology of kidney stone formation, and propose that chemical inhibitionof MTOR could be a prospective strategy for disease suppression.
(2) The expression of M2Mφ-related genes was lower whereas that of M1Mφ-related genes
was higher in papillary tissue of CaOx stone formers. Overall, our results suggest that
renal crystal development is facilitated by M1Mφs, but suppressed by M2Mφs.
(3) Our study revealed that FABP4 plays an important, previously unrecognized role inkidney stone formation, providing a feasible mechanism to explain the link between
nephrolithiasis and metabolic syndrome.

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References
Taguchi K, Yasui T, et al. Kidney Int. 2020 May;97(5):1042-1056.
Okada A, Yasui T, et al. Active Phagocytosis and Diachronic Processing of Calcium
Oxalate Monohydrate Crystals in an in vitro Macrophage Model. Kidney Blood Press Res.
2019;44(5):1014-1025.
Yasui T, et al. Pathophysiology-based treatment of urolithiasis. Int J Urol. 2017
Jan;24(1):32-38.

一般の皆様へ
尿路結石の再発率は 5 年で 50％と高いが、 予防法は確立していない。 尿路結石の形成機序
は、 結石内に 90 数％含まれる無機成分からのアプローチが主体であった。 私たちは結石内にわずか数％しか含まれていない有機物質 （マトリックス） が結石形成に重要な作用をしていると考え、 細胞傷害を介し、 マトリックスの発現を介して尿路結石が形成される分子機構を解明した。
尿路結石モデルマウスでの腎結石が自然消失する」 という現象を捉え、 この現象に着目し、 治
療法開発にむけた尿細管上皮細胞 ・ M φ ・ 脂肪細胞らの免疫応答と細胞間ネットワークの機序解明を行った。

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The model chicken of cardiac septum defect ; A new tool for
analysis of structural heart disease
Saori Tani-Matsuhanatmsaori@port.kobe-u.ac.jp
Kobe University

Summary Abstract
The cardiac neural crest cells arise in the caudal hindbrain and then migrate to the heart
through the pharyngeal arches. These cells contribute to heart formation, including theoutflow tract, and are unique to this neural crest population. MafB is a transcription factor expressed specifically in early migrating cardiac neural crest cells in addition to being
expressed in rhombomeres (r) 5 and 6. We showed that MafB is a novel critical player inproper cardiac neural crest development, and propose that MafB is part of a gene regulatory subcircuit that is unique to the cardiac neural crest.
Key Words ： Cardiac neural crest, Heart development, Cardiac defect, Gene regulatory
mechanism

Introduction
The neural crest is an embryonic multipotent cell population characterized by migratory anddifferentiation abilities. They differentiate into peripheral neurons, pigment cells, craniofacialchondrocytes and connective tissue, and so on. Cardiac neural crest is a subpopulation of
the neural crest and is positioned from the post-otic vesicle to the 3rd somite. They migrate
through pharyngeal arches 3, 4 and 6 and contribute to the formation of the heart, including
the septum of the aortic and pulmonary trunks, aortic arches and cardiac ganglions.

Results
To reveal the genome-wide profile in cardiac neural crest cells, we have used a
FoxD3 neural crest enhancer to isolate a pure population of cardiac neural crest cells fortranscriptome analysis. This has led to the identification of transcription factors, signaling
receptors/ligands, and cell adhesion molecules upregulated in the early migrating cardiac
neural crest. The transcription factor MafB, previously best known for its expression andfunction in rhombomeres 5 and 6, was isolated as the highest upregulated genes in the
cardiac neural crest by the transcriptome analysis (ref.1).
1. Effects of MafB loss on other cardiac
neural crest genes
To know the role of MafB in cardiac
neural crest gene regulatory network, we
examined the effects of MafB-loss of function
on expression of other neural crest marker
genes, Sox10, Krox20, Ets1 as well as MafB

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itself. Expression of Sox10 was reduced in the cardiac neural crest stream on the loss offunction embryos, whereas cranial neural crest cells were unaffected. Expression of Krox20,
Ets1 and MafB itself was also reduced. We also analyzed the long-term effects of MafBloss of function in later stage embryo. In migrating cardiac neural crest cells of pharyngeal
arches (PA) 3 and 4, which is a known pathway of cardiac neural crest cell migration into
the developing heart, MafB-loss of function embryo shows slightly reduced expression of
Crabp1 which is a later neural crest marker gene (Fig. 1).
2. MafB is a critical input for Sox10E2 enhancer activity in the cardiac neural crest
Sox10 is a neural crest marker gene expressed at all axial levels including the cardiac
neural crest. The Sox10E2 enhancer is specifically expressed in the cranial plus postotic neural crest, but absent from the trunk neural crest. We found that three previously
unrecognized putative MafB binding sites, MafB (a), MafB (b) and MafB (c), within the
Sox10E2 enhancer. To test the necessity of MafB sites to mediate reporter expression, we
individually mutated each motif within the enhancer. Wild-type or mutant constructs driving
GFP were electroporated onto chicken embryos together with wild-type Sox10E2 driving
mCherry as a control reporter. Mutation of binding motifs MafB(b) and MafB(c) resulted in
reduced GFP expression in cardiac neural crest cells. This result showed that MafB is a
critical input for Sox10E2-driven Sox10 expression in cardiac neural crest (ref. 1).
3. The transcriptional regulation mechanism to express MafB in cardiac neural crest cells
To know the transcriptional mechanism of cardiac neural crest specific expression in
MafB gene, we have determined a cis-regulatory region controlling MafB expression by
an in vivo reporter assay using chicken embryos. A reporter driven by this region can
trace migrating cardiac neural crest cells and the activity is unique to cardiac neural crest
cells among neural crest cell population. Taken this advantage of this reporter, we also
succeeded to trace the migrating cardiac neural crest cells into the pharyngeal arches inlater stages (Tani-Matsuhana and Inoue, under review).

Discussion & Conclusion
These results lead us to conclude that MafB is required for proper cardiac neural crest
development and provided molecular insight into what makes the cardiac neural crest
unique. The reporter gene driven by MafB cis-regulatory region will lead to a moleculartechnique tracing cell linage in the cardiac neural crest population of the heart. It canalso be developed for cardiac neural crest-specific gene knockdown or overexpression
experiments, which can be expected as a new tool for analysis of structural heart disease.
Thus, the specificity of the MafB regulatory element in cardiac neural crest has potential for
promoting our understanding of cardiac neural crest and heart development.

References(1) Tani-Matsuhana, S., Vieceli, F. M., Gandhi, S., Inoue, K., Bronner, M. E.
(2018) Transcriptome profiling of the cardiac neural crest reveals a critical role for MafB.
Dev. Biol. 444, S209-S218.

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一般の皆様へ
心臓は生命維持に必須な器官です。 心臓流出路は、 初めは総動脈幹という一本の管ですが、次第に内部に隔壁ができ、 大動脈と肺動脈の２本に分離します。 この隔壁は心臓神経堤細胞によって形成されています。 心臓神経堤細胞の欠損 ・ 発生異常は総動脈幹が分離しない総動脈
幹遺残症 (PTA) を引き起こします。 心臓神経堤細胞の重要性は明らかであるにも関わらず、 心
臓神経堤細胞形成に関わる遺伝子制御機構についてはほとんどわかっていません。 これまでの私の研究で、 心臓神経堤細胞で特異的に発現する MafB 遺伝子が心臓神経堤細胞の初期発生に必須であることを明らかにしました。 ニワトリ胚内で MafB の機能を欠失させることで、 心臓神経堤細胞由来の隔壁を失った総動脈幹遺残症 (PTA) モデルニワトリ胚を作り出し、 それを利用して心臓隔壁欠損を引き起こす分子機序の解明を目指しています。

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Visualizing aging individuality
Yukinori Hirano
yukinori@ust.hk
Kyoto University

Summary Abstract
We aimed to understand the mechanism underlying aging variability as we define, aging
individuality. To this end, we developed the visualizing tools of aging individuality, in whichanimals produces light when the aging individuality emerges.
Key Words ： Aging, Individuality, Drosophila

Introduction
As our population rapidly ages, the promotion of healthy aging is of critical importance formodern society. Previous studies have identified conserved aging factors among different
species, including Sirtuin family proteins (ref. 1), the mammalian target of rapamycin(mTOR) signaling (ref. 2), and the insulin/insulin-like growth factor 1 (IGF-1) signaling (ref.
3). However, age-related dysfunctions vary significantly among individuals. Therefore, in
addition to searching for common factors related to aging, we must try to understand “aging
individuality (the mutable variability in age-related dysfunctions)”.

Results
To investigate the gene expression profile of individual flies, we analyzed the heads
and bodies separately from 36 aged males and 6 young males of Drosophila by RNAseq analysis. We combined data analysis from well-known statistical analysis in RNA-seq(DESec2) to determine the significantly altered genes, and clustering analysis (WGCNA,
ref. 4) which reveals the genes showing correlated expression pattern, and therefore
indicates the specific pathway acting in the specific individuals. Though data analysis using
our platform, we determined that aged flies display the multiple clusters of gene with alteredexpression only in the subset of individuals, which are different in male and females, andalso in heads and bodies, suggesting that these genes could act as the genetic marker
to define aging individuality. To visualize the aging individuality, representative genes
from each cluster were selected, and transcriptional activators, GAL4, LexA, or QF were
knocked into the C-terminus of each gene via CRISPR/Cas9 together with a T2A selfcleaving peptide, allowing the endogenous gene and activator to be synthesized separatelyfrom the same transcript. The transcriptional activators then induced expression of thebioluminescent proteins, Nanoluc, Eluc, and Akaluc, which emit light at the wavelengthsof 460, 536, and 650 nm, respectively. Upon feeding a specific substrate for eachbioluminescent protein on 96-well plates, the emitted light wan captured with band-passfilters in the luminometer. A maximum of three different reporters can be monitored in an
individual at a time, enabling the visualization of the proportion (how many animals in agiven population express the marker genes), the dynamics (when the marker genes are

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expressed), and the complexity (how the marker gene expressions are overlapped) of
aging individuality. To date, a total of 12 transgenic lines were constructed as the candidate
genetic markers for aging individuality in male heads. The additional genes selected frommale body, female head/body will be targeted similarly to below. Representative resultsof 4 lines with GAL4 insertion in rdgB (retinal function), CG14419 (unknown function),
SPH93 (immune response), and PEPCK2 (gluconeogenesis) crossed with UAS-Eluc flies
were shown (Fig. 1A-D). Consistent with the RNA-seq data, only a subset of the aged
flies emitted light after substrate feeding (Fig. 1A-D, left panels). An EMCCD camera
demonstrated the stereotyped localization of the signals: eyes in rdgB::GAL4 flies, mouthand genitalia in CG14419::GAL4 flies, and sparse, unspecified tissues in SPH93::GAL4
and PEPCK2::GAL4 flies (Fig. 1A-D, right panels). It is noteworthy that heterogenous
light emission was observed in the stereotyped organs, indicating stochastic features; in
rdgB::GAL4 flies, light was only emitted from one eye, but not from both eyes (Fig. 1A).
In addition, the SPH93::GAL4 flies died 2-3 days after light emission, suggesting that thisreporter is linked to life expectancy, while no such link was observed in other reporters. We
are now trying to link the marker genes to the aging phenotypes, in addition to longevity,
such as behavioral change (sleep, locomotor, sensation, or feeding), and metabolic change.
We will further try to understand mechanism underlying the aging individuality by forward
genetic screening in fly.

Discussion & Conclusion
Although aging is ubiquitous in all animals, the related dysfunctions differ between
individuals; conditions in humans range from cancers, cardiovascular diseases, cognitive
disorders, and diabetes. The central question here is “why age-related dysfunctions vary
among individuals”. We now obtained a tool to access the aging individuality. After we
conduct a forward genetic screen in Drosophila to identify the genes or pathways that
regulate aging individuality, hopefully we could determine the entry point to tackle theage-related multiple dysfunctions. The long-term goal of this study is to elucidate themechanisms underlying aging individuality, which could contribute to the development of
personalized therapies for age-related dysfunction.

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References
1, Kaeberlein, M., McVey, M. & Guarente, L. The SIR2/3/4 complex and SIR2 alone
promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes &development 13, 2570-2580, doi:10.1101/gad.13.19.2570 (1999).
2, Kaeberlein, M. et al. Regulation of yeast replicative life span by TOR and Sch9 in
response to nutrients. Science 310, 1193-1196, doi:10.1126/science.1115535 (2005).
3, Kenyon, C. The plasticity of aging: insights from long-lived mutants. Cell 120, 449-460,
doi:10.1016/j.cell.2005.02.002 (2005).
4, Zhang, J. et al. Weighted Gene Co-expression Network Analysis for RNA-Sequencing
Data of the Varicose Veins Transcriptome. Frontiers in physiology 10, 278, doi:10.3389/
fphys.2019.00278 (2019).

一般の皆様へ
老化は各器官、 例えば神経系や心血管系、 消化器系、 運動器系といった様々な器官の機能障害を引き起こし、 高齢者の社会的活動を著しく制限する。 老化に伴う機能低下は、 各個体で訪れる時期は様々で、 また均一にすべての器官で機能低下を示すわけではない。 従って老化に伴う器官機能不全に立ち向かうためには、 特定の器官で機能低下を示した個体を識別した上で、 解析し、 さらには機能低下を改善させる方法論を見出していかなければならない。 そこで私は、 個体間の老化プロセスの違い 「老化個性」 に焦点を当て、 それを可視化する方法論の確立が必要不可欠であると考え、 老化個性を可視化する本研究を行った。

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In vivo imaging the brain defines new mechanisms of leukocytes
migration cascade in Central Nervous System Lupus
Yoshishige Miyabe
y-miyabe@nms.ac.jp
Department of Cell Biology, Institute for Advanced Medical Sciences, Nippon Medical
SchoolSummary Abstract
How chemoattractants (CAs) control leukocytes entry into central nervous system (CNS)
is largely unknown. We recently established the technique of in vivo imaging the brain to
define new mechanism of leukocyte migratory behavior in CNS lupus.
Key Words ： chemoattractant, leukocytes migration, central nervous system lupus

Introduction
Chemoattractant-induced arrest of circulating leukocytes and their subsequent diapedesis
is a fundamental component of inflammation1. Thus, the control of leukocyte entry into thetissue represents a major point at which new therapeutics could be developed to attenuate
inflammation1. Central nervous system (CNS) lupus is clinically involved in approximately
40% of all systemic lupus erythematosus patients. How leukocytes infiltrate into CNS in
the pathogenesis of CNS lupus is not well understood. Recently, we have developed thetechnique of in vivo imaging the brain to elucidate the mechanism how leukocytes entry into
the CNS in the pathogenesis of CNS lupus.

Results
Recent advances in imaging technology have provided unprecedented views into immune
cell function in live animals, providing entirely new paradigms for immune cell function. In
fact, the applicant has developed techniques to apply intravital imaging technology to study
the arthritic joint2. Our new joint imaging technique has discovered new unexpected biology
concerning the specific functions of chemoattractants (CAs) in regulating leukocyte entry injoints in vivo. For example, we have found that classical complement C5a receptor C5aR1
plays a unique role in capturing neutrophils on the endothelium in the inflamed joints3.
In addition, we have demonstrated that atypical C5a receptor C5aR2 on endotheliumtransports C5a to initiate neutrophil adhesion and inflammation in the inflamed joints4.
The aim of our study is that we will apply the technique of in vivo imaging the brain totake our understanding of CNS lupus to an entirely new level by allowing the real timevisualization of the events that lead to encephalitis in live mice.
It has reported that CAs, such as chemokines, lipid mediators and complement
components in central nervous fluids (CFs) derived from CNS lupus patients are increasing
than those of healthy donors, suggesting that CAs might play important roles of leukocytes
recruitment into CNS in the pathogenesis of CNS lupus. However, the mechanism how
CAs in CFs promote leukocytes entry into CNS in the pathogenesis of CNS lupus remain

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unclear.
Thus, we hypothesized that CAs were transported into CFs from blood and might control
leukocytes recruitment into CNS in the pathogenesis of CNS lupus. Our project consistsof (1) study if CAs can entry into CFs from blood, (2) establish in vivo brain imaging and(3) define the mechanism how leukocytes can entry into CNS in an animal model of CNS
lupus, and has been conducted since 2020, with a 3 year plan.
In 2020, we have determined whether CAs in blood could entry into CFs. We
intravenously (i.v.) injected with several dose of CAs or sterile PBS as control, into wildtype (WT) mouse and isolated CFs from WT mice at several time course. The concentrationof CAs in CFs was measured by ELISA. We found that the concentration of CAs in CFs
derived from WT mice injected with CAs i.v. was increased than those of WT mice injected
PBS, and a dose dependent manner. Also, we observed some subsets of leukocyte
infiltration into brain when we intravenously injected with CAs into WT mouse.
To develop the technique of in vivo brain imaging, mice were anesthetized and positioned
on a stereotactic surgical stage, and the skull immobilized. Hair in the frontal and parietal
regions of the skull was cleaned with an electrical hair clippers and chemical depilatory
agent. Skin was excised from top of the skull, and the underlying periosteum scraped off.
Using a high-speed air-turbine drill with a burr tip size of 0.8mm in diameter, a groove wasmade to form a circle of approximately 4mm in the parietal bone of the skull. This area wasmade thinner by cautious and continuous drilling of the groove until the bone flap became
loose. Using a blunt microblade, the bone flap was separated from dura mater underneath,leaving the dura intact. After removal of the bone flap, the dura mater was continuously kept
moist with PBS. The window was then sealed by adhering a cover glass to the bone using
a tissue glue. After the cranial window surgery, in vivo brain imaging was performed under aconfocal microscopy. We could observe leukocytes migratory behavior in brain.

Discussion & Conclusion
Our recent work has demonstrated that CAs were generated within the tissue, and thentransported by atypical CA receptors into vessel lumen. Transported CAs bind with CA
receptors on circulating leukocytes and induce to migration into the tissue in inflammation 2,3.
In contrast, the mechanism how CAs control leukocytes entry into CNS in CNS lupus islargely unknown.
Our preliminary data has demonstrated that CAs in blood might entry into CFs in WT
mice and then promote leukocytes entry into CNS. However, the mechanism how CAs aretransported into CNS from blood remains unclear. Also, if transported CAs could directly
promote circulating leukocytes entry into CNS is unknown. We will apply the technique of invivo imaging the brain to clarify the mechanism of CAs-mediated leukocytes migration in an
animal model of CNS lupus.

References
1. Miyabe Y, Lian J, Miyabe C, Luster AD. 2019. Chemokines in Rheumatic Diseases andTherapeutic Implications. Nature Reviews Rheumatology 15(12), 731-746.
2. Miyabe Y, Kim ND, Miyabe C, Luster AD. 2016. Studying Chemokine Control of

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Neutrophil Migration In Vivo in a Murine Model of Inflammatory Arthritis. Methods in
Enzymology 570: 207-31.
3. Miyabe Y, Miyabe C, Mani V, Mempel TR, Luster AD. 2019. Atypical complement receptor
C5aR2 transports C5a to initiate neutrophil adhesion and inflammation.
Science Immunology 4, eaav5951.
4. Miyabe Y, Miyabe C, Murooka TT, Kim YE, Newton GA, Kim ND, Haribabu B, Luscinskas
FW, Mempel TR, Luster AD. 2017. Complement C5a Receptor is the Key Initiator of
Neutrophil Adhesion Igniting Immune Complex-induced Arthritis. Science Immunology 2,
eaaj2195.

一般の皆様へ近年、 サイトカイン阻害を目的とした生物学的製剤が続々と開発され、 効果的に活性化免疫細胞を抑制可能となり、 自己免疫疾患の予後は劇的に改善しました。 一方で、 これらは全身性に免疫細胞の “活性化” を阻害する為、 重篤な感染症や二次発癌などの副作用が問題となっています。 生命現象の根底にある細胞の “動き” は炎症病態の根底にあり、 その機序を解明する事で細胞の “動き” を臓器特異的に制御可能な次世代免疫療法の開発につながると期待されます。

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Molecular Pathology of Moyamoya Disease
Daisuke Morito
morito@med.showa-u.ac.jp
Showa University School of Medicine

Summary Abstract
Mysterin, also known as RNF213 or ALO17, is a large cellular protein that contains locomotive ATPase modules and ubiquitin ligase domain(s). Mutations found in the ubiquitin
ligase domain elevate the risk of cerebrovascular moyamoya disease to a varying degree.
We demonstrated its unexpected role and pathogenic effects of the moyamoya mutations.
Key Words ： moyamoya disease, mysterin, RNF213, ubiquitin, AAA+ ATPase

Introduction
Moyamoya disease is a unique cerebrovascular disorder characterized by bilateralstenosis/occlusion of the terminal region of internal carotid arteries and following brain
infarction and hemorrhage. I performed the molecular cloning of its causal gene andnamed it mysterin (also known as RNF213 or ALO17) as a final touch of genetic analysis
of moyamoya disease (#Liu, #Morito et al., PLOS ONE, 2011; patent 5854423). I havedemonstrated the ATPase and ubiquitin ligase activities of mysterin protein and its
physiological function in cells (i.e., metabolic regulation of lipid droplets; Sci Rep, 2014,2015, 2019; J Cell Biol, 2019). Mutations found in the ubiquitin ligase domain of mysterin
elevate the risk of moyamoya disease to a varying degree, while molecular mechanism
underlying moyamoya disease remained elusive.

Results
Precise imaging analysis for the first time revealed that mysterin protein rapidly shuttles
between the cytosol and the lipid droplets (LDs) that are the organelles specialized forcellular lipid storage and metabolism, suggesting the physiological contribution of mysterin
to the lipid metabolism (Figure). Localization of mysterin to the LDs resulted in specificelimination of several LD surface proteins including ATGL, the primary lipase that regulates
the stability of the LDs, and led to immobilization (stabilization) of the LDs. Both ATPase
activity and ubiquitin ligase activity of mysterin are indispensable for this process (i.e., LD
localization and immobilization). Ubiquitin ligase activity of mysterin is partly spoiled by
the patient mutations in the ubiquitin ligase domain, resulting in the lack of LD localizationof mysterin. Instead, the mutant mysterin protein forms aggregate-like structure in cells,suggesting the previously-unexpected link between the LD metabolism and moyamoya
disease.

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Recent structural analysis demonstrated that mysterin’s ATPase domain forms a dyneinlike ATPase core and may have a motor-like (locomotive) activity (Clausen and colleagues,
eLife, 2020). Given the lack of LD localization of ATPase mutant mysterin, the putative
locomotive ATPase activity possibly underlies the dynamic shuttling of mysterin between
the cytosol and the LDs.
In addition, the study suggested that mysterin protein contains two independent ubiquitin
ligase domains. We examined each involvement in mysterin’s intracellular behavior
and found that the two domains have distinct functions, one of which is associated with
mysterin’s intracellular localization.
Furthermore, a study recently reported showed a surprising function of mysterin (Otten,
Randow and colleagues, Nature, 2021). It was observed that mysterin ubiquitylates nonproteinous substrate LPS decollating the surface of Salmonella that invades the cells. Given
the LD localization and ubiquitylation (unpublished observation) by mysterin, mysterin could
be a ubiquitin ligase which has an affinity to cellular/exogenous membranes and triggers
ubiquitin-related pathways on the membrane (e.g., autophagic defense against Salmonella).
We are now evaluating the mechanism by which mysterin recognizes specificmembranes and mechanistic involvement of dynein-like ATPase and potential two ubiquitin
ligase activities. Also, we are evaluating the potential cytotoxic effect of the aggregation ofmutant mysterin.

Discussion & Conclusion
Mysterin is the 21st largest protein within those encoded in the human genome (Uniprot),which has a unique combination of enzymatic activities (ATPase and ubiquitin ligase). It is
specifically targeted to the LDs and the invading Salmonella and ubiquitylates their membrane surfaces, which further leads to downstream events (e.g., autophagy). Many of mutations associated with moyamoya disease have been found in mysterin’s ubiquitin ligase
domain and led to the aggregate-like pattern of mysterin in cells. One of the currently mostimportant question is what is the exact role of each potential ubiquitin ligase activity of mysterin. We are promoting functional analyses on mysterin, focusing on several points including this question.

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References
1. Sugihara M, Morito D (corresponding), Shiori Ainuki, Yoshinobu Hirano, Kazutoyo Ogino,
Akira Kitamura, Hiromi Hirata, Kazuhiro Nagata. AAA+ ATPase/ubiquitin ligase mysterinstabilizes cytoplasmic lipid droplets. J Cell Biol. 2019 Mar 4; 218(3):949-960.
2. Kotani Y, Morito D (corresponding), Kenshiro Sakata, Shiori Ainuki, Munechika Sugihara,
Tomohisa Hatta, Shun-ichiro Iemura, Seiji Takashima, Tohru Natsume, Kazuhiro Nagata. Alternative exon skipping biases substrate preference of the deubiquitylase USP15
for mysterin/RNF213, the moyamoya disease susceptibility factor. Sci Rep. 2017 Mar
9;7:44293
3. Morito D (co-corresponding) and Nagata K. Molecular Biology of Mysterin/RNF213. Moyamoya Disease Explored Through RNF213, Book Chapter, Springer, pp45-57 (2017)
4. Morito D (co-corresponding) and Nagata K. Physiological Role of Mysterin/RNF213 inZebrafish. Moyamoya Disease Explored Through RNF213, Book Chapter, Springer,
pp59-67 (2017)
5. Kotani Y, Morito D (co-corresponding), Satoru Yamazaki, Kazutoyo Ogino, Koichi
Kawakami, Seiji Takashima, Hiromi Hirata, Kazuhiro Nagata. Neuromuscular regulationin zebrafish by a large AAA+ ATPase/ubiquitin ligase, mysterin/RNF213. Sci Rep. 2015
Nov 4;5:16161
6. Morito D, Kouki Nishikawa, Jun Hoseki, Akira Kitamura, Yuri Kotani, Kazumi Kiso, Masataka Kinjo, Yoshinori Fujiyoshi, Kazuhiro Nagata. Moyamoya disease-associated protein
mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomericstate. Sci Rep. 2014 Mar 24;4:4442
7. Liu W, Morito D (co-first) , Seiji Takashima, Yohei Mineharu, Hatasu Kobayashi, Toshiaki
Hitomi, Hirokuni Hashikata, Norio Matsuura, Satoru Yamazaki, Atsushi Toyoda, Kenichiro Kikuta, Yasuchi Takagi, Kouji H. Harada, Asao Fujiyama, Roman Herzig, Boris
Krischek, Liping Zou, Jeong Eun Kim, Masafumi Kitakaze, Susumu Miyamoto, Kazuhiro
Nagata, Nobuo Hashimoto, Akio Koizumi Identification of RNF213 as a susceptibility
gene for moyamoya disease and its possible role in vascular development. PLoS One.
2011;6(7):e22542.

一般の皆様へ
もやもや病は東アジア地域の子どもに多い脳血管疾患です。 脳の真下のあたりの動脈が狭くなり、 血が通りにくくなります。 その結果、 脳への血液供給が不足し、 脳梗塞などが起こります。
なぜ動脈が狭くなるのか、 なぜこの箇所で起こるのかなど、 ほとんど謎のままです。 私たちはもやもや病の原因遺伝子と考えられる新たな遺伝子ミステリンを物質として取り出すことに成功しま
した （分子クローニング、 2011）。 以来、 解析を進め、 この遺伝子から合成されてくるタンパク
質ミステリンの分子としてのはたらきや、 体の中での機能、 またもやもや病患者さんから見つかった遺伝子変異により機能がどのように変化するかを順番に明らかにしてきました。 まだ研究の途上ですが、 もやもや病発病メカニズムのヒントをつかみつつあります。 本財団や公的組織をふくむ各方面からの支援を受けながら、 近い将来の病態解明を目指して研究を続けています。

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Role of actin cytoskeletal fluctuation in retinal organoid formation
Satoru Okuda
satokuda@staff.kanazawa-u.ac.jp
Kanazawa University

Summary Abstract
Epithelial cell extrusion is observed in organoid formation. Various molecular factors
have been reported as key drivers of epithelial extrusion; however, little is known about how
these factors are linked to the mechanical process. We developed a simple mathematicalmodel that explains cell extrusions under a wide range of physiological and pathophysiological conditions. The model shows that cells can be extruded from homogeneous sheets,
owing to the inherent mechanical instability of the 3D foam geometry of the epithelial monolayer.
Key Words ： Cell extrusion, cytoskeletal contractility, organoid

Introduction
In cell extrusion, a cell embedded in an epithelial monolayer loses its apical or basal
surface and is subsequently squeezed out of the monolayer by neighboring cells. Cell
extrusions occur during apoptosis, epithelial-mesenchymal transition, or precancerous cell
invasion. They play important roles in embryogenesis, homeostasis, carcinogenesis, andmany other biological processes. Although many of the molecular factors involved in cell
extrusion are known, little is known about the mechanical basis of cell extrusion.

Results
We used a three-dimensional (3D) vertex model (Ref. 1) to investigate the mechanicalstability of cells arranged in a monolayer with 3D foam geometry. We created a geometricmodel of a cell sheet in which the cells are represented as polyhedrons with averagevolume within a planar sheet with homogeneous thickness. Here, we parameterized thethickness by introducing cell density into the sheet.
Cell extrusions occasionally occur even without the active generation of force by the
extruding cells, suggesting that mechanical instability inherent in the 3D geometry of theepithelial monolayer might be sufficient to drive cell extrusion. As a result, the energy
landscape led to mechanical instability and subsequent extrusion. For example, thedecrease in ka destabilized the dual-sided state and stabilized the apical extrusion state.
We found that when the cells composing the monolayer have homogeneous mechanicalproperties, cells are extruded from the monolayer when the symmetry of the 3D geometryis broken because of an increase in cell density or a decrease in the number of topologicalneighbors around single cells. Those results suggest that mechanical instability inherent in
the 3D foam geometry of epithelial monolayers is sufficient to drive epithelial cell extrusion.
In the situation in which cells in the monolayer actively generate contractile or adhesive
forces under the control of intrinsic genetic programs, the forces act to break the symmetry

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The Novartis Foundation(Japan)
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Discussion & Conclusion
We presented a theoretical model that describes the mechanical stability of cellularmonolayers. The model shows that the cellular monolayer has inherent mechanicalinstability that is sufficient to cause cell extrusion without any additional provoking factors,
even when the cells have homogenous mechanical properties. Analytical calculations showthat the instability depends on the cell density, topology, and mechanical properties within
the monolayer. The active generation of forces by an extruding cell can provoke extrusionand direct the extrusion to the apical or basal side of the monolayer, suggesting that cells
utilize the mechanical instability of the epithelial geometry to regulate extrusion.

References
1. Okuda, S., Inoue, Y., & Adachi, T. (2015). Three-dimensional vertex model for simulating
multicellular morphogenesis. Biophysics and physicobiology, 12, 13-20.

一般の皆様へ
上皮組織では、 多数の細胞が密に並んだシート状の組織であり、 シート内の各細胞は、 脳の初期発生等の過程に置いて多層化したり脱離したりします。 この細胞脱離の仕組みの理解は、胚発生 ・ 組織再生 ・ がん疾患などの幅広い現象の解明にとって重要な課題です。 本研究では、上皮組織のシート構造を簡単化して捉え、 力学エネルギーに基づいた理論的な解析を行うこと
により、 上皮組織に内在する力学的な不安定性が細胞脱離を引き起こす仕組みを明らかにしま
した。

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Elucidating the system to generate dormancy that conferchemotherapy-resistance to cancer cells
Keisuke Nimura
nimura@gts.med.osaka-u.ac.jp
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University

Summary Abstract
Most ovarian cancers respond to initial chemotherapy, but some cancer cells that havedeveloped resistance to treatment remain and can cause recurrence. In order to overcome
this problem, the underlying causes of cancer cell resistance must be identified and targeted for treatment. One of the major mechanisms of resistance to chemotherapy is the entryof cancer cells into a quiescent state. However, the system by which the quiescent stateof cancer cells is formed and maintained is largely unknown.In this study, we will identify
quiescent cancer cells, which are resistant to chemotherapy, by single cell sequencing andother methods, and clarify the entire tumor microenvironment including these cells. Basedon these results, we aim to develop cancer therapeutics targeting quiescent cancer cells.
Key Words ： ovarian cancer, dormant cancer cell

Introduction
Cancer cells that have acquired resistance to treatment exist in cancers that recur after
anticancer drug treatment, but the full picture of the mechanism is unknown. Previous in
vitro studies using cancer cell lines have shown that some cancer cells remain by entering
a quiescent phase and becoming tolerant to anticancer drugs, and then repopulate byacquiring resistance. On the other hand, the applicants have clarified the dynamic chromatin
structure conversion mechanism during the transition from quiescent to proliferative phaseusing B cells as a model. In the quiescent phase, naïve B cells have a highly aggregatedchromatin structure, but upon receiving an activation signal, they quickly convert to aloose chromatin structure by transcription factor Myc and histone acetylation to enablerepopulation. This suggests that a dynamic chromatin structure conversion mechanism isrequired for the repopulation of quiescent cells in cancer, but the molecular mechanism bywhich quiescent cancer cells convert to a chromatin structure that allows repopulation has
not yet been clarified.

Results
We analyzed single-cell gene expression of 10,000 cells from three cases, two casesof primary serous adenocarcinoma and one case of recurrent serous adenocarcinoma,
respectively. 10,116 and 8,968 single-cell gene expression data were obtained from thelibrary of primary serous adenocarcinoma using cellranger. We obtained single cell gene
expression data for 10,116 cells and 8,968 cells from the library of first-episode serous
adenocarcinoma. We obtained single-cell gene expression data for 10,296 cells from thelibrary of recurrent serous adenocarcinoma, with an average of 61.7k reads per cell. We were

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for the Promotion of Scienceable to detect the expression of 2500 genes per cell. The obtained data was analyzed by
t-SNE and visualization was performed. As a result, we were able to identify EPCAM- andPAX8-positive epithelial cancer cells, CD4- and CD8-positive T cells, macrophages, anddendritic cells. The immune cells showed relatively similar gene expression patterns among
the cases, whereas the epithelial cancer cells showed slightly different gene expression
patterns among the cases. In addition, 8% of the cells were found to be EPCAM-negative,
oncogene MYC-positive, and mesenchymal marker THY1-positive mesenchymal carcinoma
cells. Furthermore, these mesenchymal carcinoma cells were negative for MKI67,
suggesting that they are in a quiescent state compared to epithelial carcinoma cells.
High-grade serous adenocarcinoma is the most frequent form of ovarian cancer, andalthough it responds well to initial chemotherapy, it recurs in most cases. We performed
flow cytometric analysis of high-grade serous adenocarcinoma tissues from more than 10
cases, including both first-episode and recurrent cases, using three antibodies: CD45, amarker of blood cell lineage; EpCAM, a marker of epithelial lineage; and THY1, a marker
of mesenchymal lineage. The results showed that high-grade serous adenocarcinomas
contain about 10% cells that express extremely high levels of THY1. Similar analysis ofrecurrent high-grade serous adenocarcinoma tissues showed that the number of cells
expressing THY1 was increased in the tumor. Furthermore, gene expression analysis ofseven cell types separated by CD45, EpCAM, and THY1 expression showed that THY1expressing cells had higher expression of ABCG2, which is involved in drug efflux, andNOTCH3, which is involved in cancer progression, while preliminary results showedthat THY1-expressing cells had significantly lower expression of the cell proliferation
marker MKI67. The preliminary results showed that the expression of the cell proliferation
marker MKI67 was markedly low while the expression of ABCG2, which is responsible
for cancer efflux, and NOTCH3, which is involved in cancer progression, were high. Next,
we searched for transcription factors that are specifically expressed in THY1-expressing
cells and identified several mesenchymal-related transcription factors. Next, we performed
SNP analysis using genes that were commonly highly expressed in the seven isolated
cells in cases where normal tissue could be recovered. Clustering analysis revealed that
THY1-positive cells, unlike normal tissues, had a SNP pattern similar to that of tumor cells.
These results strongly suggest that THY1 high-expressing cells are not just a population ofmesenchymal cells.
The mouse ovarian cancer cell line ID8 forms peritoneal dissemination nests andproduces ascites when administered into the peritoneal cavity of mice. To verify thepresence of slow-growing mesenchymal carcinoma cells in the seeded nests formed by ID8,
we generated a cell line capable of detecting slow-growing cells. We expressed the BFP
gene fused with histone H2B at the tetracycline promoter, which can transiently express thegene in a doxycycline-inducible manner. This construct was knocked into the ROSA locus
to avoid the effects of expression of other genes. In addition, this ID8 retains the luciferase
gene, so that the IVIS system can measure tumor growth in the abdominal cavity overtime without killing the mice. Now, by adding doxycycline to these ID8-tet-H2B-BFP cells,
we were able to construct cells expressing H2B-BFP. First, under culture conditions, H2BBFP is transiently expressed; since H2B-BFP is incorporated into chromatin, it does notdecrease without division. By collecting cells that retain H2B-BFP after long-term culture, it
is possible to recover slow-growing cells.

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Discussion & Conclusion
In this study, single cell analysis of ovarian cancer clinical specimens and gene
expression analysis of cell populations isolated and collected by flow cytometry
suggested the presence of ~10% mesenchymal carcinoma cells in ovarian cancer.
These mesenchymal carcinoma cells expressed the mesenchymal marker THY1 andthe oncogene MYC, but not the epithelial marker EPCAM or the fibroblast marker
FSP1. Furthermore, although preliminary, THY1-positive cells were found in peritoneal
disseminated nests collected after chemotherapy, suggesting that some of these cells
may persist after chemotherapy and lead to recurrence. To prove that these mesenchymal
carcinoma cells are truly cancer cells, we also transplanted an ovarian cancer cell line
into mice and collected THY1-expressing cells from the tumor. After confirming that thiscell population shows the same gene expression pattern as the clinical samples, we will
implant them into mice to prove their tumorigenic potential. Through these experiments,
we will prove that THY1-expressing cells are mesenchymal carcinoma cells and that they
are likely to persist after chemotherapy. If we can prove the existence of these THY1expressing cells, it will provide a basis for the development of new therapies targeting thiscell population.

一般の皆様へ
がん治療上の問題点の 1 つとして、 がん細胞が抗がん剤の抵抗性を獲得することがあげられます。 抗がん剤の多くは細胞増殖が盛んな細胞に対して効果を示します。 がんの中には細胞増殖が極めて遅いがん細胞が存在すると考えられており、 このような細胞増殖の遅いがん細胞には
抗がん剤は効きにくいと考えられます。 ところが、 このようながん細胞がどのような性質を持つのかあまりはっきりしていません。 私達は、 細胞増殖の遅いがん細胞の性質を明らかにして、 この細胞を除去する治療法の開発を進めています。

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Mechanism and targeting therapy for TGF pathway-dependent
diffuse-type gastric cancer
Yoku Hayakawa
hayakawayoku@gmail.com
The University of Tokyo Hospital
Key Words ： Gastric cancer, TGF pathway, mouse model, LRG1, CD38

Introduction
Gastric cancer is a disease with a high morbidity and mortality rate. Signet ring cell typegastric cancer, so-called scirrhous gastric cancer, has a particularly poor prognosis among
gastric cancers. In this cancer, signet ring cell carcinoma invades the stomach wall diffusely,
causing early metastasis to the whole body including peritoneal dissemination, and areresistant to many cancer therapies. Since signet ring cell carcinoma is hereditary in families
with mutations in the CDH1 gene, the importance of the CDH1 gene has been suggested.
In addition, genomic analysis revealed frequent abnormalities in the TGF / SMAD pathway
in this subtype. We attempted to establish a mouse model that mimics scirrhous gastriccancer by combining a gastric epithelial-specific labeled mouse (TFF1-Cre) with Credependent genetically modified mice. As a result, we established a mouse model ofscirrhous gastric cancer that causes early mucosal infiltration and leads to cancer death bycombining three gene mutations, CDH1, TGFBR2, and p53. Using these TFF1-PTC mice,
we pursued a new treatment method for scirrhous gastric cancer.

Results
We performed RNAseq analyses using TFF1PTC stomach and gastric organoid samples.
Compared to healthy stomach, TFF1-PTC
stomach is enriched with genes associated with
angiogenesis and fibrosis. Among these genes,
we found that both TFF1-PTC stomach and gastricorganoid express markedly higher level of CD38
and LRG1, compared to normal samples. CD38 is
known to bind to CD31, an endothelial cell surface
marker, and regulate immune cell migration.
LRG1 is known to bind to CD105 (also known as endoglin), and regulate angiogenesis
and fibrosis. In vitro experiments suggest that LRG1 and CD38 upregulation is caused
specifically by simultaneous knockout of CDH1 and Tgfbr2, and also modulated by BMPdependent signals. By performing scRNAseq, we revealed that CD105 is expressed in a
subset of endothelial cells.
We examined the effects by CD38 and CD105 inhibition by using our TFF1-PTC mousemodel. TFF1-PTC mice were treated with specific antibodies that target CD38 or CD105 for

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2 weeks, and gastric tumor sections were pathologically compared. As a result, both antiCD38 and anti-CD105 antibodies significantly decreased tumor invasion in the stomach,with reduction of fibroblasts and endothelial cells in tumor stroma. Thus, these data suggestthat LRG1 and CD38 are critical factors that regulate robust angiogenesis and fibrosisin this type of gastric cancer, and can be a therapeutic target by blocking tumor-stromalinteraction.

Discussion & Conclusion
Among all cancers, scirrhous gastric cancer is the most deadly cancer. Furthermore, it
is difficult to identify high-risk groups for this type of gastric cancer, given that this cancer
occurs even in young people who are not infected with H. pylori. Thus, our discovery that
CD38 and LRG1 may be a promising treatment target for scirrhous gastric cancer would
greatly contribute to improving prognosis of patients with advanced gastric cancer. In
addition, since a similar mechanism may exist in other cancer cases with same mutations,this might contribute to establish precision medicine for cancers with mutations in CDH1
and TGF pathways.

一般の皆様へ
本研究では、 予後不良の癌であるスキルス胃癌の新規治療標的を、 独自のマウスモデルを用いて同定した。 樹立したスキルス胃癌マウスサンプルの網羅的遺伝子発現解析によって、 この癌で特徴的に発現上昇する遺伝子として LRG1 と CD38 を同定した。 これらの分子は腫瘍内の血管新生や線維化に重要であり、 特異的抗体によるマウスモデルの治療によって、 スキルス胃癌の進展を抑制することが可能であった。 LRG1 ・ CD38 標的治療は、 腫瘍間質相互作用を阻
害することでスキルス胃癌の治療に有用であると考えられる。

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Mechanisms involved in regulation of osteoclast formation by
glucan recognition via immune receptors
Wataru Ariyoshi
arikichi@kyu-dent.ac.jp
Division of Infections and Molecular Biology, Kyushu Dental University

Summary Abstract
Our previous studies reported that β-glucans suppress osteoclast formation by suppressing NFATc1, an essential transcriptional factor for osteoclastogenesis. In this study,
we focused on the molecular mechanisms by which β-glucans suppress NFATc1 expression mediated by interaction with dectin-1. β-glucans reduced RANKL-induced Blimp1
expression and upregulated Irf-8 expression, suggesting that suppression of the RANKLstimulated Blimp1 expression leading to the upregulation of transcriptional repressors might
be involved in the inhibitory effect of β-glucans on NFATc1 expression. Our findings indicatethat further research on β-glucans may be useful in the treatment of osteoclast-related diseases, such as osteoporosis.
Key Words ： β-glucan, dectin-1, osteoclast, blimp-1

Introduction
Various signaling cascades mediated by immunoreceptors have been found to regulate
osteoclast differentiation, suggesting that immune cells interact with osteoclasts throughseveral signaling factors [1]. Dectin-1 is a type II membrane receptor which recognizes
β-glucans. We have demonstrated the obvious inhibitory effect of curdlan, laminarin,lichenan, glucan from baker’s yeast, and β-1,3-glucan from Euglena gracilis on osteoclastdifferentiation. However, glucan from black yeast and β-D-glucan from barley showed alesser inhibitory effect on osteoclast differentiation compared with other β-glucans.
To explore these discrepancies, we performed structural analyses of β-glucans.
Furthermore, we investigated the effects of β-glucans on the expression of negative
regulators of osteoclastogenesis.

Results
It is possible that diverse effect of β-glucans on osteoclast differentiation reflect
differences of purity and three-dimensional structure (e.g., β-(1,6)-linked side chains) ofeach of the β-glucans (Table 1, [2]).

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Source and structure of β-glucans [Ref 2]
To prepare the dectin-1 retrovirus vector, cDNA-encoded mouse dectin-1 was cloned
into the pFB-Neo plasmid. GP2-293 packaging cells were transfected by a plasmid mixture
containing dectin-1/pFB-Neo and pVSV-G with Lipofectamine LTX. The culture supernatantof GP2-293 cells was harvested as a retrovirus fluid for dectin-1 transfection. RAW264.7
cells were cultured with the virus vector in the presence of 8 μg/ml Polybrene for 48 h. Cells
expressing dectin-1 (d-RAW) were maintained in α-MEM containing 10% FBS, penicillin G(100 units/ml), streptomycin (100 μg/ml) and G418 disulfate aqueous solution (50 mg/ml).
For the negative control of d-RAW cells, a control virus vector without dectin-1 cDNA wasused for infection (c-RAW).
c-RAW cells and d-RAW cells were cultured with 40 ng/ml of osteoclast differentiation
factor (receptor activator of nuclear factor-kappa B ligand; RANKL) for 7 days. On Day
7, cells were subjected to TRAP analysis. Remarkably, β-glucan (25 μg/ml) inhibited theosteoclast differentiation of d-RAW cells more effectively than c-RAW cells (Figure 1).
These data suggested that β-glucan directly binds to dectin-1 expressed on the surface ofosteoclast progenitors and suppresses their differentiation into osteoclasts.

Figure 1 Effect of β-glucan on osteoclast differentiation [Ref 3]
Previous studies have shown that nuclear factor of activated T cell c1 (NFATc1) is strongly
induced by RANK-RANKL signaling and plays an important role as a key transcriptional
regulator in osteoclast terminal differentiation [4]. Given that NFATc1 has been suggested
to be a target of the essential transcription factor of osteoclast differentiation, we asked theeffect of β-glucans on the induction of NFATc1 expression. We found that although RANKLThe Novartis Foundation(Japan)
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upregulated NFATc1 protein level in d‐RAW cells, β-glucan also suppressed this induction(Figure 2).

Figure 2 Effect of β-glucan on NFATc1 expression [Ref 3]
Recently, various molecules that control osteoclast differentiation have been identified.
Several transcription repressors that suppress osteoclast differentiation, such as v-maf
musculoaponeurotic fibrosarcoma oncogene homolog B (MafB), interferon regulatory
factor-8 (Irf-8), and B cell lymphoma 6 (Bcl6) are rapidly downregulated by RANKL
stimulation [5-7]. Blimp-1 binds to the promoters and suppresses the transcription of thesefactors [8]. These previous studies have suggested that the RANK-RANKL interaction
enhances transcription factors that promote osteoclast differentiation, while inhibits theactivity of transcription repressors for osteoclastogenesis. Thus, we investigated the effectsof β-glucans on these negative regulators of osteoclastogenesis.
d-RAW cells were incubated with RANKL and β-glucan (25 μg/ml) for the indicated times.
The mRNA level of Blimp1, MafB, Irf‐8, and Bcl6 were assessed using real‐time RT‐qPCR.
Protein level of Blimp1 in whole‐cell lysates were detected by western blot analyses. β‐actin
served as loading control.
Glucan from baker’s yeast significantly downregulated Blimp1 and showed a slightincrease in the expression of Irf-8 gene. The RANKL‐induced upregulation of Blimp1 protein
was also suppressed by glucan from baker’s yeast (Figure 3).

Effect of β-glucan on Blimp1‐mediated negative regulators of osteoclastogenesis [Ref 3]

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Discussion & Conclusion
As for the structure of β-glucans, it is known that a certain amount of molecular weightis required for the biological activity of β-glucans. A recent study reported that a splitluciferase complementation assay is useful strategy to characterize the side chain structureof β-glucans [9]. Structural analyses of β-glucans are also currently under investigation in
our laboratory.
Next, we focused on the inhibitory factors of osteoclast differentiation as the candidates
involved in the downregulation of NFATc1 by the interaction of β-glucans and dectin-1.
β-glucans significantly reduced RANKL-induced Blimp1 expression and upregulated Irf-8
expression. Although additional research is needed to elucidate the molecular mechanisms
by which β-glucans regulate Blimp1 expression, our data suggest that the suppressionof the RANKL-stimulated Blimp1 expression leading to the upregulation of transcriptional
repressors might be involved in the inhibitory effect of β-glucans on NFATc1 expression.
We demonstrated that β-glucans attenuate RANKL-induced osteoclast differentiation by
downregulating NFATc1 activation. Our findings indicate that further research on β-glucans
may be useful in the treatment of osteoclast‐related diseases, such as osteoporosis.

References
1. Shinohara M, Takayanagi H (2007) Novel osteoclast signaling mechanisms. Curr
Osteoporos Rep, 5, 67–72.
2. Ariyoshi W, Hara S, Koga A, Nagai-Yoshioka Y, Yamasaki R (2021) Biological Effects of
β-Glucans on Osteoclastogenesis. Molecules, 26, 1982.
3. Hara S, Nagai-Yoshioka Y, Yamasaki R, Adachi Y, Fujita Y, Watanabe K, Maki K,
Nishihara T, Ariyoshi W (2021) Dectin-1-mediated suppression of RANKL-induced
osteoclastogenesis by glucan from baker’s yeast. J Cell Physiol, 236, 5098-5107.
4. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M,
Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T (2002) Induction andactivation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling interminal differentiation of osteoclasts. Dev Cell, 3, 889-901.
5. Kim K, Kim J, Lee J, Jin HM, Kook H, Kim KK, Lee SY, Kim N. (2007) MafB negatively
regulates RANKL-mediated osteoclast differentiation. Blood, 109, 3253–3259.
6. Miyauchi Y, Ninomiya K, Miyamoto H, Sakamoto A, Iwasaki R, Hoshi H, Miyamoto K,
Hao W, Yoshida S, Morioka H, Chiba K, Kato S, Tokuhisa T, Saitou M, Toyama Y, Suda
T, Miyamoto T (2010) The Blimp1‐Bcl6 axis is critical to regulate osteoclast differentiation
and bone homeostasis. J Exp Med, 751-762.
7. Zhao B, Takami M, Yamada A, Wang X, Koga T, Hu X, Tamura T, Ozato K, Choi Y,
Ivashkiv LB, Takayanagi H, Kamilio R (2009) Interferon regulatory factor‐8 regulatesbone metabolism by suppressing osteoclastgenesis. Nat Med, 15, 1066-1071.
8. Nishikawa K, Nakashima T, Hayashi M, Fukunaga T, Kato S, Kodama T, Takahashi
S, Calame K, Takayanagi H, Rao A (2010) Blimp 1-mediated repression of negative
regulators is required for osteoclast differentiation. Proc Natl Acad of Sci U S A, 107,
3117–3122.
9. Yamanaka D, Kurita S, Hanayama Y, Adachi Y (2021) Split Enzyme-Based Biosensors
for Structural Characterization of Soluble and Insoluble β-Glucans. Int J Mol Sci, 22,
1576.

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一般の皆様へ
真菌や細菌、 海藻などが含有する糖鎖であるβ -glucan は、 免疫系細胞が持つ受容体である dectin-1 に認識されることで、 種々の生物学的機能を発揮します。 私達はβ -glucan が、 破骨細胞前駆細胞上の dectin-1 に結合し、 破骨細胞への分化や骨吸収活性を低下させることを証明しました。 そこで本申請では、 β -glucan の破骨細胞の分化抑制メカニズムに着目し、 破骨細胞分化に重要な分子に対する負の調節機能を発揮することを見出しました。 β -glucan の創薬への応用が、 骨粗鬆症や歯周病などに対して、 破骨細胞の骨吸収活性の低下を介した効
果的な治療法の提案へと繋がると考えており、 今後も研究を続けて参ります。

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Weighted gene co-expression network analysis in Drosophila
Michiko Sekiya
mmsk@ncgg.go.jp
National Center for Geriatrics and Gerontology

Summary Abstract
The fruit fly Drosophila melanogaster has been used as an efficient genetic model inbasic biology research. Recently, various human pathological conditions including neurodegenerative diseases have been modeled in Drosophila. These fly models are widely used
to investigate disease mechanism and for drug discovery. In this study, we aimed to unravel
the similarities and differences between human and Drosophila at a gene regulatory network level using weighted gene co-expression network analysis.
Key Words ： Weighted gene co-expression network, Drosophila

Introduction
The fruit fly Drosophila melanogaster has been used to model various humanpathological conditions including neurodegenerative diseases. These Drosophila models
are widely used to investigate disease mechanisms and to perform drug screen. Recently,
an integrative network analysis that use multi-omics data sets are used to identify thedisease mechanisms and novel therapeutic targets (Wang M. Li, A., Sekiya, M. et al.
Neuron, 2021). To further utilize and integrate Drosophila models into a drug discovery
pipeline, it is important to understand the similarities and differences between human andDrosophila at a gene regulatory network level.

Results
To construct the weighted gene co-expression networks in Drosophila, we extracted
the RNA from the heads of flies and performed the RNA sequencing (RNA-seq). The
expression data was obtained by the mapping of the sequences.
Drosophila
Wild type Canton-S line was used in this study. Flies were maintained in standard
cornmeal media at 25°C, 40–50% humidity with 12 hr light/dark cycle. More than 2000 flies
were collected for and flash-frozen in liquid nitrogen at the age of 7 days.
RNA extraction
Eighteen group were prepared with more than 100 flies as one group. Heads of flies weremechanically isolated, and total RNA was extracted using TRIzol Reagent (Thermo Fisher
Scientific) according to the manufacturer’s protocol with an additional centrifugation step
(16,000 x g for 10 min) to remove cuticle membranes prior to the addition of chloroform.
The quality of RNAs were evaluated by Bioanalyzer (Agilent Technologies) and the RNAs
with RNA integrity number (RIN) more than 8 were used for construction of RNA library. We
prepared 18 RNAs and the RIN ranged 8.2~9.1.
Library preparation
Preparation of samples for RNA-seq analysis was performed using the TruSeq

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Stranded mRNA Library Prep kit (Illumina). Briefly, polyA containing mRNAs were isolated,fragmented and used for cDNA synthesis. Synthesized double strand cDNAs were ligatedwith appropriate adaptors for sequencing and amplified. The size and concentration of theRNA-seq libraries were measured by Bioanalyzer. All 16 samples met the quality required
for sequence analysis.
RNA sequence and data analysis
Paired-end sequencing reads (150bp) were generated on a NovaSeq 6000 platform.
Average read number was 68 million reads/sample and the average percentage of baseswith a quality Score greater than 30 was 90.2%. The raw sequencing reads were aligned
to fly genome BDGP6 using HISAT2 aligner (v2.1.0) and gene-level expression were
quantified by StringTie (v2.1.2) based on Ensembl BDGP6.22.98 annotation model.
Visualization of weighted gene co-expression network
Using iDEP (integrated Differential Expression and Pathway) analysis application,weighted gene co-expression networks in Drosophila were visualized (Figure 1).

We found that a gene network can be constructed by 16 sets of transcriptome data from
Drosophila. Depending on the setting, the number and size of networks required to build
a gene network were changed significantly. As the next step, we will perform the network
analysis using the WGCNA package (R package), which is capable of extracting gene
members in the network and detailed information. After that analysis, we will compare thegenes from human gene co-expression networks and conduct verification experiments
using Drosophila.

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Discussion & Conclusion
In this study, we obtained gene expression data of the 32870 gene from the head of wildtype Drosophila by RNA sequencing. Based on the variations of gene expressions, we are
currently constructing co-expression networks. If necessary, we will integrateadditional
RNA sequencing data to build a more reliable network. In the future, we will compare these
Drosophila networks with the gene networks constructed from human gene expression
data. Such information will be helpful to appropriately integrate genetic or pharmacologic
screening data from Drosophila into drug discovery in human diseases. We expect thatcomparing gene regulatory networks are important to further understand the similarities
and differences between Drosophila and humans and to best utilize fly models of humandiseases. Disease models have different phenotypes for each model organism, which may
also be explained by the differences in the genetic network of each organism.

References
Wang M. Li, A., Sekiya, M. et al. Neuron., 2021, 109(2):257-272.

一般の皆様へ
キイロショウジョウバエは有用な遺伝学モデルとして使用されていますが、 最近では神経変性疾患をはじめとした様々なヒト病態モデルが開発され、 疾患研究、 創薬分野においても多用されています。 一方、 生体内 （特に脳内） での変化を包括的に捉え、 疾患の発症機序解明、 治療薬標的の同定へとつなげる 1 つのアプローチとして、 遺伝子ネットワーク解析が有用であることが分かってきました。 疾患研究において、 さまざまなモデル動物と遺伝子ネットワーク解析を用いることで、 疾患 ・ 創薬研究は飛躍的に進むことが予想されます。 遺伝子ネットワークレベルでのショウジョウバエとヒトとの類似性と違いを解析し理解することで、 疾患研究におけるショウジョウ
バエの有用性も高まると考えられます。

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Elucidation of the promotion mechanism in plant shootregeneration by environmental stimuli.
Sachihiro Matsunaga
sachi@edu.k.u-tokyo.ac.jp
Department of Integrated Biosciences, Graduate School of Frontier Sciences
The University of Tokyo

Summary Abstract
The environmental stimuli influence plant regeneration, but the molecular mechanism
has not been elucidated. Our in vitro shoot regeneration via dedifferentiation of roots
demonstrates that shoot regeneration occurs a few weeks later after the callus formationfrom roots. Using this system, we elucidated the mechanism of shoot regeneration by
the gamma-ray irradiation as an environmental stimulation. We successfully developed
the method to promote shoot regeneration by irradiation and identified the stress genes
involved in shoot regeneration.
Key Words ： shoot regeneration, gamma-ray, environmental stimuli, stress response

Introduction
Our de novo shoot regeneration is based on callus formation from roots (1, 2). Callus
is a pluripotent cell mass derived from an explant placed in an auxin-rich callus inducing
medium (CIM). Subsequently, de novo shoots are regenerated from callus cultured on
cytokinin-rich shoot inducing medium (SIM). It is known that cell fate changes during thisregeneration processes. It is unknown whether the accumulation of DNA damage impacts
on shoot regeneration.

Results
We found that γ-ray irradiation before CIM transfer enhanced shoot regeneration. First,
we irradiated A. thaliana with gamma-ray at several irradiation doses. We successfully
determined an irradiation dose of gamma-ray, which most efficiently enhanced shootregeneration. Second, we investigated whether the period before irradiation impacts
shoot regeneration efficiency. The gamma-ray irradiation enhanced shoot regeneration
irrespective of the developmental stage of seedlings. Third, we investigated whether theperiod after irradiation affects shoot regeneration efficiency. Callus at a particular time
for incubation after irradiation enhanced shoot regeneration. Fourth, we investigatedwhether the CIM period affects shoot regeneration efficiency. Callus for a particular time ofincubation also enhanced shoot regeneration.
Based on the above condition, we investigated whether DNA repair impacts shootregeneration efficiency. In A. thaliana, two kinases, Ataxia Telangiectasia and Rad3 Related
Protein (ATR) and Ataxia Telangiectasia Mutated (ATM), are involved in DNA damage
response. However, atr and atm mutants irradiated with gamma-ray exhibited enhancementof shoot regeneration compared with the control. Thus, the DNA repair pathway involved in

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ATM and ATR did not regulate the enhancement of shoot regeneration by gamma-ray.
Thus, to find the novel regulators for the enhancement of shoot regeneration, we
performed the transcriptome analysis by RNA-seq at the onset of callus induction gammaray irradiation 6 days prior to callus induction alters the gene expression pattern at both
callus periods, contributing to the enhancement of the shoot regenerative competency.
We performed Gene Ontology (GO) enrichment analysis among up-regulated genes
in the CIM 0 and 6 days. GO analysis demonstrated that at the 6 days after irradiation,
the expression of genes associated with stress response was upregulated. We identified
the stress response genes, which are probably responsible for the enhancement of shootregeneration.
We also performed ChIP-seq analyses of H3K4me2. We got consistent data with theabove RNA-seq data. Our integrated studies revealed the molecular and 1 day before shootinduction. In CIM 0days and CIM 6days, 409 and 415 genes, were up-regulated, and 512
and 777 genes were down-regulated as shown in graphs below. The result indicated thatthe mechanism by regulators of the stress response.
Furthermore, we examined the enhancement in shoot regeneration by other DNA
damage stress except for the irradiation. We tried to add heavy metal, including excess
copper, to the medium. We found a mutant responsible for shoot formation under theexcess copper (3). We also developed a novel live-imaging technique to analyze the shootregeneration via the environmental stimuli (4). Based on the research, we will do our best
to publish the primary data about the relationship between shoot regeneration and gamma
irradiation as an academic paper in the future.

Discussion & Conclusion
We revealed that several stress response genes regulated the enhancement in shootregeneration by environmental stimuli. We used gamma-ray irradiation as environmental
stimuli, which induce DNA damage. Our data showed that the initiation kinases of DNA
repair, ATR and ATM were not involved in the enhancement of shoot regeneration,suggesting that the enhancement is not related to DNA repair pathway. Our seq-analyses
demonstrated that some stress response genes regulate the mechanism. The molecular
genetic analyses of these genes will contribute to developing new breeding techniques forenhancement in shoot regeneration or shoot formation in plants.

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References
1. Ishihara, H.#, Sugimoto, K.#*, Tarr, P. T, Temman, H., Kadokura, S., Inui, Y., Sakamoto,T., Sasaki, T., Aida, M., Suzuki, T., Inagaki, S., Morohashi, K., Seki, M., Kakutani, T.,
Meyerowitz, E. M., and Matsunaga, S.* (2019) Primed histone demethylation regulates
shoot regenerative competency. Nature Commun., 10, 1786. #These authors equallycontributed to this work.
2. Sugimoto, K., Temman, H., Kadokura, S., and Matsunaga, S.* (2019) To regenerate or
not to regenerate: factors that drive plant regeneration. Curr. Opin. Plant. Biol., 47, 138150.
3. Sakamoto, Y., Sato, M., Sato, Y., Harada, A., Suzuki, T., Goto, C., Tamura, K., Toyooka,
K., Kimura, H., Ohkawa, Y., Hara-Nishimura, I., Takagi, S. and Matsunaga, S.* (2020)
Subnuclear gene positioning through lamina association affects copper tolerance. Nature
Commun., 11, 5914.
4. Shibuta, M. K., Sakamoto, T., Yamaoka, T., Yoshikawa, M., Kasamatsu, S., Yagi, N.,
Fujimoto, S., Suzuki, T., Uchino, S., Sato, Y., Kimura, H. and Matsunaga, S.* (2021) A live
imaging system to analyze spatiotemporal dynamics of RNA polymerase II modificationin Arabidopsis thaliana. Commun. Biol., 4, 580.

一般の皆様へ
植物が環境刺激を受けることで器官再生が促進するメカニズムを明らかにすることができた。 ヒトや動物とは異なり脳や神経系を持たない植物が、 外部環境の変動を捉えて、 器官再生にどのように結びつけているのかを分子的メカニズムの一端がわかった。 今後、 このメカニズムを利用して、 植物の再生促進法の開発が可能になることが期待される。

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Analyses of pain regulation mechanisms by IL-27
Hiroki Yoshidayoshidah@cc.saga-u.ac.jp
Saga University Faculty of Medicine

Summary Abstract
IL-27 is an anti-inflammatory cytokine. Recently we reported that IL-27 has a pain
regulatory role. To address IL-27-producing cells and/or cells responsible for IL-27mediated pain regulation, we generated IL-27-reporter mice. By taking advantage of thisreporter mouse, we tried to identify IL-27-producing cells in the pain sense transmission.
Unfortunately, we were unable to detect IL-27-producing cells in the skin, neuron fibers, andbrain. However, by electrophysiological study, three types of neuronal fibers were shown to
be possibly responsible for pain regulation.
Key Words ： IL-27, pain, neuralgia

Introduction
Interleukin 27 (IL-27) is a member of IL-12 cytokine family and is composed of p28
subunit and EBI-3 subunit. IL-27 has immunosuppressive effects on various immune cells
although it was originally reported as a Th1-inducing cytokine. Lack of IL-27 signaling (p28deficient mice and IL-27 receptor (WSX-1)-deficient mice), surprisingly demonstrated lower
threshold to various pain and neuralgia symptom while administration of IL-27 in p28deficient mice restored the susceptibility to normal levels. These data indicate that IL-27 has
a novel function to regulate pain sensitivity. Precise mechanisms by which IL-27 regulates
pain is unknown.

Results
IL-27 is reportedly produced by macrophages and dendritic cells stimulated by microbial
products, such as LPS. However, both p28-deficient and WSX-1-deficient mice showedspontaneous neuralgia and lower threshold to pain without any additional stimulation orpriming to pain. This indicates that IL-27 is constitutively produced and exerts its painregulation effects. To address the question where and in which type of cells IL-27 is
produced in the pathway of pain sense transmission, we have developed an IL-27-reportermouse in which Venus (a fluorescent protein) is produced under p28 promoter regulation.
The reporter mice work well in that macrophages/dendritic cells from LPS-administrated
mice emit sufficient fluorescent signal by flow cytometric detection. We assume that either
keratinocytes and/or Langerhans cells in the skin, neurons to sense various types of pain,or brain cells including glia cells, are the producer of IL-27 in terms of pain regulation.
However, we have been unable to detect p28-Venus signal in any of above-mentioned cellsby fluorescence microscopic examination. These results indicate that constitutive productionof IL-27 p28 is very weak for detection by this method. Interestingly, although dendritic cells
isolated from LPS-administrated mice expressed p28/Venus ex vivo, dendritic cells fromThe Novartis Foundation(Japan)
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untreated mice failed to respond to in vitro LPS stimulation by p28/Venus production. Thissuggests that IL-27 production by dendritic cells and/or cells involved in pain regulation may
require additional stimulation.
Besides the detection of p28-Venus signal, we performed a functional assay where
p28-deficient or WSX-1-deficient neurons were isolated and tested for (hyper-) sensitivity
to stimulation. In our previous report, live mice were tested for their sensitivity against
mechanical stimulation, heat/cold stimulation, and chemical stimulations. In the current
study, hindlimb skin-saphenous nerve preparations were isolated ex vivo and used forelectrophysiological analysis of nociceptors. C-fibers from WSX-1-deficient and p28deficient mice showed hyper discharge rate over wild-type neurons against mechanicalstimulation with lower threshold for excitation. Repeated experiments for confirmation andfor statistical analysis, however, is required. In the isolated nerve specimen, we were unable
to detect p28 nor WSX-1 gene expression by RT-PCR method.
We have not examined the direct pain-regulatory effects of IL-27 in the brain in thecurrent study. Lack of IL-27 signaling does not affect brain development or behavior in themice. IL-27 is known to be abundantly expressed in the brain (and in the central nervous
system) of mice. Recently, IL-27 is reported to be expressed by astrocytes and microglia
cells in inflammatory settings. The precise role of IL-27 in the brain has yet to be clarified,although some reports showed that IL-27 is pro-inflammatory rather than anti-inflammatory
in this context. We have confirmed the expression of IL-27 in astrocytes and microglia. The
pain regulatory role of IL-27 in these cells is completely unknown.
To address the above-mentioned, unsolved questions of IL-27-producing cells in thepain sensation, we started conditional p28 knockout mice in which cell (such as neurons,
Langerhans cells, and astrocyte)-specific gene destruction is possible. With this strainof mice, further clarification of responsible cells and responsible mechanisms of IL-27mediated pain regulation will be possible.

Discussion & Conclusion
We have successfully generated IL-27 (p28)-Venus reporter mice, which is one of theaims of this study. Unfortunately, we were unable to detect IL-27 production in the assumed
cells assumed to be responsible in pain regulation in unstimulated mice. This suggeststhat the constitutive expression of IL-27 is too low to be detected by this method and/or IL27 expression required additional stimulation in this pathway. Electrophysiological study
indicates neurons (C fibers) may be responsible, but further examination is necessary.
For the further analyses, we started generating p28 conditional mice. By taking
advantage of this strain of mice, identification of responsible cells and mechanisms in IL-27mediated pain regulation will be possible.

References
1. Sukhbaatar, O., D. Kimura, M. Miyakoda, S. Nakamae, K. Kimura, H. Hara, H. Yoshida,
S. I. Inoue, and K. Yui. 2020. Activation and IL-10 production of specific CD4(+) T cells
are regulated by IL-27 during chronic infection with Plasmodium chabaudi. Parasitol Int
74: 101994.
2. Ishikawa, A., Y. Miyake, K. Kobayashi, Y. Murata, S. Iizasa, E. Iizasa, S. Yamasaki, N.

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Hirakawa, H. Hara, H. Yoshida, and T. Yasaka. 2019. Essential roles of C-type lectin
Mincle in induction of neuropathic pain in mice. Sci Rep 9: 872.
3. Kanai, K., A. M. Park, A. Watanabe, T. Arikawa, T. Yasui, H. Yoshida, I. Tsunoda, and O.
Yoshie. 2018. Murine gamma-Herpesvirus 68 Induces Severe Lung Inflammation in IL27-Deficient Mice with Liver Dysfunction Preventable by Oral Neomycin. J Immunol 200:
2703-2713.
4. Sasaguri, T., T. Taguchi, Y. Murata, K. Kobayashi, S. Iizasa, E. Iizasa, M. Tsuda, N.
Hirakawa, H. Hara, H. Yoshida, and T. Yasaka. 2018. Interleukin-27 controls basal pain
threshold in physiological and pathological conditions. Sci Rep 8: 11022.

一般の皆様へインターロイキン 27 （IL-27） は、 免疫抑制作用を持つサイトカインとして、 自己免疫疾患や感染時の過剰な炎症 ( サイトカインストームなど ) に対する治療応用に関する研究が進んでいます。 今回、IL-27 による全く新しい痛み知覚制御作用を発見し、現在その詳細を解析しています。
これは、 モルヒネや消炎鎮痛剤とは異なる作用によるものであり、 新しい疼痛の治療法の開発につながることが期待されます。

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Molecular and neural mechanism associated with social
behavior
Teiichi Furuichi
tfuruichi@rs.tus.ac.jp
Tokyo University of Science

Summary Abstract
Autism spectrum disorder (ASD) affects approximately 1 in 40 children and is highly inheritable. As there is no cure for ASD, understanding its pathophysiology is an important
health issue. In this study, we performed a comprehensive brain transcriptome and behavioral analyses of BTBRTF/ArtRbrc (BTBR/R) mice. Our data identified potentially important
genes, non-coding RNAs, and RNA interactions which are probably critical to brain development and/or function. Collectively, we showed that BTBR/R have mild but significant deficits
in social behavior and is an excellent animal model that can contribute to the understandingof the genetic underpinnings of autism susceptibility.
Key Words ： autism spectrum disorder; ASD; transcriptome; social behavior; mouse model

Introduction
Autism spectrum disorder (ASD) is a complex neurodevelopmental disordercharacterized by social and communication deficits, and stereotyped repetitive behaviors
with restricted interests. ASD is highly heritable and is also affected by environmental
factors. Gene transcriptional profiling in ASD has been performed by utilizing post-mortem
brain samples. The restricted availability of brain tissue from humans with ASD represents asignificant challenge. From this point of view, animal models are valuable for analyzing themolecular and pathological mechanisms of ASD at the gene and protein expression level.
In this study, we performed a comprehensive brain transcriptome analysis in the cerebral
cortex and striatum, in which altered cortico-striatal connectivity has been suggested to bepresent in patients with ASD, between BTBR/R mice and C57BL/6J (B6) mice with highsociality. Our results revealed the differential transcription profiles in microarray expression
levels as well as cellular-regional levels in the cerebral cortex and striatum of BTBR/R mice
and B6 mice, and suggested that BTBR/R mice have a slight autistic-like tendency in termsof social novelty recognition and stereotypic behavior.

Results
To compare the transcriptome features in the cerebral cortex and striatum between
BTBR/R and B6 mice, we performed DNA microarray analysis. The data indicated that
BTBR/R mice exhibited the differential expression of 1,280 transcripts (974 downregulated,
306 upregulated) in the cerebral cortex and striatum compared to B6 mice (Fig. 1A). Using
the bioinformatics databases, we classified types of the differentially expressed genes(DEGs) identified, as shown in Fig. 1B. Downregulated and upregulated transcript groups
consisted of 524 and 184 protein-coding transcripts, 387 and 111 non-coding transcripts,

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33 and 6 pseudogenes, and 30 and 5 other transcripts, respectively. Using RNA–RNA
interaction network analysis tool, we further suggested the differential regulation oftranscription and translation in the cerebral cortex and striatum between two strains via
RNA–RNA interactions. To gain an insight into the biological significance in altered gene
expression between BTBR/R and B6 mice, we performed the functional gene classificationof DEGs by the gene ontology (GO) enrichment, the KEGG pathway and weighted
correlation WGCNA analyses. Overall, our comprehensive bioinformatics analyses ofgene expression highlighted functional alterations in the cerebral cortex and striatumof BTBR/R mice. We next explored whether BTBR/R mice DEGs were the known ASD
candidate genes by utilizing the autism gene database AutDB, and identified 53 genes (40downregulated and 13 upregulated) out of 1,280 DEGs to be ASD candidate genes. We
comprehensively analyzed the spatial cellular expression of DEGs in the BTBR/R and B6
mice brains in a qualitative manner using the in situ hybridization (ISH) method (Fig. 2A, B).
Finally, we analyzed ASD-related social behavior of BTBR/R and showed that, compared toB6 mice, BTBR/R mice have a mild impairment in social recognition (Fig. 3A-C) as well as
increased stereotypic behavior (Fig. 3D).

Discussion & Conclusion
In conclusion, we characterized the transcriptomic features of the cerebral cortex andstriatum of BTBR/R mice in comparison with B6 mice, using microarray and ISH analyses
together with comprehensive bioinformatics approaches. We identified DEGs (upregulated
and downregulated) and co-expression as well as interaction RNA networks in BTBR/
R mice brains. In addition, the BTBR/R mice data were comprehensively compared tothose reported in the previous studies of subjects with ASD as well as ASD animal models,
including another BTBR subline T+ Iptr3tf/J (BTBR/J) mice. Our results allow us to propose
potentially important genes and ncRNAs that may be associated with brain function andbehaviors characteristic to BTBR/R mice that are indicative of an autistic-like phenotype.
Taken together, the results of our study suggest the genetic aspects of BTBR/R micebrain function, which is informative to the understanding of the genetics of the observed
behavioral defects. To contribute further to the understanding of ASD genetics and biology,
further studies regarding detailed cellular expression patterns as well as functional aspects

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for the Promotion of Scienceof the DEGs in BTBR/R mice brain are required, considering the differences and/or
similarities with socially impaired BTBR/J mice and highly social B6 mice.
*Remarks: We also studied mouse models for oxytocin release and associated social
behavior as well as brain-derived neurotrophic factor (BDNF) release.

References
Mizuno S, Hirota JN, Ishii C, Iwasaki H, Sano Y, and Furuichi T. Comprehensive
Profiling of Gene Expression in the Cerebral Cortex and Striatum of BTBRTF/ArtRbrc
Mice Compared to C57BL/6J Mice. Front Cell Neurosci. 2020, 14:595607. doi: 10.3389/
fncel.2020.595607.
Kojima M, Ishii C, Sano Y, Mizui T, and Furuichi T. Journey of brain-derived neurotrophic
factor: from intracellular trafficking to secretion. Cell Tissue Res. 2020, 382(1):125-134. doi:
10.1007/s00441-020-03274-x.

一般の皆様へ自閉スペクトラム症 （ASD） は、 社会的コミュニケーションの欠損と常同行動 ・ 興味の限局を特徴とし世界各国で高い有病率を示します。 原因が不明で完治療法がないため、 病因究明や治療法の確立が課題となっています。 遺伝要因が強い多因子疾患と考えられていますが、 ヒト脳での遺伝子解析には限界があります。 本研究では、 高社会性を示す B6 マウスと比較して、
BTBR/R マウスの社会性が低下していること、 両マウスの脳内の遺伝子発現 （トランスクリプトーム） の比較解析とバイオインフォマティクス解析により ASD の遺伝因に示唆的な情報を示すことができました。 今後、 BTBR/R マウスが ASD 関連の研究開発に寄与する有用な動物モデルとなると期待されます。

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Development of H2S-Activatable Near-Infrared-dye Probes:
Bioimaging and Therapy
Yusuke Ishigaki
yishigaki@sci.hokudai.ac.jp
Hokkaido University

Summary Abstract
Zigzag bisquinodimethanes (BQDs) with eight aryl groups were designed as redoxswitchable molecules that undergo four-electron oxidation to produce doubly twisted
tetracationic pentacenes. In contrast to one-stage four-electron oxidation of BQDs, stepwise
two-electron reduction of tetracationic pentacenes occurs to give the original BQDs via
dicationic anthracenes. Since both tetracations and dications exhibit near-infrared (NIR)
absorptions (-1400 nm) based on an intramolecular charge-transfer interaction, changes
in not only their structures but also their UV-Vis-NIR spectra can be controlled by redox
stimuli. Such NIR absorbing cationic dyes are potential candidates for H2S-activatableprobes, which will be clarified in due course.
Key Words ： Near-Infrared Absorption, Organic Redox Systems, Cationic Dyes

Introduction
Recently, much attention has been focused on developing electrochromic material(EM), by which the electric potential as an input is transduced into the color change as an
output. With regard to the use in biological systems, NIR absorption properties are of great
importance for bioimaging and phototherapy.[1–3] However, only limited numbers of such NIR
EM have been reported so far. In this study, we designed and synthesized novel NIR EM,
which can be applicable for H2S-activatable probes.

Results
In this study, we proposed a new approach for preparing extended [n]acenes byelectrochemical oxidation, and demonstrated unprecedented hysteretic three-state redox
interconversion among neutral bisquinodimethanes (BQDs), tetracationic pentacenes, anddicationic anthracenes, in which both cations exhibit significantly red-shifted absorptions in
the near-infrared (NIR) region based on an intramolecular charge-transfer (CT) interaction.
The target BQs were prepared from 5,7,12,14-pentacenetetrone over 4 steps, where
stepwise introduction of aryl groups was conducted by Suzuki-Miyaura cross-coupling
reaction following dibromoolefination. According to X-ray analyses, these BQDs with three
non-fused benzene rings adopt a zigzag conformation. By treatment with four equivalents of(4-BrC6H4)3N+•SbCl6– (Magic Blue), tetracationic (SbCl6–)4 salts were obtained quantitatively.
X-ray analyses revealed that the tetracations have a fully conjugated planar pentacenecore, which is almost orthogonal to the diarylmethylium moieties. The original BQDs werecompletely recovered when tetracations were treated with an excess amount of Zn powder,

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which demonstrates that reversible redox interconversion between neutral donors andtetracations can proceed.
To investigate the redox properties in detail, we conducted cyclic voltammetry in CH2Cl2.
For methoxyphenyl derivative, the voltammogram showed a one-wave four-electronoxidation peak at +1.12 V (vs. SCE). The one-stage four-electron process for oxidation
was verified by using ferrocene as an external standard and confirmed by differential pulse
voltammetry. As a result of the change in structures, return peaks, where two-stage twoelectron reduction occurs via intermediate dication, appeared in the far cathodic region (Ered/
V: +0.55 for tetracation and +0.27 for dication). Such a cathodic peak shift is a characteristicfeature of dynamic redox (dyrex) systems,[4,5] in which the steady-state concentration ofintermediate radical species is negligible. Similar redox behavior was observed for tertbutylphenyl derivative (Eox/V: +1.26 for neutral donor; Ered/V: +0.89 for tetracation and +0.76
for dication).
To obtain further information regarding the structure of the intermediary dications as
expected by the voltammetric analyses of BQDs, we performed a titration experiment on thechemical reduction of tetracations, which was monitored by UV-Vis-NIR spectroscopy. Upon
the addition of aliquots of Bu4NI (TBAI) in CH2Cl2, the UV-Vis-NIR spectrum of tetracation
with tert-butylphenyl groups changed to that of neutral species via the intermediate dication
in a stepwise fashion, where several isosbestic points were observed in both stages.
Especially in the first stage, the absorption maxima at 498 and 1094 nm for tetracationin the visible and NIR region shifted to 509 and 1005 nm for dication (Figure 1). Similar
results were obtained by using methoxyphenyl derivative with decamethylferrocene. The
NIR absorption of dications suggested that localized dication with an anthracene skeleton
fused with an anthraquinodimethane (AQD) unit stabilized by intramolecular CT interaction
would be likely, and this idea is supported by the fact that the DFT method gave no energyminimized structure for the quinoidal form.

Figure 1. Change in UV-Vis-NIR spectrum of
tetracationic (SbCl 6–) 4 salt (6.00 µM) upon the
addition of several aliquots of TBAI in CH2Cl2.

After many trials, dications could be isolated in pure form. Upon two-electron reductionof tetracationic (SbCl 6–) 4 salts in CH 3CN with two equivalents of reducing reagent,dicationic (SbCl6–)2 salts were isolated quantitatively. These structures of dications are

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for the Promotion of Sciencebis(diarylmethylium)-substituted anthracene fused with an AQD unit, as characterized by
1
H NMR spectra and finally determined by single-crystal X-ray analyses. Additionally, when
dications were treated with two equivalents of Magic Blue and with an excess amount of
Zn powder, tetracations and original BQDs were isolated quantitatively for both reactions.
Therefore, unprecedented hysteretic three-state redox interconversion among tetracationicpentacenes, dicationic anthracenes, and neutral BQDs, as shown by cyclic voltammetry,
was demonstrated on a preparative scale (Scheme 1).[6]Scheme 1

Discussion & Conclusion
In conclusion, we designed and synthesized zigzag-structured BQD derivatives with
non-fused benzene rings, which undergo one-stage four-electron oxidation to produce
tetracationic pentacene derivatives with a doubly twisted conformation. When tetracationicpentacenes were reduced, two-stage two-electron reduction occurs via dicationicanthracenes to the original BQDs. Such hysteretic three-state redox interconversion among
them demonstrates perfect control of the number of fused benzene rings ([1] -> [5] -> [3]-> [1]) in the [n]acene structure by redox conversion. It is noteworthy that cationic species
can be isolated even for pure hydrocarbons. Furthermore, since these cations exhibit
significantly red-shifted NIR absorptions (-1400 nm) based on intramolecular CT interaction,changes in structure as well as UV-Vis-NIR absorptions can be controlled by redox
switching. These results provide a new tactic for creating [n]acene-based redox switcheswith fully controlled multiple-electron transfer, and for dynamically interconverting theirstructures and properties in a unimolecular fashion. Such NIR absorbing cationic dyes arepotential candidates for H2S-activatable probes, which will be clarified in due course.

References[1] L. Wu, Y. Sun, K. Sugimoto, Z. Luo, Y. Ishigaki, K. Pu, T. Suzuki, H. Chen, D. Ye, J. Am.
Chem. Soc. 2018, 140, 16340–16352.
[2] L. Wu, Y. Ishigaki, Y. Hu, K. Sugimoto, W. Zeng, T. Harimoto, Y. Sun, J. He, T. Suzuki, X.
Jiang, et al., Nat. Commun. 2020, 11, 446.
[3] Y. Ishigaki, T. Harimoto, K. Sugimoto, L. Wu, W. Zeng, D. Ye, T. Suzuki, Chem. – An
Asian J. 2020, 15, 1147–1155.

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for the Promotion of Science[4] T. Suzuki, Y. Ishigaki, M. Takata, J. Nishida, T. Fukushima, Heterocycles 2021, 102,
419–450.
[5] Y. Ishigaki, Y. Uchimura, T. Shimajiri, T. Suzuki, Bull. Chem. Soc. Jpn. 2021, 94, 1385–
1393.
[6] Y. Ishigaki, T. Harimoto, K. Sugawara, T. Suzuki, J. Am. Chem. Soc. 2021, 143, 3306–
3311.

一般の皆様へ
電気化学的な刺激によって色調が変化するエレクトロクロミック材料 （EM） は様々な応用の観点で注目を集めています。 目に見える波長 （約 400-780nm） よりもエネルギーの弱い近赤外光（約 780-2500nm） を吸収する EM は、 バイオイメージングなどへ応用できますが、 重金属が入っていない有機分子で作ることは容易でないため、 報告例が限られていました。 そこで本研究では、 近赤外光を吸収する新たな有機 EM を設計 ・ 合成しました。 電気化学的な刺激によって生じる近赤外色素では、 1400nm に及ぶ光を吸収するため、 バイオイメージングと医療への応用が期待されます。

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Identification of molecular mechanism underlying drug
resistance by tumor-stroma derived exosome
Takatsugu Ishimototaka1516@kumamoto-u.ac.jp
The International Research Center for Medical Sciences (IRCMS), Kumamoto
University

Summary Abstract
This study elucidates a novel molecular mechanism through which Annexin A6 in CAFEV activates FAK-YAP by stabilizing b1 integrin at the cell surface of gastric cancer cells
and subsequently induces drug resistance.
Key Words ： tumor microenvironment, cancer-associated fibroblasts (CAFs), gastric cancer

Introduction
Cancer-associated fibroblasts (CAF) are found among stromal cells in solid tumors andplay a central role in cancer progression, and the molecular mechanisms of CAFs havebeen extensively investigated. Extracellular vesicles (EV) from CAFs are composed ofdiverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer
cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated.

Results
To quantify the amount of Cancer associated fibroblasts (CAFs) in gastric cancer (GC)tissues, resected tissues from 335 GC patients were subjected do immunohistochemical
staining using an antibody specific for alpha-smooth muscle actin. CAF-high GC patients
exhibited significantly shorter overall survival. Moreover, stage IV GC patients with CAFhigh tumors who received chemotherapy had remarkably shorter overall survival. Based onthese results, we first hypothesized that CAFs in the tumor stroma are involved in mediating
the drug resistance of GC cells.
At first, we cultured GC cells with
CAF-conditioned medium (CM) in
extracellular matrix (ECM) exhibitedcharacteristic network formation andremarkable drug resistance. Next, we
conducted RNA sequencing to identify
unique molecular patterns in GC cells
in the ECM after CAF-CM treatment.
Based on the results from theseanalyses, we presumed that CAF
derived extracellular vesicles (EVs)might be involved in network formation

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for the Promotion of Scienceand drug resistance. Thus, we extracted CAF-EVs by ultracentrifugation, and showed thatEVs contained in CAF-CM play a central role in network formation and drug resistance inGC cells.
We conducted a comprehensive proteomic analysis using GC-EVs or CAF-EVs and focused on annexinA6 most specifically expressed in CAF-EVs. AnnexinA6-depleted CAFEVs diminished both the network formation and drug resistance of GC cells in the ECM(Figure 1).
According to the results from the network analysis of the RNA sequencing data, we
focused on β1 integrin and showed that the CAF-EV-induced increase in cell surface β1
integrin expression in GC cells is critical for network formation and drug resistance in the
ECM.
Annexin A6 and β1 integrin have been found to be important in GC cell network formationin the ECM; thus, we predicted that annexin A6 and β1 integrin interact with one another.
We conducted such as protein stability assay, flow cytometry and immunofluorescenceanalyses and revealed that β1 integrin expression at the surface of GC cells in the ECM isenhanced by annexin A6 in CAF-EVs.
Because signaling pathways
downstream of integrin are regulated byprotein-protein interactions, we further
performed a comprehensive proteomic
analysis of GC cells treated with andwithout CAF-CM as well as RNA
sequencing. Differentially expressed
genes related to focal adhesion and
the PI3K-AKT and Hippo signaling
pathways were identified in GC cells
treated with CAF-CM according to apathway analysis of the comprehensive
transcriptomics and proteomics data.
Further functional analyses indicatet h a t i n t e g r i n - FA K s i g n a l i n g a n dsubsequent YAP activation play a
critical role in network formation in the ECM and drug resistance of GC cells treated with
CAF-EVs.
To verify the significance of CAF-EVs in GC drug resistance in an in vivo model, GC
cells were injected into the peritoneal cavities of nude mice. This model elucidated thatannexinA6 in CAF-EVs play a role in the drug resistance of GC cells in vivo as well as
in vitro. We then examined whether FAK and YAP inhibitors could attenuate the cisplatin
resistance of GC cells mediated by CAF-EVs. The antitumor effect of cisplatin wasrecovered by additional treatment with the FAK and YAP inhibitors (Figure 2).

Discussion & Conclusion
Annexin A6 in EVs derived from CAFs stabilizes b1 integrin at the plasma membrane ofgastric cancer cells in the ECM. Subsequently, phosphorylated FAK leads to AKT activation

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for the Promotion of Scienceand YAP accumulation, which results in the network formation of gastric cancer cells in the
ECM and their substantial drug resistance. Moreover, blockade by an FAK or a YAP inhibitor
but not an AKT inhibitor effectively inhibited the drug resistance of gastric cancer cells. The
findings of this study provide evidence demonstrating that FAKYAP signaling activated byAnnexin A6 in CAF-EVs from the tumor stroma is a novel target for combination therapy
with currently available anticancer drugs.

References
1, Bu L, Baba H, Yoshida N, Miyake K, Yasuda T, Uchihara T, Tan P, Ishimoto T*.
Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor
microenvironment. Oncogene. 38(25):4887-4901. 20192, Bu L, Baba H, Yasuda T, Uchihara T, Ishimoto T*. Functional diversity of cancerassociated fibroblasts in modulating drug resistance. Cancer Sci. 111(10):3468-3477.
2020
3, Uchihara T, Miyake K, Yonemura A, Komohara Y, Itoyama R, Koiwa M, Yasuda T, Arima
K, Harada K, Eto K, Hayashi H, Iwatsuki M, Iwagami S, Baba Y, Yoshida N, Yashiro M,
Masuda M, Ajani J, Tan P, Baba H, Ishimoto T*. Extracellular Vesicles from CancerAssociated Fibroblasts Containing Annexin A6 Induces FAK-YAP Activation by Stabilizing
β1 Integrin, Enhancing Drug Resistance. Cancer Res. 15;80(16):3222-3235. 2020

一般の皆様へ
胃がんに対して抗がん剤治療が効きにくくなる 「治療抵抗性」 に関わる因子として、 CAFs が分泌する細胞外小胞 (EVs) 中のアネキシン A6 が胃がん細胞に取り込まれることで、 抗がん剤の効果が低下することが分かりました。 アネキシン A6 タンパク質や EVs を分泌する CAFs をターゲットにした新たな癌治療法の可能性を見出しました。

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Stabilization mechanism of amyloid aggregates revealed
by protein engineering techniques
Koki Makabe
makabe@yz.yamagata-u.ac.jp
Yamagata university

Summary Abstract
Amyloid is a proteinous aggregate which involves several neurodegenerative diseases
such as Alzheimer’s. Thus, the basic understanding of amyloid biophysical properties is important for understanding such diseases. Although important, insoluble and heterogeneous
nature of amyloid humpers its characterization. Here, we report the structural analysis ofamyloid edge using a model protein system.
Key Words ： Amyloid, beta-sheet, model protein, protein engineering

Introduction
Amyloid is a proteinous aggregate which involves several neurodegenerative diseases
such as Alzheimer’s. Thus, the basic understanding of amyloid biophysical properties
is important for understanding such diseases. Although important, insoluble andheterogeneous nature of amyloid humpers its characterization. To overcome this limitation,
we have developed a protein model system to capture the amyloid core structure into a
globular protein, termed peptide self-assembly mimic (PSAM). Here, we evaluated thestructural property of the elongation edge of beta-sheet which mimics the edge of amyloid.

Results
To mimic the elongation edge of amyloid, we constructed a PSAM variant whose
C-terminal beta-sheet edge was exposed to the solvent. To do this, the C-terminal domainof the original PSAM was deleted by gene engineering method. By deleting the C-terminal
domain, the C-terminal beta-strand of the central single layer beta-sheet was no longer
caped by the C-terminal domain. We termed this variant as PSAM-edge. PSAM-edge wasexpressed in E.coli and purified by His tag affinity chromatography.
We confirmed that PSAM-edge is monomeric in a solution buffer by size exclusion
chromatography (SEC). Because beta-sheet edge of PSAM-edge is exposed to solvent, it
is surprising that PSAM-edge does not form any aggregate.
Stability of the PSAM-edge was evaluated by urea-induced chemical denaturation
method by monitoring the CD values. The resulting titration denaturation curve of PSAMedge showed clear sigmoidal transition with a single transition. We fitted this denaturation
curve with a two-state unfolding model and the experimental data well fitted with thetwo-state model suggesting that the beta-sheet region co-operatively unfolded with the
N-terminal domain.
SEC and chemical denaturation experiments clearly demonstrate that PSAM-edge
behaves as a general globular protein. It means the elusive beta-sheet edge properties can

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for the Promotion of Sciencebe observed by using this PSAM variants. Thus, we next tried to determine the molecular
structure of PSAM-edge by x-ray crystal structure analysis. We successfully obtained single
crystals for x-ray diffraction experiments. One of the crystals diffracts to the resolution of
2.2 angstrom. The phase of the diffraction data was determined by molecular replacement
method using a wildtype OspA structure without the C-terminal domain, which was theoriginal scaffold for PSAM construction. There were four molecules in the asymmetric unit
and these four molecules were almost identical with a root mean square deviation (RMSD)
values below 1.3 angstrom. Thus, we analyzed the one of the four structures (denoted as
molA) for further analysis. The electron density map of PSAM-edge is clear to the final betastrand. Only the C-terminal four residues are disordered. This observation indicated thateven the absence of the C-terminal capping domain, the central beta-sheet is stably formed
except for the terminal four residues. We initially expected that terminal few beta-strand
must be disordered because of the flexible nature of the terminal structure. The crystal
structure unexpectedly demonstrated that the initial our expectation was wrong. Thus, in
actual amyloid, the elongation edge of the fibril must be stable to attach and incorporate
the next molecule. At the same time, if the terminal structure is stable, it is also a dangerof intermolecular aggregation. From the PSAM-edge structure, the flexible nature of theterminal four residues may play a key role to prevent the aggregation. Fluctuating terminal
portion may exclude the attaching molecule and preventing the association.
To observe the dynamic movement of PSAM-edge, we performed the MD simulation.
The MD simulation period was 50 ns and we evaluated the molecular fluctuation of PSAMedge. In MD trajectory, we found that the central beta-sheet is stably formed same as the
crystal structure. During the simulation, only the terminal four residues fluctuated which isalso same with the crystal structure observation where these residues are disordered. The
striking consistency between the crystal structure and MD simulation suggests the exposed
beta-sheet stability is general.

Discussion & Conclusion
We successfully modeled beta-sheet edge by deleting the capping C-terminal domainfrom original PSAM. In general, natural proteins prevent the exposure of the beta-sheetedge because of its high tendency for the aggregation. In this viewpoint, it is interesting to
see the structure of the exposed beta-sheet edge of PSAM-edge. Although PSAM-edge
exposes the beta-sheet edge, it is highly soluble and monodisperse. The aggregation
resistant property of PSAM-edge could be attributable to the fluctuation of the C-terminal
residues observed in the crystal structures and MD simulation. The fluctuation should
prevent edge-to-edge interaction. In the future study, it is interesting to compare the edge
property of actual amyloids and PSAM edge. In addition to that, the knowledge about theedge property of PSAM-edge will expand the designability of beta-sheet rich protein inprotein design field.

References
1. Shiga S. and Makabe K.
Structural analysis of the β-sheet edge of peptide self-assembly using a model protein.
Proteins, in press, 2021 doi: 10.1002/prot.26063.

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一般の皆様へ
この研究ではアルツハイマー病などの病変部位で見つかる、 蛋白質凝集体の性質を明らかにするために、 病変蛋白質そのものではなくて、 別の扱いやすい蛋白質に凝集体の構造を移植して評価を行いました。 特に、 βシートと呼ばれる蛋白質の分子構造が集まって、 病気の凝集体になるところをモデル系によって、 詳細な構造で評価しました。 詳細な構造から、 そのようなβシートは単独でも安定に構造ができていることがわかりました。 これは病気を引き起こす凝集が非常に安定なことと関連があるかもしれません。

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Identification of the lipid species which regulate epidermis
differentiation in Arabidopsis.
Mitsutomo Abe
mabe@bio.c.u-tokyo.ac.jp
Department of Life Sciences, Graduate School of Arts and Sciences,
The University of Tokyo

Summary Abstract
The differentiation of distinct cell types in appropriate patterns is a fundamental process
in the development of multicellular organisms. The protodermal cell is an ideal system forstudying position– and lineage–dependent cell differentiation in plants. In this study, I foundthat very-long-chain fatty acid-containing ceramides which are predominantly synthesizedin the outermost cells, bind to a lipid-binding domain of ATML1 and stabilize the ATML1 protein only in the outermost cells.
Key Words ： Arabidopsis thaliana, epidermis, protoderm, ATML1, VLCFA-ceramide

Introduction
Sessile lifestyle imposes a bewildering array of biotic and abiotic stresses on vascular
land plants. To defend against a hostile environment on land, plants have a specialized
defensive cell layer called the epidermis at the interface between the plant body andthe external environment. In Arabidopsis, ATML1 plays an essential role in protoderm
differentiation and effectively orchestrate a variety of protoderm-specific events (Abe et al.,2001, 2003). However, little is known about the molecular basis of the positional signal that
confines the expression of this key regulator to the outermost cell layer.

Results
Forming the crucial interface between the plant and the outside world, the outermost
cell layer derives from a meristem called the protoderm. While protoderm differentiation
is known to be driven by the homeodomain-containing transcription factor ARABIDOPSISTHALIANA MERISTEM LAYER 1 (ATML1), the positional signals that restrict protodermal
differentiation to the outermost position are still poorly understood. I and my colleagues
first found that ATML1 expressed in the outermost cells of the lateral root primordia andis required for lateral root formation. Interestingly, when ATML1-EGFP is expressedubiquitously, it is only retained in the nuclei of outermost layer of lateral root but not in thoseof the inner cells. These results indicate the existence of an unidentified signal that relays
the information of cell position and is required to maintain ATML1 expression.
ATML1 protein is characterized by a steroidogenic acute regulatory protein (StAR)related lipid-transfer (START) domain. In order to identify the amino acid residue crucial forthe lipid binding, I compared the amino acid sequence of the START domains of ATML1
with the known ligand contact site of mammalian START domains. Based on the sequencealignments, Tryptophan 471 (W471) residue of ATML1 protein exhibits strong similarity with

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The Novartis Foundation(Japan)
for the Promotion of Sciencethe ligand contact site of mammalian START domains. Thus, we focused on W471 as aputative lipid contact site in ATML1.
To analyze the function of W471 in the position-dependent modulation of ATML1, I
introduced a W471L point mutation into ATML1 based on the previous study by usingsite-directed mutagenesis methods and generated transgenic Arabidopsis ubiquitously
expressing ATML1W471L-EGFP. Interestingly, when I induced ATML1W471L-EGFP in all cells ofroot tissue, EGFP fluorescence was not observed even in the nuclei of the outermost cells.
These observations indicate that W471 is crucial for the stability of ATML1 protein in theoutermost cells.
In order to screen for potential lipid species that interact with the ATML1 START domain,
I next performed in vitro protein–lipid binding assays and found that the recombinant GSTtagged START domain of ATML1 binds to very-long-chain fatty acid (VLCFA)-containing
ceramides (VLCFA-Cers), ceramide-1-phosphates, phosphatidic acids, phosphatidylserines,
and several phosphatidylinositol phosphates. Interestingly, further evaluation of theeffect of the W471L mutation revealed that the W471L mutation specifically attenuates
the interaction between GST-START and VLCFA-Cers. Thus, VLCFA-Cers emerge as a
candidate lipid mediating the positional information and affecting ATML1 function.
To determine whether the post-translational regulation of ATML1 is affected by VLCFACers, I treated transgenic Arabidopsis seedlings, expressing ATML1 WT–EGFP, with an
inhibitor of VLCFA biosynthesis (cafenstrole). The cafenstrole treatment resulted indisappearance of the fluorescence of ATML1WT-EGFP in outermost cells, confirming thatthe stability of ATML1 protein is indeed affected by VLCFA-Cers. We also analyzed theexpression pattern of PASTICCINO2 (PAS2), encoding an essential enzyme for VLCFA
biosynthesis. In gPAS2-EGFP containing Arabidopsis, the fluorescence of PAS2–EGFP wasrestricted to the outermost cells. Collectively, these findings indicate that VLCFA-Cers in theoutermost cells play a essential role in the position-dependent stability of ATML1 protein.

Discussion & Conclusion
Here, I have provided an initial characterization of VLCFA-Cers-mediated positionalsignaling in Arabidopsis. I also found that stabilization of the ATML1 protein is dependent
on the interaction with VLCFA-Cers and that the interaction between ATML1 and VLCFACers is crucial for the function of ATML1 in the outermost cells. Therefore, VLCFA-Cers are
considered as an essential component of cell position-dependent signaling in Arabidopsis.
The START domain is also conserved in another class of homeodomain proteins, which
are key transcription factors in vascular development and leaf polarity. Given that theassociation between the START domain and the homeodomain is confined to the plant
kingdom, such a plant-specific lipid-protein complex that mediates positional signaling might
represent an inevitable evolutionary strategy for sessile plants.

References
Abe, M., Takahashi, T. and Komeda, Y. (2001). Identification of a cis-regulatory elementfor L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain
protein. Plant J. 26, 487-494.

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Abe, M., Katsumata, H., Komeda, Y. and Takahashi, T. (2003). Regulationof shoot epidermal cell differentiation by a pair of homeodomain proteins in
Arabidopsis. Development 130, 635-643.
Nagata, K., Ishikawa, T., Kawai-Yamada, M., Takahashi, T. and Abe, M. (2021).
Ceramides mediate positional memory in Arabidopsis thaliana protoderm differentiation.
Development 148, dev194969.

一般の皆様へ陸上植物は、 乾燥をはじめとする環境ストレスや病害を引き起こす有害な生物に囲まれて生きています。 そこで、 植物は特殊化した細胞 （表皮細胞） を外部環境との境界に発達させ、 自らを保護しているのです。 本研究によって、 外部環境との境界、 すなわち 「位置」 を認識し、 正しい場所に表皮細胞を分化させる際に、 特殊な脂質が重要なはたらきをしていることがわかりました。 今後、 地球環境の劇的な変化が予想されることから、 過酷な環境でも丈夫に生育可能な植物の作出につながると期待しています。

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Publication - the 33rd (FY2019) Grant Recipients
Title of the research
project

Evolution of membrane morphogenesis in Asgard archaea

Recipient (Institution)

Yosuke Senju （Research Institute for Interdisciplinary Science (RIIS),Okayama
University）

Journal article / othermaterial

Springer Science+Business Media, LLC, part of Springer Nature 2021
doi.org/10.1007/978-1-0716-1142-5_14

Title of the paper

Liposome Co-sedimentation and Co-flotation Assays to Study Lipid–Protein
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Title of the research
project

Functional mechanisms and dynamics of oxytocin in autism spectrum disorder

Recipient (Institution)

Teruhiro Okuyama （The University of Tokyo,Institute for Quantitative
Biosciences (IQB),Associate professor）

Journal article / othermaterial

bioRxiv June 25, 2021
doi.org/10.1101/2021.06.25.449869

Title of the paper

Disrupted social memory ensembles in the ventral hippocampus underlie social
amnesia in autism-associated Shank3 mutant mice

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doi.org/10.1016/j.conb.2020.12.008

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Journal article / othermaterial

Development, Growth & Differentiation, December 2020
doi: 10.1111/dgd.12697

Title of the paper

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Title of the research
project

Cell senescence effects on transcriptional regulation and differentiation in beige
preadipocytes

Recipient (Institution)

Kenji Ikeda （Department of Molecular Endocrinology and Metabolism,Tokyo
Medical and Dental University）

Journal article / othermaterial

Front Endocrinol (Lausanne). 2020 Jul 28; 11: 498
doi: 10.3389/fendo.2020.00498

Title of the paper

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Adipocytes

Title of the research
project

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

Recipient (Institution)

Takashi Ebihara （Department of Medical Biology,Akita University Graduate
School of Medicine）

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Journal article / othermaterial

Cells 2020, 9, 1193;
doi:10.3390/cells9051193

Title of the paper

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doi.org/10.1016/j.alit.2020.11.007

Title of the paper

Trained innate lymphoid cells in allergic diseases

Title of the research
project

In vivo Imaging of Brain in CNS Lupus Defines Unique Roles for
Chemoattractants

Recipient (Institution)

Yoshishige Miyabe （Institute for Advanced Medical Sciences,Nippon Medical
School）

Journal article / othermaterial

JMA J. 2020;3(3):doi: 10.31662/jmaj.2020-0019

Title of the paper

Targeting the Chemokine System in Rheumatoid Arthritis and Vasculitis

Title of the research
project

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

Recipient (Institution)

Wataru Ariyoshi （Division of Infections and Molecular Biology,Kyushu Dental
University）

Journal article / othermaterial

Molecules 2021, 26, 1982
doi.org/10.3390/molecules26071982

Title of the paper

Biological Effects of β-Glucans on Osteoclastogenesis

Title of the research
project

Elucidation of the promotion mechanism in plant shoot regeneration byenvironmental stimuli

Recipient (Institution)

Sachihiro Matsunaga （Matsunaga Laboratory, Department of Applied Biological
Science, Faculty of Science and Technology,Tokyo University of Science）

Journal article / othermaterial

NATURE COMMUNICATIONS (2020) 11:5914
doi.org/10.1038/s41467-020-19621-z

Title of the paper

Subnuclear gene positioning through lamina association affects copper
tolerance

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Communications biology 14, May 2021
doi.org/10.1038/s42003-021-02106-0

Title of the paper

A live imaging system to analyze spatiotemporal dynamics of RNA polymerase
II modification in Arabidopsis thaliana

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Title of the research
project

Molecular and neural mechanism associated with social behavior

Recipient (Institution)

Teiichi Furuichi （Department of Biological Science, Faculty of Science andTechnology,Tokyo University of science）

Journal article / othermaterial

Front. Cell. Neurosci. 2020, 14: 595607.
doi: 10.3389/fncel.2020.595607.

Title of the paper

Comprehensive Profiling of Gene Expression in the Cerebral Cortex andStriatum of BTBRTF/ArtRbrc Mice Compared to C57BL/6J Mice.

Journal article / othermaterial

Cell Tissue Res. 2020, 382(1):125-134.
doi: 10.1007/s00441-020-03274-x.

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Journey of brain-derived neurotrophic factor: from intracellular trafficking tosecretion

Journal article / othermaterial

The Journal of Neuroscience, May 19, 2021
doi.org/10.1523/JNEUROSCI.3240-20.2021

Title of the paper

CAPS2 Deficiency Impairs the Release of the Social Peptide Oxytocin, as Wellas Oxytocin-Associated Social Behavior

Title of the research
project

Identification of molecular mechanism underlying drug resistance by tumorstroma derived exosome

Recipient (Institution)

Takatsugu Ishimoto （Division of Translational Research and Advanced
Treatment Against Gastrointestinal Cancer,Kumamoto University Hospital）

Journal article / othermaterial

Cancer Res. 2020; 80(16): 3222-3235
doi: 10.1158/0008-5472.CAN-19-3803

Title of the paper

Extracellular Vesicles from Cancer-Associated Fibroblasts Containing Annexin
A6 Induces FAK-YAP Activation by Stabilizing b1 Integrin, Enhancing Drug
ResistanceTitle of the research
project

The ultrastabilization mechanism of amyloid revealed by a protein engineering
technique

Recipient (Institution)

Koki Makabe （School of science and engineering, Makabe lab.,Yamagata
university）

Journal article / othermaterial

2021 Wiley Periodicals LLC, 8 February 2021
doi: 10.1002/prot.26063

Title of the paper

Structural analysis of the β-sheet edge of peptide self-assembly using a model
proteinTitle of the research
project

Identification of the lipid species which regulate epidermis differentiation in
Arabidopsis

Recipient (Institution)

Mitsutomo Abe （Graduate School of Arts and Sciences,The University ofTokyo）

Journal article / othermaterial

The Company of Biologists, Development (2021) 148, dev194969.
doi:10.1242ldev.194969

Title of the paper

Ceramides mediate positional signals in Arabidopsis thaliana protoderm
differentiation

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The Novartis Foundation(Japan)
for the Promotion of Science＜論文掲載＞第 33 回 （2019 年度） 受賞者
助成タイトル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

受賞者

千住 洋介 （岡山大学異分野基礎科学研究所）

論文掲載誌 ・ 書誌事項

Springer Science+Business Media, LLC, part of Springer Nature 2021
doi.org/10.1007/978-1-0716-1142-5_14

論文タイトル

Liposome Co-sedimentation and Co-flotation Assays to Study Lipid–Protein
Interactions

助成タイトル

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

受賞者

奥山 輝大 （東京大学定量生命科学研究所）

論文掲載誌 ・ 書誌事項

bioRxiv June 25, 2021
doi.org/10.1101/2021.06.25.449869

論文タイトル

Disrupted social memory ensembles in the ventral hippocampus underliesocial amnesia in autism-associated Shank3 mutant mice

論文掲載誌 ・ 書誌事項

Current Opinion in Neurobiolog, June 2021
doi.org/10.1016/j.conb.2020.12.008

論文タイトルDistinct functions of ventral CA1 and dorsal CA2 in social memory

論文掲載誌 ・ 書誌事項

Development, Growth & Differentiation, December 2020
doi: 10.1111/dgd.12697

論文タイトルDiverse sensory cues for individual recognition

助成タイトル

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

受賞者

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）

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Front Endocrinol (Lausanne). 2020 Jul 28; 11: 498
doi: 10.3389/fendo.2020.00498

論文タイトルUCP1 Dependent and Independent thermogenesis in Brown and Beige
Adipocytes

助成タイトル

メモリー様 2 型自然リンパ球の分化制御機構の解析

受賞者

海老原 敬 （秋田大学大学院医学系研究科微生物学講座）

論文掲載誌 ・ 書誌事項

Cells 2020, 9, 1193;
doi:10.3390/cells9051193

論文タイトル

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for the Promotion of Science

論文掲載誌 ・ 書誌事項

Allergology International 70 (2) 2021, 174-180
doi.org/10.1016/j.alit.2020.11.007

論文タイトル

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助成タイトル

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

受賞者

宮部 斉重 （日本医科大学先端医学研究所細胞生物学部門）

論文掲載誌 ・ 書誌事項

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論文タイトル

Targeting the Chemokine System in Rheumatoid Arthritis and Vasculitis

助成タイトル

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

受賞者

有吉 渉 （九州歯科大学感染分子生物学分野）

論文タイトル

Molecules 2021, 26, 1982
doi.org/10.3390/molecules26071982

論文掲載誌 ・ 書誌事項

Biological Effects of β-Glucans on Osteoclastogenesis

助成タイトル

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

受賞者

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）

論文掲載誌 ・ 書誌事項

NATURE COMMUNICATIONS (2020) 11:5914
doi.org/10.1038/s41467-020-19621-z

論文タイトル

Subnuclear gene positioning through lamina association affects copper
tolerance

論文掲載誌 ・ 書誌事項

Communications biology 14, May 2021
doi.org/10.1038/s42003-021-02106-0

論文タイトルA live imaging system to analyze spatiotemporal dynamics of RNApolymerase II modification in Arabidopsis thaliana

助成タイトル

社会性行動に関連する分子 ・ 神経機構の解明

受賞者

古市 貞一 （東京理科大学理工学部応用生物科学科）

論文掲載誌 ・ 書誌事項

Front. Cell. Neurosci. 2020, 14: 595607.
doi: 10.3389/fncel.2020.595607.

論文タイトルComprehensive Profiling of Gene Expression in the Cerebral Cortex andStriatum of BTBRTF/ArtRbrc Mice Compared to C57BL/6J Mice.

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for the Promotion of Science

論文掲載誌 ・ 書誌事項

Cell Tissue Res. 2020, 382(1):125-134.
doi: 10.1007/s00441-020-03274-x.

論文タイトル

Journey of brain-derived neurotrophic factor: from intracellular trafficking tosecretion

論文掲載誌 ・ 書誌事項

The Journal of Neuroscience, May 19, 2021
doi.org/10.1523/JNEUROSCI.3240-20.2021

論文タイトルCAPS2 Deficiency Impairs the Release of the Social Peptide Oxytocin, as Wellas Oxytocin-Associated Social Behavior

助成タイトル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

受賞者

石本 崇胤 （熊本大学病院消化器癌先端治療開発学）

論文掲載誌 ・ 書誌事項

Cancer Res. 2020; 80(16): 3222-3235
doi: 10.1158/0008-5472.CAN-19-3803

論文タイトル

Extracellular Vesicles from Cancer-Associated Fibroblasts Containing Annexin
A6 Induces FAK-YAP Activation by Stabilizing b1 Integrin, Enhancing Drug
Resistance

助成タイトル

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

受賞者

真壁 幸樹 （山形大学大学院理工学研究科）

論文掲載誌 ・ 書誌事項

2021 Wiley Periodicals LLC, 8 February 2021
doi: 10.1002/prot.26063

論文タイトルStructural analysis of the β-sheet edge of peptide self-assembly using a model
protein

助成タイトル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

受賞者

阿部 光知 （東京大学大学院総合文化研究科）

論文掲載誌 ・ 書誌事項

The Company of Biologists, Development (2021) 148, dev194969.
doi:10.1242ldev.194969

論文タイトル

Ceramides mediate positional signals in Arabidopsis thaliana protoderm
differentiation

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Ⅲ.
Reports from the Recipients of Grants
for International Meetings

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
Sept. 30, 2021
1. Name of Research Meeting / Conference
The International Centenarian Consortium: ICC 2021
2. Representative
Yasumichi Arai, Professor, Faculty of Nursing and Medical Care, Keio University/
Center for Supercentenarian Medical Research, Keio University School of Medicine
35, Shinnaomachi, Shinjuku-ku, Tokyo, Japan
P.O. 160-8582
Tel: +81 3 5269 2468
e-mail: yasumich@keio.jp
3. Opening period and Place
Web site conference: June 22, 2021 ~ July 3, 2021
On line session: June 22, 2021 ~ July 3, 2021
4. Number of participants / Number of participating countries and areas
Number of participants: 82
Number of participating countries and areas: 12(Japan, Korea, Switzerland, USA, Spain, Australia, Netherland, UK, Portugal, Italia,
Sweden, Hong Kong.)
5. Total cost
740,371Yen
6. Main use of subsidy
Outsourcing fee for web site and on-line session.
7. Result and Impression
The International Centenarian Consortium (ICC) meeting was originally scheduledin June 2020; however, it was postponed for one year due to the COVID-19 pandemic.
In this year, ICC2021 was held online from June 22 to July 3, 2021, by the Center for
Supercentenarian Medical Research at the Keio University School of Medicine and the
Osaka University Graduate School of Human Sciences.
In this website conference, about 20 researchers presented their centenarian researchas on-demand presentation with voice.
During this period, the ICC2021 Live Conference was also held on June 29 from9:00 p.m. to 12:00 a.m. Japan Standard Time, with approximately 80 researchers in thefields of longevity and centenarians participating from around the world. The numberof participants in ICC2021 was quite larger than usual, as the number of participantsin previous years' ICCs has been around 30-50. At first, Prof. Nobuyoshi Hirose (KeioUniv.) and Prof. Peter Martin (Iowa State University), the leader of the ICC, gave opening

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remarks, including the history of the ICC and the history of the online conference due tothe corona pandemic.
Next was the Biomedical Session chaired by Professor Yasumichi Arai (Keio
University), in which the three prominent researchers gave lectures. Next was thePsychosocial Session chaired by Prof. Yasuyuki Gondo (Osaka University), whichfeatured lectures by the three prominent researchers. The next session, chaired by Prof.
Yasumichi Arai (Keio Univ.), was the Presentation Award session, which was attended
by newcomers Prof. Kwangsung Park (Chonnam National University, South Korea) andstudent award winners Dr. Giulia Accardi (University of Palermo, Italy) Dr. Giulia Accardi(University of Palermo, Italy), Dr. Niccolo Tesi (Amsterdam UMC, The Netherlands), and
Dr. Rotem Arieli (Iowa State University, United (University of Palermo, Italy), and Dr.
Rotem Arieli (Iowa State University, United States) gave 10-minute presentations, whichwere followed by a Q&A session.
In the last session, Prof. Ingmar Skoog (University of Gothenburg, Sweden)
announced the next ICC meeting, which will be held on May 25-28 in Marstrand, near
Gothenburg, Sweden. The meeting ended with closing remarks by Prof. Yasuyuki
Gondo.
Although it was only three hours long, it was a dense three hours with six keynote
speakers, four award sessions, and many questions and answers. While online
conferences are a great tool for simultaneous participation and discussion from many
regions, including Europe, the Americas, Oceania, and Asia, I also felt the desire ofmany people to have face-to-face meetings after the COVID19 pandemic.
8. Additional description
ICC2021 conference site is assessable with this link;
https://activenet-tv.jp/icc2021/
We have acknowledged The NOVARTIS Foundation (Japan) for the Promotion of
Science on the website.
ICC2021 WEB conference is supported by Keio Global Research Institute (KGRI),
Osaka University Graduate School of Human Sciences Center for collaborative future
creation project aging and death study Lab (CCFC), and The NOVARTIS Foundation(Japan) for the Promotion of Science.

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Report on Research Meeting
1. Name of Research Meeting / Conference
3rd Mini-symposium on the blood-brain barrier: from basic to clinical research
2. Representative
President: Kentaro Hayashi (Shimane University)
Vice President Yoichi Morofuji (National Nagasaki Medical Center)
Advisers: Masami Niwa (PharmaCo-Cell Company Ltd.)
Yasufumi Kataoka (Fukuoka University)
Takayuki Matsuo (Nagasaki University)
William A. Banks (University of Washington)
Maria A. Deli (Biological Research Centre)
3. Opening period and Place
International lounge, Faculty of Medicine, Shimane University, Izumo, Shimane, Japan
March 26th (Fri) & 27th (Sat), 2021
4. Number of participants / Number of participating countries and areas
Total number of participants: 109
Web 50, Shimane University 22, Nagasaki Satellite 25, Fukuoka Satellite 11
Number of participating countries and areas: Japan, USA, Hungary, Slovakia, Korea,
China, Bangladesh
5. Total cost
JPY 1,400,000
6. Main use of subsidy
Printing cost and travel expenses for inviting presenters
7. Result and Impression
Mini-symposium on the blood-brain barrier: from basic to clinical research is aconference designed for both scientists and physicians with common interests withblood-brain barrier and drug development for central nervous system. It was foundedby three research groups: Nagasaki University (Japan), Biological Research Centre(Hungary), and University of Washington (USA) in 2019. Facing the COVID-19 pandemic
and its various effects, cities around the world had to take actions towards the safety
and health of their habitants. Responding to the pandemic, we made a decision that our
symposium should be held on a reduced scale and apply web conference style. 3rd Minisymposium on the blood-brain barrier: from basic to clinical research was successfully
held at International lounge, Faculty of Medicine, Shimane University, Izumo, Shimane,
Japan from March 26th to 27th, 2021 (Conference Chair: Professor Kentaro Hayashi,
Shimane University, Japan). In total 108 participants from 7 countries joined this eventon site or on line.
The symposium covered a wide range of topics in blood-brain barrier, fromThe Novartis Foundation(Japan)
for the Promotion of Science

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The Novartis Foundation(Japan)
for the Promotion of Science

fundamental researches, such as in vitro modeling, HIV infection, tryptophan kynurenine
pathway, to translational researches including SARS-CoV-2, stroke, and epilepsy. All
participants were satisfied with a friendly conference and enjoyed fruitful discussion in allpresentations.
8. Additional description
We are grateful to the NOVARTIS Foundation (Japan) for its generous financial support.

The Novartis Foundation(Japan)
for the Promotion of Science

131

The Novartis Foundation(Japan)
for the Promotion of Science

34th Grant Report (FY2020)

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 under authorization of the Ministry of Education, Science, Sports andCulture, followed by a transition to a public interest incorporated foundation on Apr. 1,
2012. The grants conducted in FY 2020 are as follows.
34th Novartis Research Grant:
Research Meeting Grant:

39 Researchers (JPY 1 mil.), Subtotal JPY 39.0mil.
7 Meetings (JPY 0.4 mil.), Subtotal JPY 3.6mil.
Total
JPY 42.6mil.

34th Novartis Research Grant (FY2020)
The Grant is to aim supporting creative research in Japan in the field of Bio, life scienceand relevant chemistry.
#

Name

Institution

Title

Research Project

1 Koji Tokoyoda

Department of Immunology,
Professor Roles of memory helper T cells in vivo
Tottori University

2 Akihide Yoshimi

Cancer RNA Research
Unit, National Cancer
Center Research InstituteSectionHead

3 Minako Ito

Division of Allergy and
Immunology, Medical
Institute of Bioregulation,
Kyushu University

Significance of acquired immune cells
Associate
during embryonic and developmental
Professor
stages in brain formation

4 Chiaki Maruyama

Developmental
Neuroscience Project,Tokyo Metropolitan Instituteof Medical Scienece

Project
leader

5 Daisuke Kurotaki

International ResearchCenter for Medical
Science,
Kumamoto University

Specially Molecular basis for early dendritic cell
Appointed lineage specification during pathogen
Associate
Professor infection

6 Naoki Takahashi

Department of Nephrology,
University of Fukui

Elucidation of the mechanismsof new tubulointerstitial nephritis
Associatewith IgM-positive plasma cells andprofessor
comprehensive analysis of IgM-typeautoantibodies in the serum

7 Ken Ohmori

Department of Chemistry,
School of Science,
Tokyo Institute ofTechnology

Synthesis and development of flavanAssociate derived polyphenol with protective
Professor effect against amyloid-beta-induced
toxicity

8 Yuki Yoshida

Faculty of Advanced
Science and Technology,
Kumamoto University

Project
Molecular genetic analysis of plant
Assistantsub-stomatal chamber development
Professor

9 Jun Ishihara

School of pharmaceuticalsciences, Pharmaceuticalorganic chemistry
department, Nagasaki
University

Professor

Targeting Spliceosomal Mutant
Leukemias by a Novel ASO Therapy

Functional analysis of subplate
layer in neocortical formation of thedeveloping mouse cortex

Highly Efficient Synthesis of Rare
Macrolides, Directing Drug Discovery

The Novartis Foundation(Japan)
for the Promotion of Science132

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

10 Kazuaki Ishihara

Graduate School ofEngineering,
Nagoya University

Catalytic Enantioselective
α-Halogenation of Acylpyrazoles
Professor
Directed towards Synthesis of Chiral
Drugs

11 Midori Arai

Chemical Biology Lab.,
Department of Bioscience& Informatics Faculty of
Science and Technology,
Keio University

Professor

12 Fuun Kawano

Graduate school of Arts
and Sciences, Laboratoryof Prof. Moritoshi Sato,The University of Tokyo

Development and evolution of an
Assistant
optogenetic gene expression system
Professor
with red-light.

13 Keiichiro Ishiguro

Institute of Molecular
Embryology and Genetics,
Kumamoto University

Associate
Research on infertility
Professor

14 Jafar SHARIF

Developmental Genetics
Laboratory, Center for
Integrative Medical
Sciences (IMS), RIKEN

Multidimensional regulation of DNA
Seniorreplication by a genome-epigenomeResearchtranscriptome (GET) network during
Scientist
cellular senescence

15 Kanae Ando

Graduate school ofscience, Department of
Biological Sciences, Tokyo
Metropolitan University

A chemical genetics approach
Associate to identify direct substrates ofprofessor Alzheimer’s disease-associatedkinase MARK4 in neurons

16 Kazunori Kanemaru

School of Medicine,
Division of Cellular andMolecular Pharmacology,
Nihon University

Establishment of in vivo Ca2+
Associateimaging method for analysis of insulin
Professor
dynamics

17 Ko Matsui

Graduate School of Life
Sciences, Super-network
Brain Physiology,
Tohoku University

Professor

18 William Addison

Genome wide analysis of DNA
Division of Molecular
Assistantmethylation and transcription
Signaling and Biochemistry,
Professor
dynamics in ICF syndrome
Kyushu Dental University

19 Kenichi Noma

Institute for Genetic
Medicine,
Hokkaido University

Elucidating the 3D genome structure
Professor and its related protein composition inhuman senescent cells

20 Kosei Takeuchi

Department of Medical
Biology,
Aichi Medical University

Elucidation of the hyper-adaptation
mechanism by reconstruction ofsynapse-formations and AI motion
Professor
capture systems - Challenging forthe hyper-amelioration from spinal
cord injury-

21 Shinobu Takizawa

The Institute of Scientificand Industrial Research,
Osaka University

Exploration of pharmaceutical
Associate resources based on rapid synthesis
Professor and machine learning analysis of
optically active compounds

22 Nobutaka Fujieda

Graduate School of Life andEnvironmental Sciences,
Associate Development of Protein Ligand Library
Biophysical Chemistry,
Professor for Stereodivergent Synthesis
Osaka Prefecture
University

23 Yuki Takamatsu

Department of Virology
I, National Institute of
Infectious Diseases

Invasive evolution of pathogenic
bacteria and cryptic gene activation

Glial control of behavioral disorders
induced by social distancing

Senior
Revealing the intra-cellular dynamics
Researcher of SARS-CoV2.

The Novartis Foundation(Japan)
for the Promotion of Science

133

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

24 Takao Iwawaki

Molecular and cell biological approach
Division of Cell Medicine,to understanding the mechanisms
Medical Research Institute,
Professor of fatigue by studying the cellular
Kanazawa Medical
stress response and the inflammatory
University
response

25 Hisayuki Amano

Department of
Sirt1-NAD+ pathway represses
Biochemistry,
Assistant fibrosis in inflammatory bowel disease
Kindai University, Faculty of Professor and colitis-associated colorectal
Medicinecancer

26 Ken Takai

Breast Oncology,
Saitama Cancer Center

RNA molecular medicine,
27 Katsutomo Okamura Nara Institute of Scienceand Technology

Targeting the host factors essential
Chief
for breast cancer metastasis in PDX
physicianmouse models
Professor

Identification of RNA-binding proteinsthat regulate miRNA processing
by integrated computational andbiochemical analysis

28 Hideaki Fujiwara

Department of Hematology
and Oncology,
Okayama University
Hospital

Development of novel GVHD
Assistant mechanism and therapy targeting
Professor metabolic disturbance of intestinal
epithelial cells that leads to dysbiosis.

29 Shinya Kimura

Development ofpharmaceutical chemistry,
Meiji Pharmaceutical
University

Development of supramolecular
Assistanthydrogel that enable drug delivery into
Professor
central nervous system

30 Aya Tanatani

Faculty of Core Research,
Natural Science Division,
Ochanomizu University

Development of potent nonProfessor secosteroidal vitamin D derivatives
based on the structure of bile acid

32 Takeshi Yamamoto

Blood Purification Center,
Osaka University Hospital

Clinical
Fellow

33 Mashito Sakai

Department of Biochemistry
Deciphering hepatic feeding-stateand Molecular Biology,
Professor dependent metabolic gene regulation
Nippon Medical School
coordinated by intercellular crosstalk

34 Takuya Imamura

Biomedical Program,
Graduate School ofIntegrated Sciences for
Life,
Hiroshima University

Systems approaches towards theProfessor non-coding RNA-based production ofhumanized mice

35 Tomoe Nishitani

Department ofPharmacology, Wakayama
Medical University

Novel mechanism of energy
Professor metabolism by Ca2+ regulatory
proteins

36 Yoshito Yamashiro

Life Science Center for
Survival Dynamics, TARA,
University of Tsukuba

The Role of fibulin-4 in endothelial
Assistantfunctions and application for clinical
Professor
study

37 Ritsuko Suyama

Graduate School of Frontier Specially Identification of the molecularAppointed mechanisms in the microbes
Biosciences, Osaka
Assistantuniversity
Professor enhanced oogenesis

38 Hiroshi Kurosaka

Department of Orthodontics
Investigating novel
and Dentofacial
Associate
treatment strategies for
Orthopedics, Osaka
Professor
Pseudohypoparathyroidism
University Graduate Schoolof Dentistry

39

Hiroshi Inoue

Metabolism and Nutrition
Research Unit, Institute for
Frontier Science Initiative,
Kanazawa University

Unraveling the pathophysiology of AKI
to CKD transition by integrating single
cell RNA sequencing and lipidomic
analyses

Investigation of hepatic metabolic
homeostasis and its deterioration
Professor by physiological and bioinformatic
analyses of vagus nerve-mediatedorgan-crosstalk

The Novartis Foundation(Japan)
for the Promotion of Science

134

The Novartis Foundation(Japan)
for the Promotion of Science

FY2020 Research Meeting Grant(JPY 400 thousand x 9 = 3.6 million)#

Meeting

Place /
Date

Institution / Title

Name

2021 Asia Pacific Drosophila
Neuroscience Conference

Saitama /2023.3

Laboratory for
Neurodiversity, RIKEN
Center for Brain Science

AdrianW Moore2

The International Centenarian
Consortium

Tokyo /
2021.6.22–
7.03

Center for
Supercentenarian Medical
Research,
Keio University School of
Medicine

Yasumichi Arai

3

International Symposium on the
Biosynthesis of Natural Products

2022

Graduate School of
Pharmaceutical Sciences,
The University of Tokyo

Ikuro Abe

1

Minisymposium on the blood4 brain barrier: from basic to clinicalresearch

Department ofNagasaki /Neurosurgery, Nagasaki
2021.3.26-27
University

Yoichi Morofuji

Division of Carcinogenesis,
Japanese Foundation for
Takuro Nakamura
Cancer Research5

The 39th Sapporo International
Cancer Symposium

Sapporo /2021.7.5-7

6

The 2nd International Symposiumon Hybrid Catalysis

Aichi /
2022.7

Institute of Transformative
Bio-Molecules,
Nagoya University

Takashi Ooi

13th AFMC International Medicinal
7 Chemistry Symposium (AIMECS
2021)

Tokyo/
2021.11.2912.2

Laboratory of Synthetic
Organic Chemistry,
Graduate School of
Pharmaceutical Sciences,
The University of Tokyo

Motomu Kanai

864th Annual Meeting of Japanese
Neurochemistry Society

Nara /
2021.9.2910.1

Department of Anatomy and
Neuroscience,
Akio Wanaka
Nara Medical University

9

39th JES Summer on
Endocrinology & Metabolism

Chiba /
2021.7.8-10

Department of MolecularDiagnosis, Graduate
School of Medicine, Chiba
University

Tomoaki Tanaka

The Novartis Foundation(Japan)
for the Promotion of Science

135

The Novartis Foundation(Japan)
for the Promotion of Science

第34期 （2020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。 2020年度は、 下記の総額4,260万円の助成事業を実施しました。
第34回ノバルティス研究奨励金
研究集会助成39件 （1件100万円）
9件 （1件 40万円）

3,900万円
360万円
総額 4,260万円

第34回ノバルティス研究奨励金 （2020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

属

職位

1

常世田好司

鳥取大学 医学部 免疫学分野

2

吉見 昭秀

国立研究開発法人国立がん研究センター 研究所
がん RNA 研究ユニット

独立
スプライシング変異白血病に対する新規 ASO
ユニット長 治療法の開発

3

伊藤美菜子

九州大学 生体防御医学研究所
アレルギー防御学分野

准教授

4

丸山 千秋

公益財団法人
東京都医学総合研究所
脳神経回路形成プロジェクト

プロジェクトリ 胎生期マウス大脳新皮質形成におけるサブプレーダー
ート層の機能解明

5

黒滝 大翼

熊本大学
国際先端医学研究機構

特任准教授

感染防御における樹状細胞系譜早期運命決定
の機序解明

6

髙橋 直生

福井大学 医学系部門
腎臓病態内科学分野

診療准教授

IgM 陽性形質細胞を伴う新腎炎のメカニズムの解明と血中に存在する IgM 型自己抗体の網羅
的解析

7

大森

東京工業大学 理学院化学系

准教授

アミロイドβ凝集抑制作用を有するフラバンポリ
フェノール類の合成と機能開拓

8

吉田 祐樹

熊本大学
大学院先端科学研究部

特任
助教

植物の気孔腔形成メカニズムの分子遺伝学的
解析

9

石原

長崎大学 薬学部
薬品製造化学研究室教授

高効率的合成に基づく稀少多環性マクロリド天然物の創薬展開10石原 一彰

名古屋大学 工学研究科
有機 ・ 高分子化学専攻
触媒有機合成学研究室教授

光学活性医薬品合成を指向したアシルピラゾールの触媒的不斉α - ハロゲン化反応の開発

11

荒井慶應義塾大学
理工学部生命情報学科
ケミカルバイオロジー研究室

教授

病原微生物の浸潤進化に学ぶ休眠遺伝子活性
化法の開発

12

河野 風雲

東京大学
大学院総合文化研究科
佐藤守俊研究室

助教

赤色光作動性の遺伝子発現光操作技術の開発
と改良研究

13

石黒啓一郎

熊本大学 発生医学研究所准教授

不妊不育に関わる原因遺伝子の解明

14

シャリフ
ジャファル

理化学研究所 生命医科学研究センター （IMS）
免疫器官形成研究チーム

専任
研究員

ゲノム ・ エピゲノム ・ トランスクリプトーム （GET）
による DNA 複製の多層的な制御及び細胞老化におけるその生物学的な機能

建

淳

緑

教授

研 究 課 題
生体内における記憶ヘルパーＴ細胞の役割

脳形成における胎生期 ・ 発達期の獲得免疫細胞の意義

The Novartis Foundation(Japan)
for the Promotion of Science

136

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

属

職位

研 究 課 題アルツハイマー病発症機構解明に向けた神経細胞内 MARK4 基質の網羅的な同定

15

安藤香奈絵

東京都立大学
大学院理学研究科
神経分子機能研究室

准教授

16

金丸 和典

日本大学
医学部細胞分子薬理学部門

准教授 新規生体内β細胞 Ca2+ イメージング法による（研究所） 生体内インスリン動態解析

17松井東北大学大学院
生命科学研究科
超回路脳機能分野

教授

社会的隔離による行動障害と脳内グリア機能の
連関の解明

18

アディソン
ウィリアム

九州歯科大学
分子情報生化学助教

ICF 症 候 群 の 間 葉 系 幹 細 胞 モ デ ル に お け る
DNA メチル化と転写制御のゲノムワイド解析

19

野間 健一

北海道大学
遺伝子病制御研究所教授

ヒト老化細胞に形成される 3D ゲノム構造とその形成に関わるタンパク質組成の解明

20

武内 恒成

愛知医科大学
医学部生物学教授

AI モーションキャプチャーによる中枢神経損傷
後生理機能回復 - シナプスコネクターと再生阻害因子制御を利用した脊髄損傷後超回復モデ
ル-

21

滝澤

大阪大学
産業科学研究所准教授

光学活性化合物の迅速合成と機械学習を基盤とする医薬資源の探索

22

藤枝 伸宇

大阪府立大学
大学院生命環境科学研究科
生物物理化学研究室准教授

立体分岐型合成を実現するタンパク質配位子ライブラリーの開発

23

高松 由基

国立感染症研究所
ウイルス第一部

主任
研究官

新型コロナウイルスの細胞内動態の解明

24

岩脇 隆夫

金沢医科大学
総合医学研究所
細胞医学研究分野

教授

細胞ストレス応答と炎症反応の解析から迫る 「疲労メカニズム」 の分子細胞生物学的な解明

25

天野 恭志

近畿大学医学部 生化学教室

助教

Sirt1-NAD+ 経路による炎症性腸疾患とその関連大腸がんの抑制機構の解明

26

高井

埼玉県立がんセンター
乳腺腫瘍内科医長

PDX モデルにおける乳癌転移に必須な宿主因子の解析と標的治療の探索

27

岡村 勝友

奈良先端科学技術大学院大学
RNA 分子医科学研究室

教授

miRNA 生合成制御に関わる RNA 結合蛋白質の網羅的同定のための数理学生化学解析的アプローチの開発28藤原 英晃

岡山大学病院 血液 ・ 腫瘍内科

助教

Dysbiosis に基づく腸管上皮細胞代謝異常を標的とする新規 GVHD 機序の解明と治療応用

29

木村 真也

明治薬科大学 薬化学研究室

助教

中枢への薬物輸送を可能にする超分子ヒドロゲ
ルの開発

30

棚谷

綾

お茶の水女子大学
基幹研究院自然科学系

教授

胆汁酸をリードとした高活性な非セコステロイド型ビタミン D 誘導体の創製

31

鈴木

匠

茨城大学
発生生物学研究室

助教

神経幹細胞が多種多様な神経を作り分ける分子機構の解明32

山本 毅士

大阪大学医学部附属病院
血液浄化部

医員

シングルセル解析とリピドーム解析を用いた、 急性腎障害が慢性腎臓病へ移行する機序の解明

33

酒井真志人

日本医科大学
大学院医学研究科
分子遺伝医学分野

教授

肝臓における摂食依存性の遺伝子発現の細胞間相互作用を介した制御機構の解明

34

今村 拓也

広島大学
大学院統合生命科学研究科
生命医科学プログラム

教授

ヒト形質賦与マウス作製に向けたノンコーディング
RNA 情報活用システムズアプローチ

35

西谷 友重

和歌山県立医科大学
薬理学講座

教授

Ca2+ シグナル制御因子を介した新規エネルギ
ー代謝調節機構の解明

36

山城 義人

筑波大学
生存ダイナミクス研究センター

助教

細胞外マトリクスを介した血管機能を制御する新しい分子メカニズムの解析とマウスモデルを用いた治療効果の検証

広忍健The Novartis Foundation(Japan)
for the Promotion of Science

137

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

37

須山 律子

38

黒坂

39

井上

所

属

職位

研 究 課 題大阪大学
大学院生命機能研究科
生殖生物学研究室

特任
助教

マイクロバイオームが亢進する生殖機能とその分子機構の解明寛

大阪大学大学院歯学研究科
顎顔面口腔矯正学教室

講師

胎生時期に着目した偽性副甲状腺機能低下症の新規治療方法の開発

啓

金沢大学
新学術創成研究機構栄養 ・ 代謝
研究ユニット

教授

神経性臓器連関のメカニズムから読み解く肝代
謝恒常性とその破綻の解明

The Novartis Foundation(Japan)
for the Promotion of Science138

The Novartis Foundation(Japan)
for the Promotion of Science

2020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。 2020年度は9 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

開催地／
開催日

研 究 集 会 名

2021 Asia Pacific Drosophila
Neuroscience Conference1ムーア
エイドリアン

理化学研究所 脳神経科学研究センター
神経細胞多様性研究
チーム

チーム
リーダー

埼玉県／
2023.32新井 康通

慶應義塾大学
医学部百寿総合研究
センター

専任講師

東京／
2021.6.22-7.3

国際百寿者研究会

3

阿部 郁朗

東京大学大学院
薬学系研究科教 授

2022

天然物生合成国際
シンポジウム

4

諸藤 陽一

長崎大学
脳神経外科

助 教

長崎／
2021.3.26-27

血液脳関門シンポジウム―基礎から臨床まで―

5

中村 卓郎

公益財団法人
がん研究会
がん研究所発
がん研究部

副所長

札幌／
2021.7.5-7

第 39 回札幌国際がんシンポジウム

6

大井 貴史

名古屋大学
トランスフォーマティブ
生命分子研究所

教 授

愛知／2022.7

第 2 回ハイブリッド触媒に関す
る国際会議

7

金井

東京大学
大学院薬学系研究科
有機合成化学教室

教 授

東京／
2021.11.29-12.2

第 13 回
アジア医薬化学連合
国際メディシナルケミストリーシン
ポジウム

8

和中 明生

奈良県立医科大学
解剖学第二講座教 授

奈良／2021.9.29-10.1

第 64 回日本神経化学会大会

9

田中 知明

千葉大学大学院
医学研究院
分子病態解析学

教 授

千葉／
2021.7.8-10

第 39 回内分泌代謝学サマーセミナー

求

The Novartis Foundation(Japan)
for the Promotion of Science

139

The Novartis Foundation(Japan)
for the Promotion of Science

Promotion Results according to the PROGRAM(Unit : 10k yen)
Research Grants
Year

1987

# of
people18

Amount1,800

Meeting Grants# ofpeopleAmount8400

Japan-Europe
Research Exchange# ofpeople# ofpeopleAmount0Oversea Reseach Trip0

Amount
0

0

1988

39

3,900

8

400

7

1,740

0

0

1989

42

4,200

8

400

9

2,260

0

0

1990

51

7,650

10

500

8

2,440

0

0

1991

55

11,000

11

550

9

2,710

9

250

1992

50

10,000

10

500

10

3,315

8

265

1993

50

10,000

10

500

11

3,511

9

300

1994

50

10,000

10

500

8

2,530

6

155

1995

50

6,500

10

500

7

2,020

6

170

1996

45

5,850

10

500

6

1,600

4

120

1997

41

4,920

10

500

6

1,610

2

55

1998

41

4,920

10

500

4

1,070

8

160

1999

41

4,920

10

500

4

710

8

160

2000

41

4,100

8

400

3

660

0

0

2001

41

4,100

7

350

2

440

0

0

2002

40

4,000

8

400

0

0

0

0

2003

40

4,000

4

200

0

0

0

0

2004

45

4,500

5

200

0

0

0

0

2005

45

4,500

5

200

0

0

0

0

2006

46

4,600

6

240

0

0

0

0

2007

50

5,000

6

240

0

0

0

0

2008

45

4,500

7

280

0

0

0

0

2009

30

3,000

6

240

0

0

0

0

2010

38

3,800

5

200

0

0

0

0

2011

41

4,100

6

240

0

0

0

0

2012

40

4,000

6

240

0

0

0

0

2013

42

4,200

5

200

0

0

0

0

2014

42

4,200

6

240

0

0

0

0

2015

35

3,500

6

240

0

0

0

0

2016

35

3,500

5

200

0

0

0

0

2017

41

4,100

5

200

0

0

0

0

2018

37

3,700

5

200

0

0

0

0

2019

37

3,700

5

200

0

0

0

0

2020

39

3,900

9

360

0

0

0

0

1,347

163,060

236

10,76094

26,616

60

1,635

Total

The Novartis Foundation(Japan)
for the Promotion of Science

140

The Novartis Foundation(Japan)
for the Promotion of Science

Promotion Results according to the PROGRAM(Unit : 10k yen)Year

Travel Expense to
Japan
# of
peopleSpecial Grant# ofpeopleAmountTotal # ofpeopleTotal
AmountAmount

1987

0

0

0

0

26

2,200

1988

0

0

0

0

54

6,040

1989

0

0

0

0

59

6,860

1990

0

0

0

0

69

10,590

1991

0

0

0

0

84

14,510

1992

0

0

0

0

78

14,080

1993

0

0

0

0

80

14,311

1994

0

0

2

110

76

13,295

1995

0

0

1

50

74

9,240

1996

0

0

0

0

65

8,070

1997

0

0

1

30

60

7,115

1998

0

0

0

0

63

6,650

1999

0

0

4

130

67

6,420

2000

0

0

3

142

55

5,302

2001

0

0

3

120

53

5,010

2002

0

0

0

0

48

4,400

2003

0

0

0

0

44

4,200

2004

0

0

0

0

50

4,700

2005

0

0

0

0

50

4,700

2006

0

0

0

0

52

4,840

2007

5

1,000

0

0

61

6,240

2008

5

1,000

0

0

57

5,780

2009

3

600

0

0

39

3,840

2010

0

0

0

0

43

4,000

2011

0

0

0

0

47

4,340

2012

0

0

0

0

46

4,240

2013

0

0

0

0

47

4,400

2014

0

0

0

0

48

4,440

2015

0

0

0

0

41

3,740

2016

0

0

0

0

40

3,700

2017

0

0

0

0

46

4,300

2018

0

0

0

0

42

3,900

2019

0

0

0

0

42

3,900

2020
Total

0

0

0

0

48

4,260

13

2,600

14

582

1,854

213,613

The Novartis Foundation(Japan)
for the Promotion of Science

141

The Novartis Foundation(Japan)
for the Promotion of Science

助成金実績一覧表（単位：万円）
研究奨励金
年号

人数

研究集会

助成額

人数

日欧研究交流

助成額

人数

海外出張助成

助成金額

人数

助成金額

1987

18

1,800

8

400

0

0

0

0

1988

39

3,900

8

400

7

1,740

0

0

1989

42

4,200

8

400

9

2,260

0

0

1990

51

7,650

10

500

8

2,440

0

0

1991

55

11,000

11

550

9

2,710

9

250

1992

50

10,000

10

500

10

3,315

8

265

1993

50

10,000

10

500

11

3,511

9

300

1994

50

10,000

10

500

8

2,530

6

155

1995

50

6,500

10

500

7

2,020

6

170

1996

45

5,850

10

500

6

1,600

4

120

1997

41

4,920

10

500

6

1,610

2

55

1998

41

4,920

10

500

4

1,070

8

160

1999

41

4,920

10

500

4

710

8

160

2000

41

4,100

8

400

3

660

0

0

2001

41

4,100

7

350

2

440

0

0

2002

40

4,000

8

400

0

0

0

0

2003

40

4,000

4

200

0

0

0

0

2004

45

4,500

5

200

0

0

0

0

2005

45

4,500

5

200

0

0

0

0

2006

46

4,600

6

240

0

0

0

0

2007

50

5,000

6

240

0

0

0

0

2008

45

4,500

7

280

0

0

0

0

2009

30

3,000

6

240

0

0

0

0

2010

38

3,800

5

200

0

0

0

0

2011

41

4,100

6

240

0

0

0

0

2012

40

4,000

6

240

0

0

0

0

2013

42

4,200

5

200

0

0

0

0

2014

42

4,200

6

240

0

0

0

0

2015

35

3,500

6

240

0

0

0

0

2016

35

3,500

5

200

0

0

0

0

2017

41

4,100

5

200

0

0

0

0

2018

37

3,700

5

200

0

0

0

0

2019

37

3,700

5

200

0

0

0

0

2020

39

3,900

9

360

0

0

0

0

1,347

163,060

236

10,76094

26,616

60

1,635

Total

The Novartis Foundation(Japan)
for the Promotion of Science

142

The Novartis Foundation(Japan)
for the Promotion of Science

助成金実績一覧表（単位：万円）
海外受入
年号

特別助成
人数

助成金額

人数計

金額合計

助成人数

助成金額

1987

0

0

0

0

26

2,200

1988

0

0

0

0

54

6,040

1989

0

0

0

0

59

6,860

1990

0

0

0

0

69

10,590

1991

0

0

0

0

84

14,510

1992

0

0

0

0

78

14,080

1993

0

0

0

0

80

14,311

1994

0

0

2

110

76

13,295

1995

0

0

1

50

74

9,240

1996

0

0

0

0

65

8,070

1997

0

0

1

30

60

7,115

1998

0

0

0

0

63

6,650

1999

0

0

4

130

67

6,420

2000

0

0

3

142

55

5,302

2001

0

0

3

120

53

5,010

2002

0

0

0

0

48

4,400

2003

0

0

0

0

44

4,200

2004

0

0

0

0

50

4,700

2005

0

0

0

0

50

4,700

2006

0

0

0

0

52

4,840

2007

5

1,000

0

0

61

6,240

2008

5

1,000

0

0

57

5,780

2009

3

600

0

0

39

3,840

2010

0

0

0

0

43

4,000

2011

0

0

0

0

47

4,340

2012

0

0

0

0

46

4,240

2013

0

0

0

0

47

4,400

2014

0

0

0

0

48

4,440

2015

0

0

0

0

41

3,740

2016

0

0

0

0

40

3,700

2017

0

0

0

0

46

4,300

2018

0

0

0

0

42

3,900

2019

0

0

0

0

42

3,900

2020

0

0

0

0

48

4,260

13

2,600

14

582

1,854

213,613Total

The Novartis Foundation(Japan)
for the Promotion of Science143

The Novartis Foundation(Japan)
for the Promotion of Science

34th Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2021

From April 1 st, 2020 to March 31, 2021
（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

21,599,548

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

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

Fixed Assets Total
Assets Total

120,594

80,035,442

15,088,926

Donation

40,330,000

Other Income

560,827
55,979,753(2) Ordinaty Expenditure
Project Expense

50,367,712

Grant Expense

42,600,000

Novartis Research Grant

39,000,000

1,201,755,584

Research Meeting Grant

3,600,000

Administrative Expense

3,592,833

Ordinary Expenditure Total

53,960,545

41,354,175

Ordinary Balance without Appraisal Profit or Loss

2,019,208

41,354,175

2. Nonrecurring Profit & Loss

1. Current Liabilities

Liabilities Total

Interest from basic fund

1,180,156,036

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

01. Designated Net Assets

General Net Assets Ending Balance

160,280,815

Designated Net Assets Total（Amount Appropriating to basic Fund）(Amount Appropriating to specific assets)

1,000,120,594

Ⅱ Designated Net Assets Changes(1,000,000,000)

Designated Net Assets Change(330,000)(120,494)

Designated Net Assets Ending Balance

1,000,120,594

2.General Net Assets

160,280,815

Ⅲ Net Assets Balance Ending Balance

1,160,401,409（Amount Appropriating to）(100,000,000)

Equity Total（Net Assets）

1,160,401,409

Liabilities & Equity Total

1,201,755,584

The Novartis Foundation(Japan)
for the Promotion of Science

144

The Novartis Foundation(Japan)
for the Promotion of Science

第34期 （2020年度） 財務報告

貸借対照表

正味財産増減計算書

2021 年 3 月 31 日現在

2020 年 4 月 1 日から 2021 年 3 月 31 日まで（単位：円）

科

目

金 額

Ⅰ資産の部

21,599,548⑴ 基本財産1,100,000,000⑵ 特定資産特定資産合計固定資産合計
資産合計

120,594

80,035,442

55,979,753⑵ 経常費用

支払助成金

50,367,712
42,600,000

3,600,000

管理費

3,592,833

経常費用 計53,960,545

41,354,175

当期経常増減額

2,019,208

41,354,175

２． 経常外増減の部
当期経常外増減額
一般正味財産期末残高

1,000,120,594（ う ち 特 定 資産 へ の 充 当 額 ）（120,494）

負債及び正味財産合計

560,827

研究集会助成金（1,000,000,000）

正味財産合計雑収益

1,201,755,584（ う ち 基 本 財産 へ の 充 当 額 ）（ う ち 基 本 財産 へ の 充 当 額 ）40,330,000

39,000,000

１ ． 指 定 正 味財 産

２ ． 一 般 正 味財 産

受取寄付金

ノバルティス研究奨励金

Ⅲ正味財産の部

指定正味財産合計

15,088,926

1,180,156,036

１． 流動負債

負債合計

基本財産運用益

事業費

Ⅱ負債の部

流動負債合計金 額⑴ 経常収益

経常収益 計⑶ その 他 固 定 資 産
その 他 固 定 資 産 合 計

目

１． 経常増減の部

２． 固定資産

基本財産合計科

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）

160,280,815

0160,280,815

Ⅱ指定正味財産増減の部
当期指定正味財産増減額
指定正味財産期末残高
Ⅲ正味財産期末残高（330,000）
1,000,120,594
1,160,401,409（100,000,000）
1,160,401,409
1,201,755,584

The Novartis Foundation(Japan)
for the Promotion of Science

145

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
Post
Chairman

Trustee

Name

As of July 1, 2021
Title

Kuniaki Takata, Ph.D. President, Gunma Prefectural Public University Corporation
Sadayoshi Ito, M.D.,
Ph.D.

Special Administrator, Katta General Hospital
Professor Emeritus, Tohoku University

Akimichi Kaneko,
M.D., Ph.D.

Professor Emeritus, Keio University

Fujio Murakami, Ph.D. Professor Emeritus, Osaka University
Leo LeePresident, Novartis Pharma K.K.

Tokuzo Nakajima, CPA Representative, Tokuzo Nakajima CPA Firm
AuditorMasanori Fuse

Financial Advisor, Novartis Pharma K.K.

[Board of Councilors] 10 councilors
Post
Chairman

Name

As of July 1, 2021
Title

Takao Shimizu, M.D., Project Head, National Center for Global Health and Medicine
Ph.D.
Professor Emeritus, University of Tokyo
Senior Professor, International Research Center for
Masamitsu Iino, M.D.,
Neurointelligence, University of Tokyo
Ph.D.
Professor Emeritus, University of Tokyo
Hiroyuki Kawashima,
Ph.D

Former Professor, Niigata University

Tsuneyoshi Kuroiwa,
Ph.D.

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

Masakatsu Shibasaki, President, Microbial Chemistry Research Foundation
Ph.D.
Professor Emeritus, University of Tokyo
Councilor

Akihiko Nakano, Ph.D.

Deputy Director, RIKEN Center for Advanced Photonics
Professor Emeritus, University of Tokyo

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

Trustee, Foundation for Biomedical Research and Innovation at
Kobe
Director, Institute of Biomedical Research and Innovation
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

146

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

Name

As of July. 1, 2021
Title

Hiroshi Ito, M.D., Ph.D. Professor, Keio University School of Medicine,
Takashi Ohshima,
Ph.D.

Professor, Kyushu University Graduate School of Pharmaceutical
Sciences

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.,
Professor, Juntendo University Graduate School of Medicine
Ph.D
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.
MemberProfessor, 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

Fumitoshi Kakiuchi,
Ph.D.

Professor, Keio University School of Fundamental Science andTechnology

Hiroshi Kawasaki,
M.D., Ph.D.

Professor, Kanazawa University Faculty of Medicine

Shoen Kume, Ph.D.

Professor, Tokyo Institute of Technology School of Life Science andTechnology

Kiyoshi Takeda, M.D.,
Professor, Osaka University Graduate School of Medicine
Ph.D.
Kazuhiro Nakamura,
Ph.D.

Professor, Nagoya University Graduate School of Medicine

Tetsuya Higashiyama, Professor, Nagoya University Institute of Transformative BioPh.D.
Molecules
Sachiko Miyake, M.D.,
Professor, Juntendo University Graduate School of Medicine
Ph.D.
Hozumi Motohashi,
M.D., Ph.D.

Professor, Tohoku University Institute of Development, Aging andCancer

The Novartis Foundation(Japan)
for the Promotion of Science

147

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［理事 ・ 監事］
任期2020年6月17日～ 2022年6月
2020年11月26日～ 2022年6月

2021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

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

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

レオ リー

ノバルティス ファーマ株式会社 代表取締役社長 *

理 事

現

職

任期 2020年6月17日～ 2024年6月
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

監 事

［評議員］
任期 2020年6月17日～ 2024年6月

2021年7月1日現在 （敬称略）

職 名

氏 名

評議員長

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

飯野 正光

東京大学ニューロインテリジェンス国際研究機構 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

黒岩 常祥

日本学士院会員
東京大学名誉教授

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

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

森 美和子

北海道大学名誉教授

廣瀬

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

評議員

徹

現

職

The Novartis Foundation(Japan)
for the Promotion of Science

148

The Novartis Foundation(Japan)
for the Promotion of Science［選考委員］
2021年7月1日現在 （敬称略）
職 名
選考委員長

選考委員

氏 名
伊藤

裕

現

職

慶應義塾大学 医学部 教授

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

順天堂大学 大学院医学研究科 教授

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

倉永英里奈

東北大学 大学院生命科学研究科 教授

斎藤 通紀

京都大学 高等研究院 教授

垣内 史敏

慶應義塾大学 理工学部 教授

河崎 洋志

金沢大学 医学系 教授

粂昭苑

東京工業大学 生命理工学院 教授

竹田潔

大阪大学 大学院医学系研究科 教授

中村 和弘

名古屋大学 大学院医学系研究科 教授

東山 哲也

名古屋大学 トランスフォーマティブ生命分子研究所 教授

三宅 幸子

順天堂大学 大学院医学研究科 教授

本橋ほづみ

東北大学 加齢医学研究所 教授

The Novartis Foundation(Japan)
for the Promotion of Science149

事 務 局 便 り
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。
法人 ： 支出した寄付金は、 通常一般の寄付金の損金算入限度額とは別枠で、 下記の範囲内で損金の算入できます。
（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。
（E-メール ： foundation.japan@novartis.com）

事務局より
2021年度もお陰様で財団年報を発行する運びとなりました。 2020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したが、 ２年も近く経ちますと、 何が日常で何が非日常だったのかがだんだんと区別がつかなくなってきました。 10月になり、 ワクチ
ンの効果がでてきたせいでしょうか感染者数が急激に減ってきました。 考えてみれば、 このmRNAワクチンも、 多くの基礎研究が積み上げられた結果であることに思いを致しております。
このような日常の中でも、 当財団の事業である研究奨励金助成 ・ 研究集会助成事業に対して多くの応募があり、 その中から選ばれた研究に助成が行われ、 それぞれに成果を上げられていることを嬉しく思います。
当財団は1987年９月の財団設立以来、 助成件数は総数で1854件、 総額21億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ていることも、 偏に、 助成事業にご理解 ・ ご支援をいただいた方々、 助成を受けられて研究を継続さ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。
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
E-メール：foundation.japan@novartis.com
ホームページ：http://japanfoundation.novartis.org/